Surgical Management of Vitiligo

Surgical Management of Vitiligo EDITED BY

Somesh Gupta,

MD, DNB

Department of Dermatology and Venereology All India Institute of Medical Sciences New Delhi, India

Mats J. Olsson,

PhD

Department of Medical Sciences Section of Dermatology and Venereology University Hospital Uppsala, Sweden

Amrinder J. Kanwar,

MD

Department of Dermatology, Venereology and Leprology Postgraduate Institute of Medical Education and Research Chandigarh, India

Jean-Paul Ortonne, Department of Dermatology Nice University Hospital Nice, France

MD

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Surgical Management of Vitiligo

Dedicated to my parents, Dr. Ram Pratap Gupta and Indra Gupta, who nurtured my interest in Medicine, to my wife Dr. Meenu who was with me all along the way, and to my yet unborn children (twins) through whom I see my vision for the future. Somesh Gupta To the patients and to my most highly respected friends, colleagues, and mentors Dr. Aaron B. Lerner (Yale University) and the late Dr. Lennart Juhlin (Uppsala University) for always supporting my thoughts and development and for an absolutely reliable friendship. Mats J. Olsson Dedicated to vitiligo patients Amrinder J. Kanwar

Surgical Management of Vitiligo EDITED BY

Somesh Gupta,

MD, DNB

Department of Dermatology and Venereology All India Institute of Medical Sciences New Delhi, India

Mats J. Olsson,

PhD

Department of Medical Sciences Section of Dermatology and Venereology University Hospital Uppsala, Sweden

Amrinder J. Kanwar,

MD

Department of Dermatology, Venereology and Leprology Postgraduate Institute of Medical Education and Research Chandigarh, India

Jean-Paul Ortonne, Department of Dermatology Nice University Hospital Nice, France

MD

© 2007 by Blackwell Publishing Ltd Blackwell Publishing, Inc., 350 Main Street, Malden, Massachusetts 02148-5020, USA Blackwell Publishing Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UK Blackwell Publishing Asia Pty Ltd, 550 Swanston Street, Carlton, Victoria 3053, Australia The right of the Author to be identified as the Author of this Work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. First published 2007 1 2007 Library of Congress Cataloging-in-Publication Data Surgical management of vitiligo / edited by Somesh Gupta ... [et al.] p. ; cm. Includes bibliographical references. ISBN-13: 978-1-4051-4521-3 (alk. paper) ISBN-10: 1-4051-4521-8 (alk. paper) 1. Vitilio—Surgery. I. Gupta, Somesh. [DNLM: 1. Vitiligo—Surgery. 2. Cell Transplantation—methods. 3. Skin Transplantation—methods. WR 265 S961 2007] RL790.S87 2007 616.5’5—dc22 2006015410 ISBN-13: 978-1-4051-4521-3 ISBN-10: 1-4051-4521-8 A catalogue record for this title is available from the British Library. Set in (9/12 pts Meridien) by Charon Tec Ltd (A Macmillan Company), Chennai, India www.charontec.com Printed and bound in Singapore by C.O.S. Printers Pte Ltd Commissioning Editor: Stuart Taylor Editorial Assistant: Jennifer Seward Development Editor: Elisabeth Dodds Production Controller: Kate Charman For further information on Blackwell Publishing, visit our website: http://www.blackwellpublishing.com The publisher’s policy is to use permanent paper from mills that operate a sustainable forestry policy, and which has been manufactured from pulp processed using acid-free and elementary chlorine-free practices. Furthermore, the publisher ensures that the text paper and cover board used have met acceptable environmental accreditation standards.

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Contents

List of contributors, viii Foreword, xi Preface, xiii

Section 1 Pathogenesis and medical treatment 1 Pathogenesis of vitiligo, 3

Jean-Paul Ortonne 2 Understanding the mechanism of repigmentation in vitiligo, 14

Amrinder J. Kanwar and Davinder Parsad 3 Classification of vitiligo, 20

Sang Ju Lee, Sung Bin Cho and Seung-Kyung Hann 4 Medical treatment of vitiligo, 31

Thierry Passeron and Jean-Paul Ortonne

Section 2 Overview of surgical management 5 History and chronology of development of surgical therapies for vitiligo, 41

Rafael Falabella 6 The concept of stability of vitiligo, 49

Koushik Lahiri and Subrata Malakar 7 Patient selection and preoperative information in surgical therapies for vitiligo, 56

Nanny van Geel and Jean Marie Naeyaert 8 Classification of surgical therapies for vitiligo, 59

Philippe Bahadoran and Jean-Paul Ortonne 9 Surgical management of vitiligo and other leukodermas: evidence-based

practice guidelines, 69 Somesh Gupta, Tarun Narang, Mats J. Olsson and Jean-Paul Ortonne 10 Evaluation of outcome in surgical therapies for vitiligo, 80

Nanny van Geel and Jean Marie Naeyaert

v

vi

Contents

Section 3 Tissue grafting 11 Minigrafting for vitiligo, 87

Subrata Malakar and Koushik Lahiri 12 Suction blister epidermal grafting, 96

Somesh Gupta and Ashima Goel 13 Thin split-thickness skin grafts for vitiligo, 108

Niti Khunger 14 Treatment of leukoderma by transplantation of ultra-thin

epidermal sheets 115 Mats J. Olsson 15 Transplantation of hair follicles for vitiligo, 123

Subrata Malakar, Gun Yoen Na and Koushik Lahiri 16 Mesh grafts for vitiligo, 128

C.R. Srinivas, Reena Rai and M. Sinha 17 Flip-top pigment transplantation, 134

Brent E. Pennington, Jean L. Bolognia and David J. Leffell 18 Ultrasonic abrasion and seed grafts for vitiligo, 139

Katsuhiko Tsukamoto, Reiko Kitamura and Osami Takayama 19 Complications and limitations of melanocyte transplantation, 144

Yvon Gauthier

Section 4 Cellular grafting 20 Treatment of leukoderma by transplantation of basal cell layer

suspension, 151 Mats J. Olsson 21 Setting up a tissue culture laboratory, 161

Rafal Czajkowski, Tomasz Drewa and Waldemar Placek 22 Treatment of leukoderma by transplantation of cultured autologous

melanocytes, 168 Mats J. Olsson 23 Transplantation of in vitro cultured epithelial grafts for vitiligo and

piebaldism, 180 Liliana Guerra, Sergio Bondanza and Desanka Raskovic 24 Simplifying the delivery of cultured melanocytes and keratinocytes for

grafting patients with vitiligo, 191 Sheila MacNeil and Paula Eves 25 Safety concerns in transplantation of in vitro cultured cellular grafts, 203

Liliana Guerra, Elena Dellambra and Patrizia Paterna

Section 5 Special issues 26 Post-surgery patient information, 209

Mats J. Olsson

Contents 27 Surgical management of lip vitiligo, 211

Somesh Gupta, Ashima Goel and Amrinder J. Kanwar 28 Surgical management of vitiligo of eyelids and genitals: special issues, 220

Somesh Gupta 29 Surgical management of acral vitiligo, 225

Sharad Mutalik 30 Surgical management of leukotrichia, 229

Karoon Agrawal and Aparna Agrawal 31 Surgical treatments of leukodermas other than vitiligo vulgaris, 238

Mats J. Olsson

Section 6 Miscellaneous 32 Micropigmentation, 249

Gurvinder P. Thami 33 Laser for repigmenting vitiligo, 255

Thierry Passeron and Jean-Paul Ortonne 34 Application of lasers in transplantation procedures for vitiligo, 259

Cengiz Acikel, Ersin Ulkur and Bahattin Celikoz 35 Combining medical and surgical therapies, 267

Alain Taïeb and Yvon Gauthier 36 Surgical depigmentation of vitiligo: bleaching cream, laser and cryosurgery, 273

Monique R.T.M. Thissen 37 Future directions in surgical management of vitiligo, 277

Yvon Gauthier 38 Informed consent, 281

Mats J. Olsson Index, 283 Color plate section appears after page 114

vii

List of contributors

Editors Somesh Gupta, MD, DNB Department of Dermatology and Venereology All India Institute of Medical Sciences New Delhi, India

Karoon Agrawal, MS, MCh(Plastic Surgery) Department of Plastic Surgery Jawaharlal Institute of Postgraduate Medical Education and Research Pondicherry, India

Philippe Bahadoran, MD, PhD Mats J. Olsson, PhD Department of Medical Sciences Section of Dermatology and Venereology University Hospital Uppsala, Sweden

Amrinder J. Kanwar, MD Department of Dermatology, Venereology and Leprology Postgraduate Institute of Medical Education and Research Chandigarh, India

Jean-Paul Ortonne, MD Department of Dermatology Nice University Hospital Nice, France

Department of Dermatology University Hospital Nice, France

Jean L. Bolognia, MD Department of Dermatology Yale University New Haven, CT, USA

Sergio Bondanza, BSc Laboratory of Tissue Engineering and Cutaneous Physiopathology Istituto Dermopatico dell’Immacolata, IDI-IRCCS Rome, Italy

Bahattin Celikoz, MD

Contributors

Department of Plastic Reconstructive and Aesthetic Surgery Gulhane Military Medical Academy Haydarpasa Hospital Istanbul, Turkey

Cengiz Acikel, MD Department of Plastic Reconstructive and Aesthetic Surgery Gulhane Military Medical Academy Haydarpasa Hospital Istanbul, Turkey

Sung Bin Cho, MD

Aparna Agrawal, MD

Rafal Czajkowski, MD, PhD

Department of Plastic Surgery Jawaharlal Institute of Postgraduate Medical Education and Research Pondicherry, India

Departments of Dermatology and Tissue Engineering Nicolaus Copernicus University in Torun Ludwik Rydygier Medical College in Bydgoszcz Bydgoszcz, Poland

viii

Department of Dermatology Yonsei University College of Medicine Seoul, South Korea

List of contributors Elena Dellambra, PhD Laboratory of Tissue Engineering and Cutaneous Physiopathology Istituto Dermopatico dell’Immacolata, IDI-IRCCS Rome, Italy

ix

Koushik Lahiri, MBBS, DVD, DNBI, FAAD, PhD(Scholar) Pigmentary Disorder Unit Rita Skin Foundation Salt Lake, Kolkata, India

Tomasz Drewa, MD, PhD

Sang Ju Lee, MD

Department of Tissue Engineering Nicolaus Copernicus University in Torun Ludwik Rydygier Medical College in Bydgoszcz Bydgoszcz, Poland

Department of Dermatology Yonsei University College of Medicine Seoul, South Korea

David J. Leffell, MD Paula Eves, PhD Department of Engineering Materials The Kroto Research Institute University of Sheffield Sheffield, UK

Department of Dermatology Yale University New Haven, CT, USA

Sheila MacNeil, PhD

Department of Dermatology Universidad del Valle and Hospital Universitario del Valle Cali, Colombia

Department of Engineering Materials and Division of Clinical Sciences (North) The Kroto Research Institute University of Sheffield Sheffield, UK

Yvon Gauthier, MD

Subrata Malakar, MBBS, DCH, MD

Department of Dermatology Pigmentary Disorders Outpatient Clinic Hôpital Saint André Bordeaux, France

Pigmentary Disorder Unit Rita Skin Foundation Salt Lake, Kolkata, India

Rafael Falabella, MD

Sharad Mutalik, MBBS, DVD Ashima Goel, MD Department of Dermatology, Venereology and Leprology Postgraduate Institute of Medical Education and Research Chandigarh, India

Consultant Dermatologist, Dermatologic and Laser Surgeon Skin and Cosmetology Clinic Pune, India

Gun Yoen Na Liliana Guerra, MD Laboratory of Tissue Engineering and Cutaneous Physiopathology Istituto Dermopatico dell’Immacolata, IDI-IRCCS Rome, Italy

Department of Dermatology School of Medicine Kyungpook National University Hospital Daegu, South Korea

Jean Marie Naeyaert, MD, PhD Seung-Kyung Hann, MD Drs. Woo & Hann’s Skin Clinic Seoul, South Korea

Department of Dermatology Ghent University Hospital Ghent, Belgium

Niti Khunger, MD, DNB, DDV

Tarun Narang, MD

Safdarjang Hospital & V. M. Medical College New Delhi, India

Department of Dermatology and Venereology Postgraduate Institute of Medical Education and Research Chandigarh, India

Reiko Kitamura, MD, PhD Department of Dermatology University of Yamanashi Faculty of Medicine Yamanashi, Japan

Davinder Parsad, MD Department of Dermatology, Venereology and Leprology Postgraduate Institute of Medical Education and Research Chandigarh, India

x

List of contributors

Thierry Passeron, MD

Osami Takayama, MD

Department of Dermatology University Hospital of Nice Nice, France

Takayama Dermatology and Plastic Surgery Clinic Yamanashi, Japan

Patrizia Paterna, BSc Laboratory of Tissue Engineering and Cutaneous Physiopathology Istituto Dermopatico dell’Immacolata, IDI-IRCCS Rome, Italy

Alain Taïeb, MD Department of Dermatology Hôpital St André Centre Hospitalier et Universitaire de Bordeaux Bordeaux, France

Brent E. Pennington, MD

Gurvinder P. Thami, MD

Nashville Skin and Cancer Nashville, TN, USA

Department of Dermatology and Venereology Government Medical College and Hospital Chandigarh, India

Waldemar Placek, MD, PhD Department of Dermatology Nicolaus Copernicus University in Torun Ludwik Rydygier Medical College in Bydgoszcz Bydgoszcz, Poland

Monique R.T.M. Thissen, MD, PhD Department of Dermatology and Venereology University Hospital Maastricht Maastricht, The Netherlands

Reena Rai, MD Department of Dermatology, Venereology, and Leprology PSG Institute of Medical Sciences and Research Coimbatore, Tamil Nadu, India

Katsuhiko Tsukamoto, MD, PhD Department of Dermatology Yamanashi Prefectural Central Hospital Yamanashi, Japan

Desanka Raskovic, MD VI Division of Dermatology Istituto Dermopatico dell’Immacolata, IDI-IRCCS Rome, Italy

M. Sinha, MS, MRCSEd Department of Plastic and Reconstructive Surgery Canniesburn Plastic Surgery Unit Glasgow Royal Infirmary Glasgow, UK

C.R. Srinivas, MD Department of Dermatology, Venereology, and Leprology PSG Institute of Medical Sciences and Research Coimbatore, Tamil Nadu, India

Ersin Ulkur, MD Department of Plastic Reconstructive and Aesthetic Surgery Gulhane Military Medical Academy Haydarpasa Hospital Istanbul, Turkey

Nanny van Geel, MD, PhD Department of Dermatology Ghent University Hospital Ghent, Belgium

Foreword

To raise new questions, new possibilities, to regard old problems from a new angle, requires creative imagination and marks real advance in science.

Albert Einstein Many researchers throughout the world have dedicated their time and lives in search of the etiology of vitiligo, an important skin pathology that although physically asymptomatic in most patients, provokes profound changes in psychological behavior and social interaction with other individuals in daily life. And most important, in spite of multiple known factors involved in its pathogenesis that have been published in many journals devoted to different disciplines – from the laboratory to the ambulatory patient – finding the cause of this condition has been elusive. To our satisfaction, remarkable progress has been made in the past two decades. When medical therapy is no longer useful for treating depigmented lesions, melanocyte transplantation, performed judiciously, provides acceptable repigmentation for

an important proportion of appropriately selected patients presenting with stable disease. We are fortunate that Professors Somesh Gupta, Mats J. Olsson, Amrinder J. Kanwar and Jean-Paul Ortonne have provided the readers with a superb book consisting of 38 chapters written by 48 international authorities from 13 countries, in their fields of expertise. This magnificent book is the most comprehensive scientific work written to date, in which all topics of surgical management of vitiligo are covered by a panel of well known authors. Not only are all pertinent surgical topics dealt with in fine detail, but also the current understanding of pathogenic mechanisms and clinical aspects of the disease are described, providing the foundations for determining which patients may be candidates for surgical repigmentation therapy. Finally, after reading the contents and concepts of Surgical Management of Vitiligo, I was inspired to express the following words: “Innovation increases human knowledge though sometimes originates controversy, which in turn stimulates improvement.” Rafael Falabella, MD

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Preface

The concept of surgically treating vitiligo was first proposed by a few pioneers in the 1960s. Over the years, this technique has developed considerably and is becoming progressively more sophisticated. Vitiligo is characterised by a lack of melanocytes, which are destroyed by various unidentified mechanisms. Initial attempts to repigment vitiligo skin were aimed at mobilising melanocytes from unaffected skin and/or hair follicles to depigmented skin lesions. This strategy, which is often tedious, does not consistently provide the desired results, even though a little more is now known about the mechanisms that control the migration and differentiation of melanocyte precursors found in hair follicles. Several autologous pigmented skin graft techniques for vitiligo patients have been developed in order to facilitate the proliferation and migration of melanocytes in grafts so that they colonise the vitiligous skin in which they were implanted. These techniques were micronised in order to reduce and avoid any skin abnormalities that could result from the healing of these grafts. They can be used for mucous membranes and on sensitive areas such as the eyelids, and areas that are resistant to repigmentation, such as the fingers. The transplantation of pigmented hair follicles to vitiligous skin has also been successfully explored. Cellular grafts of epidermal cell suspensions (keratinocytes and melanocytes) or autologous melanocytes cultivated prior to grafting have also been used successfully. Several groups even suggest grafting epidermis that is reconstructed in vitro,

consisting of both melanocytes and epidermal keratinocytes. What is the future for the surgical treatment of vitiligo? It is likely that these “biotherapies” will become part of future vitiligo treatments, either by using cytokines, or growth factors that facilitate the differentiation, proliferation and migration of melanocytes. Melanocyte progenitors and/or human embryonic stem cells will most likely have a role in future vitiligo biotherapies, to the extent that the tolerance and efficacy of factors that modulate pigment cells will have been demonstrated. Also, a U.S. team has recently reported that it is possible to grow functional melanocytes from human embryonic stem cells in vitro. One can therefore hope that the production of melanocytes for the repigmentation of vitiligous skin will become easier and more productive in the future. Cellular graft techniques also need to evolve. It is currently possible to prepare the ‘bed’ for melanocyte grafts by destroying the epidermis using lasers, while avoiding damage to the basal membrane at the epidermal-dermal junction. It is hoped that future techniques will improve this step further. I would like to thank Somesh Gupta and Amrinder J. Kanwar for inviting me to be involved in the publication of this book with Mats J. Olsson, one of the pioneers in melanocyte grafting for the treatment of vitiligo. The resulting monograph, the first publication that is dedicated to the surgical treatment of vitiligo, is a true encyclopedia containing contributions from most of the global teams

xiii

xiv

Preface

that surgically treat vitiligo. It will not be the last and should be regularly updated as progress is made in this area. I hope you enjoy reading it and I hope it meets your expectations. Perhaps in the future, effective therapies that block the processes that cause skin

depigmentation will become available. In the meantime, we need to continue the progress being made on repigmenting techniques. For several skin sites, surgical and biotherapeutic approaches will be the optimal choice. Jean-Paul Ortonne, MD

SECTION 1

Pathogenesis and medical treatment

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CHAPTER 1

Pathogenesis of vitiligo Jean-Paul Ortonne

Introduction Vitiligo is an acquired cutaneous hypomelanosis with a 0.5–2% incidence worldwide, without predilection for sex or ethnicity. The clinical presentation is characterized by well-circumscribed white macules. Several clinical phenotypes have been identified. Generalized vitiligo is characterized by acquired depigmentation due to melanocyte loss, in a pattern that is non-focal and generally bilateral across the midline, though not necessarily symmetric [1]. This definition differentiates generalized vitiligo from segmental vitiligo and other localized forms of vitiligo whose true pathogenic relationship to generalized vitiligo is as-yet unknown. This chapter is a review of the recent developments of vitiligo pathogenesis. There are three major hypotheses for the pathogenesis of vitiligo that are not exclusive of each other – the autoimmune hypothesis, the neuronal dysfunction hypothesis, and the melanocyte self-destruction hypothesis. Several other hypotheses have been recently proposed.

Vitiligo: a melanocyte disorder or more? Melanocytes present or absent in vitiligo macules? Vitiligo is characterized by a disappearance of epidermal and/or follicular melanocytes. It is likely that melanocytes are destroyed by an as-yet unknown process. Indeed, melanocyte destruction has never been clearly demonstrated [2]. One recent study reports that melanocytes are never completely absent in the skin [3]. Melanocyte cultures were successfully established from depigmented epidermal suction

blister roof of 12 randomly selected vitiligo patients. These “vitiligo” melanocytes produced melanin in vitro. Although interesting, these observations in a small group of patients cannot be generalized. The persistence of melanocytes within vitiligo macules was already reported in 1956 [4]. Besides the so-called “absolute” type of vitiligo in which there are no dopa-positive melanocytes in the vitiliginous epidermis, there are “relative” types of vitiligo in which melanocytes remain in the white macules with a decreased dopa-positivity. It is likely that the disappearance of epidermal melanocytes in vitiligo macules is not an immediate process, but is a progressive one. Indeed, it may be suggested that the “relative” types of vitiligo are considered possible forerunners of the “absolute” types. Thus, it would not be surprising to find melanocytes in the epidermis of the white macule of “relative” vitiligo. An immunohistological study of vitiliginous skin using a panel of melanocyte markers, related and unrelated to the melanogenic pathway, could not detect identifiable epidermal melanocytes [5]. From the presently available data, it is generally agreed that there are no longer functional melanocytes in vitiligo skin and that this loss of histochemically recognizable melanocytes is the result of their destruction. The impact of vitiligo on melanocyte stem cells (MSC) is not known. MSC are present in the bulge region of hair follicles in the adult skin in mice. Undifferentiated MSCs in the bulge have been shown to express the three transcription factors, PAX 3, SOX 10, and MITF. These factors play a key role in controlling the balance between MSC maintenance and differentiation. Studies to evaluate MSCs in vitiligo-pigmented and depigmented hair follicles are strongly required to better understand the mechanism of poliosis in vitiligo patients and

3

4

Chapter 1

the cellular event underlying perifollicular repigmentation of vitiligo [6].

Keratinocytes Several observations suggest that epidermal cells other than melanocytes are also altered in vitiligo involved and uninvolved skin. Epidermal keratinocytes produce several factors that support the growth and differentiation of neighboring melanocytes, such as basic fibroblast growth factor (b-FGF) and stem cell factors (SCF). A recent study demonstrated that the expression of SCF (P
0.001) and b-FGF was usually reduced in the depigmented compared with the normally pigmented vitiligo epidermis [7]. SCF has been demonstrated to prevent TRAIL-induced melanocyte apoptosis in vitro. These results suggest that melanocyte cell death in vitiligo can result from deprivation of keratinocyte-derived SCF. Cytoplasmic vacuolization and/or the presence of an extracellular granular material that may be derived from the cytoplasm of altered keratinocytes have been reported mainly in the adjacent normalappearing vitiligo skin, but also in the perilesional skin and rarely in the lesional skin [8]. Focal areas of vacuolar degeneration in the lowest layers of the epidermis, especially in the basal layer in association with mild mononuclear cell infiltrate, have also been observed [9]. The significance of these morphological observations is not known, but several hypotheses can be proposed. They may be related to architectural disturbances induced by a local immunological reaction. They may be due to toxic intermediate metabolites of melanogenesis, which destroy not only the pigment cell from which they originate [8], but also the adjoining keratinocytes. The recently proposed theory of a breakdown in the detoxification mechanisms in vitiligo skin fits very well with these observations.

these quantitative changes of Langerhans cells, a functional impairment of these cells has also been documented in vitiligo skin. How this functional impairment of Langerhans cells is related to the pathogenesis of vitiligo remains to be established. All these observations suggest that vitiligo affects the entire keratinocyte–Langerhans cell-melanocyte unit (KLM) [11]. In the epidermis there is a complex exchange of messages between these three cell types that is just beginning to be understood. No doubt a better clarification of these epidermal cell interactions will help in understanding the basic mechanisms involved in vitiligo.

Genetics of vitiligo Epidemiological data Familial studies have shown the increased prevalence of vitiligo in close relatives of affected individuals. In a large series performed in India, this increase was about 4.5-fold in close biological relatives [12]. Another study performed on 160 white kindred living in US shows a relative risk (RR) for vitiligo of about 7 for parents, about 12 for siblings, and about 36 for children [13]. The pattern of relationship between RR and degree of kinship indicates involvement of genetic factors, although it is not consistent with single-locus Mendelian transmission. The major genetic component in vitiligo pathogenesis and also the role of environmental factors were recently emphasized [14]. In this epidemiological study the frequency of vitiligo in probands’ siblings was 6.1%, about 18 times that of the population frequency. Nevertheless, the concordance of vitiligo in monozygotic twins was only 23%, indicating that a non-genetic component also plays an important role. Moreover, probands with earlier disease onset tended to have more relatives affected with vitiligo, suggesting a greater genetic component in early onset families.

Langerhans cells The role of Langerhans cells in vitiligo has been open to controversy. The Langerhans cell density evaluated either by histochemical techniques (ATPase) or with the monoclonal antibodies OKT6 and anti-HLADR has been variably reported as decreased, normal, or increased [10]. In addition to

One vitiligo or several vitiligos? For most authors, vitiligo is a unique disorder with several clinical presentations but one physiopathology. Indeed, almost all the recent genetic studies have ignored the clinical presentation of patients. However, recent data strongly suggest that there is not one

Pathogenesis of vitiligo vitiligo but several vitiligos. A complex segregation analysis was performed on 2247 Chinese patients and their families. For the first time the results were analyzed according to the clinical manifestations [15]. The results showed a different age of disease onset depending on the subtypes of vitiligo. More interestingly, a polygenetic additive model was found to be the best model for segmental, localized, acrofacial, and generalized vitiligo whereas the best model for universal vitiligo was an environmental model. All of these data suggest that heterogeneous pathogeneses underlie different phenotypes of vitiligo.

Genetic aspects of vitiligo The earliest genetic studies of vitiligo were casecontrol association studies of the major histocompatibility complex (MHC). They were carried out by testing various different vitiligo phenotypes versus controls in many different populations. Genetic association of vitiligo with alleles of MHC loci appeared to be strongest in patients and families with various vitiligo-associated autoimmune/autoinflammatory disorders versus patients and families with only generalized vitiligo. Thus, it is not clear whether the MHC association is with vitiligo, vitiligo-associated autoimmune/autoinflammatory disorders, or both. Allelic association between vitiligo and a number of other candidate genes has also been described (Table1.1).

Table 1.1 Susceptibility loci for vitiligo. References

Susceptibility locus

Mapping

[73]

SLEV1

17p13

[16]

AIS1

1p31.3–p32.2

[18]

AIS2

7p

[18]

AIS3

8q

[18]

[74]

6p21.3–21.4

[75]

4q13–q21

Modified from Passeron T, J Autoimmun 2005;25:63–8, and Spritz RA, J Dermatol Sci 2006;41:3–10.

5

Which gene(s) for vitiligo? Two large genome-wide screens for generalized vitiligo showed significant linkage of an oligogenic autoimmune susceptibility locus, termed AIS1 (1p31.3–p32.2) [16,[17]. An additional seven signals on chromosome 1,7,8,11,19, and 22 met genome-wide criteria for “suggestive linkage.” In an extended study with a cohort of 102 multiplex families the localization of AIS1 was confirmed and two new susceptibility loci have been found. AIS2 is located on chromosome 7 and AIS3 on chromosome 8. Additionally, the locus SLEV1 on chromosome 17 was confirmed and two new potential linkages on chromosome 9q and on 13q are also reported (Table 1.1) [18]. Interestingly, all loci except AIS3 derive principally from the autoimmunityassociated family subgroup. These loci may predispose to a vitiligo-associated autoimmunity diathesis. On the other hand, analyses suggest a linkage to SLEV1 in the autoimmune families and nonlinkage in the non-autoimmune families. Thus, linkage to SLEV1 in these families indicates that SLEV1 confers susceptibility to a broader range of autoimmune diseases than just lupus and vitiligo. A genome-wide linkage analysis in Chinese families identified interesting linkage evidence in 1p36, 4q13–21, 6p21–p22, 6q24–q25, 14q12–q13, and 22q12. These findings in the Chinese population shared a minimal overlap with the linkage findings in the Caucasian population. Such little overlap between the linkage findings of this two populations may suggest that vitiligo is associated with a strong genetic heterogeneity [19]. Many candidate genes for vitiligo have been proposed so far (Table 1.2). However, most of the loci described do not correspond to positions of these proposed biological candidate genes. Finally, one of the best candidate genes could be FOXD3 (“Forkhead box” D3). FOXD3 is located on chromosome 1 (1p32–p31) and is a transcription factor that suppresses melanoblast development from the neural crest [20]. Therefore, dysregulated (over-)expression might harm melanocytes. Moreover FOXD3 also regulates endodermal differentiation including thyroid, pancreas, adrenal, and gut [21] and other FOX factors are involved in autoimmune syndromes [22]. Mutations in FOXD3

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Chapter 1

Table 1.2 Vitiligo candidate genes. Gene

Mapping

Product

Disease

PTPN 22

1p13

Lymphoid protein tyrosine phosphatase

Vitiligo vulgaris

FOXD3

1p32–p31

Transcriptor factor involved in melanoblast differentiation

Early and progressive vitiligo

VIT 1/FBX 011

2p21

?

Vitiligo vulgaris

CTLA 4

2q33

Antigen-4 of T-cytotoxic lymphocytes

Vitiligo vulgaris

MITF

3p14.1–p12.3

Transcription factor

Vitiligo vulgaris

KIT

4q12

Transmembrane tyrosine kinase

Vitiligo vulgaris

MHC (HLA-DRB1, HLA-DRB4, HLA-DQB1)

6p21.1

Major MHC

Vitiligo vulgaris

ESR 1

6p25.1

Oestrogen receptor 1

Vitiligo vulgaris

CAT

11p13

Catalase

Vitiligo vulgaris

GTPCH (GTP-cyclohydoxylase I gene)

14q22.1–q22.2

Rate-limiting enzyme of the tetrahydrobiopterin pathway

Vitiligo vulgaris

ACE

17q23

Angiotensin converting enzymes

Vitiligo vulgaris

AIRE

21q22.3

Transcriptor factor

APECED

COMT

22q11.2

Catecholamine O methyl transferase

Vitiligo vulgaris

Modified from Passeron T, J Autoimmun 2005;25:63–8, and Spritz RA, J Dermatol Sci 2006;41:3–10.

leading to elevated FOXD3 transcription have been recently reported in one AIS1-linked family [23]. Thus, FOXD3 is worth further investigation and represents a serious candidate gene in AIS1-linked autoimmune disease.

Pathogenesis The classic hypotheses

Vitiligo is an autoimmune disease This theory is the most long-standing and popular hypothesis for the pathogenesis of vitiligo. It proposes that melanocytes are killed by autoimmune effector mechanisms.

Association with autoimmune disease Sporadic generalized vitiligo is associated with autoimmune thyroid disease, pernicious anemia, Addison’s disease, systemic lupus erythematosus [14]. Familial generalized vitiligo is also characterized

by a broad repertoire of associated autoimmune diseases, such as thyroiditis, rheumatoid arthritis, psoriasis, adult-onset-dependent diabetes mellitus, pernicious anemia, and Addison’s disease [24]. Furthermore generalized vitiligo is a component of the APECED (APS1) and Schmidt (APS2) multiple autoimmune disease syndromes. These same vitiligoassociated autoimmune/autoinflammatory disorders also occur, at increased frequencies, in patients’ firstdegree relatives, regardless of whether or not those relatives have vitiligo themselves. These observations suggest that specific genes predispose to a specific group of autoimmune diseases that includes generalized vitiligo, autoimmune thyroid disease, rheumatoid arthritis, psoriasis, adult-onset insulin-dependent diabetes mellitus, and pernicious anemia.

Cellular immunity Recent evidence has emerged for a role for cellmediated immunity in vitiligo pathogenesis. The

Pathogenesis of vitiligo discovery of a T-cell infiltrate in the margin of inflammatory vitiligo was the first clue for participation of cellular immunity in vitiligo pathogenesis. Infiltrating activated CD4 and CD8 T-cells, but not the B-cells, have been observed at the periphery of vitiligo lesions [25]. A more recent study of vitiligo lesional skin noted a high frequency of cutaneous lymphocyte antigen-positive-activated cytotoxic T-cells clustered in perilesional skin in the vicinity of disappearing melanocytes [26]. Furthermore, melanocytes in close proximity to activated lymphocytes focally expressed HLA-DR and intercellular adhesion molecule-1, suggesting a major role for skin-homing T-cells in melanocyte death [26]. The reports of an increase of CD45RO memory T-cells, increased levels of soluble interleukin-2 receptors and expression of the cutaneous lymphocyte antigen in number infiltrating T-cells, all suggest an activation of circulating T-cells and their recruitment to the vitiligo skin [27–29]. In vitiligo skin the CD4/CD8 ratio is reversed with a predominant presence of CD8 T-cells. Further evidence for a role played by cytotoxic T-cells in vitiligo stems from studies of melanoma patients. Vitiligo-like depigmentation has been observed following successful immunotherapy of melanoma, including high-dose IL-2 therapy, infusion of peptide pulsed dendritic cells, and Melan A/MART-1 specific CTL clones [30]. A specific cellular immune response predominantly directed against the melanosomal protein Melan-A/MART-1 was observed in HLA-A2-positive vitiligo patients where CD8 T-cells displaying Melan-A/Mart-1-specific reactivity ex vivo were demonstrated in the peripheral blood of these patients. Another study reported evidence of an association between CD8  T-lymphocyte reactivity to the melanocyte antigen gp100 and to a lesser extent Melan-A/MART-1 and vitiligo [30]. These findings support the concept of an immunopathological mechanism in vitiligo in which cell-mediated play a crucial part. Recently, new understanding for the requirements for CD8 T-cell mediated destruction of melanocytes was brought [31]. CD4 T-cell help induced by systemic immunization and a local inflammation are both required to break MHC class-I-restricted T-cell tolerance.

7

Table 1.3 Identified target autoantibodies for vitiligo

antibodies. Autoantigen Function Tyrosinase

Melanogenic enzyme

TRP-2

Melanogenic enzyme

TRP-1

Melanogenic enzyme

Pmel-17

Melanocyte-specific protein

MCHR1

Melanin concentrating hormone receptor 1

SOX 9

Transcription factor

SOX 10

Transcription factor

Humoral immunity Several circulating autoantibodies (Table 1.3) have been found in sera of vitiligo patients. These include antibodies to non-pigment cell antigens (common tissue antigens), cytoplasmic pigment cell antigens, and pigment cell surface antigens [32]. The heterogeneity of this antibody response is surprising and does not fit with a selective destruction of melanocytes. One reasonable explanation is that this humoral response could be secondary to a primary melanocyte destruction mediated by other mechanisms. The incidence and serum level of antibodies was found to correlate with the disease activity and the extent of the cutaneous depigmentation. Functional in vitro assays have shown the ability of antibodies to damage melanocytes, both by complement activation and by ADCC [33]. Furthermore injections of IgG fractions of serum from patients with vitiligo have a destructive effect on melanocytes of the human skin grafted onto nude mice [34]. Antibody-dependent immunity against the mélanosome membrane protein-1 (TYRP-1) of melanocytes leads to autoimmune hypopigmentation. Hypopigmentation occurred in mice deficient in activating FcR containing the common  subunit and in mice deficient in the C3 complement but not in mice doubly deficient in both FcR and C3 [35].

The neural hypothesis There are several observations, clinical findings, and laboratory evidence that suggest the involvement of the nervous system in the pathogenesis of

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vitiligo. Embryologically, melanocytes are derived from the neural crest. There are isolated reports of vitiligo associated with viral encephalitis and transverse myelitis. Communication between the nervous system and epidermal melanocytes has been proved [36]. Ultrastructural studies demonstrate frequent direct contacts between dermal nerve endings and melanocytes in vitiligo skin [37] or structural alterations (swelling of axons, duplication of the basement membrane, etc.) [38,39], but the significance of these morphological findings is unknown. The neural hypothesis is based in the first place on the presence of segmental vitiligo. The distribution of segmental vitiligo is often said to be dermatomal, suggesting the role of regional nerves in this condition. In actuality it is unilateral, but not dermatomal (i.e. it does not follow a specific pattern of cutaneous sensory nerves) [40]. Thus, the role of the nervous system in the pathogenesis of vitiligo is still undefined. A few physiological studies have demonstrated altered bleeding times, epinephrine vasoconstrictor effect, and abnormal sympathetic skin responses in lesions of vitiligo [41,42]. Other studies have shown altered neuropeptides in vitiligo. Aberrations in -endorphin and met-enkephalin secretion have been reported [43]. The plasma met-enkephalin levels were generally higher in vitiligo patients, especially in those with active vitiligo, than in controls. Immunohistological observations suggest that the immunoreactivity to neuropeptide Y and vasoactive intestinal polypeptide is increased at the marginal areas or within vitiligo macules. These observations support the hypothesis of neural involvement and neuro-immunomodulation in vitiligo [39]. Still other studies have demonstrated a reduction in the immunoreactive nerve growth factors (NGFr-IR) [44] and an absence of Merkel cells [45]. Several studies demonstrating abnormalities of acetylcholine, catecholamines, or related enzymes (catechol-o-methyltransferase (COMT) and monoamino oxidase [46,47]) have also been reported with conflicting results [48–50]. A reduced acetylcholinesterase activity in vitiliginous skin as compared to adjacent normal skin has been reported [51]. Increased urinary levels of catecholamines have been found during the active phase of vitiligo [52,53].

The autocytotoxic theory In 1971, Lerner [54] postulated that melanocytes have a genetically based protective mechanism that eliminates toxic products like DOPA, DOPAchrome, and 5,6-dihydroxyindole, manufactured during melanogenesis. Individuals who are deficient in this mechanism have accumulation of these melanotoxic products, which results in depigmentation. Another possible mechanism could be damage by genetic mechanisms or by perioxidation [55] to the membranes of melanosomes, which prevent leakage of these compounds into the cellular milieu [56,57]. The oxidative stress theory proposes that melanocyte death results from an intrinsic increased sensitivity to oxidative stress either from toxic intermediates of melanin precursors or from other sources. In vivo and in vitro evidence for hydrogen peroxide (H2O2) accumulation in the epidermis of vitiligo patients has been reported, resulting from low epidermal catalase levels [58]. Several studies suggest that (a) cultured vitiligo melanocytes exhibit increased sensitivity to oxidative stress and (b) catalase helps to establish vitiligo melanocyte cultures and to restore melanocyte functions after exposure to H2O2 [2]. According to Schallreuter and her group, the origin of the epidermal H2O2 accumulation and low epidermal catalase levels within the entire skin of vitiligo patients may arise from several potential sources: (1) perturbed (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH4) de novo synthesis/recycling/regulation; the absence of catalase leads to accumulation of toxic superoxide radicals. One such mechanism involves a group of compounds called pteridines. L-tyrosine is the central substrate for catechol synthesis in keratinocytes and melanin synthesis in melanocytes L-tyrosine itself is produced from L-phenylalanine and the reaction is regulated by the enzyme phenylalanine hydroxylase. This enzyme is under the control of pteridines including 5,6,7,8-tetrahydrobioopterin (6BH4). Defects in the production of pteridines lead to the accumulation of epidermal phenylalnine and shortage of L-tyrosine. A clinical study using a loading oral dose of L-phenylalanine showed slower turnover of L-phenylalanine to L-tyrosine in patients compared with controls [59]. The defective deranged synthesis of pteridins leads to the concomitant accumulation of H2O2;

Pathogenesis of vitiligo (2) impaired catecholamines synthesis with increased monooxidase A activities; (3) low glutathione peroxidase activities; (4) “oxygen burst” via NADPH oxidase from a cellular infiltrate [58]. Under in vitro conditions, vacuole of vitiligo melanocytes has been demonstrated which was reversible upon exogenous addition of bovine catalase to the culture medium [3]. Until now, this interesting concept has not yet been validated in vivo in patients with vitiligo. Whether H2O2 is the cause or the consequence of vitiligo remains to be identified. Another mechanism involves the ion calcium. In the epidermis there is an efficient antioxidant mechanism of thioredoxin/thioredoxin reductase (T/TR) which reduces H2O2 to water. The activity of this system is allosterically regulated by ionic calcium and defective uptake of calcium could result in altered redox status and accumulation of H2O2 [60]. Estrogens can also contribute to hydrogen peroxide [61]. Successful removal by a UVB-activated pseudocatalase has also been reported. However, this conclusion arises from an open trial including only 33 patients and the results have not been confirmed by further studies. The efficacy of pseudocatalase to promote vitiligo repigmentation is still a matter of debate. However, there is no abnormality in blood antioxidant status in patients with vitiligo. Blood levels of superoxidase dismutase, glutathione peroxidase, glutathione reductase, non-enzymatic oxidants such as -tocopherol (Vit E), retinol (Vit A), ascorbic acid (Vit C) have been found to be normal. In the context of the oxidative stress theory, selenium is widely prescribed to stabilize and to repigment vitiligo. However, two distinct studies demonstrated that there is an increase in total blood antioxidant status (high serum selenium levels) in vitiligo patients [62,63]. As a consequence, oral supplementation should not be practiced in patients who exhibit a spontaneous increase in selenium levels, as it could be potentially harmful (selenium toxicity).

The new hypotheses

A disorder of melanocyte survival The active mechanism by which melanocytes are destroyed in vitiligo skin has not yet been determined. Several morphological observations suggest

9

the involvement of melanocyte apoptosis and of the SCF/c-kit/MITF/Bcl-2 pathway in the pathogenesis of vitiligo. This pathway plays a key role in the maintenance of melanocyte survival. SCF of keratinocyte origin strongly protects melanocytes from TNF-related apoptosis inducing ligand (TRAIL) [64]. Bcl-2, a MITF-dependent kit transcriptional target in melanocytes, is essential for the maintenance of an appropriate lifetime for melanocytes. The decrease of Bcl-2 expression of melanocytes increases their susceptibility to apoptosis. Bcl-2/ mice develop graying and whitening of hair early in life during the second hair cycle, due to disappearance of follicular melanocytes. Levels of SCF expression (P
0.001) are reduced in the depigmented epidermis of vitiligo patients compared to normally pigmented paired epidermis [7]. A reduction in the number of kit-positive melanocytes in the perilesional skin of vitiligo patients has also been reported. Immunohistochemistry with antibodies to melanocyte markers revealed that at the edges of the lesional vitiligo epidermis, melanocytes do not express the kit-protein and the melanocyte-specific microphthalmia transcription factor (MITF-M) [65]. Western blotting confirmed down-regulated expression of c-kit and MITF-M proteins at the edge of the lesional epidermis in vitiligo. These findings strongly suggest a deficiency of the melanocyte survival pathway SCF/c-kit/MITF/Bcl-2 which might be responsible for dysfunction and/or loss of melanocytes in vitiligo epidermis. Interestingly, we have observed a marked progression of a vitiligo which was stable since many years after treatment with tyrosine kinase inhibitors that inhibit c-kit [66]. Moreover, several cases of vitiligo-like depigmentation occurring after treatment with new tyrosine kinase inhibitors that inhibit c-kit (STI-571 and SU 11428) have been reported [67,68].

The melanocyte growth factor deficient theory Defective growth and passage capacities of vitiligo melanocytes derived from uninvolved and perilesional skin in vitro have been described [69]. Interestingly, these growth defects of vitiligo melanocytes could be partially corrected in vitro by

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the adjunction of fetal lung fibroblast-derived growth factors. In addition, melanocytes taken from actively repigmenting vitiligo macules grow correctly, suggesting a correction of the growth defect. Based on these results, it has been suggested that a decreased concentration of melanocyte growth factor(s) could play a role in the pathogenesis of vitiligo [70].

Viral infections Viral infections have been implicated in the pathogenesis of autoimmune diseases. In one study, CMV DNA was detected in the involved and uninvolved skin of 38% of vitiligo patients and 0% of control subjects. EBV, CMV, Herpes simplex, varicella-zoster, and human T-lymphotropic virus were negative. There are no definitive data to confirm or refute the viral hypothesis. Additional studies are needed to confirm these results [71].

Melanocyte defective adhesion Interaction between melanocytes and the dermoepidermal basement membrane are mediated by integrins (61). Interactions between melanocytes and keratinocytes are mediated by cadherins in association with -catenin. Repeated friction in non-lesional skin of vitiligo patients induces detachment and transepidermal elimination of melanocytes [72]. This suggests that minor mechanical trauma in non-lesional vitiligo skin is probably the cause of depigmentation occurring in the Köbner’s phenomenon. Transepidermal elimination of melanocytes in vitiligo may be a possible mechanism of chronic loss of melanocytes, perhaps previously damaged by another process [2].

Conclusion From the available data, it is likely that the loss of epidermal and follicular melanocytes in vitiligo results in melanocyte death. The identification of at least two different clinical phenotypes of vitiligo suggests that melanocyte destruction may be the result of several different pathogenetic mechanisms. Many different hypotheses have been proposed. Recent developments of the genetics of generalized vitiligo strongly suggest a role of immunological factors in generalized vitiligo susceptibility. Besides

genetic and immunologic factors, the environment is likely to be involved in the pathogenesis of vitiligo in ways that are not yet known. There are now probably too many hypotheses of vitiligo. All hypotheses are not mutually exclusive. A “consequence” theory suggests that genetic factors, stress, accumulation of toxic compounds, infection, autoimmunity, altered cellular environment, and impaired melanocyte migration and proliferation can all contribute to the phenomenon of vitiligo.

References 1 Spritz RA. The genetics of generalized vitiligo and associated autoimmune diseases. J Dermatol Sci 2006; 41:3–10. 2 Gauthier Y, Cario Andre M, Taieb A. A critical appraisal of vitiligo etiologic theories. Is melanocyte loss a melanocytorrhagy? Pigment Cell Res 2003; 16:322–32. 3 Tobin DJ, Swanson NN, Pittelkow MR, Peters EM, Schallreuter KU. Melanocytes are not absent in lesional skin of long duration vitiligo. J Pathol 2000;191:407–16. 4 Jarrett A, Szabo G. The pathogenesis varieties of vitiligo and their response to treatment with meladinine. Br J Dermatol 1956;68:313. 5 Le Poole IC, van den Wijngaard R, Westerhof W, et al. Loss of melanocytes in vitiligo lesions. J Invest Dermatol 1992;98:541. 6 Sommer L. Checkpoints of melanocyte stem cell development. Sci STKE 2005;2005:pe42. 7 Lee AY, Kim NH, Choi WI, Youm YH. Less keratinocyte-derived factors related to more keratinocyte apoptosis in depigmented than normally pigmented suction-blistered epidermis may cause passive melanocyte death in vitiligo. J Invest Dermatol 2005;124:976–83. 8 Moellmann G, Klein-Angerer S, Scollay DA, et al. Extracellular granular material and degeneration of keratinocytes in the normally pigmented epidermis of patients with vitiligo. J Invest Dermatol 1982;79: 321–30. 9 Bhawan J, Bhutani LK. Keratinocyte damage in vitiligo. J Cutan Pathol 1983;10:207–12. 10 Ortonne JP, Bose SK. Vitiligo: where do we stand? Pigment Cell Res 1993;6:61–72. 11 Nordlund JJ, Ortonne JP. Vitiligo and depigmentation. In: Weston WL, Mackie RM, and Provost TT (eds.) Current Problems in Dermatology, Vol. 4. St. Louis, Missouri: Mosby-Yearbook, 1992;3–30.

Pathogenesis of vitiligo 12 Das SK, Majumder PP, Chakraborty R, et al. Studies on vitiligo. I. Epidemiological profile in Calcutta, India. Genet Epidemiol 1985;2:71–8. 13 Majumder PP, Nordlund JJ, Nath SK. Pattern of familial aggregation of vitiligo. Arch Dermatol 1993;129: 994–8. 14 Alkhateeb A, Fain PR, Thody A, et al. Epidemiology of vitiligo and associated autoimmune diseases in Caucasian probands and their families. Pigment Cell Res 2003;16:208–14. 15 Zhang XJ, Liu JB, Gui JP, et al. Characteristics of genetic epidemiology and genetic models for vitiligo. J Am Acad Dermatol 2004;51: 383–90. 16 Alkhateeb A, Stetler GL, Old W, et al. Mapping of an autoimmunity susceptibility locus (AIS1) to chromosome 1p31.3–p32.2. Hum Mol Genet 2002;11:661–7. 17 Fain PR, Gowan K, LaBerge GS, et al. A genomewide screen for generalized vitiligo: confirmation of AIS1 on chromosome 1p31 and evidence for additional susceptibility loci. Am J Hum Genet 2003;72:1560–4. 18 Spritz RA, Gowan K, Bennett DC, Fain PR. Novel vitiligo susceptibility loci on chromosomes 7 (AIS2) and 8 (AIS3), confirmation of SLEV1 on chromosome 17, and their roles in an autoimmune diathesis. Am J Hum Genet 2004;74:188–91. 19 Zhang XJ, Chen JJ, Liu JB. The genetic concept of vitiligo. J Dermatol Sci 2005;39:137–46. 20 Kos R, Reedy MV, Johnson RL, Erickson CA. The winged-helix transcription factor FoxD3 is important for establishing the neural crest lineage and repressing melanogenesis in avian embryos. Development 2001; 128:1467–79. 21 Guo Y, Costa R, Ramsey H, et al. The embryonic stem cell transcription factors Oct-4 and FoxD3 interact to regulate endodermal-specific promoter expression. Proc Natl Acad Sci USA 2002;99:3663–7. 22 Jonsson H, Peng SL. Forkhead transcription factors in immunology. Cell Mol Life Sci 2005;62:397–409. 23 Alkhateeb A, Fain PR, Spritz RA. Candidate functional promoter variant in the FOXD3 melanoblast developmental regulator gene in autosomal dominant vitiligo. J Invest Dermatol 2005;125:388–91. 24 Laberge G, Mailloux CM, Gowan K, et al. Early disease onset and increased risk of other autoimmune diseases in familial generalized vitiligo. Pigment Cell Res 2005; 18:300–5. 25 Badri AM, Todd PM, Garioch JJ, et al. An immunohistological study of cutaneous lymphocytes in vitiligo. J Pathol 1993;170:149–55. 26 van den Wijngaard R, Wankowicz-Kalinska A, Le Poole C, et al. Local immune response in skin of generalized vitiligo patients. Destruction of melanocytes

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is associated with the prominent presence of CLA+ T cells at the perilesional site. Lab Invest 2000;80: 1299–309. Le Poole IC, van den Wijngaard RM, Westerhof W, Das PK. Presence of T cells and macrophages in inflammatory vitiligo skin parallels melanocyte disappearance. Am J Pathol 1996;148:1219–28. Yeo UC, Yang YS, Park KB, et al. Serum concentration of the soluble interleukin-2 receptor in vitiligo patients. J Dermatol Sci 1999;19:182–8. Mahmoud F, Abul H, Haines D, et al. Decreased total numbers of peripheral blood lymphocytes with elevated percentages of CD4CD45RO and CD4CD25 of T-helper cells in non-segmental vitiligo. J Dermatol 2002;29:68–73. Mandelcorn-Monson RL, Shear NH, Yau E, et al. Cytotoxic T lymphocyte reactivity to gp100, MelanA/ MART-1, and tyrosinase, in HLA-A2-positive vitiligo patients. J Invest Dermatol 2003;121:550–6. Steitz J, Bruck J, Lenz J, et al. Peripheral CD8 T cell tolerance against melanocytic self-antigens in the skin is regulated in two steps by CD4 T cells and local inflammation: implications for the pathophysiology of vitiligo. J Invest Dermatol 2005;124:144–50. Ongenae K, Van Geel N, Naeyaert JM. Evidence for an autoimmune pathogenesis of vitiligo. Pigment Cell Res 2003;16:90–100. Norris DA, Kissinger RM, Naughton GM, Bystryn JC. Evidence for immunologic mechanisms in human vitiligo: patients’ sera induce damage to human melanocytes in vitro by complement-mediated damage and antibody-dependent cellular cytotoxicity. J Invest Dermatol 1988;90:783–9. Gilhar A, Zelickson B, Ulman Y, Etzioni A. In vivo destruction of melanocytes by the IgG fraction of serum from patients with vitiligo. J Invest Dermatol 1995;105:683–6. Trcka J, Moroi Y, Clynes RA, et al. Redundant and alternative roles for activating Fc receptors and complement in an antibody-dependent model of autoimmune vitiligo. Immunity 2002;16:861–8. Hara M, Toyoda M, Yaar M, et al. Innervation of melanocytes in human skin. J Exp Med 1996;184: 1385–95. Morohashi M, Hashimoto K, Goodman Jr. TF, et al. Ultrastructural studies of vitiligo, Vogt-Koyanagi syndrome, and incontinentia pigmenti achromians. Arch Dermatol 1977;113:755–66. Breathnach AS, Bor S, Wyllie LM. Electron microscopy of peripheral nerve terminals and marginal melanocytes in vitiligo. J Invest Dermatol 1966;47:125–40.

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39 Al’Abadie MS, Senior HJ, Bleehen SS, Gawkrodger DJ. Neuropeptide and neuronal marker studies in vitiligo. Br J Dermatol 1994;131:160–5. 40 Nordlund JJ. The pigmentary system, 2006. 41 Dutta AK, Mandal SB. A study of non-nervous vasoconstrictor responses. Int J Dermatol 1972;11:177–80. 42 Merello M, Nogues M, Leiguarda R, et al. Abnormal sympathetic skin response in patients with autoimmune vitiligo and primary autoimmune hypothyroidism. J Neurol 1993;240:72–4. 43 Mozzanica N, Villa ML, Foppa S, et al. Plasma alphamelanocyte-stimulating hormone, beta-endorphin, met-enkephalin, and natural killer cell activity in vitiligo. J Am Acad Dermatol 1992;26:693–700. 44 Liu PY, Bondesson L, Lontz W, Johansson O. The occurrence of cutaneous nerve endings and neuropeptides in vitiligo vulgaris: a case-control study. Arch Dermatol Res 1996;288:670–5. 45 Bose SK. Probable mechanisms of loss of Merkel cells in completely depigmented skin of stable vitiligo. J Dermatol 1994;21:725–8. 46 Ma QY, Zeng LH, Chen YZ, et al. Ocular survey of deafmute children. Yan Ke Xue Bao 1989;5:44–6. 47 Schallreuter KU, Wood JM, Pittelkow MR, et al. Increased monoamine oxidase A activity in the epidermis of patients with vitiligo. Arch Dermatol Res 1996;288:14–8. 48 Lerner AB. Vitiligo. J Invest Dermatol 1959;32:285–310. 49 Bamshad J, Lerner AB. S-Adenosylmethionine in skin. J Invest Dermatol 1964;43:115–7. 50 Durneva SG. [Changes in the content of epinephrine and norepinephrine in the blood of patients with vitiligo]. Vestn Dermatol Venerol 1973;47:33–6. 51 Iyengar B. Modulation of melanocytic activity by acetylcholine. Acta Anat (Basel) 1989;136:139–41. 52 Morrone A, Picardo M, de Luca C, et al. Catecholamines and vitiligo. Pigment Cell Res 1992;5:65–9. 53 Morrone A, Piccardo M, De Luca C, et al. Urinary levels of catecholamine metabolites in children affected with vitiligo. Pigment Cell Res 1992;5:90. 54 Lerner AB. On the etiology of vitiligo and gray hair. Am J Med 1971;51:141–7. 55 Riley PA. Mechanism of pigment-cell toxicity produced by hydroxyanisole. J Pathol 1970;101:163–9. 56 Yohn JJ, Norris DA, Yrastorza DG, et al. Disparate antioxidant enzyme activities in cultured human cutaneous fibroblasts, keratinocytes, and melanocytes. J Invest Dermatol 1991;97:405–9. 57 Medrano EE, Nordlund JJ. Successful culture of adult human melanocytes obtained from normal and vitiligo donors. J Invest Dermatol 1990;95:441–5.

58 Schallreuter KU, Moore J, Wood JM, et al. In vivo and in vitro evidence for hydrogen peroxide (H2O2) accumulation in the epidermis of patients with vitiligo and its successful removal by a UVB-activated pseudocatalase. J Investig Dermatol Symp Proc 1999;4:91–6. 59 Schallreuter KU, Zschiesche M, Moore J, et al. In vivo evidence for compromised phenylalanine metabolism in vitiligo. Biochem Biophys Res Commun 1998;243: 395–9. 60 Schallreuter KU, Pittelkow MP. Defective calcium uptake in keratinocyte cell cultures from vitiliginous skin. Arch Dermatol Res 1988;280:137–9. 61 Schallreuter KU, Chiuchiarelli G, Cemeli E, et al. Estrogens can contribute to hydrogen peroxide generation and quinone-mediated DNA damage in peripheral blood lymphocytes from patients with vitiligo. J Invest Dermatol 2006;126:1036–42. 62 Boisseau-Garsaud AM, Garsaud P, Lejoly-Boisseau H, et al. Increase in total blood antioxidant status and selenium levels in black patients with active vitiligo. Int J Dermatol 2002;41:640–2. 63 Beazley WD, Gaze D, Panske A, et al. Serum selenium levels and blood glutathione peroxidase activities in vitiligo. Br J Dermatol 1999; 141:301–3. 64 Larribere L, Khaled M, Tartare-Decker S, et al. P13K mediates protection against TRAIL-induced apoptosis in primary human melanocytes. Cell Death Differ 2004;11:1084–91. 65 Kitamura R, Tsukamoto K, Harada K, et al. Mechanisms underlying the dysfunction of melanocytes in vitiligo epidermis: role of SCF/KIT protein interactions and the downstream effector, MITF-M. J Pathol 2004;202: 463–75. 66 Legros L, Cassuto JP, Ortonne JP. Imatinib mesilate (Glivec): a systemic depigmenting agent for extensive vitiligo? Br J Dermatol 2005;153:691–2. 67 Raanani P, Goldman JM, Ben-Bassat I. Challenges in oncology. Case 3. Depigmentation in a chronic myeloid leukemia patient treated with STI-571. J Clin Oncol 2002;20:869–70. 68 Hasan S, Dinh K, Lombardo F, et al. Hypopigmentation in an African patient treated with imatinib mesylate: a case report. J Natl Med Assoc 2003;95:722–4. 69 Puri N, Mojamdar M, Ramaiah A. In vitro growth characteristics of melanocytes obtained from adult normal and vitiligo subjects. J Invest Dermatol 1987;88:434–8. 70 Ramaiah A, Puri N, Mojamdar M. Etiology of vitiligo. A new hypothesis. Acta Derm Venereol 1989;69:323–6. 71 Grimes PE, Sevall JS, Vojdani A. Cytomegalovirus DNA identified in skin biopsy specimens of patients with vitiligo. J Am Acad Dermatol 1996;35:21–6.

Pathogenesis of vitiligo 72 Gauthier Y, Cario-Andre M, Lepreux S, et al. Melanocyte detachment after skin friction in non lesional skin of patients with generalized vitiligo. Br J Dermatol 2003;148:95–101. 73 Nath SK, Kelly JA, Namjou B, et al. Evidence for a susceptibility gene, SLEV1, on chromosome 17p13 in families with vitiligo-related systemic lupus erythematosus. Am J Hum Genet 2001;69:1401–6.

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74 Arcos-Burgos M, Parodi E, Salgar M, et al. Vitiligo: complex segregation and linkage disequilibrium analyses with respect to microsatellite loci spanning the HLA. Hum Genet 2002;110:334–42. 75 Chen JJ, Huang W, Gui JP, et al. A novel linkage to generalized vitiligo on 4q13–q21 identified in a genomewide linkage analysis of Chinese families. Am J Hum Genet 2005;76:1057–65.

CHAPTER 2

Understanding the mechanism of repigmentation in vitiligo Amrinder J. Kanwar and Davinder Parsad

The basic pathogenesis of vitiligo in general, or for any of the putative subsets of vitiligo, is not fully known. Upon spontaneous or medically induced improvement in vitiligo patches, repigmentation spreads inwards from the borders of lesion. In some color appears diffusely in depigmented areas, but more commonly the pigment spread is perifollicular. The nature of repigmentation can be classified into three types: 1 perifollicular when predominant repigmentation is follicular; 2 marginal when predominant repigmentation is from the borders of patches; 3 diffuse pigmentation when there occurs generalized darkening across the patches of vitiligo. Melanocytes in human skin reside both in the epidermis and in the matrix and outer root sheath (ORS) of anagen hair follicles. Staricco [1] described two types of melanocytes in hair follicles: the amelanotic or inactive type and melanotic or active melanocytes. Epidermal and amelanotic hair follicle melanocytes proliferated well in culture, whereas the melanotic hair follicle melanocytes did not. Amelanotic hair follicle melanocytes have been shown to differ from epidermal melanocytes in being less differentiated, and expressing less mature melanosome antigens. Nishimura et al. [2] identified the stem cells of melanocyte lineage in the lower permanent portion of mouse hair follicle throughout the hair cycle. They also analyzed the repigmentation process in Tg/
mice and found that these bulge stem cells are the source of melanocytes in epidermis. Nishimura et al. [3] suggested in another publication that hair graying is due to loss of melanocyte stem cells. Commo et al. [4] also found

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specific depletion of bulb and ORS melanocytes in graying human hair.

Mechanisms of medically induced repigmentation Perifollicular repigmentation The mechanism of perifollicular repigmentation pattern has been researched extensively and many authors believe that it is the only mode of repigmentation in vitiligo [5]. Repigmentation of vitiligo lesions is thought to occur by the migration of undifferentiated melanocytes from the ORS of the hair follicles to the intrafollicular epidermis. Ortonne et al. [6] demonstrated the mechanism of psoralen plus ultraviolet A (PUVA)-induced repigmentation of vitiligo based on a histochemical and ultrastructural study. They divided the repigmentation process into three stages: 1 proliferation of hypertrophic melanocytes in the lower portion of hair follicle; 2 migration of hypertrophic melanocytes along the hair follicle toward the infundibulum; and 3 migration of melanocytes to the adjacent epidermis. Similarly Cui et al. [7] have also shown that during repigmentation, treatment stimulated the inactive melanocytes in the middle and/or lower parts of the ORS of hair follicles to divide, proliferate, and migrate upward along the surface of the ORS to the nearby epidermis, where the melanocytes continued to migrate radially to form the pigmented island visible clinically in repigmented vitiligo lesions. It is conceivable that successful repigmentation depends on the availability of migratory factors as well as

Understanding the mechanism of repigmentation in vitiligo mitogens that allow melanocytes to thrive and proliferate in order to repopulate the depigmented epidermis. After exposure to the therapeutic agents and especially UV light exposure, it is believed that keratinocytes as well as other inflammatory cells in vitiligo lesions may release different melanocyte growth factors to stimulate the inactive melanocyte reservoir in the ORS of hair follicles surrounding the vitiliginous patch [7]. Three melanocyte mitogens – basic fibroblast growth factor, stem cell factor, and endothelin-1 – have been shown to stimulate melanocyte migration which may be either chemotactic or chemokinetic [8]. At present effective approaches to activate melanoblasts in ORS are not available, but would be promising approaches to treat vitiligo patients. Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteolytic enzymes involved in extracellular matrix remodeling and in cell migration during a variety of physiological and pathological processes. Epidermal melanocytes are localized in the basal cell layer of epidermis just above the basement membrane zone. Melanocytes migrating into depigmented vitiligo skin from the ORS in the dermis would need to penetrate the existing extracellular matrix tissue barrier. Recently, a study by Lei et al. [9] showed the presence of MMP-2 in melanoblast and found that this expression is induced by 8methoxypsoralen.

Diffuse repigmentation Jarrett and Szabo [10] suggested that reactivation of dihydroxyphenylalanine (DOPA) negative melanocytes which persist in the center of lesions might be responsible for diffuse type of repigmentation. Treatment of early vitiligo lesion can also show diffuse repigmentation because of stimulation of remaining melanocytes in the lesion. However, there are some reports indicating that vitiligo lesions are not completely devoid of melanocytes. A study by Tobin et al. [11] and Bartosik et al. [12] provided evidence that melanocytes are still present in the depigmented epidermis of stable vitiligo of as long as 25 years’ duration. We have also shown the presence of melanin in depigmented lesion of vitiligo of 5 years’ duration [13].

15

Marginal repigmentation There is a large reservoir of normal melanocytes surrounding the depigmented patch of vitiligo. These activated melanocytes which are attracted by the higher concentration of growth factors in the vitiligo patches, gradually migrate and actively produce melanin [14]. Moreover it can be hypothesized that if a topically applied drug has chemo-attractant action for melanocytes, it can attract melanocyte from surrounding normal skin. This could possibly explain the marginal type of repigmentation. Recently we conducted a study to correlate repigmentation patterns with different treatment modalities [15]. PUVA and narrowband UVB predominantly exhibit a perifollicular pattern of repigmentation (Fig. 2.1A). Diffuse type of repigmentation (Fig. 2.1B) was seen with topical corticosteroids. Marginal repigmentation (Fig. 2.1C) was seen in only few patients who were treated with PUVA and topical calcipotriol. The speed of repigmentation is much faster when repigmentation starts in a diffuse pattern as compared to the follicular repigmentation. However, perifollicular repigmentation (91.7%) was observed to be more stable than diffuse type of repigmentation (58.7%).

Mechanisms of surgically induced repigmentation Sometimes there can be repigmentation of depigmented lesion even after simple dermabrasion [16]. It has been hypothesized that with dermabrasion, there is altered milieu of epidermal cytokines which can stimulate inactive melanocytes of ORS. The usual type of repigmentation is perifollicular. The melanocytes which appear in the epidermis 8–10 days after dermabrasion are 2–3 times larger than normal melanocytes with a hypertrophic cellular body, elongated dendrites, and intense DOPA oxidase activity [1]. The aim of surgical induction of repigmentation is to replenish melanocytes in the depigmented lesions of vitiligo which either have no reservoir or fail to activate melanocyte in ORS with known treatment modalities. Most surgical procedures, like epidermal grafting and transplantation of noncultured melanocytes, restore normal epidermal

16

Chapter 2

(A)

(C)

Fig. 2.1 Types of repigmentation patterns.

(B)

(A) perifollicular, (B) diffuse, and (C) marginal.

Understanding the mechanism of repigmentation in vitiligo

17

(A)

(B) Fig. 2.2 A vitiligo patch (A) treated with minigrafting, showing perigraft pigmentation and (B) resembling perifollicular

repigmentation. (Courtesy: Somesh Gupta, MD, DNB, New Delhi, India.)

melanocytes. The usual repigmentation seen with such procedures is diffuse type. Arrunategui et al. [17] have shown that melanocytes in the lower third of hair follicle can repigment depigmented skin in vitiligo. They demonstrated perifollicular repigmentation by grafting the lower portion of the hair bulb and upper hair shaft and peri-infundibular epidermis in achromic skin in vitiligo. In minigrafting, the tiny miniature grafts act as islands of melanocyte reservoir, which simulates hair follicle reservoir. The perigraft pigmentation originating from 1 to 2 mm minigrafts resembles perifollicular pigmentation (Fig. 2.2). The spread generally occurs 5–10 mm beyond the graft margin [18,19]. The spread is more in skin phototypes IV–VI than in phototypes I–III. Melanocytes have the ability to migrate. When the suction blister epidermal grafts remain in contact

with the denuded surface of the recipient vitiliginous area for about a week, the melanocytes readily migrate to the graft bed resulting in repigmentation. Several authors have described that in epidermal grafting the graft “take” is not necessary for successful repigmentation [20]. Contact of grafts for 7–8 days with denuded recipient vitiliginous area is sufficient for successful outcome, as during this period melanocytes migrate from the graft to the graft bed. Therefore the epidermal grafts act only as a carrier of melanocytes. The initial repigmentation in epidermal grafting is confined to and corresponds with the grafted area. Subsequently, with or without photochemotherapeutic stimulation, melanocytes migrate from the grafted area to the surrounding depigmented skin leading to diffuse repigmentation. There is some resistance to pigment spread from the epidermal grafts at the

18

Chapter 2

References

Fig. 2.3 Achromic fissure at the periphery of the vitiligo

macule persisting 3 years after epidermal grafting suggesting resistance to repigmentation at the margins. (Courtesy: Somesh Gupta, MD, DNB, New Delhi, India.)

periphery of vitiligo macules. This may result into an achromic fissure at the periphery of the vitiligo lesion (Fig. 2.3). In transplantation of melanocyte–keratinocyte cell suspension and in cultured “pure” melanocytes, repigmentation is diffuse and resembles natural pigmentation as melanocytes repopulate the area evenly. The stability of surgically induced repigmentation depends on the type of vitiligo being treated. It will be most likely to be stable in the unilateral segmental vitiligo. Many published papers comment on short-term results only and long-term stability of repigmentation in generalized vitiligo is largely unknown. Olsson and Juhlin [21] based on their long-term follow-up study demonstrated that patients with extensive vitiligo vulgaris more often showed incomplete repigmentation with any surgical modality. They also had a lower chance of retaining their repigmentation compared with those with less extensive vitiligo. In all techniques, imperfect color matching of the graft may be seen initially. Hypopigmentation of the grafted area may be due to reactivation of the disease or due to insufficient concentration of grafted melanocytes. Hyperpigmentation of the grafted area can be explained by the fact that during re-epithelization, there is release of cytokines which can act as melanocyte stimulants.

1 Staricco RGJ. The melanocytes and the hair follicle. J Invest Dermatol 1960;35:185–94. 2 Nishimura EK, et al. Dominant role of the niche in melanocyte stem-cell fate determination. Nature 2002;416:854–60. 3 Nishimura EK, Granter SR, Fisher DE. Mechanisms of hair graying: incomplete melanocyte stem cell maintenance in the niche. Science 2005;307:720–4. 4 Commo S, et al. Human hair greying is linked to a specific depletion of hair follicle melanocytes affecting both the bulb and the outer root sheath. Br J Dermatol 2004;150:435–43. 5 Nordlund JJ, Ortonne JP. Vitiligo vulgaris. In: Nordlund JJ, Boissy RE, Hearing VJ, King RA, and Ortonne JP (eds.) The Pigmentary System: Physiology and Pathophysiology. New York: Oxford University Press, 1998;513–51. 6 Ortonne JP, Schmitt D, Thivolet J. PUVA-induced repigmentation of vitiligo: scanning electron microscopy of hair follicles. J Invest Dermatol 1980;74:40–2. 7 Cui J, Shen LY, Wang GC. Role of hair follicles in the repigmentation of vitiligo. J Invest Dermatol 1991;97: 410–16. 8 Horikawa T, Norris DA, Yohn JJ, et al. Melanocyte mitogens induce both melanocyte chemokinesis and chemotaxis. J Invest Dermatol 1995;104:256–9. 9 Lei TC, Vieira DW, Hearing VJ. In vitro migration of melanoblasts require matrix metalloproteinase-2: implication to vitiligo therapy by photochemotherapy. Pigm Cell Res 2002;15:426–32. 10 Jarrett A, Szabo G. The pathological varieties of vitiligo and their response to treatment with meladinine. Br J Dermatol 1956;68:313–17. 11 Tobin DJ, Swanson NN, Pittelkow MR, Peters EM, Schallreuter KU. Melanocytes are not absent in lesional skin of long duration vitiligo. J Pathol 2000;191: 407–16. 12 Bartosik J, Wulf HC, Kobayasi T. Melanin and melanosome complexes in long standing stable vitiligo – an ultra structural study. Eur J Dermatol 1998;8:95–7. 13 Parsad D, Wakamatsu K, Kanwar AJ, et al. Eumelanin and phaeomelanin contents of depigmented and repigmented skin in vitiligo patients. Br J Dermatol 2003; 149:624–6. 14 Cui J. The melanocyte reservoir and its necessity. In: Hann SK and Nordlund JJ (eds.) Vitiligo – A Monograph on the Basic and Clinical Science, 1st edn. New York: Blackwell Science, 2000;163–7. 15 Parsad D, Pandhi R, Dogra S, Kumar B. Clinical study of repigmentation patterns with different treatment

Understanding the mechanism of repigmentation in vitiligo modalities and their correlation with speed and stability of repigmentation in 352 vitiliginous patches. J Am Acad Dermatol 2004;50:63–7. 16 Savant SS. Therapeutic spot and regional dermabrasion in stable vitiligo. Indian J Dermatol Venereol Leprol 1996;62:139–45. 17 Arrunategui A, Arroyo C, Garcia L, et al. Melanocyte reservoir in vitiligo. Int J Dermatol 1994;33:484–7. 18 Barman KD, Khaitan BK, Verma KK. A comparative study of punch grafting followed by topical corticosteroid versus punch grafting followed by PUVA therapy in stable vitiligo. Dermatol Surg 2004;30:49–53.

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19 van Geel N, Ongenae K, Naeyaert J-M. Surgical techniques for vitiligo: a review. Dermatology 2001;202: 162–6. 20 Shenoi SD, Srinivas CR, Pai S. Treatment of stable vitiligo with epidermal grafting and PUVA. J Am Acad Dermatol 1997;36:802–3. 21 Olsson MJ, Juhlin L. Long-term follow-up of leucoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol 2002;147: 893–904.

CHAPTER 3

Classification of vitiligo Sang Ju Lee, Sung Bin Cho and Seung-Kyung Hann

Vitiligo is a relatively common acquired depigmenting disorder that affects approximately 0.5–2% of the population without racial, sexual, or regional differences [1,2]. Although many studies have concentrated on its pathogenesis, the hypothesis concerning the pathogenesis of vitiligo has not yet been fully elucidated. Vitiligo has various clinical features, which have been reported by many investigators, but the number of patients studied is limited and there is no uniform classification that all clinicians agree upon. However, understanding these characteristics can give us valuable information not only in differentiating vitiligo from other pigmentary disorders, but also in treating vitiligo with the proper modalities and predicting the prognosis of vitiligo up to a certain degree. Currently, a classification describing vitiligo as generalized or localized type according to the distribution of lesions is widely used (Table 3.1). Localized type is subdivided into focal, segmental, and mucosal subtypes, while generalized type is subdivided into acrofacial, vulgaris, and universal subtypes. An overlap of various types can be classified as mixed type [3]. In 1977, Koga performed a sweat secretion stimulation test using physostigmine, and accordingly reclassified vitiligo into non-segmental type (type A) and segmental type (type B) [4]. He proposed that the non-segmental type results from immunological mechanisms, while segmental type results from dysfunction of the sympathetic nervous system in the affected skin [4]. After Koga’s report suggesting that these two types differed in pathogenesis and clinical presentation, many clinicians began to take an interest in segmental vitiligo, since most of these cases did not cross the midline and were distributed along a unilateral dermatome, thus enabling prediction of the prognosis [5,6]. Segmental

20

Table 3.1 Classification of vitiligo according to the

distribution of lesions. 1. Localized (a) Focal (b) Segmental (c) Mucosal 2. Generalized (a) Acrofacial (b) Vulgaris (c) Universal (d) Mixed

type is usually localized to one dermatome, shows relatively stable disease activity after its initial rapid-spreading phase, and is associated with a significantly lower rate of autoimmune diseases than non-segmental type [5,6]. According to these characteristics, vitiligo can also be classified into three major clinical types: segmental (unilateral); nonsegmental (bilateral); and mixed (Table 3.2). The segmental form generally does not cross the midline and does not have a classical dermatomal distribution but affects one segment of the integument. The segment might be composed of several or parts of several adjacent dermatomes or have no relationship to dermatomes at all, nor any other lines such as Blaschko’s line or acupuncture lines. The progression is usually limited to months or a few years [6,7]. The non-segmental form is characterized by bilateral, usually symmetrical, depigmented macules. It is further subdivided into focal and mucosal subtypes, localized form that is limited to small areas of the integument, and into generalized type including acrofacial, vulgaris, and universal subtypes. The latter is characterized by widespread

Classification of vitiligo Table 3.2 Classification of vitiligo with emphasis on

segmental vitiligo. 1. Segmental (unilateral) 2. Non-segmental (bilateral) (i) Localized (a) Focal (b) Mucosal (ii) Generalized (a) Acrofacial (b) Vulgaris (c) Universal 3. Mixed: segmental and non-segmental

extensive depigmentation that most commonly spreads throughout the life of the individual. In addition, there is a very rare variety of generalized vitiligo that seems to be a manifestation of a systemic autoimmune disease. This disorder is manifested by vitiligo, as well as multiple endocrine failures such as diabetes mellitus, adrenal insufficiency, thyroid dysfunction, and gonadal dysfunction. All of the latter endocrine abnormalities seem to be caused by autoantibodies but the cause of the loss of melanocytes remains unidentified.

21

Today’s focal vitiligo can change to a generalized type tomorrow. Therefore, prevention of spreading is an important management strategy for bilateral vitiligo. Areolar or nipple involvement in nonsegmental vitiligo usually appears bilaterally as a late manifestation. Hann et al. [11] reported the most common initial lesion site as the face (39.0%), followed by the anterior trunk (23.6%), neck (10.4%), and posterior neck (9.1%).

Focal vitiligo Focal vitiligo exhibits one or more macules in one area, but not clearly in a segmental or zosteriform distribution. Focal vitiligo is a starting point leading to other types of vitiligo. When focal vitiligo appears initially as a single lesion, it may not be easy to determine the type of vitiligo. However, it can often spread in a linear fashion, very slowly manifesting the features of segmental vitiligo. Although early treatment does not always prevent spread of vitiligo, focal vitiligo frequently spreads to the whole body without treatment. Moon et al. [12] reported systemic steroid effectively prevents the spreading of vitiligo (focal type as well as generalized type). It is yet to be known what percentage of focal vitiligo progresses to other types of vitiligo.

Mucosal vitiligo

Clinical characteristics of non-segmental (bilateral) vitiligo Bilateral vitiligo comprises all types of vitiligo other than segmental, such as focal, mucosal, acrofacial, vulgaris, and universal vitiligo. Confetti type vitiligo or vitiligo fulminans may appear initially as generalized type. The incidence of generalized, nonsegmental type of vitiligo varies from 50% to 90% [8–10]; Song et al. [10] have reported this type in 1315 patients. There were 660 cases (50.2%) of generalized type, which consisted of acrofacial (14.4%) and vulgaris (35.8%) varieties, and one case of universal type. There were 654 cases (49.7%) of localized type consisting of focal (33.7%), segmental (15.4%), and mucosal (0.6%). Clinically, localized type of vitiligo, with the exception of segmental type, may be an intermediary stage and evolve into the generalized or, rarely, into the universal type.

Mucosal vitiligo shows vitiligo of the mouth and mucous membranes, including the genitalia (Fig. 3.1). Mucous membrane involvement in vitiligo is not mentioned in most published reports or is said to be rare. The mucous membranes of Caucasians are pink in color and loss of pigment is very difficult to detect even with Wood’s lamp. In contrast, the labial and oral mucosa of those with darker skin is pigmented and the patchy loss of pigmentation is readily apparent to the attentive observer. In a study of 45 patients living in Tanzania, 75% had patchy loss of pigment from the gums or mucosa of the inner lips. Mucosal involvement occurs rather frequently around body orifices such as lips, genitals, gingival, areolae, and nipples [11]. Significant progression of vitiligo in patients with mucosal involvement indicates that it is a poor prognostic factor. In addition to the mucous membranes of the mouths, vitiligo often occurs on the genitals [10].

22

Chapter 3 vulgaris was the most common form of the disease in 1002 (69.8%) out of 1436 patients with vitiligo.

Universal vitiligo Universal vitiligo implies loss of pigment over the entire body surface area and complete or nearly complete depigmentation can be noted.

Factors affecting vitiligo progression Physical trauma, sunburn, psychological stress, inflammation, pregnancy, and contraceptives may be the precipitating factors of non-segmental vitiligo. Fig. 3.1 Mucosal vitiligo on the mouth.

Differences between segmental and non-segmental vitiligo Non-segmental vitiligo is about 2.4-fold more common than segmental vitiligo. In patients with nonsegmental vitiligo, the age of onset is later, mean duration is longer, and depigmented area is larger than in the patients with segmental vitiligo. The incidences of Köbner’s phenomenon, progressiveness, and mucosal involvement are known to be more common in non-segmental vitiligo. There are no differences in sex, blood typing, family history, and associated disorders between segmental and non-segmental vitiligo [16]. Fig. 3.2 Acrofacial vitiligo on the fingertips and around

the mouth.

Acrofacial vitiligo Acrofacial vitiligo encompasses depigmentation of the distal parts of the extremities (hands rather than feet) and facial orifices, the latter in a circumferential pattern (Fig. 3.2) [13]. The body surface areas contiguous to the initial sites usually show the highest rates of progression. However, when the hands are the initial site, vitiligo most commonly progressed to the face. This could be explained clinically by the fact that one-third of the patients whose lesions started from the hands were of the acrofacial type [14].

Vitiligo vulgaris Vitiligo vulgaris is composed of several scattered macules. Handa and Kaur [15] showed that vitiligo

Trichrome vitiligo Trichrome vitiligo is a rare type of vitiligo, in which an intermediate hue of varying width exists between the normal and totally depigmented area (Fig. 3.3) [17–19]. Among 21 patients with trichrome vitiligo, 20 patients (95.2%) showed vitiligo vulgaris and one focal vitiligo [20]. Of the trichrome lesions, 85.5% were localized to the trunk region, including abdomen, back, and buttock, leading to the assumption that trichrome vitiligo predominates in unexposed skin. Actually the lesion of the trichrome vitiligo is composed of four distinct areas with an intermediate hue and brown-colored broadband, peripherally surrounded by a dark-brown-colored border, and normal skin. Thus Hann et al. [21] suggested that the name “quadrichrome vitiligo” is more appropriate than trichrome vitiligo, which was first suggested by Fitzpatrick [17]. Some authors regard “quadrichrome vitiligo” as another variant of vitiligo [3,22]. Trichrome vitiligo has also been

Classification of vitiligo

23

Fig. 3.4 Marginal inflammatory vitiligo showing an

erythematous, raised rim at the periphery of the hypopigmented patch.

trichrome vitiligo had histopathological findings of active-spreading vitiligo; therefore trichrome vitiligo was regarded as a phenomenon occurring in restricted areas of active vitiligo. Trichrome vitiligo is a variant of active vitiligo. The changes of melanocytes, keratinocytes, and Langerhans cells may be involved in the pathogenesis of depigmentation in trichrome vitiligo. Fig. 3.3 Trichrome vitiligo showing characteristic light

brown skin between normal and vitiliginous skin.

Marginal inflammatory vitiligo

described as an isomorphic Köbner’s phenomenon. In one patient, trichrome concentric rings or lines surrounding an achromic line corresponding to former scratches were observed [23]. The significance of trichrome vitiligo is unknown. However, it is clearly a metastable or transitional pigmentary state, though it may persist for months to years with minor change [3]. A reasonable interpretation is that trichrome corresponds to a gradual centrifugal spread of hypomelanosis or stepwise depigmentation [17,19]. However, the sharp demarcation between the three areas is not consistent with such an active centrifugally spreading lesion [3]. Therefore, whether trichrome vitiligo is a temporary phenomenon of active-spreading vitiligo or a hypomelanosis showing an unusual progression pattern remains to be clarified. Recently, Hann et al. [20] reported that

Inflammatory vitiligo is an unusual variant of vitiligo which is described as having an erythematous, raised rim at the periphery of the hypopigmented or depigmented patch (Fig. 3.4) [3]. Most of the red, raised inflammatory lesions precede or appear simultaneously with the onset of classic vitiligo [24], but they may appear several months or years later [25]. A mild pruritus may be present and, as in classic vitiligo, the patients with inflammatory vitiligo may exhibit Köbner’s phenomenon [24]. The neck is the most common site of the inflammatory vitiligo (44%). The significance of this localized inflammatory reaction is unknown and it still remains to be debated whether inflammatory vitiligo represents a distinct form of vitiligo or simply shows an exaggerated status of the usual inflammatory process occurring in vitiligo [26,27]. Histologically, the presence of a mild lymphocytic infiltrate at the border of active vitiligo may be seen even in the clinically

24

Chapter 3

non-inflammatory lesion. However, Pincus [19] reported that biopsy specimens of inflammatory vitiligo consistently showed greater degrees of inflammation as compared to hypopigmented or normally pigmented skin.

Clinical features of segmental vitiligo The clinical features of segmental vitiligo markedly differ from those of non-segmental vitiligo. Segmental vitiligo usually has an onset early in life and spreads rapidly within the affected area limited to one segment of the integument, that is, part of the face, part of the trunk and extremity, or one extremity [4]. The lesions stop abruptly at the midline of the affected segment. The progression of the segmental type can cease and the depigmented patches can remain without any change for the life of the patient. This feature differs remarkably from the chronic progressive course of non-segmental vitiligo [5,6].

Incidence The incidence of segmental type is not well established but the variable percentage of those with vitiligo having the segmental form has been reported. el-Mofty and el-Mofty [28] reported 5% of those with vitiligo had the segmental type, while Koga and Tango [5] reported 27.9% had this type of depigmentation. Results of previous Korean studies have shown a range between 5.5% and 16.1% [10,29].

that the onset of segmental vitiligo generally affects young children whereas non-segmental vitiligo occurs at all ages. In Hann and Lee’s report [6], segmental vitiligo developed before 30 years of age in 87.0% of cases, and before 10 years of age in 41.3%, and the mean age of onset was 15.6 years. These observations are in accordance with the report that segmental vitiligo occurs in young people before age 30 years [5].

Family history Family history of segmental vitiligo was reported by approximately 7.4–11.5%, which is similar to that of non-segmental vitiligo [6,29].

Site of involvement The face is the most common development site of the segmental vitiligo regardless of the gender of the patient [6]. The trunk, neck, extremities, and scalp are involved in descending frequency in males. In females, the neck is more frequently involved than the extremities. Lerner [2] reported that segmental vitiligo occurs as a single lesion in 75% of patients, a finding confirmed by the study of Hann and Lee [6], who found that 87% of patients had a single lesion. Among the 31 patients with segmental vitiligo studied by Park et al. [29], the abdomen, neck, face, and chest, in descending order of frequency, were the most commonly involved sites. The results in this report differ from those of Hann and Lee’s study [6].

Involvement of body and scalp hair Age of onset Segmental vitiligo appears at all ages, but most of the lesions occur in younger people between ages of 5 and 30 years [30]. Koga and Tango [5] reported

Fig. 3.5 Five patterns of segmental vitiligo on the face.

Poliosis occurs in 48.6% of cases of segmental vitiligo [6], mostly involving the eyebrows and scalp hair (46.7%). Due to the fact that segmental vitiligo tends to involve the face, neck and scalp, poliosis of the

Classification of vitiligo

25

eyebrows and scalp hair is commonly present. White hair indicates that the reservoir for repigmentation has been destroyed. Leukotrichia of the eyebrows can be treated with a combination of epidermal grafting and psoralen plus ultraviolet A (PUVA) [31].

Distribution of lesions Studies on the distribution of segmental vitiligo revealed that the skin innervated by the trigeminal nerve was most frequently involved, followed by the thoracic, cervical, lumbar, and sacral nerves [6]. Although the skin is innervated by certain sensory nerves, the actual distribution of the depigmentation did not correspond very well to a true dermatome as seen in other cutaneous disorders like herpes zoster. Most of the patterns of segmental vitiligo listed above did not follow a dermatomal distribution. It has been suggested that segmental vitiligo might follow Blaschko’s lines or acupuncture lines [32]. The distribution of segmental vitiligo possibly follows an unknown pathway which may be a group of identical clonal cells.

(A)

Classification of segmental vitiligo on the face Segmental vitiligo of the face does not always follow dermatomes, nor any other lines including Blaschko’s line or acupuncture lines. The distribution of segmental vitiligo on the face is classified into five patterns (Fig. 3.5) [33]. Type 1a represents the lesion which initiates from the right side of the forehead, crosses the midline of the face, and spreads down to the eyeball, nose, and cheek of the left side of the face (Fig. 3.6). Type 1b shows a mirror image of 1a. The lesion starts from the left side of the face and spreads down the right side of the face, crossing the midline. In type 2, the lesion starts from the area between the nose and lip, then arches to the preauricular area (Fig. 3.7). In type 3, the lesion initiates from the lower lip and spreads down to the chin and neck (Fig. 3.8). In type 4, the lesion originates from the right side of the forehead and spreads down to the eyeball, nose, and cheek areas without crossing the midline (Fig. 3.9). In type 5, the lesion is confined to the left cheek area (Fig. 3.10). Some segmental vitiligo on the face cannot be classified by this system. Type 1 is the most common and type 5 is

(B) Fig. 3.6 Type 1a represents the lesion which starts from

the right side of the forehead and crosses the midline of the face and spreads down to the eyeball, nose, and cheek of the left side of the face.

the least common. There are no significant differences in age, sex, duration of initial lesions, progression pattern, and clinical type. This classification of facial segmental vitiligo can provide some indication

26

Chapter 3

(A)

(B) Fig. 3.7 Type 2 shows a lesion that starts from the area between the nose and lip, arching to the preauricular area.

(A)

(B) Fig. 3.8 Type 3 shows a lesion that starts from the lower lip and spreads down to the chin and cheek.

Classification of vitiligo

27

(A)

(B) Fig. 3.9 Type 4 shows a lesion that originates from the right side of the forehead and spreads down to the eyeball,

nose and, cheek areas without crossing the midline of the face.

as to the future distribution of early lesions if they begin to spread. Since the face is a commonly involved site of vitiligo and it is the area that causes psychological impact, most patients are willing to undergo intensive treatment. Therefore, knowledge about the exact spreading pattern and prognosis is of great interest to both patients and doctors.

Progression of lesions Segmental vitiligo is not an intermediary stage of vitiligo. Most vitiliginous patches of segmental type remain unchanged for the rest of the patient’s life after rapid spreading in the affected dermatomal area [6]. However, rarely it can progress again after being quiescent for several years. Segmental vitiligo usually spreads over the affected area, so the

progression pattern can be easily predicted. However, in very rare cases, lesions may become bilateral and generalized called mixed type. Early segmental vitiligo usually appears as a solitary oval shaped white macule or as a patch, which is difficult to differentiate from focal type vitiligo until a typical distribution of lesions has appeared. A white macule on the nipple or areola appearing as the initial lesion can be assumed to be an early indication of segmental vitiligo [6,21]. Nipple or areolar involvement as the initial lesion in non-segmental vitiligo is very rare and is not uncommonly bilateral.

Factors affecting vitiligo progression Physical trauma, sunburn, psychological stress, inflammation, pregnancy, and contraceptives are

28

Chapter 3

Fig. 3.11 Bilateral segmental vitiligo distributed in a lin-

Fig. 3.10 Type 5 represents a lesion that is usually

confined to the cheek area.

known to be the precipitating factors of vitiligo [6]. But some authors reported that sunburn, trauma, or pregnancy were considered aggravating factors in only about 4.8% of patients with segmental vitiligo [6,29].

Köbner’s phenomenon Koga and Tango [5] reported that Köbner’s phenomenon, the appearance of vitiligo after trauma or scratches on the normal skin, was not found in 134 patients with segmental vitiligo. However, some authors reported Köbner’s phenomenon in 5.3% of the patients with segmental vitiligo [6].

Associated diseases In 1980, el-Mofty and el-Mofty [28] suggested that segmental vitiligo is not associated with other autoimmune diseases. However, Park et al. [29] showed that

ear pattern on both the right and left thoracic dermatome. The right side lesions are located on the shoulder and arm, while the left side lesions are at the lower chest and upper abdomen, which do not cross the midline.

about 9.5% of cases with segmental vitiligo were associated with other autoimmune disorders. Koga and Tango [5] asserted that an autoimmune disease associated with vitiligo occurred more frequently in non-segmental vitiligo than in segmental type and that this difference might be due to different pathogenic mechanisms. Hann and Lee [6] reported 6.7% of patients out of 208 patients had an associated diseases suspected of being allergic or immunological. The most common disease associated was atopic dermatitis (3.4%) and also noted were thyroid diseases, diabetes mellitus, pernicious anemia, and halo nevus. Considering the prevalence of autoimmune disorders reported to be as high as 15% among general population in European countries, whether these findings are pathogenetically associated or are incidental is still debatable.

Classification of vitiligo

Bilateral segmental vitiligo If segmental vitiligo occurs bilaterally, following the contralateral same or different dermatomes, it may confuse the definition of the vitiligo type (Fig. 3.11). As segmental vitiligo rarely appears on bilateral dermatomes and its distribution is not completely understood, it may resemble some of the other types of non-segmental vitiligo or white patches of both legs seen in piebaldism. Hann et al. [11] reported that 5 out of 240 patients who had segmental vitiligo, exhibited two different depigmented segments on the same or opposite sites of the body. The clinical course of bilateral segmental vitiligo seems to be the same as unilateral segmental vitiligo, although only five cases have been followed for up to maximum of 3 years. PUVA therapy and steroid treatment could induce repigmentation or stop progression of vitiliginous lesions in bilateral segmental vitiligo.

Treatment response and prognosis Segmental vitiligo was previously considered to be resistant to treatment. However, recent studies reported good results in the treatment of segmental vitiligo if done correctly. Segmental vitiligo has an excellent prognosis for cure since it responds well either to medical or surgical therapies.

Conclusion Vitiligo has various clinical features but can be classified into two major clinical types: segmental and non-segmental. Segmental vitiligo has clinical features that are different from those of bilateral vitiligo. Understanding these characteristics can give us valuable information in differentiating vitiligo from other hypopigmentary disorders, in treating vitiligo with the proper methods and in predicting the prognosis of vitiligo to some extent.

References 1 Porter JR, Beuf A, Lerner A, Nordlund J. Response to cosmetic disfigurement: patients with vitiligo. Cutis 1987;39:493–4. 2 Lerner AB. Vitiligo. J Invest Dermatol 1959;32:285–310. 3 Ortonne JP, Mosher DB, Fitzpatrick TB. Vitiligo and Other Hypomelanoses of Hair and Skin. New York: Plenum Publishing, 1983;147–8.

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4 Koga M. Vitiligo: a new classification and therapy. Br J Dermatol 1977;97:255–61. 5 Koga M, Tango T. Clinical features and courses of type A and type B vitiligo. Br J Dermatol 1988;118: 223–8. 6 Hann SK, Lee HJ. Segmental vitiligo: clinical findings in 208 patients. J Am Acad Dermatol 1996;35:671–4. 7 Barona MI, Arrunategui A, Falabella R, Alzate A. An epidemiologic case–control study in a population with vitiligo. J Am Acad Dermatol 1995;33:621–5. 8 Howitz J, Brodthagen H, Schwartz M, Thomsen K. Prevalence of vitiligo: epidemiological survey on the Isle of Bornholm, Denmark. Arch Dermatol 1977;113: 47–52. 9 Moscher DB. Vitiligo: etiology, pathogenesis, diagnosis, and treatment. In: Fitzpatrick TB, Eisen AZ, Wolff K, Freedberg IM and Austen KF (eds.) Fitzpatrick’s Dermatology in General Medicine, Vol. I, 4th edn. New York: McGraw-Hill, 1993;923–33. 10 Song MS, Hann SK, Ahn PS, Im S, Park YK. Clinical study of vitiligo: comparative study of type A and type B vitiligo. Ann Dermatol 1994;6:22–30. 11 Hann SK, Park YK, Chun WH. Clinical features of vitiligo. Clinic Dermatol 1997;15:891–7. 12 Moon TK, Im S, Hann SK, Cho SH, Park YK. The effect of small doses of oral corticosteroids in vitiligo patients. Korean J Dermatol 1995;33:880–5. 13 Kovacs SO. Vitiligo. J Am Acad Dermatol 1998;38: 647–66. 14 Hann SK, Nordlund JJ. Clinical features of generalized vitiligo. In: Hann SK and Nordlund JJ (eds.) Vitiligo, 1st edn. Oxford: Blackwell Science, 2002;35–48. 15 Handa S, Kaur I. Vitiligo: clinical findings in 1436 patients. J Dermatol 1999;26:653–7. 16 Bang JS, Lee JW, Kim TH, et al. Comparative clinical study of segmental vitiligo and non-segmental vitiligo. Korean J Dermatol 2000;38:1037–44. 17 Fitzpatrick TB. Hypomelanosis. S Med J 1964;57: 995–1005. 18 Kim YS, Hann SK. Clinical and histopathologic characteristics of trichrome vitiligo. Korean J Dermatol 1997;32:720–8. 19 Pincus H. Vitiligo: What is it? J Invest Dermatol 1959; 32:281–94. 20 Hann SK, Kim YS, Yoo JH, Chun YS. Clinical and histopathologic characteristics of trichrome vitiligo. J Am Acad Dermatol 2000;42:589–96. 21 Hann SK, Lee HS, Park YK. Clinical features of vitiligo. Ann Dermatol 1998;10:217–28. 22 Mosher DB, Fitzpatrick TB, Ortonne JP, et al. Vitiligo. In: Fitzpatrick TB, Eisen AZ and Wolff K (eds.)

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Chapter 3 Fitzpatrick’s Dermatology in General Medicine, Vol. I, 3rd edn. New York: McGraw-Hill, 1987;810–12. Dupre A, Christol B. Cockade-like vitiligo and linear vitiligo a variant of fitzpatrick’s trichrome vitiligo. Arch Dermatol Res 1978;262:197–203. Lee D, Lazova R, Bolognia JL. A figurate papulosquamous variant of inflammatory vitiligo. Dermatology 2000;200:270–4. Ortonne JP. Special features of vitiligo. In: Hann SK and Nordlund JJ (eds.) Vitiligo, 1st edn. Oxford: Blackwell Science, 2002;70–5. Eng AM. Marginal inflammatory vitiligo. Cutis 1970; 6:1005–8. Gokhale BB, Mehta LN. Histopathology of vitiliginous skin. Int J Dermatol 1983;22:477–80.

28 el-Mofty AM, el-Mofty M. Vitiligo. A symptom complex. Int J Dermatol 1980;19:237–44. 29 Park KC, Youn JI, Lee YS. Clinical study of 326 cases of vitiligo. Korean J Dermatol 1988;26:200–5. 30 Seghal VN. A clinical evaluation of 202 cases of vitiligo. Cutis 1974;14:439–45. 31 Hann SK, Im S, Park YK, Hur W. Repigmentation of leukotrichia by epidermal grafting and systemic psoralen plus UV-A. Arch Dermatol 1992;128:998–9. 32 Bolognia JL, Orlow SJ, Glick SA. Lines of Blaschko. J Am Acad Dermatol 1994;31:157–90. 33 Hann SK, Chang JH, Lee HS, Kim SM. The classification of segmental vitiligo on the face. Yonsei Med J 2000;41:209–12.

CHAPTER 4

Medical treatment of vitiligo Thierry Passeron and Jean-Paul Ortonne

Individuals affected by vitiligo have a vast reduction of quality of life. The color contrast between healthy pigmented skin and the depigmented vitiligo patches can give patients psychological problems [1,2]. Although up to now no treatment provides truly satisfactory results, physicians have a large variety of therapeutic approaches, both medical (including depigmenting therapies and camouflaging) and surgical [3]. This chapter will focus on medical treatment of vitiligo. A meta-analysis of the literature has shown that the best non-surgical therapies for localized and generalized vitiligo are topical corticosteroids and phototherapy, respectively [4]. However, comparison of all of the studies must be undertaken carefully, as great variability is apparent in the patient population (particularly localization, age, and skin type) and in the duration of treatment. New medical therapeutic approaches are also discussed.

Phototherapy Phototherapy is considered one of the most effective treatments for vitiligo [5]. Several strategies are used including ultraviolet A (UVA) phototherapy, photochemotherapy (oral and topical) such as psoralen plus UVA (PUVA), psoralen plus sunlight (PUVASOL), broad- and narrowband UVB (BB- and NB-UVB, respectively) phototherapy, 308-nm excimer light and combination phototherapy. There is increasing evidence that UVB therapy is superior to UVA in treating vitiligo. Many studies have demonstrated the effectiveness of PUVA therapy in this indication; however, there are specific contraindications and a higher risk of side effects, including skin carcinomas, associated with it [6–9]. It is important to note that long-term follow-up of UVB therapy is much more limited than UVA, but,

by means of a dose–response model, it has been calculated that long-term NB-UVB therapy may carry substantially less risk for skin cancer than PUVA therapy [10].

UVA phototherapy

Topical PUVA (paint) photochemotherapy Most investigators agree that topical psoralen photochemotherapy should be restricted to vitiligo patients with an involvement of less than 20% of the body surface. This treatment can be used in children. Topical PUVA is difficult to perform because of the high risk of phototoxicity from the topical psoralen formulations. However, advantages of topical PUVA include lower cumulative UVA doses than oral PUVA and lack of ocular and systemic toxicity. Low concentrations of psoralens should be used. A 0.1% concentration of 8-methoxypsoralen (8-MOP) has the same effectiveness as higher concentrations (0.5% or 1%), but a lower risk of toxicity. The commercially available 1% 8-MOP lotion should be diluted from 1:10 to 1:100. The patient should be exposed to UVA approximately 20–30 minutes after application of the topical preparation (preferentially cream or ointment to avoid “running” leading to streaks of hyperpigmentation) with a cotton-tipped applicator by a physician or a nurse to avoid a rim of hyperpigmentation around the lesions if the psoralen is applied on the surrounding normally pigmented skin. The initial dose should not exceed 0.25 J/cm2. This treatment is performed once or twice a week, never on consecutive days with increments by 0.12–0.25 J/cm2/week, until mild erythema is achieved at the treated sites. Following treatment, the area is washed, a broad-spectrum sunscreen is

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applied, and excessive sun exposure is avoided for at least 24 hours [11]. The mean clinical response is about 60% repigmentation, depending on the anatomic site. Due to the risk of severe blistering reactions, topical photochemotherapy should never be used with sunlight as an UVA source. However, a home topical PUVA protocol using very dilute 8-MOP (0.001%) has been proposed for the treatment of vitiligo. In a large cohort of patients (n
125), only 3% had blistering reactions [12].

Oral PUVA photochemotherapy PUVA involves the use of psoralens followed by exposure to long-wavelength UVA irradiation. Oral PUVA is used most commonly in patients with extensive vitiligo. There are numerous psoralens occurring naturally in plant species. Of these, only a few are used therapeutically, including methoxsalen (8-MOP), trioxsalen (4,5,8-trimethylpsoralen), and bergapten (5-MOP), almost exclusively for vitiligo. Trioxsalen is no longer available and 5-MOP is pending approval in the USA. By far the most commonly used oral psoralen is 8-MOP (0.4–0.6 mg/kg) and treatments are typically administered 2 times/week. For patients with vitiligo, the initial dose of UVA is usually 0.5–1.0 J/cm2. This dose is gradually increased until minimal asymptomatic erythema of the involved skin occurs. 5-MOP has about the same response rate as 8-MOP in repigmenting vitiligo. The former appears to be more suitable for the treatment of vitiligo because of its lower incidence of adverse effects, in particular a reduced phototoxicity of depigmented skin as well as less nausea and vomiting. The response rate of PUVA is variable, and complete repigmentation is achieved in only a few patients. Some degree of repigmentation is seen in about 60–80% of patients. A satisfactory cosmetic result is usually obtained in less than 20% of cases. According to a recent meta-analysis, the mean success rate in treating vitiligo for oral methoxsalen plus UVA was 51%. As with other forms of phototherapy and topical corticosteroids, the areas that respond most favorably are the face, the mid-extremities, and the trunk [4]. The total number of PUVA treatments

required is between 50 and 300. Evidence of repigmentation is usually first seen after 1–4 months of treatment, but complete repigmentation usually requires 100–300 treatments. Repigmentation, as with NB-UVB, usually appears in a perifollicular pattern and/or from the periphery of the lesions. The former represents the repopulation of the interfollicular epidermis with melanocytes from the follicular reservoir, and pigmented hairs are a better prognostic sign than depigmented hairs. General contraindications to oral PUVA include photosensitivity disorders, pregnancy and lactation, a history of skin cancer, arsenic exposure or cutaneous radiation therapy, cataract and retinal disease. To date, only a few vitiligo patients with PUVA-induced cutaneous carcinomas have been reported. This may reflect a smaller cumulative UVA dose, but large follow-up studies have not yet been done in PUVAtreated vitiligo patients. Until more data are available, it seems wise to recommend a maximum cumulative PUVA dose and a maximum number of PUVA treatments of 1000 J/cm2 and 300 treatments, respectively, to vitiligo patients. The rate of repigmentation with oral PUVA varies, depending upon the anatomic site. Schematically two groups of lesions can be identified: 1 the UV-responsive lesions that include the face and neck, the trunk, and the proximal extremities; and 2 the UV-resistant areas, including the bony prominences, the distal digits, and the lips. Children with vitiligo tend to respond somewhat better to PUVA than adults. Darkly pigmented patients often achieve more repigmentation with PUVA than patients with lighter skin, probably because they tolerate higher UVA doses. Retention of PUVA-induced repigmentation has been observed in more than 90% of patients, 14–15 years after discontinuation of treatment. Retention of repigmentation seems to be more common in areas with complete repigmentation.

UVB phototherapy

BB-UVB Very few studies to evaluate the potential of BB-UVB in the treatment of vitiligo are available. One of the studies reported that 75% repigmentation

Medical treatment of vitiligo was achieved in 8 of 14 patients after 12 months of treatment, mostly in patients with skin phototypes IV–VI [13]. These results are not confirmed by a recent intra-individual comparative study, which showed 22% of patients treated by NB-UVB achieved a repigmentation of at least 75% after 12 months of treatment versus none with BB-UVB [14]. The latter study involved only 10 patients, and the localization of the treated lesions was different between the two groups (upper part of the body for NB-UVB and lower part for broadband). Larger studies are still needed but NB-UVB should be preferred to BB-UVB in treating vitiligo.

PUVB (with BB-UVB) A left–right comparative study suggests that PUVB is equally effective as PUVA therapy [15] in the treatment of vitiligo. Each patient received 0.7 mg/kg 8-MOP 2 hours before the session. BB-UVB initial dose of 0.03 J/cm2 was increased by 0.03 J/cm2 every session whereas UVA was started at 0.5 J/cm2 and increased by 0.5 J/cm2 every session. All the patients were of skin types III and IV. After 30 sessions, both PUVA and PUVB produced 50–60% of repigmentation with similar incidences of side effects. Unfortunately, no comparative study of PUVB versus UVB monotherapy is yet available.

NB-UVB Narrowband fluorescent tubes (Philips TL01/ Waldman) with an emission spectrum of 311 nm are used for this therapy. A meta-analysis of the literature concludes that NB-UVB therapy is the most effective and safe therapy for generalized vitiligo [4]. The starting dose varies from 100 to 250 mJ/cm2, with increments of 10–20% at each subsequent exposure and then held once a mild erythema develops. Treatments are administered 2–3 times/week, never on 2 consecutive days. Several studies have demonstrated the effectiveness of NB-UVB as monotherapy. About 60% of patients obtain greater than 75% repigmentation. Short-term side effects include pruritus and xerosis. Long-term side effects are unknown. The advantages of NB-UVB over oral PUVA include shorter treatment times, no drug cost, no or less side effects such as nausea, phototoxic reactions. There is no need for post-treatment

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photoprotection. This treatment can be used in children, pregnant or lactating women, and in individuals with hepatic and kidney dysfunctions. Furthermore, there is less contrast between depigmented and normally pigmented skin, and possibly less long-term side effects. NB-UVB therapy is becoming the first choice of therapy for adults and for children (over 6 years of age) with vitiligo [16]. NB-UVB has been used in combination with pseudocatalase. However, no controlled studies have been performed to validate the beneficial effect of adjuncting pseudocatalase [17].

Focused micro-phototherapy In the past few years new devices delivering UVB light have been developed for the treatment of localized vitiligo. NB-UVB micro-phototherapy utilizes a device that delivers a focused beam with spectrum from 300 to 320 nm with a peak emission of 311 nm. Two studies report excellent results [18,19], but comparative studies with 308-nm excimer laser are still lacking. Monochromatic excimer light 308 nm can also be delivered by lamps. A pilot study using such a device reports that 18 out of 37 vitiligo patients achieved 75% or more repigmentation after 6 months of treatment. These observations should be confirmed by a comparative trial (excimer laser versus excimer lamp) in a larger population [20].

Other photochemotherapies Khellin (topical or systemic) plus UVA (KUVA) or phenylalanine plus UVA have also been proposed for the treatment of vitiligo. There have been conflicting reports regarding these treatment strategies, and there is a concern about the hepatic toxicity of khellin. For these reasons, these modalities are not recommended for the treatment of vitiligo.

Immunomodulators In the recent past, several preliminary studies have reported the efficacy of immunomodulatory drugs, including levamisole, anapsos, isoprinosine, and suplatast, as repigmenting agents for the treatment of vitiligo. Unfortunately, none of these initial observations have been followed by clinical studies

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demonstrating the efficacy of these compounds in the treatment of vitiligo. As a consequence, these agents have not been widely used by vitiligo patients. However, recent advances in vitiligo research provide information strengthening the autoimmune theory of vitiligo. Corticosteroids and topical immunomodulators, such as tacrolimus, demonstrate some efficacy for the treatment of vitiligo.

Corticosteroids Topical steroids are useful for the treatment of localized vitiligo. Marked or almost complete repigmentation can be obtained with potent corticosteroids (e.g. betamethasone valerate, fluticasone propionate) and very potent corticosteroids (e.g. clobetasol, betamethasone). However, corticosteroids of low potency show no therapeutic effect at all. A recent meta-analysis concluded that potent and superpotent topical steroids are effective treatment for localized vitiligo [4]. Steroid-induced repigmentation occurs within 1–4 months of treatment in a perifollicular pattern and from the margins of the lesions. Side effects include dermal atrophy, steroid-induced acne, rosacea, telangiectasia, ecchymoses, and striae. Furthermore, suppression of the hypothalamic– pituitary–adrenal axis may occur after prolonged applications on large areas. To minimize the incidence of these side effects, it is recommended to use topical steroids on limited skin areas, to avoid prolonged use on “sensitive” areas such as face and body folds, and to use them once or twice daily for only 6–8 weeks followed by a treatment-free interval of several weeks as mild steroid-induced skin atrophy is reversible. No repigmentation after 3 months of treatment should lead to discontinuation of treatment. The mechanism of steroid-induced repigmentation is unknown, although several hypotheses are proposed: suppression of immunity-driven melanocyte destruction? Stimulation of melanocyte proliferation and migration? Intra-lesional corticosteroids must be avoided. Systemic steroids (high-dose pulsed therapy, minipulsed regimen, or daily oral low-dose) have been claimed to rapidly arrest spreading vitiligo and induce repigmentation. In most of these studies, a response to systemic corticosteroid therapy has

been limited to patients with rapidly progressive generalized vitiligo. Given the significant potential for serious side effects of systemic corticosteroid therapy, the role of these drugs in the treatment of vitiligo remains controversial. We do not recommend this therapy for vitiligo patients.

Calcineurin inhibitors Preliminary observations suggest that tacrolimus and pimecrolimus may be effective treatments for both localized and generalized vitiligo [21,22]; 0.1% tacrolimus ointment is applied twice daily for about 3 months [23]. Unfortunately, these studies are open label involving a very small number of patients. A 2-month double-blind randomized trial compared 0.1% tacrolimus and 0.05% clobetasol propionate in children with vitiligo [24]. This study confirmed the initial observation that tacrolimus stimulates vitiligo repigmentation, however tacrolimus ointment was not superior to clobetasol in terms of repigmentation. The same results were recently obtained in an open intra-individual study performed with 1% pimecrolimus cream [25]. Once again 0.05% clobetasol propionate induces comparable rate of repigmentation as that with topical calcineurin inhibitor. Interestingly enough the best results were observed on sun-exposed areas suggesting that UV may also be involved in tacrolimus- and pimecrolimus-induced repigmentation of vitiligo. Further studies are required to establish the safety and efficacy of topical calcineurin inhibitors in the treatment of vitiligo. Recent personal observations suggest that tacrolimus monotherapy in the absence of UV has little or no repigmenting potential in vitiligo [26].

Combination therapies Interest in combination treatments was first clearly demonstrated with the combination of UVA and topical steroids. In this prospective, randomized, controlled, left–right comparison study, it was shown that the combination of UVA and fluticasone propionate was much more effective than UVA or topical steroid alone [27]. To the best of our knowledge, the combination of UVB therapy with topical steroids

Medical treatment of vitiligo has not yet been evaluated, although some series have studied the combination of UVB and other synergistic drugs. Oxidative stress has been shown to be involved in the pathogenesis of vitiligo. Pseudocatalase has the ability to remove hydrogen peroxide and so could be interesting in the treatment of vitiligo. The combination of topical pseudocatalase with UVB has shown very promising results in a pilot study (complete repigmentation on the face and the dorsum of the hands in 90% of patients) [17]. Unfortunately, these results were not confirmed in a later study [28]. The occurrence of repigmentation of vitiligo in patients treated with calcipotriol (a vitamin D3 analog) for psoriasis has suggested that it might be efficacious in treating vitiligo. The use of calcipotriol with sun or PUVA therapy has provided some interesting rates of repigmentation. However, the results are very controversial [29–31]. Combination of calcipotriol and UVB also provides controversial data. However, two of the three studies clearly showed that the combination of calcipotriol with UVB had no enhancing effect on repigmentation suggesting the absence of interest of adding calcipotriol to UV [14,32,33]. Tacrolimus ointment has shown some interesting results in the treatment of vitiligo, but the best results were achieved in sun-exposed areas. So far, two studies have evaluated whether the combination of 308-nm excimer laser and topical tacrolimus could be synergistic. These series have compared the efficiency of 308-nm excimer combined with tacrolimus ointment to 308-nm excimer laser monotherapy [34] or associated with placebo ointment [35]. In both cases, a total of 24 sessions were done and tacrolimus ointment was applied twice a day. The results were similar and showed a greater efficiency with the combined treatment as compared to laser alone. Tolerance was good and side effects were limited to constant erythema, sticking, and rarely bullous lesions. These encouraging results are corroborated by two others reports associating UVB light and topical tacrolimus [36,37]. However, the increased risk of skin cancers promoted by combining of two immunosuppressive treatments cannot be excluded. So, pending a long-term follow-up, this combination should be reserved to control studies.

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Others Systemic antioxidant therapy The rationale for this approach rests on the hypothesis that vitiligo results from a deficiency of natural antioxidant mechanisms. Although to date not validated by a controlled clinical trial, selenium methionine, tocopherols, ascorbic acid, and ubiquinone are widely prescribed by dermatologists to arrest spreading of vitiligo and to promote its repigmentation.

Topical calcipotriol Topical calcipotriol as monotherapy has no effect on vitiligo.

Prostaglandin Prostaglandin has been shown to play a role in melanocytes proliferation and melanogenesis. A pilot study has evaluated the topical applications of prostaglandin E2 (PGE2) in treating localized vitiligo [38]. After 6 months of daily applications, 15 of 24 patients achieved marked to complete repigmentation. Side effects were limited to mild irritation in two cases. However, these encouraging results have not been confirmed so far.

Depigmentation therapies Patients who have widespread disease with only few areas of normally pigmented skin on the face or other exposed areas can be treated with depigmenting agents. The patients must be carefully chosen: adults who recognize that their appearance will be altered significantly and who understand that depigmentation also requires lifelong care of the skin (sunscreens, protective clothing, etc.). The guidelines for using permanent depigmentation in vitiligo are as follows [39]: 1 desire for permanent depigmentation, 2 age over 40 years, 3 more than 50% of depigmentation of the sites to be treated, and 4 willingness to accept the fact that repigmentation will no longer be possible. A psychological evaluation of the readiness of patients undergoing procedures such as full skin bleaching is highly desirable. Previous studies have demonstrated such a procedure as valuable [40].

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The most commonly used agent for further depigmenting vitiligo patients with an extensive involvement is monobenzylether of hydroquinone (MBEH) 20% applied twice daily to the affected areas for 9–12 months or more. MBEH is a potent irritant and/or allergenic compound. A patch-test to detect contact sensitivity to MBEH should be performed before starting therapy. It normally takes 1–3 months to initiate a response. Loss of pigment can also occur at distant sites of applications. Although depigmentation from MBEH is considered permanent, repigmentation following a sunburn or even intense sun exposure may occur. Monomethylether of hydroquinone (MMEH), also named 4-hydroxyanisole or 4-methoxyphenol, in a 20% cream can be used as an alternative for MBEH. Side effects include contact dermatitis, pruritus, exogenous ochronosis, and leukomelanoderma en confetti. Depigmentation by Q-switched ruby laser therapy is reported to achieve faster depigmentation compared with depigmentation using a bleaching agent [41].

Photoprotection Photoprotection may be useful to prevent sunburn of susceptible vitiligo skin or induction of Köbner phenomenon. Furthermore, sun exposure stimulates tanning of uninvolved skin and increases the contrast with lesional skin. On the other hand, sun exposure can promote repigmentation of vitiligo. Sun block preparations containing zinc oxide or titanium dioxide are claimed to be more effective than other sunscreen preparations. However, the new broad-spectrum sunscreens providing both UVA and UVB protection are very efficient provided they are reapplied every 2 hours. Use of sun-protective clothing such as wide-brimmed hats should also be recommended.

Camouflaging The goal of camouflage is to normalize the appearance of a patient suffering from a disfigurement. This is usually done on lesions in the exposed areas, such as the face and the dorsal regions of the hands. This approach is very interesting in areas resistant to medical and surgical treatments such as the

extremities of hand and feet. A recent study demonstrates that cosmetic camouflage advice improves quality of life in patients with vitiligo [42]. Unlike traditional cosmetics, cover creams are used because of their unique properties. They are waterproof and opaque and offer wide varieties of cosmetic shades. Corrective cosmetics are available in various shades, allowing a perfect match to normal skin color in most patients. Synthetic melanins have been incorporated into cover-ups that may be useful in patients with vitiligo [43]. The use of DHA (dihydroxyacetone 1,3-dihydroxydimethylacetone) to camouflage the depigmented lesions of patients with vitiligo vulgaris and segmental vitiligo has been proposed recently [44]. DHA preparations color the stratum corneum brown owing to its oxidative properties and provide temporary pigmentation resembling a UV-induced tan. In general, DHA pigmentation is not considered to be photoprotective. Recent investigations suggest that manipulation of the extent of hydration, pH, and availability of certain amino acids in the stratum corneum might produce DHA-induced pigmentation with greater photoprotection. For the camouflage of vitiligo lesions, DHA was stabilized at optimal levels. Five percent DHA was prepared with 10% ethanol and 1% sodium citrate buffer with 0.1% ethylene diamine tetracetic acid (EDTA) at pH 4.5 at 4°C. After application with a sponge swab, the result appeared after a reaction time of approximately 6 hours. The pigmentation cannot be rubbed off on clothes or be removed by washing and remains for about 3–4 days. The color fades slowly with desquamation of skin. The main disadvantage is that it does not give a uniform color to the skin. Beta-carotene and canthaxanthin (Phenoro-carotene 10 mg and canthaxanthin 15 mg per capsule) oral preparations have been used to treat cosmetic defects in vitiligo. By darkening vitiliginous skin, they reduce the contrast between involved and normally pigmented skin. Good cosmetic results are seen in vitiligo patients with skin types I and II. In one study, 10–35% of patients gave very satisfactory responses, with the rest unsatisfactory. There is increased resistance to sun exposure in vitiligo. As canthaxanthin is reported to produce retinopathy, proper ophthalmic consultations are mandatory.

Medical treatment of vitiligo Eye shadows, mascaras, and liners accentuate patients’ eyes and draw attention to them to further distract from facial cosmetic defects while lipsticks can be used to cover vitiligo of lips.

Psychological support Vitiligo may cause a considerable level of distress due to its disfiguring nature and the quality of life of a majority of patients is very severe. Vitiligo patients often experience indifference from the doctors toward their skin problem and do not feel adequately supported by them. About 50% of vitiligo patients feel that they are not adequately informed about their disease and its treatment [45]. Only 36% of physicians encourage their patients to treat the disease, being pessimistic concerning expected treatment results [44].

Conclusion Patients suffering from vitiligo need a global therapeutic approach. The disease and its course need to be fully explained to the patients and all therapeutic options discussed. Medical treatments bring very useful ways to repigment or decrease the contrast with healthy skin. Potent topical steroids and NB-UVB are considered to be the best first choices for localized and generalized vitiligo, respectively. However, treatments such as topical calcineurin inhibitors and focused phototherapy provide interesting new options. Finally, the combination therapies clearly show better rates of repigmentation and their use should certainly increase in the next few years.

References 1 Kent G, Al’Abadie M. Psychologic effects of vitiligo: a critical incident analysis. J Am Acad Dermatol 1996;35: 895–8. 2 Parsad D, Pandhi R, Dogra S, et al. Dermatology Life Quality Index score in vitiligo and its impact on the treatment outcome. Br J Dermatol 2003;148:373–4. 3 Ortonne JP, Passeron T. Melanin pigmentary disorders: treatment update. Dermatol Clin 2005;23:209–26. 4 Njoo MD, Spuls PI, Bos JD, et al. Nonsurgical repigmentation therapies in vitiligo. Meta-analysis of the literature. Arch Dermatol 1998;134:1532–40.

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5 Passeron T. UVB therapy for pigmentary disorders. In: Nordlund JJ, Boissy RE, Hearing VJ, King RA, and Ortonne JP (eds.) The Pigmentary System. 2nd ed. New York: Oxford University Press, 2006;1183–7. 6 Lindelof B, Sigurgeirsson B, Tegner E, et al. PUVA and cancer risk: the Swedish follow-up study. Br J Dermatol 1999;141:108–12. 7 Stern RS, Nichols KT, Vakeva LH. Malignant melanoma in patients treated for psoriasis with methoxsalen (psoralen) and ultraviolet A radiation (PUVA). The PUVA follow-up study. N Engl J Med 1997;336:1041–5. 8 Stern RS. The risk of melanoma in association with long-term exposure to PUVA. J Am Acad Dermatol 2001; 44:755–61. 9 Garland CF, Garland FC, Gorham ED. Epidemiologic evidence for different roles of ultraviolet A and B radiation in melanoma mortality rates. Ann Epidemiol 2003;13:395–404. 10 Slaper H, Schothorst AA, van der Leun JC. Risk evaluation of UVB therapy for psoriasis: comparison of calculated risk for UVB therapy and observed risk in PUVA-treated patients. Photodermatology 1986;3: 271–83. 11 Schaffer JV, Bolognia JL. The treatment of hypopigmentation in children. Clin Dermatol 2003;21:296–310. 12 Grimes PE. Psoralen photochemotherapy for vitiligo. Clin Dermatol 1997;15:921–6. 13 Koster W, Wiskemann A. [Phototherapy with UV-B in vitiligo]. Z Hautkr 1990;65:1022–4, 1029. 14 Hartmann A, Lurz C, Hamm H, et al. Narrow-band UVB 311 nm vs. broad-band UVB therapy in combination with topical calcipotriol vs. placebo in vitiligo. Int J Dermatol 2005;44:736–42. 15 Mofty ME, Zaher H, Esmat S, et al. PUVA and PUVB in vitiligo – Are they equally effective? Photodermatol Photoimmunol Photomed 2001;17:159–63. 16 Ortonne J. Vitiligo and other disorders of hypopigmentation. In: Bolognia J, Jorizzo J and Rapini R (eds) Dermatology, Vol. I. New York: Mosby, 2003;947–73. 17 Schallreuter KU, Wood JM, Lemke KR, Levenig C. Treatment of vitiligo with a topical application of pseudocatalase and calcium in combination with short-term UVB exposure: a case study on 33 patients. Dermatology 1995;190:223–9. 18 Lotti TM, Menchini G, Andreassi L. UV-B radiation microphototherapy. An elective treatment for segmental vitiligo. J Eur Acad Dermatol Venereol 1999; 13:102–8. 19 Menchini G, Tsoureli-Nikita E, Hercogova J. Narrowband UV-B micro-phototherapy: a new treatment for vitiligo. J Eur Acad Dermatol Venereol 2003;17:171–7.

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20 Leone G, Iacovelli P, Paro Vidolin A, Picardo M. Monochromatic excimer light 308 nm in the treatment of vitiligo: a pilot study. J Eur Acad Dermatol Venereol 2003;17:531–7. 21 Smith DA, Tofte SJ, Hanifin JM. Repigmentation of vitiligo with topical tacrolimus. Dermatology 2002;205: 301–3. 22 Mayoral FA, Gonzalez C, Shah NS, Arciniegas C. Repigmentation of vitiligo with pimecrolimus cream: a case report. Dermatology 2003;207:322–3. 23 Grimes PE, Soriano T, Dytoc MT. Topical tacrolimus for repigmentation of vitiligo. J Am Acad Dermatol 2002;47:789–91. 24 Lepe V, Moncada B, Castanedo-Cazares JP, TorresAlvarez MB, Ortiz CA, Torres-Rubalcava AB. A doubleblind randomized trial of 0.1% tacrolimus vs 0.05% clobetasol for the treatment of childhood vitiligo. Arch Dermatol 2003;139:581–5. 25 Coskun B, Saral Y, Turgut D. Topical 0.05% clobetasol propionate versus 1% pimecrolimus ointment in vitiligo. Eur J Dermatol 2005;15:88–91. 26 Ostovari N, Passeron T, Lacour JPh, Ortonne JP. Lack of efficacy of tacrolimus in the treatment of vitiligo in the absence of UVB exposure in difficult to treat sites. Arch Dermatol 2006;142:252–3. 27 Westerhof W, Nieuweboer-Krobotova L, Mulder PG, Glazenburg EJ. Left-right comparison study of the combination of fluticasone propionate and UV-A vs. either fluticasone propionate or UV-A alone for the long-term treatment of vitiligo. Arch Dermatol 1999; 135:1061–6. 28 Patel DC, Evans AV, Hawk JL. Topical pseudocatalase mousse and narrowband UVB phototherapy is not effective for vitiligo: an open, single-centre study. Clin Exp Dermatol 2002;27:641–4. 29 Parsad D, Saini R, Verma N. Combination of PUVAsol and topical calcipotriol in vitiligo. Dermatology 1998; 197:167–70. 30 Ermis O, Alpsoy E, Cetin L, Yilmaz E. Is the efficacy of psoralen plus ultraviolet A therapy for vitiligo enhanced by concurrent topical calcipotriol? A placebo-controlled double-blind study. Br J Dermatol 2001;145:472–5. 31 Baysal V, Yildirim M, Erel A, Kesici D. Is the combination of calcipotriol and PUVA effective in vitiligo? J Eur Acad Dermatol Venereol 2003;17:299–302. 32 Kullavanijaya P, Lim HW. Topical calcipotriene and narrowband ultraviolet B in the treatment of vitiligo. Photodermatol Photoimmunol Photomed 2004;20:248–51.

33 Ada S, Sahin S, Boztepe G, Karaduman A, Kolemen F. No additional effect of topical calcipotriol on narrowband UVB phototherapy in patients with generalized vitiligo. Photodermatol Photoimmunol Photomed 2005; 21:79–83. 34 Passeron T, Ostovari N, Zakaria W, et al. Topical tacrolimus and the 308-nm excimer laser: a synergistic combination for the treatment of vitiligo. Arch Dermatol 2004;140:1065–9. 35 Kawalek AZ, Spencer JM, Phelps RG. Combined excimer laser and topical tacrolimus for the treatment of vitiligo: a pilot study. Dermatol Surg 2004;30:130–5. 36 Castanedo-Cazares JP, Lepe V, Moncada B. Repigmentation of chronic vitiligo lesions by following tacrolimus plus ultraviolet-B-narrow-band. Photodermatol Photoimmunol Photomed 2003;19:35–6. 37 Tanghetti EA, Gillis PR. Clinical evaluation of B clear and protopic treatment for vitiligo. Laser Surg Med 2003;32:37. 38 Parsad D, Pandhi R, Dogra S, Kumar B. Topical prostaglandin analog (PGE2) in vitiligo – a preliminary study. Int J Dermatol 2002;41:942–5. 39 Mosher DB, Parrish JA, Fitzpatrick TB. Monobenzylether of hydroquinone. A retrospective study of treatment of 18 vitiligo patients and a review of the literature. Br J Dermatol 1977;97:669–79. 40 Silvan M. The psychological aspects of vitiligo. Cutis 2004;73:163–7. 41 Njoo MD, Vodegel RM, Westerhof W. Depigmentation therapy in vitiligo universalis with topical 4-methoxyphenol and the Q-switched ruby laser. J Am Acad Dermatol 2000;42:760–9. 42 Ongenae K, Dierckxsens L, Brochez L, van Geel N, Naeyaert JM. Quality of life and stigmatization profile in a cohort of vitiligo patients and effect of the use of camouflage. Dermatology 2005;210:279–85. 43 Levy SB. Tanning preparations. Dermatol Clin 2000; 18:591–6. 44 Suga Y, Ikejima A, Matsuba S, Ogawa H. Medical pearl: DHA application for camouflaging segmental vitiligo and piebald lesions. J Am Acad Dermatol 2002; 47:436–8. 45 Ongenae K, Van Geel N, De Schepper S, et al. Management of vitiligo patients and attitude of dermatologists towards vitiligo. Eur J Dermatol 2004;14: 177–81.

SECTION 2

Overview of surgical management

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CHAPTER 5

History and chronology of development of surgical therapies for vitiligo Rafael Falabella

Introduction Skin grafts in ancient times The history of skin grafts is, as frequently occurs in mankind, a result of creativity and imagination. Transferring skin from a donor site to a recipient area in order to enhance healing of wounds was a human dream that started many centuries ago. Sanskrit texts, 2500–3000 BC, in ancient India, describe skin transplants done by potters and tile makers of the Koomas caste in individuals with mutilated noses after punishment for theft or adultery [1]. Grafts were harvested from buttock skin and adapted to the new recipient site for nasal reconstruction [2,3].

Switzerland described the first autograft with very thin pinch grafts that stimulated granulation tissue [5], and in 1872 Oilier in France observed better results with “split-thickness skin grafts” when some dermis was included in the grafted tissue [6]. Following this method, in 1875, Wolfe in Scotland, introduced the full-thickness skin graft for the treatment of ectropion [7] and in 1886, Karl Thiersch in Germany reported the use of thin split-thickness skin grafts in clinical practice [8], though it had been previously initiated by Oilier 14 years before. In 1944, Brown in England developed the electric dermatome, for rapid, thinner, and homogeneous harvesting of split-thickness skin grafts [8], a method that was improved and perfected in modern times.

Skin grafts during and after renaissance Many centuries went by before further developments for skin grafting occurred. Early work in Europe at the beginning of the Renaissance was done by Brancas, an Italian surgeon, who developed a novel technique for nasal reconstruction by using skin from the arm; this method was also credited one century later to Gasparo Tagliacozzi, also from Italy, who published his work in 1597 in his treatise “De curtorum chirurgia per insitionem,” where he described a similar method for reconstruction of nasal deformities inflicted by syphilis and war wounds [1]. In 1823, Bunger in Germany successfully described the first autograft by repairing nasal defects using full thickness skin grafts from the patient’s thigh [4]. Later, in 1869, Reverdin in

Depigmentation is a physical and social disease Depigmented disorders correspond to diverse dermatoses in which melanocytes are absent since birth or disappear because of trauma or gradual destruction. Many of these conditions are still labeled as “idiopathic” since their pathogenesis is not yet completely understood. However, modern technology and meticulous research have contributed to an enormous amount of knowledge, which constitute an important platform for unraveling the secrets of most pigmentary disturbances within a reasonable time in the forthcoming future. From ancient times, depigmented disorders were known in different cultures and were addressed in

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medical or history books. Egyptians were the first to recognize depigmentation, and probably vitiligo was one of the most common ailments affecting this civilization; they treated these disorders with a plant extract from Ammi majus, which was usually grown on the lands near the Nile river and was a source of the important molecule psoralen, still in use. In some cultures, these disorders were recognized for many centuries, and in addition, they might have also been confused with leprosy, pinta or other infectious diseases of the skin, leading to severe social problems for affected individuals, such as discrimination, familial stigmatization, lack of job opportunities, and even divorce or social rejection [9]. Today, we frequently see vitiligo patients having similar problems and the clinician should be aware that vitiligo is not only a cosmetic ailment, but also a source of social problems in affected individuals, and that patients deserve treatment whenever possible.

First attempts on vitiligo grafting: exchange grafts By the middle of the 20th century, in 1952, original work by Spencer and Tolmach [10] was done with exchange grafts; punch grafts harvested from normally pigmented skin were implanted onto leukodermic areas of patients with vitiligo. Inconsistent results were frequently observed in these trials, since sometimes repigmentation around the grafted skin was seen in some patients, whereas in other individuals repigmentation failure was the rule. Later on, in 1986, studies with punch grafts were done by Beck and Schmidt [11], who disclosed similar findings and the question at this time was why repigmentation occurred in some patients and repigmentation failure was frequently observed.

Development of surgical methods for repigmentation of vitiligo Long before most of the findings known today about the pathogenesis of vitiligo had been accomplished, different investigators were trying to solve the problem of depigmentation in patients with refractory vitiligo not responding to medical therapies, with grafting and transplantation techniques. Basically, five groups of procedures have been described since

initial trials were done, and they will be described in the following section in chronological order, as they were reported in the literature.

Thin dermo-epidermal grafts This method was the first one described as a surgical technique in 1964 by Behl in India [12]. This method consisted of using dermo-epidermal grafts harvested with knife or dermatome in patients with different types of vitiligo, who successfully recovered their pigmentation after grafting. Split-thickness grafts were easily harvested with this technique and placed onto achromic recipient surfaces, previously denuded with dermabrasion. Slight to moderate scarring was a possible side effect, depending on the thickness and unevenness of grafts. In 1973 the same author reported additional patients treated by this method [13], and after more than 20 years, he used this method for treating several hundred patients with similar success [14]. This method was continued and improved by Kahn et al. [15] and Kahn and Cohen [16] by obtaining very thin dermo-epidermal sheets which were harvested with a technologically advanced motorized dermatome and implanted onto superficially dermabraded recipient surfaces; the achieved results were impressive with no visible scarring and excellent repigmentation, but stability of vitiligo was an important issue in order to reach good results. In 1995, Agrawal and Agrawal [17] expanded this experience in more patients, and achieved high figures of repigmentation. In 1996, Kahn et al. [18] reported the use of a short-pulse carbon dioxide laser for de-epithelialization of achromic lesions in vitiligo and demonstrated that this laser does not cause sufficient thermal necrosis on the surface of the papillary dermis to interfere with a satisfactory skin graft take and excellent repigmentation. Very thin split-thickness grafts were also used in 1997 by Olsson and Juhlin [19], and again, very good results occurred in the treated patients. Today, this method is well recognized by international experts in repigmentation and if carried out properly yields excellent results [20]. A miniature modification of this technique was reported with success in 1999 by McGovern et al.

History of repigmentation surgery [21], by grafting very small thin pieces of skin harvested by thin shaving, and inserting them under a small dermo-epidermal flap raised by shaving. They described this technique as flip-top transplantation.

Epidermal grafting Less than 10 years after thin dermo-epidermal grafts were initiated, epidermal grafting with suction blisters was successfully used for the first time in 1971 by Falabella for repigmentation of leukoderma in a patient with depigmentation post-burn and another one with segmental vitiligo, initially diagnosed as a depigmented nevus [22,23]. In 1984, this new technique for repigmentation was successfully tried by Falabella [24] in additional patients with vitiligolike leukoderma occurring after thermal trauma, a response that was expected because of the stable nature of this condition. In 1985, a group of patients with vitiligo were treated with suction epidermal grafting by Suvanprakorn et al. [25], and interesting results disclosed that although repigmentation with this method was possible in these patients, a good number of them had unsuccessful results. His results were expanded in 1988 by Koga [26], who found that the Köbner phenomenon is a limitation for performing this procedure in patients with active vitiligo; in addition, in 1993, Mutalik [27] found excellent repigmentation in 48 of 50 patients with stationary vitiligo. In two different publications in 1992 and 1995, Skouge et al. [28] and Skouge and Morison [29] reported that harvesting epidermal grafts could be accelerated by heat within the suction device, and that psoralen photochemotherapy psoralen plus ultraviolet A (PUVA) was an important complement for this treatment to obtain faster and even repigmentation. Furthermore, in 1996, Suga et al. [30] demonstrated the high efficacy of epidermal grafting and topical PUVA in 28 patients with localized and segmental vitiligo not responding to other therapies. Finally, refractory areas such as lips were successfully repigmented in 2004 by Gupta et al. [31] with epidermal grafting followed by photochemotherapy. At present, epidermal grafting is one of the most popular techniques used with great success for vitiligo repigmentation.

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Minigrafting This repigmentation technique was described in 1983 by Falabella [32] in three patients with segmental (unilateral) vitiligo. This method was designed to simulate the pigmentation response occurring during perifollicular repigmentation in vitiligo after medical treatment, a method that is particularly successful for patients with stable vitiligo. Additional patients were treated successfully in 1986 and 1988 by the same author [33,34]. Small minigrafts of 1–1.2 mm were harvested with a small punch from hidden donor sites, such as the gluteal region, and implanted at recipient sites, where similar perforations were made to prepare the recipient site. Repigmentation of affected skin disclosed a similar repigmentation to that originated by migration of melanocytes around hair follicles, with subsequent coalescence and complete recovery of depigmented lesions. An interesting test for evaluation of vitiligo stability, the minigrafting test, was described in 1995 by Falabella et al. [35] by transplanting 4–6 minigrafts on recipient sites; repigmentation around minigrafts allowed detection of patients with stable vitiligo that might be candidates for treatment by surgical methods. So far, this is the most powerful tool described for detecting stability of vitiligo before a decision on surgical therapy is made [36]. After this report, in 1995, Boersma et al. [37], found high percentages of repigmentation when grafting 59 patients with stable vitiligo vulgaris (bilateral) that had been selected with the minigrafting test, before performing minigrafting. Minigrafting is the simplest so far described, but its success depends on the small size of minigrafts. When larger grafts have been used, that is, punch grafts of 2.0–2.5 mm, high percentages of repigmentation have also been achieved, but less acceptable cosmetic results occur with this method, as reported in 1999 by Malakar and Dhar [38] and in 2001 by Sarkar et al. [39]. Malakar and Lahiri [40] demonstrated the usefulness of punch grafts in lip vitiligo, but he reported cobblestoning as a side effect in 30% of his group of 108 patients, most probably because grafts larger than 1.2 mm were used in the procedure.

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Today, minigrafting is the simplest method providing a high percentage of repigmentation in many patients with stable vitiligo that can be performed in serial sessions, if required.

Epidermal suspensions An interesting and efficacious method for repigmentation using epidermal suspensions was initially described in 1992 by Gauthier and Surleve-Bazeille [41]. The epidermal cell suspension was obtained by exposing a thin dermo-epidermal sheet harvested from the scalp of a patient with vitiligo to a 0.25% trypsin solution. After enzymatic digestion, the epidermal cells became separated individually forming a cell suspension that was seeded on the achromic area, denuded by liquid nitrogen freezing. The grafted cells gradually produced a homogeneous repigmentation of the treated area. This method was improved in 1998 by Olsson and Juhlin [42] by adding a melanocyte culture medium for additional stimulation of melanocyte growth in order to enhance the repigmentation yield of treated areas. Further on, the technique was modified in 2001 by van Geel et al. [43], by complementing the cell suspension with hyaluronic acid, which allowed a better adherence of cells to the grafted surface and permitted a faster and easier procedure. van Geel et al. [44] performed the first prospective, randomized, double-blind, placebo-controlled study with epidermal suspensions in symmetrically distributed vitiligo lesions. In 28 patients resistant to medical therapy, a strong difference between epidermal suspensions and placebo was found, demonstrating that repigmentation was primarily caused by the transplanted melanocytes. In another study done in 2003 by Mulekar [45] with epidermal suspensions, remarkable success was achieved in 184 patients with high percentages of repigmentation in unilateral vitiligo. More recently, in 2004, a relatively good response in bilateral vitiligo was observed by the same author [46] who demonstrated a high repigmentation percentage in 50 patients with unilateral vitiligo followed-up for up to 5 years post grafting. Epidermal suspension transplantation is an excellent repigmentation technique that causes no scarring at recipient site and yields a high repigmentation rate.

In vitro cultures of epidermis with melanocytes and melanocyte suspensions In 1989, epidermis with melanocytes was cultured for the first time for repigmentation purposes by Falabella et al. [47]. Epidermal layers successfully grown on Eagle’s minimal essential medium (MEM) supplemented with pituitary extract and hormones, were applied initially to a patient with segmental vitiligo with satisfactory results. Shortly thereafter, three additional patients with unilateral and bilateral stable vitiligo were treated by the same authors with the same method, and 100% repigmentation was also observed [48]. The achieved improvement in these patients remained unchanged during the following 7 years, suggesting that repigmentation was permanent. Almost simultaneously, in the same year, Brysk et al. [49] also reported a small group of patients treated by a similar method with epidermis and melanocytes grown on a 3T3 cell medium, with successful repigmentation. A few months later, Plott et al. [50] reported further, although incomplete repigmentation, in three out of four patients with vitiligo using the same method as described previously by Brysk [49]. In 1992, Falabella et al. [51] described three additional patients with refractory vitiligo who achieved satisfactory repigmentation with the original method reported previously. In 1993, a modification of the epidermal suspensions method was suggested by Jha et al. [52], to enhance the number of melanocytes and epidermal cells; the epidermal suspension was cultured for a short period of time and then grafted onto denuded recipient sites, achieving good repigmentation of the treated patients. Not too long after these successful trials, in 1992, pure melanocyte suspensions were cultured and implanted in patients with vitiligo by Olsson and Juhlin [53] and Lontz et al. [54] who achieved good repigmentation in their patients. Shortly thereafter, in 1994, Olsson et al. [55], reported the use of cultured melanocyte suspensions that were kept in cryostorage for several months, and successful repigmentation was observed in four patients with vitiligo, demonstrating the potential of this technique for treating patients with extensive vitiligo in several

History of repigmentation surgery sessions, with melanocytes harvested from a single donor site. Additional work with autologous cultured melanocytes, ultra-thin epidermal sheets, and basal cell layer suspensions done later in 2002 by Olsson and Juhlin [56], for evaluating the efficacy and permanency of repigmentation, disclosed no significant difference with these different methods. Other modifications in the culture cell methods were carried out in the following years by different authors. In 1995, Falabella et al. [57] added minigrafting as a complement for repigmentation to those areas of vitiligo not resolved completely by in vitro cultured epidermis. In 1998, Andreassi et al. [58] developed a novel method to improve melanocyte cultures by incorporating a supporting layer of biomaterial to cultured keratinocytes and melanocytes that were used to cover depigmented skin in vitiligo. In 2003, Guerra et al. [59] modified epidermal cultures and obtained epidermis with melanocytes using porcine dermis as a supporting collagen material, to facilitate handling of cultured grafts during surgery. Cultured epidermis with melanocytes and pure melanocyte suspensions are important technological advances for refractory vitiligo therapy. Although still at a developmental stage, these methods have proved highly efficacious, so far free of serious side effects, and constitute a great hope for the future. Simplification of such techniques and reasonable costs will gradually make them the method of choice for treatment of vitiligo with extensive depigmentation in future.

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A different surgical option for repigmentation described in 1998 by Na et al. [64] was performed by implanting the upper two-thirds of scalp hair follicles separated individually by microdissection, into depigmented patches of vitiligo. The aim was to obtain repigmentation from melanocytes located primarily at the follicular ostium. The elimination of the lower third of the hair shaft prevented hair growth on the treated surfaces. Lastly, the ruby laser was introduced in 1997 by Thissen and Westerhof [65] for the purpose of eliminating the unsightly residual pigmentation in extensive vitiligo not responding to depigmentation with medical therapy.

Repigmentation of leukotrichia Repigmentation of leukotrichia was initially observed in 1989 and in 1992 by Falabella et al. [47,49] after transplantation of in vitro cultured epidermis with melanocytes in achromic areas of vitiligo with depigmented hairs. The most possible explanation of this phenomenon was interpreted as a migration of pigment cells in a reversal pathway from the follicular ostium, downwards through the outer root sheath and finally to the hair bulb of depigmented hairs, where melanocytes transferred melanosomes to the newly formed hair shaft, resulting in hair repigmentation. In 1992, Hann et al. [66] successfully repigmented leukotrichia in vitiligo patients with epidermal grafting and PUVA. Later, in 1995, Agrawal and Agrawal [67] performed thin split thickness grafts to repigment leukotrichia in a group of eight patients, with partial pigment recovery. They achieved better results in eyebrows.

Other procedures Different types of lasers were proposed for preparing an adequate graft recipient bed, particularly for irregular lesions on difficult sites which could not be approached by liquid nitrogen blistering or dermabrasion; for this purpose, in 1996 the shortpulse carbon dioxide laser was successfully used by Kahn et al. [60], and in 1998, the pulsed ErbiumYAG laser was introduced by Yang and Kye [61] and by Kaufmann et al. [62]. Another method for preparing a good recipient site by using sonic abrasion for epidermal removal was described in 2002 by Tsukamoto et al. [63].

Conclusions From ancient to modern times a long history of vitiligo therapy has elapsed. Although this condition is yet a disease of unknown etiology, therapy and prognosis have improved remarkably during the past 50 years, after unraveling most of the histological, ultra-structural, biochemical, immunological, and molecular events occurring within and around the pigment cell. Surgical attempts to treat a disease that appeared initially as an exclusive medical condition began in the past century around the 1960s, and different approaches with many refinements have been

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successfully reported since then. Nevertheless, more work with the modern sophisticated molecular biology techniques available today is required to find the ultimate cause of vitiligo and render this condition completely stable; only then can all patients with vitiligo have a total recovery with a combination of medical and surgical therapies, by making melanocytes available in every depigmented area of skin [68].

References 1 Davis JS. The story of plastic surgery. Ann Surg 1941;113:641–56. 2 Chick LR. Brief history and biology of skin grafting. Ann Plast Surg 1988;21:358–65. 3 Hauben DJ, Baruchin A, Mahler D. On the history of the free skin graft. Ann Plast Surg 1982;9:242–6. 4 Chick LR. Brief history and biology of skin grafting. Ann Plast Surg 1988;21:358–65. 5 Reverdin JL. Greffe epidermique. Bull Soc Imperiale Chir Paris 1869;493. 6 Ollier L. Sur les greffes cutanees ou autoplastiques. Bull Acad Med Paris 1872;2:243. 7 Wolfe JR. A new method of performing plastic operations. Br J Med 1875;2:360. 8 Herman AR. The history of skin grafts. J Drugs Dermatol 2002;1:298–301. 9 Porter JR, Beuf AHJ. Racial variation in reaction to physical stigma: a study of degree of disturbance by vitiligo among black and white patients. Health Soc Behav 1991;32:192–204. 10 Spencer GA, Tolmach JA. Exchange grafts in vitiligo. J Invest Dermatol 1952;19:1–5. 11 Beck HI, Schmidt H. Graft exchange in vitiligo. Studies on the outcome of exchanging biopsies from vitiliginous skin to normal, pigmented skin and vice versa. Acta Derm Venereol 1986;66:311–5. 12 Behl PN. Treatment of vitiligo with homologous thin Thiersch skin grafts. Curr Med Pract 1964;8:218–21. 13 Behl PN, Bhatia RK. Treatment of vitiligo with autologous thin Thiersch’s grafts. Int J Dermatol 1973;12:329–31. 14 Behl PN. Repigmentation of segmental vitiligo by autologous minigrafting (letter to the editor). J Am Acad Dermatol 1985;12:118–9. 15 Kahn AM, Cohen MJ, Kaplan L, Highton A. Vitiligo: treatment by dermabrasion and epithelial sheet grafting—a preliminary report. J Am Acad Dermatol 1993;28:773–4.

16 Kahn AM, Cohen MJ. Vitiligo: treatment by dermabrasion and epithelial sheet grafting. J Am Acad Dermatol 1995;33:646–8. 17 Agrawal K, Agrawal A. Vitiligo: repigmentation with dermabrasion and thin split-thickness skin graft. Dermatol Surg 1995;21:295–300. 18 Kahn AM, Ostad A, Moy RL. Grafting following shortpulse carbon dioxide laser de-epithelialization. Dermatol Surg 1996;22:965–7. 19 Olsson MJ, Juhlin L. Epidermal sheet grafts for repigmentation of vitiligo and piebaldism, with a review of surgical techniques. Acta Derm Venereol 1997;77: 463–6. 20 Kahn AM, Cohen MJ. Repigmentation in vitiligo patients. Melanocyte transfer via ultra-thin grafts. Dermatol Surg 1998;24:365–8. 21 McGovern TW, Bolognia J, Leffell DJ. Flip-top pigment transplantation: a novel transplantation procedure for the treatment of depigmentation. Arch Dermatol 1999;135:1305–7. 22 Falabella R. Epidermal grafting: an original technique and its application in achromic and granulating areas. Arch Dermatol 1971;104:592–600. 23 Falabella R. Suction blister device for separation of viable epidermis from dermis. Clin Exp Dermatol 2004;29:105–6. 24 Falabella R. Repigmentation of leukoderma by autologous epidermal grafting. J Dermatol Surg Oncol 1984;10:136–44. 25 Suvanprakorn P, Dee-Ananlap S, Pongsomboon C, Klaus SN. Melanocyte autologous grafting for treatment of leukoderma. J Am Acad Dermatol 1985;13:968–74. 26 Koga M. Epidermal grafting using the tops of suction blisters in the treatment of vitiligo. Arch Dermatol 1988;124:166–8. 27 Mutalik S. Transplantation of melanocytes by epidermal grafting. An Indian experience. J Dermatol Surg Oncol 1993;19:231–4. 28 Skouge JW, Morison WL, Diwan RV, Rotter S. Autografting and PUVA. A combination therapy for vitiligo. J Dermatol Surg Oncol 1992;18:357–60. 29 Skouge JW, Morison WL. Vitiligo treatment with a combination of PUVA therapy and epidermal autografts. Arch Dermatol 1995;131:1257–8. 30 Suga Y, Butt KI, Takimoto R, Fujioka N, Yamada H, Ogawa H. Successful treatment of vitiligo with PUVApigmented autologous epidermal grafting. Int J Dermatol 1996;35:518–22. 31 Gupta S, Sandhu K, Kanwar A, Kumar B. Melanocyte transfer via epidermal grafts for vitiligo of labial mucosa. Dermatol Surg 2004;30:45–8.

History of repigmentation surgery 32 Falabella R. Repigmentation of segmental vitiligo by autologous minigrafting. J Am Acad Dermatol 1983; 9:514–21. 33 Falabella R. Repigmentation of stable leukoderma by autologous minigrafting. J Dermatol Surg Oncol 1986;12:172–9. 34 Falabella R. Treatment of localized vitiligo by autologous minigrafting. Arch Dermatol 1988;124:1649–55. 35 Falabella R, Arrunategui A, Barona MI, Alzate A. The minigrafting test for vitiligo: detection of stable lesions for melanocyte transplantation. J Am Acad Dermatol 1995;32:228–32. 36 Falabella R. The minigrafting test for vitiligo: validation of a predicting tool. J Am Acad Dermatol 2004;51: 672–3. 37 Boersma BR, Westerhof W, Bos JD. Repigmentation in vitiligo vulgaris by autologous minigrafting: results in nineteen patients. J Am Acad Dermatol 1995;33:990–5. 38 Malakar S, Dhar S. Treatment of stable and recalcitrant vitiligo by autologous miniature punch grafting: a prospective study of 1,000 patients. Dermatol 1999;198:133–9. 39 Sarkar R, Mehta SD, Kanwar AJ. Repigmentation after autologous miniature punch grafting in segmental vitiligo in North Indian patients. J Dermatol 2001;28:540–6. 40 Malakar S, Lahiri K. Punch grafting for lip leucoderma. Dermatol 2004;208:125–8. 41 Gauthier Y, Surleve-Bazeille JE. Autologous grafting with noncultured melanocytes: a simplified method for treatment of depigmented lesions. J Am Acad Dermatol 1992;26:191–4. 42 Olsson MJ, Juhlin L. Leucoderma treated by transplantation of a basal cell layer enriched suspension. Br J Dermatol 1998;138:644–8. 43 van Geel N, Ongenae K, De Mil M, Naeyaert JM. Modified technique of autologous noncultured epidermal cell transplantation for repigmenting vitiligo: a pilot study. Dermatol Surg 2001;27:873–6. 44 van Geel N, Ongenae K, De Mil M, et al. Double-blind placebo-controlled study of autologous transplanted epidermal cell suspensions for repigmenting vitiligo. Arch Dermatol 2004;140:1203–8. 45 Mulekar SV. Melanocyte-keratinocyte cell transplantation for stable vitiligo. Int J Dermatol 2003;42:132–6. 46 Mulekar SV. Long-term follow-up study of segmental and focal vitiligo treated by autologous, noncultured melanocyte-keratinocyte cell transplantation. Arch Dermatol 2004;140:1211–15. 47 Falabella R, Borrero I, Escobar C. In vitro culture of melanocyte-bearing epidermis and its application in

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the treatment of vitiligo and the stable leukodermas. Med Cutan Ibero Lat Am 1989;17:193–8. Falabella R, Escobar C, Borrero I. Transplantation of in-vitro cultured epidermis bearing melanocytes for repigmenting vitiligo. J Am Acad Dermatol 1989;21: 257–66. Brysk MM, Newton RC, Rajaraman S, et al. Repigmentation of vitiliginous skin by cultured cells. Pigm Cell Res 1989;2:202–7. Plott RT, Brysk MM, Newton RC, et al. A surgical treatment for vitiligo: autologous cultured-epithelial grafts. J Dermatol Surg Oncol 1989;15:1161–6. Falabella R, Escobar C, Borrero I. Treatment of refractory and stable vitiligo by transplantation of in-vitro cultured epidermal autografts bearing melanocytes. J Am Acad Dermatol 1992;26:230–6. Jha AK, Pandey SS, Gulati AK, et al. Inoculation of a cultured autologous epidermal suspension containing melanocytes in vitiligo. Arch Dermatol 1993;129: 785–6. Olsson MJ, Juhlin L. Melanocyte transplantation in vitiligo. Lancet 1992;340(8825):981. Lontz W, Olsson MJ, Moellmann G, Lerner AB. Pigment cell transplantation for treatment of vitiligo: a progress report. J Am Acad Dermatol 1994;30:591–7. Olsson MJ, Moellmann G, Lerner AB, Juhlin L. Vitiligo: repigmentation with cultured melanocytes after cryostorage. Acta Derm Venereol 1994;74:226–8. Olsson MJ, Juhlin L. Long-term follow-up of leucoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol 2002;147: 893–904. Falabella R, Barona M, Escobar C, et al. Surgical combination therapy for vitiligo and piebaldism. Dermatol Surg 1995;21:852–7. Andreassi L, Pianigiani E, Andreassi A, Taddeucci P, Biagioli M. A new model of epidermal culture for the surgical treatment of vitiligo. Int J Dermatol 1998;37:595–8. Guerra L, Primavera G, Raskovic D, et al. Erbium: YAG laser and cultured epidermis in the surgical therapy of stable vitiligo. Arch Dermatol 2003;139:1303–10. Kahn AM, Ostad A, Moy RL. Grafting following shortpulse carbon dioxide laser de-epithelialization. Dermatol Surg 1996;22:965–7. Yang JS, Kye YC. Treatment of vitiligo with autologous epidermal grafting by means of pulsed erbium: YAG laser. J Am Acad Dermatol 1998;38:280–2. Kaufmann R, Greiner D, Kippenberger S, Bernd A. Grafting of in vitro cultured melanocytes onto

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laser-ablated lesions in vitiligo. Acta Derm Venereol 1998; 78:136–8. 63 Tsukamoto K, Osada A, Kitamura R, et al. Approaches to repigmentation of vitiligo skin: new treatment with ultrasonic abrasion, seed-grafting and psoralen plus ultraviolet A therapy. Pigment Cell Res 2002;15:331–4. 64 Na GY, Seo SK, Choi SK. Single hair grafting for the treatment of vitiligo. J Am Acad Dermatol 1998;38:580–4. 65 Thissen M, Westerhof W. Laser treatment for further depigmentation in vitiligo. Int J Dermatol 1997;36: 386–8.

66 Hann SK, Im S, Park YK, Hur W. Repigmentation of leukotrichia by epidermal grafting and systemic psoralen plus UV-A. Arch Dermatol 1992;128:998–9. 67 Agrawal K, Agrawal A. Vitiligo: surgical repigmentation of leukotrichia. Dermatol Surg 1995;21:711–5. 68 Falabella R. Surgical treatment of vitiligo: why, when and how. J Eur Acad Dermatol Venereol 2003;17: 518–20.

CHAPTER 6

The concept of stability of vitiligo Koushik Lahiri and Subrata Malakar

If doubt is challenging you and you do not act, doubts will grow. Challenge the doubts with action and you will grow. Doubt and action are incompatible.

Table 6.1 Minimum period of stability in different studies. Author and reference

Year

Period of stability

Das and Pasricha [11]

1992

4 months

Boersma and Westerhof [12]

1995

6 months

Jha et al. [13]

1992

1 year

Savant [14]

1992

2 years

Falabella [15]

2003

2 years

Falabella [19]

1995

2 years

Falabella [7]

1992

3 years

John Kanary

Vitiligo has continued to elude researchers and patients over the years in spite of the considerable advancement in basic understanding of the disease process and the treatment protocol. Conventional medical therapy remains beleaguered by unpredictable and inadequate outcomes. When the disease becomes refractory to conservative therapy, transplantation techniques are the only options left to replenish the lost melanocytes. Various surgical modalities and transplantation techniques have evolved during last few decades [1–9]. But, to date none of the medical or surgical therapeutic choices assure success consistently in all the cases. This is primarily because of the obscure etiopathogenesis and elusive activity profile of the disease itself. Not only with medical therapy but also with any of the surgical modus operandi, proper selection of cases is of paramount importance. The specific criteria for selection have been well defined [6]. Any endeavor to define norms or principles for selection is based on one single criterion, that is, stability of the disease. It is taken as the most important parameter before opting for any transplantation technique to treat vitiligo [10]. Stability is the decisive factor, the cornerstone of surgical therapy for vitiligo.

has been little consensus regarding the optimal required period of stability. The recommended period of stability in different studies varies widely from 4 months to 3 years (Table 6.1) [7,11–13]. Unfortunately, none of these criteria were based on evidence obtained from the research. They were merely conceived on subjective basis and documented arbitrarily [15]. Even the 2-year time frame used to decide whether lesions are progressive or stable has been a matter of divergence of opinion [15]. Due to a dearth of a better alternative this criterion of a minimal period of stability still holds good for selecting patients for surgical procedure. The basic concept of stability in vitiligo is itself still not transparent and defined beyond doubt [16]. A few years back some unanswered questions were raised about this concept by us [16].

Period of stability

Different status of stability in different patches

The significance of stability in vitiligo surgery has been recognized for the last three decades, yet there

The first and foremost goal for vitiligo surgery is to induce complete and permanent repigmentation in

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depigmented skin. But due to some inexplicable reason simultaneous depigmentation and repigmentation have been noticed in different areas of the same patient [15]. Variable results were obtained over the donor site and recipient site of the same patient [15,16,18]. Depigmentation of grafts was documented in herpes-labialis-induced lip leukoderma [21–27]. Concurrent donor site repigmentation and depigmentation of grafts at the recipient site (Fig. 6.1) or donor site depigmentation with complete repigmentation of the recipient area with pigment spreading out from each minigraft have also been noticed. It was suggested to observe the donor site as well to rule out Köbnerization as a side effect of surgical manipulation while deciding on the stability status of the patient by minigraft test [18]. Simultaneous perigraft spread of pigment in one area and spread of vitiligo in another area can be found in the same patient (Fig. 6.2). Concurrence of surgical repigmentation from the grafts and increase in size of an existing lesion in the same anatomical location was also observed (Fig. 6.3A–C). This area based variable status of stability is not related to the conventional refractory behavior of vitiligo in some “resistant” anatomical sites, which lack melanocyte reservoir in the form of hair follicles, such as palm, sole, lips, nipples, areola, glans penis, and bony prominences. Neither is this related to the type of vitiligo such as unilateral (segmental/focal) or bilateral (symmetric, vulgaris, or generalized).

(A)

Assessment of stability Before any surgical intervention, evaluation of stability status of vitiligo is an essential prerequisite. Clinically, this can be judged by three simple indicators: 1 history, 2 Köbner phenomenon, and 3 test grafting (TG). Falabella proposed some criteria to assess the stability of the disease [6,19]. These are: 1 lack of progression of old lesions within the past 2 years (in unilateral vitiligo may be shorter, and in bilateral vitiligo, stability establishes after several years);

(B) Fig. 6.1 (A) Depigmentation of grafts in the recipient

area of stable vitiligo (shown with arrows). (B) Repigmentation of the donor site in the same patient.

The concept of stability of vitiligo

(A)

(B) Fig. 6.2 (A) Perigraft spread of pigment (shown with

arrow). (B) Simultaneous spread of disease over thumb and knee (shown with starburst sign).

2 no new lesions developing within the same period; 3 absence of a recent Köbner phenomenon (either from history or experimentally produced); 4 repigmentation of depigmented areas by medical treatment or sometimes spontaneous repigmentation; 5 a positive minigrafting test; and 6 lack of Köbnerization at donor site. On the backdrop of a pervasive incongruity about the minimal period of stability an attempt was made for the first time by Falabella in 1995 to fathom stability before surgery by introducing minigrafting test [17]. The purpose of this test was to serve several purposes: • establishing the stability of the depigmenting process;

51

• determining a means by which patients could be selected; • identifying patients who may respond to pigment cell transplantation; and • anticipating the response to surgical repair. In the original suggested procedure a few grafts (1.0–1.2 mm) were placed in the center of the depigmented lesion to be scrutinized. Dressing was done by Micropore® adhesive tape and kept for a couple of weeks. After removal of the tape the area was exposed to sunlight for 15 min daily for a period of 3 months. No treatment was permitted during this test period. All test sites are visualized under Wood’s light. The test was considered positive if unequivocal repigmentation took place beyond 1 mm from the border of the implanted grafts. On the other hand, if less than 1 mm or no repigmentation was observed the test was considered as negative [17]. In some of the biggest series this evaluation has been termed as “test grafting” (TG) and found this to be a more reliable criteria than the period of stability alone [20–22]. Over the years this “test” has been acknowledged as a powerful tool for detecting stable vitiligo, which anticipates the repigmentation success in vitiligo when surgery becomes a therapeutic option. But, simultaneously some skepticism occurred regarding this recognition. In the original article there was no mention of the donor site. One of the opinions was to take the outcome of the donor site as well into consideration. That will give a more comprehensive idea about the status of stability [18]. But, doubts were registered over overdependence on TG per se as that attaches a quite a few fallacies [16,20]: 1 TG positivity is not consistently associated with a favorable outcome in all cases even after successful graft take. 2 Encouraging results with transplantation were obtained in a few TG negative cases. 3 Simultaneous donor site repigmentation and depigmentation of grafts at the recipient site or donor site depigmentation with complete repigmentation of the recipient area has been noted. Juhlin echoed this apprehension and observed, “… neither the history nor test graft is a complete help to obtain good effect. It seems we have to wait until the method of identification and isolation of skin-homing

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(A)

(B)

Fig. 6.3 (A) Minigrafting on dorsum of foot, first session

(shown with arrows). (B) Second session, perigraft repigmentation from grafts of previous session (shown with arrows). (C) Appreciable repigmentation (arrow). Reactivation of disease on the same anatomical site (shown with starburst).

(C)

melanocyte-specific cytotoxic T-lymphocytes is available” [28]. None other than Falabella himself expressed doubts on the comprehensiveness of minigraft test. He observed, “even when the minigrafting test is positive, ‘depigmenting activity’ may still be present and prevent satisfactory repigmentation” [29]. More recently Njoo et al. explored the association between the experimentally induced Köbner phenomenon (KP-e) and the Köbner phenomenon by history (KP-h), disease activity, and therapeutic responsiveness in vitiligo vulgaris [10]. They proposed to measure disease activity on a 6-point scale from
1 to 4 (vitiligo disease activity [VIDA] score) and therapy-induced repigmentation grade. But the test was lacking in application potential. The authors have observed and concluded: 1 The VIDA score did not always predict a positive KP-e. 2 The KP-e may function well as a clinical factor to assess present disease activity and may also predict

the responsiveness to fluticasone propionate plus UV-A therapy but not to UV-B (311 nm) therapy. In this context, it can be concluded that overdependence on KP or TG may be sometimes misleading in this enigmatic disease, because these two criteria reveal the clinical stability only, and not the stability status of the disease at the cellular level [20].

How stable is stability? Another important indicator is the durability of the stability of vitiligo. It is often hard to predict how long the disease will remain stable. Similarly difficult to envisage is when it will start to become unstable. Repigmentation has been successfully induced in previous graft failure cases under NB-UVB (narrowband-UVB) (311 nm) phototherapy [30]. It was also proposed that NB-UVB might have played the pivotal role in stabilizing the disease by promoting apoptosis of the perilesional and circulating melanocyte-specific cytotoxic CD8 cells [31,32].

The concept of stability of vitiligo After transplantation, the appearance of spontaneous repigmentation in distant non-grafted vitiligo patches (Fig. 6.4) points toward a possible release and local absorption of fresh cytokines from the transplanted donor skin while stimulating the vitiliginous patches and hair follicles of the grafted sites may have played some role in the repigmentation at the distant sites [34,35]. Another theory was that the immunogenic mechanism which was originally responsible for development of vitiligo may have been lost because of loss of antigenicity due to the autologous grafts [33]. Successful split-thickness skin graft (STSG) in minigrafting failure cases further complicates the picture [36]. Very recently the period of stability of repigmentation has been proposed as 1 year; to qualify as stable the repigmentated status must be maintained for a period of not less than 1 year [37].

Understanding stability from within The exact pathomechanism of stability is as vague as the pathogenesis of the disease itself. The incompletely understood mechanism may involve a combination of various factors. The only definite and consistent fact regarding the pathogenesis of vitiligo is the absence of melanocytes in the lesion. So, it can be deducted that destruction of melanocytes is the keystone for unfolding of the disease process. Classically, there have been three hypotheses to elucidate vitiligo: the neural hypothesis, the selfdestruct hypothesis, and the immune hypothesis of which the last one is gaining grounds. Aberrations of both humoral and cell-mediated immunity have been observed. Most of the patients with generalized vitiligo were found to have circulating antibodies to cell surface antigens on normal human melanocytes; these antibodies are cytotoxic to normal melanocytes and to melanoma cells in tissue culture. Perilesional and circulating T-cells strongly suggest melanocyte-specific cytotoxic T-cells are involved. The response of vitiligo to UVB is possibly mediated by T-cell apoptosis [31,32]. In 1986 Grimes first reported a reduction in the number of lymphocytes and helper T-cells and an increase in number of natural killer cells in vitiligo

53

Fig. 6.4 Punch grafting on right leg (shown with

arrows). Spontaneous repigmentation over left leg (shown with arrows).

patients. She also noticed decreased helper/suppressor ratios, particularly among patients with vitiligo of less than a year’s duration and among those whose serum assayed positive for autoantibodies [38]. But D’Amelio in 1990 came up with just the reverse observation. A study of 22 patients with vitiligo stable for a year or more demonstrated high helper T-cell levels and high helper/suppressor ratios (also in 23 first-degree relatives) compared to controls. He also reported loss of circadian rhythm of helper T-cells in patients with active diseases and no loss of circadian rhythm of suppressor T-cell [39]. Cui et al. observed that cytotoxic antibodies are more common in patients with active disease than in those with inactive disease [40]. An attempt was made by Hann in 1994 to compare active and inactive lesion of vitiligo by immunohistochemical methods. He documented that the percentage of B-cells was higher in those with disease of recent onset (under a year) than in controls (those with later onset disease). He also noticed an increase in number of CD4 lymphocytes (especially in active lesions), but not CD8 lymphocytes, in marginal skin of active lesions. Epidermal ICAM-1 was expressed in active but not in stable epidermal lesions [41]. In a subsequent study in 1995, Hann detected decreased helper T-cell levels with a reversed helper/suppressor ratio [42]. In the margins of lesions, Badri found increase in CD3, CD4, and CD8 T-cells; many were

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activated and expressed the cutaneous lymphocyteassociated antigen (HECA-452 ) typical of infiltrating T-cells. These changes were most marked within 0.6 mm of the lesional edge. He couldn’t find any association between T-cell numbers and disease activity [43]. Very recently some interesting observations by Wankowicz-Kalinska have opened a new horizon in the basic understanding of the pathogenesis of the disease [44]. The detection of melanocyte-reactive cytotoxic T-cells in the peripheral blood of vitiligo patients and the observed correlation between perilesional T-cell infiltration and melanocyte loss in situ suggest the important role of cellular autoimmunity in the pathogenesis of this disease. T-cells have been isolated from both perilesional and non-lesional skin biopsies of vitiligo patients, then cloned and analyzed their profile of cytokine production. Perilesional Tcell clones (TCC) derived from patients with vitiligo revealed a predominant Type-1-like cytokine secretion profile, but the degree of Type-1 polarization in uninvolved skin-derived TCC correlated with the process of microscopically observed melanocyte destruction in situ. Furthermore, CD8 TCC derived from two patients also were analyzed for reactivity against autologous melanocytes. The antimelanocyte cytotoxic reactivity was observed among CD8 TCC isolated from perilesional biopsies of patients with vitiligo.

Conclusion So, where do we stand? It is obvious that in any attempt to predict the outcome of any vitiligo surgery, along with the clinical stability, assessment of “cellular” stability is important and the cellular parameters should also be taken into consideration. Ultrastructural investigation with electron microscopy and histoenzymological analysis of the peri- and non-lesional skin of vitiligo patients may help in deciding cellular stability, though this remains at an experimental stage. Probably some growth factors which are responsible for both mitogenic and melanogenic stimulation of melanocytes should also be taken into account. Before embarking on any surgical alternative no preconceived, arbitrary formulae should guide us to assess stability of vitiligo.

The understanding of the area based variable status and limited period of inertia or activity might necessitate TG at various sites at different time intervals. Until those unexplored areas are charted and the cellular parameters come into existence, we should not refrain the surgical option. We should continue surgical intervention based on logical application of our clinical knowledge.

References 1 Orentriech N, Selmanwitz VJ. Autograft repigmentation of leucoderma. Arch Dermatol 1972;105:734–6. 2 Falabella R. Repigmentation of segmental vitiligo by autologous minigrafting. J Am Acad Dermatol 1983; 9:514–21. 3 Halder RM, Breadon JY, Johnson BA. Micropigmentation for the treatment of vitiligo. J Dermatol Surg Oncol 1989;15:1092–8. 4 Behl PN. Treatment of vitiligo with homologous Thiersch’s skin grafts. Curr Med Pract 1964;8:218–21. 5 Kiistala U, Mustakallio KK. In vivo separation of epidermis by production of suction blisters. Lancet 1964;1:1444–5. 6 Falabella R. Grafting and transplantation of melanocytes for repigmenting vitiligo and other types of leucodermas. Int J Dermatol 1989;28:363–9. 7 Falabella R, Escobar C, Borrero I. Treatment of refractory and stable vitiligo by transplantation of in vitro cultured epidermal autografts bearing melanocytes. J Am Acad Dermatol 1992;26(2 Pt 1):230–6. 8 Malakar S, Dhar S, Malakar RS. Single-hair transplant: a novel technique. Dermatology 1999;199:370. 9 Olsson MJ, Juhlin L. Long-term follow-up of leucoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol 2002;147:893–904. 10 Njoo MD, Das PK, Bos JD, Westerhof W. Association of the Köbner phenomenon with disease activity and therapeutic responsiveness in vitiligo vulgaris. Arch Dermatol 1999;A135:414. 11 Das SS, Pasricha JS. Punch grafting as a treatment for residual lesions in vitiligo. Ind J Dermatol Venereol Leprol 1992;58:315–19. 12 Boersma BR,Westerhof W. Repigmentation in vitiligo vulgaris by autologous minigrafting: results in 19 patients. J Am Acad Dermatol 1995;33:990–5. 13 Jha AK, Pandey SS, Shukla VK. Punch grafting in vitiligo. Ind J Dermatol Venereol Leprol 1992;58:328–30.

The concept of stability of vitiligo 14 Savant SS. Autologous miniatures punch grafting in vitiligo. Ind J Dermatol Venereol Leprol 1992;58:310–14. 15 Falabella R. Surgical treatment of vitiligo: why, when and how. J Eur Acad Dermatol Venereol 2003;17:518–20 [Editorial]. 16 Malakar S, Lahiri K. How unstable is the concept of stability in surgical repigmentation of vitiligo? Dermatology 2000;201:182–3. 17 Falabella R, Arrunategui A, Barona MI, Alzate A. The minigrafting test for vitiligo: detection of stable lesions for melanocyte transplantation. J Am Acad Dermatol 1995;32:228–32. 18 Westerhof W, Boersma B. The minigrafting test for vitiligo: detection of stable lesions for melanocyte transplantation. J Am Acad Dermatol 1995;33:1061. 19 Falabella R. Reply. J Am Acad Dermatol 1995;33:1061. 20 Lahiri K, Malakar S, Banerjee U, Sarma N. Clinicocellular stability of vitiligo in surgical repigmentation: an unexplored frontier. Dermatology 2004;209:170–1. 21 Malakar S, Dhar S. Rejection of punch grafts in three cases of herpes labialis induced lip leucoderma, caution and precaution. Dermatology 1997;195:414. 22 Malakar S, Dhar S. Acyclovir can abort rejection of punch grafts in herpes-simplex induced lip leucoderma. Dermatology 1999;199:75. 23 Malakar S, Lahiri K. Successful repigmentation of six cases of herpes labialis induced lip leucoderma by micropigmentation. Dermatology 2001;203:194. 24 Lahiri K, Malakar S. Herpes simplex induced lip leucoderma: revisited. Dermatology 2004;208:182. 25 Malakar S, Dhar S. Treatment of stable and recalcitrant vitiligo by autologous miniature punch grafting: a prospective study of 1,000 patients. Dermatology 1999;198:133–9. 26 Malakar S, Lahiri K. Punch grafting for lip leucoderma. Dermatology 2004;208:125–8. 27 Lahiri K, Malakar S, Sarma N, Banerjee U. Repigmentation of vitiligo with punch grafting and narrow-band UV-B (311 nm) – a prospective study. Int J Dermatol 2006;45:649–55. 28 Juhlin L. How unstable is the concept of stability in surgical repigmentation of vitiligo. Dermatology 2000; 201:183 [Editorial comment]. 29 Falabella R, Escobar C, Borrero I. Treatment of refractory and stable vitiligo by transplantation of in vitro cultured epidermal autografts bearing melanocytes. J Am Acad Dermatol 1992;26(2 Pt 1):230–6. 30 Lahiri K, Malakar S. Inducing repigmentation by regrafting and phototherapy (311 nm) in punch failure

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cases of lip vitiligo – a pilot study. Ind J Dermato Venereol Leprol 2004;70:156–8. Ortonne JP. Vitiligo pathogenesis: what’s new? J Eur Acad Dermatol Venereol 2003;17:30. van den Wijngaard RM, Aten J, Scheepmaker A. Expression and modulation of apoptosis regulatory molecules in human melanocytes: significance in vitiligo. Br J Dermatol 2000;143:573–81. Malakar S. Spontaneous repigmentation of vitiligo patches other than the grafted site. Ind J Dermatol 1997;47:68–70. Malakar S, Dhar S. Spontaneous repigmentation of vitiligo patches distant from the autologous skin graft sites: a remote reverse Köbner’s phenomenon? Dermatology 1998;197:274. Malakar S, Lahiri K. Spontaneous repigmentation in vitiligo: why it is important. Int J Dermatol 2006; 45:478–9. Malakar S. Successful split thickness skin graft in stable vitiligo not responding to autologous miniature punch grafts. Ind J Dermatol 1997;42:215–18. Parsad D, Pandhi R, Dogra S, Kumar BJ. Clinical study of repigmentation patterns with different treatment modalities and their correlation with speed and stability of repigmentation in 352 vitiliginous patches. J Am Acad Dermatol 2004;50:63–7. Grimes PE, Ghoneum M, Stockton T, et al. T cell profiles in vitiligo. J Am Acad Dermatol 1986;14:196. D’Amelio R, Frati C, Fattorossi A, Aiuti F. Peripheral T-cell subset imbalance in patients with vitiligo and in their apparently healthy first degree relatives. Ann Allergy 1990;65:143. Cui J, Arita Y, Bystryn JC. Cytolytic antibodies to melanocytes in vitiligo. J Invest Dermatol 1993;100:812. Ahn SK, Choi EH, Lee SH, et al. Immunohistochemical studies from vitiligo – comparison between active and inactive lesions. Yonsei Med J 1994;35:404. Hann SK, Kim JB. Detection of antibodies to human melanoma cell in vitiligo by western blot analysis. Yonsei Med J 1995;36:457. Badri AM, Todd PM, Garioch JJ. An immunohistological study of cutaneous lymphocytes in vitiligo. J Pathol 1993;170:149–55. Wankowicz-Kalinska A, van den Wijngaard RM, Tigges BJ, et al. Immunopolarization of CD4 and CD8 T cells to Type-1-like is associated with melanocyte loss in human vitiligo. Lab Invest 2003;83:683–95.

CHAPTER 7

Patient selection and preoperative information in surgical therapies for vitiligo Nanny van Geel and Jean Marie Naeyaert

Introduction Vitiligo afflicts millions of people worldwide. Although vitiligo in general is a benign disorder, the cosmetic disfigurement may lead to considerable emotional distress and impairs the patients’ private and professional lives. This depigmenting disorder should therefore be taken seriously by all physicians involved in the treatment of vitiligo patients. A large variety of therapeutic agents are used in the treatment of vitiligo, such as psoralen plus ultraviolet A (PUVA), narrowband ultraviolet B (NB-UVB), and local steroids [1–3]. Transplantation of autologous melanocytes can be considered as a secondor third-line therapeutic approach. The choice of surgical intervention should be individualized, as vitiligo type, disease activity, total disease extension, localization of lesions, and the motivation of the patient are of major importance in this decision-making process. Before surgical treatment is initiated, several points of attention should be considered and discussed with the patient.

Selection for autologous melanocyte transplantation Unfortunately, only a minority of vitiligo patients are suitable for melanocyte transplantation. However, a strict selection of patients is an essential requirement for successful repigmentation. General selection criteria for autologous transplantation methods (Table 7.1) are as follows.

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Table 7.1 General selection criteria for autologous

transplantation. 1. Vitiligo type 2. Disease stability 3. Surface area to be treated 4. Köbner phenomenon 5. Localization of lesions 6. Motivation of the patient 7. Age

Vitiligo type The best indications are a stabilized segmental or focal vitiligo. In a segmental vitiligo accompanied by leukotrichia it may be considered as a first treatment option as a reservoir of melanocytes is lacking. Only a minority of patients with a generalized vitiligo can be selected for a surgical intervention, as in general this type of vitiligo extends over time.

Disease stability Distinction between active and stable phases of the disease is the most important selecting factor for surgical therapy. Active vitiligo usually requires medical therapy such as PUVA, narrowband UVB, local steroids, and topical immunomodulators, whereas in stable vitiligo, surgical treatment may be considered.

Patient selection and preoperative information Table 7.2 Establishing the stability. 1. Photographs 2. Minigrafting test 3. Köbner phenomenon 4. Segmental or generalized vitiligo 5. Extension of lesions

57

in which the size and number of lesions are stationary for several years and the Köbner phenomenon is absent. This stage of disease is therefore also referred to as “stable” vitiligo. • Extension of lesions: Extensive depigmentations are in general linked to an progressive generalized vitiligo [7]. These patients will therefore not be suitable for transplantation, as the chance for a successful result will be low.

Surface area to be treated According to the majority of authors, vitiligo can be classified as being “stable,” when progression of old lesions and/or development of new lesions are/is absent in the past year [4]. However, this is often a rough and subjective assessment of the activity status, which reduces its reliability. Unfortunately, no serological or clinical markers are available to estimate the disease activity objectively. However, several factors might be helpful in establishing stability (Table 7.2): • Photographs: Images from different time points are very useful for the investigator to evaluate the course of disease. In case of extensive depigmentations, it may be easier to follow only some selected reference lesions over time. The advantage of “digital” images is that an accurate and objective measurement of the surface can be performed as well (see Chapter 10). • Minigrafting test: The minigrafting test has been introduced by Falabella et al. to identify patients with stable vitiligo who may respond to melanocyte transplantation [5]. The test is considered positive when a repigmentation halo occurs around small 1–2-mm minigrafts implanted 3–4 mm apart within the recipient area. It is recommended that only patients with positive tests should be selected for treatment. • Köbner phenomenon: The appearance of white macules after trauma (Köbner phenomenon) has been mentioned being a sign of disease activity [6]. • Segmental or generalized vitiligo: Segmental vitiligo tends to be more stable than generalized vitiligo, because causative factors (probably neurochemical mediators from nearby nerve endings) usually seem to disappear. On the other hand, generalized vitiligo can enter long phases of clinical quiescence,

Surgical management of vitiligo can be prolonged. Extensive depigmentations are therefore difficult to restore by melanocyte transplantation. Even in smaller lesions, complete repigmentation can require additional sessions to obtain acceptable cosmetic results. The surgical technique used will be mainly determined by the maximum treatable surface area. Tissue grafting (punch grafting, bullae, split-thickness grafts) can be considered in small-to medium-sized lesions, whereas for extensive depigmentations culture techniques or epidermal suspension techniques are better options.

Köbner phenomenon It has been observed by many investigators that the presence of Köbner phenomenon negatively influences surgical treatment results [5,8]. This was clearly demonstrated in our double-blind placebocontrolled study evaluating the transplantation of autologous non-cultured epidermal cell suspension [8]. Our results did demonstrate that the appearance of the Köbner phenomenon at the donor site after the treatment was accompanied with a negative treatment result. The presence of the Köbner phenomenon can be predicted anamnestically, clinically, or more objectively and reliably by evaluating the donor site after performing the minigrafting test (see above).

Localization of lesions The localization of the treated area clearly influences the mean percentage of repigmentation [7]. In general, best results are observed in the neck and presternal region. On the contrary, joint areas showed rather disappointing results. These poor results may be caused by the difficulty in immobilizing these regions, with poor attachment of the graft. It has also

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Chapter 7

been suggested in the literature that repigmentation of these areas correlates inversely with the dermal component of the transplant. Transplanting minigrafts to vitiliginous areas on the fingers has been successful, but the success of repigmentation in these areas decreased when split-thickness grafts or epidermal blister roofs were used [9]. Although the number of reported cases is low, these results suggest that acral connective tissue is less capable of maintaining melanocytes in grafts, compared with connective tissue in other parts of the body. The preferential lesions to be treated are those located on exposed and more visible areas. Difficult to treat areas are joints, lips, eyelids, genitals, cutaneous folds, dorsum of hands and feet, especially fingers and toes.

Motivation of the patient The motivation and expectations of the patient are important to discuss before treatment. It can be helpful to provide images of treatment results to avoid confusion or misunderstanding. It has to be pointed out that the cause of the disease will not be cured and that despite the treatment the natural course of vitiligo will remain the same. Besides, the results can vary inter-individually and a successful result cannot be guaranteed. It has to be ascertained that the patient is well informed about the technical procedure, the costs and the possible side effects. Possible side effects or limitations that should be acceptable for the patient are incomplete repigmentation, lack of graft survival, slight hyperor hypopigmentation, and minor scar formation at donor or recipient sites. In our experience most patients seem to accept the minor imperfections easily, and are satisfied with the results.

Age So far no accepted rule exists concerning the minimal age of patients to undergo a surgical treatment for vitiligo. However, in general surgical procedures are not recommended in children.

Contraindications As in other surgical treatments, patients should have no history of hypertrophic scars, keloid, or bleeding

disorders. In case there is a tendency toward hyperpigmentation after trauma, one has to take special consideration as well. Other contraindications are blood-borne virus infections, such as hepatitis C and human immunodeficiency virus (HIV). A blood test should always be taken and the laboratory answer be in the hand of the surgeon before transplantation is conducted. This is to minimize the risk of carry over aerosol virus in the surgical part of the procedure. Both dermabrasion and laser vaporization of the skin is known to cause airborne-particlescarrying viruses.

References 1 Rodriguez MC, Farrando A, Kon F, Gatti CF. Psoralen plus ultraviolet A (PUVA) therapy for vitiligo. Report of 50 patients. Prensa Medica Argentina 2001;88:761–6. 2 Westerhof W, Nieuweboer-Krobotova L. Treatment of vitiligo with UV-B (311 nm) versus topical PUVA. Arch Dermatol 1997;133:1525–8. 3 Clayton R. A double-blind trial of 0.05% clobetasol propionate in the treatment of vitiligo. Br J Dermatol 1977;96:71–3. 4 van Geel N, Ongenae K, Vander Haeghen Y, Naeyaert JM. Autologous transplantation techniques for vitiligo: how to evaluate treatment outcome? Eur J Dermatol 2004;14:46–51. 5 Falabella R, Arrunategui A, Barona MI, Alzate A. The minigrafting test for vitiligo: detection of stable lesions for melanocyte transplantation. J Am Acad Dermatol 1995;32:228–32. 6 Njoo MD, Das PK, Bos JD, Westerhof W. Association of the Köebner phenomenon with disease activity and therapeutic responsiveness in vitiligo vulgaris. Arch Dermatol 1999;135:407–13. 7 Olsson M, Juhlin L. Long-term follow-up of leucoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Brit J Dermatol 2002;147: 893–904. 8 van Geel N, Ongenae K, De Mil M, et al. Double blind placebo controlled study of autologous transplanted epidermal cell suspensions for repigmenting vitiligo. Arch Dermatol 2004;140:1203–8. 9 Yaar M, Gilchrest BA. Vitiligo. The evolution of cultured epidermal autografts and other surgical treatment modalities. Arch Dermatol 2001;137:348–9.

CHAPTER 8

Classification of surgical therapies for vitiligo Philippe Bahadoran and Jean-Paul Ortonne

Introduction

Tissue grafts

Surgical therapies of vitiligo [1–5], generally based on melanocyte transplantation, represent an interesting option for vitiligo lesions that cannot be treated properly by medical therapies, provided that: (1) the lesions are stable (see Chapter 6: “The concept of stability of vitiligo”) and (2) that patients are otherwise carefully selected (see Chapter 7: “Patient selection”). This chapter will provide a classification and an overview of the different surgical techniques for vitiligo. Each technique will be discussed in detail in a specific chapter. Surgical transplantation techniques for vitiligo all rely on the same basic principle: to repopulate depigmented skin lesions with functional melanocytes arising from normal epidermis. Normal epidermis can be obtained from different anatomical regions. The most convenient is the gluteal region where any change induced by the harvesting procedure can be easily covered. Other common donor regions include the medial aspects of the thighs or of the arms. Surgical transplantation techniques for vitiligo are classified according to the nature of the graft. There are two main groups of grafts. Tissue grafts consist of the simple transfer of epidermis or skin sampled and implanted as is. Cellular grafts consist of the transplantation of disaggregated epidermal cells. Cellular grafts can be further classified in two categories according to whether or not cells have been cultivated between harvesting and grafting (Fig. 8.1).

There are three main types of tissue grafts for vitiligo: suction blister grafts, split-thickness grafts, and fullthickness punch grafts. A common point of tissue grafts is that they cannot (suction blister grafts, punch grafts) or can hardly (split-thickness grafts) be expanded. Hence they cannot be used for the treatment of large areas. A difference between tissue grafts is that suction blister grafts and split-thickness grafts consist exclusively (suction blister grafts) or almost exclusively (split-thickness grafts) of epidermis while punch grafts include both the epidermis and the dermis.

Full-thickness punch grafts (“minigrafts”) Procedure A punch biopsy, encompassing both the epidermis and the dermis, is used as a graft [6–13].

Preparation of recipient site Small perforations are performed after local anesthesia with a biopsy punch of 1.2–2.5 mm. The perforations should be of 0.5–1-mm deep and be separated from each other by 3–5 mm. The achromic plugs are pulled out and the recipient area is covered with normal saline compresses. When the treated area is large and many perforations are anticipated, a motor driven mini-punch device is helpful to prepare the recipient site. Pulsed Er:YAG (erbium: yttrium–aluminum–garnet) laser was proposed as

59

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Chapter 8

Surgical therapies for vitiligo

Autologous grafting techniques

Cellular grafts

Tissue grafts Full thickness punch graft/minigrafting

Nongrafting techniques

Laser

Micropigmentation

Non-cultured-keratinocyte and melanocyte cell suspension

Split-thickness grafts

Cultured autografts

Thin-manual dermatome

Cultured melanocytes

Ultra-thin-motorized dermatome

Cultured epithelial grafts

Suction blister grafts

Seed grafts

Hair follicle grafts

Flip-top graft Fig. 8.1 Classification of various surgical techniques employed in patients with vitiligo and other leukodermas.

an alternative to punch biopsy to prepare the recipient site [12,13].

Harvesting of grafts and grafting Perforations are performed with a punch of similar size as for recipient site, at a distance of about 1 mm from each other. Some recommend taking a punch biopsy 0.25–0.5 mm larger in diameter than the recipient pits because of the risk of retraction of the graft [13]. Grafts are freed from the donor site and excess adipose tissue is trimmed to achieve a thickness equal to the depth of recipient pits. Grafts are inserted into the small perforations, held in place with steri-strips and covered by a non-adherent dressing for approximately 1 week. Sunlight exposure or psoralen plus ultraviolet A (PUVA) is important to stimulate pigment spread.

Results Repigmentation around punch grafts usually starts 2–3 weeks after graft placement. Coalescence of pigment from all punch grafts occurs within 4–6 months. Good repigmentation has been achieved in 68–82% of cases [3]. To get the best results, some authors recommend selecting eligible patients according to the results of test grafting with 5–10 punch grafts [10]. Despite the lack of controlled comparative studies, punch grafts seem slightly less effective than suction blisters or split-thickness grafts [1]. Cosmetic complications at the recipient site seem to be more frequent than with suction blisters or splitthickness grafts. Cobblestoning or variegated appearance occur in about 30% of cases [3,11,14,15]. Scar formation at the donor site occurs in about 40% of cases [3].

Classification of surgical therapies for vitiligo

Advantages This is believed to be the easiest and least expensive method of surgical repigmentation since it does not require any special equipment. Punch grafting is suitable for difficult locations such as lips, palms and soles, and fingers.

Disadvantages The method is time consuming. For large lesions, serial procedures performed at 3–4-week intervals are recommended. Regrafting between the transplanted grafts may be necessary. Punch grafting is not suitable for body folds.

Split-thickness grafts Procedure A sheet of skin of 0.2–0.3-mm thickness, thus consisting almost exclusively of epidermis, is used as a graft [16–19].

Harvesting of grafts The split-thickness graft is harvested after local anesthesia, with a manual or motorized dermatome.

Preparation of recipient site and grafting The recipient area is denuded with by dermabrasion [16,17,19] or by CO2 laser [18]. Grafts of approximately 4–5 cm in length are placed over the denuded recipient site close to another with slight overlap. Grafts are held in place with staples or steri-strips, and covered with a non-adherent sterile dressing for approximately 1 week. Meshing has been used to expand the split-thickness graft to cover an area 2 or 3 times larger than the donor area. The skin is passed through a machine that makes small slits, which allows expansion similar to that in fish netting. Pigmentation occurs at the spaces between the mesh, called the intricities, which fills in with new epithelial skin growth [19].

Results According to the literature, split-thickness grafts have a high success rate of 78–91%, which is comparable to that of suction blisters [1,17,19]. The success rate relies greatly on the thinness of the graft since a thick graft will leave additional cosmetic damage to both recipient and donor area. Repigmentation of

61

leukotrichia is possible [16]. The most common adverse effects at recipient sites are thick margins, partial loss of graft and milia-like cysts in about 5%, 11%, and 13% of cases. The most common adverse effects at donor sites are scar formation and hypopigmentation in about 12% and 16% of cases, respectively [1].

Advantages This is a suitable method to cover multiple lesions or large areas at one time. Split-thickness grafting is suitable for difficult areas such as eyelids and lips.

Disadvantages Taking split thickness grafts of uniform thickness requires skill and dexterity. It is not possible to cover palms, soles, and body folds with this technique.

Suction blister grafts Procedure The tops of blisters induced by suction are used as grafts [6,12–14,20–33]. The mechanical split created by suction is exactly at the dermoepidermal junction, so the tops of blisters are purely epidermal and very thin.

Harvesting of grafts At present most centers performing suction blister epidermal grafting for vitiligo use a machine consisting of a vacuum pump connected by flexible tubing to suction cylinders. The tops of blisters are carefully removed to obtain 5–8-mm large epidermal sheets. The epidermal sheets can be cut into smaller fragments [20].

Preparation of recipient site and grafting Liquid nitrogen freezing is the simplest way of deepithelializing the recipient site [6]. Other possible methods are dermabrasion [6], CO2 laser [30,33], Er:YAG laser [13,26,34], suction blisters [28], and PUVA [21]. Grafts are placed dermal side down in the previously de-epithelialized recipient beds. Grafts are held in place with sterile non-adherent dressings for approximately 1 week. Sunlight exposure or PUVA therapy, starting 1 week after healing, is recommended to stimulate pigment spread. Pigmentation can also be enhanced by preoperative PUVA therapy of the donor site [25].

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Chapter 8

Results

Hair follicle grafts [36,37]

Repigmentation in the grafted epidermis occurs within few weeks. Good repigmentation is achieved in 73–88% of cases [3]. Repigmentation of leukotrichia has also been noted [23]. Temporary hyperpigmentation and mottled pigmentation can be seen in the grafted sites in 2–65% [3]. In a retrospective series of 117 cases, the success rate was better in segmental/focal vitiligo and in individuals
20 years of age [32]. Localization of the vitiligo patch did not seem to influence the outcome significantly. Hyperpigmentation was observed in 32% of cases [32].

A small strip of hairs is taken from the occipital area. Single hairs are separated from the strip and are implanted into vitiligo patches by an 18-gauge needle 3 to 5 mm apart from each other. The interest of hair follicle grafting is to act as a filler for residual spots left over by other surgical techniques. However, this technique can only be used in hair-bearing areas.

Advantages Since the graft is strictly epidermal, there is no scarring at donor or recipient site. The donor site can therefore be used more than once. Suction blister grafting is suitable for difficult areas such as eyelids, lips, and bony prominences. Altogether, suction blister grafting is considered to be effective, easy, safe, and inexpensive.

Disadvantages Harvesting blisters is time consuming. Since the epidermal sheets are small, large areas cannot be covered unless the procedure is repeated. It is difficult to cover palms, soles, and body folds with this technique.

Other types of tissue grafts Differing from full-thickness punch grafts, split-thickness grafts, and suction blister grafts which have been widely used in patients with vitiligo, the data available for the following techniques is very limited.

Flip-top grafts [38] In the “flip-top” technique, a 2–4 mm donor epidermis sample containing minimal dermis is shaved with a razor blade and sectioned into smaller 1–2-mm grafts. At the recipient site, a 5-mm flap of epidermis containing minimal dermis is raised with a razor blade and the grafts are placed underneath this flap [28]. The potential interest of flip-top grafting is to improve the healing process.

Cellular grafts The potential advantage of techniques based on cell separation and/or culture is to allow the treatment of larger lesions than techniques based on whole tissue. On the other hand, the main limitation of these techniques is that they require special personnel and equipment, increasing cost and restricting the treatment to academic settings. In addition the recent enforcement of US and European health agencies regulations mandating good manufacturing practices (GMP) for autologous cultured cells used in patient care may hamper the development of cell culture therapies of vitiligo even in academic settings.

Transplantation of autologous epidermal cell suspensions Procedure

Seed grafts [35] A very thin piece of epidermal graft skin (5–8/1000 inches) is taken from a normally pigmented donor site with a hand dermatome. The graft skin (about 2–4 cm) is minced into fragments
1 mm2. These minced skin pieces are placed on the epidermalabraded vitiligo lesions. The advantage of epidermal seed-grafting is to cover more area than sheetgrafting.

A shaved biopsy skin sample is taken with a dermatome after local anesthesia. The skin sample is immersed in a trypsin solution, the epidermis is separated from the dermis, and after additional laboratory procedures a cellular suspension of keratinocytes and melanocytes is obtained. The recipient area can be denuded by inducing blisters with liquid nitrogen [39,40], or by dermabrasion [41–45], or by CO2 laser [46,47]. The cellular suspension is then

Classification of surgical therapies for vitiligo either injected in each blister at the recipient area, or spread onto the denuded recipient area. In the latter case, the adhesion of the cellular suspension onto the recipient area can be increased by coverage with a collagen film [41–45] or by the addition of hyaluronic acid to make the suspension viscous [46,47].

Results Good repigmentation has been achieved in 67–85% of cases [3]. The best results seem to occur in segmental and focal vitiligo [43]. In addition, the effectiveness of this surgical technique was strongly confirmed by a prospective, randomized, doubleblind placebo-controlled study [47]. No adverse effects have been reported, except erythema at recipient site caused by dermabrasion.

Advantages The most important advantage is that large areas can be treated by cellular grafts since the recipient area can be 5–10-fold larger than the donor area. Laboratory procedures are short and simple since cell culture is not required.

63

denuded by liquid nitrogen application [40,48], dermabrasion [49–52], CO2 laser [53–55] or Er:YAG laser [56].

Results Success rates vary between 22% and 75% [3,55]. In a recent study, it was shown that this technique is more efficient in patients with stable localized vitiligo than in patients with stable generalized vitiligo [55]. Hyperpigmentation is quite common during the first few weeks, and linear hypopigmentation may appear at the junction of the transplant site and normal skin [55].

Advantages The main advantage is the possibility of repigmenting large vitiligo areas using a very small shave biopsy since melanocytes are expanded in vitro. As compared with the transplantation of uncultured melanocyte–keratinocyte suspensions, the transplantation of cultured melanocytes can provide more cells for the treatment of large lesions. Using this approach, lesions of up to 500 cm2 can be treated with melanocytes generated from biospy specimens as small as 1 cm2.

Disadvantages Taking split-thickness grafts of uniform thickness requires skill and dexterity. It is not possible to cover palms, soles, and body folds with this technique. Laboratory procedures require specific equipment and personnel.

Transplantation of cultured autologous melanocytes

Disadvantages This method requires laboratory setup, specialized personnel, culture media and growth factors, and is therefore expensive. The procedure is time consuming. The presence of TPA, a tumor promoter, in the traditional culture media of melanocytes, is a matter of concern. However the use of TPA-free medium supplemented with other growth factors provides a conceptual solution to this potential problem.

Procedure Lerner et al. first described the use of cultured pure autologous human melanocytes in a patient with piebaldism [48]. A cell suspension is obtained by trypsinization from a superficial shave biopsy or from a suction blister. Melanocytes are expanded in vitro for 15–30 days by the addition of chemical media with various growth factors. Melanocytes are detached from the culture plates and the suspension is transferred to the superficially denuded recipient area and spread with a density of 1000– 2000 melanocytes/mm2. The recipient site can be

Transplantation of cultured autologous epidermal grafts Some potential advantages were pointed out to suggest that epidermal grafts might be more appropriate than melanocyte cultures for surgical therapy of vitiligo [57]: (1) keratinocytes regulate melanocyte growth and differentiation, (2) melanocytes organize themselves into the basal layer of cultured keratinocytes in the same way as in normal epidermis, (3) melanocyte–keratinocyte cultures allow the

Shave biopsy

Vacuum pump

Split-thickness grafts

Suction blister grafts

Shave biopsy

Shave biopsy

Cultured melanocyte suspension

Cultured epidermal grafts

D: Donor area; R: Recipient area

Shave biopsy

Epidermal cell suspension

Variants

Punch biopsies

Harvesting of grafts

Full-thickness punch grafts

Technique

73–88%

Limited lesions Segmental lesions Facial lesions Eyelids, lips, bony prominences

22–75%

Extensive lesions

33–80%

67–85%

Extensive lesions

See Text

78–91%

68–82%

Success rate (%)

Multiple/large lesions Extremities Eyelids, lips

Limited lesions Segmental lesions Lips, palms/soles, fingers

Indications

Liquid nitrogen Extensive lesions Dermabrasion Diathermosurgery Laser

Liquid nitrogen Dermabrasion Laser

Liquid nitrogen Dermabrasion Laser

Liquid nitrogen Dermabrasion Laser

Dermabrasion Laser

Punch biopsies Laser

Preparation of recipient area

R: Hyperpigmentation (transient)

R: Hyperpigmentation (transient)

R: Long lasting erythema (dermabrasion)

R: Hyperpigmentation

R: Thick margins, partial loss, milialike cysts

D: Scarring R: Cobblestoning or variegated appearance

Side effects

Table 8.1 Summary of various surgical techniques employed in patients with vitiligo and other leukodermas.

Tissue grafts

Cellular grafts

Special equipment Time consuming

Special equipment Time consuming

Larger areas treatable No scar formation

Special device Time consuming Regrafting between blisters may be needed

Easy, safe, inexpensive No scar formation

Larger areas treatable No scar formation

Requires dexterity for harvesting a thin biopsy

No scar formation

Special equipment

Time consuming Regrafting between punches may be needed

Easiest and least expensive method

Larger areas treatable No scar formation Easiest of cellular techniques

Disadvantages

Advantages

57–67

48–56

39–47

35–38

6, 12–14, 20–34

16–19

6–15

Reference

Classification of surgical therapies for vitiligo production of large quantities of cultured autografts in a shorter time than pure melanocyte cultures, and (4) cultured epidermal grafts have been safely used for the treatment of thousands of patients with burns while the experience of pure melanocyte cultures is still limited.

Procedure A shave biopsy of normally pigmented skin is the source for epidermal cell culture. After separating the epidermis from the dermis, the cells are seeded in a medium that allows co-cultivation of keratinocytes and melanocytes. The melanocyte– keratinocyte ratio is evaluated routinely in order to maintain a proper melanocyte concentration into the epidermal grafts. After a few weeks, a cultured epidermal sheet is obtained, released by dispase, and attached to a petrolatum gauze. The recipient site can be denuded by liquid nitrogen application [58,59], dermabrasion [60–62], diathermo surgery (“Timed”) [57], CO2 laser [63–65], or Er:YAG laser [66]. The gauze to which the cultured epidermis adheres is applied onto the superficially denuded recipient site and covered with dressings. The cultured epidermis can be seeded on a supporting layer (membrane of hyaluronic acid or collagencoated sheet) before application on the recipient site [60,63]. Sun-avoidance is required for at least 1 month [58,59] and up to 2–3 months [57,66] after grafting. In an attempt to perfect the co-culture conditions a recent study compared different culture media and several kinds of chemically defined plasma polymer substrates [67].

65

patients. Variation of melanocyte concentration in cultured grafts was not correlated with the percentage of repigmentation [66]. Hyperpigmentation of the recipient site is frequent but disappears after several months.

Advantages The main advantage of this technique is the expansion of cells in culture, which allows the treatment of a wide area of vitiligo with a small piece of donor skin. The procedure is non-scarring at both donor and recipient sites.

Disadvantages The main disadvantage of this technique is the requirement of special personnel and equipment hence a high cost, and the need to comply with complex GMP procedures. Complications are the same as in other minor surgical procedures such as infection, bleeding, and graft failure.

Conclusion The surgical techniques for vitiligo can be classified according to the nature of the graft (Table 8.1). Based on this classification, the choice of a method should take into account both the extension and the location of the lesion to treat. The choice also depends on the experience of the user and the facilities available. Comparative studies with similar criteria of evaluation are needed to assess precisely the effectiveness and safety of each method [1,68].

Acknowledgements Results Good repigmentation was reported in 33–80% of patients [57,59,63,66]. Recently, Guerra et al. conducted the two largest studies on transplantation of cultured epidermis for vitiligo. In the first study of 105 vitiligo lesions from 32 patients, the average repigmentation was 77% and there was good repigmentation in 60% of patients. Repigmentation of the periorificial region and the extremities was poor [57]. In the second study of 49 vitiligo lesions from 21 patients, the average repigmentation was 76% and there was good repigmentation in 80% of

Dr. P. Bahadoran is grateful to Dr. Y. Gauthier and Dr. N. van Geel for sharing their invaluable experience in melanocyte grafting.

References 1 Njoo MD, Westerhof W, Bos JD, et al. A systematic review of autologous transplantation methods in vitiligo. Arch Dermatol 1998;134:1543–9. 2 Mutalik S, Ginzburg A. Surgical management of stable vitiligo: a review with personal experience. Dermatol Surg 2000;26:248–54.

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3 van Geel N, Ongenae K, Naeyaert JM. Surgical techniques for vitiligo: a review. Dermatology 2001;202: 162–6. 4 Pianigiani E, Andreassi A, Andreassi L. Autografts and cultured epidermis in the treatment of vitiligo. Clin Dermatol 2005;23:424–9. 5 Falabella R. Surgical approaches for stable vitiligo. Dermatol Surg 2005;31:1277–84. 6 Falabella R. Surgical therapies for vitiligo and other leukodermas, Part I: minigrafting and suction epidermal grafting. Dermatologic Ther 2001;14:7–14. 7 Falabella R. Repigmentation of stable leukoderma by autologous minigrafting. J Dermatol Surg Oncol 1986; 12:172–9. 8 Falabella R. Treatment of localized vitiligo by autologous minigrafting. Arch Dermatol 1988;124:1649–55. 9 Boersma BR, Westerhof W, Bos JD. Repigmentation in vitiligo vulgaris by autologous minigrafting: results in nineteen patients. J Am Acad Dermatol 1995;33:990–5. 10 Falabella R, Arrunategui A, Barona MI, et al. The minigrafting test for vitiligo: detection of stable lesions for melanocyte transplantation. J Am Acad Dermatol 1995; 32:228–32. 11 Malakar S, Dhar S. Treatment of stable and recalcitrant vitiligo by autologous miniature punch grafting: a prospective study of 1,000 patients. Dermatology 1999; 198:133–9. 12 Sachdev M, Shankar DS. Dermatologic surgery: pulsed erbium:YAG laser-assisted autologous epidermal punch grafting in vitiligo. Int J Dermatol 2000;39:868–71. 13 Pai GS, Vinod V, Joshi A. Efficacy of erbium YAG laserassisted autologous epidermal grafting in vitiligo. J Eur Acad Dermatol Venereol 2002;16:604–6. 14 Gupta S, Jain VK, Saraswat PK. Suction blister epidermal grafting versus punch skin grafting in recalcitrant and stable vitiligo. Dermatol Surg 1999;25:955–8. 15 Khandpur S, Sharma VK, Manchanda Y. Comparison of minipunch grafting versus split-skin grafting in chronic stable vitiligo. Dermatol Surg 2005;31:436–41. 16 Agrawal K, Agrawal A. Vitiligo: repigmentation with dermabrasion and thin split-thickness skin graft. Dermatol Surg 1995;21:295–300. 17 Olsson MJ, Juhlin L. Epidermal sheet grafts for repigmentation of vitiligo and piebaldism, with a review of surgical techniques. Acta Derm Venereol 1997;77:463–6. 18 Kahn AM, Ostad A, Moy RL. Grafting following shortpulse carbon dioxide laser de-epithelialization. Dermatol Surg 1996;22:965–7; discussion 7–8. 19 Kahn AM, Cohen MJ. Repigmentation in vitiligo patients. Melanocyte transfer via ultra-thin grafts. Dermatol Surg 1998;24:365–7.

20 Suvanprakorn P, Dee-Ananlap S, Pongsomboon C, et al. Melanocyte autologous grafting for treatment of leukoderma. J Am Acad Dermatol 1985;13:968–74. 21 Koga M. Epidermal grafting using the tops of suction blisters in the treatment of vitiligo. Arch Dermatol 1988;124:1656–8. 22 Skouge JW, Morison WL, Diwan RV, et al. Autografting and PUVA. A combination therapy for vitiligo. J Dermatol Surg Oncol 1992;18:357–60. 23 Hann SK, Im S, Bong HW, et al. Treatment of stable vitiligo with autologous epidermal grafting and PUVA. J Am Acad Dermatol 1995;32:943–8. 24 Skouge J, Morison WL. Vitiligo treatment with a combination of PUVA therapy and epidermal autografts. Arch Dermatol 1995;131:1257–8. 25 Suga Y, Butt KI, Takimoto R, et al. Successful treatment of vitiligo with PUVA-pigmented autologous epidermal grafting. Int J Dermatol 1996;35:518–22. 26 Yang JS, Kye YC. Treatment of vitiligo with autologous epidermal grafting by means of pulsed erbium:YAG laser. J Am Acad Dermatol 1998;38:280–2. 27 Tang WY, De Han J, Lu NZ, et al. Surgical pearl: fine gauze is a useful carrier for epidermal graft in the treatment of vitiligo by means of the suction blister method. J Am Acad Dermatol 1999;40:247–9. 28 Kim HY, Kang KY. Epidermal grafts for treatment of stable and progressive vitiligo. J Am Acad Dermatol 1999;40:412–7. 29 Gupta S, Shroff S. Modified technique of suction blistering for epidermal grafting in vitiligo. Int J Dermatol 1999;38:306–9. 30 Oh CK, Cha JH, Lim JY, et al. Treatment of vitiligo with suction epidermal grafting by the use of an ultrapulse CO2 laser with a computerized pattern generator. Dermatol Surg 2001;27:565–8. 31 Ozdemir M, Cetinkale O, Wolf R, et al. Comparison of two surgical approaches for treating vitiligo: a preliminary study. Int J Dermatol 2002;41:135–8. 32 Gupta S, Kumar B. Epidermal grafting in vitiligo: influence of age, site of lesion, and type of disease on outcome. J Am Acad Dermatol 2003;49:99–104. 33 Acikel C, Ulkur E, Celikoz B. Carbon dioxide laser resurfacing and thin skin grafting in the treatment of “stable and recalcitrant” vitiligo. Plast Reconstr Surg 2003;111:1291–8. 34 Sachdev M, Krupashankar DS. Suction blister grafting for stable vitiligo using pulsed erbium:YAG laser ablation for recipient site. Int J Dermatol 2000;39: 471–3. 35 Tsukamoto K, Osada A, Kitamura R, et al. Approaches to repigmentation of vitiligo skin: new treatment with

Classification of surgical therapies for vitiligo

36

37

38

39

40

41

42

43

44

45

46

47

48 49

ultrasonic abrasion, seed-grafting and psoralen plus ultraviolet a therapy. Pigment Cell Res 2002;15:331–4. Malakar S, Dhar S. Repigmentation of vitiligo patches by transplantation of hair follicles. Int J Dermatol 1999; 38:237–8. Sardi JR. Surgical treatment for vitiligo through hair follicle grafting: how to make it easy. Dermatol Surg 2001;27:685–6. McGovern TW, Bolognia J, Leffell DJ. Flip-top pigment transplantation: a novel transplantation procedure for the treatment of depigmentation. Arch Dermatol 1999;135:1305–7. Gauthier Y, Surleve-Bazeille JE. Autologous grafting with noncultured melanocytes: a implified method for treatment of depigmented lesions. J Am Acad Dermatol 1992;26:191–4. Issa CM, Rehder J, Taube MB. Melanocyte transplantation for the treatment of vitiligo: effects of different surgical techniques. Eur J Dermatol 2003;13:34–9. Olsson MJ, Juhlin L. Leucoderma treated by transplantation of a basal cell layer enriched suspension. Br J Dermatol 1998;138:644–8. Olsson MJ, Juhlin L. Long-term follow-up of leucoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol 2002;147: 893–904. Mulekar SV. Melanocyte–keratinocyte cell transplantation for stable vitiligo. Int J Dermatol 2003;42: 132–6. Mulekar SV. Long-term follow-up study of segmental and focal vitiligo treated by autologous, noncultured melanocyte–keratinocyte cell transplantation. Arch Dermatol 2004;140:1211–5. Mulekar SV. Long-term follow-up study of 142 patients with vitiligo vulgaris treated by autologous, non-cultured melanocyte–keratinocyte cell transplantation. Int J Dermatol 2005;44:841–5. van Geel N, Ongenae K, De Mil M, et al. Modified technique of autologous noncultured epidermal cell transplantation for repigmenting vitiligo: a pilot study. Dermatol Surg 2001;27:873–6. van Geel N, Ongenae K, De Mil M, et al. Double-blind placebo-controlled study of autologous transplanted epidermal cell suspensions for repigmenting vitiligo. Arch Dermatol 2004;140:1203–8. Lerner AB, Halaban R, Klaus SN, et al. Transplantation of human melanocytes. J Invest Dermatol 1987;89:219–24. Olsson MJ, Juhlin L. Repigmentation of vitiligo by transplantation of cultured autologous melanocytes. Acta Derm Venereol 1993;73:49–51.

67

50 Lontz W, Olsson MJ, Moellmann G, et al. Pigment cell transplantation for treatment of vitiligo: a progress report. J Am Acad Dermatol 1994;30:591–7. 51 Olsson MJ, Moellmann G, Lerner AB, et al. Vitiligo: repigmentation with cultured melanocytes after cryostorage. Acta Derm Venereol 1994;74:226–8. 52 Olsson MJ, Juhlin L. Transplantation of melanocytes in vitiligo. Br J Dermatol 1995;132:587–91. 53 Chen YF, Yang PY, Hung CM, et al. Transplantation of autologous cultured melanocytes for treatment of large segmental vitiligo. J Am Acad Dermatol 2001;44:543–5. 54 Chen YF, Chang JS, Yang PY, et al. Transplant of cultured autologous pure melanocytes after laserabrasion for the treatment of segmental vitiligo. J Dermatol 2000;27:434–9. 55 Chen YF, Yang PY, Hu DN, et al. Treatment of vitiligo by transplantation of cultured pure melanocyte suspension: analysis of 120 cases. J Am Acad Dermatol 2004; 51:68–74. 56 Kaufmann R, Greiner D, Kippenberger S, et al. Grafting of in vitro cultured melanocytes onto laserablated lesions in vitiligo. Acta Derm Venereol 1998; 78:136–8. 57 Guerra L, Capurro S, Melchi F, et al. Treatment of “stable” vitiligo by Timedsurgery and transplantation of cultured epidermal autografts. Arch Dermatol 2000; 136:1380–9. 58 Falabella R, Escobar C, Borrero I. Transplantation of in vitro-cultured epidermis bearing melanocytes for repigmenting vitiligo. J Am Acad Dermatol 1989;21: 257–64. 59 Falabella R, Escobar C, Borrero I. Treatment of refractory and stable vitiligo by transplantation of in vitro cultured epidermal autografts bearing melanocytes. J Am Acad Dermatol 1992;26:230–6. 60 Brysk MM, Newton RC, Rajaraman S, et al. Repigmentation of vitiliginous skin by cultured cells. Pigment Cell Res 1989;2:202–7. 61 Plott RT, Brysk MM, Newton RC, et al. A surgical treatment for vitiligo: autologous cultured-epithelial grafts. J Dermatol Surg Oncol 1989;15:1161–6. 62 Kumagai N, Uchikoshi T. Treatment of extensive hypomelanosis with autologous cultured epithelium. Ann Plast Surg 1997;39:68–73. 63 Andreassi L, Pianigiani E, Andreassi A, et al. A new model of epidermal culture for the surgical treatment of vitiligo. Int J Dermatol 1998;37:595–8. 64 Toriyama K, Kamei Y, Kazeto T, et al. Combination of short-pulsed CO2 laser resurfacing and cultured epidermal sheet autografting in the treatment of vitiligo: a preliminary report. Ann Plast Surg 2004;53:178–80.

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65 Pianigiani E, Risulo M, Andreassi A, et al. Autologous epidermal cultures and narrow-band ultraviolet B in the surgical treatment of vitiligo. Dermatol Surg 2005; 31:155–9. 66 Guerra L, Primavera G, Raskovic D, et al. Erbium:YAG laser and cultured epidermis in the surgical therapy of stable vitiligo. Arch Dermatol 2003;139:1303–10.

67 Eves PC, Beck AJ, Shard AG, et al. A chemically defined surface for the co-culture of melanocytes and keratinocytes. Biomaterials 2005;26:7068–81. 68 van Geel NA, Ongenae K, Vander Haeghen YM, et al. Autologous transplantation techniques for vitiligo: how to evaluate treatment outcome. Eur J Dermatol 2004;14:46–51.

CHAPTER 9

Surgical management of vitiligo and other leukodermas: evidence-based practice guidelines Somesh Gupta, Tarun Narang, Mats J. Olsson and Jean-Paul Ortonne

Introduction Both non-surgical and surgical therapies have been employed in the management of vitiligo. However, there are no uniformly agreed guidelines or recommendations available for surgical management of vitiligo and other leukodermas. Current surgical treatment practices are based on informal consensus meetings, expert opinions, results from uncontrolled clinical trials, and personal and/or institutional experiences and infrastructures [1]. There is only one randomized study available showing superiority of non-cultured melanocyte suspension over placebo [2]. In the absence of randomized clinical trials, the guidelines will be formulated on the basis of meta-analysis of published uncontrolled clinical trials. Here a word of caution is necessary that this is only an attempt to draw some conclusions from metaanalysis of a broad variety of reports published over a long period, by different individuals. Often the results are reported as subjective observations without real measurement tools or statistical analyses. The procedures are performed using different surgical equipment and bandage materials in distinct clinical settings. The patient populations are from different parts of the world, so are genetically heterogeneous. All these factors may bias the conclusions. A Consensus Report on the definition and assessment of vitiligo has recently been worked out by the Vitiligo European Task Force. This report will be a valuable tool to make research outcomes on

this subject more uniform and comparable, but it will still take many years until a sufficient number of new publications has used the consensus assessment to make future studies more comparable and meta-analyses more appropriate. In spite of inherent biases, meta-analyses of published literature have been accepted as useful means to formulate evidence-based practice guidelines [3].

Method We searched two databases, Medline and Embase, from 1966 to 2005 with Keywords “Vitiligo,” “Leukoderma,” “Transplantation,” “Grafting,” and “Surgery” in various combinations. A few leading researchers were contacted to retrieve reports under publication. Some Asian journals not indexed in the literature were hand-searched for relevant reports. Cross-references of some leading reviews on the subject were also studied. All the studies pertaining to transplantation procedures in vitiligo and other leukodermas were reviewed and evaluated for the data. Those studies providing inadequate data about patients or outcome or analyzing data of “patches” or “procedures” but not of “patients” were excluded. Follow-up studies of previously published case series were not included to avoid duplication of data. Data for two different subtypes of vitiligo, namely bilateral (generalized and acrofacial) and localized (segmental and focal), was compared. Data for certain “difficult to treat” sites was also analyzed. Adverse effects and total treated area were also looked into.

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The successful outcome was defined as greater than 75% repigmentation, “excellent” repigmentation, repigmentation of “most” of the treated area, or “complete” or “almost complete” repigmentation. The percentage of patients with successful outcome and 95% confidence intervals (CI) were calculated. The mean number of adverse events per patient was calculated to find out which procedures have a better safety profile.

Results A total of 96 studies were identified which were suitable for inclusion of some data in the analyses [4–99].

Procedures The following transplantation procedures have been found to be commonly used for hypopigmentation disorders. Tissue grafts: 1 Thin and ultra-thin split-thickness skin grafts (STSG) 2 Suction blister epidermal grafts (SBEG) 3 Mini-punch grafts (MPG) 4 Hair follicular grafts (HFG) Cellular grafts: 5 Non-cultured epidermal cell suspension (NCES) 6 Cultured “pure” melanocytes (CM) 7 Cultured epithelial grafts (CE) In addition, two procedures, namely ultrasonic abrasion and seed grafts and flip-top technique of grafting have also been tried in one study each [100,101]. In older literature, epidermal shave grafts have also been used. These studies were not included in the meta-analysis of the literature due to paucity of the data. There were 14 studies in STSG, 27 studies in SBEG, 20 studies in MPG, 12 studies in NCECS, 12 studies in cultured melanocytes, 12 studies in cultured epidermis/co-cultures, and 2 studies in follicular transplantation in patients with vitiligo.

seem to be the most effective procedures, with overall success rates of 80.3% (CI 76.4–84.2%) and 77.9% (CI 72.2–83.6%), respectively (Fig. 9.1). The least successful method was hair grafting, with an overall success rate of 58.3% (CI 38.6–78%); however, this technique was especially useful in management of leukotrichia. Among cellular grafts, all techniques seem to be equally effective with success rates of 61.1% (CI 56.1–66.1%), 63.6% (CI 57.2–70%), and 63.6% (CI 55.8–70.6%) for non-cultured epidermal cell suspension, cultured melanocytes, and cultured epidermis, respectively. The success rates in bilateral vitiligo including generalized and acrofacial vitiligo were less than those in segmental or focal vitiligo with all the procedures. The success rates varied from 25% (CI 0–67.4%) for hair grafting to 70.4% (CI 55.1–85.7%) for thin and ultra-thin split-thickness grafts in bilateral vitiligo, while in segmental vitiligo, these ranged from 70.6% (CI 48.9–92.3%) for hair grafting to 100% (CI 100–100%) for thin and ultrathin split-thickness skin grafts. Overall, the transplantation procedures were successful in 73.1% (CI 71.5–74.7%) of all cases of vitiligo, 84.4% (CI 81.8–87%) in patients with segmental or focal vitiligo and 58.5% (CI 54.4–62.6%) in patients with bilateral vitiligo.

Vitiligo on “difficult to treat” sites As data pertaining to individual sites was not provided in most of the studies, the number of patients for specific sites was too small to enable us to apply methods of statistical analysis (Table 9.1).

Fingers and toes Cultured melanocytes, thin or ultra-thin splitthickness grafts, mini-punch grafts, and suction blister epidermal grafts are all highly successful (
80% success rate) on fingers and toes, which are otherwise very difficult to treat with medical therapies and excimer laser. Cultured epidermis is not as successful on this site, with a success rate of 30.8%.

Palms and soles

Outcome Vitiligo Among all procedures, suction blister epidermal grafts and thin and ultra-thin split-thickness grafts

There is only a single case report of successful minigraft transplant on palm [68]. The donor site used for this case was the skin of sole (instep). In the literature, there are no reports of any other method used for vitiligo of this site.

2154/2946

632/749 14/24 325/556

117/184

178/280

71

28/67

50

12/17

88/116

95/108 217/355 41/68

98/175

60

67/85

404/499

82/138

Success rate (%)

70

1065/1400

19/27

80

57/78

90

159/204

34/34

186/208

100

150/181

Practice guidelines

40

1/4

30

20

10

0 STSG

SBEG

MPG

NCES

CM

CE

Hair

All methods

Transplantation method Bilateral vitiligo

Segmental/focal vitiligo

All types

Fig. 9.1 Success rates of various transplantation methods in vitiligo, including its subtypes. The values given on

each bar represent number of successful patients/total number of patients. Error bars represent 95% CI.

Table 9.1 Summary of the outcome of literature analysis for surgical treatment of vitiligo at “difficult to treat” sites.

Site

STSG (successful/ total)

SBEG (successful/ total)

MPG (successful/ total)

NCES (successful/ total)

CM (successful/ total)

CE (successful/ total)

All methods (successful/ total)

Fingers and toes

15/16

13/15

24/27

15/25

6/6

4/13

77/102

Lips

2/2

40/45

102/132

6/14

–

–

150/193

Eyelids

7/9

14/14

1/2

8/10

–

–

30/35

Nipples and areolas

–

2/2

–

2/4

–

–

4/6

Elbows and knees

1/1

–

4/6

39/78

12/27

1/1

57/113

Genitals

0/1

1/1

1/1

3/3

1/1

–

6/7

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Chapter 9

Lips

Table 9.2 Repigmentation of leukotrichia in vitiligo after

Thin or ultra-thin split-thickness grafts and suction blister epidermal grafts are most successful in treatment of vitiligo of the lips, though very few cases treated with the former procedure have been reported in the literature. There is also a risk of mismatch in texture with dermo-epidermal grafts; therefore, presently the simplest and the most suitable procedure for this limited mucocutaneous transition area is pure epidermal grafts obtained through suction blisters. In terms of repigmentation, minigrafts are also successful at this site, but high complication rates in the form of cobblestoning which are associated with that method make it less desirable for this cosmetically highly prominent site. Non-cultured melanocyte suspension is less effective than tissue grafts on lips (42.9% success rate). Concerning cultured cellular grafts no published data for procedures on lips are available but sometimes the lips and eyelids are included in the data of face as a whole, which makes it difficult to analyze when scrutinizing the reports.

transplantation.

be successful on genitals, though data from very few patients is available [70,81].

Eyelids

Leukotrichia in vitiligo

Suction blister epidermal grafts, thin or ultra-thin split-thickness grafts, and NCEC are among the most effective surgical treatments for eyelids [51]. No data for cultured cellular grafts are available for this specific site.

The course of leukotrichia after transplantation has been specifically studied in only a few studies (Table 9.2) [13,14,43]. STSG and SBEG appear to be more successful in repigmentation of leukotrichia than MPG. Sufficient data is not available for cellular grafts. Transplantation of hair has been found to be successful in about half of the patients in the treatment of leukotrichia.

Nipples and areolas Suction blister epidermal grafts are reported to be successful on the nipple and areola, though data from very few patients is available. Non-cultured melanocyte suspension has been found to be successful in about half of the patients.

Elbows and knees These sites are difficult to treat because of their mobility. Inadequate data are available for thin and ultra-thin split-thickness skin grafts, SBEG, and cultured epidermis. Minigrafts and non-cultured melanocyte suspension appear to be successful in about half of the patients.

Genitals Suction blister epidermal grafts, non-cultured cellular suspension and cultured melanocytes are reported to

Number of patients with successfully repigmented white hair

Transplantation method

Total number of patients

STSG

10

8

SBEG

28

27

96.4

MPG

63

16

25.4

NCES

1

1

100

CM

–

–

–

CE

1

1

100

13

6

Hair

Success rate (%) 80

46.2

Size of the treated area Cases with more extensive vitiligo vulgaris, involving greater than 30% body surface area, are generally considered unsuitable for transplantation procedures as chances of retention of the pigment are less [102]. This is not the case in segmental vitiligo, which seems to respond equally well regardless of the size of the involved area. Extensive areas may be best treated with cellular grafts – theoretically, culture methods would provide an unlimited number of cells/tissue for transplantation, while NCEC would provide up to 8–10 times donor-to-recipient expansion. Among tissue grafts, minigrafts are also based on the principle of pigment spread and a 1.25-mm

Practice guidelines graft is expected to provide a pigmented halo of 5–10 mm, depending on the skin type of the patient (more in darker skin type). This means a grafted area of approximately 5 mm2 (or a graft of 1.25 mm) will result in repigmentation of approximately 20 mm2 (pigment spread of 5-mm diameter) to 80 mm2 (pigment spread of 10-mm2 diameter) – 4–15 times donor-to-recipient expansion. Therefore, moderately large areas may be treated with NCEC, thin split-thickness grafts, and minigrafts. Small areas do not require donor skin expansion through cultures or suspension, and therefore should be treated with less complex tissue grafting procedures such as suction blister epidermal grafts or thin split-thickness grafts. In literature, the mean area treated with each procedure is as follows: STSG: 69.3 cm2, SBEG 4 cm2, MPG 21 cm2, NCES 31.6 cm2, CM 34.7 cm2, and CE 161.5 cm2. In SBEG, data was available from only one study. Therefore, it seems that larger areas may be treated with cellular grafts and thin and ultra-thin split-thickness grafts and moderate areas may be treated cellular grafts and minigrafts. Smaller areas may be easily treated with suction blister epidermal grafts which gives good esthetic results and is technically less challenging.

Adverse events No serious adverse events have been reported with any of the transplantation methods. Cellular grafts appear to have the least frequency of adverse events. Cultured melanocytes, cultured epidermis, and NCEC have a mean of 0, 0.02, and 0.08, respectively, adverse events at recipient site, and 0.01, 0, and 0.009, respectively, at the donor site (Tables 9.3 and 9.4). Tissue grafts are reported to be associated with more adverse effects and the maximum number of adverse events on the recipient site are seen when MPG and STSG methods are used.

Other leukodermas/hypopigmentation disorders (Table 9.5) Lip leukoderma due to recurrent herpes labialis It was found to be manageable with thin or ultra-thin split-thickness grafts. Minigrafting has been reported

73

Table 9.3 Mean number of adverse events per patient. Recipient

Donor

STSG

0.5

0.5

SBEG

0.2

0.3

MPG

0.7

0.05

NCES

0.08

0.009

CM

0

0.01

CE

0.02

0

to be unsuccessful for this condition and reactivation of herpes simplex virus infection has been thought to be responsible for graft rejection [103]. Even when minigrafts were successfully transplanted with the help of concurrent acyclovir therapy, there was no pigment spread from the grafts, which soon also became depigmented. Micropigmentation (tattooing) has been successful in such cases [104].

Piebaldism In piebaldism, thin and ultra-thin split-thickness grafting, NCEC, cultured melanocytes, and cultured epidermis have been tried and all these methods yielded 100% success rates.

DLE leukoderma In discoid lupus erythematosus (DLE) leukoderma, thin split-thickness grafts, suction blister epidermal grafts, and minigrafts gave 100% success rates [48]; however, the number of treated patients were few (1, 4, and 1, respectively). These patients were given antimalarial therapy to prevent reactivation of disease or köbnerization.

Halo nevus Recent studies have shown evidence that halo nevus is a different entity from vitiligo [105]. Threequarters (75%) of patients with halo nevus will show successful outcome with transplantation procedures. Non-cultured melanocyte suspension, cultured melanocytes, and cultured epidermis have been found to be successful in the treatment of halo nevi.

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Chapter 9

Table 9.4 Nature of adverse events in transplantation procedures. STSG (n
76) Recipient area

SBEG (n
441)

NCES (n
318)

Hyperpigmentation

2

53

312

3

Hypopigmented borders

4

1

5

20

Cobblestoning

CM (n
259)

CE (n
170)

Hair (n
21)

1

397

Contact dermatitis to topical medication Inclusion cysts

6

3

7

244

9

3

20

Thick margins/stuck-on appearance

9

Variegated appearance Infection

2

Scarring

Donor area

MPG (n
1295)

1

19

All adverse events

37

76

Hyperpigmentation

1

131

Scarring

30

983

2 24

3

60

Köbner phenomenon

4

Infection

3

3

1

All adverse events

31

135

64

3

Table 9.5 Summary of the outcome of literature analysis in other leukodermas.

Type of leukoderma

STSG (successful/ total)

SBEG (successful/ total)

MPG (successful/ total)

NCES (successful/ total)

CE (successful/ total)

All methods (successful/ total)

–

–

10/18

2/2

7/7

39/39

Lip leukoderma due to herpes

10/10

–

0/8

Piebaldism

14/14

2/2

4/4

DLE leukoderma

1/1

4/4

1/1

–

–

–

6/6

Contact leukoderma

–

1/1

1/1

1/1

1/1

–

4/4

Halo nevus

1/0

–

–

2/2

1/1

–

3/3

Nevus depigmentosus

–

1/1

–

1/2

–

–

2/3

Albinism

–

–

–

–

–

0/1

0/1

4/4

–

–

1/1

76/76

–

–

–

–

Post-burn leukoderma Ideopathic guttate hypomelanosis

60/60 –

11/11 4/4

–

CM (successful/ total)

10/10

4/4

Practice guidelines

75

Table 9.6 Guidelines for surgical management of vitiligo and other leukodermas. First choice(s)

Alternative

Acral (fingers and toes)

CM, SBEG, STSG

MPG, NCES, CE

Palms

MPG

–

Lips

SBEG, STSG

MPG, NCES

Eyelids

SBEG, NCES, STSG

MPG

Nipple and areola

SBEG, NCES

Genitals

NCES, SBEG, CM

–

Small

SBEG, STSG

MPG, Hair

Moderate

NCES, STSG

MPG

Extensive

CM, CE, NCES

STSG –

A. Vitiligo Site

Area

B. Herpes lip leukoderma

STSG

C. Piebaldism

Any – choice depends on the size of the lesions. All are likely to be successful

D. DLE leukoderma

SBEG, STSG, MPG

E. Contact leukoderma

Any – all are likely to be successful

F. Halo nevus

NCES, CM, CE

G. Nevus depigmentosus

SBEG

NCES

H. Post-burn leukoderma

Any – STSG, SBEG, and MPG have been tried with consistent success

–

I. Ideopathic guttate hypomelanosis

Any – SBEG has been tried successfully

–

J. Albinism

Should never be treated with autologous transplantation methods

–

–

Nevus depigmentosus

Leukoderma after partial thickness burn injuries

There were only three cases with nevus depigmentosus reported to be treated with transplantation procedures – two with melanocyte suspension and one with epidermal grafts [52,76,77]. It seems that there is no pigment spread phenomenon in these patches and the whole depigmented area needs to be replaced with normally pigmented epidermal sheets [52].

Post-burn leukoderma is almost always successfully managed with transplantation procedures. In the literature, reports of use of tissue grafts are available and most reported cases have been treated with split-thickness grafts [106]. This may be due to familiarity of plastic surgeons with this procedure, as most of the reported studies have been done in

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that setup. Cultured epidermis has also been used in post-burn leukoderma in a single case report.

Idiopathic guttate hypomelanosis This has been successfully treated with suction blister epidermal grafts (4/4) [45].

Albinism Albinism should never be managed with transplantation procedures as it is not likely to be successful. Only one case of albinism has been reported in the literature, in which cultured melanocyte transplantation was tried without any success [92].

Leukoderma due to contact with chemicals It is manageable with transplantation methods – cell suspension, suction blister epidermal grafts, and minigrafts were all found to be 100% successful in these patients.

Guidelines Based on reported success rates, adverse events, technical complexities, anatomical location, and size of the areas, the guidelines have been developed (Table 9.6). Yet every patient should be evaluated individually to find out the best possible options. This depends on expertise of the treating surgeon, economy of the institute, infrastructure available, and patient’s preference. Patient should be counseled for all possible therapeutic options and should be given liberty to choose from the available modalities.

References 1 Njoo MD, Bossuyt PMM, Westerhof W. Management of vitiligo. Results of a questionnaire among dermatologists in the Netherlands. 2 van Geel N, Ongenae K, De Mil M, et al. Doubleblind placebo-controlled study of autologous transplanted epidermal cell suspensions for repigmenting vitiligo. Arch Dermatol 2004;140:1203–8. 3 Pai M, McCulloch M, Gorman JD, et al. Systematic reviews and meta-analyses: an illustrated, step-bystep guide. Natl Med J India 2004;17:86–95. 4 Gupta S, Handa S, Kumar B. A novel scoring system for evaluation of results of autologous transplantation methods in vitiligo. Indian J Dermatol Venereol Leprol 2002;68:33–37.

5 Malakar S. Successful split thickness skin grafts in stable vitiligo not responding to autologous miniature skin grafts. Indian J Dermatol 1997;42:215–8. 6 Olsson MJ, Juhlin L. Epidermal sheet grafts for repigmentation of vitiligo and piebaldism, with a review of surgical techniques. Acta Derm Venereol 1997;77:463–6. 7 Kahn AM, Ostad A, Moy RL. Grafting following short-pulse carbon dioxide laser de-epithelialization. Dermatol Surg 1996;22:965–7. 8 Bose SK. Modified Thiersch grafting in stable vitiligo. J Dermatol 1996;23:362–4. 9 Kahn AM, Cohen MJ. Vitiligo: treatment by dermabrasion and epithelial sheet grafting. J Am Acad Dermatol 1995;33:646–8. 10 Khandpur S, Sharma VK, Manchanda Y. Comparison of minipunch grafting versus split-skin grafting in chronic stable vitiligo. Dermatol Surg 2005;31:436–41. 11 Agrawal K, Agrawal A. Vitiligo: repigmentation with dermabrasion and thin split-thickness skin graft. Dermatol Surg 1995;21:295–300. 12 Bose SK. Thiersch grafting in recurrent herpesinduced lip depigmentation. J Dermatol 2005;32: 432–5. 13 Agrawal K, Agrawal A. Vitiligo: surgical repigmentation of leukotrichia. Dermatol Surg 1995;21:711–5. 14 Bose SK. Is there any treatment of leukotrichia in stable vitiligo? J Dermatol 1997;24:615–7. 15 Al-Qattan MM. Surgical management of post-burn skin dyspigmentation of the upper limb. Burns 2000;26:581–6. 16 Kahn AM, Cohen MJ. Treatment for depigmentation following burn injuries. Burns 1996;22:552–4. 17 Kahn AM, Cohen MJ, Kaplan L, Highton A. Vitiligo: treatment by dermabrasion and epithelial sheet grafting – a preliminary report. J Am Acad Dermatol 1993;28:773–4. 18 Acikel C, Ulkur E, Celikoz B. Carbon dioxide laser resurfacing and thin skin grafting in the treatment of “stable and recalcitrant” vitiligo. Plast Reconstr Surg 2003;111:1291–8. 19 Behl PN. Treatment of vitiligo with autologous thin Thiersch’s grafts. Int J Dermatol 1973;12:329–31. 20 Achauer BM, Le Y, Vander Kam VM. Treatment of vitiligo with melanocytic grafting. Ann Plast Surg 1994;33:644–6. 21 Njoo MD, Nieuweboer-Krobotova L, Westerhof W. Repigmentation of leucodermic defects in piebaldism by dermabrasion and thin split-thickness skin grafting in combination with minigrafting. Br J Dermatol 1998;139:829–33.

Practice guidelines 22 Onur Erol O, Atabay K. The treatment of burn scar hypopigmentation and surface irregularity by dermabrasion and thin skin grafting. Plast Reconstr Surg 1990;85:754–8. 23 Gupta S, Sandhu K, Kanwar A, Kumar B. Melanocyte transfer via epidermal grafts for vitiligo of labial mucosa. Dermatol Surg 2004;30:45–8. 24 Gupta S, Goel A, Kanwar AJ. Autologous melanocyte transfer via epidermal grafts for lip vitiligo. Int J Dermatol 2006;45:747–50. 25 Czajkowski R. Comparison of melanocytes transplantation methods for the treatment of vitiligo. Dermatol Surg 2004;30:1400–5. 26 Gupta S, Kumar B. Epidermal grafting in vitiligo: influence of age, site of lesion, and type of disease on outcome. J Am Acad Dermatol 2003;49:99–104. 27 Pai GS, Vinod V, Joshi A. Efficacy of erbium YAG laser-assisted autologous epidermal grafting in vitiligo. J Eur Acad Dermatol Venereol 2002;16:604–6. 28 Gupta S, Kumar B. Epidermal grafting for vitiligo in adolescents. Pediatr Dermatol 2002;19:159–62. 29 Oh CK, Cha JH, Lim JY, Jo JH, Kim SJ, Jang HS, Kwon KS. Treatment of vitiligo with suction epidermal grafting by the use of an ultrapulse CO2 laser with a computerized pattern generator. Dermatol Surg 2001;27: 565–8. 30 Kim CY, Yoon TJ, Kim TH. Epidermal grafting after chemical epilation in the treatment of vitiligo. Dermatol Surg 2001;27:855–6. 31 Kim HU, Yun SK. Suction device for epidermal grafting in vitiligo: employing a syringe and a manometer to provide an adequate negative pressure. Dermatol Surg 2000;26:702–4. 32 Sachdev M, Shankar DS. Dermatologic surgery: pulsed erbium:YAG laser-assisted autologous epidermal punch grafting in vitiligo. Int J Dermatol 2000;39:868–71. 33 Sachdev M, Krupashankar DS. Suction blister grafting for stable vitiligo using pulsed erbium:YAG laser ablation for recipient site. Int J Dermatol 2000;39: 471–3. 34 Gupta S, Shroff S, Gupta S. Modified technique of suction blistering for epidermal grafting in vitiligo. Int J Dermatol 1999;38:306–9. 35 Gupta S, Jain VK, Saraswat PK. Suction blister epidermal grafting versus punch skin grafting in recalcitrant and stable vitiligo. Dermatol Surg 1999;25: 955–8. 36 Kim HY, Kang KY. Epidermal grafts for treatment of stable and progressive vitiligo. J Am Acad Dermatol 1999;40:412–7.

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37 Yang JS, Kye YC. Treatment of vitiligo with autologous epidermal grafting by means of pulsed erbium:YAG laser. J Am Acad Dermatol 1998;38:280–2. 38 Tang WY, Chan LY, Lo KK. Treatment of vitiligo with autologous epidermal transplantation using the roofs of suction blisters. Hong Kong Med J 1998;4: 219–24. 39 Suga Y, Butt KI, Takimoto R, et al. Successful treatment of vitiligo with PUVA-pigmented autologous epidermal grafting. Int J Dermatol 1996;35: 518–22. 40 Skouge J, Morison WL. Vitiligo treatment with a combination of PUVA therapy and epidermal autografts. Arch Dermatol 1995;131:1257–8. 41 Hann SK, Im S, Bong HW, Park YK. Treatment of stable vitiligo with autologous epidermal grafting and PUVA. J Am Acad Dermatol 1995;32:943–8. 42 Mutalik S. Transplantation of melanocytes by epidermal grafting. An Indian experience. J Dermatol Surg Oncol 1993;19:231–4. 43 Hann SK, Im S, Park YK, Hur W. Repigmentation of leukotrichia by epidermal grafting and systemic psoralen plus UV-A. Arch Dermatol 1992;128: 998–9. 44 Koga M. Epidermal grafting using the tops of suction blisters in the treatment of vitiligo. Arch Dermatol 1988;124:1656–8. 45 Suvanprakorn P, Dee-Ananlap S, Pongsomboon C, Klaus SN. Melanocyte autologous grafting for treatment of leukoderma. J Am Acad Dermatol 1985;13: 968–74. 46 Shenoi SD, Srinivas CR, Pai S. Treatment of stable vitiligo with autologous epidermal grafting and PUVA. J Am Acad Dermatol 1997;36:802–3. 47 Zachariae H, Zachariae C, Deleuran B, Kristensen P. Autotransplantation in vitiligo: treatment with epidermal grafts and cultured melanocytes. Acta Derm Venereol 1993;73:46–8. 48 Gupta S. Epidermal grafting for depigmentation due to discoid lupus erythematosus. Dermatology 2001; 202:320–3. 49 Falabella R. Repigmentation of leukoderma by autologous epidermal grafting. J Dermatol Surg Oncol 1984;10:136–44. 50 Falabella R. Epidermal grafting. An original technique and its application in achromic and granulating areas. Arch Dermatol 1971;104:592–600. 51 Nanda S, Relhan V, Grover C, Reddy BS. Suction blister epidermal grafting for the management of vitiligo of the eyelid: special considerations. Dermatol Surg 2006;32:387–91.

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52 Gupta S, Goel A. Letter to the editor: nevus depigmentosus needs transplant of epidermal sheets. Dermatol Surg 2005;31:1746–7. 53 Malakar S, Lahiri K. Punch grafting for lip leucoderma. Dermatology 2004;208:125–8. 54 Sarkar R, Mehta SD, Kanwar AJ. Repigmentation after autologous miniature punch grafting in segmental vitiligo in North Indian patients. J Dermatol 2001;28:540–6. 55 Falabella R, Barona M, Escobar C, Borrero I, Arrunategui A. Surgical combination therapy for vitiligo and piebaldism. Dermatol Surg 1995;21:852–7. 56 Singh AK, Bajaj AK. Autologous miniature skin punch grafting in vitiligo. Indian J Dermatol Venereol Leprol 1995;61:77–80. 57 Boersma BR, Westerhof W, Bos JD. Repigmentation in vitiligo vulgaris by autologous minigrafting: results in nineteen patients. J Am Acad Dermatol 1995;33:990–5. 58 Rathi TM, Singh AK. Punch grafting in the treatment of stable vitiligo. Indian J Dermatol Venereol Leprol 1994;60:188–91. 59 Savant SS. Autologous miniature punch skin grafting in stable vitiligo. Indian J Dermatol Venereol Leprol 1992;58:310–4. 60 Falabella R. Treatment of localized vitiligo by autologous minigrafting. Arch Dermatol 1988;124:1649–55. 61 Falabella R. Repigmentation of segmental vitiligo by autologous minigrafting. J Am Acad Dermatol 1983; 9:514–21. 62 Falabella R. Repigmentation of leukoderma by minigrafts of normally pigmented, autologous skin. J Dermatol Surg Oncol 1978;4:916–9. 63 Garg T, Khaitan BK, Manchanda Y. Autologous punch grafting for repigmentation in piebaldism. J Dermatol 2003;30:849–50. 64 Falabella R. Repigmentation of stable leukoderma by autologous minigrafting. J Dermatol Surg Oncol 1986; 12:172–9. 65 Hallaji Z, Daneshpazhooh M, Rezai-khiabanloo S. Successful treatment of vitiligo with punch graft followed by outdoor topical psoralen plus ultraviolet A radiation. Arch Iran Med 2003;6:86–90. 66 Shah BH, Joshipura SP, Thakkar JK. Surgical treatment in acrofacial vitiligo. Indian J Dermatol Venereol Leprol 1994;60:26–27. 67 Malakar S, Dhar S. Acyclovir can abort rejection of punch grafts in herpes-simplex-induced lip leucoderma. Dermatology 1999;199:75. 68 Kumar P. Autologous punch grafting for vitiligo of the palm. Dermatol Surg 2005;31:368–70.

69 Malakar S, Dhar S. Acyclovir can abort rejection of punch grafts in herpes-simplex-induced lip leucoderma. Dermatology 1999;199:75. 70 Mulekar SV, Al Issa A, Al Eisa A, Asaad M. Genital vitiligo treated by autologous, noncultured melanocyte–keratinocyte cell transplantation. Dermatol Surg 2005;31:1737–9. 71 Xu AE, Wei XD, Cheng DQ, et al. Transplantation of autologous noncultured epidermal cell suspension in treatment of patients with stable vitiligo. Chin Med J (Engl) 2005;118:77–9. 72 Mulekar SV. Long-term follow-up study of segmental and focal vitiligo treated by autologous, noncultured melanocyte–keratinocyte cell transplantation. Arch Dermatol 2004;140:1211–5. 73 Tegta GR, Parsad D, Majumdar S, Kumar B. Efficacy of autologous transplantation of noncultured epidermal suspension in two different dilutions in the treatment of vitiligo. Int J Dermatol 2006;45:106–10. 74 Mulekar SV. Long-term follow-up study of 142 patients with vitiligo vulgaris treated by autologous, non-cultured melanocyte–keratinocyte cell transplantation. Int J Dermatol 2005;44:841–5. 75 van Geel N, Ongenae K, De Mil M, Naeyaert JM. Modified technique of autologous noncultured epidermal cell transplantation for repigmenting vitiligo: a pilot study. Dermatol Surg 2001;27:873–6. 76 Olsson MJ, Juhlin L. Leucoderma treated by transplantation of a basal cell layer enriched suspension. Br J Dermatol 1998;138:644–8. 77 Gauthier Y, Surleve-Bazeille JE. Autologous grafting with noncultured melanocytes: a simplified method for treatment of depigmented lesions. J Am Acad Dermatol 1992;26:191–4. 78 Pandya V, Parmar KS, Shah BJ, Bilimoria FE. A study of autologous melanocyte transfer in treatment of stable vitiligo. Indian J Dermatol Venereol Leprol 2005;71:393–7. 79 Chen YF, Chang JS, Yang PY, et al. Transplant of cultured autologous pure melanocytes after laserabrasion for the treatment of segmental vitiligo. J Dermatol 2000;27:434–9. 80 Lontz W, Olsson MJ, Moellmann G, Lerner AB. Pigment cell transplantation for treatment of vitiligo: a progress report. J Am Acad Dermatol 1994;30:591–7. 81 Olsson MJ, Juhlin L. Transplantation of melanocytes in vitiligo. Br J Dermatol 1995;132:587–91. 82 Kaufmann R, Greiner D, Kippenberger S, Bernd A. Grafting of in vitro cultured melanocytes onto laserablated lesions in vitiligo. Acta Derm Venereol 1998;78:136–8.

Practice guidelines 83 Olsson MJ, Moellmann G, Lerner AB, Juhlin L. Vitiligo: repigmentation with cultured melanocytes after cryostorage. Acta Derm Venereol 1994;74:226–8. 84 Olsson MJ, Juhlin L. Repigmentation of vitiligo by transplantation of cultured autologous melanocytes. Acta Derm Venereol 1993;73:49–51. 85 Lerner AB, Halaban R, Klaus SN, Moellmann GE. Transplantation of human melanocytes. J Invest Dermatol 1987;89:219–24. 86 Chen YF, Yang PY, Hung CM, Hu DN. Transplantation of autologous cultured melanocytes for treatment of large segmental vitiligo. J Am Acad Dermatol 2001; 44:543–5. 87 Jha AK, Pandey SS, Gulati AK, et al. Inoculation of a cultured autologous epidermal suspension containing melanocytes in vitiligo. Arch Dermatol 1993;129:785–6. 88 Pianigiani E, Risulo M, Andreassi A, et al. Autologous epidermal cultures and narrow-band ultraviolet B in the surgical treatment of vitiligo. Dermatol Surg 2005;31:155–9. 89 Guerra L, Primavera G, Raskovic D, et al. Erbium:YAG laser and cultured epidermis in the surgical therapy of stable vitiligo. Arch Dermatol 2003;139:1303–10. 90 Guerra L, Capurro S, Melchi F, et al. Treatment of “stable” vitiligo by Timedsurgery and transplantation of cultured epidermal autografts. Arch Dermatol 2000;136:1380–9. 91 Andreassi L, Pianigiani E, Andreassi A, Taddeucci P, Biagioli M. A new model of epidermal culture for the surgical treatment of vitiligo. Int J Dermatol 1998;37:595–8. 92 Kumagai N, Uchikoshi T. Treatment of extensive hypomelanosis with autologous cultured epithelium. Ann Plast Surg 1997;39:68–73. 93 Falabella R, Escobar C, Borrero I. Transplantation of in vitro-cultured epidermis bearing melanocytes for repigmenting vitiligo. J Am Acad Dermatol 1989;21: 257–64. 94 Falabella R, Escobar C, Borrero I. Treatment of refractory and stable vitiligo by transplantation of in vitro cultured epidermal autografts bearing melanocytes. J Am Acad Dermatol 1992;26:230–6. 95 Toriyama K, Kamei Y, Kazeto T, et al. Combination of short-pulsed CO2 laser resurfacing and cultured

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epidermal sheet autografting in the treatment of vitiligo: a preliminary report. Ann Plast Surg 2004;53: 178–80. Guerra L, Primavera G, Raskovic D, et al. Permanent repigmentation of piebaldism by erbium:YAG laser and autologous cultured epidermis. Br J Dermatol 2004;150:715–21. Stoner ML, Wood FM. The treatment of hypopigmented lesions with cultured epithelial autograft. J Burn Care Rehabil 2000;21:50–4. Malakar S, Dhar S. Repigmentation of vitiligo patches by transplantation of hair follicles. Int J Dermatol 1999;38:237–8. Na GY, Seo SK, Choi SK. Single hair grafting for the treatment of vitiligo. J Am Acad Dermatol 1998;38: 580–4. Tsukamoto K, Osada A, Kitamura R, et al. Approaches to repigmentation of vitiligo skin: new treatment with ultrasonic abrasion, seed-grafting and psoralen plus ultraviolet A therapy. Pigm Cell Res 2002;15:331–4. McGovern TW, Bolognia J, Leffell DJ. Flip-top pigment transplantation: a novel transplantation procedure for the treatment of depigmentation. Arch Dermatol 1999;135:1305–7. Olsson MJ, Juhlin L. Long-term follow-up of leucoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol 2002;147:893–904. Lahiri K, Malakar S, Sarma N. Herpes-simplexinduced lip leucoderma revisited. Dermatology 2004; 208:182. Malakar S, Lahiri K. Successful repigmentation of six cases of herpes-labialis-induced lip leucoderma by micropigmentation. Dermatology 2001;203:194. de Vijlder HC, Westerhof W, Schreuder GM, et al. Differences in pathogenesis between vitiligo vulgaris and halo nevi associated with vitiligo is supported by an HLA association study. Pigm Cell Res 2004;17: 270–4. Grover R, Morgan BD. Management of hypopigmentation following burn injury. Burns 1996; 22:627–30.

CHAPTER 10

Evaluation of outcome in surgical therapies for vitiligo Nanny van Geel and Jean Marie Naeyaert

Introduction Many studies have been performed and are still ongoing to investigate the efficacy of several available and new treatment modalities. Among these, a lot of studies are concentrating on transplantation techniques for vitiligo [1]. In these surgical vitiligo trials a variety of assessment methods have been used to evaluate the response to therapy [2]. Many of these evaluation methods rely on the subjective assessment of repigmentation by the investigator. However, to allow an objective and reliable evaluation of results a consensus about an adequate scoring system is needed. It will enable accurate and appropriate data collection usable for both direct comparison and for pooling of treatment results from different clinical trials. For many skin diseases (e.g. acne vulgaris, eczema, mycosis fungoides, and psoriasis) a system for quantification of clinical symptoms has been developed [3–6]. So far, however, there is no generally accepted and validated measurement tool or index for vitiligo.

Current evaluation methods in transplantation studies To gain more insight into currently used evaluation methods in surgical vitiligo studies our group conducted a systematic literature survey [2]. Forty-three clinical vitiligo trials concerning surgical treatment modalities, published in 1966–2002, were selected. We could demonstrate that there was no consensus at all about the choice of the evaluation methods and outcome parameters used in surgical vitiligo studies. These differences resulted in incompatible and noncomparable data. Secondly, as most methods have

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been based on visual assessment, a high inter- and intraobserver variability may be suspected, leading to a highly subjective outcome. Only three included studies did mention the use of a more objective measurement tool. In the study of Boersma et al. [7] and Guerra et al. [8] planimetric measurement was used based on photographs and transparent films, respectively, while the group of Hatchome mentioned the use of a “microbalance” [7–9]. As interobserver variation can flaw accurate assessment, one study mentioned more than one observer evaluating the same patient to increase the reliability [10].

Outcome parameters The most important parameter in evaluating treatment in vitiligo is undoubtedly the amount of repigmentation. However, according to our recent literature survey 20 different final scoring systems were used in our 43 selected reports to evaluate repigmentation capacity [2]. In 14 studies (33%) repigmentation was recorded as an exact percentage. Ten studies (23%) used this parameter in a broader, less-defined sense, varying from “more than 75% repigmentation” to “less than 30%, 31–50%, 51–75%, 76–90%, and 91–100%.” Eleven authors (26%) only stated the presence or absence of repigmentation or acceptance of the graft. Four other studies (9%) qualified repigmentation as “zero,” “partial,” or “complete” and three (7%) mentioned the presence of “poor,” “moderate,” “fair,” “good,” or “excellent” results. In one study results are classified on a grading scale from 0 to 3, of which grade 0 means no response and grade 3 almost complete response [2].

Evaluation of outcome in surgical therapies for vitiligo For comparing or pooling of treatment results, exact percentages are the easiest to handle. Making subdivisions in groups is then still possible.

Description of patient population For correct interpretation and comparison of results in vitiligo studies it is also important to have complete information about the included patient population (e.g. patients’ skin type, type of vitiligo, disease activity and treated localization). Previous observations could demonstrate a tendency that darker skin types (Fitzpatrick skin type IV or more), segmental vitiligo, stable disease, and lesions on the face and trunk may have a better therapeutic response to surgical interventions [11,12]. Besides, the definition of important parameters such as disease activity should be precisely defined and universally agreed on. Only this will enable correct comparison and pooling of outcome from different clinical trials.

Different assessment methods 1 Visual assessment 2 Photographic image analysis 3 Transparent sheets 4 Evaluation of treatment from the patient’s point of view

Visual assessment Visual estimation is a quick method, but has the major disadvantage of being subjective. A commonly used visual method to roughly assess repigmentation of the body surface involved is “the rule of nine”- and the “hand palm
1%”- method. The first assumes that the total body surface area comprises 9% for head and neck, 9% for each arm, 18% for each leg, 36% for the trunk, leaving 1% for the genitalia. In the second method a flat hand palm represents 1% of the total body surface area. However both methods are very subjective. A high inter- and intraobserver variability in the calculation of the body surface area among clinicians has already been demonstrated in several psoriasis studies [13,14]. Later, the so-called Dermatological Global Assessment (DGA) system has been developed and allows a much more detailed description

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of the disease extent spread over seven specific body parts on a scale of 0–4 [15]. We investigated the variance in visual surface estimations of small vitiligo lesions both interindividually and between different observers, respectively. We observed a huge spread on visual assessment which indicates a high inter- and intraobserver variability for the visual estimation [16].

Photographic image analysis Photographic image analysis may be considered a far more objective tool than visual assessment, although it may underestimate the real affected surface as it is based on two-dimensional (2D) measurements. Therefore, its use is limited to the estimation of surface changes over time of some target lesions and is therefore useful for the evaluation of pigment transplantation. When using photographs for treatment evaluation it is very important to use a standardized technique to take the pictures. It can be helpful to take all photographs at a fixed working distance, using a “spacer” attached to the camera. Furthermore in case of digital images, a color card (see Plate 10.1, facing p. 114) can be used for color calibration of the picture. The color calibration procedure eliminates most variations in the images due to camera settings and other outside influences like extraneous lighting, and ensures that images can be compared. Our group introduced a new digital image analysis system that might be useful in consistently measuring surfaces of vitiligo lesions both before and after melanocyte transplantation [16]. The system is based on a semi-automatic color segmentation technique. The most important difference with currently used techniques is that this system is capable of measuring a surface from a digital image without the need of a manual tracing procedure on the skin. This makes the procedure much easier and less time-consuming. We could demonstrate that the system was accurate and that the reproducibility was significantly improved compared to the visual estimation of surfaces.

Transparent sheets The use of tracing lesions (point counting grids) on a transparent sheet will probably be the most

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accurate measurement method at the moment. This method has the advantage of taking the local curvature into account, thereby avoiding possible underestimation of the lesion surface due to the move from three to two dimensions. However, it is a very time-consuming method, which limits its use mainly to clinical studies. The surface measurement of the transparent sheets can be performed manually or with an image processing program and is very accurate and exhibits no intra- or interobserver variability.

Conclusion

Evaluation of treatment from the patient’s point of view

1 van Geel N, Naeyaert JM, Ongenae K. Surgical Techniques for vitiligo: a review. Dermatology 2001; 202:162–6. 2 van Geel N, Ongenae K, Vander Haeghen Y, Naeyaert JM. Autologous transplantation techniques for vitiligo: how to evaluate treatment outcome? Eur J Dermatol 2004;14:46–51. 3 Pochi PE, Shalita AR, Strauss JS, et al. Report of the Consensus Conference on Acne Classification. Washington, DC, March 24 and 25, 1990. J Am Acad Dermatol 1991;24:495–500. 4 Bahmer FA. ADASI score: atopic dermatitis area and severity index. Acta Derm Venereol Suppl (Stockh.) 1992;176:32–3. 5 Lamberg SI, Green SB, Byar DP, et al. Clinical staging for cutaneous T-cell lymphoma. Ann Intern Med 1984;100:187–92. 6 Fredriksson T, Pettersson U. Severe psoriasis: oral therapy with a new retinoid. Dermatologica 1978; 157:238–44. 7 Boersma B, Westerhof W, Bos J. Repigmentation in vitiligo vulgares by autologous minigrafting: results in nineteen patients. J Am Acad Dermatol 1995;33:990–5. 8 Guerra L, Capurro S, Melchi F, et al. Treatment of “stable” vitiligo by Timedsurgery and transplantation of cultured epidermal autografts. Arch Dermatol 2000; 136:1380–9. 9 Hatchome N, Kato T, Tagami H. Therapeutic success of epidermal grafting in generalized vitiligo is limited by the Köebner phenomenon. J Am Acad Dermatol 1990; 22:87–91. 10 Özdemir M, Cetinkale O, Wolf R, et al. Comparison of two surgical approaches for treating vitiligo: a preliminary study. Int J Dermatol 2002;41:135–8. 11 Falabella R. Treatment of localized vitiligo by autologous minigrafting. Arch Dermatol 1988;124:1649–55. 12 Olsson M, Juhlin L. Long-term follow-up of leucoderma patients treated with transplants of autologous

“Quality of Life” and “global assessment” are interesting additional tools in the evaluation of treatment outcome, because the percentage of repigmentation alone may not always be a good indicator of the satisfaction of the patient. A good repigmentation of a small but well exposed area (e.g. face, hands) may be more satisfying to a patient than good repigmentation on large but totally covered areas of the body. To measure quality of life, a validated multidimensional concept (Dermatology Life Quality Index, DLQI) has been developed by Finlay [17]. It encompasses the physical, social, and psychological well-being of an individual. For the “global assessment” no standardized questionnaire is available yet for vitiligo, but usually one uses a visual scale, grading from 0 to 10. Evaluating the quality of life of vitiligo patients is also important to convince physicians, insurance companies, and governmental institutions that vitiligo is not “only a pure cosmetic skin disorder” but may have severe psychosocial impact and that different forms of treatment in these cases may be both helpful and effective in selected patients. It is also interesting to evaluate the clinical relevance of an achieved result from the patient’s point of view, as the interpretation of a “successful” treatment can differ between dermatologists and vitiligo patients. The clinical relevance can be investigated by asking some simple questions to the patient, for example, (a) Are you satisfied with the obtained result? (b) Do you find the treatment worthwhile? (c) If you had to make the choice for this treatment, would you choose it again?

One can conclude that there is an urgent need for an universally accepted, objective, reliable, and useful measurement method to evaluate the efficacy of surgical vitiligo studies. In addition, evaluating treatment results the clinical relevance from the patient’s point of view deserves special attention. A combination of both a clinical and a psychological evaluation is probably the most appropriate assessment.

References

Evaluation of outcome in surgical therapies for vitiligo cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol 2002;147: 893–904. 13 Ramsay B, Lawrence CM. Measurement of involved surface area in patients with psoriasis. Br J Dermatol 1991;124:565–70. 14 Tiling-Grosse S, Rees J. Assessment of area of involvement in skin disease: a study using schematic figure outlines. Br J Dermatol 1993;128:69–74. 15 Gupta G, Long J, Tillman DM. The efficacy of narrowband ultraviolet B phototherapy in psoriasis using

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objective and subjective outcome measures. Br J Dermatol 1999;140:887–90. 16 van Geel N, Vander Haeghen Y, Ongenae K, Naeyaert JM. A new digital image analysis system useful for surface assessment of vitiligo lesions in transplantation studies. Eur J Dermatol 2004;14:150–5. 17 Finlay AY, Khan GK. Dermatology Life Quality Index (DLQI) – a simple practical measure for routine clinical use. Clin Exp Dermatol 1994;19:210–16.

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SECTION 3

Tissue grafting

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CHAPTER 11

Minigrafting for vitiligo Subrata Malakar and Koushik Lahiri

Great things are not done by impulse, but by a series of small things brought together. Vincent Van Gogh

Vitiligo, the most significant form of cutaneous achromia, has been an illusive, if not enigmatic problem through the ages. The treatment has undergone an enormous evolutionary change from the Vedic days of “Vasuchika” to the most modern transplantation techniques. But the ultimate goal remains the same – to achieve complete and durable repigmentation. Many patients respond to standard medical treatment options, but several patients remain recalcitrant or respond only partially. Any attempted repigmentation of these resistant patches with conventional medicinal modalities is often unsuccessful and sometimes exasperating, indicating the absence of melanocyte reservoirs to induce repigmentation. Under these circumstances, melanocytes’ repopulation of the achromic areas is not possible unless a new source of pigment cells is placed by surgical methods within the depigmented lesion(s). Different corrective surgical methods have evolved during the last four decades. Some of these are thin Thiersch’s graft [1], suction blister epidermal graft [2], punch graft [3], mini-punch graft [4,5], cultured melanocytes graft [6,7], cultured epidermal graft [8], autografting and psoralen plus ultraviolet A (PUVA) [9,10], single hair transplant [11,12], ultrathin epidermal sheets [13], and basal cell layer suspension, minigrafting and narrowband ultraviolet B (NB-UVB) [14]. Among all these methods, minigrafting using skin punch is the easiest, fastest, least aggressive, and minimally expensive method.

The punch instrument The skin punch or surgical punch is an instrument which is used almost solely by dermatologists. It is

interesting to note that originally it was used as a trephine to cut through the skull bone. Its use was documented in abscess removal from tibia as early as 1852 [15]. In 1878 Watson described its use in the correction of accidental gunpowder disfigurement [16]. The importance of cutaneous punch instruments in dermatology was first established by Keyes in 1887 [17]. The Keyes punch (Fig. 11.1A) has been used in dermatology since then for diagnostic purpose. Its rounded sharp cutting end and thick handle make it very much appropriate for small skin biopsies. Due to the thick walls with angled sides above the cutting edge, tissue leans to be pushed away as the punch is made, causing less dermis to be cut through (in diameter) than overlying epidermis. This is also a function of the bevel, which is outside the barrel of the Keyes punch [18]. To overcome these difficulties other punches have been developed [19]. The walls of the Loo trephine (Fig. 11.1b) are thinner and less slanted than those of the Keyes punch, making it advantageous to use in correction of depressed scars or for minor autotransplants where a straight vertical incision is needed. The newer disposable punches (Fig. 11.1c) are excellent for punch biopsies or excisional work on cysts. The razor-sharp edge is a great benefit. The punch is made in a number of different dimensions.

Evolution of mini-punch grafting In the history of skin grafting, a couple of observations can be mentioned as a preamble to further discussion of punch grafting. The first documented successful result of experimental skin grafting was described in sheep by Baronio in 1804 [20].

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(A)

(B)

(C)

Fig. 11.1 (A) Keyes punch, (B) Loo trephine, and

(C) disposable punch.

But it took another almost one and a half centuries to get the first recorded autograft response of dark-skinned autografts when transplanted to light areas in spotted guinea pigs by Lewin and Peck in 1941 [21]. In 1972 Norman Orentriech first reported autograft repigmentation in human. He treated a Black woman with longstanding leukoderma that followed a chemical burn many years back when she was treated with a home remedy which included a copper penny dipped in vinegar for presumptive tinea infection on her cheek. Orentriech deployed nine 1 and 2-mm diameter normal skin autografts and observed the “pigment spread phenomenon.” He reported a maximum of 1-mm pigment spread from both 1 and 2-mm grafts [3]. In 1976 Labuono and Shatin made a similar observation after transplanting hair bulbs with hair punch grafting within the leucodermic scars of discoid lupus erythematosus (DLE) [22]. Falabella in 1978 reported a novel method of repigmenting leukoderma. With the help of a powerdriven dermabrasion unit he used dental burrs to create abrasion 2–3 mm in diameter and less than 1 mm in depth, and 5 mm apart. In the donor area skin was raised by means of a curved needle, and was snipped off just below it to harvest 1–2-mm size minigrafts. He reported about a 3-mm perigraft pigment spread by this technique. Three patients,

one with piebaldism, another with chemical leukoderma, and a third with post-burn depigmentation were treated by this method [23]. He observed that these superficial split thickness grafts evoked a much better outcome than the full thickness hair punch grafting by Labuono. In the same article, interestingly, it was concluded that “… true vitiligo is not treatable by transplantation of grafts of normally pigmented, autologous skin” [23]. In 1983, miniature punches of 1.5 mm diameter were used by Falabella in three patients with segmental vitiligo [24]. Behl (1995) expressed some reservation while commenting on Falabella’s work on minigrafting and claimed that results were better with thin Thiersch grafting. In a rejoinder Falabella reiterated his faith in miniature punch grafting and countered with his sets of reasons and logics in favor of punch grafting [25,26]. In the following years Falabella reported success with mini-punch grafting in chemical leukoderma, post-dermabrasion leukoderma, and focal and segmental vitiligo [27–29]. While repigmenting stable leukoderma with autologous minigrafting Falabella made an important observation regarding the relationship between the donor graft area and the area of surgical repigmentation and found that a 1-mm donor graft can originate a pigmented spot 25 times larger than its size [27]. In 1995 it was Falabella again who combined epidermal grafting and minigrafting in the treatment of vitiligo and piebaldism [30]. Westerhof in 1994 reported success with punch grafting in stable vitiligo and observed a maximum of 5 mm of pigment spread [31]. In the subsequent year (1995) Boersma stressed on the importance of proper selection of cases before minigrafting [32]. Various studies point towards the high effectiveness of the procedure [33–36]. An assortment of different evaluation parameters of mini-punch grafting has also evolved over the years [37–40]. Very recently mini-punch grafting has been combined with NB-UVB (311 nm) and documented encouraging results [14].

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Minigrafting test/test grafting Before embarking on any surgical intervention in vitiligo, proper assessment of the stability status is of paramount importance. This concept has been discussed in detail separately (Chapter 6). Clinically, stability can be judged by three simple indicators: 1 History: Lack of progression of old lesions and absence of development of any new lesion within a specified period (6 months to 2 years). 2 Köbner phenomenon: Absence of a recent Köbner phenomenon either from history or experimentally induced. 3 Test grafting (TG): On the backdrop of a pervasive incongruity about the minimal period of stability an attempt was made for the first time by Falabella in 1995 to fathom stability before surgery by introducing minigrafting test [41]. The objective of this test was to serve several purposes: • establishing the stability of the depigmenting process; • determining a means by which patients could be selected; • identifying patients who may respond to pigment cell transplantation; and • anticipating the response to surgical repair. In the original suggested procedure a few grafts (1.0–1.2 mm) were placed in the center of the depigmented lesion to be scrutinized. Dressing was done by Micropore® adhesive tape and kept for a couple of weeks. After removal of the tape the area was exposed to sunlight for 15 minutes daily for a period of 3 months. No treatment was permitted during this test period. All test sites were visualized under Wood’s light. The test was considered positive if unequivocal repigmentation takes place beyond 1 mm from the border of the implanted grafts. On the other hand, if less than 1 mm or no repigmentation was observed the test was considered as negative. In some of the biggest series this evaluation has been termed as “test grafting” and found to be a more reliable exercise than the dependence on period of stability alone [14,36,42].

Fig. 11.2 Instrument required for minigrafting.

Over the years this “test” has been vindicated and acknowledged as a powerful tool for detecting stable vitiligo, which anticipates the repigmentation success in vitiligo when surgery becomes a therapeutic option.

Method [24,34–36] After proper assessment of the stability status and routine physical examination and investigations an informed consent is taken from the patient. The donor and recipient areas are surgically prepared. 1 The instruments required are 1.5 or 1.2-mm punches, small Jeweler’s or graft-holding forceps and small curved tip scissors (Fig. 11.2). 2 Recipient area is prepared first; 2% lignocaine with or without adrenaline is infiltrated as local anesthetic. 3 To minimize the chance of developing achromic fissure, the initial recipient chambers are made on or very close to the border of the lesion. The punched out chambers are spaced according to the result of TG or at a distance of 5–10 mm from each other. 4 The donor area is either upper lateral portion of thigh or gluteal area. Punch should be held between thumb and index and middle fingers (Fig. 11.3). Punch impressions are made very close to each other so that a maximum number of grafts can be taken from a small area. Same sized punches are used for both donor and recipient area. 5 The grafts are placed directly from donor (buttock/ upper thigh) to the recipient areas. This speeds up

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the procedure and lessens the chance of infection. Care is taken so that the graft edges are not folded, the tissue is not crushed or placed upside down. The needle of the syringe or the tip of the scissors is used for proper placement of grafts in the recipient chambers. 6 Hemostasis is achieved by pressing a saline soaked gauze piece over the area. 7 For the recipient area three layers of dressing from inside out are: Paraffin-embedded non-adherent sterile gauze (Jelonet®), sterile Surgipad®, and bioocclusive Micropore®. 8 For the donor area only Surgipad® and Micropore® are used. 9 The recipient area may be immobilized if necessary. Proper instructions for special areas like lips are necessary. To secure grafts in the recipient area these patients are advised to take liquid diet for the first 24 hours, preferably with a straw. Patients are allowed normal diet after this period. 10 Sometimes dressings are opened after 24 hours to look for any dislodgement of grafts; if any are found, they are replaced. 11 Finally after 4–7 days the dressings are removed.

Follow up and course of events Post surgically the patients are exposed to psoralen plus ultraviolet A (PUVA) [9,10] or sunlight (PUVASOL) [35,36], or NB-UVB [14] or not combined with any medical therapy in some studies [36]. The patients are followed up fortnightly for an initial 2 months and then monthly until complete repigmentation is achieved. Minimal superficial scarring is expected at the donor site after healing with secondary intention and acceptable. Scabs may fall off from the recipient site within 7–14 days, though in many instances there may not be any scab formation. Perigraft repigmentation is expected to start in around 3–4 weeks [14,35–37]. The entire depigmented and grafted area is expected to be completely repigmented within 3–6 months, based on the area of grafting and body part involved (Figs. 11.4–11.7).

Fig. 11.3 Correct positioning of the hand while taking

grafts with a miniature punch.

Fig. 11.4 Segmental vitiligo.

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Fig. 11.7 Complete repigmentation after two sessions.

Fig. 11.5 Complete repigmentation after 6 months.

Fig. 11.8 Cobblestoning due to bigger grafts.

• • • • • •

Variegated appearance and color mismatch Static graft (no pigment spread) Depigmentation of graft Perigraft halo/achromic fissure Graft dislodgement/rejection Hypertrophic scar and keloid formation

Fig. 11.6 Depigmented nipple and areola.

Donor site

Complications [14,34–36] Figs. (11.8–11.10)

Recipient site • Cobblestoning • Polka dot appearance

• Keloid and hypertrophic scar • Superficial scar • Depigmentation/spread of disease • Contact dermatitis to adhesive tapes By proper selection of cases most of these complications are avoidable.

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Advantages • Easiest, fastest, and least expensive method. • High rate of success with very few preventable/ manageable side effects. • Can be performed anywhere on any site (except angle of the mouth).

Discussion

Fig. 11.9 Variegated appearance.

Fig. 11.10 Depigmented grafts.

Cobblestoning is regarded as the commonest of all [14,35,36,42]. It was observed that with time it was corrected in most of the cases [35]. In resistant cases corrective electrofulguration may be needed [43]. In this regard it is only apt to conclude that grafting should not be performed with punches more than 1.5 mm in diameter. On face and lips it should be even smaller (1.2 mm or 1 mm) [14,44]. Herpes labialis induced lip leukoderma (HILL) is another dicey entity bearing the risk of rejection of grafts [45–48].

Surgical correction of vitiligo and other cutaneous achromia has come a long way in the last four decades. But among all other methods, autologous miniature punch grafting has already established its place as the easiest, fastest, safest, and least aggressive means of vitiligo surgery. When the graft is taken off, the piece of tissue is completely detached from the donor site and then it is placed on the vascular bed in the recipient holes. From this vascular bed it derives its blood supply. Initially the graft adheres to its new bed by fibrin. There is diffusion of nutrients through this fibrinous layer which keep the graft alive initially. Within 2–3 days, capillary linkage occurs with vascularization of the graft. The thinner the graft the denser the capillary network in the superficial dermis and thus earlier is the process of vascularization [49]. Phototherapy induced stimulation of melanocyte migration from the hair follicle reservoir is now a well-established fact. They spread centrifugally from the infundibulum to the basal cell layer and recolonize the epidermis with active and functional melanocytes [50,51]. But the presence of pilosebaceous apparatus within the minigrafts is not necessary for the repigmentation process, as in suction blister grafts only epithelial cells present in the grafts are capable of inducing repigmentation [52]. In 1970 Billingham and Silvers have demonstrated the phenomenon of melanocyte migration from graft’s edge within the achromic skin to recolonize and replenish the area with functional and active melanocytes [53]. Falabella in 1988 tried to establish a histological/ histochemical background of surgical repigmentation [29]. In another study consistent and comparable status of melanization was noted over both normal and

Minigrafting for vitiligo surgically repigmented area using Masson Fontana stain [38]. Even after recognizing the significance of stability and after three decades of experience in vitiligo surgery, there is little consensus regarding the optimal required period of stability. In one study the minimal period of stability as a prerequisite for grafting was mentioned to be as little a period as 4 months [54], while on the other side of the spectrum in another study it was taken as 3 years [13,14]. Other variable figures like 6 months, 1 year, and 2 years can easily be obtained from some other studies as well [32,33,55,56]. Even the same author has taken different periods of stability into consideration in different articles [8,26]. It is often hard to predict how long the disease will remain stable. Similarly difficult is to envisage when it will start to become unstable [57,58]. Repigmentation has been successfully induced in previous graft failure cases under NB-UVB (311 nm) phototherapy [59]. The observation of spontaneous repigmentation of non-grafted vitiligo patches indicates a possible release of fresh cytokines from the donor skin, while stimulating the vitiliginous patches and hair follicles of the grafted sites may have played some role at the distant sites by local absorption [60,61]. Another theory was the immunogenic mechanism which was originally responsible for development of vitiligo may have lost its antigenicity due to the autologous grafts [62]. The size of the grafted lesions varied between 15 and 144 cm2 in different studies. Likewise the size of the punch instruments differed in different studies. But now there is a consensus towards using smaller punches such as 1.2 or 1.5 mm. Falabella even recommends 1-mm grafts for the facial region and 1.2 mm for other body parts [63]. In this way, cobblestoning, the commonest complication of punch grafting can also be avoided. Though the rate of cobblestoning was substantial in most of the studies, it was found that with time it was corrected. In resistant cases electrofulguration is helpful [64]. Very recently repigmentation of leucotrichia with minigrafting and NB-UVB was reported [14]. The same was also observed and documented before with PUVASOL and minigrafting [44]. Another important parameter is the post-graft appearance of repigmentation time. It was found to

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Table 11.1 MPS in different studies. Author and reference number

Year

MPS (in mm)

Orentriech and Selmanwitz [3]

1972

1

Falabella [23]

1978

3

Falabella [29]

1988

4

Westerhof et al. [31]

1994

5

Savant [33]

1992

15

Lahiri and Sengupta [35]

1997

10

Malakar and Dhar [36]

1999

10

Lahiri et al. [14]

2005

12

MPS: maximum pigment spread.

be between 2 and 6 weeks in different studies. After minigrafting and PUVASOL, appearance of repigmentation time in different regions varied between 14 and 39 days, with an overall average being approximately 21.6 days as shown in one study [35]. With the deployment of NB-UVB along with minigrafting appearance of repigmentation time in different regions varied between 14 and 32 days, with an overall average being approximately 20.6 days [14]. Orentriech in his original article (1972) observed that whether 1- or 2-mm grafts were employed, the pigment spread was consistently 1 mm [3]. Various other results can be found in the literature. (Table 11.1). The pigment spread may vary in patients with different skin types, probably more in those with darker skin types. Falabella while establishing a relationship between donor graft and the area of surgical repigmentation, found that a 1-mm donor graft can repigment an area 25 times larger than the graft itself [27]. In a recent study with 1.5-mm grafts and NB-UVB in skin type IV and V subjects this value was found to be more than double of that (56.21 times) [14]. Previously in one study with PUVASOL this relationship was found to be 42 times [40]. Darker skin types and deployment of NB phototherapy all may have accounted for this high statistical value.

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Considering all the indicators and parameters, minigrafting is not only the easiest, safest, and least expensive method, but it is one of the most effective treatment options in treating stable and recalcitrant vitiligo.

References 1 Behl PN. Homologous thin Thiersch’s grafts in treatment of vitiligo. Curr Med Pract 1964;8:218–21. 2 Falabella R. Epidermal grafting: an original technique and its application in achromic and granulating areas. Arch Dermatol 1971;104:592–600. 3 Orentriech N, Selmanwitz VJ. Autograft repigmentation of leucoderma. Arch Dermatol 1972;105:784–6. 4 Falabella R. Repigmentation of leucoderma by minigrafts of normally pigmented, autologous skin. J Dermatol Surg Oncol 1978;4:916–19. 5 Falabella R. Repigmentation of segmental vitiligo by autologous mimigrafting. J Am Acad Dermatol 1983; 9:514–21. 6 Lerner AB, Halaban R, Klaus SN, et al. Transplantation of human melanocytes. J Invest Dermatol 1987;89: 219–24. 7 Lerner AB. Repopulation of pigmented cells in patients with vitiligo. Arch Dermatol 1988;124:1701–2. 8 Falabella R, Escobar C, Borrero I. Treatment of refractory and stable vitiligo by transplantation of in vitro cultured epidermal autografts bearing melanocytes. J Am Acad Dermatol 1992;26:230–6. 9 Skouge JW, Morison WL, Diwan RV, Rotter S. Autografting and PUVA. A combination therapy for vitiligo. Dermatol Surg Oncol 1992;18:357–60. 10 Hann SK, Im S, Bong HW, Park YK. Treatment of stable vitiligo with autologous epidermal grafting and PUVA. J Am Acad Dermatol 1995;32:943–8. 11 Na GY, Seo SK, Choi SK. Single hair grafting for the treatment of vitiligo. J Am Acad Dermatol 1998; 38:580–4. 12 Malakar S, Dhar S, Malakar RS. Repigmentation of vitiligo patches by transplantation of hair follicles. Int J Dermatol 1999;38:237–8. 13 Achauer BM, Le Y, Vander Kam VM. Treatment of vitiligo with melanocytic grafting. Ann Plast Surg 1994;33:644–6. 14 Lahiri K, Malakar S, Sarma N, Banerjee U. Repigmentation of vitiligo with punch grafting and narrowband UV-B (311 nm) a prospective study. Int J Dermatol 2006;45:649–55.

15 Thompson CJS. The evolution and development of surgical instruments. IV. The trepan. Br J Surg 1937;25:726. 16 Watson BA. Gunpowder disfigurements. St. Louis Med Surg J 1878;35:145. 17 Keyes EL. The cutaneous punch. J Cutan Genitourin Dis 1887;5:98. 18 Hagerman D, Wilson H. The skin biopsy punch: evolution and modification. Cutis 1970;6:1139. 19 Stegman SJ. Commentary: the cutaneous punch. Arch Dermatol 1982;118:943. 20 Baronio G. Degli innesti animali, Milano, 1804, Stamperia e Fonderia del genio. 21 Lewin ML, Peck SM. Pigment studies in skin grafts on experimental animals. J Invest Dermatol 1941;4:504. 22 Labuono P, Shatin H. Transplantation of hair bulbs and melanocytes into leucodermic scars. J Dermatol Surg Oncol 1976;2:53–5. 23 Falabella R. Repigmentation of leucoderma by minigrafts of normally pigmented, autologous skin. J Dermatol Surg Oncol 1978;4:916–18. 24 Falabella R. Repigmentation of segmental vitiligo by autologous minigrafting. J Am Acad Dermatol 1983; 9:514–21. 25 Behl PN. Repigmentation of segmental vitiligo by autologous minigrafting. J Am Acad Dermatol 1985; 12:118–19. 26 Falabella R. Reply. J Am Acad Dermatol 1985;12:119. 27 Falabella R. Repeigmentation of stable leucoderma by autologous minigrafting. J Dermatol Surg Oncol 1986;12:172–9. 28 Falabella R. Post dermabrasion leucoderma. J Dermatol Surg Oncol 1987;13:44–8. 29 Falabella R. Treatment of localized vitiligo by autologous minigrafting. Arch Dermatol 1988;124:1649–55. 30 Falabella R, Barona M, Escobar C, et al. Surgical combination therapy for vitiligo and piebaldism. Dermatol Surg 1995;21:852–7. 31 Westerhof W, Boersma B, et al. Grafting techniques in leucoderma. Book of Abstract. 7th International Congress of Dermatology, New Delhi, India. 1994;15. 32 Boersma BR, Westerhof W, et al. Repigmentation in vitiligo vulguris by autologous minigrafting: results in nineteen patients. J Am Acad Dermatol 1995;33: 990–5. 33 Savant SS. Autologous miniatures punch grafting in vitiligo. Ind J Dermatol Venereol Leprol 1992;58: 310–14. 34 Malakar S. Punch grafting. In: An Approach to Dermatosurgery, 1st edn. Calcutta: A Paul, 1996;44–6. 35 Lahiri K, Sengupta SR. Treatment of stable and recalcitrant depigmented skin conditions by autologous punch grafting. Ind J Dermatol Venereol Leprol 1997;63:11–14.

Minigrafting for vitiligo 36 Malakar S, Dhar S. Treatment of stable and recalcitrant vitiligo by autologous miniature punch grafting: a prospective study of 1,000 patients. Dermatology 1999;198:133–9. 37 Lahiri K, Sengupta SR. A regionwise comparative study of post punch graft appearance of repigmentation (AOR) time in cutaneous achromia. Indian J Dermatol 1998;43:13–15. 38 Lahiri K, Sengupta SR. A clinico-microscopic corroboration of surgical repigmentation – a study of 30 cases. Indian J Dermatol 1998;43:99–101. 39 Lahiri K, Sengupta SR. A regionwise comparative study of the extent of post punch graft surgical repigmentation in cutaneous achromia. Ind J Dermatol Venereol Leprol 1998;64:173–5. 40 Lahiri K, Sengupta SR. Relationship between donor graft area and area of surgical repigmentation. Indian J Dermatol 1999;44:11–14. 41 Falabella R, Arrunategui A, Barona MI, Alzate A. The minigrafting test for vitiligo: detection of stable lesions for melanocyte transplantation. J Am Acad Dermatol 1995;32:228–32. 42 Malakar S, Lahiri K. Punch grafting for lip leucoderma. Dermatology 2004;208:125–8. 43 Malakar S, Lahiri K. Electrosurgery in cobblestoning. Indian J Dermatol 2000;45:46–7. 44 Malakar S, Dhar S. Repigmentation of leucotrichia over vitiligo patches after punch grafting. Indian J Dermatol Venereol Leprol 1998;64:252–3. 45 Malakar S, Dhar S. Rejection of punch grafts in three cases of herpes labialis induced lip leucoderma, caution and precaution. Dermatology 1997;195:414. 46 Malakar S, Dhar S. Acyclovir can abort rejection of punch grafts in herpes-simplex induced lip leucoderma. Dermatology 1999;199:75. 47 Malakar S, Lahiri K. Successful repigmentation of six cases of herpes labialis induced lip leucoderma by micropigmentation. Dermatology 2001;203:194. 48 Lahiri K, Malakar S. Herpes simplex induced lip leucoderma: revisited. Dermatology 2004;208:182. 49 Burge S, Rayment R. Free skin grafts. In: Simple skin Surgery, 1st edn. Bombay: Blackwell Scientific Publications, 1986:71–84. 50 Parrish JA, Fitzpatrick TB, Shea C, et al. Photochemotherapy of vitiligo: use of orally administered

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psoralen and a high-intensity long-wave ultraviolet light system. Arch Dermatol 1976;112:1531–4. Ortonne JP, Schmnitt D, Thivolet J, et al. PUVA induced repigmentation of vitiligo, scanning electron microscopy of hair follicles. J Invest Dermatol 1980;74:40–2. Suvanprakorn P, Dee-Analap S, Pongsomboon CH, et al. Melanocyte autologous grafting for treatment of leukoderma. J Am Acad Dermatol 1985;13:968–74. Billingham RE, Silvers WK. Studies on the migratory behaviour of melanocytes in guinea pig skin. I. J Exp Med 1970;131:101–17. Das SS, Pasricha JS. Punch grafting as a treatment for residual lesions in vitiligo. Ind J Dermatol Venereol Leprol 1992;58:315–19. Jha AK, Pandey SS, Shukla VK. Punch grafting in vitiligo. Ind J Dermatol Venereol Leprol 1992;58:328–30. Singh KG, Bajaj AK. Autologous miniature skin punch grafting in vitiligo. Ind J Dermatol Venereol Leprol 1995;61:77–80. Malakar S, Lahiri K. How unstable is the concept of stability in surgical repigmentation of vitiligo? Dermatology 2000;201:182–3. Lahiri K, Malakar S, Banerjee U, Sarma N. Clinicocellular stability of vitiligo in surgical repigmentation: an unexplored frontier. Dermatology 2004;209:170–1. Lahiri K, Malakar S. Inducing repigmentation by regrafting and phototherapy (311 nm) in punch failure cases of lip vitiligo – a pilot study. Indian J Dermato Venereol Leprol 2004;70:156–8. Malakar S, Dhar S. Spontaneous repigmentation of vitiligo patches distant from the autologous skin graft sites: a remote reverse Köebner’s phenomenon? Dermatology 1998;197:274. Malakar S, Lahiri K. Spontaneous repigmentation in vitiligo: why it is important. Int J Dermatol http:// www.blackwell-synergy.com/doi/abs/10.1111/j.136 5–4632.2005.02657.x Malakar S. Spontaneous repigmentation of vitiligo patches other than the grafted site. Indian J Dermatol 1997;47:68–70. Falabella R. Surgical treatment of vitiligo: why, when and how (Editorial). J Eur Acad Dermatol Venereol 2003;17:518–20. Malakar S, Lahiri K. Electrosurgery in cobblestoning. Indian J Dermatol 2000;45:46–7.

CHAPTER 12

Suction blister epidermal grafting Somesh Gupta and Ashima Goel

Introduction Most melanocyte replenishment techniques using non-cultured melanocyte-bearing donor skin involve transplantation of dermo-epidermal grafts (e.g. miniature punch, thin Thiersch’s, and pinch) with the exception of epidermal grafts obtained by suction blistering. The dermis as a whole has a well-known regulatory influence over epidermal morphogenesis and differentiation. Studies using embryonic tissue recombinants prepared by annealing the epidermis from one source (e.g. age, species, or region) with a dermis from another, which were implanted and allowed to differentiate in organ culture or grafted on nude mice, have shown that the morphological characteristics (thickness, architecture, and pattern of differentiation) of the epidermis conform to the region of the body from which the dermis was obtained [1]. Thus dermo-epidermal grafts retain characteristics of their site of origin, and this may lead to mismatch in the texture and thickness with the surrounding skin; however, pure epidermal grafts (in the absence of donor dermal tissue) adopt most of the characteristics of the recipient area. Obviously, among all non-culture transplantation techniques, the most satisfactory aesthetic results are likely to be obtained with pure epidermal grafts and melanocyte resuspension procedures [2]. Presently the only reliable technique to harvest “pure” epidermal grafts is through suction blistering [3,4]. Light and electron microscopic studies have shown that in suction blisters, cleavage most often takes place between the base of the basal cells and the basal lamina, so that the latter usually remains on the floor of the blister. There is some loss of melanocytes in suction blistering and the number of melanocytes in blistered epidermis is only about half of the normal number for that site. Occasionally, keratinocytes

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and melanocytes are seen in the blister fluid or lying on the floor of the blister, which is partly responsible for the loss [5]. Melanocytes in blistered epidermis are also depleted around the hair follicles.

Suction blistering Some 125 years ago Unna observed and documented for the first time that blisters could occasionally be produced by certain types of dry cupping on intact skin [6]. The first in vitro separation of epidermis from dermis by the use of suction was achieved by Blank and Miller [7] in 1950. The first in vivo induction of blister was reported by Slowey and Leider [8] in 1961 and subsequently in 1964 by Kiistala and Mustakallio [9,10]. Falabella [11] first used this technique for transplantation of viable epidermis in achromic lesions. Several years later, in 1985, Suvanprakorn et al. [12] reported successful transplantation of epidermis obtained through suction blistering for repigmentation of lesions of vitiligo. Over the years, various types of suction apparatus have been developed including the angiosterrometer [9], the conventional respiratory or gastrointestinal suction pump [11], the oil rotatory pump [13], and manually operated suction unit [14]. The initial suction blister device consisted of a motor-driven suction pump with cups, vacuum bottle, and a mercury manometer, which was expensive, heavy, and cumbersome. Various modifications of the suction blister device are now available. They are all aimed at simplifying the apparatus and the procedure. Of great significance was the modification made by Mukhtar et al. [15] in which they used disposable syringes for producing the blister. They cut the needle end of a 10-ml syringe and introduced the plunger through this end. To generate negative pressure they

Suction blister epidermal grafting pulled the plunger which was held in position by a small metal rod. This was an extreme simplification of the device. However, they had erroneously concluded that the height of the column of vacuum within the syringe had no effect on the magnitude of the suction generated, such that the final height of the piston is arbitrary. Lewis [16] objected to this by applying Boyle’s law. He suggested that the height of the piston within the syringe is directly related to the pressure gradient thereby generated. Therefore in the simple syringe introduced by Mukhtar et al. [15], the negative pressure inside the syringe could not be measured. To overcome this drawback, Gupta et al. [17] developed a modified suction blister device consisting of a disposable syringe of 5, 10, or 20 ml attached to a three-way tap with a latex rubber tube (taken from a drip set), a 50-ml syringe for aspiration of air to produce the vacuum, and a manometer to measure the negative pressure (Fig. 12.1). The vacuum was produced by connecting the suction syringe end of three-way tap and 50-ml syringe end by changing the position of three-way stopcock and pulling the plunger of the 50-ml syringe. The pressure was measured by connecting the suction syringe end and manometer end by altering the position of the stopcock. Once adequate suction was achieved, the suction syringe end was locked and 50-ml syringe and the manometer could be removed because no further pressure monitoring was essential after that. Syringe-induced blister formation in a given patient is mostly dependent on the anatomic site, the pressure gradient generated, and the length of time the gradient is applied. Subsequently, in another study, Gupta et al. [18] calculated the column of vacuum or the amount of aspirated air that is necessary for the production of a specific pressure gradient. Therefore the pressure could be known by measuring the volume of aspirated air and there is no need of a manometer (Fig. 12.2). The ideal pressure gradient required to produce suction blisters varies according to age of the patient. For example, for an adult,
400 mmHg is appropriate, whereas an elderly patient with age-related weakening of dermo-epidermal junction (DEJ) requires less (
300 mmHg) pressure. Conversely, an adolescent has stronger DEJ and requires higher negative pressure (
500 mmHg).

97

Fig. 12.1 Suction syringe with manometer to measure

the pressure (reproduced from Gupta et al. [18]).

Fig. 12.2 Suction syringe without a manometer that can

be used in conjunction with reference to Table 12.1 (reproduced from Gupta et al. [18]).

Syringes of 5, 10, or 20 ml can be used for producing the blisters of various sizes according to the need. Generally, 10-ml syringes are the most suitable (Fig. 12.3A, B). The mean amount of air that needs to be aspirated from the syringes of various sizes to produce the desired pressure and their confidence intervals are given in Table 12.1. In a larger syringe (20 ml), application of higher pressure (
500 mmHg) can lead to formation of hemorrhagic or defective blisters. Hence this is not included in reference table. Suction blisters were raised over the lateral aspect of the thigh. Burm et al. [19] developed another suction device consisting of only two units: a portable suction pump and suction cups containing an independent one-way check valve. The suction pump is commercially available (Medi-Pump, Model 1130D,

98

Chapter 12 Table 12.1 Reference for required volume of air to

be aspirated from the syringe to achieve a particular pressure (adapted from Gupta et al. [18]).

Size of suction syringe (ml)

(A)

(B) Fig. 12.3 (A) Suction syringes in place. (B) Unilocular

suction blisters.

Volume of aspirated air to achieve
300 mm

Volume of aspirated air to achieve
400 mm

Volume of aspirated air to achieve
500 mm

5

8.7  9.4

12.9  2.1

19.4  2.3

10

13.9  2.7

20.4  3.2

30.3  3.2

20

24.6  4.2

38.4  6.7

–

within each cup reaches up to 250–300 mmHg. Small vesicles begin to appear 1–11⁄2 hours later, which coalesce to form a single bulla 3–4 hours later (Fig. 12.4B). The maximum size of a harvested epidermal sheet is approximately 1.7 cm in diameter for small cup and 2.5 cm for a large one. A negative pressure cutaneous suction chamber system (NPCSCS, Electronic Diversities, MD, USA) has been developed to induce blisters rapidly (Fig. 12.5) [20]. The apparatus consists of temperature-controlled suction chambers connected to a control unit incorporating a diaphragm pump, which can generate up to
508 mmHg of pressure. A slightly higher (40°C) temperature facilitates blister formation. In this device heat is produced by applying a low a.c. voltage to incandescent lamps within the chambers. Each of the chambers is attached to an orifice plate that has multiple openings. This plate is applied directly to the subject’s skin and secured in place by a belt placed around the limb/trunk [21]. The combination of high negative pressure and thermal regulation allows for rapid blister formation.

Suction blister induction time Tomas Ind., Inc., USA) and contains a manometer to permit readings up to 760 mmHg. The suction cups are cylindrical plastic cups with a one-way check valve (Fig. 12.4A). These are available in two sizes: 15 and 22 mm in inside diameter. Suction cups are placed on the donor skin, which is moisturized with tap water. These cups are individually connected with every suction pump until the negative pressure

In various studies, the reported time for suction blistering varies widely between 15 minutes to more than 3 hours [13,21–24]. Various factors that can affect suction blister induction time (SBIT) [24] are discussed as below: 1 Diameter of the cup: Gupta and Kumar [24] showed in their study that the average SBIT was about 3 times more with the 50-ml syringe than the

Suction blister epidermal grafting

99

(A)

(B) Fig. 12.4 (A) Suction cups containing an independent one-way check valve. (B) Unilocular suction blisters. (Courtesy:

Dr. Jin Sik Burm, South Korea.)

250 208 SBIT (min)

200 150

123 98

100 63

72

50 0 1

1.4 1.7 2.2 3.2 Diameter of the suction syringe

Fig. 12.6 Relationship of mean SBIT and diameter of

suction syringe in five subjects (based on data from Gupta and Kumar [24]).

Fig. 12.5 Negative pressure cutaneous suction chamber

system.

2-ml syringe. The graphical presentation suggested a directly proportional increase in the SBIT with an increase in the diameter of the suction syringe (Fig. 12.6). They found that 10- and 20-ml syringes are appropriate for grafting procedures, as they produce sufficiently large blisters in a reasonable time (1–2.5 hours). Large syringes (50 ml) take more time, but the large roof of the blister can be used for grafting of a single patch of 3.5–4 cm in size. Very

small blisters (induced by 5- or 2-ml syringes) can be generated quickly, and are useful in experiments and in grafting of smaller geographical extensions of achromic areas in to the pigmented skin. However, such small blisters are difficult to handle. 2 Site of suction blistering: Falabella [11] used the inner aspect of the thigh as the suction blister site in his original study. Others have used the inner portion of the arm, the abdomen, buttocks, and the extensor surface of thigh [25,26]. Later, however, it was observed that more rapid induction of blisters could be achieved on the skin over a hard or firm base, like bony prominences [13]. Close proximity of the skin to the bone and making the skin tense help in reducing SBIT [17]. The skin overlying the shin is a good site for rapid induction

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Chapter 12

of suction blisters [13], because in addition to the close proximity to the bone, its distensibility and elasticity are also lower. However, as there is a small but definite risk of Köbnerization, it is recommended to choose a covered site like skin overlying greater trochanter of femur. 3 Age of the subject: Experiments have shown that the DEJ weakens as the person grows older [27]. There is progressive decrease in the vertical resistance at the DEJ. This results in easier and faster separation of the DEJ by means of suction in older subjects [27] and SBIT is usually inversely proportional to the age of the individual. 4 Negative pressure: Various investigators have used a negative pressure ranging from
200 to
500 mmHg [13,26,28]. Gupta and Kumar [24] observed in their study that for suction syringes or cups with larger diameter, the ideal negative pressure is toward the lower side (i.e.
300 to
400 mmHg). Very high pressure (
500 mmHg) in a large suction area often results in bruising and failure of blister formation [14]. In suction syringes or cups of lesser diameter (1 cm), the negative pressure can be increased up to
500 mmHg to expedite the process of suction blistering [20]. 5 Temperature: van der Leun et al. [29] first reported the facilitating effect of heat on the suction blister formation. The optimum temperature in the suction area is about 40–45°C [17,23]. 6 Intradermal injection of saline: The normal saline injected in the suction area not only acts as a local anesthetic, but also reduces SBIT, probably by acting as a tissue expander [25,26]. Intradermal edema produced by saline hastens the accumulation of fluid at the DEJ. 7 Other maneuvers: Pre-treatment of the suction blister site with topical psoralen plus ultraviolet A (PUVA) for about 2 weeks reduces the extensibility of the skin by causing photosclerosis and making the base firm, thereby reducing SBIT. It also stimulates melanogenesis in the donor epidermis and helps in achieving better pigmentation in the grafted achromic area [26]. We have observed that once the small vesicles have appeared in the suction area, increasing the negative pressure by another
50 mmHg speeds up the process without causing hemorrhages in the suction area.

8 Pathological variations: Diseases which increase the skin extensibility (ability to be elongated), like cutis laxa and Ehlers–Danlos syndrome, may require a longer time for suction blister induction. Corticosteroid atrophy may reduce the SBIT by weakening the DEJ. 9 Individual variations: Finally, there is a great person-to-person variation in SBIT. We observed in one of our patients (a 23-year-old woman), while applying five suction cups in series, the blister in the first cup had appeared by the time we had applied suction in the fifth cup (unpublished observation). This observation defied all the abovedescribed principles of SBIT. In an occasional patient, blisters may not form at all, though it is rare. In summary, use of 10- or 20-ml syringes as suction cups and a negative pressure of
400 mmHg is an appropriate choice for suction blistering, which requires about 1.5–2.5 hours for formation of suction blisters. Some additional measures may reduce the SBIT to about 1–1.5 hours.

Harvesting the grafts After a unilocular blister is ready, the periphery of the blister is cut with a curved iris scissors, except a small arc is left attached to help identifying the dermal and epidermal side. Due to the flimsiness of the epidermal sheet and its tendency to curl toward either surface (dermal or epidermal), extra care has to be taken in harvesting it. In such thin grafts both surfaces are difficult to distinguish and application of an upside-down graft with epidermal surface facing the graft bed will invariably result in failure of repigmentation. One edge of a sterile glass slide, smeared with an antibiotic ointment, is kept near the blister. A pair of fine tipped or Jeweler’s forceps is placed beneath the graft. The graft is lifted gently and is everted on the glass slide with the dermal side facing upwards. A fine gauze [30] and acetate sheet (transparency sheets used in overhead projectors) [31] are other alternatives for graft carrier. The ideal material for transferring epidermal grafts should be firm yet flexible and transparent. The acetate sheets fulfill these criteria [31]. Once the graft is transferred on the carrier, the remaining attached arc is also cut. The fibrin clot

Suction blister epidermal grafting

101

attached to the graft is removed and the graft is spread to its full size.

Preparation of the recipient site Recipient site preparation can be done with one of the following methods: 1 Dermabrasion: Dermabrasion is simply the controlled mechanical abrasion of the epidermis and a variable segment of the upper dermis. There are a variety of abrasive instruments available to perform dermabrasion (e.g. wire brush, diamond fraise, curette, manual dermabrader, sandpaper, and others) [32]. The instruments can be hand-held or power driven. Diamond-impregnated cylindrical fraises are preferred over wire brushes as the latter cause more deeper destruction than required for transplantation procedures for leukoderma/vitiligo. Hand-held micromotor is available which is shock-proof and has better control. The variable speed of the motor is controlled by a foot pedal rheostat. The heads are driven at rotational speed between 12,000 and 15,000 rpm to obtain slow, gradual controlled denudation [33]. For smaller areas or areas with thinner epidermis, such as eyelids, manual dermabraders are more useful. These are available in different sizes and shapes (Fig. 12.7). Sandpaper is another alternative. To avoid scarring at recipient site, dermabrasion should be stopped on appearance of fine bleeding points (Fig. 12.8). The disadvantage of the procedure is the risk of transmission of human immunodeficiency virus and hepatitis viruses to the operating room staff from airborne and aerosolized particles during the procedure. Appropriate protective measures like gloves, gowns, masks, and protective eyewear reduce the risk. Manual dermabraders are safer. The advantage of dermabrasion lies in its simplicity. There is no lag period of days unlike in liquid nitrogen and phototoxic blisters. 2 Suction blistering: Denuding of recipient vitiliginous patch can be done by producing suction blisters. The blisters are produced by the same technique as described for the donor site. The advantage is that only epidermis is removed and there is no risk of scarring. However, it is not always possible to

Fig. 12.7 Manual dermabraders.

Fig. 12.8 Appropriate depth of superficial dermabrasion

of vitiliginous recipient area.

produce suction blisters on skin over curved surfaces, eyelids and lips, and on the skin covering loose connective tissue. The roof of recipient site blister can be used as an autologous biological dressing. It is elevated like a flap, the denuded area is covered with grafts and then flap re-placed to cover the grafts. The periphery of the flap is sealed with tissue glue (cyanoacrylate). This retains the moisture, reduces chances of infection, and helps in faster healing [34]. There is no risk of dressing adhering to the grafts, leading to detachment. 3 Liquid nitrogen-induced blisters: These can be produced by freezing the recipient site by liquid nitrogen. Liquid nitrogen is applied using a large cotton applicator for 5–10 seconds, usually 2–3 days prior to

102

Chapter 12

the date of surgery. The entire area should be treated with slightly overlapping margins of the frozen sections in order to ensure adequate blistering [35]. Excessive freezing should be avoided as it may lead to failure of graft uptake and later scarring. Blisters will develop within few hours through the separation of epidermis from dermis, though grafting is performed 48 hours later when edema and inflammation subside. The blisters are left intact until the day of grafting and just before grafting, the depigmented bullous epidermis is unroofed with scissors and wound bed (floor of the blister) is cleaned. If blistering does not occur, it can be produced with 15–20 minutes of suction [35]. Although liquid nitrogen is more convenient than suction blister at recipient sites, it can cause inflammation and subsequent hypertrophic scarring and post-inflammatory hyperpigmentation [36]. Sometimes it is associated with perilesional hypopigmentation [37]. 4 Laser ablation: Lasers can be used to de-epithelialize the recipient site in vitiligo. A variety of lasers have been reported to be suitable for this. These include continuous wave, pulsed, and ultrapulse CO2 laser, and Er:YAG (erbium:yttrium– aluminum–garnet) laser (see Chapter 34 for more details). Laser ablation has several advantages over other methods: it is a faster procedure, there is less thermal damage to the perilesional normal skin and de-epithelialization can precisely be done on irregular shapes and surfaces [36]. However, it adds significantly to the overall cost of the procedure. 5 Phototoxic blisters: The lesions are painted with 0.75% 8-methoxypsoralen. The surrounding skin is protected with a physical sunscreen. The site is then exposed to 15 J/cm2 of UVA. The procedure is repeated after 4 hours. The lesional blisters develop in about 24 hours [38]. This technique is simple. It removes only epidermis. There is little or no bleeding. As only depigmented skin blisters, it enables easy demarcation of margins [13,37].

epidermal sheets from the recipient bed and interfere with the effective transfer of melanocytes from grafted epidermis. Epidermal grafting after hair plucking is time consuming and may show uneven results because of unremoved hairs [25,39,40]. Hence chemical epilation may be a better option than plucking or shaving. It is readily available, simple, painless to use, comfortable, safe, and effective. It does not disturb the normal hair growth [41]. The most commonly used chemical depilatories are mercaptans, particularly salts of thioglycolic acid. Thioglycolate depilatory works by hydrolyzing disulfide bonds in cysteine, which maintains the structural integrity of hair, to turn hair into a jelly-like consistency [41]. The most common side effect of thioglycolate depilatory is irritant dermatitis (1–5%), which can be controlled by decreased application or by a different mode of application. The effect of a chemical depilatory lasts up to 2 weeks, which is enough for successful “take” of epidermal grafts.

Transfer of grafts and dressing The grafts are transferred on the recipient area with dermal side facing toward the graft bed. Generally grafts are placed 0.5–1 cm away from each other as pigment cells from the grafts migrate to cover the intergraft area. The area is covered with a non-adherent antibiotic gauze (Sofra tulle®, Aventis Pharmaceuticals, Mumbai, India). The skin over joints should be immobilized using an adhesive plaster. Sometimes a splint is needed on the joints like ankle (Fig. 12.9). The dressing is removed on the 8th day.

Chemical epilation before epidermal grafting on hair-bearing skin When epidermal grafting by suction blister is done on a hairy area such as the scalp and eyebrows, underlying hairs grow so fast as to raise the grafted

Fig. 12.9 A splint is needed on the joints for immobiliz-

ing the area to prevent the displacement of grafts.

Suction blister epidermal grafting

Course of grafts Grafts are taken up by 8–10 days. The grafted area looks mostly hyperpigmented, due to the epidermal cyanosis resulting from the lack of associated dermis and thus its blood supply. Some of the areas, however, resemble normal skin color while the areas in-between adjacent grafts still appear depigmented. Gradually, over the next 2–3 months, these intergraft depigmented tissues become pigmented while the grafted area becomes lighter in color, so as to finally match the surrounding skin color. In most areas, the grafts detach in about 1–2 weeks time after the removal of dressing; however, this usually does not affect the outcome adversely [42]. During the period of contact between grafts and the recipient bed, melanocytes migrate to the depigmented recipient skin resulting in pigmentation. Therefore,

103

the procedure is actually “melanocyte transfer” and epidermis obtained through suction blistering is only a carrier [43]. The pigment spreads from the graft to the surrounding area and up to 46% increase in the pigmentation has been recorded [44]. Maximum pigmentation generally occurs within first 3–4 months [44].

Efficacy and safety of epidermal grafting Epidermal grafting is one of the most effective methods among all transplantation procedures (Figs 12.10–12.13; Plate 12.1, facing p. 114) [22]. A large patient series has revealed an overall success rate of 64% in all patients, 91% in patients with segmental/ focal vitiligo, and 53% in patients with generalized

(A)

(B) Fig. 12.10 (A) A segmental vitiligo on face. (B) Two years after epidermal grafting.

(A)

(B) Fig. 12.11 (A) Acral vitiligo 2 weeks after epidermal grafting. Note faint pigmentation appearing in the grafting area. (B) One year post-transplantation, nearly complete repigmentation.

104

Chapter 12

(A)

(B) Fig. 12.12 (A) Lip-tip vitiligo on areola of the breast. (B) More than 90% repigmentation 2 months after epidermal

grafting.

(A)

(B) Fig. 12.13 (A) A focal vitiligo on face. (B) Almost complete repigmentation 14 months after transplantation.

vitiligo [45]. A meta-analysis of the literature, published in the same article, has shown an overall success rate of 87% in all patients, 88% in patients with segmental/focal vitiligo, while 61% in patients with generalized disease (Fig. 12.14) [45]. The success rate was higher in patients younger than 20 years than in those aged 20 years or more. There was no statistically significant difference in outcomes on different body areas. The most common adverse effect reported was hyperpigmentation at both donor and recipient site (Fig. 12.15). This is probably more common in patients with skin phototypes IV–VI. Scarring at donor or recipient sites is very rare. No serious side effects have been reported to date.

Advantages and limitations Epidermal grafting is an easy, safe, inexpensive, and effective treatment option for patients with stable

vitiligo. The results are rapid. The biggest advantage of the procedure is that it is a “scarless” surgery. Both recipient and donor sites heal without the slightest scarring. We have been using the same donor area for several sessions of the procedure. Being a purely epidermal graft, there is no stuck-on effect. Color and texture match with the surrounding skin is generally good. The advantage over splitthickness graft is that there is no shrinkage of the graft as “pure” epidermal grafts are devoid of dermal elastic tissue. There is no inward rolling of the margins, as seen in split-thickness grafts. Peripheral beading and depigmented halo, commonly seen with split-thickness grafts [46], are unusual. On the other hand, the procedure gives considerably superior results to minigrafting, as the latter is often associated with cobblestoning. In one comparative study, the success rate of suction blister epidermal grafting (SBEG) was 82% while that of minigrafting

Suction blister epidermal grafting

100

100 91 90

92

88 79

Success rate (%)

80 66

70

87

81

82

60

105

67 71

68

61

61

58

64

56

53

50 40 30 20 10

Type of the disease

Age of the subject

Lower limbs

Upper limbs

Trunk

Head and neck

20 years

20 years

Focal/ segmental

Vulgaris

0

Site of grafting

Gupta and Kumar

All patients

Literature

Fig. 12.14 Success rates of epidermal grafting in a study by Gupta and Kumar (n  117) and a meta-analysis of literature (n  301) (based on data from [45]).

100

100 90

70 60 50 32

30

24

17 6 0

1 1

Recipient area Gupta and Kumar

0 Hyperpigmentation

0

Scarring/keloid formation

0 0.6

Contact dermatitis

10

Infection

20

1.7

Köbner phenomenon

40

Hyperpigmentation

Incidence (%)

80

Donor area Literature

Fig. 12.15 Adverse effects of epidermal grafting as reported in a case series by Gupta and Kumar (n  117) and a meta-analysis of literature (n  462) (based on data from [45]).

106

Chapter 12

was only 67%, though this difference was not statistically significant [2]. A study comparing epidermal grafting with transplantation of cultured autologous melanocytes found no difference in the outcome in both the groups [47]. The author of this study considered epidermal grafting advantageous over cultured melanocytes transplantation because of its simplicity [47]. Epidermal grafting is a tissue grafting procedure that has all the merits of autologous cellular transplantation without the disadvantages due to procedural complexities of “pure” cultured melanocyte or non-cultured melanocyte–keratinocyte cell suspension transplantation. Furthermore, it does not have the limitations of other tissue grafts (minigrafts, thin split-thickness grafts, and pinch grafts), which often lead to mismatch in texture and color, and may not be acceptable on the cosmetically prominent sites such as eyelids and lips. SBEG has several limitations as well. Obtaining large epidermal sheets is not feasible with this technique; therefore, it requires several sessions to cover large areas. Handling of the grafts is difficult due to their extreme thinness. As the grafts are not affixed to the treated area, they may easily slip off. Donor site blistering requires few hours. Some patients complain of unbearable pain during the blistering, though most tolerate it well. Music or television in the procedure room helps divert the attention of the patient from the blistering process. The procedure is difficult to perform on certain areas, such as body folds, palms, and soles.

Conclusion SBEG is one of the simplest, although time consuming, surgical methods for treating stable vitiligo and other secondary leukodermas. It does not require special surgical skills and small areas can be easily treated by this method. Since the split is physiological, the graft obtained is very thin, with excellent cosmetic results especially on mucosae like lips. However, large areas will require more number of grafts and multiple sessions; hence, other surgical methods may be more useful in such circumstances. The desired future direction for this procedure is

the development of more effective equipments to raise large suction blisters, rapidly and reliably.

References 1 Chu DH, Hawke AR, Holbrook K, Loomis CA. The structure and development of skin. In: Freedberg IM, Eisen SAZ, Wolff K, Austin FK, Goldsmith LA, Katz SI, et al. (eds.) Fitzpatrick’s Dermatology in General Medicine, 6th edn. New York: McGraw-Hill, 2003;58–88. 2 Gupta S, Jain VK, Saraswat PK, Gupta S. Suction blister epidermal grafting versus punch skin grafting in recalcitrant and stable vitiligo. Dermatol Surg 1999;25:955–8. 3 Kiistala U. Suction blister device for separation of viable epidermis from dermis. J Invest Dermatol 1968;50:129–37. 4 Lee A-Y, Jang J-H. Autologous epidermal grafting with PUVA-irradiated donor skin for the treatment of vitiligo. Int J Dermatol 1998;37:551–4. 5 Hunter JAA, McVittie E, Comaish JS. Light and electron microscopic studies of physical injury to the skin. Br J Dermatol 1974;90:481–90. 6 Unna P. Zur anatomic der blasenbildung an der manschlichen haut. Vjschr Derm Syph 1878;5:1–4. 7 Blank H, Miller OG. A method for separating the epidermis from dermis. J Invest Dermatol 1950;15:9–12. 8 Slowey C, Leider M. Abstract of a preliminary report: the production of bulla by quantitated suction. Arch Dermatol 1961;83:1029–30. 9 Kiistala U, Mustakallio KK. In-vivo separation of epidermis by production of suction blisters. Lancet 1964; 1:1444–5. 10 Kiistala U, Mustakallio KK. Dermo-epidermal separation with suction: electron microscopic and histochemical study of initial events of blistering of human skin. J Invest Dermatol 1967;48:456–77. 11 Falabella R. Epidermal grafting: an original technique and its application in achromic and granulating areas. Arch Dermatol 1971;104:592–600. 12 Suvanprakorn P, Dee-Ananiap SP, et al. Melanocyte autologous grafting for the treatment of leukoderma. J Am Acad Dermatol 1985;13:968–74. 13 Koga M. Epidermal grafting using tops of suction blisters in the treatment of vitiligo. Arch Dermatol 1988; 124:1656–8. 14 Kiistala U. Suction blister device for separation of viable epidermis from dermis. J Invest Dermatol 1968; 50:129–37. 15 Mukhtar M, Singh S, Shukla VK, Pandey SS. Surgical pearl: suction syringe for epidermal grafting in vitiligo. J Am Acad Dermatol 1997;37:638–9.

Suction blister epidermal grafting 16 Lewis EJ. Mechanism of “suction”. J Am Acad Dermatol 1998;39:664. 17 Gupta S, Shroff S, Gupta S. Modified technique of suction blistering for epidermal grafting. Int J Dermatol 1999;38:306–9. 18 Gupta S, Ajith C, Kanwar AJ, Kumar B. Surgical pearl: standardized suction syringe for epidermal grafting. J Am Acad Dermatol 2005;52:348–50. 19 Burm JS. Simple suction device for autologous epidermal grafting. Plast Reconstr Surg 2000;106:1225–6. 20 Alexis AF, Wilson DC, Todhunter JA, Stiller MJ. Reassessment of the suction blister model of wound healing: introduction of a new higher pressure device. Int J Dermatol 1999;38:613–7. 21 Skouge JW, Morison WL, Diwan RV, Rotter S. Autografting and PUVA. J Dermatol Surg Oncol 1992; 18:357–60. 22 Njoo MD, Westerhof W, Bos JD, Bossuyt MM. A systematic review of autologous transplantation methods in vitiligo. Arch Dermatol 1998;134:1543–9. 23 Falabella R. Grafting and transplantation of melanocytes for repigmenting vitiligo and other types of leukoderma. Int J Dermatol 1989;28:363–9. 24 Gupta S, Kumar B. Suction blister induction time: 15 minutes or 150 minutes? Dermatol Surg 2000;26:754–6. 25 Hann SK, Im S, Bong HW, Park YK. Treatment of stable vitiligo with autologous epidermal grafting and PUVA. J Am Acad Dermatol 1995;32:943–8. 26 Suga Y, Butt KI, Takimoto R, Fujioka N, Yamada H, Ogawa H. Successful treatment of vitiligo with PUVApigmented autologous epidermal grafting. Int J Dermatol 1996;35:518–22. 27 Yaar M, Gilchrist BA. Aging of skin. In: Freedberg IM, Eisen AZ, Wolff K, Austen KF, Goldsmith LA, and Katz SI (eds.) Fitzpatrick’s Dermatology in General Medicine, 6th edn. New York: McGraw Hill, 2003;1386–98. 28 Yang JS, Kye KC. Treatment of vitiligo with autologous epidermal grafting by means of pulsed erbium: YAG laser. J Am Acad Dermatol 1998;38:280–2. 29 van der Leun JC, Beerens ED, Lowe LB. Repair of dermal–epidermal adherence: a rapid process observed in experiments on blistering with interrupted suction. J Invest Dermatol 1974;63:397–401. 30 Tang WY, De Han J, Lu NZ, Chan LY, Lo KK. Surgical pearl: fine gauze is a useful carrier for epidermal graft in the treatment of vitiligo by means of the suction blister method. J Am Acad Dermatol 1999;40:247–9. 31 Albert S, Shenoi SD. Acetate sheets in the transfer of epidermal grafts in vitiligo. J Am Acad Dermatol 2001; 44:719–20.

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32 Orentreich N, Orentreich DS. Dermabrasion as a complement to dermatology. Clin Plast Surg 1998;25:63–80. 33 Baker TM. Dermabrasion. As a complement to aesthetic surgery. Clin Plast Surg 1998;25:81–8. 34 Gupta S, Kumar B. Surgical pearl: autologous biological dressing for epidermal grafting in vitiligo and other achromic disorders. J Am Acad Dermatol 2003;48: 430–1. 35 Falabella R. Repigmentation of leukoderma by autologous epidermal grafting. J Dermatol Surg Oncol 1984;10:136–144. 36 Kim HY, Kang KY. Epidermal grafts for treatment of stable and progressive vitiligo. J Am Acad Dermatol 1999;40:412–7. 37 Oh C-K, Cha J-H, Lim J-Y, et al. Treatment of vitiligo with suction epidermal grafting by the use of an ultrapulse CO2 laser with a computerized pattern generator. Dermatol Surg 2001;27:565–8. 38 Albert S, Srinivas CR, Shenoi SD, et al. Phototoxic blister induction in vitiligo surgery. Br J Dermatol 1999;141:30–31. 39 Mutalik S, Ginzburg A. Surgical management of stable vitiligo: a review with personal experience. Dermatol Surg 2000;26:248–54. 40 Na GY, Seo SK, Choi SK. Single hair grafting for the treatment of vitiligo. J Am Acad Dermatol 1998;38: 580–4. 41 Kim C-Y, Yoon T-J, Kim T-H. Epidermal grafting after chemical epilation in the treatment of vitiligo. Dermatol Surg 2001;27:855–6. 42 Gupta S, Sandhu K, Kanwar AJ, Kumar B. Melanocyte transfer via epidermal grafts for vitiligo of labial mucosa. Dermatol Surg 2004;30:45–8. 43 Gupta S. Commentary on: epidermal grafting over the eyelid-special considerations. Dermatol Surg 2006;32: 391–2. 44 Tang WYM, Chan LY, Lo KK. Treatment of vitiligo with autologous epidermal transplantation using the roofs of suction blisters. Hong Kong Med J 1998;4: 219–24. 45 Gupta S, Kumar B. Epidermal grafting in vitiligo: influence of age, site of lesion, and type of disease on outcome. J Am Acad Dermatol 2003;49:99–104. 46 Malakar S, Malakar RS. Surgical pearl: composite film and graft unit for the recipient area dressing after split thickness skin grafting in vitiligo. J Am Acad Dermatol 2001;44:856–7. 47 Czajkowski R. Comparison of melanocyte transplantation methods for the treatment of vitiligo. Dermatol Surg 2004;30:1400–5.

CHAPTER 13

Thin split-thickness skin grafts for vitiligo Niti Khunger

Introduction Surgical treatment by autologous thin split-thickness skin grafts provides a therapeutic option for obtaining pigmentation in recalcitrant patches of vitiligo in a short period of time. Thin split-thickness skin grafts were first introduced by Ollier in 1872 and Thiersch in 1874 [1], and are now known after their names. Haxthausen [2], in 1947, transplanted Thiersch–Ollier grafts from normal to vitiliginous skin in three patients, however pigmentation persisted only in one. In 1964, Behl [3] treated 107 patients with vitiligo surgically using Thiersch–Ollier grafts. He obtained good results in 70% of patients with a complication rate of 19%. Subsequently there have been several reports of using thin split-thickness grafts for the surgical treatment of recalcitrant vitiligo [4–9].

Basic principles and biology of skin grafting In skin grafting, a section of the skin of variable size and thickness is detached from its blood supply and donor site and placed on a new recipient site. The skin graft can be either a split-thickness or a full-thickness graft. Split-thickness grafts are further classified into thin or Thiersch–Ollier (0.125–0.275 mm), intermediate or Blair–Brown (0.275–0.4 mm) and thick or Padgett (0.4–0.75 mm) split-thickness grafts [10]. The outcome of skin grafts depends on their thickness. In the surgical treatment of vitiligo, thin split-thickness (Thiersch–Ollier) grafts are used. There are three biological changes which follow skin grafting [1]: 1 Graft take adherence: The first phase begins with placement of the graft and continues for 72 hours.

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In this phase, the adherence of the graft is due to fibrin bonding and a process of plasmatic imbibitions nourishes the transplanted tissue. The graft appears pink. The first 72 hours are most crucial for graft uptake. Bleeding, infection, and mechanical movement due to improper immobilization can lead to graft failure at this stage. Therefore, strict immobilization in the first 3 days is essential. The second phase begins with the onset of vascular anastomosis and fibrovascular growth. 2 Graft revascularization: In this stage there is formation of endothelial buttons and then capillaries that rapidly penetrate into the deeper part of the graft. Connection of the graft and host vessels takes place. Insufficient vascular proliferation, development of a thick layer of fibrin or hematoma or seroma can lead to failure of graft uptake. 3 Contracture: There is initial contracture of the graft when it is harvested because of contraction of the elastin fibers. Contracture also occurs at the recipient site. These two factors may lead to perigraft halo and achromic fissures. Overlapping of graft edges at the recipient site and applying 1–2 mm larger graft than the recipient vitiligo patch can prevent these complications.

Selection of patient A careful selection of patients is important to achieve a successful result. The disease should be stable for at least 1 year. However there is no consensus regarding the duration of stable disease and according to different authors it varies from 6 months [11] to 2 years [12], but most authors report a duration of 1 year to be adequate [13–16]. Lahiri and

Thin split-thickness skin grafts for vitiligo Malakar [17] have raised questions regarding stability of the disease. They suggested that overdependence on the incidence of Köbner phenomenon or test grafting can sometimes be misleading and clinical stability of the disease may not correlate with stability at the cellular level. WankowiczKalinska et al. [18] reported that antimelanocyte cytotoxic reactivity was observed among CD8
perilesional T cell clones (TCC) in the biopsy of two patients with stable vitiligo suggesting that histoenzymological and ultrastructural analysis of perilesional and non-lesional skin of vitiligo patients would be useful to predict stability of disease. Unfortunately, no blood test is available to detect stability of disease. Olsson and Juhlin [12] have observed that the chances of recurrence or loss of pigmentation after transplantation are higher in patients with widespread, progressive vitiligo vulgaris and in those patients with hypothyroidism. Therefore, such patients should preferably not be selected for surgery until prognosis is clearly explained. A careful history and examination are the only means available at present to judge the stability of disease.

History There should be no new lesions or increase in size of the existing lesions for at least 1 year. There should be no history of recent Köbner phenomenon.

Examination Borders of the lesions should be well defined, hyperpigmented, or showing marginal repigmentation. Ill-defined borders merging with surrounding normal skin suggest that the lesion may be expanding. Lesions showing follicular repigmentation suggest a stable disease. There should be no evidence of Köbnerization. Segmental vitiligo is more stable as compared to vitiligo vulgaris [12,14]. Patients with stable, localized, or segmental lesions are the ideal candidates for surgery.

Counseling Proper counseling of the patient is very important before attempting surgical treatment. It should be emphasized that skin grafting cannot stop progression of the disease but only provides a pigmentary cover. The disease may recur. Immediate results

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may not be cosmetically acceptable and color matching with surrounding normal skin can take up to 1–6 months.

Method Harvesting the graft

Site The gluteal regions are the most preferred site for cosmetic reasons; however, posterior, lateral, anterior, and medial surfaces of the thigh are more suitable when a larger size graft is required. Arms have also been used as a donor site [5]. The donor site is shaved and, after surgical cleansing, the required area is marked with sterile marking ink or a surgical pen. At least a 10–25% larger area than the size of the vitiligo patch is marked, as there will be some contraction of the graft due to attached dermal elastic fibers.

Anesthesia Topical anesthesia using a mixture of lidocaine 2.5% w/w and prilocaine 2.5% w/w cream (EMLA® Astra, Zeneca, Sweden) under occlusion for at least 2 hours is usually sufficient [19]. After the cream is wiped off, a pin-prick test is performed to judge the degree of anesthesia. If the effect is not satisfactory, field block with 1% lignocaine at the margins of the donor area should be given. Infiltration anesthesia of the entire area must be avoided because it can result in a uneven surface of the skin leading to graft of uneven thickness.

Instruments The graft can be harvested freehand, using either a sterile razor blade mounted on a Kocher’s forceps or a blade-holding instrument. A silver knife, which holds a razor blade, is also useful to obtain smaller grafts [16]. The Humby knife, which has a roller mechanism, provides larger size grafts of consistent thickness (Fig. 13.1). With the Humby knife, the graft thickness can be controlled by adjusting the distance between the roller and the blade. The Humby knife can only be used on convex surfaces [20]. A motordriven Zimmer dermatome [7] (Zimmer Inc., Warsaw, IN, USA) can be used to obtain very thin grafts, depending on the expertise and availability (see Chapter 14 for more details).

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Fig. 13.1 Graft harvesting with Humby knife. (Courtesy:

Fig. 13.2 Fine bleeding pattern on donor area in thin

Somesh Gupta, MD, DNB, New Delhi, India.)

split-thickness graft. (Courtesy: Somesh Gupta, MD, DNB, New Delhi, India.)

Method The blade-holding instrument moves to and fro smoothly on the skin surface. Ideally, skin should not move with the knife. Lubrication with mineral oil helps in stabilizing the skin. An assistant applies counter-traction on the area in front of the knife by hand or using a wooden board, which also moves with the forward movement of the knife (Fig. 13.1). The skin is firmly stretched at one end by the assistant and a thin split-thickness graft is harvested. Some surgeons recommend application of a sheet of polyurethane on the donor skin to make donor skin more rigid and harvesting easier. This 0.05-mm thick sheet remains adhered to the graft and prevents wrinkling and curling of the edges. The harvested skin is immediately submerged in normal saline in a sterile Petri dish. The bleeding pattern on the donor area gives a fair idea of thickness of the graft. A thin graft would produce closely placed fine bleeding points (Fig. 13.2), while a thicker graft would produce sparsely placed, coarse bleeding points. The donor area is covered with a non-adherent dressing (framycetin tulle or Tegaderm® (3M, St. Paul, MN)) and a pressure dressing is given.

Preparing the recipient area The aim of preparing the recipient area is to denude the epidermis for proper graft uptake. Clinically, this level is achieved when pinpoint bleeding is seen. One study has shown that the depth to which the areas of vitiligo were excised influenced the final outcome of split-thickness grafting [21]. Optimum

results were obtained with excision of full-thickness skin, sparing subcutaneous fat, using a No. 15 scalpel blade. Loss of graft and cosmetic adverse effects like retention cysts and prominent edges were greater when only epidermis or epidermis with a variable thickness of dermis was dermabraded. Probably the residual dermis in the recipient area influences the melanocyte activity in the graft. However, these results need to be confirmed in a larger study. Özdemir et al. [16] found that thin split-thickness graft applied on a recipient site prepared with shaving with a silver knife was associated with higher rates of repigmentation (90%) than when thin splitthickness grafts were applied on recipient site prepared with suction blister method (65%). These two studies suggest that in thin split-thickness grafting for vitiligo, the recipient area needs deeper denudation and removal of epidermis alone does not give the optimum results. Although interesting, these observations in small groups of patients cannot be generalized.

Anesthesia Topical anesthesia using EMLA® cream is usually sufficient if it is applied under occlusion 2 hours before the procedure, depending on the area involved [19]. Infiltration anesthesia using 1% lignocaine without adrenaline may be used. For larger areas, the procedure can be done under general anesthesia. Preoperative medication with diazepam, 5 mg the previous night and 2 hours before the procedure may be given to an anxious patient.

Thin split-thickness skin grafts for vitiligo

Method After surgical cleansing, the area is first marked with a surgical pen. The commonest method used is to abrade the skin with a diamond fraise attached to a high-speed electric dermabrader at 10,000 rpm until pinpoint bleeding is seen. Liquid nitrogen cryosurgery [5], suction blistering [16], and shaving with scalpel or silver knife [16] have also been used but they have their limitations. The recipient site can also be prepared using a pulsed Er:YAG (erbium:yttrium–aluminum–garnet) laser [22,23] or ultrapulse carbon dioxide (CO2) laser (Coherent Ultrapulse 5000 C Coherent Inc., Palo Alto, CA, USA) set at 300 mJ/pulse [24]. Kahn et al. [25] have demonstrated that there was no histological difference in adherence of the skin graft using the shortpulsed CO2 laser versus a dermabrader. According to Oh et al. [24], the ultrapulse CO2 laser has an advantage over the Er:YAG laser because it achieves better hemostasis and causes an epidermal–dermal split in a single pass (see Chapter 34 for more details). The denuded surface is cleaned properly to remove epidermal remnants and blood clots. The graft is then placed over the denuded area, taking care to ensure that the dermal surface is facing down. There should be no wrinkles on the graft surface. The free edge of the graft should be stretched evenly at the periphery. In a large graft, fenestrations are made on the graft surface with a No. 11 blade or 22-gauge needle to allow drainage of the exudate. Proper immobilization and securing the graft are the most important criteria to achieve a successful result. Octyl-2-cyanoacrylate adhesive can be used to secure the graft [26]. It also has antimicrobial properties against staphylococci, Pseudomonas and Escherichia coli [26]. In a study of 50 patients [27] with stable, recalcitrant vitiligo treated with splitthickness skin grafting over 180 lesions, cyanoacrylate adhesive was effective in immobilizing the grafts even over mobile areas and prevented wrinkling of the graft. No adverse effects were noted. It was quick, easy to apply, and could be removed easily by peeling with a forceps, with many advantages over existing methods. It is best applied in an interrupted manner at the edges of the graft and in the center over mobile areas such as eyelids to prevent wrinkling of the graft. This is followed by a non-adherent dressing,

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such as paraffin dressing, silicone netting (Mepitel, Molnlycke AB, Molnlycke Sweden) or framycetin tulle (Soframycin® tulle, Aventis, Mumbai, India). Malakar and Malakar [28] suggested a composite film and graft unit (FGU) to immobilize the graft effectively. They placed an adhesive semipermeable film neatly over the entire graft as well as a small area beyond the graft. Due to the adhesive on the film, the graft is fixed to the recipient bed. The surface of the composite is punctured with a 22-gauge needle.

Postoperative care The dressing is removed at 24 hours to observe for any serous collection or hematoma, which is drained. Subsequently dressing is changed after a week. The dressing at the donor site is removed on 8th day; by that time the healing is almost complete. Prophylactic oral antibiotics are given for 1 week to prevent postoperative infection.

Modifications at “difficult to treat” sites In order to optimize results the procedure can be modified at certain sites.

Eyelids Strict immobilization of the upper eyelid is essential for graft uptake. This is achieved by spreading a thin layer of the cyanoacrylate adhesive which secures and prevents wrinkling of the graft. Application of neomycin eye ointment before closing the eye prevents dryness. The eye is then bandaged firmly.

Beard area, eyebrows, and hairy areas Do not shave the hair prior to grafting. It has been observed that plucking the hair with a forceps after giving anesthesia delays hair growth and prevents the graft from being lifted up [29]. Alternatively the hair may be removed by chemical epilation prior to grafting [30].

Lips The lips are a difficult site to treat surgically; however, split-thickness skin grafting gives excellent results [14]. Proper immobilization of the graft is essential especially of the lower lip. The lower lip may be everted with a suture when the inner

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mucosa is involved. The patient should be advised to take liquid diet with a straw for a period of 7 days to prevent contamination of the graft. There are very few studies focusing on treatment of lip vitiligo with thin split-thickness skin grafts. In a study of 14 patients with lip vitiligo, involving the upper lip in five patients and lower lip in nine patients, excellent response (75–100%) was seen in seven patients (50%), good response (50–75%) in two patients, fair (25–50%) in two patients, and poor response (25% repigmentation) in three patients [31]. Hyperpigmentation and hypertrophy may be initial adverse effects, which resolve gradually.

Areola The areola is not an uncommon site for vitiligo. The entire areola should be grafted even if a small area is involved to prevent color mismatch. It is an interesting observation that the color of the graft is recipient dominant in a majority of cases [32]. Local factors, probably of dermal origin, play an important role in regulating melanocyte activity [33].

abraded area. Milia are common on the face and neck (Plate 13.1, facing p. 114), but are temporary. They resolve spontaneously or can be expressed out. They are caused by remnants of epidermis following dermabrasion. An even dermabrasion and thorough cleaning of the abraded site before applying the graft reduce the incidence. Hypertrophic scarring, if it occurs, subsides spontaneously over 3–4 months. Topical retinoids may be used to hasten the response. Scarring at the donor site can occur. Njoo et al. [34], in a meta-analysis of the literature, reported an incidence of 12% at the donor site. Recurrence at the grafted site or Köbner phenomenon at the donor site may occur in unstable cases or if the disease becomes active. In my personal experience depigmentation over the grafted site is uncommon. Fresh activity of the disease can be seen by the appearance of new lesions at the edge of the graft or at distant sites.

Efficacy

• Disease should be stable. • Grafts should be even and appropriately thin. • Strict immobilization of transplanted sites is essential.

In the study of 8000 cases and 50,000 grafts by Behl et al. [9], smaller lesions showed a good response in 95% and the larger lesions showed a good response in 70.8% cases. However, details of response on various sites and in various subtypes of disease were not given. Malakar [35] successfully treated seven patients with split-thickness grafts, who had failed to respond to minigrafting. A comparative study by Özdemir et al. [16] showed that thin split-thickness grafting (repigmentation rates 65–90%) is superior to the suction blister epidermal grafting (repigmentation rate 45%).

Course and adverse effects

Advantages and limitations

The side effects are generally few. Secondary infection is not very common. Behl et al. [9] reported an incidence of 0.2%. Hyperpigmentation is common on the lower legs and in dark-skinned patients. Peripheral depigmentation (halo) and achromic fissures may be seen at the edges of the graft because of graft shrinkage. It is less common in split-thickness skin grafting as compared to epidermal cell suspensions and responds readily to treatment with topical or systemic psoralens [12]. To prevent this complication, the graft should extend 1–2 mm beyond the

Split-thickness skin grafting is the most successful technique among all methods for surgical repigmentation of vitiligo [7,13,14,34,35]. It has the advantage of providing pigmentary cover to a relatively large area in a short period of time (Fig. 13.3; Plate 13.2, facing p. 114). Pigmentation is uniform and cobblestoning, common with minigrafting, does not occur. Post-transplant systemic photochemotherapy is not required routinely. Difficult areas such as eyelids, inner canthus of eyes, areola, nipples, and genitals are easier to treat. Repigmentation of leukotrichia is

Fingers Split-thickness skin grafting is not always successful in the acral areas [13]. Strict immobilization using splints is required.

Prerequisites for optimum results

Thin split-thickness skin grafts for vitiligo

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It has some limitations also. Color and texture matching can take time especially on exposed areas and in dark-skinned patients. When large areas need to be covered, limitation of the donor site is a disadvantage.

Conclusion Autologous melanocyte transplantation by thin split-thickness epidermal grafts is a safe, effective, and inexpensive treatment modality in stable vitiligo, unresponsive and refractory to medical therapy. It has the advantage of providing uniform pigmentation over a short period of time.

(A)

References

(B)

(C) Fig. 13.3 (A and B) Vitiligo on the ankle treated with

thin split-thickness grafts. (C) Significant improvement in appearance after 1 year.

also possible [36]. As compared to non-cultured or cultured melanocytes suspensions, no reagents or expensive equipment or elaborate laboratory facilities are required.

1 McCarthy JG. Introduction to plastic surgery. Plastic Surgery. Philadelphia, PA: W.B. Saunders, 1990:8. 2 Haxthausen H. Studies on the pathogenesis of morphea, vitiligo and acrodermatitis atrophicans by means of transplantation experiments. Acta Derm Veneoreol (Stockh) 1947;27:352–67. 3 Behl PN. Treatment of vitiligo with homologous thin Thiersch grafts. Curr Med Pract 1964;8:218–21. 4 Agrawal K, Agrawal A. Vitiligo: repigmentation with dermabrasion and thin split thickness skin grafting. Dermatol Surg 1995;21:295–300. 5 Kahn A, Cohen M. Vitiligo: treatment by dermabrasion and epithelial sheet grafting. J Am Acad Dermatol 1995;33:646–8. 6 Bose S. Modified Thiersch grafting in stable. Vitiligo J Dermatol 1996;23:362–4. 7 Olsson M, Juhlin L. Epidermal sheet grafts for repigmentation of vitiligo and piebaldism, with a review of surgical techniques. Acta Derm Veneoreol (Stockh) 1997;77:463–6. 8 Behl PN, Bhatia RK. Treatment of vitiligo with autologous thin Thiersch grafts. Int J Dermatol 1973; 12:329–31. 9 Behl PN, Azad O, Kak R, Srivastava G. Autologous thin Thiersch’s grafts in vitiligo: experience of 8000 cases 50000 grafts (1959–98) with modified technique in 198 cases in the year 1997–98. Indian J Derm Venereol Leprol 1999;65:117–21. 10 Andreassi A, Bilenchi R, Biagioli M, D’Aniello C. Classification and pathophysiology of skin grafts. Clinics Dermatol 2005;23: 332–7. 11 Halder RM, Young CM. New and emerging therapies for vitiligo. Dermatol Clin 2000;18:79–89.

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12 Olsson M, Juhlin L. long-term follow-up of leukoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol 2002;47:893–904. 13 van Geel N, Ongenae K, Naeyert JM. Surgical techniques for vitiligo: review. Dermatology 2001;202:162–6. 14 Gupta S, Kumar B. Epidermal grafting in vitiligo. Influence of age, site of lesion and type of disease on outcome. J Am Acad Dermatol 2003;49:99–104. 15 van Geel N, Ongenae K, de Mil M, et al. Double-blind placebo-controlled study of autologous transplantation epidermal cell suspensions for repigmenting vitiligo. Arch Dermatol 2004;140:1203–8. 16 Özdemir M, Cetinkale O, Wolf R, et al. Comparison of two surgical approaches for treating vitiligo: a preliminary study. Int J Dermatol 2002;41:135–8. 17 Lahiri K, Malakar S. Clinico-cellular stability of vitiligo in surgical repigmentation: an unexplored frontier. Dermatology 2004;209:170–1. 18 Wankowicz-Kalinska A, van den Wijngaard RM, Tigges BJ, et al. Immunopolarization of CD4
and CD8
T cells to Type 1 like is associated with melanocytes loss in vitiligo. Lab Invest 2003;83:683–95. 19 Juhlin L, Olsson MJ. Optimal application times of eutectic mixtures of local anaesthetics (EMLA) cream before dermabrasion of vitiliginous skin. Eur J Dermatol 1995;5:365–70. 20 McGregor IA, McGregor AD. Free skin grafts. Fundamental Techniques of Plastic Surgery, 9th edn. Edinburgh: Churchill Livingstone, 1995;35–59. 21 Vadodaria S, Kangesu T. Preparation of recipient areas when skin grafting for camouflage of static localized vitiligo. Indian J Plastic Surg 1995;28:13–18. 22 Yang JS, Kye YC. Treatment of vitiligo with autologous epidermal grafting by means of pulsed erbium: YAG laser. J Am Acad Dermatol 1998;38:280–2. 23 Sachdev M, Krupashankar DS. Suction blister grafting for stable vitiligo using pulsed erbium:YAG laser ablation for recipient site. Int J Dermatol 2001;39:471–3. 24 Oh CK, Cha JH, Lim LY, et al. Treatment of vitiligo with suction epidermal grafting by the use of an ultrapulse CO2 laser with a computerised pattern generator. Dermatol Surg 2001;27:563–8.

25 Kahn AM, Ostad FA, May RL. Grafting following shortpulse carbon dioxide laser de-epithelization. Dermatol Surg 1996;22:965–8. 26 Torium DM, o Grody K, Desai D, Bagal A. Cyanoacrylate adhesive. Plast Reconstr Surg 1998;102:2209–19. 27 Khunger N. Cyanoacrylate adhesive in split thickness skin grafts for resistant vitiligo – an experience in 50 cases. Presented at 31st National Conference of Indian Association of Dermatologist Veneorologist and Leprologist, Kolkata, January 30–February 2, 2003, Book of Abstracts, p. 180. 28 Malakar S, Malakar RS. Surgical pearl. J Am Acad Dermatol 2001;44:856–8. 29 Khunger N. Vitiligo surgery at difficult sites. Presented at the 5th Biennial Conference of Association of Cutaneous Surgeons of India, Chandigarh, India, November 22–24, 2002, Book of Abstracts, p. 40. 30 Kim CY, Yeon TJ, Kim TH. Epidermal grafting after chemical epilation in the treatment of vitiligo. Dermatol Surg 2001;27:855–6. 31 Khunger N, Misra RS. Refractory vitiligo on the lips: surgical treatment by split thickness skin grafting. Presented at the 2nd National Conference of Association of Dermatological Surgeons of India, January 25–26, 1998, Book of Abstracts, p. 30. 32 Khunger N, Kapoor S, Pall A, Jain RK. Cosmetic results of surgical treatment of vitiligo of the nipple–areolar complex. A clinical study of melanocyte activity. Presented at the 1st Conference of Asian Society for Pigm Cell Research, February 1–2, 2005, Book of Abstracts, p. 31. 33 Yaar M, Gilchrest BA. Vitiligo. The evolution of cultured epidermal autografts and other surgical treatment modalities. Arch Dermatol 2001;137:348–9. 34 Njoo MD, Westerhof W, Bos TD, Bossuyt MM. A systematic review of autologous transplantation methods in vitiligo. Arch dermatol 1998;134:1543–9. 35 Malakar S. Successful split thickness skin graft in stable vitiligo not responding to autologous miniature skin grafts. Indian J Dermatol 1997;42:215–18. 36 Agrawal K, Agrawal A. Vitiligo: surgical repigmentation of leukotrichia. Dermatol Surg 1995;135:1305–7.

Plate 10.1 Digital image with color card for color calibration.

(A) Plate 12.1 (A) A large segmental vitiligo on face. (B) More than 90% repigmentation persisting 4 years after the

epidermal grafting.

(B)

(A)

(B) Plate 13.1 Before (A) and after (B) thin split thickness grafting on eyelid. Note: milia formation.

(A)

(B) Plate 13.2 (A) Segmental vitiligo on the neck and lower lip. (B) Treated in a single session of split thickness grafting

with excellent cosmetic outcome.

(A)

(B) Plate 14.1 (A) Before the treatment. (B) One year after the transplantation of ultra-thin split grafts showing a nice

take and a good color matching.

Plate 15.1 The examples of the clinical result after hair grafting on the vitiligo patients.

Plate 15.2 First the vitiliginous patch was repigmented and then the poliotic hairs repigmented.

(A)

(B) Plate 16.1 (A) Before meshed graft transplant. (B) Following repigmentation.

(A)

(B) Plate 17.1 (A) Vitiligo patch prior to fliptop pigment cell transplantation on the chin and lip of an 18-year-old Indian

male. (B) After approximately 5 transplant sessions, the chin is largely repigmented as is the upper lip area. The black marks from an indelible marker denote where new grafts will be placed to fill in the areas of the pigmentation between existing grafts. No scarring is present and the color closely matches that of the surrounding skin.

(A)

(B) Plate 17.2 (A) Vitiligo of the dorsal hand and knuckle area. (B) Vitiligo of the dorsal hand and knuckle area after two

sessions of flip-top transplantations. Immobilization of the hand during healing is helpful in ensuring graft take.

(A)

(B)

(C)

(D)

Plate 18.1 (A) Segmental vitiligo on the face. (B) Abrasion with ultrasonic aspirator. (C) Seed grafting onto this lesion

(D) Four months after operation.

(A)

(B) Plate 18.2 (A) Segmental vitiligo on finger. (B) Six months after seed grafting.

(A)

(B)

Plate 19.1 (A) Before melanocyte transplant.

(C)

(B) Inflammatory hyperpigmentation 2 months after melanocyte transplantation. (C) Normal pigmentation after 6 months.

(A)

(B) Plate 20.1 (A) Vitiligo lesion on the lower abdomen before the treatment. (B) Three and one-half months after the

transplantation with basal cell layer suspension showing a nice take and a good color matching.

Plate 22.1 Cultured adult human melanocytes seen in

phase contrast microscopy. Notice the long and branched dendrites on these “neural crest derived cells.”

(A)

(B) Plate 22.2 (A) Before the treatment. (B) One year after the transplantation with cultured autologous melanocytes

showing a nice take and a good color matching.

(A)

(B)

(D)

(C)

(E)

(F)

(G)

Plate 23.1 Skin biopsy (A) is minced (B) and embedded in a trypsin-EDTA solution (C). Isolated epidermal cells are

plated onto the feeder-layer of 3T3-J2 cells (D): 3T3 cells support keratinocyte growth up to the reconstitution of a stratified squamous epithelium (E: phase-contrast microscopy). Secondary cultures are used for transplantation procedures: epidermal grafts are detached by enzymatic treatment, mounted onto vaseline gauzes (basal side down) (F), and placed in sterile boxes (G).

(A)

(B)

(C)

(D) Plate 23.2 (A) Colony with small proliferating keratinocytes. (B) Colony with large and flattened terminally differenti-

ated keratinocytes. (C) Colony forming efficiency (CFE) of keratinocytes isolated from secondary cultures ready for transplantation: the blue arrow indicates a large and regular growing colony; the black arrow indicates a small and irregular aborted colony. (D) Melanocytes in the reconstituted epidermis (DOPA staining; phase-contrast microscopy).

(A)

(B)

(C)

(D)

Plate 23.3 An achromic lesion (A) is deepithelialized without bleeding or inflammation (B) and transplanted with

in vitro cultured epithelial grafts (C), resulting in complete repigmentation with excellent color match (D).

using S100 antibody (using a red end point). Melanocytes have been cultured alone in MCDB (A), KDM (B) and M2 medium (C). Keratinocytes were grown alone in Greens (D), KDM (E) and M2 medium (F). 1:1 co-cultures of keratinocytes and melanocytes were grown in Greens (G), KDM (H) and M2 medium (I).

Plate 24.1 Photomicrographs of melanocytes (stained red) and keratinocytes alone and in co-culture in various media. Cell cultures have been stained

Transfer of M:K (1:2 ratio) from amine coated carrier dressing

Transfer of M:K (1:2 ratio) from acid coated carrier dressing

D

B

S100 staining

Plate 24.2 Use of an in vitro human wound bed model to demonstrate transfer of melanocytes and keratinocytes from chemically defined carriers. Histological sections have been stained with hematoxylin and eosin (H&E) to illustrate keratinocyte morphology (A&C) and S100 to illustrate the presence of melanocytes (B&D – brown endpoint).

C

A

H&E staining

(A)

(B) Plate 27.1 Before (A) and 3 months after (B) the melanocyte transfer via epidermal grafts. (Reproduced from Gupta

et al. Dermatol Surg 2004;30:45–8, with permission from Blackwell Publishing.)

(A)

(B) Plate 27.2 Before (A) and 3 months after (B) the mini-punch grafting. (Reproduced from Lahiri et al. Int J Dermatol

2006;45:649–55, with permission from Blackwell Publishing.)

(A)

(B)

Plate 28.1 (A) Vitiligo on upper eyelid before epidermal

(C)

grafting. (B) After dermabrasion, placement of epidermal grafts. (C) Complete repigmentation with good color match at 6 months.

(B)

(A) Plate 28.2 (A) Twenty-nine-year-old man with focal vitiligo on genitals of 5 years’ duration. (B) Almost complete

regimentation 2 months after autologous non-cultured melonocyte-keratinocyte cell transplantation. (Courtesy: Sunjeev V. Mulekar and Ahmed Al Issa, National Center for Vitiligo & Psoriasis, Riyadh, Saudi Arabia.)

(A)

(B) Plate 29.1 Vitiligo over dorsum of hand before treatment. (B) Pigmentation seen 3 months after epidermal grafting.

(A)

(B)

Plate 32.1 (A) Pre-treatment photograph of HIV-infected

(C)

patient demonstrating complete loss of gingival pigmentation in the maxillary and mandibular arches. (B) After complete tattooing has been done. Note that margins of the gingiva, near the teeth, are not tattooed to resemble the normal distribution of pigmentation that is present in naturally pigmented gingiva. (C) Final result at about one month after tattooing. (Courtesy: Dr. Howard Tenenbaum; reproduced with permission from: Center JM et al. J Periodontol 1998;69:724–728.)

(A)

(B)

(C)

(D)

Plate 34.1 (A) Preoperative view of a stable and recalcitrant vitiliginous hand. (B) Bloodless and smooth recipient site

after laser-assisted superficial dermabrasion. (C) Thin skin grafts were applied and affixed with a skin stapler and sterile tapes. (D) Postoperative view 10 months after the operation. (Reprinted with permission from Acikel C et al. Plast Reconstr Surg 2003;111:1291–98.)

(A) Plate 34.2 (A) Preoperative view. (B) Postoperative view after 6 months. Note the hairs on the skin grafted area.

Although pigmentation of the achromic area is achieved, the final color match is not ideal on the leg.

(B)

CHAPTER 14

Treatment of leukoderma by transplantation of ultra-thin epidermal sheets Mats J. Olsson

Introduction Several inborn or acquired disorders result in hypoor depigmented areas of the skin lacking melanocytes. In many cases medical therapies and ultra-violet (UV) treatment may be of benefit (e.g. those with generalized vitiligo). However, some of the disorders with a lack of melanocytes in the epidermis as well as in the hair follicles do not respond to these approaches (i.e. piebaldism, segmental vitiligo, and depigmentation after burn injury). Also, many cases of generalized vitiligo do not respond to medical or UV treatment; especially those with lesions on hands, fingers, feet, and toes (i.e. areas with reduced numbers of hair follicles). To overcome this lack of response to non-invasive methods, surgical techniques have been developed. There are a number of different surgical methods for restoring the pigmentation of leukodermic skin. In this chapter, I will exclusively discuss the use of very thin epidermal sheets obtained by a high-speed air-driven dermatome. This is entirely a surgical method for restoring the epidermal melanin unit, not requiring any high-tech laboratories, or support from specially trained technical staff. To get some perspective on the matter, it is important to keep in mind that different surgical approaches in vitiligo began long ago, later on leading to the development of the more refined method with transplantation of ultra-thin sheets, which will be discussed in the following section.

In 1947, a Danish dermatologist, Haxthausen, transplanted Thiersch grafts from normal to vitiliginous skin lesions and vice versa on small areas in three patients. At the follow-up inspection after 9–12 months the normal skin grafted to the depigmented areas remained pigmented only in one out of three patients [1]. Five years later, good repigmentation of vitiligo was obtained in a Black patient by Spencer and Tolmach, who used fullthickness grafts [2]. Their findings were consistent with those in an experimental study in which exchange autografts were transplanted in white and dark patches in guinea pigs [3] and were later also confirmed by others using Thiersch and splitthickness grafts to restore pigmentation in vitiligo patches [4,5]. A reasonable explanation for the total lack of response in two of the three patients treated by Haxthausen is probably that the disease was in an active phase, which underlines the importance of a careful selection of patients [4,6,7]. More recently, modern technology has made it possible to harvest ultra-thin epidermal sheets with highly controllable machine-powered dermatomes. Kahn et al. (1991) were first to treat depigmented lesions resulting from burn injuries with this new precision equipment [8]. This technique has later been used by us in a slightly modified form and employed in the treatment of generalized vitiligo, segmental vitiligo, halo nevi, and piebaldism [7,9]. The general aim in each patient chosen to undergo a transplant treatment always should be to select a

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specific transplantation method likely to be most effective for that particular patient. While selecting a specific transplantation method, the following points should be taken into consideration: • the locations of the anatomical areas to be treated, • extent of the white areas, • whether there are several small or one large lesion, • type of leukoderma, • what is the texture of the skin on the recipient area, • are there coarse hairs growing in the recipient area, • texture of the skin in possible harvest area, • history of Köbner phenomenon, and • history of hypertrophic scars and keloids. Once all these variables have been evaluated the decision for the most suitable method can be made. Treatment of leukoderma with ultra-thin epidermal sheets can be implemented in all types of vitiligo and piebaldism. The method can also be used to treat depigmentation after burns and chemical injuries. As with all kinds of surgical approaches one has to be extra careful in selecting appropriate candidates among patients with generalized vitiligo (vitiligo vulgaris). Generalized vitiligo is progressive in most cases and the lesions increase in size and number during the patient’s lifetime. It has involvement of autoimmune components and if the disease is not completely stable or in spontaneous regression, there is always a risk that the melanocytes in the transplanted tissue will be attacked by the immune system and not survive. Ultra-thin epidermal sheet transplantation is quick, effective, and appropriate in flat, large, coherent areas with not too much movement. Areas such as those over the joints, eyelids, and the corners of one’s mouth are more difficult to treat with epidermal sheets than with cellular grafts (cell suspension). Areas of the face should also be avoided due to the increased risk of hyperpigmentation (more about that in discussion). Lesions in areas with coarse hair should also be avoided due to the reason that the growing hair will elevate the sheet, interfering with graft uptake. Methods like this, which do not involve preparation of free cells or culturing steps, do not require any special trained lab technicians or laboratories.

This reduces the invested time, facility, and personnel costs, which usually makes the method less expensive in comparison to technically more complicated methods. But compared with the methods utilizing free cell suspensions or culture expanded cells, a direct method like transplantation of ultrathin sheets needs much larger donor areas, theoretically limiting the possibilities to treat extensive areas. The possibilities to store frozen samples, and/or to ship the donor tissue and use it in a later session or at another location is also more limited compared to techniques utilizing free cells (see Chapter 22, “Treatment of leukoderma by transplantation of cultured autologous melanocytes”).

Ultra-thin epithelial sheet grafting principles Step-by-step the procedure in the surgical treatment of leukoderma with the use of autologous ultra-thin donor sheets obtained with an air-pressure powered high-speed dermatome and transplanted to denuded recipient areas will be discussed in the following section.

Calculation of areas The borders of the chosen recipient areas are marked with a pen (in Wood’s light on fair skin, if needed). A transparent film (overhead-projector plastic sheets) is put over the marked areas and the markings are transferred to the film. The film is copied in a copy machine (Xeroxed) and the outlined areas on the paper are cut out and weighed on an electronic balance. The total area is calculated. This is an important maneuver that ensures that excessive donor tissue is not harvested or excessive recipient area is not denuded.

Premedication A still and calm patient is important for success of the procedure. We therefore usually give 5–10 mg of diazepam and/or 10 mg of ketobemidone, orally, 60–90 minutes before transplantation, plus 500– 1000 mg of paracetamol 40–50 minutes before the surgery. Erythromycin or a similar antibiotic is given for 8 days, starting on the day of surgery.

Treatment of leukoderma by transplantation of ultra-thin epidermal sheets

117

Anesthesia of recipient site The recipient area is anesthetised with eutectic mixture of local anaesthesia (EMLA®, AstraZeneca, Södertälje, Sweden) under plastic foil occlusion for 1–2 hours depending on the anatomical area to be treated [10] and then immediately prior to the surgery also locally anesthetised with injection of a mixture of equal parts of 1% lidocaine and Tribonate®-buffer (Fresenius Kabi, Uppsala, Sweden). A thin and long needle is inserted outside the white lesion and then parallel to the skin surface thrust into the white lesion. This is to avoid interfering bleeding from needle stick in the area to be treated. In large areas it may not be possible to reach into the center of the lesions from the borders, but a combination of ring-block effect of peripheral injection and EMLA® cream will provide satisfactory anesthesia in the center of the lesion. Small areas can be easily infiltrated, and there is no need to apply EMLA® prior to the injection. Total nerve block can be used to achieve satisfactory anesthesia, but it requires some skill to put the injections of local anesthetic in the right spot and should, therefore, preferably be handled by an anesthesiologist. Local anesthesia with a freezing spray such as ethyl chloride or fluor-ethyl (Gebauer Pharmaceutical Preparations, Cleveland, OH, USA) can also be used immediately prior to the dermabrasion. The sprays give some anesthesia but are not sufficient to give total pain relief. However, when skin is chilled it becomes more firm and easy to dermabrade. It is also much easier for an inexperienced eye to detect remaining epidermal remnants on the chilled denuded surface.

Preparation of the recipient site The recipient area is cleaned with alcohol, outlined with a sterile surgical marker pen and the epidermis is removed down to the dermal–epidermal junction, using a high-speed dermabrader (20,000 rpm), fitted with a diamond fraise (Fig. 14.1). A diamond fraise wheel, pear, and/or cone suitable for the size and location of the area to be treated are chosen. Normally one can handle most of the lesions with a 6 mm wide regular fraise wheel, but on rough skin such as on the knees one might need a coarse wheel

Fig. 14.1 Dermabrader. The handpiece is fitted with a

regular 17 mm
6 mm wheel.

(A)

(B)

(C)

(D)

Fig. 14.2 Diamond fraises: (A) wheel, (B) cone, (C) pear, and (D) handle fitted with a cone for manual abrasion in delicate locations.

and in delicate areas such as around the nostrils, corners of the mouth, and on the eyelids you might need a small pear-shaped fraise. For the eyelids, we have a special made hand-tool fitted with a regular cone; this is to ensure not to damage the eyelid or the eye during the procedure (Fig. 14.2). Make sure to go over the area with the dermabrasion from a minimum of two different directions. An uniform punctate capillary bleeding from the dermal papilla can be seen (Fig. 14.3) and light freezing with fluor-ethyl spray reveals if there are any islands of epidermis left. The denuded areas are washed with phosphate buffered saline (PBS) or saline solution and kept under moistened gauze until the donor

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Fig. 14.4 The Zimmer air dermatome with a piece of

thin donor tissue. The shave biopsy can be stretched out on the machine front and be cut into appropriate sizes. Fig. 14.3 Dermabrasion de-epithelialized recipient area.

Notice the small pinpoint bleedings.

area is harvested and the sheet ready to be transplanted, or for some minutes, to ensure that all bleeding has stopped. In cases with some remaining punctate bleeding, electrocoagulation with a finepointed electrocate-needle is performed.

Donor area Normally pigmented gluteal or thigh skin is shaved and cleaned with alcohol. An area of the size of the recipient patch(es) is marked with a sterile surgical marking pen. The marked area is anesthetized with EMLA®-cream (AstraZeneca, Södertälje, Sweden) under occlusion for 1–2 hours prior to a ring-block injection with a mixture of equal parts of 1% lidocaine and Tribonate®-buffer (Fresenius Kabi, Uppsala, Sweden). It is important not to inject inside the lines of the marked area due to the risk of buckled areas with embossed lesions within the donor area. An uneven surface makes it impossible to harvest an ultra-thin coherent sheet. The skin is stretched hard by an assistant and a very shallow sheet of skin is taken with a Zimmer®air driven ultra-dermatome (Zimmer Inc. Warsaw, IN, USA) (Fig. 14.4). Depending on the anatomical area to be harvested, the nature of the skin and the total graft size needed, the machine is fitted with a special shield plate, controlling the width of the sheet. The machine is delivered in a special autoclavable metal box including a set of four different bottom plates with width gaps from 1 to 4 in. The

level control is set at a shallow marking. In principle, it is possible to harvest thinnest possible grafts, but in practice, a coherent melanocyte containing sheet, strong enough to be transferred to and fastened in the recipient area, is needed. Each machine and operator has his/her own small individual peculiarities and nature, and therefore, the user has to set right his/her own individual settings, depending on personal angle approach (about 25°), forward speed, and pressure. The harvested sheet is immediately moistened with s-MEM, that is, Joklik’s modified minimal essential medium (GIBCO BRL, Life Technology, Gaitersburg, MD, USA), so as not to dry out before it is grafted to the recipient site. The donor area is covered for 8–9 days with semipermeable Tegaderm™ (3M, St. Paul, MN, USA), plus a layer of the air and water vapor permeable stretch fabric tape Fixomull®, extending beyond the margins of the Tegaderm with a few centimeters, to ensure that the blister of fluid that will build up under the dressing will not break. The Tegaderm and the Fixomull are glued in place with Mastisol® (Ferndale Laboratories, Inc, Ferndale, MI, USA). This special glue ensures that the dressing will be secured for the period needed.

Transplantation phase The thin epidermal sheets are applied to the dermabraded recipient areas (Fig. 14.5). For large areas the sheets are cut in to somewhat smaller pieces to allow the drainage of the exudate. The sheet can be

Treatment of leukoderma by transplantation of ultra-thin epidermal sheets

119

Fig. 14.5 Application of the thin donor skin onto the

dermabraded vitiligo lesion. Notice that several smaller pieces are put together, allowing exudates to leak through the joints.

moistened and stretched out on the machine itself or in a large Petri dish and easily be cut in suitable pieces with an ordinary scalpel. The epidermal sheet is transferred to the recipient area with the help of two forceps holding the corners or for smaller pieces on the blade of a scalpel or sterilized microscope slide. A metal-spatula or curved iris scissors is used to scrape the surface of the sheet, to ensure that no bubbles or fluid are left within between the wound bed and the epidermal sheet. The grafts are secured with a silicone netting (Mepitel®, Mölnlycke AB, Mölnlycke, Sweden) extending about 1 cm onto the nondermabraded surrounding locking it in place and then covered with saline-moistened sterile gauze-pads and bandaged. In all cases the patient should stay at least for 4 h in a hospital bed after the procedure has been completed, if possible longer. Immobilization improves the chances for a good outcome. Hands should be splinted and if legs or feet are treated the patient should be transported in a wheelchair to the car for home transportation. The bandages are removed after 8–9 days.

Follow-up inspection An overall follow-up evaluation at 5–8 months after transplantation is recommended. During the first 2 weeks, the transplanted areas are erythematous. Some crust formations of overlapping

Fig. 14.6 One week after the transplantation at bandage

removal. Notice the crusts of dried serum and overlapping skin in the borders.

skin or dry oozed serum in the corners of the areas and small punctual bleedings under the sheets can be seen during this period (Fig. 14.6). Often the pigmentation can be seen immediately, but in fairskinned individuals it may take some time before the outcome can be judged. Wood’s light or diascopy pressure can help when evaluating at an early stage. During the first year a slight hyper- or hypopigmentation in some of the treated areas is not uncommon but the color gradually matches with the complexion of the surrounding skin and most often blends in well in about 1–2 years (Plate 14.1, facing p. 114). Long-lasting hyperpigmentations can occur. A white, 1–2 mm halo between the transplant and normally pigmented skin can sometimes be seen. This is seen less often in patients with piebaldism or segmental vitiligo, compared with generalized vitiligo. Most often this achromic border is filled after some time of repeated sun exposure.

Evaluation and documentation The final result can be seen about 1 year after the surgery. The transparent plastic film, used prior to the surgery to calculate the recipient areas, is stored in the patient’s file and used for evaluation at follow-up inspection. The film is put over the lesions to evaluate the outcome/progress. It is important to

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mark the angle of an elbow, knee, or neck on the sheets since a change in position can alter the size. Total body chart drawings and pictures of the treated lesions should be taken before the treatment and at follow-ups. An estimation of the total extension of the white areas (treated and not treated) is also important to document. This is important for planning of further sessions in future in those patients with extensive areas and for scientific outcome evaluation when comparing the results in extensive versus nonextensive generalized vitiligo [7]. Files with anamneses and treatment charts are kept in the hospital’s central records.

Discussion Depigmented skin with white hair and glabrous skin respond poorly or not at all to attempts of medical repigmentation. This indicates that hair follicles are the normal and most important reservoir for spontaneous and medical repigmentation, and that surgical transplants are the only effective method to replace the lost epidermal melanocytes and the follicular reservoir. The sheets are very thin and therefore there is no risk of contraction of the graft due to dermal elastin fibers. Therefore the sheets can be placed side by side within the lesion and only have to overlap about 1 mm beyond the dermabraded area onto the normally pigmented skin. The order of various steps in the surgical procedure depends on the number of people in the staff and extension and locations of the areas to be treated, but normally it is best to dermabrade the recipient areas before the donor area is harvested. This gives some valuable time for the bleeding to stop at the recipient areas and provides absolutely fresh graft. Topical thrombin can be used at both donor and recipient site to achieve hemostasis, though it is seldom needed. Patients are told not to use any medicine containing salicylates (e.g. Aspirin) for 10 days before surgery and 1 week after the surgery. Kahn et al. (1996) showed in a study that de-epithelialization of the recipient site with the help of a short-pulse carbon dioxide laser can give similar results as with dermabrasion [11].

Excellent results have been seen in most patients at a 6-month follow-up inspection, however on long-term follow-up it is obvious that the type of leukoderma influences the final outcome. At a mean follow-up time of 4.5 years it was clear that patients with segmental vitiligo and piebaldism retained all repigmentation but patients with generalized vitiligo retained an average of 59% pigmentation of the treated surface areas. This is related to the nature of the disease and it underscores the need for careful selection of patients in the group with generalized vitiligo. Patients with extensive generalized vitiligo and those who have not had completely stable, non-progressive vitiligo for at least 2 years should not be chosen for any type of transplantation procedure. The predicted long-term outcome with ultra-thin sheet transplantation method is as good as with other methods [7]. Eyelids, fingers, and perioral locations are the most difficult areas in which to achieve satisfying results. It is common to see small epithelial miliaria-like cysts in the recipient areas in the first 6 months, especially on the face and neck (Fig. 14.7). Milia can be managed easily by puncturing them with a fine needle and expressing out the contents. This is only to speed up the process, as untreated milia will eventually disappear spontaneously after sometime and will not recur. Milia can develop due to remnant of epithelial cells following the dermabrasion, but a

Fig. 14.7 Epithelial milia seen at 4-week follow-up.

Notice also the slight initial hyperpigmentation.

Treatment of leukoderma by transplantation of ultra-thin epidermal sheets more likely reason for the milia formation is the occlusion of the sweat-ducts where the outflow is blocked, in combination with a rapid proliferation of the epithelial cells at the end of the ducts. We have not seen milia formation when methods with transplantation of cellular grafts have been used. In the patients with light skin complexion the transplanted areas may appear pink in the first month. In our patients, no scarring has been seen in any of the patients, both at the donor or the recipient sites. All donor areas have repigmented nicely and were imperceptible already at the 6-month follow-up. The final color of the transplanted area can be the same as that of the surrounding skin, lighter or darker, and this does not seem to be related to the skin type of the patient or the anatomical location of the treated area. When the initial shade of repigmentation in all kinds of leukoderma, was compared with the outcome from two other methods, it was evident that the ultra-thin epidermal sheet method more often resulted in slight hyperpigmentation (Table 14.1) [7]. With this risk in mind, one has to think twice before using this method on the face. The patients should be informed about the risk of hyperpigmentation before undertaking the procedure. The ultra-thin epidermal sheet method does not require laboratories or laboratory or technical expertise, but needs an operating theater equipped with an outlet for high-quality compressed air, and an autoclave large enough to fit in a machine box of

about 45 cm
35 cm. The elbows and knees are not suited for this method and the outcome of the results lies in the technical skill of the operating personnel. The method requires practical training and should be carried out only by a dedicated staff that has decided to perform the procedure on a regular basis.

Conclusions • This method does not require any laboratory, technicians, or expensive equipments or chemicals. • Segmental vitiligo and piebaldism almost always respond with complete repigmentation in the transplant area, regardless of the transplantation method used. • Segmental vitiligo and piebaldism retain all their repigmentation for as long as we have followed them up (up to 9 years’ follow-up). • It is more difficult to achieve full repigmentation in vitiligo vulgaris, but it usually responds well if the disease is not in a progressive state. • Selection of appropriate patients in the group of vitiligo vulgaris patients is crucial for a successful outcome. • Active and extensive vitiligo vulgaris more often respond with poor repigmentation and patients with these signs should not undergo transplantation. • The ultra-thin epidermal sheet method is a fairly easy method to use, but should not be used in areas where greater movement is required with excessive mobility, such as those over the joints.

Table 14.1 The shade of repigmentation obtained by different surgical methods

used for treatment of leukoderma.

Repigmentation of treated areaa Somewhat lighter

Cultured melanocytes n
71

Epidermal sheets n
20

Basal-layer suspension n
40

7.0

5.0

15.0

The same

47.9

25.0

45.0

Somewhat darker

45.1

70.0

40.0

a

121

As compared to surrounding pigmented skin. n  numbers of treated patients with each method. The figures in each classification represent the distribution in percent.

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• Initial epithelial milia are common on face and neck regions. • Hyperpigmentation at the recipient area is not uncommon and can be permanent. • No significant side-effects from this transplantation technique have been observed.

References 1 Haxthausen H. Studies on the pathogenesis of morphea, vitiligo and acrodermatitis atrophicans by means of transplantation experiments. Acta Derm Venereol (Stockh) 1947;27:352–67. 2 Spencer GA, Tolmach JA. Exchange grafts in vitiligo. J Invest Dermatol 1952;19:1–5. 3 Orentreich N, Selmanowitz VJ. Autograft repigmentation of leukoderma. Arch Dermatol 1972;105:734–6. 4 Behl P. Treatment of vitiligo with homologous thin Thiersch’s grafts. Curr Med Pract 1964;8:218–21. 5 Agrawal K, Agrawal A. Vitiligo: repigmentation with dermabrasion and thin split-thickness skin graft. Dermatol Surg 1995;21:295–300.

6 Beck HI, Schmidt H. Graft exchange in vitiligo: studies on the outcome of exchanging biopsies from vitiliginous skin to normal, pigmented skin and vice versa. Acta Derm Venereol 1986;66:311–5. 7 Olsson MJ, Juhlin L. Long-term follow-up of leukoderma patients treated with transplants of autologous cultured melanocytes, ultra-thin epidermal sheets and basal cell layer suspension. Br J Dermatol 2002; 147:893–904. 8 Kahn AM, Cohen MJ, Kaplan L. Treatment for depigmentation resulting from burn injuries. J Burn Care Rehabil 1991;12:468–73. 9 Olsson MJ, Juhlin L. Epidermal sheet grafts for repigmentation of vitiligo and piebaldism, with a review of surgical techniques. Acta Derm Venereol 1997;77:463–6. 10 Juhlin L, Olsson M. Optimal application times of a eutectic mixture of local anaesthesia (EMLA) cream before dermabrasion of vitiliginous skin. Eur J Dermatol 1995;5:368–70. 11 Kahn AM, Ostad A, Moy RL. Grafting following shortpulse carbon dioxide laser de-epithelialization. Dermatol Surg 1996;22:965–7; discussion 7–8.

CHAPTER 15

Transplantation of hair follicles for vitiligo Subrata Malakar, Gun Yoen Na and Koushik Lahiri

Introduction

Preoperative worknote

Epidermal melanization is the end result of collective and synchronized functioning of various structures and products of the body [1]. In vitiligo when and how this progress gets deranged is not exactly known. Although several hypotheses, that is, autoimmune, self-destructing, neural and composite, are put forth to explain it, none is satisfactory. Single hair transplants were introduced to repigment vitiligo patches in 1998 [2]. This novel procedure is based on the concept of the existence of a pool of undifferentiated melanocyte stem cells in the hair that can replenish the pool of differentiated melanocytes. These stem cells are maintained in the niche microenvironment, which is thought to be located in the “bulge” area, where arrector pili muscle attaches with the hair. These stem cells would probably be the good source of melanocytes [3,4].

The patient is routinely evaluated before the grafting as is done in other surgical procedures. Special attention should be given to the donor site hair. The procedure can be done with a standard hair transplanter [2] or manually as described by Malakar [7]. If done with a hair transplanter, the hairs are cut short as it is not necessary to keep a long hair shaft. While in the latter technique, the length of the hair should be kept as long as possible to facilitate and fasten this particular procedure. If early camouflage of leukotrichial hairs is desired, they could be removed by thermolysis before transplanting hairs on vitiligo patch.

Indications Stable vitiligo is the main indication for this novel technique. A vitiligo patch of 10 cm
10 cm requires around 100 hairs. The donor area is occiput. A very small hair strip graft is required for the above patch. As the occiput is the donor site, we can completely avoid the complications of other grafting procedures (e.g. punch grafting, split thickness skin grafting, and blister technique); all these have visible scar and/or hypo- or hyperpigmentation at the donor site [5,6]. The procedure has the potential as the method of choice to repigment the vitiligo patch having leukotrichia.

The technique (Na’s method, Figs 15.1–15.5) There are four steps for transplantation of single hairs by this technique: Step I: After surgically preparing the donor site (occiput), a strip graft is harvested, the length of which is determined by the size of the recipient area (usually an elliptical incision of approximately 2–3 cm). Step II: The removed donor scalp is washed with cold normal saline, and then cut into segments. Each segment is divided into many single hairs. The length of the hair shaft is 5–10 mm preserved. Step III: The single hairs are placed into recipient area with a hair transplanter. For glabrous areas, the lower third of the follicle is cut off and only the upper two-thirds are grafted. In hairy areas, especially the eyebrows and scalp, the entire hair follicle is grafted.

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Fig. 15.1 The removed donor scalp was cut into seg-

Fig. 15.3 The prepared hair was inserted into the hair

ments, and then divided into single or follicular unit hairs with blade.

transplanter.

Fig. 15.2 Divided single hairs, and the lower third to

Fig. 15.4 Various types of hair transplanters according to

lower half of the follicle was cut off.

the caliber of the needle. Plunger

Step IV: After grafting, an occlusive dressing is applied for 1 week.

The modified technique (Malakar’s method, Figs 15.6–15.10) There are six steps of transplantation of single hairs using this technique: Step I: After surgically preparing the donor site (occiput), a strip graft is harvested. The length of the hairs, a necessary prerequisite for the procedure is kept preserved. Single hairs are separated. Step II: A 20-gauge curved cutting needle is inserted into the vitiligo patch; sharp end of the needle is pulled out of the skin keeping the eye of the needle above the cutis. Step III: Single hairs are inserted into the eye of the needle so that the eye of the needle takes arbitrarily

KNU implanter Housing Depth control unit

Guide hook

Groove Hollow needle Fig. 15.5 The schema of the principle of KNU hair

transplanter.

the position between the middle and the follicle of the whole length of the hair. Step IV: The hair is held by a small forceps just beyond the hair follicle. The needle is then taken

Transplantation of hair follicles for vitiligo

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Fig. 15.10 The hair is pulled upwards until the hair follicle comes down to deep dermis. Fig. 15.6 A strip graft.

out of the skin and is used for the transplantation of the next hair. Step V: The free end of the hair is then pulled gradually until the follicle enters into the tight cleft in the lower dermis created by the needle.

Postoperative care Fig. 15.7 A 20-gauge curved cutting needle is inserted

into the skin. The hair to be implanted is brought near the eye of the needle.

Fig. 15.8 The hair is inserted into and is pulled through

the eye of the needle.

After grafting procedure is over, both the donor and the recipient areas may be washed with antiseptic solution taking particular care not to pull the grafted hairs. The area is then mopped dry with gauze. The dressing of the donor area is done by paraffin embedded gauze and Micropore®. It is removed on 7th day. The recipient area is also dressed with paraffin embedded gauze and Micropore® and is removed on 4th or 5th day. The “take” of the graft is on the 2nd to 4th day after the procedure. Utmost care should be taken not to pull the grafted hairs out of the skin.

Appearance and spread of pigment

Fig. 15.9 The needle is pulled out of the skin. The hair is

held by a pair of small forceps just beyond the hair follicle.

The pigment usually appears on the vitiligo patch by the 4th or 5th week after single hair grafting. In contrast the pigment appears usually on the 3rd week after punch grafting [5,8]. The delay of appearance of pigment after hair grafting may be due to the fact that melanocyte has to traverse almost the whole of the dermis before it reaches the basal layer of epidermis before it starts producing melanin.

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Though there is delay in appearing the pigment, the color usually matches well with the surrounding skin (Plate 15.1, facing p. 114). Repigmentation of hair takes even more time. The diameter of the pigment spreading is 5–12 mm per hair transplanted. It usually starts appearing at the 4th to 5th week and continues up to 6 months or even longer. Therefore the distance between hair grafts is minimally 5 mm and maximally 12 mm. Thus, a vitiligo patch of 10 cm
10 cm requires around 100 hairs’ grafts.

Complications As the occiput is the donor area and a small strip graft is taken, a minor scar is formed, which naturally hides under the scalp hairs. There is no cobblestoning. Polka dot appearance is rarely seen. Utmost care is taken not to pull the hairs during dressing and on the day and next 2 days following grafting. The risk of failure is maximum during the first 48 hours. If the surgeon is skilled enough, failure rates are negligible. There is no Köbnerization of the donor site and, in case of failure, the recipient area does not show any small depigmented grafts as seen with negative test grafts routinely done before minigrafting [9,10].

Limitations Though the surgical modalities are important in the treatment strategies of vitiligo, all of them have their limitations. Each patient of vitiligo should be individualized for the technique he or she will undergo. Single hair transplant to repigment vitiligo over non-hairy areas is still a matter of controversy. According to Na et al.’s report and personal experiences, the lower third of the follicle is cut off, and then the upper two-thirds of grafting showed the pigment spread and minimal villus hair growth. Moreover, when the upper and lower halves of the hair follicle after cutting the entire follicle at midportion were grafted, the pigment spreading effects in both upper and lower halves were similar.

Discussion Single hair transplant is another option to repigment vitiligo patch. Hairs are harvested from the occiput and are grafted on the vitiligo patch. The single hair transplant by this technique is easy to perform and does not require any special skill. One can perform the operation with one or two assistants and with inexpensive and commonly available instruments. The most practical and probably the best application of this method is a vitiligo patch with leukotrichia. Some experts recommend thermoepilation of the depigmented hairs on the patch before grafting of the hairs. Consequently, vitiligo patch and leukotrichia can be dealt with at the same time. However, one should be careful as the hair grafting just after thermoepilation could influence in the survival of the grafts because the recipient site was damaged with coagulation necrosis by thermoepilation. The area of scalp which has vitiliginous patch or poliosis should not be selected as a donor site, for obvious reasons. After the recognition of the fact that hair regrows even if its lower two-third is discarded and that pigmented hair comes out of it with melanocyte reservoirs remaining, deployment of this method over non-hairy regions should be better avoided. Phototherapy induced stimulation of melanocyte migration from the hair follicle reservoir is now a well-established fact. Melanocytes spread centrifugally from the infundibulum to the basal cell layer and recolonize the epidermis with active and functional melanocytes [11,12]. But the existence of pilosebaceous apparatus within the minigrafts is not at all necessary for the repigmentation process. In suction blister grafts only epithelial cells present in the grafts are enough to induce repigmentation [13]. In 1970 Billingham and Silvers demonstrated the phenomenon of melanocyte migration from graft’s edge within the achromic skin to recolonize and replenish the area with functional and active melanocytes [14]. Though the appearance of pigment is delayed as compared to other modalities, the color match is much more acceptable than that with other methods. This could be explained by following. The skin color is controlled primarily by melanocytes, and

Transplantation of hair follicles for vitiligo theoretically skin melanocytes consist of stem and transient amplifying melanocytes. The stem melanocytes probably reside in the hair follicle and epidermal rete ridge. The stem melanocytes located in the niches probably consist of keratinocytes. The territory of one stem melanocyte could be suspected 2–5 mm, it is deduced from the “idiopathic guttate hypomelanosis” usually seen on extremities of elderly individuals. The cause of a more acceptable color match in hair transplantation than in other methods could be related to the stem cell migration from the graft, and then location specific transient amplifying cells proliferation. To add to the list of the vitiligo surgeries it is not just another method, but the procedure of choice to treat vitiligo with leukotrichia.

References 1 Moscher DB, Fitzpatrick TB, Hori Y, Ortonne JP. Disorders of pigmentation. In: Fitzpatrick TB, Eisen AZ, Wolff K, Freedberg IN and Austen KF (eds.) Textbook of Dermatology, 3rd edn. New York: McGraw-Hill, 1993;903–95. 2 Na GY, Seo SK, Choi SK. Single hair grafting for the treatment of vitiligo. J Am Acad Dermatol 1998;38:580–4. 3 Nishimura EK, Granter SR, Fisher DE. Mechanisms of hair graying: incomplete melanocyte stem cell maintenance in the niche. Science 2005;307:720–4. 4 Nishimura EK, Jordan SA, Oshima H, Yoshida H, Osawa M, Moriyama M, Jackson IJ, Barrandon Y, Miyachi Y, Nishikawa S. Dominant role of the niche in melanocyte stem-cell fate determination. Nature 2002; 416:854–60.

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5 Malakar S, Dhar S. Treatment of stable and recalcitrant vitiligo by autologous mimiature punch grafting: a prospective study of 1000 patients. Dermatology 1999; 198:133–9. 6 Lahiri K, Malakar S, Sarma N, Banerjee U. Repigmentation of vitiligo with punch grafting and narrow-band UV-B (311 nm) a prospective study. Int J Dermatol 2006;45:649–55. 7 Malakar S, Dhar S, Malakar RS. Repigmentation of vitiligo patches by transplantation of hair follicles. Int J Dermatol 1999;38:237–8. 8 Lahiri K, Sengupta SR. Treatment of stable and recalcitrant depigmented skin conditions by autologous punch grafting. Ind J Dermatol Venereol Leprol 1997;63:11–4. 9 Malakar S. Lahiri K. How unstable is the concept of stability in surgical repigmentation of vitiligo? Dermatology 2000;201:182–3. 10 Lahiri K, Malakar S. Inducing repigmentation by regrafting and phototherapy (311 nm) in punch failure cases of lip vitiligo – a pilot study. Indian J Dermatol Venereol Leprol 2004;70:156–8. 11 Parrish JA, Fitzpatrick TB, Shea C, et al. Photochemotherapy of vitiligo: use of orally administered psoralen and a high-intensity long-wave ultraviolet light system. Arch Dermatol 1976;112:1531–4. 12 Ortonne JP. Psoralen therapy in vitiligo. Clin Dermatol 1989;7:120–34. 13 Suvanprakorn P, Dee-Analap S, Pongsomboon CH, et al. Melanocyte autologous grafting for treatment of leukoderma. J Am Acad Dermatol 1985;13:968–74. 14 Billingham RE, Silvers WK. Studies on the migratory behaviour of melanocytes in guinea pig skin. I. J Exp Med 1970;131:101–17.

CHAPTER 16

Mesh grafts for vitiligo C.R. Srinivas, Reena Rai and M. Sinha

Historical background In 1907, Professor O. Lanz of Amsterdam cut a fullthickness skin graft into an expanded skin net with a metal stamp, which he named “Hautschlitzapparat.” He used this apparatus for covering the skin defect with one half and the donor site with the other half [1]. This idea came from his childhood, when children made scissor cuts at regular intervals in a paperstrip for making a simple accordion toy. In 1930 Douglas covered skin defects with a “sieve graft,” which was a full-thickness skin graft with punched out areas left at the donor site for secondary coverage [2]. It was primarily devised to promote drainage without expansion. In 1937 Dragstedt and Wilson reported a “modified sieve graft” and handmade staggered cuts to obtain increased coverage [3]. In 1991, Yanai and Hiraga gave a good description of the simple technique of obtaining a wide mesh skin graft without a specific meshing machine [4]. A split-thickness skin graft was glued to a sheet of paper; this was folded back and forth in stripes of less than 1 inch; staggered scissor cuts were made at each side unfolding gave the completed crude mesh. Such a graft can be useful at little expansion but yields large raw spaces at clinically useful expansion (above 2:1). This mesh allows almost no deformation for adapting to irregular surfaces. The technique described by Eroglu et al. [5] where the graft is wrapped around a chest tube and fixed with staples and then stabbed with a knife is a useful technique, but needs the availability of staples and a chest tube and hence has cost implications. In 1992, Vartak obtained four-row economical mesh skin grafts, even at different expansion ratios, by interrupting the cutting edges at adequate intervals and adding several wheels a side [6].

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In 1963 Tanner et al. made mesh graft with a block of printing blades with cuts, staggered over the different rows and hammered through the split-thickness skin graft on a wooden board, using an electrically driven hammer [7]. The flat models of mesh graft was changed to a roller device with staggered cuts (Mesh Dermatome Type I) and further a roller with continuous cuts (Mesh Dermatome Type II). Now interchangeable rollers for different expansions (Zimmer Skin mesher) and several others like Ampligreffe, Padgett skin graft mesher are available.

Introduction Surgical treatment is indicated when vitiligo is stable. It is difficult to treat large depigmented areas surgically when it fails to respond to medical treatment. The surgical treatment includes blister roof grafting [8], punch grafting followed by psoralen plus ultraviolet A (PUVA) [9], melanocyte culture and transplantation [10], split skin graft [11], and dermabrasion [12]. Blister roof grafting yields a limited amount of skin. Punch grafting, though effective, is time consuming and the area treated is limited by the amount of local anesthesia which can be used [13]. Split-thickness grafting covers the recipient area in the same ratio thus necessitating large grafts from the donor area in patients with extensive vitiligo [14] or in large wounds or burns [15]; when the donor area is limited, it is advantageous to enlarge the donor skin to cover a larger body surface area. Meshing enlarges the donor skin. The skin is passed through a machine that makes small slits, which allows expansion similar to that in fish netting. In a meshed skin graft, the skin from the donor site is stretched to allow it to cover an area larger than itself. The size of the mesh varies in ratios from 1:1

Mesh grafts for vitiligo to 1:6. A 1:1 mesh has small slits that allow the donor skin to expand one time its original size. Likewise, a 1:2 mesh has slightly larger slits that allow the donor to be enlarged two times. Following meshgrafting pigmentation occurs at the spaces between the mesh, called the intricities, which fill in with new epithelial skin growth. This technique enables large areas to be grafted when the pigmented donor skin is limited.

Instruments 1 Padgett dermatome: Electrically operated dermatome for harvesting. The thickness is adjustable in thousandths of an inch by setting pointer on calibrated scale. 2 Ampligreffe® (Collin, France): Machine with a slightly inclined metal plate with spiral barrels which rotate and help to mesh the graft by rotating the handle. 3 Dermabrador–Kurtin wire planning brushes (Robbins).

Preoperative workup The patient’s consent is obtained. The preoperative workup includes complete blood count, bleeding time, clotting time, prothrombin time, platelet count, blood sugar, and screening for hepatitis B, venereal disease research laboratory (VDRL), human immunodeficiency virus (HIV), and fitness for general anesthesia.

Treatment of the recipient and donor area before surgery On the vitiligenous areas 0.075% of 8 methoxy psoralen is applied and after 10 min the affected part is exposed to ultraviolet A (UVA) in full body phototherapy unit for two consecutive days before surgery. The dosage of UVA would depend on the type and thickness of skin; 10–14 J/cm2 usually induces either marked erythema with areas of blistering, or large multiple blisters over the vitiligenous skin; which facilitates easy removal of epidermis before grafting. We recommend five to seven sittings of narrowband ultraviolet B (NB-UVB) or PUVA for the donor site before surgery to stimulate the melanocytes.

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Preoperative preparation Both donor and recipient sites are shaved and pre-medication as required for general anesthesia administered.

Procedure Harvesting Povidone iodine is used to sterilize the donor and recipient sites at the beginning of the procedure. It is useful to lubricate the skin at the donor site with sterilized white petrolatum to facilitate easy gliding of the dermatome over the skin. Under general anesthesia 0.0252 inch splitthickness graft is harvested using electrically operated Padgett dermatome or Duvals dermatome. A Humby knife can also we used. The dermatome is held in the dominant hand of the operator at a 30–45° angle from the donor skin surface. Greater angulation of the dermatome leads to gouging or trenching of the donor site skin. With the nonoperating hand providing traction behind the dermatome and the assistant providing traction in front of the dermatome, the dermatome is activated and advanced in a smooth, continuous motion over the skin with gentle downward pressure. After the appropriate length has been harvested, the dermatome is tilted away from the skin and lifted off the skin to cut the distal edge of the graft and complete the harvesting. The graft then may be gently washed to remove the lubricant and wrapped in a moistened saline sponge until it is ready to be used. The donor site typically has numerous small punctate bleeding spots with thin-to-intermediate thickness grafts; thicker grafts have fewer, larger bleeding points that bleed more briskly. Any exposure of fat indicates that excision of the graft was performed too deeply, probably because of incorrect assembly of the dermatome or error in technique.

Meshing The graft is transferred to an Ampligreffe for meshing. The Ampligreffe contains a slightly inclined metal plate over which the graft is placed with the dermis facing upwards (Fig. 16.1). One end of the graft is gently inserted between the spiral barrels of the Ampligreffe. The handle of the Ampligreffe

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Fig. 16.1 Ampligreffe with graft.

Fig. 16.2 Meshed graft on Sofratulle®.

is then rotated. This results in the graft passing between the two barrels which forms diamond shaped sieves or mesh in the graft. This increases the size to two or four times depending on the size of the sieve. Different Ampligreffe is used to obtain different size. We recommend 1:4 ratio expansions for vitiligo. This meshed skin with the dermal surface facing upward is spread on a non-adherent paraffin and framycetin gauze, Sofratulle® (Fig. 16.2). In facilities where mechanical meshers are not available, or are too time consuming to unpack and assemble, a simple quick and effective way of meshing a small split-thickness skin graft using a DiscardA-Pad (Johnson & Johnson Inc. TX, USA) can be

Fig. 16.3 Using the belly of a 15 No. blade to cut through the graft placed on Discard-A-Pad. Note that the blade sinks in the pad ensuring a net through and through cut.

Fig. 16.4 The meshed graft is peeled off along with the

glossy paper aiding in its application.

employed [16]. It is universally used in theaters for sharp disposal. It has an adhesive pad covered by a glossy paper, on which the graft is placed with the dermis side facing the operator. The belly of a 15 blade on a knife handle is used to cut through the graft and the paper. The soft pad under the paper allows the blade to sink through, ensuring a neat, through and through cut (Fig. 16.3). The cuts are made in rows strategically placed so that the cuts in the next row are staggered with the cuts of the previous row. If the first series of cuts shreds the top border rather than making holes (Fig. 16.4), it ensures maximal opening up of the meshed graft. A ten blade can be used to achieve larger cuts and

Mesh grafts for vitiligo hence larger expansion of the meshed graft. To facilitate staggered rows of cuts, the donor site can be marked with parallel lines before harvesting the graft. The cuts can then be placed over these lines ensuring a neater result. The glossy paper along with the meshed graft can then be peeled off the adhesive pad of the DiscardA-Pad and the graft is directly applied over the recipient site with added ease of handling (Fig. 16.4). The Discard-A-Pad is still usable during the procedure. The vitiligenous skin with phototoxic erythema and blister is rubbed vigorously with saline-soaked gauze held between thumb and index finger to remove the epidermis. Areas of epidermis not removed by this procedure are dermabraded using Kurtins stainless steel wire planning brush (Robbin). Hemostasis is easily obtained if the phototoxic blister is well formed. If in the event a dermabrader is used the bleeding is easily controlled by saline-soaked gauze piece within 4–5 minutes. The Sofratulle® with meshed skin is placed on the dermabraded area. Once the graft has been placed, the recipient site should be re-inspected for hemostasis. The grafts are kept in position by bandaging using salinesoaked gauze. Movement of muscles and joints may lead to displacement of the graft. So when large areas are to be grafted it is better to cut the graft into smaller sizes by cutting the Sofratulle® on which the graft has been placed. Since the graft is already meshed there is no risk of lifting up of the graft due to bleeding or oozing. Attention must be paid to placing the dermal side down, as the dermis and epidermis can appear very similar without close inspection in lighter-skinned individuals. Care should also be taken to prevent wrinkling or excessive stretching of the graft. The graft must then be secured in place to provide stability during initial adherence and healing. After grafting Sofratulle® followed by saline-soaked gauze is placed to prevent drying. We have observed that chances of drying are greater when the humidity is less and this can be prevented by placing saline-soaked gauze over the Sofratulle®. The dressing is placed to provide uniform pressure over the entire grafted area. These dressings are intended to immobilize the graft. Grafts placed on the extremities may be managed with elevation

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and compression dressing for the entire extremity distal to the graft, to prevent edema. A plaster of Paris cast can be used over joints to immobilize the graft.

Donor site dressing Donor site dressing in an aseptic way is crucial for a successful re-epithelialization of the donor site avoiding any morbidity secondary to infection or poor wound healing. Immediately after harvesting the split-thickness skin graft, the donor site is covered with warm adrenaline saline gauze (500 ml normal saline with 0.5 ml of 1:1000 adrenaline). At the time of donor site dressing, a liberal amount of 0.5% bupivacaine is applied topically and the site is covered with a layer of Kaltostat [17] (Calcium alginate dressing, ConvaTec Ltd.) The bupivacaine provides a prolonged local anesthetic effect improving pain control at the donor site. A generous layer of absorbent gauze and soakage pads is put on top of this and bandaged in place with Creppe bandage securely. The donor site is inspected in 10–14 days, by which time the donor site usually re-epithelializes under the dressing which gets lifted off as a result. If the dressing is still stuck, no attempt is made to remove it or soak it, but the adherent areas are trimmed off using a pair of scissors and redressed until it comes off on its own. Should there be a foul smell or soakage in the donor site dressing, it should be taken down early and treated accordingly.

Postoperative care Adequate rest to the grafted area with antibiotics and anti-inflammatory drugs is given. Rapid re-epithelialization occurs and the dressing becomes loose after a week and it can easily be removed. The patient is advised to undergo phototherapy after 7–10 days.

Advantages and limitations Mesh grafts are primarily useful in two situations: 1 When there is insufficient skin, as in extensive vitiligo and massive burn in which the skin graft needs to be expanded.

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2 When a very convoluted surface must be covered with a graft where a sheet might not adhere well. The basic principle of meshing consists of transforming a sheet graft into a mesh. This leads to various advantages. 1 Increase in graft size so as to cover larger area. 2 The mesh graft allows adaptation to irregular contours and surface without tension. 3 The larger gaps allow adequate drainage of exudates. 4 The skin margins are multiplied. Meshing of the skin graft, with cutting of the epidermis and the dermal tissue, releases maximal amounts of cytokines (growth factors) in the proportions as present in healthy donor skin, favoring wound healing, and keratinocyte culture in vivo. The contact of exposed dermis to the receptor bed is increased accordingly. With mesh expansion, the vascular ingrowth occurs not only at the dermis of the ribbons but also at the edges of the squares. This leads to the early “take” and viability of the mesh graft. The epithelial margins from which spreading of keratinocytes occurs are vastly multiplied. At the modest expansion of 3 to 1, the maximum distance between the ribbons is 0.15 in. (3.80 mm), which is sufficiently small for a speedy epithelialization [18]. Since dermabrasion is a time consuming procedure and is difficult to achieve uniform depth by this procedure, topical psoralen followed by UVA in higher dose prior to treatment leads to the separation of the epidermis from the dermis and the epidermis can rapidly be removed by using salinesoaked gauze [19]. So the induction of phototoxic blister helps to quickly dermabrade the recipient area with no risk of scarring. There is minimal risk of scarring at the donor site as very thin grafts are taken. After healing the grafted skin may have a checkerboard or crocodile skin appearance, which improves with time (Plate 16.1, facing p. 114).

Conclusion Meshed grafting enables rapid harvesting of the graft. The induction of phototoxic blister helps to quickly dermabrade the recipient area and also cover larger

areas. There is no risk of scarring by inducing a phototoxic blister at the recipient site since there is no involvement of deeper dermis. There is a minimal risk of scarring at the donor site as very thin grafts are taken. The irregularity of pigmentation which is noticed following all surgical procedures is likely to improve in due course. Even if minimal irregularity persists the patients prefer this to the depigmentation. This procedure is used when large areas have to be grafted and pigmented donor skin is limited.

References 1 Lanz O. Over transplantatie. Ned Tijidsch Geneesk 1907; 43:1335. 2 Tanner JC, Vandeput J, Olley JF. The mesh skin graft. Plast reconstr Surg 1964;34:287–92. 3 Douglas B. The sieve graft, stable transplant for covering large skin defects. Surg Gynecol Obstet 1930;50:1018. 4 Dragstedt LR, Wilson H. A modified sieve graft, full thickness skingraft for covering large skin defects. Surg Gynecol Obstet 1937;65:104. 5 Eroglu L, Uysal OA. An easy and affective method for preparing meshed skin grafts. Plast Reconstr Surg 2001; 108:1083. 6 Yanai A, Hiraga Y. Method for preparing mesh skin grafts without using skin-graft meshers. Plast Reconstr Surg 1991;88:524–6. 7 Vartak A. New economical skin graft expansion wheel. Burn 1992;18:157–8. 8 Koga M. Epidermal grafting using the tops of suction blisters in the treatment of vitiligo. Arch Dermatol 1988;124:1656–8. 9 Hann SK, Im S, Bong HW, Park YK. Treatment of stable vitiligo with epidermal grafting and PUVA. J Am Acad Dermatol 1995;32:943–8. 10 Olsson MJ, Juhlin L. Transplantation of melanocytes in vitiligo. Br J Dermatol 1995;132:587–91. 11 Behl PN, Bhatia RK. Treatment of vitiligo with autologous thin Thiersch’s grafts. Int J Dermatol 1973;12: 329–31. 12 Roenigk Jr. HH. Dermabrasion. In: Roenigk RK and Roenigk Jr. RH (eds.) Dermatologic Surgery, Principles and Practice, 1st edn. New York: Marcel Dekker Inc., 1989;959–78. 13 Hruza GJ. Dermatologic surgery: introduction and approach. In: Freedberg IM, Austen NK, Lowell A, Goldsmith LA, Katz SI, et al. (eds.) Dermatology in General Medicine. New York: McGraw-Hill, 1999;2925–6.

Mesh grafts for vitiligo 14 Srinivas CR, Rai R, Uday Kumar P. Meshed split skin graft for extensive vitiligo. Indian J Dermatol Venereol Leprol 2004;70:165–7. 15 Papini R. Management of burns injuries of various depths. Br Med J 2004;329:15–60. 16 Sinha M. Meshing small skin grafts manually: a simple, quick, and effective method using discard-a-pad. Ann Plast Surg 2005;55:433–4.

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17 Attwood AI. Calcium alginate dressing accelerates split skin graft donor site healing. Br J Plast Surg 1989; 42:373–9. 18 Vandeput J, Nelissen M, Tanner JC, Boswick J. A review of skin meshers. Burns 1995;21:364–70. 19 Albert S, Srinivas CR, Shenoi SD, Pai SB, Sabitha L. Phototoxic blister induction in vitiligo surgery. Br J Dermatol 1998;138:644–8.

CHAPTER 17

Flip-top pigment transplantation Brent E. Pennington, Jean L. Bolognia and David J. Leffell

Introduction The surgical management of vitiligo was first reported in 1947 by Haxthausen [1]. This initial paper documented melanocyte transplantation through the transfer of split-thickness skin grafts between normally pigmented skin and depigmented vitiliginous patches. This original report stimulated the development of multiple other surgical interventions including punch grafts [2], pinch grafts [3], suction blister grafts [3], split-thickness skin grafts [4], and transfer of cultured autologous melanocytes [5,6]. Many of the traditional surgical modalities are complicated by the need for specialized equipment, the extensive time required for the procedure, or the variable results of repigmentation with associated scarring. Flip-top pigment transplantation, first developed by Leffell and colleagues [7], represents a modification of the pinch grafting technique in which thin epidermal donor grafts are placed beneath a hinged epidermal flap at the recipient site and sealed into place. This simple procedure performed with basic dermatological equipment provides excellent repigmentation with minimal associated scarring. The ease of the procedure, combined with the low cost and excellent results make this technique especially valuable for a disease that crosses social, ethnic, and economic boundaries.

Indications for flip-top pigment transplantation Prior to the procedure, patients with vitiligo must be carefully screened to determine those who are suitable candidates for surgical management. The initial screening involves identifying patients unresponsive to medical therapy who clearly have stable disease.

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In our protocol, vitiliginous patches that have remained static in size and shape in excess of 1 year are considered stable. Individuals who continue to develop new areas of vitiligo, demonstrate expansion of existing patches, or have exhibited the Köbner phenomenon (i.e. the development of new lesions at sites of trauma) should be excluded from consideration for the flip-top technique [8]. In order to screen for the possibility of Köbner phenomena, a minigraft test should be done in each patient before large-scale melanocyte transplantation is performed [9]. The flip-top procedure is valuable in the treatment of individuals with focal, segmental or generalized vitiligo. The technique may be easily employed in all regions of the body with the exception of the palms and soles. In these areas, the thickness of the epidermis makes the elevation of the flip-top and placement of the donor graft challenging. The procedure has minimal risk of scarring or “cobblestoning”, which makes it particularly useful for the management of lesions in cosmetically sensitive regions such as the face and neck.

Procedure and materials The procedure begins with the identification of a normally pigmented donor site for harvesting of minigrafts. In the majority of our patients, the medial aspect of the upper arm or axilla has been utilized. The donor site is injected with 1% lidocaine with epinephrine (1:100,000) using a 30-gauge needle such that a superficial wheal is raised. It is imperative that the anesthetic be administered as a superficial bleb. This minimizes scarring at the donor site and allows for harvesting of thin grafts with minimal dermis attached to the undersurface.

Flip-top pigment transplantation A sterile razor blade is then used to shave the donor epidermis at the dermal-epidermal junction. Depending upon the practitioner’s skill, this donor epidermis may be removed in multiple 2–4 mm segments or in one longer strip, which is later sectioned into minigrafts measuring approximately 1–2 mm in diameter. The donor grafts should immediately be placed on gauze soaked in isotonic saline in a Petri dish. The donor sites are dressed with petroleum jelly and an adhesive bandage (Band-Aid®). Multiple recipient sites are then identified in a depigmented patch. Given that, based on our observations, pigment may spread following the flip-top pigment transplantation, these recipient sites should be positioned a minimum of 7–10 mm apart from each other until the resultant pigmentation pattern (i.e. amount of subsequent pigment spread) in each individual is determined over time. The recipient sites are anesthetized in the same manner as the donor sites by raising a small superficial wheal. The razor blade is then used to elevate a 4–5-mm hinged flap, or flip-top, consisting of epidermis with minimal attached papillary dermis. This flap is folded back such that the papillary dermis of the recipient site is exposed. A donor graft is then placed with the dermis side down into the recipient site. The hinged flap is then folded back to its original position so that it covers the donor graft. Approximately 0.1 ml of cyanoacrylate (GluStitch™, GluStitch Inc., Point Roberts, WA or Indermil™ US Surgical, North Haven, CT) is applied to the roof of the flip-top and immediate surrounding skin to secure the flap and donor graft in place. In this position, the hinged flap of epidermis serves as a biological dressing for the donor graft and protects it from displacement. Once the cyanoacrylate has dried, a transparent polyurethane dressing (Tegaderm™, 3M Company, St. Paul, MN) may be applied over the recipient sites. Following the procedure, the patient should be cautioned to avoid trauma or excessive motion involving the affected area to prevent graft displacement. The transparent polyurethane dressing is removed at 1 week, and the recipient sites are examined for graft survival. If further melanocyte transplantation is required at the site, it should be delayed for a period of at least a month to allow an assessment of graft take.

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Results High rates of pigment retention at graft sites as well as pigment spread into surrounding areas of vitiligo have been observed with flip-top pigment transplantation. In the first reported series of patients treated with this method, repigmentation was observed in 22 of 25 (88%) graft sites that were not displaced by patient trauma [7]. Of these 22 successful recipient sites, all displayed continued pigment spread of 2–3 mm beyond the original graft perimeter in the 3 months following the procedure. No “cobblestoning” or uneven pigment distribution was observed in these patients. The procedure has minimal associated risk and is usually tolerated without difficulty by the patients. In the above series, no scarring was identified at the graft recipient sites in any of the patients. To date, no significant pain, bleeding, infection, or contact dermatitis to cyanoacrylate has been observed in any of the individuals treated by this method at our institution.

Clinical vignettes Case 1: The patient presented as a 44-year-old woman (Fitzpatrick Skin Type II) who had an 8-year history of vitiligo which was stable at the time of presentation to us. Her previous treatments included 1 year of psoralen plus ultraviolet A (PUVA) therapy and consultation regarding excimer laser treatment. Due to the extent of the area of involvement the patient sought consultation regarding pigment cell transplantation. On physical examination the patient had large patches of depigmentation scattered over the neck, chest, shin, axilla, and under both breasts. The patches ranged in size from 2 to 7 cm in their longest diameter. The patient identified the area of greatest concern to her which was on the right lateral neck extending over the clavicle. We focused on a strategy of trying to complete repigmentation of one complete area rather than distributing the transplantation effort over a large number of discontiguous sites. After informed consent was obtained the patient underwent flip-top pigment cell transplantation in nine separate sessions over a 4-year period. The number of transplants at each session varied from two to nine. Fig. 17.1A

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(A)

(B) Fig. 17.1 (A) Vitiligo patch on the right neck prior to

pigment cell transplantation. (B) After more than 50 grafts, the area of vitiligo is no longer identifiable as such. Further grafting will be done to fill in those areas which have not pigmented from the existing grafts. Note how the quality of the pigmentation in the transplanted area is similar to that of adjacent skin. No scarring is noted.

demonstrates the appearance of the right lateral neck prior to initial treatment and Fig. 17.1B demonstrates the appearance after a total of 52 transplants. During her treatment course the patient

occasionally used tacrolimus or pimecrolimus but no significant benefit was noted. Case 2: The patient presented as an 18-year-old college freshman (Fitzpatrick Skin Type IV) with a history of stable segmental vitiligo on the left side of his face for at least 5 years. Prior to consultation he had used strong topical steroids, PUVA, and tacrolimus The patient also attempted some Ayurvedic medication to no avail. On physical examination the patient was a healthy appearing Indian gentleman with a 40 cm2 depigmented patch over the left chin and left lip. There were a few 1–2 mm scattered areas of repigmentation around follicles. The patient was eager to treat the area of depigmentation on his face which was a major concern to him as he was beginning to embark on his college career. A total of 51 pigment cell transplants were performed in nine sessions over a period of 1 year. The minimum number of grafts was three in one session and the maximum number of grafts was eight in one session. Plate 17.1A (facing p. 114) demonstrates the patient prior to initiation of the pigment cell transplantation procedure and Plate 17.1B (facing p. 114) demonstrates the appearance of the vitiligo area approximately 1 year after treatment was initiated. Case 3: The patient is now a 55-year-old corrections department employee who presented approximately 4 years ago with vitiligo of both hands. Previous treatment was unsuccessful and he requested pigment cell transplantation. Due to the location of the vitiligo in the interdigital web space and over the knuckles, we considered that pigment cell transplantation would have a low chance of success because of the local mobility. Nonetheless we initiated treatment. The patient underwent approximately seven treatments to his right knuckle area as well as in is left dorsal thumb area. Plate 17.2A (facing p. 114) demonstrates the preoperative appearance of the vitiligo on the right hand and Plate 17.2B (facing p. 114) demonstrates improvement after his initial sessions with pigment cell transplantation.

Discussion Significant progress has been made over the last century in the medical treatment of vitiligo with the use of topical corticosteroids, topical immunomodulators,

Flip-top pigment transplantation natural sunlight, narrow-band ultraviolet B (NBUVB) light, and PUVA light. Despite these advances, a significant percentage of patients will experience only partial or no repigmentation even with a combination of medical therapies [9]. For these individuals, various surgical techniques remain a valuable alternative. Surgical options include transplantation of cultured autologous melanocytes, suction blister grafts, split-thickness skin grafts, and punch grafts. These techniques have demonstrated variable degrees of success with regard to repigmentation; complications also vary depending on the specific technique that is chosen. In our experience, the flip-top transplantation technique has proven to be a simple, reliable method for the treatment of vitiligo resistant to medical therapy. One of the major advantages of this technique, in comparison to other forms of melanocyte transplantation, is the lack of specialized equipment required for the procedure. The transplantation may be performed in its entirety utilizing inexpensive materials available in most dermatology offices worldwide. Additionally, the ease of the technique allows practitioners with various levels of surgical training to reproducibly achieve excellent cosmetic results. The simplicity also enables multiple sites to be transplanted in one visit in a reasonable amount of time. The flip-top technique appears to offer cosmetic results that are equal or superior to other forms of melanocyte transplantation. No visible scarring has been observed in long-term follow-up of our patients treated with this method. In addition, the pigment transplanted has a smooth, uniform appearance and there is no evidence of “cobblestoning” as can be observed with other skin grafting techniques. Proper positioning of both the anesthetic and the razor blade is critical in the process of harvesting the grafts and elevation of the flip-top. Extremely thin flaps consisting of epidermis alone or epidermis with only scant papillary dermis provide optimal cosmetic results with minimal or no associated scarring. The flip-top technique utilizes the hinged flap as a biological dressing over the donor epidermal graft. Some clinicians have proposed that this biological dressing effect enhances transplanted melanocyte survival leading to increased rates of repigmentation

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[10]. The biological dressing may also reduce the risk of infection at the recipient sites. Lastly, this hinged flap when sealed with cyanoacrylate offers the advantage of securing the minigraft in place. As a result, there is less risk of graft displacement and failure, which often complicates other forms of pinch grafting on mobile areas of the body (see Case 2). Growth of terminal hairs at the recipient site may lead to a limited degree of graft displacement even with the flip-top technique. Therefore, it is often beneficial to pluck any terminal hairs present at the recipient site prior to the procedure. Although flip-top pigment transplantation presents several benefits over other surgical modalities, it does have some limitations. Given the small size of the minigrafts, multiple sessions may be required for the treatment of large vitiliginous patches. Certain areas of the body, namely the palms and soles, are not ideal for the flip-top technique due to the thickness of the epidermis.

Conclusion Flip-top pigment transplantation is a simple, reliable method for the transfer of autologous melanocytes in the treatment of stable vitiligo refractory to medical therapy. This technique can be mastered by dermatologists around the world due to the straightforward nature of the procedure and the lack of any need for specialized equipment. Additionally, flip-top pigment transplantation consistently provides excellent repigmentation with minimal associated scarring.

References 1 Haxthausen H. Studies on the pathogenesis of morphea, vitiligo, and acrodermatitis atrophicans by means of transplantation experiments. Acta Derm Venereol 1947;27:352–67. 2 Falabella R. Repigmentation of stable leukoderma by autologous minigrafting. J Dermatol Surg Oncol 1986; 12:172–9. 3 Falabella R. Epidermal grafting: an original technique and its application in achromic and granulating areas. Arch Dermatol 1971;104:592–600. 4 Mutalik S, Ginzburg A. Surgical management of stable vitiligo: a review with personal experience. Dermatol Surg 2000;26:248–54.

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5 Falabella R, Escobar C, Borrero I. Transplantation of in-vitro cultured epidermis bearing melanocytes for repigmenting vitiligo. J Am Acad Dermatol 1989; 21:257–64. 6 Falabella R, Escobar C, Borrero I. Treatment of refractory and stable vitiligo by transplantation of in vitro cultured epidermal autografts bearing melanocytes. J Am Acad Dermatol 1992;26:230–6. 7 McGovern TW, Bolognia J, Leffell DL. Flip-top pigment transplantation. Arch Dermatol 1999;135:1305–7.

8 Falabella R, Arrunategui A, Barona MI, et al. The minigrafting test for vitiligo: detection of stable lesions for melanocyte transplantation. J Am Acad Dermatol 1995; 32:228–32. 9 Ortonne JP. Vitiligo and other disorders of hypopigmentation. In: Bolognia JL, Jorizzo JL and Rapini RP, (eds.) Dermatology. New York: Mosby, 2003;947–73. 10 Gupta S, Kumar B. Surgical pearl: autologous biological dressing for epidermal grafting in vitiligo and other achromic disorders. J Am Acad Dermatol 2003;48:430–1.

CHAPTER 18

Ultrasonic abrasion and seed grafts for vitiligo Katsuhiko Tsukamoto, Reiko Kitamura and Osami Takayama

Various surgical treatments have been developed to treat stable refractory vitiligo [1]. Ultrasonic abrasion and seed-grafts therapy is one of the new developments in the surgical managements [2]. In this method, only the epidermis of the vitiligo lesion is abraded with an ultrasonic surgical aspirator. This ultrasonically abraded recipient site is re-epithelialized with epithelial seed-grafts, and then treated with psoralen plus ultraviolet A (PUVA) therapy. Epidermal abrasion with an ultrasonic surgical aspirator leaves no scar. Seed-grafts can cover a wider area from a smaller donor site compared with the sheetgrafts technique. PUVA treatment then facilitates the spreading of pigmentation from the seed-grafts to the surrounding areas. This new method is an easy, safe, inexpensive, and effective treatment modality for stable vitiligo.

Ultrasonic surgical aspirator The ultrasonic surgical aspirator was first developed for the removal of dental plaque, in 1947. It was applied for the removal of cataracts in 1967. Currently the ultrasonic surgical aspirator is used in a wide range of procedures in different specialties, such as neurosurgery, general surgery, renal surgery, gynecologic surgery, and plastic surgery [3]. Cavitron Ultrasonic Surgical Aspirator (CUSA: Valleylab, Boulder, USA) is the most popular one in the world and now several other companies have developed similar equipments. The handpiece has 23,000–36,000 vibrations per second on the tip and can destroy the epidermal tissue apart and sucked by vacuum into a container (Fig. 18.1), but collagenrich tissues, such as dermis, blood vessels, and

Tissue Vibration

Tip of handpiece

Suction

Saline water

Fig. 18.1 Schematic illustration of the structure and principle of ultrasonic surgical aspirator.

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(Normal skin) Epidermis

Dermis (Conventional grinder) Loss of dermis

nerves, are not destroyed as they can absorb its vibrations. Figure 18.2 is a schematic diagram illustrating comparison between the skin denuded by conventional motor dermabrader and by ultrasonic surgical aspirator. The motor grinder (high-speed dermabrader) removes the skin in a flat plane, thus removing the dermis in some areas and retaining the epidermis at some places [4]. In contrast, the ultrasonic surgical aspirator cleanly removes only the epidermis and the dermis remains intact including hair follicles and eccrine ducts.

Remainder of epidermis

Ultrasonic abrasion and seed-grafts therapy [2]

(Ultrasonic abrasion)

Fig. 18.2 Diagrammatic representation of abraded skin

by conventional motor grinder and ultrasonic surgical aspirator. Note a clean split at dermo-epidermal junction by the later.

The sequence of this method is illustrated in Fig. 18.3. First, we use an ultrasonic surgical aspirator to abrade only the epidermis of the vitiligo lesions. A thin skin graft is taken from the donor site, minced and then placed onto the abraded vitiligo lesions. One month after the grafting, topical PUVA therapy is started in order to cover the entire area with pigmentation.

Ultrasonic Abrasion, Seed-Grafting

Thin piece of epidermal graft skin

Ultrasonic abrasion of vitiligo lesion

Minced fragments less than 1 mm2

These minced skin pieces were then placed onto the epidermal-abraded vitiligo lesions

Fig. 18.3 The sequence of ultrasonic abrasion and seed-graft therapy.

Ultrasonic abrasion and seed grafts for vitiligo Treatment: We used the CUSA Excel (Valleylab, Boulder, USA) or the Sonopet UST-2000 (Miwatec, Kawasaki, Japan) (Fig. 18.4). It is used with a short handpiece having a 2.0–2.5-mm tip diameter. A local anesthetic is first administered, and then, while holding the handpiece like a pen, the operator presses the tip onto the epidermis of the vitiligo lesion and moves it around in continuous, circular, brush like motions. A very thin piece of skin graft (0.005–0.008 inches thick) is taken from a normally pigmented donor site, usually the chest or arms, with a hand dermatome (Keisei Co., Japan). The graft skin (about 2
4 cm) is then minced using a surgical knife or scissors into fragments less than 1 mm2. The minced skin pieces are then placed onto the epidermal-abraded vitiligo lesion and are covered with Trex gauze with gentamycin, dry sterile gauze, and an adhesive bandage for at

(A)

141

least 5 days. The donor site is also covered with the same material. The wound dressing is removed 5–7 days after grafting. Topical PUVA treatment is started 1 month after the grafting, usually given twice weekly to enhance the spread of pigment cells from the graft. Several patients with stable segmental vitiligo were treated using this method. Each transplantation was successful, and excellent repigmentation without any scarring was observed at all grafting sites. No recurrences or complications of the treatment were seen.

Cases Case 1: A 12-year-old boy presented with a 6-year history of segmental vitiligo on his face (Plate 18.1A, facing p. 114). His lesion had been treated with steroid ointment initially and then with PUVA therapy for

(B) Fig. 18.4 CUSA and SONOPET ultrasonic surgical aspirators.

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Chapter 18 6 months, almost normal skin color was restored (Plate 18.2B, facing p. 114).

Comment

Fig. 18.5 Histological examination of the skin abraded

with ultrasonic surgical aspirator. The epidermis is cleanly removed along the basement membrane but the dermis, including hair follicles and eccrine ducts, is still intact. (HE X100).

3 years, but that treatment failed to achieve repigmentation. For ultrasonic abrasion and seed grafting, he was given general anesthesia. The vitiligo lesion was abraded by the surgical ultrasonic aspirator (Plate 18.1B, facing p. 114). A thin piece of thin split-thickness skin graft was taken from his arm and minced into small fragment. The minced skin pieces were then placed onto the ultrasonically abraded recipient patch (Plate 18.1C, facing p. 114). One week after the transplantation, the minced skin grafts were still attached to the recipient site. One month later, some were detached but melanocytes had already been transferred to the recipient area resulting into islands of repigmentation. The patient was then started on PUVA therapy. Four months after the transplantation, good repigmentation was seen at the grafting site without any scarring (Plate 18.1D, facing p. 114). No recurrence or other complications were seen even 4 years after the transplantation. Case 2: A 14-year-old boy presented with a 2-year history of segmental vitiligo on his finger (Plate 18.2A, facing p. 114). He had been treated with steroid ointment initially and then with PUVA therapy for more than 1 year but the treatment was not effective. A local anesthetic was first administered, and then procedure of ultrasonic abrasion and seed grafting was performed. Two months after the transplantation, good repigmentation was observed. At

Vitiligo is a common skin disease in all parts of the world, although its exact pathogenesis is not clear [5]. The most frequently used treatments are topical steroids and PUVA therapy [6]. Stable refractory vitiligo is treated with various surgical treatments, such as epidermal sheet grafting, mesh grafting, minigrafting, suction blister epidermal grafting, and transplantation of cultured melanocytes. In these procedures, the recipient site is generally denuded with a motor-driven grinder, which sometimes leaves a hypertrophic scar. Liquid nitrogen or PUVA may also be used for inducing blisters at recipient site, however, if used excessively, these can cause severe inflammation, hypertrophic scarring, and peripheral post-inflammatory hypo/hyperpigmentation [7]. The ultrasonic surgical aspirator abrades only the epidermis of the skin. Epidermal abraded areas re-epithelialize within a few weeks, and do not leave a scar. On histological examination of the skin that has been denuded using the ultrasonic surgical aspirator, it was found that the epidermis was cleanly removed but the dermis, including hair follicles and eccrine ducts, remained intact (Fig. 18.5). Another advantage is that the ultrasonic surgical aspirator with a short handpiece of 2.0–2-mm tip can be handled easily and safely to remove the vitiligo lesions, even spotty lesions and lesions in intricate regions, which are difficult to denude using a conventional motor-driven grinder, liquid nitrogen or PUVA therapy. Every vitiligo patient is not a good candidate for the ultrasonic surgical treatment and an appropriate selection of patients is the key to success. The best indication is segmental vitiligo on the face and extremities, even if lesions are small, spotty or located in intricate regions. Large areas of the vitiligo are difficult to abrade with an ultrasonic surgical aspirator, as it takes a long time to remove the epidermis covering an extensive vitiligo lesion using a handpiece having a 2.0–2.5-mm tip diameter. In such cases, CO2 laser with a computerized scanning device may be more useful because it can remove

Ultrasonic abrasion and seed grafts for vitiligo the superficial skin of a fixed depth even though it removes the skin in a flat plane [8]. In conclusion, this new method of ultrasonic abrasion and seed grafting is an effective treatment for stable vitiligo and is also an easy, safe, and inexpensive surgical treatment. Surgical therapy should be selected individually for each patient depending on the type, size, location of the vitiligo. Available technology and physician’s experience are also important for success.

References 1 Van Geel N, Ongenae K, Naeyaert JM. Surgical techniques for vitiligo: a review. Dermatology 2001;202:162–6. 2 Tsukamoto K, Osada A, Kitamura R, Ohkouchi M, Shimada S, Takayama O. Approches to repigmentation of vitiligoskin: new treatment with ultrasonic abrasion, seed-grafting and psoralen plus ultraviolet A therapy. Pigment Cell Res 2002;15:331–4.

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3 Ito Y, Kondo S, Sumiya N, et al. Derabrasion using an ultrasonic surgical aspirator. Plastic Reconstruct Surg 1996; 97:1034–9. 4 Olsson M, Juhlin L. Epidermal sheet grafts for repigmentation of vitiligo and piebaldism, with a review of surgical techniques. Acta Derm Venereol (Stockh) 1997;77:463–6. 5 Kitamura R, Tsukamoto K, Harada K, et al. Mechanisms underlying the dysfunction of melanocytes in vitiligo epidermis: role of SCF/KIT protein interactions and the downstream effector, MITF-M. J Pathol 2004;202: 463–75. 6 Njoo MD, Westerhof W, Bos JD, Bossuyt MM. The development of guidelines for the treatment of vitiligo. Arch Dermatol 1999;135:1514–21. 7 Kim HY, Kang KY. Epidermal grafts for treatment of stable and progressive vitiligo. J Am Acad Dermatol 1999;40:412–7. 8 Kahn AM, Ostad FA, Moy RL. Grafting following shortpulse carbon dioxide laser de-epithelialization. Dermatol Surg 1996;22:965–8.

CHAPTER 19

Complications and limitations of melanocyte transplantation Yvon Gauthier

Medical treatments are often ineffective in many patients with vitiligo or other leukodermas and surgical methods have therefore been developed. Several methods of autologous transplantation of melanocytes have been described to repigment achromic macules that are stable and those that are refractory to medical therapies. The aim of melanocyte transplant is to replace melanocytes that are missing in the achromic areas with melanocytes obtained from a normally pigmented donor site. Three different transplantation methods have been developed using skin transplants, basal cell layer suspension, and autologous cultured melanocytes. In the majority of cases, these techniques are safe and the complications are rare, on both the donor and the grafted sites. Nevertheless, based on data derived from a literature search [1] and from our personal experience we would like to conduct for each technique a systematic review of the complications which are observed during and after melanocytes transplantation.

Adverse effects at donor site Scar formation at the donor site seems to be the most undesirable adverse effect reported with minigrafting, so it would be preferable to select a donor site at an inconspicuous pigmented area, for example the buttocks. When minigrafting is successful, it is still possible that the donor site will not repigment as a result of the Köbner phenomenon, particularly in cases of unstable vitiligo. Falabella [2] suggests that a minigrafting test should be performed several months before melanocytes transplantation. This is to determine the stability of vitiligo.

Adverse effects at recipient site A cobblestone appearance of the acceptor area has been reported by several authors (Fig. 19.1). But it is possible to avoid this complication by making the holes in the recipient area about 1 mm deeper than the thickness of the grafts [3]. When this technique

Complications of melanocyte transplantation Adverse effects reported after skin transplant

Minigrafting technique One to two millimeters full thickness punch grafts are harvested from normally pigmented donor site and are then transplanted to depigmented recipient sites from which similar punch grafts have been removed.

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Fig. 19.1 Cobblestone appearance after minigrafting on

the ankle.

Complications and limitations of melanocyte transplantation

145

is performed on the face, it is important to do it with extra caution.

Roof epidermal blisters grafting Epidermal blister grafting involves the formation of epidermal blisters by application of a negative pressure to the normally pigmented skin. After blisters are formed, the depigmented epithelium is removed and the roofs of the pigmented donor blisters are transplanted to the denuded lesional area [4].

Adverse effects at donor site No scarring has been observed with this technique. Transitory hyperpigmentation was mostly reported. But this hyperpigmentation usually faded after some months.

Adverse effects at recipient site As with other techniques, imperfect color matching occurs in about 10% of cases.

Ultra-thin epidermal sheet transplantation With an electrical dermatome 0.8-mm ultra-thin epidermal sheet can be harvested and grafted on the denuded achromic macules. With this method larger areas of up to 200 cm2 have been treated with rapid and good results [5].

Adverse effects at donor site Transient or permanent hypopigmentation has been reported on the donor site. Permanent hypopigmentation usually occurs in unstable vitiligo. Less frequently, scars or keloid formation can occur.

Adverse effects at recipient site If the dressing or the immobilization is not correctly performed, it may result in a partial loss of grafts. A benign but frequent adverse effect is the milia formation at the surface of the graft approximately 2 months after the grafting (Fig. 19.2). That can easily be removed with a vaccinosyle. Thick margins, which have been observed in some patients undergoing split thickness graftings, can be treated with repeated dermabrasion. The local infections can occur very rarely if the patient does not keep the dressing clean.

Fig. 19.2 Milia after ultra-thin epidermal sheet

transplantation.

Adverse effects reported after basal cell layer suspension transplant A sample of superficial pigmented skin is obtained by shave biopsy either from the scalp [6] or from the buttocks [7]. The skin samples are immediately immersed in a 0.25% of trypsin solution and kept at 4°C for 18 hours (soft trypsinization), or at 37°C for 1 hour (hard trypsinization). After trypsinization, cells of the basal layer including keratinocytes and melanocytes are easily separated and concentrated in a medium which was injected into blister raised on achromic zone by freezing [6,7] or applied to dermabraded vitiliginous areas [8]. The size of the treated recipient site is about 8–10 times larger than the donor area.

Adverse effects at donor site We have never observed hypopigmentation on the donor site located on the scalp. The wound healing

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is very fast and normally pigmented hair regrows in about 1 month. When the donor site is the buttocks, there might be a greater risk of local depigmentation, especially if the vitiligo is unstable.

Adverse effects at recipient site With the use of separated cells milia-like eruptions were not observed. In approximately 20% of cases an inflammatory hyperpigmentation occurs, which fades after 4 or 6 months (Plate 19.1, facing p. 114).

Adverse effects reported after cultured autologous melanocytes or cultured epidermis transplant Transplants composed of pure cultured melanocytes have been made possible by the development of the techniques used to grow melanocytes in vitro. When enough melanocytes are obtained they are transplanted into the previously denuded recipient skin by dermabrasion or laser abrasion [9,10]. More recently the use of autologous epidermal cultured bearing melanocytes has also been proposed [11]. Large quantities of cultured epidermal grafts bearing a physiological number of melanocytes are prepared by a cell culture laboratory and applied on denuded achromic lesions [12].

Adverse effects at donor site Usually the size of skin biopsy specimen ranged from 0.5 to 4 cm2 according to the percentage of body surface to be treated. Therefore the scar at the donor site is small and not of much concern.

Adverse effects at recipient site Except failure of complete repigmentation or a transitory inflammatory hyperpigmentation observed on the grafted area, no other complications have been reported after cultured autografts.

Limitations of melanocyte transplantation Vitiligo can be treated in many ways. Melanocyte transplantation techniques are usually combined with previous or subsequent medical treatments

(corticosteroids or phototherapy). Distinction between active and stable phases of the disease is important for the selection of an appropriate therapy. Active vitiligo usually requires medical therapy. Surgical therapy is indicated only when the vitiligo is stable and when medical treatments have failed. There are many causes which can limit the use of melanocyte transplantation.

Limitations related to the vitiligo pathogenesis The pathogenesis of vitiligo remains unclear. The loss of melanocytes could be due to “melanocytorrhagy” and not to melanocyte destruction, which has never been observed in vivo. According to our hypothesis [13,14] autoimmune, oxidative, or neural processes could promote the melanocytic detachment and their transepidermal elimination after repeated mechanical traumas. Therefore, vitiligo macules at sites most likely to have strong and repeated mechanical traumas must not be selected for transplantation because these macules have lower chance of retaining their repigmentation. The other problem is that we still do not have any definitive test available to determine whether the disease is active or not. When vitiligo vulgaris is associated with hypothyroidism it is less likely to respond to the transplantation procedure. Longterm follow-up studies are therefore needed to address this issue.

Limitation related to the location of vitiligo macule While the success rate has been high in most body sites, we were unable to obtain substantial improvement of achromic lesions on upper and lower extremities or in periorificial areas of the face. The poor results could be explained by the difficulty in immobilizing these areas, interfering with the “take” of the skin grafted. On curved areas like on the eyelids, the basal cell layer transplant is impractical, whereas our experience also gave poorer results. But Olsson has here successfully performed a manual dermabrasion and basal cell layer suspension with great care and outcome [15]. Van Geel et al. [16] had increased the viscosity of the cellular grafts by adding

Complications and limitations of melanocyte transplantation hyaluronic acid, which improved adhesion of the graft to dermabraded skin.

Limitation related to the size of achromic areas According to Olsson [15] the probability of a successful transplantation outcome was 20 times higher in patients with 0–100 cm2 of achromic areas, 3 times higher in those with 101–500 cm2, and 2 times higher in 501–500 cm2 group, compared with the group of patients who had achromic areas of over 1500 cm2. Therefore large areas exceeding 500 cm2 are not suitable for several sessions of transplantation, except in cases of segmental vitiligo. Usually patients with large macules of vitiligo vulgaris lose the repigmentation more often than patients with smaller macules in post-transplantation period 15].

Limitations related to the transplantation technique The best method could be the method that allows the coverage of larger body surface areas in a shorter time and at a reasonable cost. Cultured melanocytes transplant or cultured epidermal autografts have the advantage that large areas can be covered in one session, but this is the most expensive and difficult method. Cell culture laboratories are needed, which restricts this method to a few big hospitals. The ultra-thin epidermal sheet method needs an operation theater well equipped for skin grafts. Postoperative immobilization for 1 day in an in-patient department is important to improve the adhesion of the graft. Finally, minigrafting, suction blister epidermal grafting, and basal cell layer suspension transplantations are the only methods which do not require expensive culturing conditions and high-tech laboratories. Nevertheless, basal cell layer suspension method requires a dermatologist who is familiar with the separation of human skin after trypsinization under sterile conditions. Although there has never been an increased risk of either carcinomas or melanoma reported, according to some authors, the use of pure melanocytes culture should be avoided because of the remote possibility of carcinogenicity [17].

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Conclusion In combination with medical treatments, melanocyte tranplantations are the methods of choice to repigment stable vitiligo macules. Progressive extensive vitiligo should never be selected for transplantation. Generally most of the transplantation methods are safe and the risks of adverse reactions are low.

References 1 Njoo MD, Westerhof W, Bos JD, Brossuyt M. A systematic review of autologous transplantation methods in vitiligo. Arch Dermatol 1998;134:1543–9. 2 Falabella R. Treatment of localized vitiligo by autologous minigrafting. Arch Dermatol 1988;124:1649–55. 3 Boersma B, Westerhof W, Bos JD. Repigmentation in vitiligo vulgaris by autologous minigrafting. J Am Acad Dermatol 1995;33:990–5. 4 Koga M. Epidermal grafting using the tops of succion blisters in treatment of vitiligo. Arch Dermatol 1988;124:1656–8. 5 Olsson M, Juhlin L. Epidermal sheets grafts for repigmentation of vitiligo and piebaldism, with a review of surgical techniques. Acta Derm Venerol 1997;77:463–6. 6 Gauthier Y, Surleve-Bazeille J-E. Autologous grafting of non cultured melanocytes, a simplified method for treatment of depigmented lesions. J Am Acad Dermatol 1992;26:191–4. 7 Gauthier Y. Les techniques de greffe mélanocytaire. Ann Dermatol Vénéréol 1995;122:627–31. 8 Olsson M, Juhlin L. Leucoderma treated by transplantation of a basal cell layer enriched suspension. Br J Dermatol 1998;138:644–8. 9 Olsson M, Juhlin L. Transplantation of melanocytes in vitiligo. Br J Dermatol 1995;132:587–91. 10 Kaufmann R, Greiner D, Kippenberger, Bernd A. Grafting of in-vitro cultured melanocytes onto laserablated lesions of vitiligo. Acta Derm Venereol 1998;78:136–8. 11 Falabella R, Escobar C, Borrero I. Transplantation of in vitro-cultured epidermal melanocytes for repigmenting vitiligo. J Am Acad Dermatol 1989;21:257–64. 12 Guerra L, Capurros S, Melchi F, Primavera G, De Luca M. Treatment of stable vitiligo by Timedsurgery and transplantation of cultured epidermal autografts. Arch Dermatol 2000;136:1380–9. 13 Gauthier Y, Cario-André M, Taïeb A. A critical appraisal of vitiligo etiologic theories. Is melanocyte loss a melanocytorragy? Pigm Cell Res 2003;16:322–32.

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14 Gauthier Y, Cario-André M, Lepreux S, Pain C, Taïeb A. Melanocyte detachment after friction in non lesional skin of patients with generalized vitiligo. Br J Dermatol 2003;148:95–101. 15 Olsson M, Juhlin L. Long-term follow-up of leucoderma patients treated with transplants of autologous cultured melanocytes, ultra-thin epidermal sheets and basal cell layers suspension. Br J Dermatol 2002; 147:893–904.

16 Van Geel N, Ongenae K, De Mil M, Vander Haeghen Y, Naeyaert JM. Double blind placebo-controlled study of autologous transplanted epidermal cell suspensions for repigmenting vitiligo. Arch Dermatol 2004; 140:1203–8. 17 Falabella R. Surgical therapies for vitiligo. Clin Dermatol 1997;32:228–32.

SECTION 4

Cellular grafting

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CHAPTER 20

Treatment of leukoderma by transplantation of basal cell layer suspension Mats J. Olsson

Introduction In the surgical management of vitiligo and other leukodermas where the melanocytes are missing, the general aim is to select an appropriate transplantation model to restore the epidermal melanin unit. The following issues need to be addressed, before deciding on the most suitable method for an individual patient: • the finances and infrastructure available; • the overall level of technical training and in addition to personnel with surgical skill, those specifically trained and experienced in the handling of live cells and chemical reagents; • experience of harvesting and delivering autologous melanocytes to leukodermic areas using various techniques; • age of the patient; • the locations of the anatomical areas to be treated; • the total extent of the depigmented skin and the total size of the areas to be treated; • number and size of the lesions; • type of leukoderma; • texture of the skin in the recipient area; • presence of coarse hair in the recipient area; • texture of skin in possible harvest area; • history of Köbner phenomenon; and • history of hypertrophic scars and keloids. A decision about the most suitable method can be made only after these variables have been evaluated. Methods involving separation of cells are usually more costly and technically advanced than methods

based on using the whole unprocessed tissue, but still less difficult and cheaper to perform than methods involving tissue or cell culturing steps. The method requires a laboratory equipped with the basic infrastructure, such as centrifuge, laminar-flow sterile hood, vortex mixer, incubator, refrigerator, and a well-trained technician. The advantage of suspension method is that we can cover areas up to 10 times the size of the donor area without expanding the number of cells in one session. The total area to be treated in one session is more limited in comparison to methods where the cells are culture expanded in their numbers. But when extensive areas are to be treated they can be divided into several smaller sessions instead. Suspensions of cells from the basal layer are useful in all anatomical areas including troublesome locations such as hairy areas and those with excessive movements like the joints, eyelids, and the corners of one’s mouth. Leukoderma is the term used for disorders, congenital or acquired, in which the skin becomes depigmented due to lack of melanin (pigment) in the epidermis. This may be due to absence of melanocytes in the epidermis and sometimes also in the matrix of the hair follicles. Piebaldism, different types of vitiligo, and chemical leukoderma belong to this group in which melanocytes are missing. Transplantation of melanocytes in a suspension from the basal cell layer or in any other form can serve as a treatment only in this type of leukodermas with missing melanocytes.

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Surgical methods are an important therapeutic approach to restore pigmentation in the depigmented areas. However, we should refrain from performing surgical therapies in patients with active disease who have melanocyte destroying activity (e.g. active vitiligo) [1]. Patients should be counseled so they have realistic expectations about the outcome of the procedure. They should be informed that expecting a 100% cosmetically perfect result is unrealistic, even if the whole procedure is faultless. A small area may remain depigmented after the procedure or a repigmented area may have a color darker or lighter than the surrounding skin. Careful evaluation and experience will help predicting the outcome in individual patients. Treatment of leukoderma with fresh autologous basal cell layer suspension can be implemented in all types of vitiligo and piebaldism. However, as with all surgical approaches, one should be careful in selecting patients with vitiligo vulgaris (generalized vitiligo), which has the involvement of an autoimmune component and, if the disease is not totally stable, the transplanted melanocytes may not survive. Attempts to improve the appearance of skin by some cosmetic clinics have led to two new groups of patients with a blotchy depigmentation. One of the groups consists of patients with chemically induced leukoderma. Many chemicals containing aromatic compounds like phenol- and hydroquinonederivatives, are in use for cosmetic or therapeutic purposes for the management of hypermelanosis [2] or as depigmenting agents in extensive vitiligo [2,3]. These compounds are known to cause pigment cell destruction. We have had several patients referred to us for the management of chemical leukoderma occurring as a side effect from the treatment of facial wrinkles with phenol or trichloroacetic acid (TCA) peel at the beauty parlor. Such patients have been successfully treated with transplantation of cell suspensions. The other group is comprised of patients who have developed leukoderma as an adverse effect of hair removal with laser or intense pulse light (IPL) pulse therapy. Laser and IPL can cause death of melanocytes and lasers have been used both in the treatment of melasma and for complete depigmentation in extensive vitiligo [3]. We have had several patients referred to us who

had lost their pigmentation following hair-removal therapy by light- or laser-treatment. Melanin absorbs light over a wide spectral range and the energy in the light beams is converted into heat and liberated inside the melanocytes, damaging them [3,4]. Physical trauma to the skin is known to be able to cause the Köbner reaction in patients with vitiligo vulgaris [5] and such patients are more prone to develop depigmented lesions when undergoing chemical peeling, laser peeling, or hair removal. There are no specific studies concerning evaluating the optimal density of cells for transplantation, but 5–10 times expansion, compared with the size of the donor area, can be done depending on the type of leukoderma. We experienced good outcome and color matching in patients with piebaldism and segmental vitiligo with 10–20 times dilution of the donor area. On the other hand, in generalized vitiligo we observed a lack of effect when the cells were diluted 10 times or more [6]. To my knowledge, the very first time a non-cultured cell suspension was used successfully to repigment vitiligo was in 1992 by Gauthier [7]. But at that time the technique involved a suspension from the scalp area which was injected into liquidnitrogen-induced blisters on the recipient sites [7]. Today we use a method that technically requires some more advanced equipment and also involves fast and precise de-epithelialization of the recipient area.

Methods Cell suspension applied to dermabraded lesions To apply an enriched basal cell layer suspension containing melanocytes onto extensive denuded skin lesions first became possible when we simplified the technique based on our earlier experience with transplantation of cultured human melanocytes to extensive machine dermabraded areas [8–11]. (See Fig. 20.1 for a schematic illustration of the procedure.) A defined culture medium and enzymes and inhibitors from a safe source (land of origin) or of recombinant type is used. Normally the donor cells are derived from a shallow shave biopsy, taken from a hidden area. Rarely,

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1. Shave biopsy, 2
5 cm 7. Dermabrasion and transplantation

6. Detach cells from culture flasks and wash them twice 2. Transportation to the laboratory

5. Culturing to expand the cell number under 2–3 weeks

3. Incubation for 50 minutes at 37°C in trypsin/EDTA 4. Separation of dermis from epidermis. Releasing of the basal cells

Illustration: Christina Hallden

Fig. 20.1 Schematic illustration of the operation steps. Notice that steps 5 and 6 used in culturing techniques (see

Chapter 22) are bypassed in this method, saving time and labor. Except the reduction of the culture step the methods are down in detail very similar.

in patients undergoing skin reduction surgery for excess skin, the removed skin may be used as a donor of cells to be prepared (see Chapter 22 for details of how to prepare a cell suspension from a full thickness skin sample). The method and success rate of enriched basal cell layer suspension transplantations have earlier been published in brief [6,12]. But in the following section we will discuss all the technical and procedural steps in detail, with a precise description on how to collect, prepare, and transplant a cell suspension obtained from the basal cell layer of the epidermis.

Obtaining the donor tissue Shave biopsy A normally pigmented area of approximately 3
5 cm2 in the gluteal region is marked, surgically cleansed, and anesthetized with a solution containing

equal amounts of 10 mg/ml lidocaine and Tribonat® (bicarbonate solution from Fresenius Kabi, Uppsala, Sweden). Tribonat buffers the otherwise acidic lidocaine solution, making injections less painful. A long thin needle is inserted from outside the marked donor area to avoid bleeding at the site from needle stick, and then parallel to the skin surface thrust into the marked area. It is important not to use adrenaline due to the risk of a buckled area with embossed lesions. Press the area with a sterile gauze-pad to facilitate the diffusion. An uneven surface makes it difficult to harvest a very thin and coherent sheet. A superficial shave biopsy (as thin as possible) is taken with a Goulian–Weck skin graft knife (Edward Weck & Company, Inc, Research Triangle Park, NC, USA). The Goulian knife should be equipped with a 006 shield, to ensure very shallow biopsies (Fig. 20.2).

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Fig. 20.3 Incubation of the Petri dish containing the Fig. 20.2 The Goulian–Weck knife. Notice that you can see

trypsin/EDTA floating shave biopsy.

the text on the knife right through the thin shave biopsy.

The specimen is put in a 15-ml test tube containing Joklik’s modified minimal essential medium (s-MEM, GIBCO BRL, Life Technology, Gaithersburg, MD, USA) and transferred to the laboratory for preparation. The donor area is covered for 8–10 days with semipermeable Tegaderm™ (3M, St. Paul, MN, USA), plus a layer of the air and water vapor permeable stretch fabric tape Fixomull®, extending a few centimeter beyond the margins of the Tegaderm™, to ensure that the fluid-filled blister that builds up under the dressing does not break. The Tegaderm™ and the Fixomull® are glued in place with Mastisol® (Ferndale Laboratories, Inc, Ferndale, MI, USA). The glue ensures that the dressing will remain secured for the period needed (8–10 days). If the skin specimen is immediately transported to a nearby laboratory it is sufficient with the basic medium alone, but if the lag period between harvesting and preparation of the suspension is between 1 and 4 hours, the medium is furnished with antibiotics (e.g. 50-U/ml penicillin and 0.05-mg/ml streptomycin) and 2-mM L-glutamine and kept at 8°C. If the lag period is more than 4 hours, then the biopsies are put in complete M2 melanocyte medium (PromoCell, Heidelberg, Cat. No. C-24300) and kept at 8°C to ensure an extended survival of the melanocytes and keratinocytes.

Release and preparation of free cells Under sterile conditions inside a laminar flow hood, the thin donor sample is transferred form the test

tube to a 6- or 10-cm diameter Petri dish depending on the sample size and directly washed once with 4 or 8 ml 0.20% w/v trypsin and 0.08% w/v EDTA (ethylenediamine tetraacetic acid) in 80% v/v phosphate buffered saline (PBS) (all SVA, Uppsala, Sweden) and 20% v/v Joklik’s modified minimal essential medium and refurnished with 5 or 10 ml of the trypsin/EDTA solution. The sample is turned back and forth with the help of jeweler’s forceps to ensure that it comes in complete contact with the solution, and finally with the epidermis side facing upwards, torn or cut into pieces of about 4 cm2. The air-bubbles under the thin fragments should be removed by gently pressing and scraping the surface with a curved forceps. The Petri dish is incubated at 37°C in 5% CO2 for about 50 minutes for thin shave biopsies (Fig. 20.3). After about half the incubation time the pieces are moved around and pressed on with a curved forceps, to ensure that the whole tissue gets soaked in with the trypsin/EDTA solution. The time of incubation vary in each individual case, depending on the thickness of the sheet and the activity of the enzyme. But by the gentle pressure with a curved forceps on the epidermis, adequacy of the incubation time can be judged by slightly lighter streaks of impression pattern. After incubation, the trypsin/EDTA solution is removed and 3 ml (15°C) of 0.5-mg/ml trypsin inhibitor (Soy-bean extract from Sigma, St. Louis, MO, USA) in PBS is added to the Petri dish to terminate the trypsin reaction. The epidermis is removed

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Fig. 20.4 Separation of basal epidermal cells after

Fig. 20.5 Suspended free basal cells in medium of

trypsination.

trypsin inhibitor and nutrition, ready to be transferred to a test tube for centrifugation.

from the dermis with the help of one curved finepointed forceps and one straight fine-pointed forceps, and the dermal parts are transferred to a test tube containing 5 ml of the highly balanced, serumfree, medium and vortex-mixed for 5 s. The dermal pieces are then removed with the tip of a Pasteur pipette or with the help of a hooked forceps and discarded. The basal cell layer side of the epidermal pieces is scraped with a curved jeweler’s forceps to free the cells and the pieces are then minced to smaller fragments (Fig. 20.4). The small epidermal fragments are transferred to and 2-ml fresh medium added to the test tube and vortex-mixed for 30 s. The remaining epidermal fragments, now mainly consisting of stratum corneum and stratum granulosum, are removed from the test tube and discarded. The rich cell containing solution of trypsin inhibitor in the Petri dish is transferred to the test-tube (Fig. 20.5), the Petri dish is then rinsed twice with a small volume of s-MEM, which is also transferred to the test-tube, gently mixed with a pipette and then centrifuged for 7 minutes at 190g (Fig. 20.6). Sometimes it may be difficult to pellet the cells due to free DNA and collagen, which form mucous-like structures. In such cases, the contents of the test tube should be run up and down a few times in a Pasteur pipette with sharp glass-edge. This will cut the mucous into shorter fragments which will facilitate the formation of a pellet when centrifuged. After the centrifugation the supernatant and some remaining fragments of floating stratum

Fig. 20.6 Pellet of cells ready to be resuspended in a

very small volume and transplanted.

corneum–granulosum are removed and the pellet is resuspended and centrifuged twice in 5-ml serumfree medium for 5 minutes at 180g to ensure that all residues of trypsin and trypsin inhibitors will be washed away. The final pellet is resuspended in a very small volume of medium, about 0.3–0.4 ml depending on the number of the donor cells in the pellet and the size of the recipient area. The small volume of cells is transferred to a 1-ml tuberculin syringe, without a needle or kept in the test-tube until transplantation and at that time directly transferred with the help of a pipette to the recipient areas. Avoid larger volumes because of the risk that the cells will float

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out onto the sides of the lesion. If found necessary, it is much easier in a later stage to add some more medium to the tube/syringe to produce right viscosity or be able to cover an extensive area.

Premedication It is important to have a still and calm patient during the transplantation procedure. Therefore, it is recommended to give 5–10 mg of diazepam and/or 10 mg of ketobemidone orally 1 hour before the transplantation, plus 500–1000 mg of paracetamol 40–50 minutes before the surgery. Erythromycin capsules, 500 mg twice daily or a similar antibiotic is given for 7 days, starting on the day of surgery.

Anesthesia of the recipient site The recipient areas are anesthetized with EMLA® cream (AstraZeneca, Södertälje, Sweden) applied under plastic foil occlusion (e.g. saran wrap) for 1–2 h and then also locally anesthetized with a mixture of equal parts of 1% lidocaine and Tribonate® buffer (Fresenius Kabi, Uppsala, Sweden) immediately prior to the surgery. A thin and long needle is used, which is inserted outside the depigmented lesion and then pushed parallel to the skin surface into the lesion. This is to avoid bleeding from needle stick in the area to be treated. In a large area, it may not be possible to reach the center of the lesions from the borders, but a combination of “ring-block” effect of peripheral injections and EMLA will provide satisfactory anesthesia in the center of the lesion also. In small areas, local anesthetic can be infiltrated to the whole lesion, and therefore EMLA is not required. Total nerve block can be used to achieve satisfactory anesthesia, but it requires some experience to inject the local anesthetic in the right spot, therefore it should be given by an anesthesiologist. Local anesthesia with freezing spray, such as ethyl chloride or fluor-ethyl (Gebauer Pharmaceutical Preparations, Cleveland, OH, USA) can also be used immediately prior to dermabrasion. The sprays give some anesthesia, but alone are not sufficient to give total pain-relief. However, the skin gets firm and easy to dermabrade when chilled and it is easier for an inexperienced eye to detect remnants of epithelium on a chilled denuded surface.

Transplantation and aftercare The washed cells resuspended in a small volume of s-MEM are transferred to a 1-ml syringe or kept in the test tube for direct application onto the skin with the help of a pipette. For handling details see above under the heading of “Release and preparation of free cells.” The recipient area is cleaned with alcohol, outlined with a sterile surgical marker pen and the epidermis is removed up to the dermal–epidermal junction, using a high-speed dermabrader (20,000 rpm), fitted with a diamond fraise. A wheel, pear, or cone suitable for the size and location of the area to be treated is chosen. Normally, we can dermabrade most of the lesions with a 6-mm wide regular fraise wheel but on rough skin, such as that on the knees, we may need a coarse wheel and on delicate areas such as around the nostrils, corners of the mouth, and on the eyelids, a small pear-shaped fraise may be needed. For eyelids, we have a special hand-tool fitted with a regular cone, this ensures not to damage the eyelid or the eye during the procedure (Fig. 20.7). The dermabrasion should be performed in at least two different directions. A uniform punctate capillary bleeding from the dermal papilla can be seen (Fig. 20.8). Light freezing with fluoro-ethyl spray reveals if there are any islands of epidermis left. The denuded area is washed with sterile PBS or saline solution and kept under moistened gauze for few minutes to ensure that bleeding has stopped. If some puncture bleeding points persist, electrocoagulation is performed with a fine-pointed electrocate needle. The cell suspension is then applied to the denuded areas and spread with the tip of a syringe, pipette, or a metal-spatula. The cells are secured with a silicone netting (Mepitel®, Mölnlycke AB, Mölnlycke, Sweden) or a plastic netting (Delnet® Fastec Inc., Bronx, NY, USA) extending about 1 cm onto the dry non-dermabraded surrounding skin, which locks it in place so it will not slide over the slippery woundbed (Fig. 20.9). Then two layers of salinemoistened woven gauze compresses and a semipermeable Tegaderm™ film are applied. The later is glued in place onto the surrounding skin with Mastisol® (Ferndale Laboratories, Inc, Ferndale, MI, USA). The glue ensures that it will be secured in place for the period needed.

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Fig. 20.9 Covering of the seeded area with an inert

silicone netting (Mepitel®).

Fig. 20.7 Dermabrader (power unit and handpiece) and

diamond fraises: (A) wheel, (B) cone, (C) pear, and (D) handle fitted with a cone for manual abrasion in delicate locations.

Fig. 20.8 Application of the cell suspension to the

de-epithelialized recipient area.

In all cases the patients should rest at least 4 hours, preferably longer, in a hospital bed after the procedure has been completed. The patient is told that the first 48 hours are most critical for the result and that it is important to restrict the physical activities and avoid tight clothing for the first 2 weeks.

It will build up a fluid-filled blister under the plastic film at the donor area. But the main part of this will dry out in some days. Exudation from the recipient areas in the first 2 days is common, especially in larger coherent lesions. The dressing is removed after 8–10 days. At dressing removal a sterile saline solution is first injected into the bandage and allowed to soak for a few minutes before the bandage is removed. Sterile forceps and small scissors are used. The Tegaderm™ film covering the donor site is also removed on the same day. Usually the skin heals nicely at both the recipient and donor sites, and a careful flush with sterile saline solution and light padding with dry gauze compresses is usually sufficient. This is followed by an application of a layer of pure Vaseline with a spatula. On skin exposed to friction or trauma, such as elbows, hands, and feet, the areas are after the application of Vaseline, covered with a gauze compress and secured in place with Micropore™ tape for another 2 days. The patient is advised to apply a thin layer of Vaseline once a day for 7 days after the removal of the dressing. This is to minimize the frictional trauma and desiccation to the still fragile surface. A layer of Vaseline is also applied to the donor area. If a superficial infection is suspected, the patient should be advised to apply a topical Fucidin or Bacitracin antibiotic ointment twice a day for a few days whereupon you have a check-up. The patient is advised to expose him/herself to midday sunlight for a few minutes 2 times a week

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for about 2 months, commencing at 1 week after the removal of the bandage.

Follow-up evaluation About 5–8 months after transplantation an overall follow-up evaluation is advised. During the first few weeks, the transplanted area is erythematous, but pigmentation can be seen as early as 3 weeks post-transplantation in Wood’s light or with diascopy. During the first year it is not uncommon to see a slight hyper- or hypopigmentation in some of the treated areas. But the color gradually matches with that of the surrounding skin and after a year it most often blends well with the surrounding skin (Plate 20.1, facing p. 114). Sometimes a 1–2-mm depigmented halo persists around the transplanted area. This is seen more often in patients with generalized vitiligo than in those with piebaldism or segmental vitiligo. Generally this halo is repigmented with repeated sun-exposures. The outcome can be predicted about 4 months after the surgery but the final result will usually be seen first about 1.5 years after the surgery.

Documentation Pictures and/or drawings of the lesions should be taken in all patients both before the surgery and during the follow-ups. Files with anamneses and treatment charts are kept in the hospital’s central records.

Discussion Depigmented skin with white hair and glabrous skin responds poorly or not at all to attempts of medical repigmentation. This indicates that hair follicles are the most important reservoir for spontaneous and medical repigmentation, and that surgical transplants are the only effective method to replace the lost epidermal melanocytes and the follicular reservoir. Skin graft techniques, including cultured or noncultured cells, are used widely to correct scars, restore pigmentation, or treat burn wounds. Many thousands of patients with different types of vitiligo have been treated with various types of transplantation

models since 1940s and so far no reports of malignant transformation or other serious adverse effects in the treated lesions have to my knowledge appeared in the medical literature. This indicates that transplantation procedures in vitiligo have a very high safety profile. But it should still be imperative to discuss and analyze the risk/benefit ratio for each individual patient [1]. Kaufmann et al. and Chen et al. have shown in their respective studies that de-epithelialization of the recipient site with the help of Er:YAG (erbium: yttrium–aluminum–garnet) or short-pulse carbon dioxide laser in transplantation of melanocytes can give similar results as with dermabrasion [13–15]. Laser ablations may have advantage over dermabrasion on some delicate sites, such as eyelids. Guerra et al. have successfully used Timedsurgery (programmed diathermosurgery) to de-epithelize the recipient areas [16]. However, Timedsurgery is a quite time-consuming procedure and therefore limits the practicalities of this method. Removal of the epidermis with the help of a device for ultrasonic abrasion has also been successfully used for transplanting melanocytes in vitiligo [17]. Topical thrombin can be used at both donor and recipient sites to achieve hemostasis, though normally it is not required. Patients are advised not to take any medicines interfering with the hemostasis, such as salicylates (Aspirin), for 10 days before and after the surgery. The differences in long-term outcome between the various types of leukoderma can probably be explained by etiological differences. Piebaldism and segmental vitiligo almost always respond with a nearly complete and durable repigmentation but long-term follow-ups have shown that vitiligo vulgaris can be more difficult to treat [12]. Several independent research groups have proposed that the etiology of vitiligo vulgaris involves autoimmune components [18–21]. This etiological hypothesis is today well accepted by most researchers in the field and also supported by the fact that vitiligo vulgaris responds to immunosuppressive and immunomodulating treatments. In addition, associated disorders may have an effect on the outcome of transplantation procedures in patients with vitiligo. We have experienced a worse

Treatment of leukoderma by transplantation of basal cell layer suspension outcome in patients with vitiligo vulgaris also having hypothyroidism in comparison to those without hypothyroidism. This possibly indicates a more active autoimmunity in patients afflicted with hypothyroidism. Our previously published multivariate regression analysis has clearly shown that the chance of achieving a successful outcome of transplantation increases with the decrease in the total extent of the affected areas in patients with vitiligo vulgaris [12]. These observations underscore the importance of informing the patients with vitiligo vulgaris that transplantation will not cure or affect the underlying cause or natural course of their disease and, therefore, it cannot be promised that new patches will not appear in the future. Patients with spreading vitiligo lesions have significantly poorer outcome than those with decreasing lesions or a stable disease. Therefore patients with extensive vitiligo vulgaris and those who do not have a completely stable, non-progressive disease for at least 2 years should not be chosen for any kind of transplantation. It is obvious that patients with stable forms of leukodermas, namely piebaldism, segmental vitiligo, and focal vitiligo benefit most from the surgical methods, since they generally retain full pigmentation and have minimal chances of developing new lesions in the future. Despite the fact that the initial number of melanocytes that get seeded into the affected area are fewer than the normal number in the skin, it is not uncommon to see a slight hyperpigmentation in the treated area. This hyperpigmentation can probably be explained by the involvement of hormones and inflammatory mediators released locally in the area during the procedure and wound healing process. But compared with other surgical methods we have used, the basal cell layer suspension method is the method that least often gives long-standing hyperpigmentations. Initial hypopigmentation can also be seen in some patients. Both the hyper- and hypopigmentation usually improve with time. In comparison with some of the other surgical methods, the basal layer suspension method has the advantage that a fairly large area can be covered with a not too large biopsy. Both the biopsy and the denuding process of the recipient area are supposed to be superficial and should not create

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any scars. But in addition to the operation theater the technique also requires a properly equipped laboratory and skillful personnel, specially trained in preparation and handling of live cells, which may restrict the method to larger hospitals or specialized clinics.

Conclusions Initial hyperpigmentation can sometimes be seen but is less frequent than with other methods including epithelial sheets or pure melanocytes [12]. Patients displaying darker tanning seem to be more prone to hyperpigmentation. After 6–18 months the color is usually the same or close to the same as the surrounding skin in most of the patients. The dorsal aspect of the fingers, especially skin on the joints, is most difficult to repigment. The dorsum of the hands and trunk generally shows good to excellent results. Special care has to be taken in patients who have lesions on both sides of the body, to be treated in a single session. The least important side (smallest areas) should be treated first and then the most important side after patient is turned around by 180°. These patients often show less satisfactory results at the sites facing downwards, indicating the importance of gravitation for a good cellular take. A depigmented 1–3-mm halo between the transplanted and surrounding pigmented skin is not uncommon, especially in patients with generalized vitiligo. Speckled areas of non-pigmented spots are common on the elbows and knees. This may be due to incomplete removal of depigmented epidermis in these areas of thick and uneven epidermis. Most of the other areas, such as those on the trunk, arms, legs, and face, generally develop even repigmentation with this method. In short it can be concluded that: • Since this technique includes separation, dilution, and spreading of the cells, the donor site is only 1/10–1/5 of the recipient area. This reduces the risk of depigmentation due to Köbner phenomenon at the donor site and makes healing process easier. • This method is simpler than methods involving cell culturing, but is still quite advanced and requires a laboratory setup.

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• Selection of patients is crucial to the success of the outcome. • Segmental vitiligo and piebaldism almost always respond with complete repigmentation, regardless of the method of transplantation. • Segmental vitiligo and piebaldism retain all the repigmentation as noted by us in up to 15 years of follow-up. • It is more difficult to achieve complete repigmentation in patients with generalized vitiligo, but they usually respond well if the disease is stable. • Patients with active and extensive generalized vitiligo often show poor repigmentation and such patients should not undergo transplantation. • Predictors of good prognosis in vitiligo vulgaris are spontaneous repigmentation, no new lesions during the last few years, not involving extensive body surface area, younger age, and a shorter duration of the disease.

References 1 Olsson MJ. What are the needs for transplantation treatment in vitiligo, and how good is it? Arch Dermatol 2004;140:1273–4. 2 Mosher DB, Parrish JA, Fitzpatrick TB. Monobenzylether of hydroquinone. A retrospective study of treatment of 18 vitiligo patients and a review of the literature. Br J Dermatol 1977;97:669–79. 3 Njoo MD, Vodegel RM, Westerhof W. Depigmenta-tion therapy in vitiligo universalis with topical 4-methoxyphenol and the Q-switched ruby laser. J Am Acad Dermatol 2000;42:760–9. 4 Nanni CA, Alster TS. Laser-assisted hair removal: side effects of Q-switched Nd:YAG, long-pulsed ruby, and alexandrite lasers. J Am Acad Dermatol 1999;41:165–71. 5 Gauthier Y. The importance of Köebner’s phenomenon in the induction of vitiligo vulgaris lesions. Eur J Dermatol 1995;5:704–8. 6 Olsson MJ, Juhlin L. Leukoderma treated by transplantation of a basal cell layer enriched suspension. Br J Dermatol 1998;138:644–8. 7 Gauthier Y, Surleve-Bazeille JE. Autologous grafting with noncultured melanocytes: a simplified method for treatment of depigmented lesions. J Am Acad Dermatol 1992;26:191–4. 8 Olsson MJ, Juhlin L. Melanocyte transplantation in vitiligo. Lancet 1992;340:981.

9 Olsson MJ, Juhlin L. Repigmentation of vitiligo by transplantation of cultured autologous melanocytes. Acta Derm Venereol 1993;73:49–51. 10 Olsson MJ, Moellmann G, Lerner AB, et al. Vitiligo: repigmentation with cultured melanocytes after cryostorage. Acta Derm Venereol 1994;74:226–8. 11 Olsson MJ, Juhlin L. Transplantation of melanocytes in vitiligo. Br J Dermatol 1995;132:587–91. 12 Olsson MJ, Juhlin L. Long-term follow-up of leukoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol 2002;147: 893–904. 13 Kaufmann R, Greiner D, Kippenberger S, et al. Grafting of in vitro cultured melanocytes onto laserablated lesions in vitiligo. Acta Derm Venereol 1998; 78:136–8. 14 Chen YF, Chang JS, Yang PY, et al. Transplant of cultured autologous pure melanocytes after laserabrasion for the treatment of segmental vitiligo. J Dermatol 2000;27:434–9. 15 Chen YF, Yang PY, Hu DN, et al. Treatment of vitiligo by transplantation of cultured pure melanocyte suspension: analysis of 120 cases. J Am Acad Dermatol 2004;51:68–74. 16 Guerra L, Capurro S, Melchi F, et al. Treatment of “stable” vitiligo by Timedsurgery and transplantation of cultured epidermal autografts. Arch Dermatol 2000; 136:1380–9. 17 Tsukamoto K, Osada A, Kitamura R, et al. Approaches to repigmentation of vitiligo skin: new treatment with ultrasonic abrasion, seed-grafting and psoralen plus ultraviolet A therapy. Pigm Cell Res 2002;15:331–4. 18 Baharav E, Merimski O, Shoenfeld Y, et al. Tyrosinase as an autoantigen in patients with vitiligo. Clin Exp Immunol 1996;105:84–8. 19 Kemp EH, Gawkrodger DJ, Watson PF, et al. Immunoprecipitation of melanogenic enzyme autoantigens with vitiligo sera: evidence for cross-reactive autoantibodies to tyrosinase and tyrosinase-related protein-2 (TRP-2). Clin Exp Immunol 1997;109:495–500. 20 Ogg GS, Rod Dunbar P, Romero P, et al. High frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in autoimmune vitiligo. J Exp Med 1998;188:1203–8. 21 Hedstrand H, Ekwall O, Olsson MJ, et al. The transcription factors SOX9 and SOX10 are vitiligo autoantigens in autoimmune polyendocrine syndrome type I. J Biol Chem 2001;276:35390–5.

CHAPTER 21

Setting up a tissue culture laboratory Rafal Czajkowski, Tomasz Drewa and Waldemar Placek

Laboratory design The success in the establishment of and working with a primary cell culture depends to a large degree on a correctly planned laboratory. Preparation of the tissue culture laboratory includes collection of proper equipment, which has to be placed according to basic rules. A tissue culture laboratory should have sufficient area to carry out numerous tasks, such as: • working in sterile conditions, • tissue/cell incubation, • culture media preparation, • laboratory equipment cleaning and sterilization, and • laboratory reagents storage. It is necessary to divide the tissue culture laboratory into two regions (two rooms). A laboratory with at least two rooms enables separation of operations related to cell isolation and cultivation from the area where the laboratory reagents, glass, and other equipment are kept. One room in the laboratory should be divided into three areas.

Sterile area The sterile area includes the place where the incubator (storage of living cells), the laminar-flow workbench (biosafety cabinet), and the inverted microscope are kept. Work efficiency in the sterile area depends on proper arrangement of laboratory equipment. The incubator, biosafety cabinet, and inverted microscope should be arranged in such a way so as to shorten the distance in transportation of cultured cells. Proper arrangement of equipments mentioned above along the one wall of laboratory room (in such an order: incubator, biosafety cabinet,

inverted microscope or inverted microscope, biosafety cabinet, incubator) makes it possible to achieve this aim. The incubator door should open in the opposite direction to the biosafety cabinet. After taking out the culture vessel with cells from the incubator, they are put in the biosafety cabinet and then they can be examined under the inverted microscope. Such an arrangement minimizes the risk of culture contamination.

Preparatory area The preparatory area should be located near the autoclaves, driers, sinks, cupboards, and waste containers. There is no special requirement for equipment arrangement in the preparatory area. The goal of designing this part of the laboratory is to keep the area clean and ensure easy access to each place. The comfort of staff and common sense should be the best guide.

Cell storage (room) area This part of the laboratory is not obligatory, particularly if the primary cultures without “feeder layers” for transplantation procedures are used. Primary cultures usually do not require storage in the freezers before transplanting, but if the cultures are propagated using continuous “feeder” cell lines (human or mouse fibroblasts), the Dewar flasks containing liquid nitrogen and deep freezers should be placed in the storage area. This area should be located near the sterile area. Each of these areas should preferably be in separate rooms, but if the availability of space and rooms is limited the sterile and cell storage area can be located in the same room. The air movement within the laboratory is a crucial element. The forced air movement from the sterile

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area to the non-sterile parts is the best arrangement. During opening of the door, the air should flow to the non-sterile parts of the laboratory. To avoid forced air movement, a sluice can be placed between the rooms to protect contamination. The sluice also serves as a place for changing of clothes and putting caps and masks. These precautions are essential, as cells are going to be transplanted into humans [1].

Equipment for cells and materials storage • The sterile liquids, like distilled and deionized water or buffered saline are kept at room temperature (17–20°C) in laboratory cupboards. • Media and antibiotics should be stored at 4°C. For this a common refrigerator is required. • Sera, enzymes, and antibodies used for cells identification need to be stored at low temperature, usually
20°C. This condition cannot be met in the freezer chamber of a normal fridge, where the temperature is unstable and around
3°C. It is a necessity to have
20°C freezer. Both the sera and enzymes cannot be frozen and thawed frequently. Frequent freezing and thawing cycles can be avoided by preparing only a small amount of enzyme or serum at any one time. The amount of each portion should be adequate for planned procedure; about 50-ml of serum is needed to prepare 0.5 l of culture medium (standard serum concentration is 10% v/v). The serum should be frozen in the 50-ml flasks. Enzyme solutions are frozen in fractionated amounts suitable for one isolation procedure; for example, trypsin is frozen in 5-ml flasks. The requirement of the protein portions for each procedure depends on the preferences of the laboratory staff. • To avoid the risk of contamination when preparing cell culture medium, a complete and defined ready to use medium can be bought from a company working under good manufacturing practice (GMP) standard. Complete ready to use media might be a little bit more expensive, but the quality is usually high and it saves a lot of time and effort. • Cells are kept at low temperatures. There is a direct correlation between temperature level and duration of safe storage of cells. Cell storage at the low temperatures can be a crucial point for successful

treatment using autologous cell transplantation. The frozen primary culture can serve as a cell source, if a multi-step transplantation procedure is planned. As a rule, lower temperature permits longer cell storage. The time of cell storage in deep freezers (from
80° to
100°C) is short, usually up to 6 months, but some investigators keep cells for longer periods in deep freezers without any cell damage. There are many advantages of keeping the cells in the deep freezers. Storage in the deep freezers is cheap, as most of the laboratories are already equipped with them. The freezer is usually large, so from the perspective of a small laboratory, there is an unlimited capacity. One of the main shortcomings can be machine failure. There is a need for another deep freezer in the back-up, and this can be rather expensive. A friendly laboratory, preferably in the same building, equipped with a deep freezer is of course a solution. For such emergency cell transport dry ice can be used (
78°C). Dry ice is produced from bottle with CO2, which is equipped with special sleeve. Dry ice can be made in a few minutes and this allows to transport of cells to another freezer. Dewar flask with liquid nitrogen can serve as a salvage place for cells. If there is no possibility to continue storage in low temperature, cells should be quickly thawed and cultured. Cell storage for a period longer than 6 months is performed in Dewar in liquid nitrogen or its vapors. There are many advantages to ultra-low temperature storage at
196°C. Ultra-low temperature and its stability are both very important factors in the success of cell storage. Theoretically cells may be preserved in these conditions for an unlimited time, but we usually propagate cell lines in culture from time to time and then “the renewed culture” is frozen again. Liquid nitrogen can contaminate the frozen cultures. The long-term cell storage can be performed in vapors of liquid nitrogen. The temperature in this condition ranges between
135°C and
190°C. Temperature fluctuations can be harmful for cells, particularly if the Dewar flask is opened quite often. If the cells are kept in the liquid nitrogen vapors, the most important thing is to check the nitrogen level. There is a quite real risk that nitrogen will evaporate from the Dewar and all cells will be lost [2].

Setting up a tissue culture laboratory

Laboratory glass Laboratory glass is equipment that is continuously replaced or recycled. Actually, it is not always truly glass; it is often single-use “plastic” utensils made of polypropylene or polystyrene. The term “glass” is not reserved for multi-use equipment as laboratories also use single-use glass pipettes and bottles. It is difficult to say if using strictly glass pipettes as single-use equipment is justified. The authors believe that glass equipment may be effectively sterilized for re-use. This requires access to an autoclave with a relevant control system (e.g. bars). Bottles used for mixing and storing medium may certainly be sterilized as they come in contact with sterile pipettes only. A pipette, which has had contact with cell culture medium, should not be used again before sterilization. The question of whether the pipettes should be single- or multi-use is still open. Doubtless, the decision to use single-use pipettes depends on economic conditions. Other materials like Petri dishes, centrifuge tubes, syringes, and plastic tips for pipettors of filters must be single-use only because of problems with sterilizing them. Cleaning of laboratory glass can be done with ultrasonic cleaners (for pipettes) or dishwashers (bottles and flasks). During sterilization, pipettes should be placed inside metal cylinders. Glass may also be sterilized with hot air, provided that the temperature 180°C is maintained for 4 hours [2].

Pipetting Pipette-aid (pipet-boy, pipettor) is a basic piece of equipment used in cell culture laboratory. It allows controlled suction and release of liquid to and from pipettes while working under laminar-flow table. The parts that must be frequently checked and replaced are the filter and the silicone collar (the one that holds pipettes) of the pipettor. These parts are used up very quickly and there is a need to have them in reserve. The pipettor cannot be left on the surface of table in safety cabinet, it must be placed in original handle provided by the manufacturer. This handle should be fixed to the safety cabinet wall. A pipettor handle can be made from a ring stand and column clamp. If the rod of the stand is bent and the

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angle between the pipette and table surface is 45°, the pipettor will also work in this position, and there is no need to hold it. This allows the pipettor to be operated with one hand; 10-ml pipettes are the most suitable for tissue culture. For larger liquid volumes 25-ml pipettes are required. There is no difference if the pipettes are calibrated for inlet or out flow, because all culture media are added in approximate volumes, with variation of 0.2–0.5 ml. Only when growth factors, hormones, or antibiotics are added the volume measuring should be done precisely. For these purposes the range of 20–1000 l is quite enough; one automatic pipette of 20–200 l and a second 100–1000 l are usually kept in the safety cabinet [3]. These high accuracy automatic pipettes should regularly be calibrated every year.

Waterbath All the liquids used in cell culture techniques that are to be in contact with human living cells have to be heated to the temperature 37°C. The waterbath provides an essential tool in bringing liquids like culture medium or phosphate buffered saline (PBS) to the right temperature prior to use (Fig. 21.1). It is filled with distilled water to prevent corrosion and bacterial growth, and additives (such us CuSO4 or other antimicrobial agents) are provided to decrease the risk of contamination. It is a good habit to spray bottles with 70% ethanol when removing them from the waterbath [3].

The microscope An inverted microscope with phase-contrast optics and essential filters is important and necessary equipment in the tissue engineering laboratory (Fig. 21.2). As the name suggests, an inverted microscope is upside down compared to a conventional microscope. The light source and condenser are on the top above the stage pointing down. The objectives and turret are below the stage pointing up. The only things that are “standard” are that a specimen is placed on top of the stage and the binocular or tube is in the standard position pointing at a conventional

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Chapter 21 used objectives fitted on an inverted microscope include 2.5, 4, 10, 20 and 40. The final magnification is the multiplication of the objective and eyepiece magnifications. Observation of tissue cultures should be done every day to assess cell morphology, cell proliferation rate, and culture sterility. Although macroscopic observation of culture flask surface is crucial to management during normal cell culture care, the features of bacterial or fungal contamination in early stage are visible only under the microscope. The microscope should be connected to a digital camera to enable capture of cell culture photo documentation. When the microscope is not in use, it should be covered to avoid dust settling on the optic system [3,4].

Cleanness and personal hygiene in the laboratory

Fig. 21.1 Waterbath filled with distilled water and

CuSO4.

Fig. 21.2 The inverted microscope.

viewing angle. An inverted microscope enables the researcher to observe cell culture in various culture flasks, as well as histological slides. Ultraviolet optics can also be used for fluorescence examinations. The most common and frequently

Growth medium is not only a good medium for mammalian cells, but also for bacteria, fungi, and mycoplasmas. Personal hygiene is very important in the tissue culture laboratory. Cleanness is a basic factor for decreasing cell culture contamination. This problem of contamination is of great concern if the laboratory workers are simultaneously physicians working in hospitals especially in departments of dermatology or surgery. The presence of bacteria in these departments is a known fact and these bacteria usually are resistant to many antibiotics. With increasing number of protection agents, the risk of cell culture contamination is lower. Before entering in the tissue culture laboratory, hands must be washed with detergents and dried with a hand dryer. The usage of towels is best avoided. The authors consider the changing of footwear before entering the laboratory to be mandatory, although it is not a practice applying to all tissue culture laboratories. Single-use gowns, latex or vinyl gloves, surgical masks and cauls are used in laboratory, especially in the case of cells cultured for transplantation practice (e.g. transplantation of autologous cultured melanocytes in vitiligo patients). The authors usually use only non-sterile latex surgical gloves, which are sprayed with 70% ethanol before each contact with cultured cells and before putting the hands into the biosafety cabinet. Special attention must be

Setting up a tissue culture laboratory paid to avoid contact of sprayed gloves with burner flames. It is indicated to wait till the gloves dry (about 30 seconds) under the biosafety cabinet [1].

The incubator The incubator plays a vital role in maintaining an optimal environment for cells to proliferate (optimal for human melanocytes growth is pH 7.2–7.4 and 5% CO2 environment, 95% humidity, and 37.0°C temperature). Cultured cells require a strictly controlled environment to grow. Specialist incubators are used to provide the optimal growth conditions, such as temperature, degree of humidity, and CO2 levels in a controlled and stable manner (Fig. 21.3). As cells remain in the incubator most of the time, it should have sufficient capacity (200 l per researcher) and should be fitted with temperature, CO2, and humidity controls. Some incubators also have the facility to control the O2 levels and are provided with a warning system that tells when some of the measurable parameters are out of settings.

Periodical cleaning of the incubator with 70% ethanol and antimicrobial agents is necessary to maintain a clean environment within the incubator. Copper-coated incubators are also now available. A copper-coated surface inside the incubator helps to reduce the bacterial and fungal contamination due to the microbial inhibitory activity of copper. The incubator must be easy to clean and made of stainless materials. The inclusion of waterbath treatment fluids in the incubator water trays will also reduce the risk of microbial growth [2–4].

The laminar-flow workbench (biosafety cabinet) The laminar-flow workbench is the basic workplace to handle human tissue samples and provides clean filtered air that allows cell cultures to be kept up in sterile environment. All activities concerning cell culture should be carried out in a class II biosafety cabinet where the air circulates vertically and is purified through HEPA (high-efficiency particulate air) filters with 99% efficiency in aerosols and particle removing. Class II biosafety cabinets are open fronted and the workspace is flushed with a downflow of sterile air, which is then refiltered and recirculated (Fig. 21.4). Some air is drawn in through the front of the cabinet and a corresponding amount discharged to the outside. Both work and worker are protected and the cabinet is particularly useful

Fig. 21.4 The laminar-flow workbench (biosafety Fig. 21.3 The incubator.

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cabinet).

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for tissue culture work. It is suitable for group 2 hazards and, in some cases, for group 3 hazards. In addition to the vertical laminar-flow biosafety cabinets, there are some horizontal laminar-flow cabinets. The horizontal-flow cabinets are useful for maintaining sterility of materials and can be used for purposes such as the preparation of culture fluids. The horizontal-flow cabinets must not be used with infectious or carcinogenic agents and human cell lines, since the air from the hood is directed at the operator. The proper location of the biosafety cabinet in the laboratory room is very important and areas close to the air-conditioning, doors, or windows should be avoided because air turbulences can unfavorably affect laminar flow. An appropriate placement of the equipment inside the cabinet is also of importance so as not to disturb the laminar-flow by placing something on the ventilation airways. The biosafety cabinet should have proper size (at least 1200 mm in width and 600 mm in depth), should guarantee low noise level, and should be easy to clean [2,4].

Fig. 21.5 “Neubauer”-type chamber.

Hemacytometer A hemacytometer is an etched glass chamber with raised sides that will hold a quartz coverslip exactly 0.1 mm above the chamber floor. The counting chamber is etched in a total surface area of 9 mm2. A hemacytometer can be viewed under a microscope to determine the concentration of cells in suspension. There are a numerous types of chambers and some have slight variations in style. The authors usually use a common “Neubauer”-type chamber (Fig. 21.5). The examined cell suspension has to be diluted half with 0.4% trypan blue solution before loading a hemacytometer chamber (hemacytometer is filled by capillary action). Staining of cells with trypan blue facilitates the determination of live/dead cell count. Trypan blue is a stain that is actively extruded from viable cells, but which readily enters and stains dead cells. Therefore, the cells that are blue are dead. The difference between the total number of cells and the number of dead cells would be the number of viable cells in a given aliquot of the culture.

Fig. 21.6 The hemacytometer showing a diagrammatic

representation of areas to be examined (circles) and indicating which cells should be counted (arrows).

To perform the count, determine the magnification needed to recognize the cells. Now systematically count the cells in four outer squares. Cells that lie on the lines should only be counted if they are touching the top and left-hand lines of each corner square (Fig. 21.6). The cell concentration is calculated as follows: total cells  cell counts per milliliter

Setting up a tissue culture laboratory

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(total cell count in four squares/4  104)  original volume of fluid from which cell sample was removed  dilution factor with trypan blue [3,4]. Example: 3.5  105 (cells per ml)  2 ml (original volume)  2 (1:1 dilution with trypan blue)  1.4  106 total cells. In well equipped laboratories with access to a special culture counter of flow cytometric machine (FACS) cell counting may be done with great speed and accuracy.

The centrifuge (refrigerated or non-refrigerated) The centrifuge is essential equipment in the tissue culture laboratory (Fig. 21.7). Centrifuges are routinely used in tissue culture as part of the subculture routine for most cell lines and for the preparation of cells for cryopreservation. This equipment is mainly used in isolating cells during density gradient centrifugation or when concentrating cells in suspension. Centrifuges produce aerosols and thus it is necessary to minimize this risk. This can be achieved by purchasing models that have sealed buckets. Care should always be taken not to over-fill the tubes and to balance them carefully. These simple steps will reduce the risk of aerosols being generated. A swing bucket rotor (with maximum speed of 6000–8000 rpm) is suitable even for gradient centrifugation protocols. In the case of melanocytes, the speed of 2000 rpm is sufficient and does not lead to destruction of the pigmented cells. The centrifuge should be situated to be easily accessed for cleaning and maintenance. Centrifuges should be checked frequently for signs of corrosion [2–4].

Fig. 21.7 The centrifuge.

References 1 Freshney RI. Design and layout. Culture of Animal Cells – A Manual of Basic Technique, 4th edn. Canada: WileyLiss, 2000;19–28. 2 Freshney RI. Equipment. Culture of Animal Cells – A Manual of Basic Technique, 4th edn. Canada: Wiley-Liss, 2000;31–50. 3 Moureen A. Equipment. General Techniques of Cell Culture, United Kingdom: Cambridge University Press, 1997;7–31. 4 Davis JM. The cell culture laboratory. Basic Cell Culture, 2nd edn. New York: Oxford University Press, 2002;1–29.

CHAPTER 22

Treatment of leukoderma by transplantation of cultured autologous melanocytes Mats J. Olsson

Introduction In the surgical management of vitiligo and other leukodermas where the melanocytes are missing, the general aim is to select an appropriate transplantation model to restore the epidermal melanin unit. The following issues need to be addressed, before deciding on the most suitable method for an individual patient: • the finances and infrastructure available; • the overall level of technical training, more specifically, experience in cell culture and skills of the personnel involved; • experience of harvesting and delivering autologous melanocytes to leukodermic areas using various techniques; • the locations of the anatomical areas to be treated; • the total extent of the depigmented skin and the total size of the areas to be treated; • number and individual size of the lesions; • type of leukoderma; • age of the patient; • texture of the skin in the recipient area; • presence of coarse hair in the recipient area; • texture of skin in possible harvest area; • history of Köbner phenomenon; and • history of hypertrophic scars and keloids. A decision about the most suitable method can be made only after these variables have been evaluated. Methods involving culturing of cells are usually more expensive than other methods and also require a technically better equipped laboratory and

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well-trained staff. But the advantages of such methods are that we can expand the number of cells harvested from a small area to cover extensive areas in one session. Theoretically we can cover the whole body surface from a shave biopsy of the size of a postal stamp, but it is recommended not to exceed 500 cm2/session as there may be excessive exudation from large areas and an increased discomfort to the patient. We can also freeze the cells and store them, and by those means plan and conduct transplantation in several smaller sessions. The culture may be done in a laboratory located in a distant city or country, and the cultured cells may be shipped to the hospital where the transplantation is to be performed. When we have more cells than required for the transplantation, the left-over cells can be frozen and stored for the future use, if and when the depigmented areas flare up or reappear again. Cultured cells are useful in all anatomical areas including troublesome locations such as hairy areas and those with excessive movements like the joints, eyelids, and the corners of one’s mouth. Leukoderma is the term used for disorders, congenital or acquired, in which the skin becomes depigmented due to lack of melanin (pigment) in the epidermis. This may be due to absence of melanocytes in the epidermis and sometimes also in the matrix of the hair follicles. Piebaldism, different kinds of vitiligo, and some cases of chemical leukoderma, belong to this group, in which melanocytes are missing.

Treatment of leukoderma by transplantation of cultured autologous melanocytes Transplantation of melanocytes can serve as a treatment only in this type of leukodermas with missing melanocytes. Surgical methods are an important therapeutic approach to restore pigmentation in the depigmented areas. However, we should refrain from performing surgical therapies in patients with active disease, who have melanocyte destroying activity (e.g. active vitiligo) [1]. Patients should be counseled so they have realistic expectations about the outcome of the procedure. They should be informed that expecting a 100% cosmetically perfect result is unrealistic, even if the whole procedure is faultless. A small area may remain depigmented after the procedure or a repigmented area may have a color darker or lighter than the surrounding skin. Careful evaluation and experience will help predicting the outcome in individual patients. Treatment of leukoderma with cultured autologous melanocytes can be implemented in all types of vitiligo and piebaldism. However as with all surgical approaches, one should be careful in selecting patients with generalized vitiligo, which has the involvement of an autoimmune component and, if the disease is not stable, the transplanted melanocytes may not survive. Attempts to alter the appearance of skin by some cosmetic clinics have led to two new groups of patients with a blotchy depigmentation. One of the groups is patients with chemically induced leukoderma. Many chemicals containing aromatic compounds like phenol- and hydroquinone-derivatives are in use for cosmetic or therapeutic purposes for the management of hypermelanosis [2] or as depigmenting agents in extensive vitiligo [3,4]. These compounds are known to cause pigment cell destruction. We have had several patients referred to us for the management of chemical leukoderma occurring as a side effect from the treatment of facial wrinkles with phenol peel or trichloroacetic acid (TCA) peel at the beauty parlor. Such patients have been successfully treated with transplantation of cultured autologous melanocytes. The other group is comprised of patients who have developed leukoderma as an adverse effect of hair removal with laser or intense pulse light (IPL) therapy. That light can cause death of melanocytes is well known and lasers have been used

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both in the treatment of melasma and for complete depigmentation in extensive vitiligo [4]. We have had several patients referred to us who had lost their pigmentation following hair-removal therapy by light- or laser-treatment. In one interesting case, a male patient had a chessboard-like pattern of leukoderma corresponding exactly to the site where the handpiece of the device had been placed when the light pulses were delivered. Melanin absorbs light over a wide spectral range and the energy in the light beams is converted into heat and liberated into the melanocytes, damaging them [4,5]. Physical trauma to the skin is known to be able to cause Köbner reaction in patients with vitiligo vulgaris [6] and such patients are more prone to develop depigmented lesions when undergoing chemical peeling, laser peeling, or hair removal. The appropriate number of melanocytes for transplantation is about 1000 cells/mm2. This is approximately equal to the density of the confluent cells in the culture flask, and therefore the bottoms of the culture flasks should give a rough estimate about the size of the area that can be covered with the cultured cells. The mean number of melanocytes in the epidermis of Swedish men (mean age 31 years) is 1000– 1500 cells/mm2 [7]. Racial differences in the color of the skin are not due to any significant difference in the number of epidermal melanocytes. Glimcher et al. studied biopsies of foreskins from 15 donors, and divided them into three groups based on the degree of pigmentation. No significant differences in the number of melanocytes could be established between the three groups [8]. The difference in color of the skin is thought to be due to the melanogenetic enzyme activities, type of melanin produced, and the distribution, packing, and degradation of melanosomes. In the keratinocytes of white skin, the melanosomes are aggregated and degraded faster than in black skin. Black skin has larger melanosomes, which are singly packed inside the keratinocytes. In white skin, the melanosomes are aggregated within the keratinocytes and the scattering effect of the melanin is then less. Fig. 22.1 illustrates the location of melanocytes in human skin.

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Stratum corneum Epidermis Basal layer Basal lamina Dermis Fig. 22.1 Location of melanocytes (MC) in human skin.

(Reproduced from Olsson MJ, Vitiligo and Piebaldism: Treatment of Leucoderma by Transplantation of Autologous Melanocytes. Uppsala, Sweden: Uppsala University, 2001.)

Methods Cultured melanocytes applied to dermabraded lesions To apply melanocytes expanded in their numbers onto extensive denuded skin lesions became possible first after the development of optimal culturing conditions for adult human melanocytes [9,10]. A defined melanocyte culture medium free from phorbol esters, pituitary extract, and calf sera is preferable. Normally the donor cells are derived from a shallow shave biopsy, taken from a hidden area. Rarely, in patients undergoing skin reduction surgery for excess skin the removed skin may be used as a donor of cells to be cultured. Melanocytes can also be of useful value and cultured for other reasons than in the surgical treatment of leukoderma (i.e. basic biological research as normal references to melanoma cells, etc.) and I will therefore also take the suitable opportunity to explain how to prepare cells from other tissues than shave biopsies. The method and success rate of cultured autologous melanocyte transplantation have earlier been published in brief [11,12]. But in the following section, we will discuss all the technical and procedural steps in detail, with a precise description on how to collect, culture, and transplant human pigment cells.

Donor tissue

From shave biopsies A normally pigmented area of about 2
4 cm2 in the gluteal region is marked, surgical cleansed, and

Fig. 22.2 The Goulian–Weck knife. Notice that you can

see right through the thin shave biopsy.

anesthetized with a solution containing equal amounts of 10-mg/ml lidocaine and Tribonat® (bicarbonate solution from Fresenius Kabi, Uppsala, Sweden). Tribonat buffers the otherwise acidic lidocaine solution, making injections less painful. A long thin needle is inserted from outside the marked donor area to avoid bleeding at the site from needle stick, and then parallel to the skin surface thrust into the marked area. It is important not to use adrenaline due to the risk of a buckled area with embossed lesions. Press the area with a sterile gauze-pad to facilitate the diffusion. An uneven surface makes it difficult to harvest a very thin and coherent sheet. A superficial shave biopsy (as thin as possible) is taken with a Goulian–Weck skin graft knife (Edward Weck & Company, Inc, Research Triangle Park, NC, USA), (see Fig. 22.2). The Goulian knife should be equipped with a 006 shield, to ensure very shallow biopsies. The specimen is put in a 15-ml test tube containing, Joklik’s modified minimal essential medium (s-MEM, GIBCO BRL, Life Technology, Gaitersburg, MD, USA) and transferred to the laboratory for preparation. The donor area is covered for a week with semi-permeable Tegaderm™ (3M, St. Paul, MN, USA), plus a layer of the air and water vapor permeable

Treatment of leukoderma by transplantation of cultured autologous melanocytes stretch fabric tape Fixomull,® extending a few centimeters beyond the margins of the Tegaderm,® to ensure that the fluid-filled blister that builds up under the dressing does not break. The Tegaderm® and the Fixomull® are glued in place with Mastisol® (Ferndale Laboratories, Inc, Ferndale, MI, USA). The glue ensures that the dressing will remain secured for the period needed (8–10 days). If the lag period between harvesting and culture is between 1 and 4 hours, the medium is furnished with antibiotics (e.g. 50-U/ml penicillin and 0.05mg/ml streptomycin) and kept at 8°C. If the lag period is more than 4 hours, then the biopsies are put in complete M2 melanocyte medium (PromoCell, Heidelberg, Cat. No. C-24300) and kept at 8°C.

From full-thickness skin samples Left-over skin from surgical reconstruction such as breast and abdominal reduction surgery can be used to obtain melanocytes for cultures. The sample is collected in a 50-ml or larger tube, containing lowcalcium and low-magnesium medium, such as sMEM, with 50-U/ml penicillin and 0.05-mg/ml streptomycin, and stored in refrigerator until transported to the laboratory. If it is not possible to take care of the tissue at once when it is delivered to the laboratory, it will stay fresh refrigerated in the tube for up to 30 hours. At preparation, begin with putting and stretching out the whole tissue in a large Petri dish containing 70% ethanol and let it soak for about 30 seconds. This is to eliminate possible microorganisms. Then rinse the tissue twice in sterile 10°C phosphate buffered saline (PBS solution). The epidermal side is turned downwards facing the bottom of the Petri dish and the dish is half-filled with fresh PBS solution. The tissue is trimmed with sharp eye-scissors to remove all fat and the main part of the dermis. The remaining skin sample is thoroughly trimmed to make it as thin as possible, without cutting too many holes through it. This is necessary to enable penetration of enzymes through the residual dermis to reach the dermal–epidermal junction, which is the desired site of their action. The enzymes do not pass through the stratum corneum. The remnants of trimmed fat and dermis are discarded and PBS is changed as often as necessary during the cutting process.

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From foreskin samples Foreskins are collected from the surgical department in test tubes containing s-MEM with 50-U/ml penicillin and 0.05-mg/ml streptomycin. The tube can be refrigerated for up to 48 hours before preparation of cells. The foreskin piece is dipped in 70% ethanol twice and then washed twice in sterile PBS to ensure that microbes are eliminated. The ring structure of the prepucial skin is cut-open and spread out in a Petri dish with the dermal side facing upwards. The dermis is removed by a flat-cutting technique with small eye-scissors to make the skin sample as thin as possible, without creating too many holes through it. The dermal fragments are discarded and PBS is changed as often as necessary during the cutting process.

Release and preparation of free cells The thin donor sample is kept in a Ø 6- or 10-cmdiameter Petri dish inside a laminar-flow hood and washed once with 4 or 8 ml 0.20% w/v trypsin and 0.08% w/v ethylenediamine tetraacitic acid (EDTA) in 80% v/v PBS (all SVA, Uppsala, Sweden) and 20% v/v Joklik’s modified minimal essential medium and refurnished with 5 or 10 ml of the trypsin/EDTA solution. The sample is turned back and forth with the help of jeweler’s forceps to ensure that it comes in complete contact with the solution, and finally, with the epidermis side facing upwards, torn or cut into pieces of about 4 cm2. The air-bubbles under the thin fragments should be removed by gently pressing and scraping the surface with a curved forceps. The Petri dish is incubated at 37°C in 5% CO2 for about 50 minutes for thin shave biopsies, about 3 hours for thin trimmed biopsies derived from full-thickness skin samples and about 2–3 hours for thin trimmed piece of foreskin (Fig. 22.3). At about half the incubation time the pieces are moved around and pressed on with a curved forceps, to ensure that the whole tissue gets soaked in with the trypsin/EDTA solution. If the full-thickness or foreskin specimen is delivered in the late afternoon, it can after the thintrimming procedure be incubated overnight in the trypsin/EDTA solution at 4–8°C and, the next morning, if needed be refurnished with fresh trypsin/ EDTA and incubated at 37°C for about 1 hour or

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Fig. 22.4 Separation of basal epidermal cells after trypsi-

nation. Note the removal of the small mucus-like dermal piece from the remaining epidermal sheets.

Fig. 22.3 Incubation of the Petri dish containing the

trypsin/EDTA floating shave biopsy. Also note the flat flasks of cultured cells.

until the epidermis can be removed from the dermis. Superficial shave biopsies should not be incubated overnight in trypsin/EDTA. It is difficult to give the exact incubation time, since it depends on the thickness of the sheet, but by gentle pressing with a curved forceps on the epidermis, one will, based on the impression pattern of slightly lighter streaks, be able to see if the incubation time is sufficient or not. After incubation, the trypsin/EDTA solution is removed and 3 ml (15°C) of 0.5-mg/ml trypsin inhibitor (Sigma, St Louis, MO, USA) in PBS is added to the Petri dish to terminate the trypsin reaction. The epidermis is removed from the dermis with the help of forceps (Fig. 22.4) and the dermis is transferred to a test-tube containing 5 ml of the highly balanced, serum-free, melanocyte medium M2 (PromoCell, Heidelberg, Germany, Cat. No. C-24300) and vortex-mixed for 5 seconds. The dermal pieces are then removed with the tip of a Pasteur pipette or with the help of a hooked forceps and discarded.

Fig. 22.5 Pellet of cells ready to be resuspended in fresh

culture medium and seeded into a culture flask.

The epidermal pieces are scraped with a curved jeweler’s forceps to free basal cells and then minced to smaller fragments and transferred, together with the trypsin inhibitor, to a test-tube. The tube is vortexmixed for 30 seconds. The Petri dish is rinsed twice with a small volume of s-MEM, which is also transferred to the test tube, and then centrifuged for 7 minutes at 190 g (Fig. 22.5). Sometimes it may be difficult to pellet the cells due to free DNA and collagen, which form mucus-like structures. In such case, the contents of the test tube should be run up and down a few times in a Pasteur pipette with sharp glass-edge. This will cut the mucus into shorter

Treatment of leukoderma by transplantation of cultured autologous melanocytes fragments which will facilitate the formation of a pellet when centrifuged. After the centrifugation the supernatant and the floating stratum corneum–granulosum fragments are removed and the pellet is resuspended in 5-ml M2 melanocyte medium and transferred to a 75- or 150-cm2 culture flask containing M2 medium for culturing. The empty test tube is rinsed twice with 1-ml M2 medium, which is also transferred to the culture flask, to ensure that all cells are collected. The culture flask should be kept lying flat when adding the cell suspension, so that the cells do not stick to the sides or ceiling of the flask. This is to ensure a maximum exchange rate. A 75-cm2 flask should have a total of about 15-ml medium and a 150-cm2 flask should contain about 30 ml of medium (Fig. 22.3).

Culturing of melanocytes Use of a defined culture condition with a medium free from phorbol esters and pituitary extracts and one that enables culturing of cells without a feeder cell layer or pre-coating of the culture flasks, such as the M2 melanocyte medium (PromoCell, Heidelberg, Germany) is preferable. The growth-promotion in defined media systems is enhanced by human growth factors produced with hybrid-DNA technology, where the human codes for the growth factors have been inserted in the genome of yeast cells and the polypeptides produced are then purified. All growth factors used occur naturally in the human skin. The cells should be microscopically controlled every day to ensure that the cells are morphologically looking healthy and that there are no signs of infections (Plate 22.1, facing p. 114). The medium in the culture flasks should be changed every third day. When the culture becomes confluent, the cells are lifted (trypsinated free) and subcultured. If a 100% pure melanocyte culture is needed for research purpose, 45-g/ml geniticin (G418) is supplemented to the medium for 3 days, starting from the 10th day of the culture in order to selectively suppress the fibroblasts. This incubation in geniticin is not required when culture is being done for therapeutic purposes, as the number of fibroblasts will not increase to more than 5–10% when melanocytes are

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cultured in M2 medium. Most of the keratinocytes will be lost in subcultures because of the higher Ca2 levels (about 1 mM). With this Ca2 concentration, keratinocytes will differentiate, stop dividing, and lose their ability to stick to a new culture flask when subcultured. For therapeutic purpose, there is no need to eliminate all keratinocytes, as the remaining keratinocytes support the melanocytes, and theoretically may enhance the healing process in the treated area. But if a 100% pure melanocyte culture is needed for research purposes, the level of Ca2 may be elevated to 1.6 mM for 2 days to differentiate and eliminate keratinocytes more efficiently. After 2 weeks, the number of cells cultured from one biopsy varies from 10
106 to 50
106 depending on the size of the biopsy and the age of the donor. At this stage the cells are ready for transplantation. When the cells in the flask are confluent, they are harvested immediately before the transplantation. The culture medium is removed from the flask and about 5 ml of 37°C trypsin/EDTA solution is added to each 150-cm2 flask. The flask is tilted back and forth a few times to ensure that the solution comes in contact with all cells and then incubated in 37°C for about 2 minutes. After the incubation, the flask is tapped with the palm of the hand on one side, while holding the flask with the other hand. This gives the flask a jerking acceleration sideways, releasing the cells from the plastic surface. Transfer the free cells quickly to a 15-ml test tube containing 4 ml 15°C trypsin inhibitor (Soy-bean extract from Sigma, St. Louis, MO, USA in PBS) with the help of a pipette. The remaining cells in the flask are washed with additional 5 ml 15°C trypsin inhibitor, sucked off with a pipette and added to the same test tube. A gentle spin of 180 g for 6 minutes will settle the cells down into a pellet. The supernatant is discarded and the cells are resuspended in 6-ml room-tempered s-MEM medium without any additives. Resuspension/wash is done twice if cells are to be used for transplantation and only once if the cells are to be seeded into new flasks for subculture. At resuspension the cells are only centrifuged for 4 minutes at 180 g, the supernatant is discarded, and the cells are resuspended in the desired medium for use in any research project or in an extremely small volume of s-MEM (about 0.3–0.4 ml), to be used for immediate transplantation.

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Cryopreservation Left-over cells to be long-term stored for future needs or cells to be shipped frozen are lifted (set free) with the help of the trypsin/EDTA solution as described above, centrifuged into a pellet and resuspended in 1-ml cryoprotectant for each 10 million cells. The cryoprotectant solution consists of 8% dimethyl sulphoxide (DMSO, Mallinckrodt, Inc., Paris, KY, USA) in undiluted newborn calf serum from safe source (country of origin with no currently known cattle infections). Few ready to use serum-free cryoprotectants are also available. Cells and cryoprotectant are mixed by gentle pipetting and transferred to a 1.8-ml cryotube/ml. The tubes are kept on ice for 10 minutes to allow the DMSO to penetrate the cells and then placed directly in 70°C to 85°C for storage or cells can directly after transferring into the cryotube preferably be put straight into a roomtemperature NALGENE freezing chamber (Cat. No. 5100-0001) to achieve a 1°C/minute rate of cooling when immediately placed in 70°C to 85°C. For long-term storage, transfer the frozen cells after 24 hours to a 150°C freezer or to a chamber of liquid nitrogen immersion storage.

Defrosting The frozen cells are defrosted by placing the cryotube into a 37°C water bath. Immediately after the thawing, the tube is quickly wiped with alcohol and the cell-containing solution is transferred carefully (cells are fragile immediately after they have been frozen) to a test tube containing about 10-ml culture medium and centrifuged at 150 g for 4 minutes. The supernatant is removed with a pipette and the cell-pellet carefully resuspended in culture medium, and transferred to a culture flask to resume cultivation or washed in s-MEM once more, centrifuged down to a pellet and finally resuspended in a small volume s-MEM (about 0.4 ml) for immediate transplantation. The survival rate of cells that have been frozen for 1 year is about 70% [13].

Premedication It is important to have a still and calm patient during the transplantation procedure. Therefore, it is recommended to give 5–10 mg of diazepam and/or 10 mg of ketobemidone orally 1 hour before the

transplantation, plus 500–1000 mg of paracetamol 40–50 minutes before the surgery. Erythromycin capsules, 500 mg
2, or a similar antibiotic, is given for 7 days, starting on the day of surgery.

Anesthesia of the recipient site The recipient areas are anesthetized with EMLA® cream (AstraZeneca, Södertälje, Sweden) applied under plastic foil occlusion (e.g. saran wrap) for 1–2 hours and then also locally anesthetized with a mixture of equal parts of 1% lidocaine and Tribonate®-buffer (Fresenius Kabi, Uppsala, Sweden) immediately prior to the surgery. A thin and long needle is used, which is inserted outside the depigmented lesion and then thrust parallel to the skin surface into the lesion. This is to avoid bleeding from needle stick in the area to be treated. In a large area, it may not be possible to reach the center of the lesions from the borders, but a combination of “ring-block” effect of peripheral injections and EMLA® will provide satisfactory anesthesia also in the center of the lesion. In small areas, local anesthetic can be infiltrated to the whole lesion, and therefore EMLA is not required. Total nerve block can be used to achieve satisfactory anesthesia, but it requires some experience to inject the local anesthetic in the right spot; therefore, it should be given by an anesthesiologist. Local anesthesia with freezing spray such as ethyl chloride or fluor-ethyl (Gebauer Pharmaceutical Preparations, Cleveland, OH, USA) can also be used immediately prior to dermabrasion. The sprays give some anesthesia, but are alone not sufficient to give total pain-relief. However, the skin becomes firm and easy to dermabrade when chilled and it is easier for an inexperienced eye to detect remnants of epidermal cells on a chilled denuded surface.

Transplantation and aftercare The washed small volume of s-MEM resuspended cell-pellet is transferred to a 1-ml syringe or kept in the test tube for direct application onto the skin with the help of a pipette. For handling details see above under the heading of “culturing of melanocytes.” The recipient area is cleaned with alcohol, outlined with a sterile surgical marker pen and the epidermis is removed up to the dermal–epidermal junction, using a high-speed dermabrader (20,000 rpm), fitted

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Fig. 22.7 Application of the melanocyte suspension to

the de-epithelialized recipient area.

Fig. 22.6 Dermabrasion until slight bleeding. Notice that

the left index finger is supporting the right hand grip to eliminate jerking movements. The electrical handpiece in the picture is fitted with a regular 17
6 mm diamond fraise wheel.

with a diamond fraise. A wheel, pear, or cone suitable for the size and location of the area to be treated is chosen. Normally, we can dermabrade most of the lesions with a 6-mm wide regular fraise wheel but on rough skin, such as that on the knees, we may need a coarse wheel and on delicate areas such as around the nostrils, corners of the mouth, and on the eyelids, a small pear-shaped fraise may be needed. For eyelids, we have a special made hand-tool fitted with a regular cone; this ensures not to damage the eyelid or the eye during the procedure. The dermabrasion should be performed in at least two different directions. A uniform punctate capillary bleeding from the dermal papilla can be seen (Fig. 22.6). Light freezing with fluoro-ethyl spray reveals if there are any islands of epidermis left. The denuded area is washed with sterile PBS or saline solution, and kept under moistened gauze for few

minutes to ensure that bleeding has stopped. If some puncture bleeding points persist, electrocoagulation is performed with a fine-pointed electrocate needle. The cell suspension with a melanocyte seeding density of 700–1000 cells/mm2 is then applied to the denuded areas and spread with the tip of a syringe, pipette, or a metal-spatula (Fig. 22.7). The cells are secured with a silicone netting (Mepitel®, Mölnlycke AB, Mölnlycke, Sweden) or a plastic netting (Delnet® Fastec Inc., Bronx, NY, USA) extending about 1 cm onto the dry non-dermabraded surrounding skin, which locks it in place so it will not slide over the slippery wound bed. Then two layers of saline-moistened woven gauze compresses and a semipermeable Tegaderm™-film are applied. The latter is glued in place onto the surrounding skin with Mastisol® (Ferndale Laboratories, Inc, Ferndale, MI, USA). The glue ensures that it will be secured in place for the period needed. See Fig. 22.8 for a schematic illustration of the procedure. In all cases the patients should rest at least 4 hours, preferably longer, in a hospital bed after the procedure has been completed. The patient is told that the first 48 hours are most critical for the result and that it is important to restrict the physical activities and avoid tight clothing for the first 2 weeks. The dressing is removed after 8–10 days. At dressing removal a sterile saline solution is first injected into the bandage and allowed to soak for a few minutes before the bandage is removed. Sterile

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Fig. 22.8 Schematic illustration of the operation steps.

forceps and small scissors are used. The Tegaderm® covering the donor site is also removed on the same day. Usually the skin heals nicely at both the recipient and donor sites, and a careful flush with sterile saline solution and light padding with dry gauze compresses is usually sufficient. This is followed by an application of a layer of pure Vaseline with a spatula. On skin exposed to friction or trauma, such as elbows, hands, and feet, after the application of Vaseline the area is covered with a gauze compress, which is secured in place with a Micropore™ tape for another 2 days. The patient is advised to apply a thin layer of Vaseline, once a day for 7 days after the removal of the dressing. This is to minimize the frictional trauma and desiccation to the still fragile surface. A layer of Vaseline is also applied to the donor area. The patient is advised to expose themselves to midday sunlight for a few minutes 2 times a week

for about 2 months, commencing at 1 week after the removal of the bandage.

Follow-up evaluation About 5–8 months after transplantation an overall follow-up evaluation is advised. During the first few weeks, the transplanted area is erythematous, but pigmentation can be seen as early as 2–3 weeks post-transplantation in Wood’s light or with diascopy. During the first year, it is not uncommon to see a slight hyper- or hypopigmentation in some of the treated areas. But the color gradually matches with that of the surrounding skin and after a year it most often blends well with the surrounding skin. Sometimes a 1–2-mm depigmented halo persists around the transplanted area. This is seen more often in patients with generalized vitiligo than those

Treatment of leukoderma by transplantation of cultured autologous melanocytes with piebaldism or segmental vitiligo. Generally this halo is repigmented with repeated sun-exposures. The outcome can be predicted early but the final result will usually be seen about 1.5 years after the surgery (Plate 22.2, facing p. 114).

Documentation Pictures and/or drawings of the lesions should be taken in all patients both before the surgery and during the follow-ups. Files with anamneses and treatment charts are kept in the hospital’s central records.

Discussion Depigmented skin with white hair and glabrous skin respond poorly or not at all to attempts at medical repigmentation. This indicates that hair follicles are the normal and most important reservoir for spontaneous and medical repigmentation, and that surgical transplants are the only effective method to replace the lost epidermal melanocytes and the follicular reservoir. There have been some safety concerns about the use of cultured autografts in vitiligo. It is advisable to use only defined culture media and buffer systems, which contain no currently known carcinogenic or virus-carrying components to minimize the theoretical risks. It is always safer to use welltried systems. All surgical methods and pharmacological treatments carry some risk of adverse events [1], and it is imperative to analyze the risk/benefit ratio for each individual patient. Skin grafts, cultured or non-cultured, are used widely to correct scars, restore pigmentation or treat burn wounds. Many thousands of patients with different types of vitiligo have been treated with various types of transplantation models since 1940s and so far no reports of malignant transformation or other serious adverse effects in the treated lesions have appeared in the medical literature. This indicates that various transplantation procedures in vitiligo have a very high safety profile. Kaufmann et al. and Chen et al. have shown in their respective studies that de-epithelialization of the recipient site with the help of Eri:YAG (erbium: yttrium–aluminum–garnet) or short-pulse CO2 laser in transplantation with cultured pure melanocytes

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can give similar results as with dermabrasion [14– 16]. Laser abrasion may have advantage over dermabrasion on some delicate sites, such as eyelids. Topical thrombin can be used at both donor and recipient sites to achieve hemostasis, though normally it is not required. Patients are advised not to take any medicines interfering with the hemostasis, such as salicylates (Aspirin), for 10 days before and after the surgery. The differences in transplantation results between the various types of leukoderma can probably be explained by etiological differences. In patients with segmental vitiligo and piebaldism, there are no pigment cells in the hair follicle reservoir and no autoimmunity is involved in the etiology, explaining poor response with (UV) ultraviolet therapy, corticosteroids and topical immuno-modulating treatment, while excellent response with autologous melanocyte transplantation. The involvement of melanocyte-specific T-lymphocytes and autoantibodies in vitiligo vulgaris and halo nevi seems to interfere adversely with the outcome of transplantation. In halo nevi, an infiltration of T-lymphocytes in the margin between the normally pigmented skin and the halo and in the border between the halo and the nevi has been demonstrated [17] and several independent research groups have proposed that the etiology of vitiligo vulgaris seems to have autoimmune components [18–21]. In addition, associated disorders may have an effect on the outcome of transplantation procedures in patients with vitiligo. We have experienced a worse outcome in patients with vitiligo vulgaris also having hypothyroidism in comparison to those without hypothyroidism. This possibly indicates a more active autoimmunity in patients afflicted with hypothyroidism. Medications which influence the immune system may also affect the prognosis after transplantation in vitiligo. For example, Simsek et al. reported that interferon alpha-2a may precipitate vitiligo in predisposed individuals [22]. It is difficult to comment on whether the total size of the depigmented areas in vitiligo vulgaris is an indicator of more aggressive autoimmunity or not. However, it appears that those with spreading vitiligo tend to have a larger area of involvement and patients with extensive depigmented areas more often lose the

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repigmentation after transplantation than those with limited involvement [12]. Our previously published multivariate regression analysis has clearly shown that the chance of achieving a successful outcome of transplantation increases with the decrease in the total extent of the vitiligo vulgaris [12]. These observations underscore the importance of informing the patients with vitiligo vulgaris that transplantation will not cure or affect the underlying cause or natural course of their disease and, therefore, it cannot be promised that new patches will not appear in the future. Patients with spreading vitiligo lesions have significantly poorer outcome than those with decreasing lesions or a stable disease. Therefore, patients with extensive vitiligo vulgaris and those who have not had a completely stable, non-progressive disease for at least 2 years should not be chosen for any kind of transplantation. It is obvious that patients with stable forms of leukodermas, namely piebaldism, segmental vitiligo, and focal vitiligo, benefit most from the surgical methods, since they generally retain full pigmentation and have minimal chances of developing new lesions in the future. Slight hyperpigmentation in the treated area can probably be explained by the involvement of hormones and inflammatory mediators released locally in the area during the procedure and wound healing process. The hyperpigmentation usually improves with time. Culturing of melanocytes, in comparison with other surgical methods, has the advantage that larger areas can be covered and that cells can be stored frozen for future use and/or without destruction globally shipped to and used at the best convenience by the consumer/recipient. But techniques requiring cell culturing are also the most difficult methods to use. It requires skillful personnel with special and demanding technical training. Special cell culture laboratories are needed, which restrict this method to well-equipped university hospitals or tissue laboratories such as those for burn clinics.

Conclusions Initial hyperpigmentation is common, especially on the hands and feet. Patients displaying darker

tanning seem to be more prone to hyperpigmentation. There is no conclusive evidence whether these patients have been more in the sun or are of a normally constitutive darker skin complexion. After 6–18 months, the color match is usually the same or close to the same as the surrounding skin in most of the patients. The dorsal aspects of the fingers, especially skin on the joints, are most difficult to repigment. The dorsum of the hands and trunk generally shows good to excellent results. Special care has to be taken in patients who have lesions on both sides of the body, and treatment should be planned for the same occasion. The least important side (smallest areas) should be treated first and then the most important side after patient is turned around by 180°. These patients often show fair and poor results at the sites facing downwards, indicating the importance of gravitation for a good cellular take. A depigmented, 1–3-mm halo between the transplanted and surrounding pigmented skin is not uncommon, especially in patients with generalized vitiligo. Speckled areas of non-pigmented skin are common on the elbows and knees. This may be due to incomplete removal of depigmented epidermis in these areas of thick and uneven epidermis. Most of the other areas, such as those on the trunk, arms, legs, and face, generally develop very even repigmentation with this method. Zachariae et al. [23] reported that this was the method that gave the most even repigmentation. In short it can be concluded that: • Since this technique includes an expansion of the number of cells, the donor site is much smaller than in other methods. This reduces the risk of depigmentation due to Köbner phenomenon at the donor site and makes healing process easier. • Theoretically the whole body surface can be covered with cells expanded from only a postage stamp-sized shave biopsy; however, it is recommended not to exceed 500-cm2 area in a single session for safety reasons. • Cultured cells can easily be stored frozen for a future treatment session or shipped to another hospital anywhere in the world. • Segmental vitiligo and piebaldism almost always respond with complete repigmentation, regardless of the method of transplantation.

Treatment of leukoderma by transplantation of cultured autologous melanocytes • Segmental vitiligo and piebaldism retain all the repigmentation as noted by us in up to 15 years of follow-up. • It is more difficult to achieve complete repigmentation in patients with generalized vitiligo, but they usually respond well if the disease is stable. • Patients with active and extensive generalized vitiligo often show poor repigmentation and such patients should not undergo transplantation. • Predictors of good prognosis are spontaneous repigmentation, no new lesions during the last few years, not involving extensive body surface area, younger age, and a shorter duration of the disease.

References 1 Olsson MJ. What are the needs for transplantation treatment in vitiligo, and how good is it? Arch Dermatol 2004;140:1273–4. 2 Fitzpatrick TB, Arndt KA, el-Mofty AM, et al. Hydroquinone and psoralens in the therapy of hypermelanosis and vitiligo. Arch Dermatol 1966;93:589–600. 3 Mosher DB, Parrish JA, Fitzpatrick TB. Monobenzylether of hydroquinone. A retrospective study of treatment of 18 vitiligo patients and a review of the literature. Br J Dermatol 1977;97:669–79. 4 Njoo MD, Vodegel RM, Westerhof W. Depigmentation therapy in vitiligo universalis with topical 4methoxyphenol and the Q-switched ruby laser. J Am Acad Dermatol 2000;42:760–9. 5 Nanni CA, Alster TS. Laser-assisted hair removal: side effects of Q-switched Nd:YAG, long-pulsed ruby, and alexandrite lasers. J Am Acad Dermatol 1999;41:165–71. 6 Gauthier Y. The importance of Köebner’s phenomenon in the induction of vitiligo vulgaris lesions. Eur J Dermatol 1995;5:704–8. 7 Rosdahl I, Rorsman H. An estimate of the melanocyte mass in humans. J Invest Dermatol 1983;81:278–81. 8 Glimcher ME, Kostick RM, Szabo G. The epidermal melanocyte system in newborn human skin. A quantitative histologic study. J Invest Dermatol 1973;61:344–7. 9 Olsson MJ, Juhlin L. Melanocyte transplantation in vitiligo. Lancet 1992;340:981. 10 Olsson MJ, Juhlin L. Repigmentation of vitiligo by transplantation of cultured autologous melanocytes. Acta Derm Venereol 1993;73:49–51.

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11 Olsson MJ, Juhlin L. Transplantation of melanocytes in vitiligo. Br J Dermatol 1995;132:587–91. 12 Olsson MJ, Juhlin L. Long-term follow-up of leucoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol 2002; 147:893–904. 13 Olsson MJ, Moellmann G, Lerner AB, et al. Vitiligo: repigmentation with cultured melanocytes after cryostorage. Acta Derm Venereol 1994;74:226–8. 14 Kaufmann R, Greiner D, Kippenberger S, et al. Grafting of in vitro cultured melanocytes onto laserablated lesions in vitiligo. Acta Derm Venereol 1998; 78:136–8. 15 Chen YF, Chang JS, Yang PY, et al. Transplant of cultured autologous pure melanocytes after laserabrasion for the treatment of segmental vitiligo. J Dermatol 2000;27:434–9. 16 Chen YF, Yang PY, Hu DN, et al. Treatment of vitiligo by transplantation of cultured pure melanocyte suspension: analysis of 120 cases. J Am Acad Dermatol 2004;51:68–74. 17 Akasu R, From L, Kahn HJ. Characterization of the mononuclear infiltrate involved in regression of halo nevi. J Cutan Pathol 1994;21:302–11. 18 Baharav E, Merimski O, Shoenfeld Y, et al. Tyrosinase as an autoantigen in patients with vitiligo. Clin Exp Immunol 1996;105:84–8. 19 Kemp EH, Gawkrodger DJ, Watson PF, et al. Immunoprecipitation of melanogenic enzyme autoantigens with vitiligo sera: evidence for cross-reactive autoantibodies to tyrosinase and tyrosinase-related protein-2 (TRP-2). Clin Exp Immunol 1997;109:495–500. 20 Ogg GS, Rod Dunbar P, Romero P, et al. High frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in autoimmune vitiligo. J Exp Med 1998;188:1203–8. 21 Hedstrand H, Ekwall O, Olsson MJ, et al. The transcription factors SOX9 and SOX10 are vitiligo autoantigens in autoimmune polyendocrine syndrome type I. J Biol Chem 2001;276:35390–5. 22 Simsek H, Savas C, Akkiz H, et al. Interferon-induced vitiligo in a patient with chronic viral hepatitis C infection. Dermatol 1996;193:65–6. 23 Zachariae H, Zachariae C, Deleuran B, et al. Autotransplantation in vitiligo: treatment with epidermal grafts and cultured melanocytes. Acta Derm Venereol 1993;73:46–8.

CHAPTER 23

Transplantation of in vitro cultured epithelial grafts for vitiligo and piebaldism Liliana Guerra, Sergio Bondanza and Desanka Raskovic

Cell therapy is going to be an important alternative to conventional surgical methods in the treatment of pigmentary disorders. Autologous epidermal cells have been utilized in the treatment of pigmentary disorders either as a non-cultured epidermal cell suspension [1–4] or once they have been cultured to expand the original cell population [2,5–18]. Non-cultured autologous epidermal cells are utilized to repigment areas 5–10-fold greater than donor areas [1–4], whereas cultured epidermal cells allow to treat areas 50–500-fold larger than donor ones [2,5–18]. Cultured melanocytes can either be inoculated as a pure melanocyte population [2,5–8] or they can be transplanted as a co-culture with keratinocytes, in the in vitro reconstituted epidermis [9–18]. Several considerations prompted us to choose the in vitro reconstituted epidermis-bearing melanocytes for the treatment of pigmentary disorders. Indeed, in the cultured epidermal sheet: (i) keratinocytes regulate melanocyte growth and differentiation, particularly because keratinocytes are a source for a variety of growth and survival factors for melanocytes [19–21]; (ii) keratinocytes regulate the proper melanocyte to keratinocyte (M/K) ratio [20,22]; (iii) melanocytes organize themselves into the basal layer of the cultured epidermis, develop dendrite arborization with melanosome-containing processes and transfer melanosomes into basal keratinocytes [20,22–24], hence melanocytes maintain their physiological characteristics when co-cultured with keratinocytes; (iv) when cultured epidermal autografts are grafted onto full-thickness burn sites,

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excised to the fascia and therefore lacking hair follicles or other sources of local melanocytes, the transplanted melanocytes expand to repopulate and hence repigment the recipient site [25,26]; (v) keratinocyte cultivation allows for easy and rapid production of large quantities of cultured autografts (up to 2 m2) [25–29,31]; and (vi) lastly, but most importantly, cultured epidermal grafts have been widely used all over the world for the last 20 years for the treatment of thousands of patients suffering from large skin and mucosal defects [25,26,28–31], and increased risks of neither carcinoma nor melanoma have ever been reported. Ultrastructural studies demonstrated that keratinocytes of achromic lesions in vitiligo patients show degenerative changes even in long-standing stable vitiligo [32]. Moreover, an impaired keratinocyte-derived cytokine production was shown in vitiligo-affected body sites [33]. In the end, the depigmented suction-blistered epidermis displays a larger number of apoptotic keratinocytes when compared with the normally pigmented epidermis in vitiligo patients [34,35]. These sets of evidence reinforce the hypothesis that an abnormal keratinocyte environment could be the major cause of vitiligo [36], and that melanocyte destruction could be a secondary phenomenon. The epidermal-melanin unit is based on the close interaction between melanocytes and keratinocytes and several keratinocyte-derived cytokines affect melanocyte migration, proliferation, and differentiation [19,21,37]. All together, these findings further convinced us to perform transplantation of co-cultured

Transplantation of in vitro cultured epithelial grafts for vitiligo and piebaldism epidermal cells, particularly in the case of vitiligo patients. By grafting melanocytes and keratinocytes together, both of which have been isolated from a healthy normally pigmented skin area, we may be able to avoid the remote possibility that altered resident keratinocytes in vitiligo lesions could affect the physiological melanocyte function when only a pure melanocyte population has been grafted.

The in vitro reconstituted epidermis-bearing melanocytes in the treatment of pigmentary disorders Owing to the pioneer work of Rheinwald and Green [38], normal human epidermal cells can be serially propagated in vitro to reconstitute several sheets of a stratified squamous epithelium, which maintains biochemical, morphological, and functional characteristics of an authentic epidermis [20,22,23,30]. Full-thickness skin biopsies (Plate 23.1A, facing p. 114) are taken from unaffected body areas, minced (Plate 23.1B, facing p. 114) and treated with a solution containing the enzyme trypsin (Plate 23.1C, facing p. 114), which breaks down junctions between cells. Isolated cells are collected and plated on a feeder layer of 3T3–J2 cells (Plate 23.1D, facing p. 114). These 3T3–J2 cells are established lines of mouse fibroblasts, which, after being lethally irradiated to prevent their growth, support keratinocyte proliferation by production of growth factors [38]. Once plated on 3T3 cells at precise cell densities, keratinocytes start growing and forming colonies. As the colonies continue to expand, they sweep the supporting 3T3 off the vessel surface, and cells of the neighboring colonies begin to make contacts. When colonies become confluent, they form a sheet of pure epithelium (Plate 23.1E, facing p. 114). At this point, epidermal cells can again be dissociated by the enzyme trypsin and plated in new culture vessels, which result in a large increase of the original cell population. Grafts destined for transplantation in achromic disorders are prepared from confluent secondary cultures. Cultures are treated with the neutral protease dispase, which dissolves the attachments between the basal cells and the bottom of the flasks, without disturbing the desmosomal junctions

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between adjacent cells. The epithelium is backed with a sterile petrolatum dressing (basal side down) and free edges are sutured to the gauze with titanium ligating clips, to prevent shrinkage of the graft (Plate 23.1F, facing p. 114). The gauze-mounted epithelium is placed in sterile, biocompatible, and non-gas-permeable boxes (Plate 23.1G, facing p. 114), and is transferred to the operating room for transplantation procedures. After the surgical removal of the achromic epidermis, autografts are applied onto the receiving bed and secured by bandages. Optimized cryopreservation procedures permit us to plan further operations using frozen epidermal cells previously isolated from the same skin biopsy specimen.

The in vitro reconstituted epidermis-bearing melanocytes: culture conditions for optimal M/K ratio Melanocyte–keratinocyte co-cultures can reproducibly be obtained by optimizing conditions for (i) cell yield through skin biopsy treatment, (ii) density for cell plating in culture flasks, and (iii) conditions for culture amplification. Human epidermis contains approximately 4
106 keratinocytes/cm2 [39]. In fact, the optimal epidermal cell yield after trypsin treatment of the skin biopsy is 3–4
106 cells/cm2. In our experience, this high cell yield may easily be obtained when the skin biopsy is larger than 0.5 cm2 (this size refers to the biopsy specimen after its shrinkage) [15,16]. The favorable cell yield from the skin biopsy allows to reconstitute an appropriate M/K ratio in primary cultures, and to maintain it in serial subcultivations. If the M/K ratio of primary cultures is unfavorable, it will be very difficult to obtain a physiological M/K ratio in the subsequent phases of culture amplification. In addition, experience has taught us that an appropriate M/K ratio in culture can be preserved during repeated subcultivations, particularly when epidermal cells are seeded in primary or secondary cultures at high density (4
104 cells/cm2) and subcultivated 1 day after they have reached the confluence condition [15–17].

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Clinical applications of cultured epidermis in pigmentary disorders Since 1981, autologous cultured epidermal cells have been used for the permanent coverage of massive full-thickness burns [30]. In most cases, epidermal regeneration obtained with cultured keratinocytes has been proven to be life saving [25,26,28–31]. In the last few decades, cultured epidermal sheets bearing biologically active melanocytes have also been used for the treatment of patients suffering from disorders of the pigmentary system, such as stable vitiligo and piebaldism, with encouraging results [9–18]. In our opinion, the highest performance of cultured grafts in the treatment of these patients is due to: 1 thorough quality controls of cell cultures, 2 accurate selection of patients, and 3 careful preparation of the receiving bed.

Quality controls of cell cultures Our keratinocyte culture medium contains a mixture of Dulbeco’s Modified Eagle’s Medium (DMEM) and Ham’s F12 media, fetal calf serum (FCS), insulin, adenine, hydrocortisone, cholera toxin, triiodothyronine, glutamine, and epidermal growth factor (EGF) [15–17]. Following the GMP (good manufacturing practice) procedures, safety and quality of media and additives for the somatic cell therapy must be assured (see Chapter 25). In particular, antibiotics should be avoided for cultures to be used in humans, EGF should come from a recombinant source and calf serum must be sourced from countries with the lowest possible GBR (geographical BSE risk) level. An alternative to the bovine serum would be the autologous serum, isolated from the same individual who donated the cells. However, we did not consider this possibility in vitiligo patients, where a very high risk exists that circulating autoantibodies can destroy melanocytes [40]. Quality controls of cell cultures also include tests to assess the absence of adventitious agents in each step of the product manufacture and assays to indicate their suitability for transplantation.

The most important quality controls to evaluate the potential clinical efficacy of cultured grafts in vitiligo patients are (1) the evaluation of the efficiency of colony formation by cultured keratinocytes during each cultivation procedure and (2) the evaluation of the correct maintenance of a physiological M/K ratio in grafts to be transplanted. A colony-forming keratinocyte can initiate one of two types of colonies: either a colony that can proliferate for a long period of time (Plate 23.2A, facing p. 114) or a colony that will arrest its growth after a short time, that is the aborted colony (Plate 23.2B, facing p. 114). An aborted colony is a colony in which all cells have lost their capacity for proliferation, that is all cells are terminally differentiated and they are large and flattened (Plate 23.2B, facing p. 114). The aborted colony is small (5 mm2), with a very irregular shape (Plate 23.2C, facing p. 114; see black arrow), in contrast to the smooth and circular perimeter of a progressively growing colony (Plate 23.2C; see blue arrow) [41]. The latter is usually large (10–30 mm2) (Plate 23.2C; see blue arrow) and contains mostly small proliferating cells (Plate 23.2A) [41]. A simple and reliable method to evaluate the clonogenic potential of individual colony-forming keratinocytes is the colony-forming efficiency (CFE) assay (Plate 23.2C) [41]. Schematically, the CFE of cultured keratinocytes is determined by plating 1000 cells, fixing colonies with 3.7% formaldehyde 12–14 days later, and staining them with 1% Rhodamine B. Total and aborted colonies are then evaluated with the aid of a phase-contrast microscope: total colonies are calculated as a percentage of total plated cells; aborted colonies are calculated as a percentage of total colonies. The CFE assay allows to check the quality of a culture system, by using well-set control values, and to identify whether inadequate culture conditions are adopted, which may accelerate epidermal cell senescence, and even cause the cultured autograft transplantation to be useless. Melanocytes are located in the epidermal basal layer and project their dendrites into the epider-mis, where they transfer melanosomes into keratinocytes. These dendritic contacts produce interfaces with multiple keratinocytes (approximately 36 per melanocyte, in vivo), giving rise to the “epidermal-melanin unit”

Transplantation of in vitro cultured epithelial grafts for vitiligo and piebaldism [42]. The M/K ratio can be quite variable in vitro; however, the final ratio between keratinocytes and melanocytes in culture remains constant during repeated subcultivations [16,22] and reflects the ratio originally present in the skin biopsy from where the cells were cultivated [22]. Melanocytes have a high level of tyrosinase activity, which is considered to be a biochemical marker specific for this cell type and can be assessed by the DOPA (dihydroxyphenylalanine) reaction [15–17,22]. The M/K ratio by DOPA staining can be performed either on cells dissociated by trypsin or on parallel cultures treated in the same way as those destined for transplantation (Plate 23.2D, facing p. 114) [15–17]. These basic quality controls eliminate one important uncontrollable variable in the evaluation of cultured autograft performance and should always represent a starting point for improving keratinocyte cultivation, in order to achieve more positive and reproducible results.

Selection of patients Selection of patients for autologous cultured epidermis transplantation is particularly important in vitiligo, whereas any patient suffering from piebaldism may be submitted to this technology. In vitiligo patients we must follow these general indications: patients must not have responded to previous treatments and must have a stabilized form of vitiligo. The necessary conditions currently utilized to select vitiligo patients for surgery are: (1) failure of at least two standard medical approaches, (2) absence of signs of vitiligo activity, and (3) absence of autoimmune disorders and/or organ-specific circulating autoantibodies. Clinical stability of vitiligo is the main inclusion criteria for its surgery, and it affects the success of surgical therapies: active lesions do not usually respond well to surgery, whereas undoubtedly stable lesions (such as those of long-standing segmental vitiligo and of piebaldism) always show complete or almost complete repigmentation [15–17]. Unfortunately, there is no consensus regarding the clinical evaluation of disease stability in vitiligo: according to different definitions, it can range from 4 months to 2 years [43]. Moreover, the clinical evaluation of disease stability in vitiligo is a very difficult task [43,44].

183

On the basis of our previous experience [15,16], we currently ask for a stability period of more than 2 years. However, this does not seem to be sufficient to exclude “reactivation” of vitiligo, either due to the misjudgment of patients of their vitiligo stability or caused by the quite unpredictable course of this disease. The minigraft test or test grafting (TG) [45,46] may detect patients with unstable vitiligo, but this procedure may also produce false negative or positive results [43,44,47]. Furthermore, in our experience very few patients were favorably disposed toward the minigraft test: in particular, patients with focal, segmental, or limited generalized vitiligo, whose achromic lesions are mainly localized in visible body areas, are not willing to be left with scars, however minimal, such as those which can occasionally be caused by the minigraft test. Hence, we are unable to perform this test regularly. An alternative could come from data showing the association of the experimentally induced Köbner phenomenon with the activity of the disease [48]. Cryopreservation of cultured cells before transplantation can give the possibility of planning cell grafting 6 months after the biopsy, in order to evaluate the appearance of Köbner phenomenon at the site from where the biopsy was taken. Also in this case, however, false negative results can be detected: in our experience only half of the “reactivated” patients have shown a positive Köbner phenomenon at the donor site.

Preparation of the receiving bed A well-prepared receiving bed is one of the most important goals in the surgery of pigmentary disorders by using the autologous cultured epidermis. When epidermis is removed without significant bleeding or inflammation, we can be sure to obtain a high and reproducible take of cultured autografts in complete absence of scars, and a consequently high repigmentation of the treated areas (Plate 23.3, facing p. 114) [15–17]. Furthermore, a fast and safe removal of epidermis allows the treatment of large vitiligo lesions during a single surgical operation. Dermabrasion [13], chemical blisters [11,14], diathermosurgery (Timedsurgery) [15] and, more recently, laser surgery [16–18] are the techniques

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which have been used to remove achromic epidermis. Programmable TIMED-diathermocautery allows to selectively remove large areas of the epidermis from the underlying dermis in local anesthesia, while precisely controlling all operative parameters (electrical power, appropriate shape of the output wave-form, emission time, dimension of the electrode) [15]. New-generation Er:YAG (erbium: yttrium–aluminum–garnet) lasers provide a controlled epidermal vaporization with minimal thermal damage to the papillary dermis and make laser disepithelialization a reproducible and predictable procedure, giving a precise epidermal ablation even in difficult anatomical sites [16,17]. In addition, erbium laser surgery can be performed without anesthesia or after a simple pre-treatment with a topical anesthetic cream. All these characteristics have been very important in changing time-consuming, painful, or uncomfortable procedures into surgical methods, which maintain clinical benefits while considerably reducing possible side effects.

The importance of the postoperative management Once applied, grafts cannot be moved anymore, because take starts in a few minutes and any further maneuver may greatly traumatize cultured cells. Grafts must be secured onto the receiving bed by non-adherent gauzes and sterile cotton bandages. Proper immobilization of treated areas is required for 6–7 days to prevent graft loss or displacement. Antibiotic and anti-inflammatory therapies should be given for at least 7 days. After 7 days, bandages and grafts can be removed to evaluate the “take down” of the cultured epidermis. Patients should then be advised not to expose treated areas to detergents, creams, or perfumes for 1 month. Sun avoidance is advisable for at least 3 months, after which mild sun or ultraviolet A (UVA) exposure is recommended, but only for patients who did not experience hyperpigmentation of the transplanted areas. Follow-up visits should be carried out 3, 6, 9, 12, 24, 36, and 48 months after transplantation, to assess the maintenance of the achieved repigmentation. Long-term check-ups also allow to control the real

stability of vitiligo and the absence of Köbner phenomenon both on the donor site and on the transplanted body area. In our experience, repigmentation cannot be evaluated before the 6-month follow-up visit, because even non-serious inflammatory reactions may induce false positive results. Hyperpigmentation is the only side effect we detected in patients who received autologous cultured cells. This reaction is transitory, but may persist for up to 2 years after transplantation and should be avoided by using protective measures against sun-exposure.

Histological examination of the regenerated epidermis In the in vitro reconstituted epidermis, melanocytes and keratinocytes maintain their in vivo characteristics, that is melanocytes are regularly distributed among basal keratinocytes in a physiological ratio (Fig. 23.1A, arrows). After the autologous transplantation, melanocytes repopulate the grafted area, so restoring the epidermal-melanin unit (Fig. 23.1B). Previous histological and ultrastructural studies of the transplanted skin area have already demonstrated a normal distribution of melanocytes in the epidermal basal layer and a normal differentiation pattern of keratinocytes after cultured epidermis grafting [9,10]. Following transplantation of cultured autologous epidermis, Kumagai and Uchikoshi [13] evaluated at different points of time the reappearance of DOPApositive melanocytes in the grafted sites. Twelve days after grafting, only a few DOPA-positive melanocytes without melanin granules appeared in the epidermis and dermis. Thirty to forty-five days after grafts, melanocytes were observed in the basal layer of the epidermis. The number of melanocytes progressively increased, reaching a normal arrangement in the epidermal basal layer 6–8 months after grafting.

Overall success rate in the literature The amount of data available on the use of cultured epidermal grafts in the treatment of pigmentary disorders is very limited [9–18], making this technology

Transplantation of in vitro cultured epithelial grafts for vitiligo and piebaldism

(A)

(B) Fig. 23.1 (A) Histological examination of a confluent

cultured graft: melanocytes (arrows) are regularly distributed among basal keratinocytes of the in vitro reconstituted epidermis (DOPA reaction and hematoxylin staining;
40 magnification). (B) Histological examination of a transplanted skin area of the leg, 12 months after cultured epidermis grafting: MEL-5 antibody recognizes melanocytes (arrows), which are properly distributed among keratinocytes of the basal layer (fast red staining;
20 magnification).

seem to be at a developmental stage whereas for us it is a routine practice. Treatment outcome was satisfactorily analyzed only in six studies reporting more than three patients/ study (Tables 23.1 and 23.2) [11,13,15–18]. In these series, the overall success rate of autologous transplantation with the in vitro reconstituted epidermis (which can be calculated by dividing the total number of repigmented cm2 by the total number of transplanted cm2) ranged between 70% and 95% (Table 23.1). Complete or almost complete

185

repigmentation (90–100% repigmentation) was obtained in 33–61% of vitiligo patients and in 100% of piebaldism patients (Table 23.2). In two studies, however, complete repigmentation was achieved by combining the minigraft [11] or the NB-UVB (narrowband ultraviolet B) therapy [18] with the cultured graft technique. In particular, complete or almost complete repigmentation was obtained in 75–100% of segmental vitiligo patients [11,15,16], whereas the percentage of complete or almost complete repigmentation in patients with other forms of vitiligo was quite variable (Table 23.2). The face and neck, chest and abdomen, and legs were easily treatable areas, but hands were very hard to treat. One of the greatest difficulties in analyzing the literature data is to try to compare studies which showed such large differences in the culture techniques utilized (some of them add substrates for cell growth and lack parameters to relate quality controls of cell cultures and the clinical efficacy of the cultured graft), in the outcome evaluation (absence of a precise assessment of the size of repigmented/ transplanted areas and of treated area localization), and in the receiving bed preparation procedures. In our opinion, much effort must be made to identify generally accepted parameters for success rate evaluation, such as the complete clinical history of treated patients, a very detailed description of treated areas, exhaustive information about the culture system chosen and its quality controls, accurate instructions regarding surgical procedures, precise indications concerning the technique utilized to measure the treatment response, and the scoring systems used to evaluate repigmentation [49].

Conclusions In vitro cultured epidermal autografts bearing melanocytes were used in the treatment of refractory pigmentary disorders as early as 1989 [9–18]. Our experience suggests that repigmentation is obtained by means of true “take” of cultured melanocytes, as opposed to migration (potentially induced by surgical maneuvers of epidermal abrasion) of resident melanocytes from surrounding pigmented skin or from pigmented hair follicles. In

11

32

21

6

93

Andreassi et al., 1998 [14]

Guerra et al., 2000 [15]

Guerra et al., 2003 [16]

Guerra et al., 2004 [17]

Pianigiani et al., 2005 [18]

24

n.d.

18

18

24

36

Months of stability

No

No

No

No

No

Yes

Minigraft test

n.d.: not defined and n.m.: not mentioned.

9

Number of patients

Falabella et al., 1992 [11]

Author [reference]

reporting more than three patients/study.

Focal or segmental

1 segmental 8 generalized

Clinical form of vitiligo

Er:YAG laser

Er:YAG laser

Er:YAG laser

70.4 (570/810)

76.7 (4667/6078)

75.9 (1759/2315)

95.4 (2791/2924)

n.m.

Cultured autografts grown on a membrane of hyaluronic acid Cultured autografts

Cultured autografts

Cultured autografts

Cultured autografts  NB-UVB

2
3 73 810 (10–200)

0.5–3.3 105 2315 (11–317) 1.2–3 417 2924 (107–710)

0.5–4 189 6078 (14–1566)

Cultured 70.8 (835.4/1180) autografts  minigrafts (three patients)

2
5 131 1180 (80–240)

49 focal 2–4 26 segmental 18 generalized

6 piebaldism

6 focal 4 segmental 1 acrofacial 10 generalized

Timedsurgery 8 focal 4 segmental 8 acrofacial 12 generalized

Carbon dioxide

Liquid nitrogen

Preparation of the recipient site

Cultured autografts (Rheinwald and Green technique) Other treatments

Average skin biopsy cm2 Average transplanted cm2 Total transplanted cm2 (minimum– maximum size) Average percentage of repigmentation (repigmented cm2/ transplanted cm2)

n.m.

1:169

1:95

1:165

n.m.

n.m.

M/K ratio in grafts

Table 23.1 The in vitro reconstituted epidermis in the treatment of vitiligo and piebaldism: description of patients and techniques used in six clinical studies

90–100 65–89 20–64 0–19

90–100 65–89 20–64 0–19

90–100 65–89 20–64 0–19

90–100

Focal–segmental Complete Partial (50) Negligible (50) Generalized Partial (50)

Andreassi et al., 1998 [14]

Guerra et al., 2000 [15]

Guerra et al., 2003 [16]

Guerra et al., 2004 [17]

Pianigiani et al., 2005 [18]

n.m.: not mentioned.

90–100 65–89 20–64 0–19

Falabella et al., 1992 [11]

Author [reference]

30

60 30 10

100 (6/6)

61.9 (13/21) 14.2 (3/21) 4.7 (1/21) 19 (4/21)

43.7 (14/32) 15.6 (5/32) 25 (8/32) 15.6 (5/32)

54.5 (6/11) 9 (1/11) 27.2 (3/11) 9 (1/11)

33.3 (3/9) – 55.5 (5/9) 11.1 (1/9)

Percentage of repigmentation/ Percentage of repigmented patients (repigmented/ treated patients)

Vitiligo: Focal–segmental Generalized (image analysis)

Piebaldism (image analysis)

Vitiligo: Focal Segmental Acrofacial Generalized (image analysis)

Vitiligo: Focal Segmental Acrofacial Generalized (image analysis)

Vitiligo: Focal/segmental (image analysis)

Vitiligo: Segmental Generalized

60 (n.m.) –

100 (6/6)

50 (3/6) 100 (4/4) – (–/1) 60 (6/10)

75 (6/8) 75 (3/4) 12.5 (1/8) 33 (4/12)

54.5 (6/11)

100 (1/1) 25 (2/8)

Clinical forms/percentage of patients with 90–100% repigmentation (repigmented/treated patients) (analysis system)

n.m.

Forehead Chest, abdomen Knees, legs

Face, neck Chest, back, abdomen Arms Legs Hands, ankles, feet

Face Face: periorificial areas Neck Chest, abdomen Knees, legs Hands, feet

Face Neck Chest Arms, forearms Hands

Chest Legs Hands Ankles, feet

Treated body areas/ Average percentage of repigmentation (repigmented cm2/ transplanted cm2)

98 92.7 97.4

68.2 94.4 26.4 100 65.6

89.6 35.2 96.3 94.2 92.2 7.6

100 79.6 90 70.5 45

83 100 32 72

No relapses 18 months after grafts. No Köbner phenomenon at the biopsy site. Hypertrophic scars in one patient.

Transitory hyperpigmentation in two patients.

Relapses 3–6 months after grafts in three patients. Köbner phenomenon at the biopsy site in one patient. Transitory hyperpigmentation in two patients.

Relapses 6–9 months after grafts in seven patients.

No relapses 18 months after grafts. No Köbner phenomenon at the biopsy site. Local sepsis in one patient.

Transitory hyperpigmentation. No relapses 1–2 years after grafts.

Relapses and side effects

Table 23.2 Overall success rate in transplanted vitiligo and piebaldism patients, including specifications of clinical forms and body areas submitted to the surgery.

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fact, complete repigmentation is easily obtained even in large achromic areas devoid of pigmented hair, where the absence of the melanocyte hair reservoir and the low potential for migration of melanocytes [50] could not have given such a positive result. In addition, the potential role of postinflammatory, spontaneous repigmentation caused by epidermal removal using laser surgery has recently been shown to be very limited, even in the presence of additional UV irradiation [51]. The main advantages of autologous cultured epidermis are (1) the possibility of producing a large amount of epithelial sheets starting from a small biopsy, so eliminating the risk of scars at multiple donor sites and (2) the possibility of transplanting a large body surface during a single operation. An additional advantage of cultured epidermal sheets is that keratinocytes regulate in vitro both proliferation and differentiation of surrounding melanocytes [20,22,23]. Therefore, in cultured epidermis, properly functioning melanocytes are interspersed within the epidermal basal layer at regular intervals and in a physiological ratio [20,22–24], hence assuring a homogeneous distribution of transplanted melanocytes. Obviously, a key issue for the successful clinical outcome of cell therapy deals with the “quality controls” of the culture system. For instance, unsatisfactory epidermal regeneration, which has been reported following the utilization of cultured epidermal autografts in full-thickness burns, might arise from depletion of epidermal stem cells, which can occur because of incorrect culture conditions or inappropriate cell culture substrates. Similarly, culture conditions must be optimized for the application of cultured grafts in “stable” vitiligo and piebaldism. In these cases, lack of a proper and reproducible melanocyte concentration within the epidermal grafts may cause repigmentation to fail or be impaired in treated areas. Therefore, the M/K ratio should be routinely evaluated in cultures before autografts. Autologous cultured epidermal sheets need safe and monitored cell culture facilities, as well as high levels of technical expertise and quality and safety controls. This determines the high cost for this type of cell therapy, which is the only disadvantage of this technology.

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41 Barrandon Y. The epidermal stem cell: an overview. Dev Biol 1993;4:209–15. 42 Halaban R, Hebert DN, Fisher DE. Biology of melanocytes. In: Freedberg IM, Eisen AZ, Wolff K, et al (eds.) Fitzpatrick’s Dermatology in General Medicine. New York: McGraw-Hill, 2003;127–48. 43 Malakar S, Lahiri K, Malakar RS. How unstable is the concept of stability in surgical repigmentation of vitiligo? Dermatology 2000;201:182–3. 44 Lahiri K, Malakar S, Banerjee U, Sarma N. Clinicocellular stability of vitiligo in surgical repigmentation: an unexplored frontier. Dermatology 2004;209:170–1. 45 Falabella R, Arrunategui A, Barona MI, Alzate A. The minigrafting test for vitiligo: detection of stable lesions for melanocyte transplantation. J Am Acad Dermatol 1995;32:228–32. 46 Falabella R. The minigrafting test for vitiligo: validation of a prediction tool. J Am Acad Dermatol 2004;51:672–3.

47 Olsson MJ. What are the needs for transplantation treatment in vitiligo, and how good is it? Arch Dermatol 2004;140:1273–4. 48 Njoo MD, Das PK, Bos JD, Westerhof W. Association of the Köbner phenomenon with disease activity and therapeutic responsiveness in vitiligo vulgaris. Arch Dermatol 1999;135:407–13. 49 van Geel NAC, Ongenae K, Vander Haeghen YMSJ, Naeyaert JM. Autologous transplantation techniques for vitiligo: how to evaluate treatment outcome. Eur J Dermatol 2004;14:46–51. 50 Falabella R. What’s new in the treatment of vitiligo. JEADV 2001;15:287–9. 51 van Geel N, Ongenae K, De Mil M, et al. Double-blind placebo-controlled study of autologous transplanted epidermal cell suspension for repigmenting vitiligo. Arch Dermatol 2004;140:1203–8.

CHAPTER 24

Simplifying the delivery of cultured melanocytes and keratinocytes for grafting patients with vitiligo Sheila MacNeil and Paula Eves

Introduction The surgical management of vitiligo has reached an interesting stage in its development as evidenced by this book. It is no longer a question of can patients with vitiligo be helped surgically but rather which patients, at what stage of their disease, and by which techniques. Several pioneering research groups, many of which have contributed to this book [1–10], have shown that transplantation of melanocytes from a pigmented site on the body to the depigmented areas will have a good chance of restoring pigmentation for patients with stable vitiligo. The issue of choosing those patients most likely to benefit from the surgery (and avoiding disappointment for those whose disease is still active and are unlikely to achieve stable pigmentation after surgery) is key and will not be discussed in this chapter. We suggest that the evidence of the last decade shows that there are also several successful techniques for surgical repigmentation of vitiligo – these can be divided into those which do not involve any culture of cells [1,2,9] and those which take a small biopsy and expand cells in the laboratory before delivering them back to patients [3–10]. In this chapter we look to apply some of the lessons of the last decade of culturing keratinocytes for grafting and propose a new approach to simplifying the delivery of cultured melanocytes to patients via a cell delivery carrier which could also be used for transport of cells over considerable distances. This

could help translate vitiligo grafting with cultured autologous cells from a clinical research project to a more routine clinical service provision. In developing this approach we have also sought to reduce the risk of delivering cultured cells to patients.

Aim In brief our aim is to develop a keratinocyte– melanocyte co-culture system that is: 1 low risk for the patient, 2 easy for the clinician to apply, and 3 capable of treating vitiligo patients who are geographically distant from the specialized culture laboratory.

Background to development of the melanocyte–keratinocyte carrier system A number of related issues have led to our development of a cell carrier system for patients with vitiligo – of greatest concern is the issue of regulating melanocyte biology so that cells expanded in vitro are transferred to the patient in the company of epidermal keratinocytes. The latter will not only ensure good and rapid re-epithelialization with reduced risk of scarring, but should regulate the number and function of melanocytes in those cultures. A few years ago we explored the use of cultured epithelial autografts (CEA) containing “passenger”

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melanocytes in the grafting of five patients with vitiligo [11]. At that time we cultured the CEA using the media which was originally developed for keratinocyte expansion by Rheinwald and Green in 1975 [12]. This media, commonly referred to as “Greens Media” consists of Dulbeco’s Modified Eagle’s Medium (DMEM) and Ham’s F12 medium in a 3:1 ratio supplemented with 10% fetal calf serum (FCS), 10 ng/ml epidermal growth factor (EGF), 0.4
g/ml hydrocortisone, 1.8  104 mol/l adenine, 5
g/ml insulin, 5
g/ml transferrin, 2  107 mol/l triiodothyronine, 0.625
g/ml amphotericin B, 100 IU/ ml penicillin, and 100
g/ml streptomycin. Our results showed that although good wound healing was achieved in all cases, we only obtained a useful degree of repigmentation in one out of the five patients [11]. Monitoring melanocyte growth and expansion in the CEA cultures in vitro prior to grafting showed that as hoped the keratinocytes did indeed regulate melanocyte number but in practice by the time the keratinocytes were fully confluent and capable of being detached as an integrated sheet there were in fact very few melanocytes remaining and even those present were now no longer expressing the pigmentation associated enzyme tyrosinase related protein 1 (TRP1). In view of the poor pigmentary results in the first five patients we stopped any further grafting and decided to examine the fate of the melanocytes in the predominantly keratinocyte cultures in some detail. The fact that keratinocytes will tightly regulate melanocyte location and biology was not new or unexpected – the importance of direct contact between melanocytes and keratinocytes in terms of organizing melanocyte number, location, dendricity, and pigmentary ability has previously been reported by De Luca and colleagues [13,14], as well as other groups [15,16]. In these studies, melanocytes in close contact with keratinocytes were highly dendritic and strongly dopa positive; whilst melanocytes grown in isolation, as well as those situated more distantly from keratinocyte colonies, lost their dendricity and showed variable dopa oxidase activity [13,15,16]. As expected in our study, melanocyte number was tightly regulated by the surrounding keratinocytes; however, what was unexpected was the finding that as keratinocytes reached confluence

they had downregulated both melanocyte number and pigmentary ability [11]. The in vitro findings from this study created a technical problem in delivering functional melanocytes to the patient, as producing CEAs involves the formation of a confluent, integrated sheet of keratinocytes which is then detached as a sheet of cells and attached to a backing dressing which is then applied to the patient. However, in allowing the keratinocytes to form a confluent, integrated sheet, it was likely that any “passenger” melanocytes in the cultures were being reduced in number as well as being downregulated with respect to pigmentary function. Accordingly, to overcome this technical problem, we decided to develop a carrier dressing that would be suitable for the delivery of co-cultures of keratinocytes (either subconfluent or geographically segregated from the melanocytes) and melanocytes. This work was for us a natural progression from our prior development of a carrier system for delivering autologous keratinocytes for grafting of patients with extensive burns or chronic wounds [17–23]. The advantage of such a system would be that we could deliver subconfluent keratinocytes with a variable number of melanocytes. In developing the system we also sought to pay attention to the media for co-culture to reduce the risk of transmission of disease for the patient from the use of animalderived materials (as detailed in earlier publications [21,24]).

Development of a cell carrier system for grafting of patients with vitiligo The aim of the delivery system was to produce a chemically defined surface which would support the culture of autologous keratinocytes and melanocytes in a low-risk clinically acceptable media. Cells should then be capable of transferring from this carrier to the patient’s wound bed to achieve reliable re-epithelialization and repigmentation. We also wished to develop a system that would be sufficiently robust so that cells could be transported on it for periods of up to 48 hours to facilitate delivery of cells to patients geographically distant from the good

Simplifying the delivery of cultured melanocytes and keratinocytes manufacturing practice (GMP) regulated laboratory required for their culture. At present we have developed a system that has been evaluated in a pre-clinical model of skin pigmentation and found to be very effective at delivering both keratinocytes and melanocytes. Future work will be required now to take this to the clinic for evaluation in an initial small clinical study. In describing the development of the system we can divide this into: 1 The production of the chemically engineered carrier by the technique of plasma polymerization. 2 The co-culture of keratinocytes and melanocytes on the carrier surfaces in a range of media. 3 The in vitro evaluation of cell transfer to a human wound bed model. During the development of the system we were very aware that the final success of the cell transport and carrier system would depend on a number of interrelated factors: (1) the media might influence both the nature of keratinocyte–melanocyte interactions and also the performance of the cells on the engineered surface; (2) the engineered surface might influence the ability or “willingness” of the cells to leave the surface and transfer to the patient’s dermis; and (3) cells in co-culture might respond differently to media and to the engineered surface than cells in monoculture. Accordingly our strategy was to produce a small range of chemically engineered surfaces and then examine culture of melanocytes, keratinocytes, and also melanocyte–keratinocyte co-cultures in several media selected for their ability to support melanocytes (MCDB153 from Sigma and M2 from PromoCell) or keratinocytes (Greens media composition and KDM from Gibco). Once this was achieved, we then went on to look at transfer of keratinocytes and melanocytes to a human dermal wound bed model examining the most promising of the surfaces and of the media. The methodologies are described in brief as follows.

Production of surface engineered carriers The technique used was that of plasma polymerization. The process involves injecting a monomer such

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Monomer Substrate Vacuum valve injected into (i.e. 24-well plate) reactor placed in reactor chamber

Radio frequency generator

Pressure gauge

Pump

Fig. 24.1 A plasma polymerization rig.

as acrylic acid or allylamine into the reactor (refer to Fig. 24.1) under low vacuum. Radio frequency energy is then used to excite the gaseous monomer to a plasma which causes polymerization. The polymer formed deposits as a thin, one molecule thick, pinhole free surface on everything inside the chamber, including the substrate in question (which is placed in the bottom of the reactor chamber – see Fig. 24.1). For initial experiments tissue culture plates (24 and 6 wells) were used and these ended up coated with the monomers we introduced. Later experiments used an inert medical grade silicone polymeric support onto which the plasma-polymerized surface was deposited. For full details see earlier reports [25,26]. The advantage of producing surfaces by plasma polymerization are that these are chemically defined (after production X-ray mass spectroscopy is undertaken to analyze the composition of all surfaces) and they are produced without needing any solvent. This solvent-free approach to produce a surface in which cells can grow on has proved successful for a number of cells including keratinocytes. The development of an acrylic acid-based surface suitable for keratinocyte attachment, proliferation, and subsequent transfer of cells to wound beds (both in vitro and in vivo) has been shown from our group in recent years – for both in vitro work [17–21] and for evaluation of cell delivery in the clinic for chronic wounds and acute burns injuries [22,23].

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Table 24.1 Media selected for cell culture – pros and cons. Pros

Cons

• Has been used clinically since 1982 for culture of keratinocytes

• Contains bovine serum and cholera toxin • Requires a feeder layer of irradiated mouse fibroblasts (3T3s) for keratinocyte expansion

KDM

• Serum-free media

• Contents not available • Has never been used clinically • Not designed for melanocyte culture

Melanocyte media MCDB153

• Provides good melanocyte expansion

• Contains bovine pituitary extract, tumor promoters, and cholera toxin – has not been used clinically

• Serum free • Provides good melanocyte expansion • Allows culture of keratinocytes without feeder layer (e.g. without collagen I or irradiated mouse fibroblasts (i3T3s)) • Has been used clinically

• Contains basic fibroblast growth factor – full contents not available

Keratinocyte media Greens

M2

In the current project we explored chemically defined substrates produced by plasma polymerization of acrylic acid, allylamine or a binary mixture of these monomers. Various mixtures of the two monomers were used, as co-polymers have previously been found to be more beneficial than individual monomers with respect to the successful culture of pig aortal epithelial cells [27]. The details of the production of these surfaces, their characterization by X-ray photoelectron spectroscopy (XPS), and their use in the culture of keratinocytes, melanocytes and co-culture of the two has been described in full in earlier publications [26,28].

Culture of keratinocytes and melanocytes on surfaces Melanocytes and keratinocytes were isolated from adult human skin (obtained from patients undergoing elective breast reduction or abdominoplasty surgery who gave informed consent for these studies) by conventional methodologies [26]. Two media designed for culture of melanocytes and two for culture of keratinocytes were explored – the pros and cons of each are listed in Table 24.1. Cells were

assessed for viability using 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide (MTT)-eluted stain assay (MTT-ESTA) and melanocytes identified by staining for S100 proteins (as detailed in an earlier publication) [26].

Assessment of cell transfer from carrier dressing to in vitro wound bed model Our previous experience with the development of a carrier surface for delivering keratinocytes to wound beds in vivo was greatly assisted by the use of a human wound bed model [20] which we also used in this study. This model consists of human split-thickness dermis which has been sterilized, de-epidermised, and made acellular while retaining some basement membrane proteins [29]. Cells were initially expanded in the laboratory and then cultured on the carrier surface for 24 hours before transfer to the wound bed model as indicated in Fig. 24.2. After 48 hours the carrier surface was removed and examined for the retention of any cells by staining with MTT-ESTA. Some experiments were sacrificed at this stage to see to what

Simplifying the delivery of cultured melanocytes and keratinocytes

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Keratinocyte melanocyte suspension

Chemically defined carrier

Silicone carrier coated with 100% acrylic acid or allylamine

Carrier  cells transferred onto human DED and incubated for 48 hours at 37°C

Cells attached to carrier dressing Carrier  cells incubated for 24 hours at 37°C

De-epidermized acellular human dermis (DED) Human skin composite Steel grid Medium Some samples sacrificed in order to assess (using MTT-ESTA) transfer of cells from the carrier dressing to human DED

Rest of the samples raised up to an air–liquid interface on steel grids and incubated for 10 days at 37°C

After 10 days skin composite is sacrificed for histological (H&E and S100) analysis

Fig. 24.2 A cartoon of cell transfer from chemically defined carriers to an in vitro model of human skin.

extent cells had transferred to the dermis (again detecting cells by staining with MTT-ESTA) others were raised to an air–liquid interface (essential to induce maturation of the epidermal layer), and then cultured for a further 10 days. At the end of this period the reconstructed skin was fixed and sectioned and examined by conventional histology hematoxylin and eosin (H&E) and melanocytes detected by immunostaining for S100. The density of melanocytes post-transfer of cells from the carrier dressing was also determined.

Results The full range of surfaces examined and the proliferation of keratinocytes and melanocytes on these have been detailed in full previously [25,26]. In this chapter we summarize the outcome of these

studies and illustrate this with examples of the morphology of the cells on the most promising surface in several media (see Plate 24.1, facing p. 114). In brief, surface preference altered depending on the cell type and the media used. A range of chemically engineered surfaces were made from 100% acrylic acid (which also contained carbonyl and alcohol functional groups) to 100% allylamine (also containing amine and secondary amine functional groups), and various co-polymers containing different ratios of acid and amine. The biggest differences with respect to cell preference for a surface were between 100% acid and 100% amine polymers. Hence, there appeared to be no advantage in pursuing the co-polymers and we focused our attention on the 100% acrylic acid and the 100% allylamine surfaces. We looked at cell performance in four media, MCDB, Greens, KDM, and M2, looking at

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cells in monoculture and co-culture. Cells grew well on both acid and amine surfaces and whilst there were slight differences in preference for one polymer surface over another, the biggest influence on successful cell expansion was the media used followed by the influence of cell–cell interactions. As illustrated in Plate 24.1 (facing p. 114), melanocytes fared better on a plasma-polymerized surface if they were cultured in either MCDB (Plate 24.1A) or M2 (Plate 24.1C) media. In the keratinocytesupporting media KDM, melanocytes were more rounded with short blunt bi-polar processes and did not grow well. Keratinocytes cultured in both Greens (Plate 24.1D) and KDM (Plate 24.1E) formed confluent, integrated sheets. Interestingly, keratinocyte cultures were also established in the melanocytesupporting media M2 (Plate 24.1F) however, the cells took longer to reach confluence in this media. When keratinocytes and melanocytes were cultured in a 1:1 ratio on the plasma-polymerized surfaces, media became less important and successful cultures containing a mix of both cell types was achieved in both keratinocyte-supporting (Plates 24.1G and H) and melanocyte-supporting media (Plate 24.1I). With respect to media, MCDB153 was included for research purposes only but was not considered for taking further into the clinic because of the inclusion of bovine pituitary extract, tumor promoters, and cholera toxin. Both Greens and M2 media have been used clinically and reassuringly whilst keratinocytes in monoculture had a preference for Greens, melanocytes in monoculture were most proliferative in M2 media. Furthermore, for the co-cultures, the greatest total number of cells was found in M2 followed by Greens and KDM (where for both the cell number was only 60% of that found in M2). Interestingly we also noted that cell proliferation in co-cultures was greater than would have been expected from the sum of the monocultures. This was a 14% increase for Greens media, a 34% increase for KDM, and a 103% increase for M2 media. This data highlights the important role direct cell–cell contact plays with respect to cell performance in vitro. A count of melanocyte density in both 1:2 co-cultures of melanocytes:keratinocytes and also freshly isolated keratinocyte cultures (containing

1–3% “passenger” melanocytes), grown in Greens and M2 media again showed M2 media to be significantly more effective for melanocyte expansion than Greens media (again as expected) by a factor of 2–3-fold. Good cell expansion was seen both on 100% acrylic acid and 100% allylamine surfaces with the acrylic acid polymer being significantly better (by a factor of approximately 25%) than the allylamine polymer [26]. At this stage we could have selected the frontrunner surface (acrylic acid) and media (M2) for development of the carrier surface but our previous experience in developing a carrier/transfer surface for keratinocytes had shown us that good transfer of cells from a surface can require a different surface to the one which achieves the greatest rate of expansion of cells [18,20]. Accordingly we then looked at transfer of cells from the two surfaces – 100% acrylic acid and 100% allylamine, under two media – Greens and M2 (at this stage we did discard KDM media as its contents are not disclosed and it has never been used clinically). M2 has been used clinically in the treatment of vitiligo (although its contents are not fully disclosed) and Greens has been used for 30 years in the treatment of patients with burns injuries. The issue of risk reduction in the use of cultured cells for patients will be discussed shortly. Transfer of cells from carriers onto the in vitro dermal wound bed model showed that both acid and amine surfaces were capable of transferring cells (over a period of 48 hours) onto the human dermis (Fig. 24.3). Plate 24.2 (facing p. 114) illustrates that if the in vitro dermal model is cultured for a further 10 days at an air–liquid interface, the transferred keratinocytes form a well integrated and progressively differentiated epidermis, whilst the melanocytes evenly space themselves out amongst the basal keratinocyte layer as would be expected in vivo. The next issue we considered was the ratio of seeding densities for establishing keratinocyte and melanocyte co-cultures. We examined two approaches: the first was to isolate melanocytes and keratinocytes into separate cultures and then combine the individual cultures (established in both Greens and M2) in a 1:2 ratio of melanocytes to keratinocytes. The second approach was to culture

MTT of acid carrier

MTT of amine carrier

MTT of DED

MTT of DED

0.02

Cell attachment (MTT-ESTA OD 540–630 nm)

Cell attachment (MTT-ESTA OD 540–630 nm)

Simplifying the delivery of cultured melanocytes and keratinocytes

0.016 0.012 0.008 0.004 0

197

0.016

0.012

0.008

0.004

0 AC carrier

DED

AA carrier

DED

Fig. 24.3 Transfer of melanocyte–keratinocyte co-cultures (1:2) onto human de-epidermised acellular dermis (DED) from

acrylic acid (AC) or allylamine (AA) coated carriers. The presence of viable cells was detected by the use of MTT staining.

freshly isolated keratinocytes (which naturally contain 1–3% “passenger” melanocytes) for 10 days in either Greens or M2 and then use these predominantly keratinocyte cultures. Our data showed that both looked promising: melanocyte numbers (counted across each section and then scaled up to establish numbers/mm2) from the 1:2 cocultures of melanocytes:keratinocytes (in Greens medium) ranged from roughly 17,000 cells/mm2 (transferred from the allylamine carriers) to 25,000 cells/mm2 (transferred from the acrylic acid carriers) – unpublished data. With the freshly isolated keratinocyte cultures melanocyte numbers varied from roughly 8000 cells/mm2 in Greens medium (for both acrylic acid and allylamine surfaces) to 17,000 cells/mm2 in M2 medium (for both acrylic acid and allylamine surfaces) – unpublished data. In summary both methodologies could be used clinically. More work needs to be carried out with respect to combining individual cultures of melanocytes and keratinocytes in order to determine

the best ratio of the two cell types to be used in combination to achieve both reliable healing and pigmentation for the patient. Alternatively, freshly isolated autologous keratinocytes could be cultured in M2 for 10–14 days to increase the melanocyte numbers before being reapplied to the patient. The key advantage to using M2 is that it not only increases melanocyte numbers in in vitro work, but this media is also serum free and contains no animal-derived products, toxins, or tumor promoters. Furthermore there is already a precedent for its use in vitiligo from the pioneering work of Olsson and Juhlin and colleagues in developing this media and using it clinically in the treatment of patients with vitiligo [3,5,6,30].

Practical issues of developing a cell carrier system for use in vitiligo The final part of this chapter will touch on the issues that must be considered to develop this carrier system for clinical use.

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In seeking to develop this system we wish to progress the pioneering work of others (which has demonstrated that cultured cells can benefit patients), by producing a cell carrier system which is as previously stated low risk for the patient, easy for the clinician to apply, and capable of treating vitiligo patients who are geographically distant from a specialist culture laboratory. The steps as we envisage them in using such a system would be: 1 The dermatologist selects the patient for surgical therapy. 2 The dermatologist takes a small biopsy and sends this to the GMP culture laboratory. 3 Keratinocyte–melanocyte co-cultures are expanded in M2 media in the GMP laboratory. – Some are frozen in liquid nitrogen for future application. – Some are applied to carrier surfaces and after 1–2 days placed cell-side down on a suitable transport media and dispatched in temperature controlled packing to the patient. 4 The dermatologist or plastic surgeon deepithelializes the recipient area on the patient and then places carrier discs plus cells cell-side down on the wound bed and bandages this lightly in place for 5 days. After 5 days the discs are discarded and the grafted areas dressed with a non-adherent dressing. 5 If required further cultures can be sent from the laboratory for grafting further areas. Obviously many of the steps in grafting remain the same, irrespective of the method of grafting, but there should be several advantages to this technology over others in current use: 1 An easier application of cells to the patient for the clinician. 2 No need to establish a culture laboratory (with inherent infrastructure and staff costs) for each treatment clinic. 3 Treatment of patients over geographically broader areas. The system we describe could allow for one regulatory authority approved GMP clean room laboratory to treat patients in several locations. This will have advantages in terms of undertaking large studies, in maintaining uniformity of culture conditions, and in terms of cost to the patients.

However, the key issues in developing a more routine approach to surgical treatment of vitiligo patients must be safety and efficacy.

Reducing risk in the use of cultured cells in vitiligo The system we describe is a simple form of tissue engineering. In looking to take this to the clinic the risks in using cultured cells on a support can be listed very simply as follows: • Risks associated with the initial biopsy. • Risks associated with expanding the cells in the laboratory – media associated. • Risks associated with tracking samples in the laboratory. • Risks associated with the polymer carrier dressing. • Risks associated with transport of cells to the patients. All of the above risks are considered by regulatory authorities and will be discussed in more detail below. Our information comes from achieving approval for the use of cultured keratinocytes for clinical use in the UK. The use of autologous cells to benefit patients is currently regulated differently in different parts of the world but with the same underlying principles. In the UK at the time of writing, this falls under the Medicines and Healthcare products Regulatory Authority (MHRA).

Risks associated with the initial biopsy Any patients receiving cultured cells in the UK must receive cells from premises approved by MHRA. The clean room laboratories required for this purpose must be run so that the chance of contamination of cultured cells with airborne particles is kept extremely low but, of equal importance, every aspect of cell culture is described by a standard operating procedure (SOP). SOPs are written to reduce the risk of patient samples being accidentally confused with others or contaminated with anything that would represent a disease risk for the recipient.

Simplifying the delivery of cultured melanocytes and keratinocytes

Risks associated with expanding the cells in the laboratory – media associated In this respect the MHRA would prefer to avoid wherever possible the use of animal cells or products or any non-defined material which could introduce a risk for the patient. This is not always possible – for example the current rapid expansion of human keratinocytes for burns patients uses a feeder layer of irradiated mouse fibroblasts and 10% bovine FCS (Greens media). The risk benefit position for patients with extensive burns is such that this is currently permitted providing bovine serum is sourced from herds without any previous incidence of bovine spongiform encephalitis (BSE) and mouse feeder cells should be from screened banks of cells. Elsewhere in Europe (e.g. France) culture of cells with bovine serum is not currently permitted for clinical use to the best of our knowledge. With respect to media constituents it is worth noting that to the best of our knowledge, there are no media for keratinocyte or melanocyte cultures that are approved by any regulatory authority (FDA or MHRA or any European Authority) for clinical use. Thus although Green’s media has been in use for culture of keratinocytes for clinical delivery since the late 1980s [31], the clinical responsibility for using the media rests with the clinician or clinical institution. Media manufacturers can in theory seek to get approval for their media to be registered with a regulatory authority for clinical use. We are aware of several companies who have looked into this procedure but as yet we are not aware of any who have engaged with regulatory authorities to produce the necessary master file that would give a full disclosure of media contents and details of sourcing of reagents that would allow the regulatory authority to approve its use for culture for clinical use. Thus several companies have developed commercial proprietary media in recent years that are serum free and are very promising for culture of keratinocytes or melanocytes (such as KDM and M2 as used in this study). However, companies do not allow a full disclosure of their media contents for proprietary reasons. One is led to conclude that there are no easy routes in this area at present.

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This is an area where patient safety could be compromised if investigators unwittingly use a commercial media which contains animal-derived proteins from unsafe sources. At present some media manufacturers will give a written undertaking that their media does not contain animal-derived proteins while pointing out that their media are not approved for clinical use. This is accordingly a difficult area at present for those who wish to use cultured cells to benefit patients and yet are very mindful of the longer-term issues that could be associated with the use of bovine or murine materials. One approach which our group has taken for the delivery of keratinocytes is to take away animal cells and products from an established culture methodology. The normal procedure requires keratinocytes to be expanded on irradiated murine feeder fibroblasts in the presence of Greens media (which contains 10% bovine FCS). Our new approach involves omitting the serum and replacing the murine fibroblasts with an irradiated human fibroblast feeder layer (MRC5s) which has been used in the production of human vaccines for the last 30 years [32]. This allows us to expand human keratinocytes using a serumfree media and with a media whose contents and source of contents are known to us. However it does require the use of a donor human fibroblast feeder layer and is therefore not entirely autologous. This is still at a pre-clinical level of development. For melanocyte–keratinocyte culture for vitiligo patients the media which performed most successfully in our studies was M2. This is serum free and does not require a feeder layer of cells and has been used clinically in the treatment of vitiligo by the Olsson group as previously stated. The one caveat in its use is that its full contents are not disclosed to the user. Greens media, sourcing FCS from BSEfree herds, with an extremely well-tested human feeder fibroblast cell line (such as MRC 5s) provides a second alternative.

Risks associated with tracking samples in the laboratory Turning to another risk – any laboratory culturing cells from several patients must ensure that the correct cells go back to the patient. Conceivably

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cells from patient A may carry viral contamination (hepatitis B, C, or acquired immunodeficiency (AIDS) virus) which would represent no particular additional threat to patient A when such cells are grafted back to the patient. However, such cells grafted to patient B would present a considerable health threat to that patient. Again, different regulatory authorities view donor sourcing and sample tracking differently. In the UK to obtain MHRA approval for delivery of autologous keratinocytes we have recently introduced the taking of a blood sample from donors at the time of skin biopsy to assist in ensuring complete traceability of cells. There is currently no contraindication to culturing cells from patients who are positive for hepatitis B, C, or AIDS to benefit that patient, but these cells must be handled in such a way that their risk of cross-contaminating other cells from other donors is negligible. This requires SOP that are extremely focused on tracking cells and preventing any risk of media or reagents from donor A cells mixing with donor B cells etc. This more than any other aspect of good laboratory practice must be monitored and demonstrated to be robust for ensuring patient safety. Whether one introduces bar coding or whatever form of sample tracking the discipline of keeping all reagents and cells separate for each patient is key to the safe running of a GMP cell culture facility.

Risks associated with the polymer carrier dressing Here the risk of disease transmission to the patient can arguably be made close to negligible. Whereas one can deliver cells from membranes coated with bovine collagen, the system we describe is entirely chemically defined and free of any animal protein. Further the plasma polymer produced carriers can be sterilized using standard accredited sterilization techniques such as gamma irradiation or ethylene oxide [20].

conventional tissue culture Petri dishes (for local use) or they can be packaged to travel for up to 48 hours by using an agar-based version of the transport media. We have developed such a media based on Greens medium (minus cholera toxin and serum) for transport of keratinocytes which allows cells to travel “face” down on the agar-based media in screw cap sterile containers. We have recently demonstrated that transport of viable keratinocytes for up to 48 hours at temperatures between 20°C and 37°C is possible this way (unpublished data). We propose to develop a similar gel-based transport media for keratinocyte and melanocyte co-cultures and to assess their ability to be transported using commercial carriers (such as FedEx or DHL) in temperature-regulated packaging.

The way forward? We propose that development of a cell carrier system will hopefully help extend the availability of cultured autologous melanocytes to more patients with vitiligo. Such a system will not impact on the key issues for clinical success in grafting of patients with cultured cells such as patient selection and surgical management. Thus, the initial biopsy and later preparation of the wound bed prior to placing carriers containing melanocytes and keratinocytes on these wound beds and the clinical follow-up and evaluation of the patients will remain critical to achieving good clinical results for the patients. However, the system will hopefully allow dermatologists, who wish to use cultured cells to benefit their patients, to have access to a cell culture and cell delivery service that is currently only available to those groups undertaking research in vitiligo grafting. We suggest that this technique has the potential to move the surgical treatment of vitiligo patients with cultured autologous cells from the realms of clinical research into a clinical service for patients.

Summary Risks associated with transport of cells to the patient Finally at the point that cells leave the laboratory cells can be transported on the carrier surface within

We describe the development of a chemically defined carrier surface for the delivery of a co-culture of autologous melanocytes and keratinocytes for grafting of patients with vitiligo.

Simplifying the delivery of cultured melanocytes and keratinocytes This technique has been evaluated in the laboratory and found to successfully transfer keratinocytes and melanocytes to a human wound bed model. The technique has the potential to extend the surgical treatment of vitiligo patients with cultured cells to more patients who could benefit from it without the need for dermatologists to establish dedicated GMP clean room facilities for each hospital.

References 1 Falabella R, Escobar C, Borrero I. Transplantation of in vitro cultured epidermis bearing melanocytes for repigmenting vitiligo. J Am Acad Dermatol 1992a; 21:257–64. 2 Falabella R, Escobar C, Borrero I. Treatment of refractory and stable vitiligo by transplantation of in vitro cultured epidermal autografts bearing melanocytes. J Am Acad Dermatol 1992b;26:230–6. 3 Olsson MJ, Juhlin L. Repigmentation of vitiligo by transplantation of cultured autologous melanocytes. Acta Derm Venereol 1993;73:49–51. 4 Lontz W, Olsson MJ, Moellmann G, Lerner AB. Pigment cell transplantation for treatment of vitiligo: a progress report. J Am Acad Dermatol 1994;30:591–7. 5 Olsson MJ, Moellmann G, Lerner AB, Juhlin L. Vitiligo: repigmentation with cultured melanocytes after cryostorage. Acta Derm Venereol 1994;74:226–28. 6 Olsson MJ, Juhlin L. Transplantation of melanocytes in vitiligo. Br J Dermatol 1995;132:587–91. 7 Kaufmann R, Greiner D, Kippenberger S, Bernd A. Grafting of in vitro cultured melanocytes onto laserablated lesions in vitiligo. Acta Derm Venereol 1998;78:136–8. 8 Guerra L, Capurro S, Melchi F, et al. Treatment of “stable” vitiligo by timedsurgery and transplantation of cultured epidermal autografts. Arch Dermatol 2000;136:1380–9. 9 Olsson MJ, Juhlin L. Long-term follow-up of leucoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol 2002; 147:893–904. 10 Chen Y-F, Yang P-Y, Hu D-N, et al. Treatment of vitiligo by transplantation of cultured pure melanocyte suspension: analysis of 120 cases. J Am Acad Dermatol 2004;51:68–74. 11 Phillips J, Gawkrodger DJ, Caddy CM, et al. Keratinocytes suppress TRP-1 expression and reduce cell number of co-cultured melanocytes – implications

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for grafting of patients with vitiligo. Pigm Cell Res 2001;14:116–25. Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975;6:331–43. De Luca M, Franzi AT, D’Anna F, et al. Co-culture of human keratinocytes and melanocytes: differentiated melanocytes are physiologically organised in the basal layer of cultured epithelium. Eur J Cell Biol 1988; 46:176–80. De Luca M, D’Anna F, Bondanza S, et al. Human epithelial cells induce human melanocyte growth in vitro but only skin keratinocytes regulate its proper differentiation in the absence of a dermis. J Cell Biol 1988;107:919–26. Donatien P, Surleve-Bazeille JE, Thody AJ, Taieb A. Growth and differentiation of normal human melanocytes in a TPA-free, cholera toxin-free, lowserum medium and influence of keratinocytes. Arch Dermatol Res 1993;285:385–92. Nakazawa K, Nakazawa H, Collombel C, Damour O. Keratinocyte extracellular matrix-mediated regulation of normal human melanocyte functions. Pigm Cell Res 1995;8:10–18. France RM, Short RD, Duval E, et al. Plasma copolymerisation of allyl alcohol/1,7-octadiene: surface characterisation and attachment of human keratinocytes. Hem Mater 1998a;10:1176–83. France RM, Short RD, Dawson RA, Mac Neil S. Attachment of human keratinocytes to plasma co-polymers of acrylic acid/octa-1,7-diene and allylamine/octa-1,7-diene. J Mater Chem 1998b;8:37–42. Haddow DB, France RM, Short RD, et al. Comparison of proliferation and growth of human keratinocytes on plasma copolymers of acrylic acid/1,7-octadiene and self-assembled monolayers. J Biomed Mater Res 1999;47:379–87. Haddow DB, Steele DA, Short RD, et al. Plasmapolymerised surfaces for culture of human keratinocytes and transfer of cells to an in vitro wound-bed model. J Biomed Mater 2003;64A:80–7. Higham MC, Dawson RA, Szabo M, et al. Development of a stable chemically defined surface for the culture of human keratinocytes under serum-free conditions for clinical use. Tissue Eng 2003;9:919–30. Moustafa M, Simpson C, Glover M, et al. A new autologous keratinocyte dressing treatment for non-healing diabetic neuropathic foot ulcers. Diab Med 2004; 21:786–9. Zhu N, Warner RM, Simpson C, et al. Treatment of burns and chronic wounds using a new cell transfer

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dressing for delivery of autologous keratinocytes. Eur J Plast Surg 2005;28:319–30. Sun T, Higham MC, Layton C, et al. Developments in xenobiotic free culture of human keratinocytes for clinical use. Wound Repair Regen 2004;12:626–34. Beck AJ, Phillips J, Smith-Thomas L, et al. Development of a plasma-polymerised surface suitable for the transplantation of keratinocyte–melanocyte co-cultures for patients with vitiligo. Tissue Eng 2003;9:1123–31. Eves PC, Beck AJ, Shard AG, Mac Neil S. A chemically defined surface for the co-culture of melanocytes and keratinocytes. Biomaterials 2005;26:7068–81. Monge S, Mas A, Hamzaoui A, et al. Improvement of silicone endothelialisation by treatment with allylamine and/or acrylic acid low-pressure plasma. J Appl Polym Sci 2003;87:1794–1802. Beck AJ, Whittle JD, Bullett NA, et al. Plasma co-polymerisation of two strongly interacting

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monomers: acrylic acid and allylamine. Plasma Process Polym 2005;2:641–9. Chakrabarty KH, Dawson RA, Harris P, et al. Development of autologous human dermal–epidermal composites based on sterilised human allodermis for clinical use. Br J Dermatol 1999;141:811–23. Olsson MJ, Juhlin L. Leucoderma treated by transplantation of a basal cell layer enriched suspension. Br J Dermatol 1998;138:644–8. Cuono CB, Langdon R, Birchall N, et al. Composite autologous-allogeneic skin replacement: development and clinical application. Plast Reconstr Surg 1987;80: 626–37. Bullock AJ, Higham MC, Mac Neil S. Development of a xenobiotic free culture and delivery system for human keratinocytes. Tissue Eng 2006;12:245–55.

CHAPTER 25

Safety concerns in transplantation of in vitro cultured cellular grafts Liliana Guerra, Elena Dellambra and Patrizia Paterna

In the last few decades, great progress has been made in the cell biology field and this has allowed the development of emerging technologies to reconstitute human tissues in vitro. On the whole, these technologies are defined as “tissue engineering.” The in vitro cultured cells have several possible clinical applications, which are included in the term of “somatic cell therapy” (SCT). The SCT is the administration to humans of autologous or allogenic or xenogenic living cells, which have been manipulated ex vivo. The ex vivo approach indicates that cells are harvested from the donor by a skin biopsy; this is followed by growth of these cells in vitro; cells are then grafted back onto the recipient. In autologous cell therapy, cells are isolated from the skin of the future recipient, allogenic cells are isolated from unrelated donors and xenogenic cells come from animals. Cells for therapeutic purposes may be delivered in several ways. For example, they may be injected or implanted in aggregated forms or along with solid substrates. Currently, all procedures for SCT are strictly regulated in the European Union (EU) and in the United States.

Regulatory environment The actively developing area of SCT requires quality and safety of these novel therapeutic approaches to be ensured, in order to safeguard public health.

European union Within the EU, laws are formulated by the Parliament and the Council, and are then implemented by the

European Commission. European laws and regulations come in two forms, either Regulations or Directives. Regulations are binding for all EU member states without the need for national legislation while Directives bind member states to the objectives of the directive within a certain timeframe, while leaving national authorities to implement the actual legislation. These Regulations and Directives are published in the Official Journal of the EU (europa. eu.int/eur-lex/en/oj/index.html). In 1993, Regulation 2309/93 created the European Agency for the Evaluation of Medicinal Products (EMEA, now named European Medicine Agency by the EC Regulation 726/2004), as the agency for regulatory control within Europe (www.emea.eu.int). All the regulatory Directives, Regulations and Guidelines are available in “The Rules Governing Medicinal Products in the European Union,” published by the Office of Official Publications of the European Communities. Currently, the most important reference texts [1,2] in the field of medicinal products for human use are the Directives 2001/20/EC and 2001/83/EC, and their following amendments. The Directive 2001/20/EC of the European Parliament and of the Council, relating to the implementation of good clinical practice (GCP) in the conduct of clinical trials on medicinal products for human use, lays down that products intended for gene therapy (GT) and cell therapy based on human cells are considered as medicinal products and, therefore, have to be manufactured in compliance with good manufacturing practices (GMPs). The Directive 2001/20/EC was approved and scheduled for

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incorporation into national legislation by the 1st May 2004. Annex 1 to the Directive 2001/83/EC of the European Parliament and of the Council, on the Community code relating to medicinal products for human use, was adapted to take into account in Part IV: Advanced therapy medicinal products, of GT and SCT products. The manufacturing process of these products must also comply with requirements of Commission Directive 2003/94/EC, laying down the principles and guidelines of GMP for medicinal products for human use.

United States Laws within the United States are formulated by Congress. Once they have been approved by both the Congress and the President, laws become part of the United States Code (USC). The USC contains two documents relevant to the pharmaceutical industry: Title 21 Food and Drugs, and Title 42 The Public Health and Welfare. Each of these titles contains several chapters dealing with food, drug and health issues. Within Title 21, the important chapter is Chapter 9 – The Federal Food, Drug, and Cosmetic Act. In this Act, section 393 authorizes the existence of the FDA (Food and Drug Administration) as part of the US Department of Health and Human Services, to carry out the duties we see it performing today. Title 42 contains Chapter 6A, the Public Health Service (PHS) Act. Regulations are required for day-to-day implementation of acts of the USC: these regulations are contained within the Code of Federal Regulations (CFR). The CFR is divided into titles: Title 21 of the CFR (Title 21 CFR) (not to be confused with Title 21 USC) deals with Food and Drugs and contains the primary regulations that govern the conduct of clinical studies (www.fda.gov/cdrh/ databases.html). Within Title 21 CFR, there are several very important regulations [3–9]: 1 part 50 provides the requirements and general elements of informed consent; 2 parts 312 and 812 cover the procedures for GCP in the conduct of clinical studies; 3 parts 210, 211, and 820 list all the GMP requirements to ensure that finished products are safe and effective;

4 part 1271 defines higher levels of GMP requirements for human cells which are more than minimally manipulated. Recommendations for appropriate human SCT application are available in the FDA’s Guidance Documents for Industry (www.fda.gov/cber/guidelines.htm).

Good Manufacturing Practice Good Manufacturing Practice (GMP) means the part of quality assurance which ensures that products are consistently manufactured in accordance with the quality standards appropriate to their intended use. The quality assurance system involves the participation of competent and appropriately qualified personnel, adequate manufacturing equipment, preestablished procedures for general manufacturing operations (standard operating procedures, SOPs) and a documentation system covering all the operations performed. Any human SCT product consisting of living cells should be manufactured in dedicated facilities and submitted to quality and safety controls during its manipulations, including control of the starting material, validation of the cell culture procedures and release tests of the cell product prior to its administration. In particular cell therapy manufacturing operations must be carried out in BL-3-type (Biosafety Level-3) cell culture laboratories and an appropriately qualified quality assurance system must be full-time operative.

BL-3-type laboratory Cell cultures must be performed in sterile conditions. All laboratories where cells for transplantation are cultured must be of independent BL-3 type (controlled humidity and temperature, air conditioning system equipped with absolute filters allowing a control of environmental contaminations, biohazard cabinets for cell cultures, controlled negative pressure in the cell culture laboratories, and controlled positive pressure in the filter zones). Cell culture rooms must be strictly utilized only for this use, and cell cultures must be physically or temporally separated, to avoid cross-contaminations among different donors. Any apparatus which is very important for cell culture quality and safety

Safety concerns in transplantation of in vitro cultured cellular grafts (refrigerators, laminar-flow hoods, CO2 incubators, liquid nitrogen containers) must be assured by an alarm system.

Source and characterization of materials and reagents Quality and safety of culture media and additives must be assured. It is recommended to avoid when possible the use of reagents with sensitization potential, such as antibiotics. Tumor promoters such as phorbol esters must be avoided in culture media. Serum-free culture media should be preferable. Nevertheless, when animal serum is required for optimal cell growth, its traceability from final container to farm of origin must be ensured. For bovine serum, the healthy status of the donor herd must be well defined and documented, with particular attention to the risk of transmitting agents causing spongiform encephalopathy (bovine spongiform encephalitis, BSE). As herd health status for exotic diseases is usually defined through the health status of the country of origin, the Office International des Epizooties (OIE) code must be followed to ensure freedom at source from such diseases (www.oie.int). Supporting cells, such as the 3T3–J2 mouse fibroblasts, must be validated for their utilization in culture procedures destined to human cell therapy. The history of cell substrates that are used in the manufacture of biotechnological products must be clearly documented. Synthetic materials for cell culture manipulation and transport must be controlled to evaluate their compatibility and durability.

Cell culture procedures During in vitro cell cultures, consideration should be given to ensure optimal growth and manipulation of isolated cells. Adequate quality controls must include verification of absence of adventitious agents, such as bacteria, yeast, fungi, mycoplasma, and viruses. Quality controls must also allow to verify whether cellular-biological parameters (morphology, growth, function, behavior, cell-to-cell interaction) are maintained in the culture conditions adopted, in order to assure the potential clinical efficacy of the final product. Consideration must be given to the possible transformation potential of cells in response to growth

205

factors. Karyotyping and soft-agar assay should be performed in order to demonstrate that cells do not reveal a transformed phenotype [10]. In the case of frozen cells, they must be stored under optimized conditions, and quality controls must be repeated at the time of their utilization to ensure cell viability, sterility, and function.

Quality assurance system A quality assurance system must be operative in the facilities where cells are collected, manipulated, stored, submitted to quality and safety controls, and packaged in the final container. Each process involved in these activities must be specified in written documentation, instructions, and procedures. The quality assurance system must include the identification of a qualified person (QP) responsible for the co-ordination and monitoring of all activities and who has the authority to stop release of the product when necessary.

Good Clinical Practice Good Clinical Practice (GCP) is a set of internationally recognized ethical and scientific quality requirements, which must be observed for designing, conducting, recording, and reporting clinical trials involving human subjects. Compliance with this good practice provides assurance that the rights, safety and well being of trial subjects are safeguarded, and that the results of the clinical trials are credible. The current thinking on GCP is indicated in the Guidelines of the International Conference on Harmonization (ICH) on Good Clinical Practice: the FDA published this text in the Federal Register (Vol. 62, No. 90, 25691–709), while the EU incorporated it into “The Rules Governing Medicinal Products in the European Union” (Vol. 3C, EU Directive 75/318/EEC). In summary, clinical trials must be conducted in accordance with the Ethical Principles for Medical Research Involving Human Subjects that have their origin in the Declaration of Helsinki (1996). Before a trial is initiated, foreseeable risks and inconveniences have to be weighed against the expected benefit for the individual trial participant and society. The right, safety and well-being of the trial participants must prevail over interests of science and

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society. The available clinical information on an investigational product must be adequate to support the proposed trial. Clinical trials have to be scientifically described in a clear detailed protocol and have to be conducted in compliance with this protocol once it has received the Ethical Committee’s approval. Freely given informed consent must be obtained from every participant before the clinical trial participation. The clinical trial sponsor and investigators must be clearly identified. Any clinical trial information must be recorded, handled, and stored in a way that allows its accurate reporting, interpretation, and verification. The confidentiality of records that could identify participants has to be safeguarded, in order to respect the individual’s privacy, in accordance with regulatory requirements. Before a trial is initiated, the sponsor must submit a valid request for authorization to the competent authority of the State in which the sponsor plans to conduct the clinical trial. A database of adverse events must allow any relevant information about suspected serious adverse reactions to be recorded

and reported to the competent authority as soon as possible.

References 1 Directive 2001/20/EC of the European Parliament and of the Council, of 4 April 2001. 2 Directive 2001/83/EC of the European Parliament and of the Council, of 6 November 2001. 3 Title 21 CFR, part 50: Protection of Human Subjects. 4 Title 21 CFR, part 210: Current Good Manufacturing Practice in Manufacturing, Processing, Packing, or Holding of Drugs. 5 Title 21 CFR, part 211: Current Good Manufacturing Practice for Finished Pharmaceuticals. 6 Title 21 CFR, parts 312: Investigational New Drug Application. 7 Title 21 CFR, part 812: Investigational Device Exemptions. 8 Title 21 CFR, part 820: Quality System Regulation. 9 Title 21 CFR, part 1271: Human Cells, Tissues, and Cellular and Tissue-Based Products. 10 Falabella R. Vitiligo. Acta Derm Venereol 1994;74:147–8.

SECTION 5

Special issues

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CHAPTER 26

Post-surgery patient information Mats J. Olsson

The post-surgery information, both verbally and in writing, is most important for a successful outcome. The information should be given to the patient several weeks before the planned surgery, so that the patient becomes aware of what the aftercare demands from him/her personally and also so that the sick leave period can be planned and the support from relatives or friends, regarding transportation and home help can be arranged in advance. It has to be stressed to the patient and also to the parents of younger patients that the final result lies in their hands as soon as they leave the hospital. They should be told that even a technically faultless surgical procedure in a correctly selected skin condition can result in a poor outcome with incomplete repigmentation if an appropriate aftercare is not followed. It would be best to keep the patient in a hospital bed for a few days after the surgery, but this is not practically possible to arrange at smaller outpatient clinics and it adds far too much to the total cost for the procedure at larger hospitals. Sometimes the patients have traveled far and cannot stay in the town or the country until the removal of the surgical dressing. So therefore we need to compromise but still do the best in the given situation, which involves the right information and instructions. The appropriate information all depends on: what kind of surgical technique was used, type of wound dressing, type of leukoderma, treated anatomical locations, extension of the areas, etc. An example of a written information for the patients is given below, which can be modified to fit depending on transplantation method and type of patients: Uppsala Vitiligo Clinic, Updated 2005-12-12

Post-wound-care information (cellular grafting) At the day of surgery • Make sure that you bring clothes that are comfortable and easy to dress and undress. • If the area to be treated is located somewhere where it might restrict your possibility to walk without disturbing the newly transplanted area, please bring a friend or a family member who can help assist you when leaving the hospital. • After the surgery you have to rest in a hospital bed for a certain time, depending on the size and location of the area(s) involved. • When leaving the hospital you should not drive yourself.

The first week • The bandage is supposed to be worn for 7–10 days depending on the area involved. • Be careful so you do not disturb the healing process. The first 48 hours are most important, but you should limit your movements the whole first week. Do not wear clothes that tighten over the treated areas. • Antibiotics may be prescribed for 5–10 days depending on the location of and the size of the treated area. • If a lot of fluid builds up under the transparent bandage-dressing the fluid can be removed with help of a needle equipped syringe, but some fluid is completely normal and not harmful for the outcome. Exudates will always build up in larger areas [1]. • The healing process on lower extremities, especially on the dorsum of the foot can be painful. This pain is instantly reduced when putting the area in a high position, with the help of a pillow or similar.

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The elevated position also reduces the build up of exudates. Over-the-counter pain relievers, such as paracetamol and ibuprofen can be used, but avoid medication containing acetyl salicylic acid, such as Aspirin. • If the bandage loosens-up at the edges you can secure it with surgical tape like Micropore® or some similar plaster. Never remove the bandage without discussing it with us first. • Do not be surprised if you sense some bad odor from the bandage at the end of the week, as it is normal. • If you get fever or experience excessive itching or pain, please contact us.

Removal of the bandage (at about 8 days post-surgery) • The bandage-dressing is removed 7–10 days after the surgery. The bandage is preferably removed at our clinic. But if you are living far away and have no possibility to come back for a dressing removal it can be removed by your local physician or in your home. Then please bring the instructions to your physician or follow the instructions carefully. • Make the bandage wet, moisten it about 10 minutes before removal. For the transparent plasticdressing, use a syringe and inject some saline solution (0.9% NaCl [aq.]) under the plastic film. For the cotton dressing put the arm, hands or feet in a bucket of water. Then remove the dressing carefully layer by layer. If some parts stick to the skin, then cut the loose parts with scissors and just leave the parts which stick. These will fall off by themselves in the shower after a couple of days. Use no force to remove. Flush carefully with 0.9% saline-solution or clean water, let air-dry and smear with a layer of perfume-free pure Vaseline (in cases of suspected infection a 2% Fucidin or Bacitracin ointment can be used).

• The bandage covering the donor-area is also removed and the area is smeared with a thin layer of pure Vaseline. • In case you choose to remove the dressing yourself and have some additional questions, please contact our clinic.

The days after removal • Be careful when you wash the lesions the first 2 weeks. Only flush with water, do not use any soap and do not rub with a towel. Let the transplanted areas dry by themselves. • Do not wear clothes that tighten over the areas and do not scrub the areas the first days. Use a thin layer of Vaseline if the skin feels too dry. • Expose the skin for UV light or natural sun light for some minutes 2–3 times a week for about 3 weeks, starting about 4th day after the removal of the bandage. • The transplanted areas remain red for a few weeks and often become slightly darker (hyperpigmented) or slightly lighter (hypopigmented) than the surrounding skin in the first few months and up to a year, but the color gradually gets closer to your normal complexion [2]. • If you would experience a purulent discharge from the lesion, a persistent swelling or erythema outside the treated lesion, or excessive itching and pain, please contact our clinic or your local physician.

References 1 Olsson MJ, Juhlin L. Transplantation of melanocytes in vitiligo. Br J Dermatol 1995;132:587–91. 2 Olsson MJ, Juhlin L. Long-term follow-up of leucoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol 2002;147: 893–904.

CHAPTER 27

Surgical management of lip vitiligo Somesh Gupta, Ashima Goel and Amrinder J. Kanwar

The lips are graceful symmetric structures that attribute certain personality traits based not only on their shape but also on their use! Lips allow the expression of the infinite shades of human emotion, ranging from affection to lust, hatred, revulsion, or anger. It displays the smile, which is unique to humans. The famed smile of the “Mona Lisa” by Leonardo da Vinci has served to convey both simplistic and more complex feelings in the imagination of art lovers since 1503 [1]. Lips are also a sexual erotic tool and are often considered as kissable, inviting, and sexually stimulating. Cosmetic disfigurement including depigmentation of the lips is embarrassing, socially stigmatizing, and has profound psychological effects on the patients and gives rise to serious emotional stress in the majority of them. The psychosocial manifestations of vitiligo range from depression and low self-esteem to job discrimination, social rejection, and even difficulty in marriage [2,3].

Anatomical considerations Lips are lined externally by skin and internally by mucosa, and there is a transition zone between these two, which is known as vermillion. Vermillion is unique to humans; it is red in color due to rich blood supply, the proximity of blood vessels to the epithelial surface, and also due to lack of keratinization in the epithelium. Epithelial covering of vermillion is characterized by a very thin stratum corneum, a prominent stratum lucidum, abundant melanocytes, numerous dermal papillae with a rich capillary supply, and absence of hair, sweat glands, and salivary glands, but numerous sebaceous glands. The internal lining of lips is stratified squamous epithelium and contains mucosal labial glands, which are situated between mucosa and orbicularis oris muscle. On the upper lip, a central dimple hollow is

formed by the philthrum and Cupid’s bow (Fig. 27.1). Boundaries of the upper and lower lips are formed by nasolabial crease and horizontal crease, respectively. In the male, keratinized external epithelium of the entire upper lip and central lower lip is hair bearing. This is important in treatment of vitiligo, as these hair-bearing areas of lip may respond to conventional medical therapies.

Vitiligo of lips Involvement of lips in vitiligo is a common occurrence in dark races and may occur in 20% of patients with vitiligo [4,5]. In White races, lip vitiligo may not be readily detected due to the deep crimson hue of the lips effectively camouflaging the vitiligo macules [6]. Diascopy and Wood’s lamp examination may help detecting clinically subtle macules of vitiligo [6]. Lip vitiligo occurs either as an extension of generalized or segmental vitiligo elsewhere on the body or as an isolated condition. In the latter group, local inflammatory conditions may play some role in inducing depigmentation. Precipitating conditions for lip leukoderma include gingivitis, smoking, recurrent herpes simplex, discoid lupus erythematosus, and fixed drug eruption [5]. More commonly, vitiligo involves the vermillion and spares the wet labial mucosa. An inverse distribution, that is sparing of vermillion and band-like involvement of the labial mucosa, can occur uncommonly, which may be associated with prolonged gingival inflammation. Another uncommon presentation is the involvement of only the lateral most part of the lower lip [7].

Surgical management The treatment of mucosal vitiligo is a therapeutic challenge. Medical management of lip vitiligo often

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Nasolabial crease Upper lip skin Philtrum Cupid’s bow

Vermilion

Lateral commissure

Lower lip skin

Horizontal crease

Fig. 27.1 Surface anatomy of lips.

results in sluggish or poor response and eventually may fail to repigment the site. During photochemotherapy, melanocytes are stimulated in vitiligo and migrate from the hair follicle reservoir, spread centrifugally from the infundibulum to the basal cell layer, and recolonize the epidermis with functional pigment cells. As the lip has no melanocyte reservoir in the form of hair follicles, photochemotherapy may not give the desirable outcome. Therefore, in published guidelines, the recommended first choice of therapy for lip vitiligo is autologous transplantation and the alternative therapy is micropigmentation [8]. This is in sharp contrast with vitiligo of other sites, which is recommended to be treated initially with UV therapy or class 3 topical corticosteroids. Hence, surgical replenishment of melanocytes is the only option available in the majority of patients. However, due to inherent histological differences between the epithelial covering of lips and that of keratinized skin, even aesthetic surgical correction is a challenge for the treating clinician.

The dermis as a whole has a well-known regulatory influence over epidermal morphogenesis and differentiation. Studies using embryonic tissue recombinants prepared by annealing the epidermis from one source (age, species, region, etc.) with a dermis from another, and implanted and allowed to differentiate in organ culture or grafted on nude mice have shown that the morphologic characteristics (thickness, architecture, and pattern of differentiation) of the epidermis conform to the region of the body from which the dermis was obtained [9]. Thus dermoepidermal grafts retain characters of their site of origin and this may lead to mismatch in the texture and thickness with the surrounding skin or mucosa, and abnormal keratinization, when applied to vermillion or labial mucosa [10]. An analysis of literature revealed that few surgical procedures have been tried for the management of lip vitiligo. We could find 10 studies and 1 metaanalysis in literature involving surgical management of lip vitiligo [11–22]. Studies included data

Surgical management of lip vitiligo

213

Table 27.1 Efficacy of various surgical procedures in the management of lip vitiligo as reported in literature.

Overall success (%)

Success in lip vitiligo (%)

8 3 10 9 31

88.9 88.7 100b 100b 87b

100 0 100 100 87

14 83

68 82

100 NM

2

NM

100

Author and reference number

Year

Total number of patients/number of patches treated

Suction blister epidermal grafts

Shah et al. [13] Hann et al. [14] Shenoi et al. [15] Gupta et al. [12]a Gupta et al. [11]a

1994 1995 1997 2004 2004

18/NM 100/142 10/10 7/9 26/31

Minigrafting

Njoo et al. [16]c Malakar and Dhar [17] Sachdev and Krupashankar [18] Malakar and Lahiri [19]

1998 1999

258 880/1715

2000

13/NM

2001

108/141

Thin split thickness skin grafts

Chitale [20] Njoo et al. [16]c

1991 1998

1/1 232

Autosomal cultured epidermal grafts

Guerra et al. [21]

2000

32/NM

NM

77

Micropigmentation

Halder et al. [22]

1989

43/NM

8

100

Procedure

Total number of lips treated

141

72b

72

1 2

100b 87

100 100 35.2

100

NM: not mentioned. a These studies used the modified technique (e.g. melanocyte transfer rather than allowing actual “take”) as described in this report. b These studies involved only lip vitiligo, therefore overall success rate and success rate in lip vitiligo were same. c Not an original study, but a meta-analysis of studies that are not included in this table to avoid duplication. (Reproduced from Gupta et al. [11], with permission from Blackwell Publishing.)

of a total of 1728 patients; out of them 312 patients had lip vitiligo, while published meta-analysis included data of 777 patients, which included 27 patients with lip vitiligo. The overall cumulative success for vitiligo of all sites and types in these studies ranged from 67% to 100%, while that for lip vitiligo varied from 0% to 100% (Table 27.1). In lip vitiligo, the average success rate with epidermal grafts was 88.5% (CI 82.3–94.8) (n
61 lips), with mini-punch grafts 77% (CI 68.8–85.2) (n
240 lips), with thin split thickness grafts 100% (CI 100–100) (n
3 lips), with cultured epidermal grafts 32.5% (CI 23.3–41.7) (n
not mentioned), and with micropigmentation, the success rate was 100% (CI 100–100) (n
8 lips) (Fig. 27.2).

The following procedures may be useful for the management of lip vitiligo: 1 suction blister epidermal grafts, 2 minigrafting, 3 thin and ultra-thin split thickness grafts, 4 autologous cultured epidermal grafts, and 5 micropigmentation or tattooing.

Suction blister epidermal grafts As discussed above, the dermo-epidermal grafts from keratinized skin are not suitable for inherently different epithelial covering of vermillion and wet labial mucosa. It has been observed that split-thickness grafts applied in the oral mucosa to cover the fullthickness defects often result in scarring and

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100%

100 90

100%

88.5%

80 72.7% Success rate (%)

70 60 50 40 32.5% 30 20 10 0 Epidermal grafts

Mini-punch grafts

Split thickness grafts

Cultured epidermal grafts

Micropigmentation

Procedure Fig. 27.2 Success rates with different surgical procedures in lip vitiligo. Data is derived from analysis of literature. Error

bars represent 95% confidence interval. (Reproduced from Gupta et al. [11], with permission from Blackwell Publishing.)

contracture at the recipient area in addition to the donor site morbidity [23,24]. The dermo-epidermal grafts retain the characteristics of the site of origin, and there have been reports of development of cutaneous diseases in these grafts, which normally do not involve oral mucosa [25]. There is a risk of development of malignant transformation in keratinized grafts applied in the wet oral mucosa. However, pure epidermal grafts adopt most of the characteristics of the recipient site and lead to a lesser degree of mismatch in texture, therefore are more suitable for mucosal vitiligo. Recently, a minor modification has been proposed to further improve the esthetic outcome of “pure” epidermal grafts. In this modified procedure, the epidermal grafts are applied on dermabraded vitiliginous lip and labial mucosa like autologous biological dressing for a period of 1 week and then discarded [11,12]. During this period of contact, melanocytes are transferred to the recipient site, which repigment without

any change in the texture and risk of abnormal keratinization resulting into better aesthetic outcome.

Method Suction blisters are raised on the lateral aspect of thigh using a device described elsewhere in this book (see Chapter 12). The affected lip is injected with saline–lignocaine mixture to produce a tumescent swelling, as well as to achieve local anesthesia. The swelling results into a partial eversion of lip and makes depigmented area more accessible. In addition, it creates a firm surface to dermabrade against. Superficial dermabrasion is done using a powerdriven dermabrader with diamond fraises or a manual dermabrader. Two stay sutures are placed through the entire thickness of the lowermost part of lower lip or uppermost part of upper lip (Fig. 27.3). The roofs of the blisters are cut and transferred on the dermabraded recipient area. The grafts are spread fully to cover the entire vitiliginous mucosa. The

Surgical management of lip vitiligo

215

Fig. 27.4 Dressing of the lip after the procedure.

(Reproduced from Gupta et al. [12], with permission from Blackwell Publishing.)

Fig. 27.3 Transfer of grafts on recipient area and place-

ment of stay sutures. (Reproduced from Gupta et al. [12], with permission from Blackwell Publishing.)

area is covered with a non-adherent gauze dressing, which is held in position with the help of stay sutures (Fig. 27.4). After 7 days the dressing along with the grafts are removed and discarded.

Advantages and limitations The results are aesthetically superior to dermoepidermal grafts (Plate 27.1, facing p. 114; Fig. 27.5). In principle, this technique is equivalent to transplantation of cultured or non-cultured pure melanocytes or melanocyte-bearing cultured epidermis; however, it is simple and, unlike with cultured melanocyte bearing autografts, does not require expensive tissue culture setup. Moreover, the usual drawbacks of cultured/non-cultured melanocyte

Fig. 27.5 Correction of vitiligo of vermillion as well as

keratinized portion of lower lip with melanocyte transfer via epidermal grafts.

transplantation, such as requirement of growth factors, and a lag period of days to weeks are absent in this technique. The major disadvantages of the technique are that it is time-consuming and it is technically difficult to keep the grafts in proper place. Several patients develop hyperpigmentation, which may persist for prolonged periods of time [11]. This may be unacceptable for some patients.

Minigrafting This is the most extensively studied transplantation procedure for vitiligo of lips. It is the easiest, fastest, and least expensive technique [8]. In this technique, small punch skin grafts are applied in the sockets

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Chapter 27

created by punch or laser in the vitiliginous mucosa. These islands of punch grafts act as melanocyte reservoirs. Under the influence of photochemotherapy, melanocytes migrate from these grafts to surrounding mucosa, resulting into repigmentation.

Method Minigrafting is usually performed with 1–1.5-mm punches. The preferred donor site for lip is retroauricular area. The grafts are taken from the donor sites and placed in the sockets created by the same size punches in the recipient area (Fig. 27.6) [19]. Hemostasis is achieved by firm pressure applied with a saline-soaked gauze piece over the area. Alternatively, the sockets can be created with pulsed erbium:yttrium–aluminum–garnet (Er:YAG) laser, with a 3-mm spot size handpiece, at a setting of 1600 mJ, frequency of 10 Hz in the burst mode of 15 pulses, giving a fluency of 16 J/cm2 per pulse [18]. There is minimal bleeding with this technique, even on the highly vascular lip. It suits for lips, which are otherwise a difficult site to treat due to profuse bleeding with conventional punch technique. The dressing comprises of three layers from inside out; these are paraffin-embedded gauze, Surgipad® and Micropore® tape. Alternatively, a transparent, semi-permeable dressing (OP-SITE®) may be used as the first layer. Minigrafting requires post-surgery photochemotherapy.

Advantages and limitations The lip has been reported to be one of the best areas for take-up of the punch grafts [19]. Minigrafting is simple to perform and does not require special training, equipments, or laboratory. The major disadvantage is cobblestoning, which is seen in 21–33% of patients [16]. This can be managed with electrofulguration [17]. Variegated appearance, which is commonly associated with minigrafting on other body parts, is uncommon on lips (Plate 27.2, facing p. 114) [19]. The grafts may be rejected in patients with oro-labial herpes [19].

Thin and ultra-thin split thickness grafts There are very few reports of use of this procedure for vitiligo of lips. This procedure is less timeconsuming and results are quick.

Fig. 27.6 Placement of punch grafts in recipient sockets

on lower lip. (Courtesy: Dr. S Malakar and Dr. K Lahiri, Kolkata, India; reproduced from Lahiri et al. (2006). Repigmentation of vitiligo with punch grafting and narrowband UVB (311 nm) – a prospective study. Int J Dermatol 2006;45:649–55, with permission from Blackwell Publishing.)

Method The lip is infiltrated with local anesthetic and adrenaline mixture. Split thickness skin is removed from the vitiliginous patch on the lip [20]. A split thickness graft of identical thickness is obtained from the donor site, which should preferably be the median aspect of arm. Alternatively, the recipient patch may be prepared using a rotating diamond fraise under topical or local anesthesia injections. The graft is transferred on the recipient area. Dressing is applied as described elsewhere. Ultra-thin grafts are obtained by a high-speed air-driven dermatome [26]. These grafts contain epidermis and a very small portion of dermis and are 0.006-in. (0.1524mm) thickness [27]. However, the use of ultra-thin split thickness grafts on lips has not been reported.

Advantages and limitations This is one of the most successful transplantation procedures for lips [11] as well as for vitiligo of other sites [16]. However, at the same time, the highest proportion of side effects has also been described with this procedure [28]. Dermo-epidermal grafts may lead to mismatch in texture, when applied on less-keratinized vermillion or non-keratinized labial mucosa due to abnormal keratinization induced by keratinized grafts. Donor site scarring is another limitation, but this is significantly less if ultra-thin grafts are used. Thick edges and hyperpigmentation are other adverse effects [16]. Milia formation may

Surgical management of lip vitiligo occur, but it is temporary and can be managed easily with needle extraction.

Autologous cultured epidermal grafts Cell therapy in vitiligo has emerged recently as an effective therapeutic option for the management of vitiligo. Its main advantage is in treating large areas from a small donor biopsy. However, technically these methods are difficult to employ in the management of lip vitiligo. Three types of cell therapies are in use in vitiligo – “pure” cultured melanocyte transplantation [29], autologous epidermal cultures bearing melanocytes [29], and non-cultured melanocyte–keratinocyte suspension transplantation [21]. Out of these, only autologous epidermal culture bearing melanocytes have been tried for the management of vitiligo of lips [21].

Method The details of this technique are described in Chapter 23. In brief, full-thickness skin biopsy specimens, taken from the pubic area, are minced and trypsinized, and cultured in 5% carbon dioxide and humidified atmosphere in keratinocyte growth medium. One day after confluence, primary cultures are trypsinized and cultivated on lethally irradiated 3T3–J2 cells. One day after confluence in secondary cultures, or 18–21 days after biopsy, the grafts are prepared from the culture. Achromic recipient mucosa or skin may be removed by dermabrasion or by programmed diathermosurgery [30]. Grafts are transferred to the recipient bed and secured with one layer of Vaseline gauze (Adaptic®; Johnson & Johnson Inc.) followed by several layers of dry gauze and bandages [21].

Advantages and limitations Transplantation of cultured epidermal grafts has several advantages over “pure” melanocyte transplantation [21]. Keratinocytes regulate melanocyte growth and differentiation. Melanocytes organize themselves into the basal layer of the cultured epidermis and maintain their physiological characteristics in co-cultures. “Pure” melanocytes are transplanted in a suspension form, while cultured epidermis in a sheet form. This is easy to retain in place especially on areas like lips. This procedure

217

does not cause scarring and texture match with the surrounding skin or mucosa is excellent. However, cultured autografts are more useful when a large area is to be treated. On smaller areas of lips, it has no advantages over suction blister “pure” epidermal grafts. The success rate on lips in the only published study was only 35%. The culture system is complex requiring a high level of expertise and high cost. There is a lag period of several weeks. Most experts and guidelines do not recommend use of cultured or non-cultured cellular grafts for the management of lip vitiligo due to either technical difficulties or poor response rates at this site [8,21,28].

Micropigmentation or tattooing Micropigmentation or tattooing may be helpful in the management of vitiligo of lips, especially in Asian and Black patients [31].

Method Tattooing can be done with manually driven needles or with needles attached to a motorized electric instrument. The later requires the Permark Enhancer II micropigmentation apparatus [22]. Recently, the watchmaker’s pin-vise has been described for use as an instrument along with sewing needles for manual tattooing in vitiligo [32]. The pin-vise is an instrument that has four jaws with a criss-cross slit. It can hold 5–15 sewing needles. Iron oxide is used as pigment, which is available in nearly 15 different colors and is hypoallergenic [22]. The affected lip is anesthetized by infiltrating with a 1–2% lignocaine solution with or without adrenaline subcutaneously. If Permark Enhancer II micropigmentation apparatus is used, the handpiece should be set at 1.75 mm and micropigmentation is carried out at maximum cycles [22]. The needle should be perpendicular to the mucosal surface and there should be repeated jabbing motions until a confluent layer of abutting pigment dots is deposited (Fig. 27.7). After the procedure, antibiotic ointment is applied and the area is dressed.

Advantages and limitations The results are immediate and permanent, although some fading may occur requiring an occasional

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Chapter 27 with the former. Micropigmentation is also successful, but has a psychological disadvantage of introducing an artificial exogenous pigment and not correcting the abnormality with natural pigment. Moreover, there is a gradual fading and discoloration requiring repeated procedures. Cultured autografts are not suitable for this site, though more studies are required. One should remember that if lip involvement in vitiligo is part of a generalized disease, the disease should be stable for at least 1 year before surgical correction is undertaken. Recurrence rates after transplantation procedures are very high in generalized vitiligo of unstable nature [29]. Other predisposing factors like smoking, gingival inflammation, and recurrent herpes simplex virus (HSV) infection should also be managed effectively to prevent recurrence of lip vitiligo.

(A)

(B) Fig. 27.7 Before (A) and immediately after (B)

micropigmentation (Courtesy: Dr. GP Thami, Chandigarh, India; reproduced from Singhal et al. [32], with permission from Blackwell Publishing.)

touch-up every 1–2 years. There is a possibility of Köbner’s phenomenon occurring in the treated lip [33]. It is very difficult to get the right color. Leaching of pigment may lead to a moderate degree of fading and discoloration. Allergic and lichenoid reaction to exogenous pigment is another potential adverse effect. Reactivation of oro-labial herpes is also common and prophylactic acyclovir is recommended in patients with a positive history.

Conclusion Lips are the most prominent parts of face and depigmentation at this site often leads to great psychological distress for the patient. Medical management is largely disappointing. Management of vitiligo of external keratinized skin of lips should be done on the same principle as described for keratinized skin. However, surgical treatment of vitiligo of vermillion and labial mucosa requires special considerations and expertise. Tissue grafts provide very high success rates varying from more than 70–100%. Pure epidermal grafts are preferred over punch or splitthickness grafts, as the aesthetic outcome is better

References 1 Greenway HT. The lips and oral cavity. In: Roenigk RK and Roenigk Jr. RH (eds.) Dermatologic Surgery, 2nd edn. New York: Marcel Dekker, Inc., 1996;313–28. 2 Porter J, Beuf AH, Lerner A, Nordlund J. Response to cosmetic disfigurement in patients with vitiligo. Cutis 1987;39:493–4. 3 Hautmann G, Panconesi E. Vitiligo: a psychologically influenced and influencing disease (review). Clin Dermatol 1997;15:879–90. 4 Ortonne JP. Depigmentation of hair and mucous membrane. In: Hann S-K and Nordlund JJ (eds.) Vitiligo, 1st edn. Oxford: Blackwell Science, 2002;76–80. 5 Coondoo A, Sen N, Panja RK. Leucoderma of the lips: a clinical study. Indian J Dermatol 1976;21:29–33. 6 Tolat SN, Gharpuray MB. Diascopy, the lips, and vitiligo (letter). Arch Dermatol 1995;131:228–9. 7 Sahoo A, Singh PC, Patnaik S, Singh N, Srichandan M. Vitiligo, lateral lower lip. Indian J Dermatol 2002;47: 15–7. 8 Njoo MD, Westerhof W, Bos JD, Bossuyt PMM. The development of guidelines for the treatment of vitiligo. Arch Dermatol 1999;135:1514–21. 9 Haake AR, Holbrook K. The structure and development of skin. In: Freedberg IM, Eisen SAZ, Wolff K, et al. (eds.) Fitzpatrick’s Dermatology in General Medicine, 5th edn. New York: McGraw-Hill, 1999;70–114. 10 Gupta S, Kumar B. Influence of age of the patient, type of the disease and site of the procedure on outcome of epidermal grafting in vitiligo. J Am Acad Dermatol 2003;49:99–104.

Surgical management of lip vitiligo 11 Gupta S, Goel A, Kanwar AJ, Kumar B. Autologous melanocyte transfer via epidermal grafts for lip vitiligo. Int J Dermatol 2006;45:747–50. 12 Gupta S, Sandhu K, Kanwar A, Kumar B. Melanocyte transfer via epidermal grafts for vitiligo of labial mucosa. Dermatol Surg 2004;30:45–8. 13 Shah BH, Joshipura SP, Thakkar JK. Surgical treatment in acrofacial vitiligo. Indian J Dermatol Venereol Leprol 1994;60:26–7. 14 Hann SK, Im S, Bong HW, Park YK. Treatment of stable vitiligo with autologous epidermal grafting and PUVA. J Am Acad Dermatol 1995;32:943–8. 15 Shenoi SD, Srinivas CR, Pai S. Treatment of stable vitiligo with autologous epidermal grafting and PUVA. J Am Acad Dermatol 1997;36:802–3. 16 Njoo MD, Westerhof W, Boz JD, Bossuyt PMM. A systematic review of autologous transplantation methods in vitiligo. Arch Dermatol 1998;134:1543–9. 17 Malakar S, Dhar S. Treatment of stable and recalcitrant vitiligo by autologous miniature punch grafting: a prospective study of 1,000 patients. Dermatology 1999;198:133–9. 18 Sachdev M, Krupashankar DS. Pulsed erbium:YAG laser-assisted autologous punch grafting in vitiligo. Int J Dermatol 2000;39:868–71. 19 Malakar S, Lahiri K. Punch grafting for lip leucoderma. Dermatology 2004;208:125–8. 20 Chitale VR. Overgrafting for leukoderma of the lower lip: a new application of an already established method. Ann Plast Surg 1991;26:289–90. 21 Guerra L, Capurro S, Melchi F, et al. Treatment of “stable” vitiligo by Timedsurgery and transplantation of cultured epidermal autografts. Arch Dermatol 2000; 136:1380–9. 22 Halder RM, Breadon JY, Johnson BA. Micropigmentation for the treatment of vitiligos. J Dermatol Surg Oncol 1989;15:1092–8.

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23 Rhee PH, Friedman CD, Ridge JA, Kusiak J. The use of processed allograft dermal matrix for intraoral resurfacing: an alternative to split-thickness skin grafts. Arch Otolaryngol Head Neck Surg 1998;124:1201–4. 24 Mitchell R. The use of collagen in oral surgery. Ann Acad Med Singap 1986;15:355–60. 25 Dimitrakopoulos I, Lazaridis N, Scordalaki A. Dermal psoriasis involving an oral split-skin graft: case report. Aust Dent J 1998;43:321–3. 26 Olsson MJ, Juhlin L. Epidermal sheet grafts for repigmentation of vitiligo and piebaldism, with a review of surgical techniques. Acta Derm Venereol 1997;77:463–6. 27 Kahn AM, Cohen MJ. Repigmentation in vitiligo patients. Melanocyte transfer via ultra-thin grafts. Dermatol Surg 1998;24:365–7. 28 van Geel N, Ongenae K, Naeyaert J-M. Surgical techniques for vitiligo. Dermatology 2001;202:162–6. 29 Olsson MJ, Juhlin L. Long-term follow-up of leucoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol 2002;147: 893–904. 30 Capurro S, Fiallo P. Epidermal disepithelialization by programmed diathermosurgery. Dermatol Surg 1997; 23:600–1. 31 Taneja A. Treatment of vitiligo. J Dermatol Treat 2002; 13:19–25. 32 Singhal A, Thami GP, Bhalla M. Watchmaker’s pinvise for manual tattooing of vitiligo. Dermatol Surg 2004;30:203–4. 33 Halder RM, Young CM. New and emerging therapies for vitiligo. Dermatol Clin 2000;18:79–89.

CHAPTER 28

Surgical management of vitiligo of eyelids and genitals: special issues Somesh Gupta

Vitiligo at mucocutaneous junctions of lips, eyelids, and genitals is a therapeutic challenge, as it is often recalcitrant to medical therapies and poses technical difficulties when planned to be treated surgically. Therefore, surgical correction of vitiligo in these areas needs special discussion. Management of lip vitiligo has been discussed in a separate chapter. This chapter deals with the vitiligo on eyelids and genitals.

Eyelids Eyelids are a common site of involvement in both generalized and segmental forms of vitiligo. Nearly 50% of segmental vitiligo on face involves eyelids [1]. The exact incidence of involvement of eyelids in generalized vitiligo is not known, though there is a common perception that when eyelids are involved they remain resistant to the conventional medical therapy due to absence of melanocyte reservoirs in the form of hair follicles. There are a limited number of medical therapeutic options available for vitiligo on eyelids. Photochemotherapy psoralen plus ultraviolet A (PUVA) on eyelids carries a risk of development of cataracts. It is recommended that all patients receiving PUVA should wear eyeglasses [2]. Potent topical steroids can readily cause atrophy of the thin skin of eyelid. Prolonged use of topical steroids on eyelids can result in exposure of the eyes to steroids, occasionally causing increased intra-ocular pressure and cataract formation [3]. However, other topical therapies, such as tacrolimus and calcipotriene have been found safe and effective on eyelids [4,5]. Various transplantation procedures have been tried for the vitiligo of the eyelids. These include ultra-thin split-thickness grafts, minigrafts, suction blister

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epidermal grafts, non-cultured melanocyte– keratinocyte cell suspension, and follicular grafts. We carried out a meta-analysis of indexed literature from 1980 to April 2005 to analyze the efficacy of transplantation procedures for eyelid vitiligo (Table 28.1) [3,6–14]. There were 10 studies describing results in eyelid vitiligo. The data of 30 patients was available, which showed a successful outcome (defined as
75% or excellent repigmentation) in 25 patients with a success rate of 83.3%. Most of the procedures gave excellent success rates (Plate 28.1, facing p. 114; Fig. 28.1; Fig. 28.2) with the exception of follicular transplantation. Malakar and Dhar [15] treated 150 patients with eyelid vitiligo with minigrafting. In their patients, the overall success rate for all sites was 79.32%; however, the success rate in eyelid vitiligo was not specified. In an attempt to create a melanocyte reservoir in the glabrous skin of the eyelids, the upper two-thirds of single hair follicles have been transplanted; however, the repigmentation was only moderate and was less satisfactory than on other areas [14]. Kawalek et al. [16] reported successful outcome with a combination of excimer laser and topical tacrolimus in a patient with eyelid vitiligo.

Special considerations The skin of the eyelid is the thinnest on the body. It is peculiar in its loose attachment and absence of fat in its corium [17]. There is no underlying bony firm support, making denudation procedures like dermabrasion difficult [3]. Therefore, it is necessary to pull the upper eyelid skin upwards to bring it over the bony orbit. The skin over eyelid can be anesthetized by infiltrating it with lidocaine 1%. Epinephrine should be avoided as vasoconstriction caused by it

Surgical management of vitiligo of eyelids and genitals

221

Table 28.1 Transplantation procedures for eyelid vitiligo. Segmental/focal

Vulgaris

All

Procedure

Authors

Total number

Successful* number (%)

Total number

Successful* number (%)

Total number

Successful* number (%)

Epidermal grafting

Suga et al. [6] Hann et al. [7] Nanda et al. [3] All studies

1 n.m. n.m. n.m.

1 (100%) n.m. n.m. n.m.

– n.m. n.m. n.m.

– n.m. n.m. n.m.

1 7 6 13

1 (100%) 7 (100%) 6 (100%) 13 (100%)

Ultra-thin split thickness

Kahn and Cohen [8] Olsson and Juhlin [9] All studies

1

1 (100%)

–

–

1

1 (100%)

–

–

5

3 (60%)

5

3 (60%)

1

1 (100%)

5

3 (60%)

6

4 (66.6%)

Non-cultured melanocyte– keratinocyte cell suspension

Olsson and Juhlin [10] Mulekar [11] All studies

–

–

3

3 (100%)

3

3 (100%)

3 3

2 (66.6%) 2 (66.6%)

– 3

– 3 (100%)

3 6

2 (66.6%) 5 (83.3%)

Minigrafting

Bonafe et al. [12] Falabella [13] All studies

n.m.

n.m.

n.m.

n.m.

1

1 (100%)

1 1

1 (100%) 1 (100%)

– –

– –

1 2

1 (100%) 2 (100%)

Single hair transplantation

Na et al. [14]

2

0 (0%)

0

0

2

0 (0%)

Total

–

8

5 (62.5%)

8

6 (75%)

30

25 (83.3%)

n.m.: Not mentioned. * Successful outcome is defined as ≥75% or excellent repigmentation.

will make it difficult to judge the depth of dermabrasion on very thin skin of the eyelids. Margin of the eyelid should not be infiltrated to avoid ectropion or destruction of the eyelashes [3]. Due to the thinness of the eyelid skin, topical eutectic mixture of lidocaine and prilocaine (EMLA®) also produces satisfactory anesthesia if applied under occlusion for about 1 hour. It is important to dress the eye firmly to prevent movements of the eyelid, which may displace the grafts. The other eye should not be covered with the dressing to enable the patient to do day-to-day work. In conclusion, tissue or cellular grafting procedures have been tried on eyelids with satisfactory outcome. However, the thin skin of eyelid should preferably

be treated with cellular grafts or very thin tissue grafts such as suction blister grafts or ultra-thin sheet grafts. Expansion of melanocytes with culture is not required for smaller areas such as eyelids [18]. Post-transplantation photochemotherapy is not necessary for uniform pigmentation. There is a risk of hyperpigmentation and color mismatch if photochemotherapy is given in post-transplantation period [18].

Genitals Genitals are a commonly affected site for vitiligo in men and sometimes it is the only site affected [19]. In non-segmental vitiligo, genitals as an initial site of

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(A) (A)

(B) Fig. 28.2 (A) Vitiligo on lower eyelid. (B) Complete

repigmentation with thin split-thickness grafting. (Courtesy: Niti Khunger, Safdarjung Hospital and VM Medical College, New Delhi, India.)

(B) Fig. 28.1 (A) Vitiligo on lower eyelid, 1 month after epidermal grafting, the grafted area is still perceptible. (B) At 6 months, almost complete repigmentation with good color match.

involvement is reported to occur in 1.6% of patients and overall, genitals are involved in about 6–10% of patients [20]. Vitiligo occurring on non-exposed skin may be considered trivial, but becomes complicated by psychological factors when it occurs on the genitals. Most patients and their sex partners ponder whether vitiligo on the penis is the consequence of a sexual act or a manifestation of a communicable disease. This belief is not entirely without substance, as in a report by Gaffoor [21] up to one-fourth of the patients with depigmentation on the genitals had a history of sexually transmitted diseases. Porter et al.

[22] studied the effect of vitiligo on sexual relationships and found that embarrassment during sexual acts was especially frequent for men with vitiligo. Mucosal vitiligo on the glans penis poses a challenge to the treating physician for several reasons [23]. One, the absence of a melanocyte reservoir in the form of hair follicles makes medical therapies less likely to be successful. Two, phototherapy or photochemotherapy, the mainstay of treatment of vitiligo at other sites, carries a significant risk of genital tumors [24] and is not recommended for the treatment of vitiligo on the genitals, a site at a greater risk for human papillomavirus-related malignancies. Three, tissue grafts like mini-punch grafts, or thin or ultra-thin split-thickness grafts from keratinized skin are not suitable for the non-keratinized

Surgical management of vitiligo of eyelids and genitals mucosa of the glans penis as these may lead to gross mismatch in texture and color [25]. In a report, Mulekar et al. [26] described successful treatment of vitiligo over glans penis with non-cultured melanocyte–keratinocyte cell suspension (Plate 28.2, facing p. 114). There was excellent restoration of normal color of the mucosa of glans penis without any change in the texture. In the absence of reports describing other methods of surgical treatment for vitiligo of the mucosa of glans penis, it is reasonable to recommend melanocyte–keratinocyte cell suspension transplantation as the treatment of choice for stable and recalcitrant vitiligo at this site. Olsson and Juhlin [9] reported successful management of keratinized skin of shaft of the penis, and scrotum with transplantation procedures, like ultrathin epidermal sheet grafts and cultured melanocyte transplantation. Among other treatment modalities, Hadi et al. [27] reported success rate of 50% with 308-nm excimer laser in four patients with genital vitiligo. There is only one report of transplantation procedure on vulva. Hann et al. [7] reported successful graft survival and repigmentation with epidermal grafting in one patient with vitiligo on vulva.

Special considerations The glans penis and inner surface of the prepuce (i.e. the preputial sac) are covered with non-keratinized epithelium. The shaft of the penis is covered by a thinly keratinized epidermis with scant numbers of pilosebaceous, eccrine, and apocrine glands. The epithelial covering over shaft of the penis has the same morphology as keratinized skin elsewhere; therefore, it needs to be treated on the same principles as described for other regions with keratinized epithelium. Scrotum skin is also keratinized but relatively thin. In female genitals, labia majora are covered with hair-bearing keratinized squamous epithelium, while labia minora are covered with less keratinized hairless squamous epithelium; the former can be treated with any of the tissue or cellular grafts, while labia minora require cellular grafts or extremely thin tissue grafts such as epidermal grafts. For a smaller area on the penis, the tissue is anesthetized using 1% lidocaine in a 1-ml syringe with a 30 gauge needle. Epinephrine is best avoided when

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injecting the penis as it may occasionally result in tissue necrosis. For superficial dermabrasion, EMLA® under occlusion for 20–30 minutes for nonkeratinized mucosa of glans penis or for 1–1.5 hours for keratinized skin of the shaft of the penis is sufficient to achieve anesthesia [28]. If a larger area is to be treated on the penis, a nerve block may be considered. The penile block requires injection around the entire circumference at the base of the penis [29]. Female external genitals are anesthetized with direct infiltration of 1% lidocaine or EMLA® cream.

References 1 Hann S-K, Chang J-H, Lee H-S, Kim S-M. The classification of segmental vitiligo on face. Yonsei Med J 2000;41:209–12. 2 Nordlund JJ. Sunscreens and sun protection. In: Hann S-K and Nordlund JJ (eds.) Vitiligo, 1st edn. Oxford: Blackwell Science, 2000;218–21. 3 Nanda S, Relhan V, Grover C, Reddy BSN. Epidermal grafting for eyelid vitiligo: special considerations. Dermatol Surg 2006;32:387–91. 4 Travis LB, Weinberg JM, Silverberg NB. Successful treatment of vitiligo with 0.1% tacrolimus ointment. Arch Dermatol 2003;139:571–4. 5 Travis LB, Silverberg NB. Calcipotriene and corticosteroid combination therapy for vitiligo. Pediatr Dermatol 2004;21:495–8. 6 Suga Y, Takimoto R, Fujioka N, Yamada H, Ogawa H. Successful treatment of vitiligo with PUVA-pigmented autologous epidermal grafting. Int J Dermatol 1996;35:518–22. 7 Hann SK, Im S, Bong HW, Park Y-K. Treatment of stable vitiligo with autologous epidermal grafting and PUVA. J Am Acad Dermatol 1995;32:943–8. 8 Kahn AM, Cohen MJ. Vitiligo: treatment by dermabrasion and epithelial sheet grafting. J Am Acad Dermatol 1995;33:646–8. 9 Olsson MJ, Juhlin L. Epidermal sheet grafts for repigmentation of vitiligo and piebaldism, with a review of surgical techniques. Acta Derm Venereol 1997;77:463–6. 10 Olsson MJ, Juhlin L. Leucoderma treated by transplantation of a basal cell layer enriched suspension. Br J Dermatol 1998;138:644–8. 11 Mulekar SV. Long-term follow-up study of segmental and focal vitiligo treated by autologous, noncultured melanocyte–keratinocyte cell transplantation. Arch Dermatol 2004;140:1211–5.

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12 Bonafe JL, Lassere J, Chavoin JP, et al. Pigmentation induced in vitiligo by normal skin graft and PUVA stimulation: a preliminary study. Dermatologica 1983; 166:113–6. 13 Falabella R. Treatment of localized vitiligo by autologous minigrafting. Arch Dermatol 1988;124:1649–54. 14 Na GY, Seo SK, Choi SK. Single hair grafting for the treatment of vitiligo. J Am Acad Dermatol 1998;38:580–4. 15 Malakar S, Dhar S. Treatment of stable and recalcitrant vitiligo by autologous miniature punch skin grafting: a prospective study of 1000 patients. Dermatology 1999;198:133–9. 16 Kawalek AZ, Spencer JM, Phelps RG. Combined excimer laser and topical tacrolimus for the treatment of vitiligo: a pilot study. Dermatol Surg 2004;30:130–5. 17 Robinson JK. The eyelid. In: Roneigk RK and Roegnik Jr. HH (eds.) Dermatologic Surgery, Principles and Practice, 2nd edn. New York: Marcel Dekker, Inc., 1996;293–312. 18 Gupta S. Commentary on: epidermal grafting for eyelid vitiligo: special considerations. Dermatol Surg 2006;32:391–2. 19 Bunker CB. Non-venereal penile dermatoses. In: Kumar B and Gupta S (eds.) Sexually Transmitted Infections, New Delhi: Elsevier, 2005;539–64. 20 Chun WH, Hann S-K. The progression of nonsegmental vitiligo: clinical analysis of 318 patients. Int J Dermatol 1997;36:908–10.

21 Gaffoor PMA. Depigmentation of the male genitalia. Cutis 1984;34:492–4. 22 Porter J, Beuf A, Lerner A, et al. The effect of vitiligo on sexual relationship. J Am Acad Dermatol 1990;22:221–2. 23 Gupta S. Commentary on: genital vitiligo treated by autologous, noncultured melanocyte–keratinocyte cell transplant. Dermatol Surg 2005;31:1740. 24 Stern RS, Bagheri S, Nichols K. The persistent risk of genital tumors among men treated with psoralen plus ultraviolet A (PUVA) for psoriasis. J Am Acad Dermatol 2002;47:33–9. 25 Gupta S, Goel A, Kanwar AJ, Kumar B. Autologous melanocyte transfer via epidermal grafts for lip vitiligo. Int J Dermatol 2006;45:747–50. 26 Mulekar SV, Al Eissa A, Marwan A, Bassel G. Genital vitiligo treated by autologous, noncultured melanocyte–keratinocyte cell transplantation. Dermatol Surg 2006:31:1737–9. 27 Hadi SM, Spencer JM, Lebwohl H. The use of the 308-nm excimer laser for treatment of vitiligo. Dermatol Surg 2004;30:983–6. 28 Lynch PJ, Edward L. Diagnostic procedures. Genital Dermatology, New York: Churchill Livingstone, 1994; 7–10. 29 Gibson LE, Perry HO. Male and female genitalia. In: Roenigk RK and Roenigk Jr. RH (eds.) Dermatologic Surgery: Principles and Practice, New York: Marcel Dekker, 1996;371–8.

CHAPTER 29

Surgical management of acral vitiligo Sharad Mutalik

Managing a case of vitiligo is a difficult task for any clinician. Over and above that, treatment of acrofacial (lip tip) vitiligo is even more difficult [1]. The natural course of this pigmentary disorder is quite unpredictable. Vitiligo lesions with acro-facial distribution are known to be resistant to the conventional medical treatment. The possible explanations are: 1 relatively less melanocytes density in these anatomical areas [2] (1130
160/mm2); 2 relatively less hair density; and 3 higher chances of Köbnerization over these friction and injury prone anatomical sites. Lip-tip vitiligo, being present on the exposed areas of the body, leads to significant cosmetic disfigurement. Localized lesions of vitiligo, which are stationary in size and stable for at least 3 years could be considered for surgical management [3]. Surgical management of lip vitiligo is covered in a separate chapter, therefore, this chapter will be restricted to the surgical management of acral vitiligo. Surgical management of acral vitiligo has several limitations. To be precise, acral vitiligo includes lesions over wrist, hand, ankle, and foot. Different surgical techniques could be applied depending on the distribution of the lesions. Acral part of the upper extremity could be broadly divided into flexor and dorsal aspects of wrist, dorsal and palmar aspects of hand, dorsal and volar aspects of fingers, ulnar and radial border of hand. Similarly acral part of the lower extremity could be broadly divided into medial and lateral aspects of ankle joint, dorsum of foot and toes and plantar aspect of the foot and toes, posterior aspect of heel and border of the entire foot.

Surgical modalities for treatment of stable acral vitiligo 1 Camouflage tattooing (micropigmentation) 2 Autologous melanocyte transplantation – Epidermal grafting by suction blister technique – Split-thickness skin grafting – Miniature punch grafting – Cultured melanocyte transplantation These techniques are described in detail in other chapters.

Camouflage tattooing This technique can be considered in very tiny macules of vitiligo, particularly in a dark-skinned individual. It is useful when the number of lesions is small, and the patient has urgency, demanding instant results [4]. Advantages: It is a simple, inexpensive method, giving immediate results. Disadvantages: This technique requires experience and skill in preparing the exact proportion of different pigments and placing the pigment at the desired depth in order to achieve acceptable matching and perfect camouflage. Inappropriate proportion of pigments can result in a color mismatch leading to poor cosmetic result. It is not suitable for the thick skin of palms and soles and lesions of larger size.

Transplantation of autologous melanocyte or melanocyte-bearing epidermis These techniques could be employed for lesions over ankles, wrists, dorsi of hands and feet up to

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the distal interphalangeal joint, as the skin over these areas has similar structural and histological characteristics as those of donor area, so the grafts are well accepted. On the other hand, epidermis over palmoplantar surfaces has specialized structural characteristics; it has compact stratum corneum [5]. Proteins like Keratin K9 are uniquely found in palmoplantar skin. Keratin K6 and K16 are found in the palmoplantar epidermis, nail plate, hair follicle, oral mucosa, sweat gland, but not in the skin covering remaining body surface. In addition, the glabrous skin over palms and soles is covered with ridges and sulci [6]. Any skin graft harvested from the gluteal region, arms, or thigh has different structural characteristics, which is naturally not accepted over palmoplantar skin.

Epidermal grafting by suction blister technique This method is best suitable for smaller lesions, on flat surfaces like dorsum of hand (Plate 29.1, facing p. 114) or foot, dorsal aspect of proximal and middle phalynx (Fig. 29.1), and lesions involving bony prominences like knuckles, medial and lateral malleoli, provided the treated area is properly immobilized with a splint. Dermabrasion is difficult, particularly over the areas like dorsi of fingers. In such cases the recipient site can be prepared by inducing a blister with liquid Nitrogen or psoralen plus ultraviolet A (PUVA), and de-roofing the blister.

Advantages: Of all the tissue grafts, epidermal graft gives the best cosmetic results, as there is no stuck-on appearance. It can also be used for postinflammatory depigmentation resulting from trauma or burns and even chemical depigmentation [7]. The donor site heals without scarring. This is a safe, simple, inexpensive office-based surgery giving excellent cosmetic results. Disadvantages: Epidermal grafts are not accepted over areas like palms, soles, volar aspect of fingers, borders of hand and foot, where the skin is quite thick. Harvesting of epidermal grafts by suction technique is quite time consuming. Multiple grafts are required to cover larger areas.

Split-thickness skin grafting A split-thickness graft of 0.1–0.2-mm thickness can be harvested using a motorized dermatome or a shaving blade held in straight hemostat. These grafts are useful for treating lesions covering flat areas like dorsi of hands and feet, medial and lateral malleoli. Advantages: This is the best method to cover larger lesions and multiple lesions in a single session, giving immediate results. Time consumed is less as compared to other methods. Disadvantages: Taking out a thin graft of uniform thickness requires skill and dexterity. Grafts which are not of uniform thickness give stuck-on appearance. Split-thickness grafts tend to remain hyperpigmented and perceptible, giving cosmetically unacceptable results.

(A)

(B) Fig. 29.1 (A) Acral vitiligo 2 weeks after epidermal grafting. Note faint pigmentation appearing in the grafting area.

(B) One year post-transplantation, nearly complete repigmentation. (Courtesy of Somesh Gupta, MD, DNB, New Delhi, India.)

Surgical management of acral vitiligo They are not suitable for dorsi of fingers and toes [8]. They are not accepted over the thick skin of palms and soles due to a remarkable histological difference in the donor and recipient skin (Fig. 29.2). Scarring and residual depigmentation can be expected at the donor site.

Miniature punch grafting Punch grafts of 1–1.5 mm size are best suitable for areas like palms, medial and lateral borders of feet, ulnar and radial borders of hands and lateral surfaces of fingers. Skin of the instep has been recently used as donor site for minigrafting on the vitiligo of palm [9]. This technique can also be used for treating the lesions over soles, but a large number of grafts are necessary to cover the entire sole. It is also difficult to secure the grafts over the plantar arches. Fortunately the sole is not visible and usually covered, therefore does not demand any surgical treatment. Advantages: This is the only effective method for treating difficult areas like palms and soles, where no other graft is accepted [7]. Being a full thickness graft, it is well accommodated in the thick skin over palmoplantar surfaces. Compared to other techniques of melanocyte transplantation, a larger number of melanocytes are available using the full thickness miniature grafting technique. This is the easiest method, technically least challenging, and least expensive of all the surgical methods. Beginners can use this technique over flat areas like dorsi of hands and feet.

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Disadvantages: This method is time consuming. Inappropriate thickness of grafts and inappropriate depth of the recipient well can result in cobblestone appearance. This technique requires postoperative medical treatment or phototherapy to facilitate pigment spread. The results are not encouraging over dorsi of fingers, toes, and are discouraging over finger tips and distal phalanges. Immobilization of the grafted areas, especially palms, is highly essential.

Transplantation of cultured melanocytes Large-sized lesions over the dorsal aspects of hands and feet show excellent results after transplantation of cultured autologous melanocytes, whereas fair to poor results were observed on dorsal aspects of phalanges and joints [10,11]. Out of the 19 transplants done on dorsi of hands by Olsson and Juhlin, excellent results were observed in 10 cases (hands), and fair to poor results were seen in 9 cases (phalanges and knuckles). Out of the 24 cases involving feet, 11 cases showed excellent, 10 cases showed good, and 3 cases showed fair results. Advantages: For larger areas, this method is the most promising amongst all the techniques. A small donor area is sufficient to cover large area, and gives excellent cosmetic results. Disadvantages: This ultramodern technique requires well-equipped infrastructure, expensive laboratory setup, and trained personnel, and is time consuming.

Conclusion

Fig. 29.2 Scarring after split-thickness skin grafting over

the palm.

There are no controlled studies on the surgical management of acral vitiligo. Acral vitiligo is refractory to medical treatment. With the help of narrowband ultraviolet B (NB-UVB) therapy, targeted UVB, and the newer immunomodulators like Tacrolimus, Pimecrolimus, some degree of repigmentation can be achieved, especially over proximal parts of the hands and feet. The task becomes difficult as one goes distally. Vitiligo involving tips of the fingers and toes is progressive and unstable in the majority of cases, hence not considered for surgical management. Anecdotal

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reports cannot be considered in laying out guidelines for surgical management of acral vitiligo. In properly selected cases, even if skin grafting is accomplished, due to structural limitations, it is very difficult to secure any kind of skin graft over the finger tip or toe tip. Grafts over periungual areas can be secured to some extent with tissue glue (cyanoacrylate) and splint, but it is impossible to secure grafts over the curved surfaces like insole and ventral aspects of the toes. Depending on the size and distribution of the lesion, treatment should be individualized (Table 29.1), in order to achieve best possible cosmetic results.

Table 29.1 Guidelines for surgical management of acral

vitiligo. Anatomical site

Surgical method

Wrist flexor/dorsal

Suction blister epidermal grafts*

Lateral and medial malleoli

Epidermal blister graft (for small lesions)* Split-thickness graft or cultured melanocyte (for large lesions)*

Dorsum of hand/foot

Epidermal blister graft (for small lesions) Split-thickness graft or cultured melanocyte culture (for large lesions) Miniature punch graft (very small lesions) Camouflage tattoo (tiny lesions in dark skin)

Palms, soles, borders of hand and foot

Miniature punch graft*

Knuckles, dorsi of fingers, up to distal interphalangeal joint

Epidermal blister graft (for small lesions)* Split-thickness graft or cultured melanocyte (for large lesions)

Periungual skin of fingers and toes

Epidermal blister graft*

Tips of fingers and toes, ??? heel posterior aspect *Splint essential for immobilization.

A long-term prospective study is needed to set up guidelines for the surgical management of vitiligo over tips of fingers and toes. Activity of the disease in these anatomical sites, fear of Köbnerization [12] and practical difficulty in securing the grafts seem to be the main hurdles in undertaking any such studies. We should be optimistic in finding out some solution in treating these difficult areas effectively.

References 1 Ortonne JP, Bahadoran P, Fitzpatrick TB, et al. Hypomelanoses and hypermelanoses. In: Freedberg IM, Eisen AZ, Wolff K, et al. (eds.) Fitzpatrick’s Dermatology in General Medicine, 5th edn. New York: McGraw-Hill, 2003;844–6. 2 Bleehen SS, Anstey AV. Disorders of skin color. In: Burns T, Breathnach S, Cox N, and Griffiths C (eds.) Rook’s Textbook of Dermatology, 7th edn. MA: Blackwell Science, 2004;39.3. 3 Mutalik S. Transplantation of melanocytes by epidermal grafting, an Indian experience. J Dermatol Surg Oncol 1993;19:231–4. 4 Gharpuray MB, Mutalik S. Manual punch for tattooing. J Dermatol Surg Oncol 1994;20:548–50. 5 Stevens HP, Leigh I. The inherited keratodermas of palms and soles. In: Freedberg IM, Eisen AZ, Wolff K, et al. (eds.) Fitzpatrick’s Dermatology in General Medicine, 5th edn. New York: McGraw-Hill, 2003;604. 6 McGrath JA, Eady RA, Pope FM. Anatomy and organization of human skin. In: Burns T, Breathnach S, Cox N, and Griffiths C. (eds.) Rook’s Textbook of Dermatology, 7th edn. MA: Blackwell Science, 2004;3.1. 7 Mutalik S, Ginzburg A. Surgical management of stable vitiligo: a review with personal experience. Dermatol Surg 2000;26:248–54. 8 Sawant S. Vitiligo surgery. In: Valia RG, and Valia A (eds.) Dermatology Update. Mumbai: Bhalani publishing, 1998;69. 9 Kumar P. Autologous punch grafting for vitiligo of the palm. Dermatol Surg 2005;31:368–70. 10 Olsson MJ, Juhlin L. Transplantation of melanocytes in vitiligo. Br J Dermatol 1995;132:587–91. 11 Falabella R. Surgical therapies for vitiligo. In: Hann SK, and Nordlund JJ (eds.) Vitiligo. London: Blackwell Science, 2000;193. 12 Olsson MJ, Juhlin L. Repigmentation of vitiligo by transplantation of cultured autologous melanocytes. Acta Derm Venereol 1993;73:49–51.

CHAPTER 30

Surgical management of leukotrichia Karoon Agrawal and Aparna Agrawal

Leukotrichia is defined as white hair. We are concerned here about the white hair within the area of vitiligo. Such leukotrichia often complicates vitiligo and is a sign of refractoriness of disease. A few authors have suggested that both the white hair and depigmentation of skin are probably the result of the same physiological disturbance [1–3]. There is no dearth of literature the world-over on the treatment of vitiligo and the results thereof. But very few authors have commented upon the leukotrichia in vitiligo areas. The present society places profound significance on appearance. In populations with dark hair, premature graying of hair is considered as a sign of ageing. Hence concerted efforts should be made to treat these patients. It is extremely important to try for permanent repigmentation of not only vitiligo but also leukotrichia to prevent psychosocial problems.

Epidemiology of leukotrichia in vitiligo Vitiligo is a very common clinical entity, however clinically leukotrichia is not seen in vitiligo of all hair-bearing areas. The incidence of leukotrichia in vitiligo of hair-bearing areas varies from 11.5% to 44% [4,5]. From India, in the pediatric age group leukotrichia in vitiligo has been reported in 12.3% patients [6]. In elderly patients with onset of disease after 50 years of age, on the other hand, it has been reported in 47.3% of vitiligo patients [7].

Histological considerations Each hair has a root within the dermis of the skin and a shaft outside the surface. It arises from an epidermal invagination called the hair follicle (Fig. 30.1). The follicle has a proximal enlargement called the hair bulb. At the base of the bulb lies a bunch of vessels known as the dermal papilla. The epidermal

cells covering the dermal papilla form the hair root. This hair root produces the medulla of the hair shaft. Peripheral root cells multiply and differentiate into keratinized compact fusiform cells forming the hair cortex. Still more peripheral cells form the heavily keratinized cuticle of the hair shaft. The outermost cells of the hair bulb form the outer and the inner root sheaths. The inner root sheath surrounds the hair shaft intradermally and it degenerates above the level of the sebaceous glands. The cellular outer root sheath is continuous with the epidermal cells of the skin [8]. A “bulge” in outer root sheath of the hair has been identified in the region of the attachment of arrector pili muscle below the level of sebaceous glands (Fig. 30.1). There is a well-demarcated area for the stemcell niche within the lower permanent portion of the hair follicle. This niche contains undifferentiated melanocyte stem cells [9] (Fig. 30.2). These stem cells have the capacity to self renew and generate differentiated progeny on activation. A portion of amplifying stem cell progeny can migrate out from the niche and retain original self-remodeling quality in new niches [10]. The interest here is in the pigmented cells of the hair follicles and their biological and biochemical behavior. Melanoblasts are the precursors of melanocytes, which are undifferentiated cells, which originate from the neural crest. There are two forms of melanocytes in the hair follicles: amelanotic melanocytes, which are biochemically inactive and devoid of pigmentary granules and dendritic or melanotic melanocytes with pigmentary granules which react specifically with Masson’s ammoniacal silver nitrate and dopa [11]. These pigmentary granules are responsible for the color of the hair and skin. These two sets of melanocytes are interchangeable. Staricco in 1963 diagrammatically

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Cuticle Cortex Medulla

Permanent portion

Hair shaft

Outer root sheath

Epidermis Dermoepidermal junction

Inner root sheath

Sebaceous gland Bulge Arrector pili muscle

Transient portion Hair bulb

Dermal papilla Fig. 30.1 Line diagram of histology of hair follicle.

⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫹

Portion A

Melanocyte stem cells Portion B Amelanotic melanocytes Melanotic melanocytes

Portion D

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

Portion C

explained that the hair follicle may be divided into four portions (Fig. 30.2). A, the upper follicle and D, the upper part of the hair bulb (in contact with the upper part of the dermal papilla) contain melanotic melanocytes. On the other hand portion B, the middle and lower follicle and portion C, the

Fig. 30.2 Line diagram depicting the melan-

otic and amelanotic zones in hair follicle (concept from Staricco, 1963).

outer root sheath of the hair bulb contain amelanotic melanocytes [11] (Fig. 30.2). The whole melanocytic population of the skin can be regarded as a bicompartmental system with two relatively distinct subpopulations: the epidermal and the follicular compartments [12]. The melanocytes in the

Surgical management of leukotrichia two compartments might be transferred from one compartment to the other when activated [12].

Normal repigmentation process In normal individuals it has been proven that repigmentation of the epidermis occurs from the regeneration of melanocytes in the amelanotic zone of the hair follicle as melanocytes are seldom seen in a phase of division in the melanotic part. Experiments have shown that whenever there is a stimulus or induced trauma by vibrapuncture [13], blistering or by dermabrasion [14] the amelanotic melanocytes of the hair follicle migrate towards the epidermis. On reaching the upper part of the hair follicle, they first become melanotic, then hyperplastic and thereafter migrate to the epidermis [11]. Nishimura et al. [9] in 2002, have thrown new light on repigmentation of epidermis by identifying a melanocyte stem cell niche in the lower permanent portion of hair follicles (Figs. 30.1 and 30.2). They further suggested that the human melanoblasts in the outer root sheath are the stem-cell source for melanocytes in the hair matrix and for epidermal melanocytes, responsible for repigmentation of hair and epidermis [9]. Loss of melanocyte stem cells can be observed preceding the loss of differentiated melanocytes in hair matrix. With aging, physiological graying of hair is due to loss of melanocyte stem cells [15]. It has been documented that hair graying is due to incomplete melanocyte maintenance and Pax3 and Mitf key molecules which help in regulating the balance between melanocyte stem-cell population and its differentiation [16].

Hypotheses of repigmentation of vitiligo and leukotrichia In vitiligo with leukotrichia there is a decline in the number of melanocytes and their activities [8]. There are basically three hypotheses of repigmentation: 1 Antegrade migration of amelanotic melanocytes (Staricco, 1962): This is similar to the mechanism of repigmentation in normal individuals. Under a stimulus like dermabrasion [11] and UV-ray exposure [17] the amelanotic melanocytes in the outer root sheath divide (Fig. 30.2). They

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increase in number and migrate gradually to become silver reactive and dopa positive and eventually become mature melanocytes and migrate to the epidermis. This, however, fails to explain the repigmentation of leukotrichia following surgery. This also does not explain the presence and proliferation of hypertrophic melanocytes in the lower and middle portions of the hair follicle. Hence the second postulate came into existence. 2 Antegrade migration from melanocyte reservoir in the hair bulb (Ortonne et al., 1980): The second postulate theorizes on the existence of a melanocyte reservoir in the region of the hair bulb, which gets activated under stimulus like PUVA therapy [18] (Figs. 30.1 and 30.2). Melanocytes dedifferentiate during the catagen and telogen phases of the hair cycle, then proliferate by mitosis, redifferentiate and populate the hair bulb at the onset of anagen phase. These melanocytes migrate to the epidermis in due course by antegrade movement [19]. But this theory fails to explain the presence of depigmented hair in vitiligo patches in the very presence of a melanocyte reservoir in the hair bulb itself. 3 Retrograde migration of melanocytes (Agrawal and Agrawal, 1995): During follow-up of surgically treated vitiligo in hair-bearing skin with leukotrichia, the repigmentation of white hair was observed a few weeks to months after the repigmentation of vitiligo [20–24]. Agrawal and Agrawal in 1995 postulated that the retrograde migration of the melanocytes from the repigmented epidermis is responsible for the repigmentation of the white hair [20]. After surgical repigmentation of vitiligo the epidermis has a high concentration of melanotic melanocytes. Only migration of these melanocytes from the area of high concentration in the epidermis to the hair follicle in the dermis, where the pigmented melanocytes are deficient, can explain the phenomenon of pigmentation of leukotrichia. This is, probably, the reason for the delayed onset of pigmentation in the shaft of white hair.

Surgical management Though leukotrichia causes a significant aesthetic concern, there are very few reports on its repigmentation: medical or surgical. Most of these articles

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report the observation of incidental repigmentation by the authors. With medical therapy the melanocytes from the hair follicles regenerate and migrate to the epidermis, resulting in the follicular pattern of repigmentation of vitiligo skin. In cases of vitiligo with leukotrichia, the melanocyte reservoirs, that is the hair bulbs, themselves are deficient in pigmented melanocytes and probably are not capable of generating pigmented melanocytes. This results in white hair, which is refractory to conservative therapy. That is why the presence of leukotrichia is a poor prognostic factor. Migration of melanocytes from epidermis to the hair follicle is the probable basis of surgical repigmentation of leukotrichia. The migration is expected only if there is a high density of melanocytes in the epidermis. Such high concentration of melanocytes can be achieved only by surgical techniques. Repigmentation of leukotrichia has been documented by few authors using following surgical techniques: 1 dermabrasion and thin split-thickness skin grafting [20]; 2 epidermal grafting and systemic PUVA [21]; 3 in vitro cultured epidermal grafting [22]; 4 punch grafting [23–24]; and 5 chemical epilation and epidermal grafting [25].

Dermabrasion and thin splitthickness skin grafting [18] This surgery should be performed in the hospital operating room under strict aseptic precautions. This procedure can be performed in children as well as in adults. It can be performed for lesions of any dimension. If the lesion is small, surgery can be performed under local analgesia in co-operative patients. If the lesion is large, general anesthesia may be required. Preoperative removal of hair is not necessary. Dermabrasion of the vitiligo area is done using wire brushes, sand paper cylinders or stainless steel burrs attached to a manual or mechanical dermabrader. The authors have used either of these with diamond tipped fraises of various sizes and shapes. For repigmentation of vitiligo by dermabrasion and thin split-thickness skin grafting, superficial dermabrasion is done to expose the vascular dermis.

However, in cases of vitiligo with leukotrichia a deeper dermabrasion is recommended by the authors. The dermabrasion should be uniform. To facilitate dermabrasion, good subcutaneous infiltration with a local anesthetic solution should be done. The infiltration solution of authors’ preference is normal saline with 0.25% lidocaine and 1:200,000 adrenaline. This provides good hemostasis. It also facilitates dermabrasion over the hard surface of the skull. Appearance of a fine punctate bleeding surface indicates the completion of the dermabrasion. The medial aspect of the arm is the preferred donor site for split-thickness skin graft. The area is infiltrated with 0.5% lidocaine with 1:200,000 adrenaline solution. After ascertaining good analgesia the thin split-thickness skin graft is harvested using an electrical dermatome or a manual skin grafting knife. The donor area is dressed with tulle grass and cotton padding. After ascertaining perfect hemostasis, the skin graft is spread over the recipient site, so that the graft overlaps the dermabraded edge. It is fixed to the surrounding skin using silk sutures. The graft is covered with tulle grass and a pressure dressing is applied. The dressing is changed on the 6th postoperative day. The overlapped skin graft is excised leaving a narrow rim of hypopigmented area around the skin graft. (For details of the procedure, see Chapters 13 and 14.) The authors do not use any adjuvant phototherapy in any of the patients. However, there is no contraindication for using it. This procedure has been performed over the eyebrow, scalp, beard, and moustache regions. No attempt was made to remove the hair preoperatively. The take of the graft has been excellent in all the patients. Graft tenting was not observed over the growing hair shaft in any patient. When the graft is very thin, the hair grows through the graft without hindering the graft survival over the dermabraded area (Figs. 30.3–30.6). Agrawal and Agrawal in 1995 presented a series of nine patients of vitiligo with leukotrichia with long-term follow-up results [20]. In their patients the repigmentation of vitiligo patch was seen during the first dressing on the 6th day. However, the

Surgical management of leukotrichia

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(A)

(B)

(C)

(D)

Fig. 30.3 (A) Preoperative photograph of a patient of segmental vitiligo having vitiligo with leukotrichia of medial segment of right eyebrow. (B) Two weeks postoperative photograph after dermabrasion and thin split-thickness split skin graft. (C) Two years postoperative photograph. (D) Eighteen years postoperative photograph.

repigmentation of hair was seen only after 2 months, which improved slowly over the years. After 2–3 years it becomes stationary. The onset of repigmentation of the hair was relatively late in scalp as compared to the eyebrow. Probably it is because of the distance the melanocytes have to travel from the basal layer of the epidermis to the hair follicle. If this assumption is true, a deeper dermabrasion in the scalp might advance the onset of repigmentation and may improve the final result [18]. There is no loss of pigmentation on long-term follow-up (Figs. 30.3–30.6). In the authors’ series the longest follow-up has been 18 years (Fig. 30.3D). This repigmentation of white hair seems to be permanent. The best result is achieved in the eyebrow, in which 80–90% of the hair becomes pigmented. On the scalp and over the beard and moustache the repigmentation is around 50% [20].

Epidermal grafting and systemic PUVA [19] Hann et al. in 1992 were the first to report the repigmentation of leukotrichia of the eyebrow in three patients [21]. They had used epidermal grafting and PUVA therapy. In their surgical technique white hair in the vitiligo site is shaved. Blisters are created using three freeze–thaw cycles with liquid nitrogen in 24 hours. Recipient areas can also be prepared by blistering with suction or CO2 laser abrasion. Suction blisters are also created over the medial aspect of the arm by using 200–300 mmHg suction for 1–2 hours. The roof of the blister is used as an epidermal graft. The roof of the blisters are removed at the recipient site and the epidermal grafts from the donor site are used as replacement grafts at the recipient site. A pressure dressing is applied [21]. (For details of the procedure, see Chapter 12.)

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(A)

(A)

(B)

(B)

(C) Fig. 30.4 (A) Preoperative picture of scalp vitiligo with leukotrichia in a patient of segmental vitiligo. (B) Ten days postoperative photograph after dermabrasion and thin split-thickness skin graft, showing good graft take in scalp. (C) Five years postoperative picture showing partial repigmentation of leukotrichia in scalp.

Postoperatively, after 7 days systemic PUVA therapy is given once a week. The repigmentation of leukotrichia is noticed after eight cycles of PUVA therapy [21]. Almost complete repigmentation of eyebrow leukotrichia was achieved in three patients.

(C) Fig. 30.5 (A) Preoperative photograph of right eyebrow vitiligo with leukotrichia. (B) Six months follow-up photograph showing partial repigmentation of eyebrow, patient still using eyebrow pencil for camouflage. (C) Five years follow-up photograph with good repigmentation of eyebrow white hair.

In vitro cultured epidermal grafting Falabella et al. in 1992 reported successful transfer of melanocytes in vitiligo patients using in vitro cultured epidermal graft bearing melanocytes [22].

Surgical management of leukotrichia

235

(A)

(B)

Fig. 30.6 (A) Preoperative photograph of vitiligo over right face. (B) Seven days postoperative photograph after dermabrasion and thin split-thickness skin graft, showing good graft take over hair-bearing area and growth of hair through the skin graft. (C) Ten years postoperative picture showing complete repigmentation of vitiligo but only partial repigmentation of leukotrichia in beard.

(C)

(For detailed procedure, see Chapter 23.) The authors have reported that in one of the nine patients with vitiligo over the leg, the white hair within the treated area regained normal pigmentation after several months of follow up after the in vitro cultured epidermal grafting [22].

Minigrafting Singh and Bajaj in 1995 noticed repigmentation of hair after skin minigrafting for vitiligo treatment in

hair-bearing areas but details of the patients were not mentioned [24]. Similarly Malakar and Dhar, in 1998, presented a preliminary report on repigmentation of leukotrichia after minigrafting for vitiligo. They noticed repigmentation of leukotrichia in three patients. The repigmentation of hair was noticed between 10 and 16 weeks [23]. (For details of the procedure, see Chapter 11.) If the hypothesis regarding the mechanism of repigmentation of leukotrichia is true, then all the methods of surgical repigmentation of vitiligo should

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result in at least partial repigmentation of leukotrichia on long-term follow-up.

Chemical epilation Some researchers expressed concern for the poor take of skin graft in hair-bearing areas [26,27]. Kim et al. in 2001 have suggested use of chemical epilator cream like salts of thioglycolic acid for 15–20 minutes, which dissolves the hair shaft. The effect of chemical epilation lasts up to 2 weeks, which is enough for successful take of the epidermal graft [25]. However Agrawal and Agrawal did not have such a problem in their series of cases (Figs. 30.3B, 30.5B, and 30.6B). No attempt was made to remove the hair preoperatively by them. During the process of mechanical dermabrasion the hair is automatically epilated.

Summary Leukotrichia in vitiligo makes the disease more obvious. Hence leukotrichia is an unacceptable element of vitiligo. Simultaneously white hair is a poor prognostic factor in the treatment of vitiligo indicating a more severe disease and such vitiligo requires surgical management. Surgical procedures involving deepithelialization using dermabrasion or liquid nitrogen and thin split-thickness skin graft have resulted in repigmentation of white hair. However, other surgical techniques used for repigmentation of vitiligo may also give the same result. The result is excellent over the eyebrow and less successful for treating leukotrichia of the scalp, beard and moustache. The repigmentation of hair is noticed much later than the repigmentation of the skin vitiligo. To notice the result of treatment one has to follow up these patients for a very long time. It has been proven that the repigmentation of skin and the hair is permanent on long-term follow-up of over 15 years. The surgical repigmentation of leukotrichia proves the point that melanocytes migrate from the skin epidermis to the hair follicles. A lot more work is needed in this field before one can predict repigmentation of leukotrichia surgically. Also the underlying mechanism of repigmentation and the given hypotheses need to be substantiated with elaborate animal and human studies using electron microscopic and biochemical techniques. Since the white

hair is a sign of ageing active management must be considered for all leukotrichia as for vitiligo.

Acknowledgment The authors sincerely thank the artists at Medical Illustration Division, JIPMER, Pondicherry for preparing the line diagrams.

References 1 Lerner AB. On etiology of vitiligo and gray hair. Am J Med 1971;51:141–7. 2 Selmanowitz VJ. Pigmentary correction of piebaldism by autografts. II Pathomechanism and pigment spread in piebaldism. Cutis 1979;24:66–73. 3 Lerner AB, Nordlund JJ. Vitiligo: the loss of pigment in skin, hair and eyes. J Dermatol 1978;5:1–8. 4 Handa S, Kaur I. Vitiligo: clinical findings in 1436 patients. J Dermatol 1999;26:653–7. 5 Jaigirdar MQ, Alam SM, Maidul AZ. Clinical presentation of vitiligo. Mymensingh Med J 2002;11:79–81. 6 Handa S, Dogra S. Epidemiology of childhood vitiligo: a study of 625 patients from north India. Pediatr Dermatol 2003;20:207–10. 7 Dogra S, Parsad D, Handa S, Kanwar AJ. Late onset vitiligo: a study of 182 patients. Int J Dermatol 2005; 44:193–6. 8 Junqueira LC, Carneiro J. Basic Histology – Text and Atlas, 10th edn. New York: Lange Medical Books, McGraw Hill, 2003;377–9. 9 Nishimura EK, Jordan SA, Oshima O, et al. Dominant role of the niche in melanocyte stem-cell fate determination. Nature 2002;416:854–60. 10 Kunisada T, Lu SZ, Yoshida H, et al. Murine cutaneous mastocytosis and epidermal melanocytosis induced by keratinocyte expression of transgenic stem cell factor. J Exp Med 1998;187:1566–73. 11 Staricco RG. Amelanotic melanocytes in the outer sheath of the human hair follicle and their role in the repigmentation of regenerated epidermis. Ann NY Acad Sci 1963;100:239–55. 12 Ortonne JP, Benedetto JP. Melanocytes epidermiques et melanocyte follicularies. Ann Genet Sel Anim 1981; 13:17–26. 13 Grinspan RC, Fairman J. Effect of vibrapuncture into areas of vitiligo. J Invest Dermatol 1956;26:243. 14 Pegum JS. Dissociated depigmentation in vitiligo. Significance and therapeutic implications. Br J Dermatol 1955;67:348–50.

Surgical management of leukotrichia 15 Staricco RG, Miller-Milinska A. Activation of the amelanotic melanocyte in the outer root sheath of the hair follicle following ultraviolet rays exposure. J Invest Dermatol 1962;39:163–4. 16 Ortonne JP, Schmitt D, Thivolet J. PUVA-induced repigmentation of vitiligo: scanning electron microscopy of hair follicles. J Invest Dermatol 1980;74:40–2. 17 Sugiyama S, Kukita A. Melanocyte reservoir in the hair follicles during hair growth cycle: an electron microscopic study. In: Kobori T and Montagna W (eds.) Biology and Disease of the Hair. Baltimore: University Park Press, 1976;181–200. 18 Agrawal K, Agrawal A. Vitiligo: surgical repigmentation of leukotrichia. Dermatol Surg 1995;21:711–5. 19 Hann SK, Im S, Park YK, Hur W. Repigmantation of leukotrichia by epidermal grafting and systemic psoralen plus UV-A.[Letter] Arch Dermatol 1992;128: 998–9. 20 Falabella R, Escobar C, Borrero I. Treatment of refractory and stable vitiligo by transplantation of in vitro cultured epidermal autografts bearing melanocytes. J Am Acad Dermatol 1992;26:230–6.

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21 Malakar S, Dhar S. Repigmentation of leukotrichia over vitiligo patches after punch grafting. [Letter] Ind J Dermatol Venereol Leprol 1998;64:252–3. 22 Singh KG, Bajaj AK. Autologous miniature skin punch grafting in vitiligo. Ind J Dermatol Venereol Leprol 1995; 61:77–80. 23 Nishimura EK, Granter SR, Fisher DE. Mechanisms of hair graying: incomplete melanocyte stem cell maintenance in the niche. Science 2005;307:720–4. 24 Lang D, Lu MM, Huang, L, Engleka KA, Zhang M, Chu EY, Lipner S, Skoultchi A, Millar SE, Epstein JA. Pax3 functions at a nodal point in melanocyte stem cell differentiation. Nature 2005;433:884–7. 25 Kim CY, Yoon TJ, Kim TH. Epidermal grafting after chemical epilation in the treatment of vitiligo. Dermatol Surg 2001;27:855–6. 26 Behl PN. Treatment of vitiligo with homologous thin Thiersch’s skin grafts. Curr Med Pract 1964;8:218–21. 27 Suvanprakorn P, Dee-Ananlap S, Pongsomboon C, Klaus SN. Melanocytes autologous grafting for treatment of leukoderma. J Am Acad Dermatol 1985; 13:968–74.

CHAPTER 31

Surgical treatments of leukodermas other than vitiligo vulgaris Mats J. Olsson

Introduction Vitiligo vulgaris is by far the most common leukoderma but not always the most suitable type of leukoderma in surgical treatment approaches. This is due to the complexity in the etiology and the instability in the disease caused by involvement of underlying immunological factors destroying the melanocytes. Vitiligo must be absolutely stable or in regression before considering any surgical treatment alternatives. On the other hand there are patients with several other types of leukoderma with an absolute stability in the condition who do not respond to pharmacological alternatives or ultraviolet (UV) treatments but are excellent candidates for surgical therapies, with an almost 100% success rate in transplant outcome. But there are also leukodermas not at all suitable for undergoing surgical approaches. If a diagnosis is not 100% clear, skin infections must be ruled out by means of Wood’s light examination, microscopy, and/or microbiological culture. I will list some of the most frequently seen leukodermas and discuss if surgical management in each of these conditions is appropriate and likely to be effective or not. What surgical method is the most suitable in each and every one of these conditions can be a question of the histological pattern of the skin in the affected lesions but is more often only dependent on the size and anatomical location of the affected lesions. The clinics’ total experience of various methods and access to technically advanced equipment will of course limit the number of methods to choose from for an individual hospital.

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Type of leukoderma and surgical method Segmental vitiligo About 5–16% of the total vitiligo population is reported to have the segmental type [1–3], which appears rather quickly, within a few months, as a unilateral area, not extending beyond the midsagittal line of the body. It follows the whole or parts of a region innervated by one or neighboring sensory nerve segments. The trigeminus region seems to be the most common location [4]. In 75–87% of the patients only a single coherent lesion is seen [4,5]. The mean age at onset is reported to be between 16 and 24 years by different groups [4,6,7]. After 6–12 months the area is stable and does not progress later in life. The follicular melanocytes are also most often lost and the hair in the lesion therefore white. Spontaneous repigmentation is uncommon. It is important not to mistake segmental vitiligo for the more common vitiligo vulgaris. Segmental vitiligo appears not to have any autoimmune involvement and should therefore not to be treated with immunosuppressive or immunomodulating therapies used in vitiligo vulgaris. Segmental vitiligo is a stable disorder that after its full appearance (growth arrest) remains unchanged. When we are sure that the white lesion has not extended in size for at least 8 months, the affected area can successfully be treated with autologous melanocyte transplantation. What method is chosen depends more on the location and size of the lesion than the expected take. Segmental vitiligo most often responds with an almost 100% repigmentation success rate and since normally no new white lesions

Surgical treatments of leukodermas other than vitiligo vulgaris are to be expected, the patient can in practice consider him/herself as cured. Direct shaves should preferably be avoided in facial lesions due to the risk of visible hyperpigmentation, noticeable square form angles and sharp borders. Transplantations of free cells applied as a suspension give less probability of hyperpigmentation and a smoother connection to the surrounding normally pigmented skin. See Section 4 for cellular grafting methods.

Piebaldism Sometimes piebaldism is wrongly called partial albinism or vitiligo. This should absolutely be avoided since the conditions are not related and the piebald patient can by mistake be put on pharmacological treatment or UV therapy which has no effect in this condition. Piebaldism is inherited as autosomal dominant, congenital, stable leukoderma affecting about 1:14,000, equally distributed among men and women, and seen in all races. The most common mutations are situated in the c-kit gene (located in 4q12), encoding the transmembrane SCF/MGF tyrosine kinase receptor [8–13] and resulting in that a subset of neural crest derived cells (melanoblasts) getting committed somewhere on their migration from the dorsal surface of the neural tube to the skin. The typical piebald maculae are stable, milkywhite areas located in the center of the forehead with a characteristic white forelock, ventral part of the trunk, and symmetrically bilateral on the midparts of the legs and arms. The forehead lesion of white skin and white forelock exists in about 90% of patients and is usually symmetrically central and forms an elongated triangular or diamond shape. Unlike vitiligo, the hands, feet, and hips are often spared. Hyperpigmented circular macules on the border, both inside the white lesions and on the normally pigmented surrounding skin are common. These macules are usually darker than the normal skin and not uniformly pigmented. As there is neither any release of cytotoxic factors in the white areas nor any melanocyte autoimmune compounds involved, only a lack of epidermal melanocytes due to a migration failure during the embryogenesis, a transplantation of autologous melanocytes to these areas of histologically normal

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skin (except for the lack of melanocytes) almost always gives an outcome of nearly 100% repigmentation success rate. Since piebaldism is a stable condition and no new white lesions will appear in the future, the patient can in practice consider his/her skin condition as cured, though the hyperpigmented macules on the borders will remain. What method is chosen depends more on the location and size of the lesions than the expected take. Direct shaves should preferably be avoided in the forehead lesion due to the risk of visible hyperpigmentation and noticeable square form angles and sharp borders. Transplantations of free cells applied as a suspension give less risk of hyperpigmentation and a smoother connection to the surrounding normally pigmented skin. Many piebald patients have extensive areas covering large sections of their arms, legs, and ventral trunk. In these cases of extensive areas culturing methods might be the only realistic approach to choose. See Section 4 for cellular grafting methods.

Waardenburg’s syndrome Waardenburg’s syndrome is a quite rare autosomal dominant disorder affecting about 1:42,000 of the general population. The disease is of neural crest abnormality, disturbing the migration capacity and settlement of melanoblasts during the embryogenesis. The manifestations and expressivity varies between individuals, affecting more locations and functions than the skin. The syndrome involves defects of various neural crest cell lineages including the melanocytes populating the skin, but also involving many other tissues derived from the neural crest [14]. There are four subgroups of Waardenburg’s syndrome classified after distinguishing features. Later the genes involved in each of these groups have been mapped. PAX3, MITF, EDN3, or SOX10 seem to be the genes most often defected [15–17]. The dermatological presentation is characterized by piebaldism-like lesions of achromic patches with well-defined irregular borders scattered with hyperpigmented islands and pigmentary abnormalities of hair including a classical white forelock is also not uncommon. The lesions are stable in size and distribution, and histochemical studies show an absence of

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melanocytes in the epidermis as well as in the hair follicles of the affected areas. This makes it unlikely that the lesions will respond to pharmacological treatments or UV therapy. Transfer of autologous melanocytes from pigmented unaffected area to affected white area seems to be the only solution. Since the condition is absolutely stable and there are no other abnormalities in the skin except the lack of melanocytes a transplantation of autologous melanocytes has a good outcome prognosis. For suggestions of methods see the paragraph about piebaldism above.

Post-burn leukoderma Post-burn leukoderma can be a result of fire but also be caused by direct applied heat or from laser or light pulse treatments. For leukoderma caused by light and laser see the paragraph below. Post-burn leukoderma has successfully been treated by several groups. If the lesion is limited the quickest approach might be direct application of split skin autograft [18]. De-epithelialization of the leukodermic recipient area has been performed by means of dermabrasion [19,20], and CO2 laser ablation [21]. Both methods showed a successful outcome. What method of de-epithelialization to choose, also including ultrasonic abrasion and chemical peeling, depends on the size of the area to be treated, the level of dermal scar tissue and the transplantation method decided to be used. If larger areas are to be treated cultured autologous cells can be applied. These cells can be transplanted as free cells, epithelial melanocyte containing sheets or as co-cultures of melanocytes and keratinocytes bound to a chemically defined carrier surface. See Section 4 for transplantation methods.

Leukoderma caused by laser Lasers have been used both in the treatment of melasma and for complete depigmentation in extensive vitiligo [22]. We have had a few patients referred to us who have lost their pigmentation after hair-removal therapy by light- or laser-treatments. One patient had got leukoderma on the arms after laser bleaching of solar lentigines. In one male patient a chessboard-like pattern on his back could be seen, indicating exactly

where the device had been placed when the light pulses were delivered to remove the hair. Melanin absorbs light over a wide spectral range and the energy in the light beams are converted into heat and liberated in the melanocytes, harming them [23]. Physical harm to the skin is known to set of Köbner reaction in patients with vitiligo vulgaris [24] and such patients most likely have an increased risk in developing white lesions when undergoing laser peeling or hair removal. Depigmentations following laser treatments are believed to increase in the coming years due to the increased popularity of tattoos and the new possibility to remove them by means of various laser treatments. Leukoderma caused by laser- or light-pulsetreatment can successfully be treated with transplantation of autologous melanocytes. But my personal opinion is that one should first wait at least 11⁄2 years after the loss of pigmentation. This is to give the skin a chance to spontaneously repigment. If no spontaneously repigmentation is seen after 11⁄2 years surgical intervention can be planned.

Chemical leukoderma (contact leukoderma) Chemicals containing aromatic structures like phenol- and hydroquinone-derivatives used in industry, cosmetics, or for therapeutic purposes are known to cause pigment cell destruction (Fig. 31.1). They have been used in treating hypermelanosis [25] as well as depigmenting agents in extensive vitiligo [22,26]. Reports exist about dark-skinned individuals getting blotchy leukoderma when they have used antiseptics (germicidal soap) or skinbleaching products containing phenol- or hydroquinone-derivatives [27,28]. This can be the reason for the high incidence of vitiligo (6%) reported in Ibadan, Nigeria during the period 1980–3 [28]. The investigator speculated that it might have a connection to the widely use of antiseptics containing phenol derivatives and hydroquinone creams used to lighten the skin tone. An extensive list of products used for bleaching the skin in the pigmented population in different countries has been put together by Scarpa and Guerci [29].

Surgical treatments of leukodermas other than vitiligo vulgaris

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OH OH

OH

OH

HO Phenol

Catechol

Hydroquinone OH

OH OH

OH CH2

H3C H3C

H3C CH3

p-tertiary butylphenol

H3C

O

CH3

p-tertiary butylcatechol

Monobenzylether of hydroquinone

Fig. 31.1 The chemical structures of these phenol derivatives illustrate some of the substances which are cytotoxic to

melanocytes. Some of them have been used in chemical bleaching of the skin. Notice the structural resemblance to the amino acid tyrosine, the basic substrate in the melanogenesis. Some phenol derivatives have the ability to bind into the active site at the melanogenitic enzyme tyrosinase, producing free radicals instead of melanin.

We have had three patients with chemical leukoderma referred to us from different cosmetic clinics, as a result of treatment for facial wrinkles. Two of the patients had undergone phenol peeling and one had trichloroacetic acid (TCA) peeling. One of the phenol-peeled patients and the TCA-peeled patients have been successfully transplanted with autologous melanocytes. But as in the case of leukoderma caused by laser treatment, I also here strongly recommend that one should first wait at least 11⁄2 years to give the skin a chance to repigment spontaneously before surgical intervention is planned.

Focal vitiligo Focal vitiligo is a term used for a phenotype of only one or a few white maculae localized in one isolated anatomical region, without clear segmental or zosteriform distribution. The pathogenesis and specific genotype in this form of vitiligo are still unclear. But new expression microarray techniques will soon group this condition and tell about a possible relationship with other groups of leukoderma. The number of patients treated with autologous melanocyte transplantations is still limited but

focal vitiligo appears to respond well to surgical treatments with an outcome success of almost 100% repigmentation.

Halo nevi Halo nevi occur in approximately 1% of the population [30]. They are found in all races and are most common in children and young adults [31]. In an examination of 1436 patients with vitiligo vulgaris, done in India, it was shown that 2% of the patients had associated halo nevi [1]. The typical halo nevus consists of a pigmented nevus surrounded by a circular, sharply outlined area of complete depigmentation. The depigmentation starts in the border of the nevi and progresses centrifugally, giving rise to approximately 2–4-cm diameter macule. The halo nevi can be one to a few in numbers and occur predominantly on the trunk. The central nevi usually regress leaving behind the white area that sometimes spontaneously repigments, with no remaining surface signs of previous nevi. When dermabrading the white area where there are no surface signs of any nevus left, we have observed scar tissue-like dermis in the center of the lesion.

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In histology, a dense lymphocytic infiltration with a relatively high proportion of CD8
cells has been observed by several groups and the density of the infiltrate changes as regression of the nevi evolves [32], with only a few or no inflammatory cells in fully regressed lesions [33]. Since there are immunological factors involved in the destruction of the pigment cells, the progression of the depigmentation must have stopped and been absolutely stable or on regression during the last year prior to a transplant treatment. A stable phase can sometime be reached earlier if the central pigment nevus, causing the autoimmune action, is removed. This can easily be done by the means of scooping (concave) the nevus out as a shave biopsy with a curved razor blade (bent between the index finger and the thumb) or excised. Removed tissue should always be sent for histological evaluation to exclude melanoma. Since these white areas are quite limited in size it is normally not necessary to use free cell suspensions or cultured cells. Autotransplants of superficial shaves or suction blister roofs transplanted to a dermabraded or laserabladed recipient site is usually sufficient.

Nevus depigmentosus Nevus depigmentosus is a congenital non-progressive hypopigmented lesion that is stable in size and distribution throughout life, and there exists no pharmacological treatment for this disorder. The pathogenesis of nevus depigmentosus is not yet fully understood. In histopathological characterization of nevus depigmentosus no alteration in the numbers of basal layer situated melanocytes identified as S-100-positive cells in the hypopigmented lesions could be established when compared with unaffected skin [34]. Ultrastructural evaluation with electron microscope on the other hand revealed a great reduction in the number of melanosomes in the melanocytes [34]. The histological and cytological characterization support the hypothesis that nevus depigmentosus is caused by a functional defect of the melanocytes. But until the fully etiology in this condition is cleared up we have to bear in mind that melanocytes can be regulated by their local environment (tissue) and

therefore the causative factor might not necessarily be in the genes of the local melanocytes themselves. This could be cleared up by culturing and comparing isolated pure melanocytes from lesion and nonlesion skin in vivo. The isolated melanocytes could be morphologically and cytologically evaluated and the mRNA expression pattern could be compared between the lesional and non-lesional melanocytes. If the causative defect is only within the local melanocytes these can easily be removed and replaced by unaffected autologous ones through a transplantation procedure. The reports of surgical treatments of nevus depigmentosus are limited to four publications and the total number of patients treated is only four [35–38]. The outcome and conclusions from these different reports are not in concordance and the statistical basis for drawing any kind of conclusion from these reports is far too small. The transplantation techniques used are not exactly the same and there might be several subgroups of nevus depigmentosus appearing similar on the surface of the skin, but with a diversified etiology. Different types might also be aggregated in different parts of the world. In other words we need to know more about the cause and have much larger statistical basis from different surgical reports before we can estimate the success rate in this condition or suggest the most appropriate surgical method.

Nevus anemicus Nevus anemicus is a congenital localized anomaly most commonly seen as a pale-colored patch on the trunk. Vascular malformations result from anomalies of embryological development, and in some of them the alteration of the involved vessels are more functional than anatomic (pharmacological nevi), as is the case of nevus anemicus [39]. Pharmacological response to certain mediators may be aberrant, with sympathetic vasoconstriction likely responsible for the pallor [40]. Several studies have shown that nevus anemicus is caused by a localized vascular hypersensitivity to catecholamines. This catecholamine sensitivity produces increased vasoconstriction and skin pallor. There are also studies showing a local hypersecretion

Surgical treatments of leukodermas other than vitiligo vulgaris of catecholamines in the lesions [41]. When diascopy is performed on the border of the patch, the differences between lesion and normal skin almost disappear. This is because the normal skin becomes blanched when the blood supply is hindered. Results of an exchange transplant study demonstrated donor dominance, suggesting that the defect in the nevus anemicus is attributable to increased sensitivity of the blood vessels to catecholamines [42]. But there are also signs of anatomical vascular malformations. Dr. Juhlin and I have earlier described a 20-yearold man with nevus anemicus on the chest where, after dermabrasion of the epidermis, enlarged telangiectatic dark-red vessels were seen within the previously pale area. They were clearly different from those seen on dermabrasion at this site in normal skin and in patients with vitiligo where the area is lighter red with only small punctual bleedings from arterial capillaries. The nevus anemicus and a port-wine stain (nevus flammeus) in the same location is a phenomenon of vascular twin spotting, which was revealed when the epidermis was removed [43]. The pale area was transplanted with an ultra-thin autologous shave. The area healed without any signs of scarring and the pale area remained [43]. That vascular twin nevi, that is telangiectatic nevus and nevus anemicus are occurring together and adjacent to each other, might be explained as twin spots resulting from a somatic recombination [44]. It is shown that nevus flammeus (port-wine stain) and nevus anemicus are associated and that the prevalence for nevus anemicus in persons with port-wine is much higher than in the average population [45,46]. To summarize this we can say that superficial transplantation methods involving only epidermal cells or tissue will not correct the pale lesions. Deeper transplants involving the whole or main parts of the skin are not recommended due to uncertain outcome and risks of scarring.

Discoid lupus erythematosus Discoid lupus erythematosus (DLE) may heal with thin, depigmented scars. Also systemic lupus erythematosus (SLE) can result in cutaneous depigmentations.

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The clinical picture of DLE is nuanced and the etiology of is not fully understood, but autoimmunity seems to play a major role in the pathogenesis. In histological sections of the lesions a degeneration of the basal layer, atrophy of the epidermis, and a lymphocytic infiltration are a few of the structural characteristics. Long-standing depigmented lesions of DLE may fail to respond to medical repigmentation attempts but seem to respond quite well to surgical treatments. Both transplantation of hair bulbs [47], minigrafting [48], and suction blisters [49] have showed good repigmentation in this condition. It is then thinkable that even other transplantation methods described in this book would be effective in DLE, provided that the lesions, with the exception of the hypopigmentation, have healed.

Albinism Albinism is an inherited most often autosomal recessive hypopigmentary disorder caused by mutation in genes involved in the production of melanin (pigment). This results in little or no pigment in the affected individual’s skin, hair, and eyes, with associated visual impairment and extreme sun sensitivity. It affects both genders in people from all ethnic groups. As in albinism when both the pigmentation of the skin and the eyes are affected it is usually referred to as oculocutaneous albinism (OCA) which as a whole group is a complex genetic disease with great clinical heterogeneity. The OCA is commonly subgrouped into four different types (OCA1, OCA2, OCA3, and OCA4). The gene affected can be the tyrosinase as is the case in OCA1, that is tyrosinase negative albinism, but OCA can also be caused by a number of other genes also including the genes involved in Hermansky–Pudlak syndrome which is a less common form of albinism. Accordingly there are different types of albinism, depending on what gene is mutated and the site of the mutation. The result is that the amount of pigment in the skin, eyes, and hair varies between different affected individuals. Some individuals with albinism can have reddish or violet eyes, due to lack of pigmentation in the iris making the iridial blood vessels visible.

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It is difficult to tell the prevalence for albinism as a group, because of the heterogeneity in the genes involved and the various prevalence between different populations. For example the tyrosinase-positive albinism (OCA2) affecting the P locus mapped to the human chromosome 15q11.2–12 has a prevalence of about 1:36,000 among European-Americans in the United States but it has a moderate to relatively high prevalence values (1:28 to 1:6500) in various Amerindian populations [50]. The same OCA2 is especially frequent where it occurs with an incidence of approximately 1:1400 in Tanzania [51], and the prevalence among schoolchildren in Zimbabwe (Harare), has been found to be 1:2833 [52]. Albinism can be classified as a functional leukoderma; that is, the number of melanocytes in the skin is about the same as in unaffected individuals but the production of melanin is altered because of an inherited mutation in one of the genes involved in the production of melanin. In other words, albino patients are not candidates for undergoing any kind of surgical transplantation of melanocytes. This is because all melanocytes are affected by the mutation and there is nowhere to harvest unaffected ones. Allogenous melanocyte transplantation is not recommended due to the great risk of rejection and the risk of transferring an infection from the donor to the recipient. In future, gene therapy or pharmacological therapy involving insertion or activation of genes involved in melanogenesis might be a treatment to offer, but until then we can unfortunately only recommend sunscreen lotions with a minimum of SPF 15 or protective clothing so people with albinism at least can enjoy outdoor activities even in summertime.

References 1 Handa S, Kaur I. Vitiligo: clinical findings in 1436 patients. J Dermatol 1999;26:653–7. 2 Park KC, Youn JI, Lee YS. Clinical study of 326 cases of vitiligo. Korean J Dermatol 1988;26:200–5. 3 Song MS, Hann SK, Ahn PS, et al. Clinical study of vitiligo: comparative study of type A and B vitiligo. Ann Dermatol 1994;6:22–30. 4 Hann SK, Lee HJ. Segmental vitiligo: clinical findings in 208 patients. J Am Acad Dermatol 1996;35:671–4.

5 Lerner AB. Vitiligo. J Invest Dermatol 1959;32:285–310. 6 Olsson MJ, Juhlin L. Long-term follow-up of leucoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol 2002; 147:893–904. 7 Seghal VN. A clinical evaluation of 202 cases of vitiligo. Cutis 1974;14:439–45. 8 Boissy RE, Nordlund JJ. Molecular basis of congenital hypopigmentary disorders in humans: a review. Pigm Cell Res 1997;10:12–24. 9 Ezoe K, Holmes SA, Ho L, et al. Novel mutations and deletions of the KIT (steel factor receptor) gene in human piebaldism. Am J Hum Genet 1995;56:58–66. 10 Fleischman RA, Gallardo T, Mi X. Mutations in the ligand-binding domain of the kit receptor: an uncommon site in human piebaldism. J Invest Dermatol 1996; 107:703–6. 11 Giebel LB, Spritz RA. Mutation of the KIT (mast/ stem cell growth factor receptor) protooncogene in human piebaldism. Proc Natl Acad Sci USA 1991;88: 8696–9. 12 Richards KA, Fukai K, Oiso N, et al. A novel KIT mutation results in piebaldism with progressive depigmentation. J Am Acad Dermatol 2001;44:288–92. 13 Spritz RA, Giebel LB, Holmes SA. Dominant negative and loss of function mutations of the c-kit (mast/stem cell growth factor receptor) proto-oncogene in human piebaldism. Am J Hum Genet 1992;50:261–9. 14 Dourmishev AL, Dourmishev LA, Schwartz RA, et al. Waardenburg syndrome. Int J Dermatol 1999;38:656–63. 15 Potterf SB, Furumura M, Dunn KJ, et al. Transcription factor hierarchy in Waardenburg syndrome: regulation of MITF expression by SOX10 and PAX3. Hum Genet 2000;107:1–6. 16 Read AP, Newton VE. Waardenburg syndrome. J Med Genet 1997;34:656–65. 17 Tomita Y, Suzuki T. Genetics of pigmentary disorders. Am J Med Genet C 2004;131C:75–81. 18 Taki T, Kozuka S, Izawa Y, et al. Surgical treatment of skin depigmentation caused by burn injuries. J Dermatol Surg Oncol 1985;11:1218–21. 19 Kahn AM, Cohen MJ. Treatment for depigmentation following burn injuries. Burns 1996;22:552–4. 20 Kahn AM, Cohen MJ, Kaplan L. Treatment for depigmentation resulting from burn injuries. J Burn Care Rehabil 1991;12:468–73. 21 Acikel C, Ulkur E, Guler MM. Treatment of burn scar depigmentation by carbon dioxide laser-assisted dermabrasion and thin skin grafting. Plast Reconstr Surg 2000;105:1973–8.

Surgical treatments of leukodermas other than vitiligo vulgaris 22 Njoo MD, Vodegel RM, Westerhof W. Depigmentation therapy in vitiligo universalis with topical 4-methoxyphenol and the Q-switched ruby laser. J Am Acad Dermatol 2000;42:760–9. 23 Nanni CA, Alster TS. Laser-assisted hair removal: side effects of Q-switched Nd:YAG, long-pulsed ruby, and alexandrite lasers. J Am Acad Dermatol 1999;41:165–71. 24 Gauthier Y. The importance of Köebner’s phenomenon in the induction of vitiligo vulgaris lesions. Eur J Dermatol 1995;5:704–8. 25 Fitzpatrick TB, Arndt KA, el-Mofty AM, et al. Hydroquinone and psoralens in the therapy of hypermelanosis and vitiligo. Arch Dermatol 1966;93:589–600. 26 Mosher DB, Parrish JA, Fitzpatrick TB. Monobenzylether of hydroquinone. A retrospective study of treatment of 18 vitiligo patients and a review of the literature. Br J Dermatol 1977;97:669–79. 27 Dogliotti M, Caro I, Hartdegen RG, et al. Leucomelanoderma in blacks. A recent epidemic. S Afr Med J 1974;48:1555–8. 28 George AO. Vitiligo in Ibadan, Nigeria. Incidence, presentation, and problems in management. Int J Dermatol 1989;28:385–7. 29 Scarpa A, Guerci A. Depigmenting procedures and drug employed by melanoderm populations. J Ethnopharmacol 1987;19:17–66. 30 Ortonne JP, Mosher DB, Fitzpatrick TB. Leukoderma Acquisitum Centrifugum: Halo Nevus and Other Hypomelanoses Associated with Neoplasm, New York and London: Plenum, 1983;567–611. 31 Wayte DM, Helwig EB. Halo nevi. Cancer 1968;22:69–90. 32 Akasu R, From L, Kahn HJ. Characterization of the mononuclear infiltrate involved in regression of halo nevi. J Cutan Pathol 1994;21:302–11. 33 Jacobs JB, Edelstein LM, Snyder LM, et al. Ultra structural evidence for destruction in the halo nevus. Cancer Res 1975;35:352–7. 34 Lee HS, Chun YS, Hann SK. Nevus depigmentosus: clinical features and histopathologic characteristics in 67 patients. J Am Acad Dermatol 1999;40:21–6. 35 Gauthier Y, Surleve-Bazeille JE. Autologous grafting with noncultured melanocytes: a simplified method for treatment of depigmented lesions. J Am Acad Dermatol 1992;26:191–4. 36 Olsson MJ, Juhlin L. Leucoderma treated by transplantation of a basal cell layer enriched suspension. Br J Dermatol 1998;138:644–8.

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37 Ravikumar B, Sabitha L, Balachandran C. Naevus depigmentosus treated with suction blister grafting. Indian J Dermatol Venereol Leprol 2000;66:89–90. 38 Gupta S. Letter to the editor: nevus depigmentosus needs transplant of epidermal sheets. Dermatol Surg 2005;31:1746–7. 39 Requena L, Sangueza OP. Cutaneous vascular anomalies. Part I. Hamartomas, malformations, and dilation of preexisting vessels. J Am Acad Dermatol 1997; 37:523–49; quiz 49–52. 40 Ahkami RN, Schwartz RA. Nevus anemicus. Dermatology 1999;198:327–9. 41 Dupre A, Bonafe JL, Jouas H. Acquired generalized anemic nevus. Dermatologica 1981;163:276–81. 42 Daniel RH, Hubler WR, Wolf JE, et al. Nevus anemicus. Donor-dominant defect. Arch Dermatol 1977;113:53–6. 43 Juhlin L, Olsson MJ. Naevus anaemicus with teleangiectatic vessels. Eur J Dermatol 2001;11:518–20. 44 Happle R. Allelic somatic mutations may explain vascular twin nevi. Hum Genet 1991;86:321–2. 45 Katugampola GA, Lanigan SW. The clinical spectrum of naevus anaemicus and its association with port wine stains: report of 15 cases and a review of the literature. Br J Dermatol 1996;134:292–5. 46 Mills CM, Lanigan SW, Hughes J, et al. Demographic study of port wine stain patients attending a laser clinic: family history, prevalence of naevus anaemicus and results of prior treatment. Clin Exp Dermatol 1997;22:166–8. 47 Lobuono P, Shatin H. Transplantation of hair bulbs and melanocytes into leukodermic scars. J Dermatol Surg 1976;2:53–5. 48 Falabella R. Repigmentation of stable leukoderma by autologous minigrafting. J Dermatol Surg Oncol 1986;12:172–9. 49 Gupta S. Epidermal grafting for depigmentation due to discoid lupus erythematosus. Dermatology 2001;202:320–3. 50 Woolf CM. Albinism (OCA2) in Amerindians. Am J Phys Anthropol 2005;(Suppl 41):118–40. 51 Spritz RA, Fukai K, Holmes SA, et al. Frequent intragenic deletion of the P gene in Tanzanian patients with type II oculocutaneous albinism (OCA2). Am J Hum Genet 1995;56:1320–3. 52 Kagore F, Lund PM. Oculocutaneous albinism among schoolchildren in Harare, Zimbabwe. J Med Genet 1995;32:859–61.

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SECTION 6

Miscellaneous

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CHAPTER 32

Micropigmentation Gurvinder P. Thami

Introduction The word “tattoo” was brought to us by Captain Cook (1796) who wrote of the Polynesian practice of inlaying black pigments under the skin, known popularly as “Tattow” in their native language [1,2]. The art of tattooing is a process of uniform implantation of minute, metabolically inert pigment granules into the dermis using manual or electrically driven needles to create artistic impressions and designs on normal skin. Tattooing has been practiced for over 4000 years as evidenced by presence of tattoos on the Egyptian mummies. Although traditionally associated with sailors and convicts, artistic tattooing is quite in fashion in today’s world. In fact, when seen in teenagers, tattooing has a strong association with high-risk behaviors like early sexual intercourse, substance abuse, interpersonal violence, school failures, etc., and thus may serve as an easily detectable permanent visual marker for an adolescent engaging in high-risk activity [3]. Among Indians, tattoos were put for ornamental purpose by roadside tattoo artists using soot collected from a small wicklamps using a bunch of sewing needles. In older times, it was believed to be a curse for a lady to die without this poor people’s ornament, which was probably the only one to remain on her dead body. The term “micropigmentation” by convention conveys the aesthetic use of tattooing for the medical purposes. Micropigmentation is, nowadays, being used commonly to esthetically camouflage various skin afflictions of cosmetic importance like vitiligo, burn scars, alopecia areata, nipple–areola reconstruction, etc. both in the dermatology and plastic surgery [1–3]. The term “dermatography” was introduced further to define Japanese tattooing techniques for medical indications using a modified apparatus

(derma injector) consisting of an electromechanical motor and a needle holder which is moved up and down in a stainless steel tube [4].

Basic principles The pigmentary substances used in the process of micropigmentation usually have a minute particle size of about 6
m; they are inert, non-toxic, nonirritating, non-allergenic, light, and tissue stable chemicals largely free from immunological destruction. Implanted intradermally between superficial and mid-dermis, these pigment particles get permanently fixed both intracellularly as well as extracellularly, within dermal mononuclear cells and collagen fibers, respectively [2,5]. Over the years, a small amount of this pigment may migrate to the regional lymph nodes with resultant fading. It has been observed on histopathology that the initial pigment, which is largely intracellular, gradually becomes extracellular and lies amongst collagen bundles, blood vessels, and hair follicles without causing foreign body inflammation in the majority of cases [5].

Pigments and preparations Iron oxide is the most common chemical substance used in micropigmentation. Various other chemical substances used for attaining different shades and colors of micropigmentation are as follows [2]: 1 titanium dioxide: white 2 cinnabar, mercuric sulfate: red 3 iron oxide: black 4 cadmium sulfate: yellow 5 iron oxide: camel yellow 6 iron oxide: light brown 7 iron oxide: dark brown.

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Pigment preparation is done by mixing various pigment powders in order to achieve the desired shade. A pigment paste is formulated using 1–2 drops of a wetting agent like normal saline or 80% alcohol or water. Colloidal suspension can also be used or alternatively a drop of glycerin may be added to paste to attain proper consistency.

Equipments and procedure Earlier physicians used the same apparatus for medical tattooing as used by the tattoo parlors. The type of needles used and the pigment deposited has also varied considerably with the time [2]. Earlier tattoo needles were crudely made of bones while pigments used were derived from soot or plant extracts. Byars in 1945 used a row of needles soldered onto a metal bar while Schmidt in 1951 used a tube tipped leather punch as a needle holder, to which a group or a row of small, sharp steel sewing needles were soldered [1,6]. Conway’s dermajector apparatus was a similar device but with a pigment cup that feeds pigment paste into the hollow shaft of needle holder [7]. Nowadays, a variety of tattoo machines and needles are available for micropigmentation. The electrically driven tattooing machines popularly known as “tattoo gun” or a “tattoo pencil” are gun-shaped machines held like a gun or a pencil (Fig. 32.1) having an assembly of three to seven stainless steel needles on a needle bar. The needles are usually of 25 gauge, with an approximate length of 36 mm and a thickness of 0.36 mm kept about 0.3 mm away from each other. The speed ranges from 1500 to 9500 strokes/min and depth of needle penetration is adjustable from 1 to 2 mm. Similarly, manual tattooing can be accomplished with a group of needles soldered on a common metal base or held together by a handle [8]. A watchmaker’s pin-vise (Fig. 32.2) is a similar instrument loaded with disposable sewing needles for manual tattooing in vitiligo. It is a cheap, effective, and sterilizable alternative, which can be procured easily where electrically driven machines are not available [9]. Under local infiltrative anesthesia with 2% lignocaine (with or without adrenaline), a thick layer of pigment paste is applied. The site to be tattooed is stretched with thumb and index finger while the

Fig. 32.1 Tattoo machine held in a gun-holding manner.

Fig. 32.2 Watchmaker’s pin-vise with disposable needles

for manual tattooing.

tattooing machine held in pen-holding manner makes repeated vertical movements of needles up and down on the surface of skin. Tattooing is done in the entire area in an overlapping manner along with maintenance of hemostasis (Fig. 32.3). A pressure dressing along with prophylactic antibiotics and antiinflammatory drugs may be given postoperatively. Follow up at 4–6 weeks and 6 months may require a further touch-up tattooing for the areas of pigment leaching or shed off (Fig. 32.4).

Micropigmentation in vitiligo Micropigmentation is quite a useful procedure for the cosmetic camouflage of vitiliginous patches especially involving mucosal and mucocutaneous areas in

Micropigmentation

251

(A) (A)

(B) (B) Fig. 32.3 Vitiligo patch on the ankle before (A) and

immediately after (B) micropigmentation.

wheatish or black races like Asians and Africans. The procedure is relatively easy, provides rapid and permanent camouflage, and is generally devoid of any significant adverse effects if universal precautions for sterilization of instruments are adhered to strictly [2]. Although the history of tattooing as a body ornament is quite long, its therapeutic value was recognized as early as 1835 when Pauli employed it in the treatment of color nevi, grafts, congenital purple plaques, and other lesions of skin. Grinspan and his co-workers used tattooing with gold salts (20%) aurothio-glucose in sesame oil for vitiligo [1]. Halder in 1989 used micropigmentation for covering recalcitrant patches of vitiligo and showed dramatic aesthetic improvements with moderate degree of fading usually within 6 weeks. He observed that as dye is

(C) Fig. 32.4 Patient 2 of mucosal vitiligo before (A),

immediately after (B), and at 6 weeks’ follow-up (C) after micropigmentation.

being applied to achromic areas the color appeared darker than the surrounding normal skin upon initial application [10]. Satisfying results have been obtained in camouflaging mucosal and gingival vitiligo [11].

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Excellent color matching has been reported in cutaneous, mucosal and mucocutaneous vitiligo, contact leukoderma, and post-inflammatory depigmentation and scarring in various studies especially in dark individuals [12–14]. The present evidence indicates its use in recalcitrant lesions of vitiligo especially over distal digits, lips, hands, wrists, axillae, elbows, hairline, perianal areas, lower legs, mucosae, and mucocutaneous junctions [8,15–20]. Extensive mucosal depigmentation in vitiligo may involve gingival tissues which may also be tattooed to obtain near normal gingival color (Plate 32.1, facing p. 114) [21]. Besides vitiligo, micropigmentation is a useful adjunctive therapy for camouflaging other disturbing discolorations and scars of the head and neck, permanent regimentation of achromic burn scars, color matching of vermilionplasty after radial forearm free flap reconstruction of the lower lip, cleft lip and cleft palate scars, senile lip rejuvenation, and an alternative treatment for disturbing corneal scars [4,6,7,15–29].

Factors affecting cosmetic results The key factor for the pigment to be retained uniformly in the dermis is its level of deposition in the dermis. An optimum level is the upper and the mid-papillary dermis at or around a depth of 1.5 mm (range 1–2 mm) from the surface of skin. The pigment deposited very high (1 mm) gets pushed out postoperatively along with the crust (pigment extrusion) at about 2 weeks while a very deep (2 mm) deposition of pigment gets carried away by the macrophages (pigment migration) after 2–3 months of the procedure [2,5]. Besides depth, the density of pigmentation achieved also depends on consistency of pigment paste (thick, moist, and even paste gives more uniform density of pigment deposited), number of needles used (more pigment deposition with greater number of needles) and uniform dispersion of pigment within the horizontal plane of dermis. The non-procedural determinants of micropigmentation include the thickness, elasticity, and laxity of skin or mucosa to be tattooed along with natural melanin content and capillary blood flow [5].

Adverse effects and limitations The immediate adverse effects of micropigmentation are few and usually result from improper technique and lack of adherence to asepsis. These include ecchymosis, crusting, edema lasting 2–3 days, reactivation of herpes simplex virus infection, secondary bacterial infection, and contact allergy to pigments in first week of procedure [1,2]. A number of infections have been known to be transmitted through the repeated use of same tattooing needles from one patient to another without proper sterilization. These include syphilis, tuberculosis, leprosy, viral infections like common warts, hepatitis B and C, and human immunodeficiency virus (HIV). Other risks involved may be: photosensitization allergy mainly to cadmium sulfate and blurring of pigment (color leaks from original site). With progression of time, implanted pigment may present an unsightly and inappropriate look (i.e. permanent make-up) which is difficult to remove even with lasers. Similarly, tattoos containing certain metal oxides can get oxidized and turn black which is even harder to remove [2]. In a phenomenon known as Köbnerization, tattoos may act as foci of localized dermatoses like psoriasis, lichen planus, sarcoidosis, and lupus erythematosus. This type of Köbnerization is especially seen around mercury dye micropigmentation. Rarely, micropigmentation has been reported to precipitate cutaneous malignant melanoma, basal cell carcinoma, and reticulohistiosarcoma [1,30]. The long-term drawbacks of micropigmentation are in the form of its life-long permanency and difficulty in removal without scarring. Despite best possible color matching, a long-standing micropigmentation tends to fade and ultimately get discolored often leaving a grayish hue needing repeated procedures to maintain an optimum level of cosmetic result. Besides this, micropigmentation also entails an inherent psychological disadvantage of having an extraneous pigment substance in place of a natural one. An acyclovir prophylaxis is justified while carrying out micropigmentation on lips in patients having a history of recurrent oro-labial herpes simplex.

Micropigmentation

Conclusions Micropigmentation is tattooing of metabolically inert pigment granules into dermis for medical indications. The pigments used are usually non-toxic, nonallergenic, tissue stable dyes having a minute particle size. Micropigmentation can be achieved by using multiple tattooing needles mounted on a manual or electrically driven equipment. Different shades of micropigmentation can be achieved by using combinations of white, yellow, black, red, and brown pigments. Tattooed pigment once impregnated is retained intracellularly as well as extracellularly within collagen bundles for many years. Cosmetic results depend on depth of pigment deposition, uniform dispersion of pigment, and vascularity of the target site. It is usually safe and inexpensive procedure without significant adverse effects if universal precautions of sterilization are followed. Micropigmentation is used to correct cosmetic defects arising out of vitiligo, post-burn and post-inflammatory hypomelanosis, and other depigmentations not amenable to medical therapy like vitiligo and not responding to medical therapies. Good cosmetic results can be achieved in mucosal and mucocutaneous vitiligo. The limitation of its irreversibility with consequent difficulty in removal of tattoo must be kept in mind while resorting to this potentially useful option.

References 1 Epstein E. Therapeutic tattooing. In: Epstein E(ed.) Skin Surgery, 2nd edn. Philadelphia, PA: Lea & Febiger Publications, 1962;308–15. 2 Garg G, Thami GP. Micropigmentation: tattooing for medical purposes. Dermatol Surg 2005;31:928–31. 3 Roberts TA, Ryan SA. Tattooing and high-risk behavior in adolescents. Pediatrics 2002;110:1058–63. 4 van der Velden EM, Wittkampf ARM, de Jong BD, et al. Dermatography, a treatment for sequelae after head and neck surgery: a case report. J Craniomaxillofac Surg 1992;20:273–8. 5 Wolfley DE, Flynn KJ, Cartwright J, Tschen A. Eyelid pigment implantation: early and late histopathology. Plast Reconst Surg 1988;82:770–4. 6 Byars LT. Tattooing of free skin grafts and pedicle flaps. Ann Surg 1945;121:644–8.

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7 Conway H. Tattooing of nevus flammeus for permanent camouflage. JAMA 1953;152:666–9. 8 Gharpuray MB, Mutalik S. Manual punch for tattooing. J Dermatol Surg Oncol 1994;20:548–50. 9 Singal A, Thami GP, Bhalla M. Watchmaker’s pin-vise for manual tattooing of vitiligo. Dermatol Surg 2004;30:203–4. 10 Halder RM, Pham HN, Breadon JY, Johnson BA. Micropigmentation for treatment of vitiligo. J Dermatol Surg Oncol 1989;15:1092–8. 11 Centre JM, Mancini S, Baker GI, et al. Management of gingival vitiligo with use of a tattoo technique. Br J Dermatol 1998;138:359–60. 12 Larson D. Micropigmentation. Ann Plast Surg 1996; 36:193. 13 Vadodaria SJ, Vadodaria BS. Tattooing for the management of white patches. Ann Plast Surg 1989;23:81–3. 14 Mahajan BB, Garg G, Gupta RR. Evaluation of cosmetic tattooing in localized stable vitiligo. J Dermatol 2002;29:726–30. 15 Malakar S, Lahiri K. Successful repigmentation of six cases of herpes-labialis-induced lip leucoderma by micropigmentation. Dermatol 2001;203:194. 16 Furuta S, Hataya Y, Watanabe T, Yuzuriha S. Vermilionplasty using medical tattooing after radial forearm flap reconstruction of the lower lip. Br J Plast Surg 1994;47:422–4. 17 Fulton JE, Rahimi AD, Helton P, et al. Lip rejuvenation. Dermatol Surg 2000;26:470–4. 18 Pitz S, Jahn R, Frisch L, et al. Corneal tattooing – an alternative treatment for disfiguring corneal scars. Br J Opthalmol 2002;86:397–9. 19 van der Velden EM, Vander Dussen MFN. Dermatography as an adjunctive treatment for cleft lip and cleft patients. J Oral Maxillofac Surg 1995;53:9–12. 20 Mazza Jr. JF, Rager C. Advances in cosmetic micropigmentation. Plast Reconst Surg 1993;92:750–1. 21 Center JM, Mancini GI, Baker GI, Mock D, Tenenbaum HC. Management of gingival vitiligo with the use of a tattoo technique. J Periodontol 1998; 69:724–8. 22 van der Velden EM, Baruchin AM, Jairath D, et al. Dermatography: a method for permanent repigmentation of achromic burn scars. Burns 1995; 21:304–7. 23 van der Velden EM, de Jong B, van der Walle HB. Tattooing and its medical aspects. Int J Dermatol 1993;32:381–4. 24 van der Velden EM, Samderubun, KoK JHC. Dermatography as a modern treatment for colouring leucoma corneae. Cornea 1994;13:349–53.

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25 van der Velden EM, Vander Dussen MFN. Dermatography as an adjunctive treatment for cleft lip and palate patients. J Oral Maxillofac Surg 1995; 53:9–12. 26 Spear SL, Convit R, Little JW. Intradermal tattoo as an adjunct to nipple–areolar reconstruction. Plast Reconst Surg 1989;83:907–11. 27 van der Velden EM, Drost Brigitte HIM, Ijsselmuiden OE, et al. Dermatography as a new treatment for

alopecia areata of the eyebrows. Int J Dermatol 1998;37:617–21. 28 Traquina AC. Micropigmentation as an adjuvant in cosmetic surgery of scalp. Dermatol Surg 2001;27:123–8. 29 Mutalik S, Ginzburg A. Surgical management of stable vitiligo. Dermatol Surg 2000;26:248–54. 30 Earley MJ. Basal cell carcinoma arising in tattoos: a clinical report of two cases. Br J Plast Surg 1983; 36:258–9.

CHAPTER 33

Laser for repigmenting vitiligo Thierry Passeron and Jean-Paul Ortonne

The use of lasers in vitiligo is usually dedicated to the depigmentation of residual pigmented areas in generalized forms [1,2] or to removing achromic epidermis before grafting [3,4]. In the past few years, new laser devices have been reported for repigmenting vitiligo lesions. First, the 308-nm excimer laser combined the selectivity of lasers and the well recognized efficacy of ultraviolet (UV) B for treating vitiligo. Although less investigated, the 632.8-nm helium–neon laser brings an innovative approach to treating vitiligo lesions.

308-nm excimer laser The xenon chloride (Xecl) excimer laser generates UVB radiation at a wavelength of 308-nm. Several reports have shown that this device is effective in the treatment of vitiligo. Patients are treated 2 or 3 times a week for 1–6 months depending on series. Low fluencies (50–200 mJ/cm2) are used. In most studies, the percentage of treated lesions achieving at least 75% repigmentation is about 30% [5–9]. As for the different phototherapies, the rate of repigmentation varies depending on the anatomic sites. The rate of repigmentation is very high on UVB responsive areas such as the face whereas the extremities and bony prominences (well-recognized UVB-resistant areas) show a statistically significant inferior repigmentation rate [9] (Figs. 33.1–33.3). Sessions can be performed 1, 2, or 3
weekly as repigmentation seems to depend on the total number of treatments, not their frequency [10]. The stability of the repigmentation with time has so far been difficult to evaluate, as follow-up of the studies is poor or nil; however, one recent series showed an absence of depigmentation of the treated lesions after 1 year [8]. Side effects are limited to mild

erythema and uncommon blistering. The major advantage of the Xecl excimer laser is its ability to confine the treatment only to the vitiliginous lesions. A pilot intra-individual comparative trial recently shows that the 308-nm excimer laser is more effective than narrowband UVB (NB-UVB) with more rapid and profound repigmentation [11]. These results need to be confirmed in a larger series but the Xecl excimer laser does represent a useful tool for the treatment of localized vitiligo. It gives the possibility to choose only a limited number of lesions without whole body irradiation. Combination of the 308-nm excimer laser and topical tacrolimus has also provided interesting results but further follow-up are still required (see Chapter 4, “Medical treatment of vitiligo”) [12,13]. Moreover, UVB phototherapy has already shown its efficacy after grafting [14]; the selectivity of the 308-nm excimer laser would be very useful in such indication and this combination worth to be further investigated.

632.8-nm helium–neon laser Another laser, the 632.8-nm helium–neon laser, was also reported being able to induce a repigmentation in segmental vitiligo [15]. In vitro studies showed that this laser increased the proliferation, and then the migration of the melanocytes. Thirty patients were treated once or twice weekly. A repigmentation of at least 75% was obtained in 20% of the patients. No side effect was noted. However, the average number of sessions needed to achieve these interesting results was very high (137 sessions, i.e. 1–2.5 years of treatment). Nevertheless, the 632.8-nm helium–neon laser represents a completely innovative therapeutic approach which is worth studying.

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(A)

(B) Fig. 33.1 Vitiligo of the face (A) before treatment, (B) 1 month after 24 sessions of 308 nm excimer laser.

(A)

(B) Fig. 33.2 Segmental vitiligo (A) before treatment, (B) 1 month after 24 sessions of 308 nm excimer laser.

Conclusion New laser devices can now be used to repigment vitiligo patches. The efficacy and good tolerance of the 308-nm excimer laser has now been demonstrated in several prospective studies and the US Food and Drug Administration (FDA) has approved this laser for the treatment of vitiligo. Although very

interesting, the results obtained with the 632.8-nm helium–neon laser still need to be further investigated. One of the main advantages of these two new devices is to treat selectively the vitiligo lesions with sparing of the surrounding healthy skin. This selectivity also limits their use to vitiligo involving less than 20% of the total surface body area. Indeed, these

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(A)

(B) Fig. 33.3 Vitiligo of the leg and knee (A) before treatment, and (B) after 30 sessions of 308-nm excimer laser

combined with twice daily applications of 0.1% of tacrolimus ointment.

lasers should not be considered as an alternative to other treatments, such as phototherapy, but as new complementary options that could possibly be combined with topical treatments or surgical grafts.

References 1 Kim YJ, Chung BS, Choi KC. Depigmentation therapy with Q-switched ruby laser after tanning in vitiligo universalis. Dermatol Surg 2001;27:969–70. 2 Rao J, Fitzpatrick RE. Use of the Q-switched 755-nm alexandrite laser to treat recalcitrant pigment after depigmentation therapy for vitiligo. Dermatol Surg 2004;30:1043–5. 3 Acikel C, Ulkur E, Celikoz B. Carbon dioxide laser resurfacing and thin skin grafting in the treatment of “stable and recalcitrant” vitiligo. Plast Reconstr Surg 2003;111:1291–8. 4 Guerra L, Primavera G, Raskovic D, et al. Erbium:YAG laser and cultured epidermis in the surgical therapy of stable vitiligo. Arch Dermatol 2003;139:1303–10.

5 Spencer JM, Nossa R, Ajmeri J. Treatment of vitiligo with the 308-nm excimer laser: a pilot study. J Am Acad Dermatol 2002;46:727–31. 6 Baltas E, Csoma Z, Ignacz F, Dobozy A, Kemeny L. Treatment of vitiligo with the 308-nm xenon chloride excimer laser. Arch Dermatol 2002;138:1619–20. 7 Taneja A, Trehan M, Taylor CR. 308-nm excimer laser for the treatment of localized vitiligo. Int J Dermatol 2003;42:658–62. 8 Esposito M, Soda R, Costanzo A, Chimenti S. Treatment of vitiligo with the 308 nm excimer laser. Clin Exp Dermatol 2004;29:133–7. 9 Ostovari N, Passeron T, Zakaria W, et al. Treatment of vitiligo by 308-nm excimer laser: an evaluation of variables affecting treatment response. Lasers Surg Med 2004;35:152–6. 10 Hofer A, Hassan AS, Legat FJ, Kerl H, Wolf P. Optimal weekly frequency of 308-nm excimer laser treatment in vitiligo patients. Br J Dermatol 2005;152: 981–5. 11 Hong SB, Park HH, Lee MH. Short-term effects of 308-nm xenon-chloride excimer laser and narrow-band

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ultraviolet B in the treatment of vitiligo: a comparative study. J Korean Med Sci 2005;20:273–8. 12 Kawalek AZ, Spencer JM, Phelps RG. Combined excimer laser and topical tacrolimus for the treatment of vitiligo: a pilot study. Dermatol Surg 2004;30:130–5. 13 Passeron T, Ostovari N, Zakaria W, et al. Topical tacrolimus and the 308-nm excimer laser: a synergistic combination for the treatment of vitiligo. Arch Dermatol 2004;140:1065–9.

14 Pianigiani E, Risulo M, Andreassi A, et al. Autologous epidermal cultures and narrow-band ultraviolet B in the surgical treatment of vitiligo. Dermatol Surg 2005;31:155–9. 15 Yu HS, Wu CS, Yu CL, et al. Helium–neon laser irradiation stimulates migration and proliferation in melanocytes and induces repigmentation in segmentaltype vitiligo. J Invest Dermatol 2003;120:56–64.

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Application of lasers in transplantation procedures for vitiligo Cengiz Acikel, Ersin Ulkur and Bahattin Celikoz

In transplantation procedures in vitiligo, recipient sites can be prepared in a number of ways, including suction blisters [1], liquid nitrogen-induced blisters [2], psoralen plus ultraviolet A (PUVA) induced blisters [3], programmed diathermal surgical procedure (Timedsurgery) [4], removal of skin by dermatome [5], mechanical dermabrasion [6], and laser-assisted de-epithelialization [7–11]. Laser-assisted dermabrasion of the leukodermic skin has gained more popularity compared to the above-mentioned skin resurfacing modalities since precise removal of epidermis is possible. Large vitiligo areas can be denuded in a short time, depth of tissue ablation can be well controlled, and a bloodless and smooth raw surface can be created by using laser for skin resurfacing procedure. Additionally, uneven surfaces such as dorsum of hand and fingers can be easily denuded. High-energy short-pulsed carbon dioxide (CO2) lasers have been used for skin de-epithelialization for more than 15 years. Prior experimental studies by Hallock and Rice [12] in rats have proven that the CO2 laser allows rapid de-epithelialization with the added advantages of minimal subsequent hemorrhage and preservation of the subdermal vascular plexus [12]. Extrapolation of this laboratory result in clinical trials was then reported to be successful for the formation of the inferior dermal pedicle in breast reduction [12,13]. Further clinical applications such as skin graft removal, partial de-epithelialization of free flaps, or de-epithelialization for mastopexy were reported by Hallock [14]. While epidermis and a portion of upper dermis is removed by laser abrasion, a residual thermal damage zone, 20–150
m in width, is created on the dermal raw surface [15,16]. These

raw surfaces sustain thin skin grafts well. The thermal damage zone is eliminated by leukocytic degradation and skin grafts adhere to dermis well. The narrow zone of coagulation necrosis neither interferes with the skin graft take nor causes infection. The fate of the skin grafts on laser-abraded surface is similar or superior to grafts applied to raw surfaces created by mechanical dermabrasion [16–21]. Er:YAG (erbium:yttrium–aluminum–garnet) laser with 2940-nm wavelength is the other ablative laser used for skin resurfacing [9,10,22]. The absorption coefficients of the Er:YAG and CO2 lasers are 12,800/cm and 800/cm, respectively, making the Er:YAG laser 12–18 times more efficiently absorbed by water-containing tissues than the CO2 laser [23]. Since the water-containing tissues such as epidermis effectively absorb the Er:YAG laser, this laser system is a more precise ablative tool. It is possible to remove 10
m epidermis with each pass. Furthermore, much narrower zones of residual thermal necrosis on the wound bed, averaging only 20–50
m, are produced by Er:YAG laser [23,24]. In the case of treating large vitiliginous areas, the recipient sites should easily be de-epithelialized in a short time while depth of tissue ablation can be well controlled. In a previous study, we treated large areas of burn scar depigmentation using CO2 laser-assisted dermabrasion and autologous thin (0.2–0.3 mm) skin grafting and achieved permanent repigmentation [16]. In this study, we showed that a smooth and bloodless raw surface could be created in a short time with minimal thermal damage and these raw surfaces could sustain thin splitthickness skin grafts well. Then we used the same surgical technique in the treatment of seven

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patients (13 anatomic sites) who had “stable and recalcitrant” vitiligo and achieved early and complete repigmentation in all patients [20].

CO2 laser resurfacing and thin skin grafting: operative procedure Safety precautions are taken in the operating room for use of the CO2 laser. After the administration of satisfactory anesthesia (general or regional), the skin of the vitiliginous skin and the skin graft donor area are shaved, prepared with a povidone iodine topical antiseptic solution, and draped in a sterile fashion. The areas are wiped with saline-soaked gauze and dried with a gauze sponge. The epidermis and a portion of the upper part of the dermis of the vitiliginous areas are ablated evenly by one pass of a flash-scanned CO2 laser (Sharplan 150 XJ SilkTouch; Sharplan Lasers Inc., Needham, MA, USA) with an F 260 Handpiece, 34 W, on SilkTouch Mode. A second pass and sometimes a third pass are required on the lateral sides of the fingers to remove the epidermis. After each pass, treated areas are wiped with saline-soaked gauze and dried with a gauze sponge. A spot size of 6 mm is used on fingers, while a large, 9-mm spot size minimizes the length of the procedure and is more effective for larger areas. The laser is set in repeat mode at 0.2 second on time and 0.4 second off time. The laser can be set in continuous mode as adequate experience gained. Small pigmented and depigmented areas over dorsum of the hands are resurfaced together and grafted as a unit to achieve more homogenous pigmentation. Pigmented epidermis, 1–2 mm in width around the achromic lesion, is also removed and grafted besides vitiliginous skin to prevent peripheral hypopigmentation. Thin split-thickness skin grafts (0.2–0.3 mm in thickness and the same size as the recipient site) are harvested from the pigmented thigh region with a Padgett electrical dermatome, meshed with a No. 15 Bard-Parker blade to avoid seroma formation, and applied over the laser-resurfaced raw surfaces. Skin grafts are fixed by skin stapler or paper tapes (Plate 34.1, facing p. 114). The grafted areas are closed with non-adherent dressing and covered with gauze

sponges moistened with saline solution. Slight pressure is applied over the grafted areas via outer bandage to avoid seroma or hematoma collection under the skin grafts. Skin graft donor areas are covered with the same dressing material. The treated sites are immobilized with plaster splints for 5 days, and dressings are changed every 3 days for 15 days. While changing the dressing of skin-grafted areas, external pressure-free time should be kept at minimum to avoid seroma formation under the skin grafts. Jobst gloves (Jobst Institute, Inc., Toledo, OH, USA) are used for 3 months in those patients who undergo surgery of their hands to protect the hands and fingers from minor trauma and sunlight. Compressive tubular bandage (Tubigrip; Seton Health Care Group, Manchester, UK) is applied over the treated extremities for 3 months for the same reasons. They are also applied to skin graft donor sites to prevent engorgement of the area with blood during movement; patients are advised to use the bandages until the color of the donor area faded. Range of motion exercises are initiated as soon as skin grafts stabilize in the early postoperative period. The patients are advised to protect their operation sites, including graft donor areas, from the sun for at least 6 months and to use broad-spectrum sunscreens and skin moisturizers. Full skin graft take is the rule if adequate immobilization and external pressure is applied in the early postoperative period. The worst case scenario is partial or total loss of the applied skin grafts requiring re-grafting. If re-grafting is not performed, spontaneous epithelialization of the raw surface will occur. The skin graft donor sites heal within 5–7 days without any complication such as scarring or Köbnerization and color match is nearly full after 6 months. However, we observed altered pigmentation in one patient and it was permanent. Complete repigmentation is observed in the early postoperative period. It takes more than 6 months to achieve final color. No additional therapy is required to enhance repigmentation. The skin grafts were reddish to pink in the first postoperative month; then they develop a temporary hyperpigmented appearance that lasted for several months. The final color establishes in the following months.

Application of lasers in transplantation procedures for vitiligo The patients receiving treatment at multiple sites have similar quality of outcomes regarding skin graft take ratio and repigmentation. However, final color match of treated sites with surrounding normal

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skin was superior in the hands and forearms compared to other anatomic sites in the same patient (Figs. 34.1–34.4). Permanent hyperpigmentation and a patchy appearance can be observed in the

(A)

(B)

(C)

(D)

(E)

(F) Fig. 34.1 (A) Preoperative view. (B) Postoperative view after 13 months (left hand) and 15 months (right hand).

(C) Preoperative view, left forearm of the same patient. (D) Postoperative view after 13 months. (E) Preoperative view, right forearm of the same patient. (F) Postoperative view after 15 months. Note that the hair pattern of the forearm has not changed. (Reprinted from Acikel C et al. [20], with permission.)

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(A)

(B) Fig. 34.2 (A) Preoperative view. (B) Postoperative view after 3 months.

(A)

(B) Fig. 34.3 (A) Preoperative view. (B) Postoperative view after 6 months.

(A)

(B) Fig. 34.4 (A) Preoperative view. (B) Postoperative view after 12 months.

Application of lasers in transplantation procedures for vitiligo treated areas if adequate protection from sunlight cannot be achieved. Skin bleaching agents such as hydroquinones may be helpful in these cases. Small keratinous cysts (milia) may be observed in the early postoperative weeks. They either spontaneously disappear or are treated definitively by opening the top of the cyst and removing the contents. These cysts do not recur. Hair growth through the thin skin graft is excellent and normal hair patterns are regained over the forearm and pretibial regions (Fig. 34.1 and Plate 34.2, facing p. 114). We have not observed any extension of the vitiligo beyond the treated areas. Any recurrence of vitiligo, development of hypertrophic scar or infection over the skin-grafted areas were not observed up to date.

Comparison of the procedure with alternative treatment methods Minigrafting and suction blister epidermal grafting are the most often used techniques. Minigrafting involves the harvesting of 1.2–2.5-mm punch grafts from the pigmented donor site, which is usually an area on the lower back below the waistline. The grafts are placed 3–4 mm apart on the recipient site, which has been prepared by using the same sized punch to make defects. Sachdev and Krupa Shankar and Pai et al. used pulsed Er:YAG laser with 3-mm spot size at 1600 mJ and 16 J/cm2 fluence to prepare recipient site defects [21,25]. The recipient sites were perfect circles with a pinpoint bleeding at the bottom of each site. The authors found that using laser to create recipient site sockets offered distinct advantages: it enabled grafting to be performed in difficult sites, for example lips, fingertips, earlobes; there is minimal blood loss with a relatively bloodless surgical field, and the procedure is more efficient and less time consuming and less dependent on operator’s skill. However, they expressed concerns that due to laser coagulation there may be less graft uptake than with conventional method [25]. Blister-induced epidermal grafts are created in 1.5–4 hours using disposable syringes of various sizes (0.7–3 cm) attached by tubing to a suction apparatus that exerts a negative pressure of 200–300 mmHg.

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Obtaining large sheets of pure epidermal grafts is not feasible; therefore, procedures must be repeated to acquire grafts to cover larger areas. The epidermal grafts have to be handled very delicately, because they roll up and tear easily. As they are not affixed to the treated area, epidermal grafts may easily slip off. Pai et al. used Er:YAG laser for recipient site preparation before transplanting suction blister epidermal grafts in patients with vitiligo. Results were better in patients who underwent laser-assisted recipient site preparation than in those who underwent phototoxic chemical dermabrasion [21]. Sachdev et al. [10] also used Er:YAG laser at 400 mJ and a fluence of 4 J to prepare recipient site before epidermal grafting in two patients with vitiligo. Oh et al. [19] used ultrapulse CO2 laser at 300 mJ and an energy density of 5 J to prepare recipient site before transplanting epidermis obtained by suction blistering in 34 lesions in 11 patients (3 generalized, 4 segmental, 4 focal). Thirty lesions showed excellent and remaining four patients showed good repigmentation. Postoperative treatment may be required for rapid spread and coalescence of the grafts [26,27]. Transplantation of autologous epidermal cell suspension can be used for treatment of up to 10 times larger area than donor skin sample. This method has been successfully used in combination with epidermal ablation of recipient site using a pulsed CO2 laser set at 300 mJ and 200 pulses/second (Coherent UltraPulse; Parallax Technology Inc., Waltham, MA) [28]. After the papillary dermis was reached the denuded lesions were treated with the epidermal cellular suspension. At 12 months, 77% of the treated lesions showed 70% or more repigmentation [28]. Surgical techniques that use autologous cultured pure melanocytes [29,30] or epidermis [31–33] seem to be most promising, in that large achromic areas can be repigmented using a very small shave biopsy specimen. Both methods are still in the development stage and require future controlled studies [34,35]. Most of the applications have been limited to small areas, and the response rate to the treatment varies depending on the patient and anatomic site. Hands, feet, and periorificial sites are the resistant sites to cultured epidermis therapy [33]. Chen et al. [36] treated 120 patients (80 with localized and 20 with generalized vitiligo) with cultured

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melanocyte and CO2 laser denudition of recipient site. The Silktouch Flashscanner attached to a Sharplan 1030 CO2 laser was used to ablate the recipient site epidermis. A setting of 4.5–7 W with a 0.2 second pulse duration was kept. Eighty-four percent of localized vitiligo patients and 54% of generalized vitiligo patients showed excellent repigmentation. In another study, Guerra et al. [22] treated 21 patients with different types of vitiligo with Er:YAG laser and cultured epidermis. Achromic epidermis was removed by using the pulsed Er:YAG laser at an energy setting of 200–500 mJ and the energy denisity ranged from 6.3 to 19.1 J/cm2. One laser pass was sufficient for most of the patients except for hand lesions, where two passes were required. The denatured residues were removed with saline moistened gauze and the area was covered with cultured epidermal sheet. Interestingly, most of their patients did not require any anesthesia. The average percentage of repigmentation was about 75%. The same authors also successfully treated six patients with piebaldism with the same technique [37]. The split-thickness skin grafting method (with various dermal thicknesses) has been used in the treatment of small vitiliginous areas with overall success rates of more than 80% complete repigmentation. Partial graft loss, inclusion cysts, hyperpigmentation, peripheral hypopigmentation, thickening of graft margin, a “stuck-on” appearance, and infection are the reported complications seen in the recipient area, while permanent depigmentation, altered pigmentation, and superficial scarring have been observed at the skin graft donor sites [5,6,38–41]. In the “CO2 laser resurfacing and thin skin grafting” procedure large achromic areas can be denuded in a short time, depth of tissue ablation can be well controlled, and a bloodless and smooth raw surface can be created by using a flashscan CO2 laser. The laser is especially useful for resurfacing of fingers and hands. At present, the major drawbacks of the CO2 laser dermabrasion are the time needed to setup the apparatus, the cost of the procedure, and the required safety precautions. In our experience, it is not easy to harvest and handle large sheet grafts thinner than 0.2 mm, for example, ultra-thin grafts [6]. We were able to harvest

and handle large sheet grafts that are 0.2–0.3 mm in thickness. If the skin graft can be taken thin enough (0.2–0.3 mm) and well secured to denuded area, no graft margin thickening develops. In the case of inadequate graft fixation, peripheral epithelialization underneath the graft will prevent graft take and result in graft edge contraction and thickening. Peripheral hypopigmentation can be prevented by removing additional pigmented epidermis around the achromic area and grafting this raw surface. Inclusion cysts have been seen occasionally and can be managed easily. Partial graft loss was due to inadequate immobilization and could be minimized with good immobilization and patient cooperation. Although the graft donor sites do repigment, the possibility of altered pigmentation should be discussed with the patient. To date, we have not observed any recurrence of vitiligo on treated areas, but longer follow-up periods are needed to see more long-term results. Kahn [42] reported one case in which a patient’s vitiligo became very active and all the skin-grafted areas spontaneously depigmented by the 61⁄2-year follow-up. Guerra et al. [22] treated 21 patients having large “stable” vitiligo lesions by means of autologous cultured epidermal grafts and three patients showed a reactivation of their vitiligo and did not show repigmentation. It should be noted that the number of treated patients was too small to detect any rare problems that might exist. The upper limit of surface area that can be treated in one session using the thin skin grafting method depends on the available skin graft donor site, blood loss from the donor sites, and length of the procedure; 6% of body surface area is the largest area that we have treated. In the “CO2 laser resurfacing and thin skin grafting” procedure the grafts completely repigment and do not need additional therapy. Nevertheless, complete repigmentation of the graft is not enough for an ideal solution. The color of the newly pigmented area ideally should be identical to that of the surrounding normal skin. The final tone of graft color is not predictable, however, especially in fair-skinned individuals. We recommend discussing slight tone differences with the patient. We would like to repeat that all of our patients were satisfied with their results compared with their preoperative appearances.

Application of lasers in transplantation procedures for vitiligo We did not use our method to treat facial vitiligo since perfect color match is critical for the face. A demarcation between skin graft and normal skin may exist, especially for smaller areas. For such situations, other modalities such as blister grafting might be a better alternative [38,39,43]. Vitiliginous hands deserve special emphasis because they are very resistant to medical and surgical therapeutic modalities and cannot be hidden. We observe that patients give their hands and face higher priority for treatment. Laser-assisted dermabrasion turned the superficial dermabrasion of the hands and fingers into an easy and short procedure, which is very difficult with other methods. We resurface the achromic areas along with the normal skin islands among them to create a single, large denuded area. Skin grafting of these raw surfaces resulted in a homogenous color tone and perfect color match because there was no normal skin around the fingers to compare. Besides, casual observers often miss subtle pigment irregularities. We believe that vitiliginous hands and larger areas over the extremities can be treated effectively by using CO2 laser resurfacing and the thin skin grafting method, and these patients may be spared the indelicate staring and glaring that is an unpleasant fact of their lives.

Conclusion Large vitiliginous areas can be resurfaced in a short time, depth of tissue ablation can be well controlled, and a bloodless and smooth raw surface can be created by using a CO2 laser or Er:YAG laser. These raw surfaces sustain thin skin grafts, and cultured and non-cultured cellular autografts well; and permanent repigmentation with good color match can be achieved, especially on the hands and upper extremities of carefully selected vitiligo patients.

References 1 Falabella R. Postdermabrasion leukoderma. J Dermatol Surg Oncol 1987;13:44–8. 2 Falabella R. Epidermal grafting: an original technique and its application in achromic and granulating areas. Arch Dermatol 1971;104:592–600.

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3 Koga M. Epidermal grafting using the tops of suction blisters in the treatment of vitiligo. Arch Dermatol 1988;124:1656–8. 4 Capurro S, Fiallo P. Epidermal disepithelialization by programmed diathermosurgery. Dermatol Surg 1997;23:600–1. 5 Behl PN, Bhatia RK. Treatment of vitiligo with autologous thin thiersch’s grafts. Int J Dermatol 1973;12:329–31. 6 Kahn AM, Cohen MJ. Repigmentation in vitiligo patients: melanocyte transfer via ultra-thin grafts. Dermatol Surg 1998;24:365–7. 7 Kahn AM, Ostad A, Moy RL. Grafting following shortpulse carbon-dioxide laser de-epithelialization. Dermatol Surg 1996;22:965–7. 8 Oh CK, Cha JH, Lim JY, et al. Treatment of vitiligo with suction epidermal grafting by the use of an ultrapulse CO2 laser with a computerized pattern generator. Dermatol Surg 2001;27:565–8. 9 Yang JS, Kye YC. Treatment of vitiligo with autologous epidermal grafting by means of pulsed erbium:YAG laser. J Am Acad Dermatol 1998;38(2 Pt 1):280–2. 10 Sachdev M, Krupashankar DS. Suction blister grafting for stable vitiligo using pulsed erbium:YAG laser ablation for recipient site. Int J Dermatol 2000;39: 471–3. 11 Pianigiani E, Risulo M, Andreassi A, et al. Autologous epidermal cultures and narrow-band ultraviolet B in the surgical treatment of vitiligo. Dermatol Surg 2005;31:155–9. 12 Hallock GG, Rice DC. Skin deepithelialization using the carbon dioxide laser. Ann Plast Surg 1987;18:283–8. 13 Becker DW, Bunn JC. Laser deepithelialization: an adjunct to reduction mammoplasty. Plast Reconstr Surg 1987;79:754–60. 14 Hallock GG. Extended applications of the carbon dioxide laser for skin deepithelialization. Plast Reconstr Surg 1989;83:717–21. 15 Alster TS, Kauvar ANB, Geronemus RG. Histology of high-energy pulsed CO2 laser resurfacing. Semin Cutan Med Surg 1996;15:189–93. 16 Acikel C, Ulkur E, Guler MM. Treatment of burn scar depigmentation by carbon dioxide laser-assisted dermabrasion and thin skin grafting. Plast Reconstr Surg 2000;105:1973–8. 17 Kahn AM, Ostad A, Moy RL. Grafting following shortpulse carbon dioxide laser de-epithelialization. Dermatol Surg 1996;22:965–7. 18 Kaufmann R, Greiner D, Kippenberger S, Bernd A. Grafting of in vitro cultured melanocytes onto laserablated lesions in vitiligo. Acta Derm Venereol 1998; 78:136–8.

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19 Oh CK, Cha JH, Lim JY, et al. Treatment of vitiligo with suction epidermal grafting by the use of an ultrapulse CO2 laser with a computerized pattern generator. Dermatol Surg 2001;27:565–8. 20 Acikel C, Ulkur E, Celikoz B. Carbon dioxide laser resurfacing and thin skin grafting in the treatment of stable and recalcitrant vitiligo. Plast Reconstr Surg 2003;111:1291–8. 21 Pai GS, Vinod V, Joshi A. Efficacy of erbium YAG laserassisted autologous epidermal grafting in vitiligo. J Eur Acad Dermatol Venereol 2002;16:604–6. 22 Guerra L, Primavera G, Raskovic D, et al. Erbium:YAG laser and cultured epidermis in the surgical therapy of stable vitiligo. Arch Dermatol 2003;139:1303–10. 23 Walsh JT, Flotte TJ, Deutsch TF. Er:YAG laser ablation of tissue: effect of pulse duration and tissue type on thermal damage. Laser Surg Med 1989;9:327–37. 24 Hohenleutner U, Hohenleutner S, Baumler W, et al. Fast and effective skin ablation with Er:YAG laser: determination of ablation rates and thermal damage zones. Laser Surg Med 1997;20:242–7. 25 Sachdev M, Krupa Shankar DS. Pulsed erbium:YAG laser assisted autologous epidermal punch grafting in vitiligo. Int J Dermatol 2000;39:868–71. 26 Mutalik S. Transplantation of melanocytes by epidermal grafting: an Indian experience. J Dermatol Surg Oncol 1993;19:231–4. 27 Hann SK, Im S, Bong HW, Park YK. Treatment of stable vitiligo with autologous epidermal grafting and PUVA. J Am Acad Dermatol 1995;32:943–8. 28 van Geel N, Ongenae K, De Mil M, et al. Double-blind placebo-controlled study of autologous transplanted epidermal cell suspensions for repigmenting vitiligo. Arch Dermatol 2004;140:1203–8. 29 Lerner AB, Halaban R, Klaus SN, Morlmann GE. Transplantation of human melanocytes. J Invest Dermatol 1987;89:219–24. 30 Chen YF, Chang JS, Yang PY, et al. Transplant of cultured autologous pure melanocytes after laser-abrasion for the treatment of segmental vitiligo. J Dermatol 2000; 27:434–9. 31 Falabella R, Escobar C, Borrero I. Transplantation of in-vitro cultured epidermis bearing melanocytes for

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repigmenting vitiligo. J Am Acad Dermatol 1987;21: 257–64. Plott RT, Brysk MM, Newton RC, et al. A surgical treatment for vitiligo: autologous cultured epithelial grafts. J Dermatol Surg Oncol 1989;15:1161–6. Guerra L, Capurro S, Melchi F, et al. Treatment of “stable” vitiligo by Timedsurgery and transplantation of cultured epidermal autografts. Arch Dermatol 2000;136:1380–9. Phillips J, Gawkrodger DJ, Caddy CM, et al. Keratinocytes suppress TRP-1 expression and reduce cell number of co-cultured melanocytes – implications for grafting of patients with vitiligo. Pigment Cell Res 2001;14:116–25. Ongenae K, van Geel N, Naeyaert JM. Autologous cellular suspensions and sheets in the treatment of achromic disorders: the need for future controlled studies. Dermatology 2001;202:158–61. Chen YF, Yang PY, Hu DN, et al. Treatment of vitiligo by transplantation of cultured pure melanocyte suspension: analysis of 120 cases. J Am Acad Dermatol 2004;51:68–74. Guerra L, Primavera G, Raskovic D, et al. Permanent repigmentation of piebaldism by erbium:YAG laser and autologous cultured epidermis. Br J Dermatol 2004; 150:715–21. Halder RM, Young CM. New and emerging therapies for vitiligo. Dermatol Clin 2000;18:79–89. Mutalik S, Ginzburg A. Surgical management of stable vitiligo: a review with personal experience. Dermatol Surg 2000;26:248–54. Agrawal K, Agrawal A. Vitiligo: repigmentation with dermabrasion and thin split-thickness skin graft. Dermatol Surg 1995;21:295–300. Njoo MD, Westerhof W, Bos JD, Bossuyt PM. A systematic review of autologous transplantation methods in vitiligo. Arch Dermatol 1998;134:1543–9. Kahn AM. Surgical treatment of vitiligo. Dermatol Surg 1999;25:669. Mosher DB, Fitzpatrick TB, Ortonne JP, Hori Y. Disorders of pigmentation. In: Freedberg IM, Eisen AZ, Wolff K,, et al. (eds.) Fitzpatrick’s Dermatology in General Medicine, Vol. 1, 5th edn. New York: McGraw Hill, 1999.

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Combining medical and surgical therapies Alain Taïeb and Yvon Gauthier When to combine medical and surgical therapies? The patient with vitiligo expects a 100% cure of his/her disease. Even if a 90% cure is achieved, the results are generally considered as poor by the patients with the darkest types of skin. Physicians with experience in this field should be able to discuss expectations versus real outcome with a given patient. Even though the pathogenesis of common forms of vitiligo is far from understood [1], accumulated experience on previous duration of disease, topography of lesions, intensity (stage) of depigmentation, existence of a possible reservoir of melanocyte precursors, and usual triggering factors make it possible to predict outcome and adapt therapy adequately. Combined therapies including surgical ones provide more treatment options than monotherapies and may offer patients a supplemental benefit in their search of a relief for this psychologically devastating condition. However, evidence-based data on the many possible combined interventions in this field are limited and most authors rely heavily on their personal experience. The place of surgery in vitiligo therapy is restricted to the repigmentation of areas where melanocytes cannot be mobilized from a local reservoir or when this reservoir is already too limited to attain the goal of achieving cosmetically acceptable repigmentation. This is the case when interfollicular melanocytes have disappeared and when no alternative source can be mobilized such as hair follicules in glabrous areas and when hair follicles are already depleted in pigment cells or their precursors [2]. In such cases, repigmentation from marginal areas would still be theoretically possible. However, marginal repigmentation is limited in most types of vitiligo when there is no central supply of pigment cells. When

this phenomenon, which suggests some migration defect in vitiligo melanocytes, will be amenable to a better understanding, novel therapies will emerge. Surgical therapies are rarely used in isolation. The typical patient, whatever his/her type of vitiligo, will be proposed a first line of medical therapy to stabilize his/her condition, and the discussion concerning surgical or combined medical/surgical therapies will be conducted following this step (Table 35.1). It is however possible to bypass previous lines of treatment Table 35.1 General outline of management for vitiligo,

situating surgical interventions.

Type of vitiligo

Usual management with reference to combined medical– surgical therapies

Segmental (includes focal and mucosal)

First line: Avoidance of triggering factors, local therapies (corticosteroids, calcineurin inhibitors). Second line: Localized UVB therapy, especially excimer lamp orlaser. Third line: Consider surgical techniques if repigmentation cosmetically unsatisfactory.

Non segmental (including acrofacial)

First line: Stabilization with UVB TL01 therapy, at least 4 months. Combination with systemic/topical therapies, including reinforcement with localized UVB therapy, possible. Second line: Consider surgical techniques in non-responding areas especially with high cosmetic impact. However, Köbner phenomenon limits the persistence of grafts. Relative contraindication in areas such as dorsum of hands.

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when staging of vitiligo indicates that no reservoir is available in a stable case of at least 1 year (some authors refer to 6 months). Once surgical treatments have been implemented, further combined approaches can be considered, based on the aforementioned principle of an interdependence of marginal repigmentation and a now replenished central reservoir of melanocytes.

What are the possible combination of medical treatments? The two main modalities used in combination with surgery are phototherapy and topical treatments, mostly topical corticosteroids (TCS) and topical calcineurin inhibitors/topical immunomodulators (TIM). The published experience of systemic treatments combined with surgery is limited and will not be covered. The immunomodulatory effects of ultraviolet (UV) radiation can explain the stabilization of the local and systemic abnormal immune responses. Besides this, it is likely that UV B (UVB) TL01, similar to psoralen plus UV A (PUVA) therapy, stimulates the dopa-negative, amelanotic melanocytes in the outer hair root sheaths, which are activated to proliferate, produce melanin, and migrate outwards to adjust depigmented skin, resulting in perifollicular repigmentation. In the interfollicular compartment, UVB has a demonstrable effect on normal melanocyte proliferation [3–4]. UVA and PUVA are currently less often used, but their action is supposed to clear the immune cells in autoimmune vitiligo and to stimulate the production of melanocytes growth and melanogenic factors released by other skin cells especially keratinocytes (bFGF, basic fibroblast growth factor; EDN1, endothelin 1) and fibroblasts (SCF, stem cell factor; HGF, hepatocyte growth factor; bFGF) [5]. Innovative developments in phototherapy such as UVB local phototherapies using either narrowband 311-nm sources [6–7] or more recently laser pulsed sources (308 nm XeCl excimer, monochromatic excimer light 308nm) which can deliver higher-fluency light impacts and stimulate quiescent cells in the hair follicle without pigmenting surrounding skin [8,9]. Excimer sources pigment faster than UVB TL01 [10]. They can be

combined with TCS or TIM. Since phototherapies, especially PUVA, have a marked effect on perifollicular repigmentation, and less on marginal repigmentation [11], it could be an advantage to combine both phototherapies and TCS/TIM. Evidence-based data comes from studies which uses a combination of UVA (without psoralens) and TCS [12] and a combination of excimer laser plus tacrolimus [13]. Topical treatments have been developed to correct epidermal and dermal abnormalities aiming at improving the quality of melanocyte environment. The effect of TCS in vitiligo is generally attributed to their anti-inflammatory component limiting the T-cell infiltrate [14]. However, an effect on decreasing pigmentation and favoring migration of melanocytes is debated. Over the last 5 years, a new class of antiinflammatory molecules, named TIMs, have been on the market and used generally off label to treat various types of vitiligo. However, the effect of calcineurin inhibitors on the pigmentary system is poorly established. Calcineurin is a serine/threonine protein phosphatase regulated by Ca2
and calmodulin. Calcineurin has been extensively studied for its role in signal transduction during T-cell activation. Calcineurin has been shown to be a common receptor for two immunophilin–immunosuppressant complexes, cyclophilin A–cyclosporin A (CyPA–CsA) and FK506–FK506 (tacrolimusbinding protein, FKBP). The binding of CyPA-CsA or FKBP-FK506 inhibits the calcium-dependent dephosphorylation of the transcription factor nuclear factor of activated T-cell (NF-AT) by calcineurin, thus blocking T-cell receptor-mediated cytokine transcription and T-cell activation. Besides immune and inflammatory responses mediated by T-cells, a variety of non-immune cells express calcineurin and members of the NF-AT family which control other functions including regulation of the second messenger cAMP pathway, Na
/K
ion transportation in the nephron, cell cycle progression in lower eukaryotes, cardiac hypertrophy, and memory formation [15]. In human skin, the presence of calcineurin has been demonstrated by immunohistochemical techniques. Its function is currently unknown, but it should participate in the balance between kinases and phosphatases critical for signal transduction [16].

Combining medical and surgical therapies Calcineurin inhibitors such as cyclosporin (CsA) and tacrolimus have been used systemically at high dosage in transplant patients for many years and their interference with the pigmentary system seems at best minimal based on clinical grounds. Dermatologists have submitted thousands of patients with psoriasis or atopic dermatitis to such agents at lower dosages without noticing obvious pigmentary side effects. However, after oral administration of CsA, the skin is exposed to high concentrations because CsA accumulates in epidermis and can reach levels more than 10-fold greater than serum trough levels. The concentration of CsA in the epidermis has been measured around 1–3 g/ml (0.83–2.4–9 M) [17]. The interpretation of cases of skin hyperpigmentation under cyclosporin is not very convincing due to simultaneous diseases or treatments [18,19]. Opposite effects have been claimed for hair pigmentation, darkening [20], or poliosis [21]. The introduction of topical formulations in this class of drugs (now referred to as TIMs) has prompted studies in orphan pigmentation disorders such as vitiligo based on a mostly T-cell putative target [22,23] and triggered the report of side effects related to the pigmentary system. Little is known concerning the effect of calcineurin inhibitors on melanocytes. In vitro data might be biased by culture systems using mitogens interfering with cell physiology as well as drug effects. In a phorbol ester containing culture media, CsA inhibited melanocyte proliferation in a dose-dependent manner in the concentration range of 0.1–10 M, as well as tyrosinase activity. It is not yet known whether CsA exerts its effect through calcineurin/ NF-AT, TPA-induced protein kinase C (PKC) pathways or other pathways [24]. Melanocytes express calcineurin A and B subunits as well as various cyclophilins and FKBP, and in melanoma cells only (not in normal melanocytes) calcineurin enzymatic activity is inhibited by CsA [16]. Due to these conflicting data, the repigmenting effects of tacrolimus observed in vitiligo have been attributed by most authors to an effect on Tlymphocytes cytokines, especially via the inhibition of TNF production which inhibits melanogenesis and could target a T-lymphocyte attack via cell surface ICAM-1 expression [23]. However, the repigmenting

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influence of tacrolimus seems restricted to thin skin, and suggests also an influence of epidermal thickness of molecular targets [22]. Along this line, an effect of TIM on keratinocytes, which could secondly influence melanocytes by paracrine factors, has been studied by Lan et al., from Taiwan [25]. In this elegant paper, the authors show that cultures of normal human keratinocytes treated with tacrolimus stimulate melanoblast and melanocyte mouse cell lines to proliferate and to migrate, and identify respectively SCF and basement membrane gelatinase MMP9 as putative intermediates. Indeed, clinical experience with both TCS and TIMs suggests that these two classes of drugs influence mostly interfollicular and to a lesser extent marginal repigmentation [11], suggesting that these are only helpful in the early stages of the disease when melanocytes are still present in the epidermis. Vitamin D analogs have been advocated on the basis of a defective calcium transport in melanocytes and keratinocytes in vitiligo skin, but their efficacy in monotherapy and even in combination with UV light is not established convincingly. The combination to surgical therapies is not sufficiently documented.

Combining surgical and medical therapies This approach is used as a synergistic treatment to enhance the repopulation by melanocytes of areas of vitiliginous skin situated between autologous grafted material, whatever its nature (thin Thiersch’s graft, grafting with the suction blister technique, minigrafting, punch grafting, epidermal sheet grafts, and skin culture – autologous or fetal containing both keratinocytes and melanocytes or melanocytes alone followed by grafting).

UVB TL01 combined with surgical treatment van Geel et al. [26] investigated the efficacy of epidermal non-cultured cellular grafting in patients with vitiligo and the role of post-inflammatory, spontaneous, or UVB TL01-induced pigmentation in obtaining repigmentation. They used a prospective, randomized, double-blind, placebo-controlled study and evaluated, in 28 patients, a total of 33 paired,

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symmetrically distributed leukodermic resions, resistant to therapy, the major criterion to enter the study. However, 19 patients were classified as stable vitiligo, whereas 9 other patients had unstable or less stable disease. After laser ablation, a hyaluronic acid-enriched cellular graft was applied to a target lesion while the paired lesion received placebo (non-cellular dressing). Three weeks later all lesions were exposed to UVB TL01 irradiation twice per week for approximately 2 months. The percentage of repigmentation was assessed at 3, 6, and 12 months using a digital image analysis system. The repigmentation pattern was also evaluated after 1 and 3 months. The authors reported a strongly significant difference between cellular grafts and placebo after 3, 6, and 12 months. In stable patients, repigmentation of at least 70% of the treated area was achieved in 55%, 57%, and 77% of the actively treated lesions at 3, 6, and 12 months after treatment, whereas repigmentation of at least 70% of the treated area was not observed at any point of time for the unstable group. The repigmentation pattern was diffuse in 94% of the responding patients. The authors conclude that after a strict preoperative selection for disease stability, transplantation resulted in repigmentation of at least 70% of the treated area. They could demonstrate with this trial design that repigmentation was primarily caused by the transplanted melanocytes, because UV treatment alone could not achieve the same level of repigmentation. This study is important because it underlines the need for a good stabilization of vitiligo before proceeding to surgery. The synergistic influence of UV is however difficult to assess due to the absence of a third arm in the trial design (grafts without UV).

PUVA versus topical corticosteroids combined with surgical treatment Barman et al. (India) reported a study designed to evaluate the efficacy of TCS in perigraft pigmentation and to compare it with perigraft pigmentation after PUVA in patients with stable vitiligo [27]. They enrolled 50 patients having static lesions of segmental vitiligo (50%), vitiligo vulgaris, and acrofacial vitiligo with a residual limited number of lesions where there was no further response to various medical treatment and without a history of

new lesions or an extension of old lesions for the last 6 months or more. All patients were punch grafted. The combinations of punches used were 2.5 mm and 2 mm or 3 and 2.5 mm for the donor and the recipient sites, respectively. Postgraft dressing was done with framycetin tulle, which was removed on the 8th day. All of the patients received analgesics and prophylactic systemic antibiotics for 3–5 days. One group (group I) received systemic PUVA and another group (group II) received TCS (fluocinolone acetonide 0.1%). PUVA therapy or TCS were started after 4 weeks of grafting in respective groups. Due to patient loss during the study, 42 patients were actually evaluated for pigment spread and side effects. In group I, the average pigment spread was 6.38 mm, whereas in group II, it was 6.94 mm, showing a slightly higher pigment spread in group II, which was statistically not significant (P  0.301). There was no difference in response to therapy in patients having segmental vitiligo as compared with non-segmental vitiligo. Cobblestoning, depigmentation of the grafts, infection, and graft displacement were the important side effects seen in some patients in both the groups. The authors concluded that the pigment spread with TCS is comparable to that with PUVA. All patients do not achieve the optimal color match in both the groups, although some amount of hypopigmentation or hyperpigmentation was considered acceptable. The authors conclude that the pigment spread with TCS gives similar results as with oral PUVA. The use of TCS is much less cumbersome, is cheap and easy to administer, and is a home therapy as compared with PUVA which can cause vomiting and nausea. Table 35.2 compares pigment spread between the two arms of treatment as reported in this study. The rate of pigment spread with photochemotherapy in this study is also comparable with the earlier non-controlled studies [28,29]. When punch grafting has been followed by PUVA/PUVASOL therapy, Savant noticed a 5–10-mm pigment spread at the end of 2.5–3 months [29], and Singh and Bajaj showed a 5–12-mm pigment spread in 71.04% cases after 6 months [30]. Like the previous study [25], the study by Barman et al. [27] lacks an arm to assess the outcome of the grafts without adjuvant therapy.

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Table 35.2 Comparison of mean pigment spread in two groups: values are expressed in millimeters. Duration of follow-up after adjuvant therapy

Punch grafting
PUVA (group I)

Punch grafting
topical fluocinolone acetonide (group II)

P-value

1st month

4.06  1.14

4.49  1.21

0.1363

2nd month

5.08  1.49

5.75  1.34

0.0716

3rd month

5.95  1.62

6.50  1.45

0.1205

4th month

6.33  1.78

6.86  1.73

0.351

5th month

6.38  1.6941

6.94  1.63

0.301

(Reprinted from Barman et al. [27], with permission from Blackwell Publishing.)

Especially in dark skin types, the risk of hyperpigmentation when transplantation procedure is combined with UVB or PUVA should be discussed with the patient. A recent Indian study showed a statistically significantly better stability and color match of repigmentation with surrounding skin was seen in UVB TL01-treated patients when compared to PUVA-treated patients [28].

Better designed studies are needed to implement evidence-based recommendations using combined modalities. It is however possible to recommend, based on indirect evidence, the adjunction of either UVB 311 nm (and by approximation excimer light 308 nm which exerts more potent effects) or of TCS/TIM to surgical techniques, in order to boost the repigmentation after surgical vitiligo procedures.

Other possible combinations with surgery and personal experience

References

Based on published data, several other options remain to be tested. Of the most recent and promising, either localized UVB delivered by excimer sources or TIM or even combined approaches need to be investigated in a controlled manner as adjuncts to surgical procedures. Surgery itself can proceed using combined steps [31]. In our department, Dr. Gauthier has already tested the combination of ultra-thin grafts [33] with the already published suction blister technique developed in Bordeaux [33]. This combinatory surgery allows to treat complicated jigsaw lesional areas with a minimal risk at donor sites, and can be combined with UV and topical treatments.

Conclusion Combining surgical and medical modalities in selected cases of vitiligo can be very effective in obtaining complete repigmentation, but large inter-individual differences occur, some of which may be partly related to the stage of the disease, which is difficult to assess and not taken into account in many reports.

1 Gauthier Y, Cario Andre M, Taieb A. A critical appraisal of vitiligo etiologic theories. Is melanocyte loss a melanocytorrhagy? Pigment Cell Res 2003;16:322–32. 2 Arrunategui A, Arroyo C, Gracia L, et al. Melanocyte reservoir in vitiligo. Int J Dermatol 1994;33:484–7. 3 Bessou S, Surleve-Bazeille JE, Sorbier E, Taieb A. Ex vivo reconstruction of the epidermis with melanocytes and the influence of UVB. Pigment Cell Res 1995;8:241–9. 4 Bessou S, Surleve-Bazeille JE, Pain C, et al. Ex vivo study of skin phototypes. J Invest Dermatol 1996;107: 684–8. 5 Imokawa G, Miyagishi M, Yada Y. Endothelin-1 as a new melanogen: coordinated expression of its gene and the tyrosinase gene in UVB-exposed human epidermis. J Invest Dermatol 1995;105:32–7. 6 Lotti TM, Menchini G, Andreassi L. UVB radiation microphototherapy: an elective treatment for segmental vitiligo. J Eur Acad Dermatol Venereol 1999;13:102–8. 7 Menchini G, Tsoureli-Nikita E, Hercogova J. Narrowband UV-B microphototherapy: a new treatment for vitiligo. J Eur Acad Dermatol Venereol 2003;17:171–7. 8 Leone G, Iacovelli P, Paro Vidolin A, Picardo M. Monochromatic excimer light 308 nm in the treatment

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18

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of vitiligo: a pilot study. J Eur Acad Dermatol Venereol 2003;17:531–7. Esposito M, Soda R, Costanzo A, Chimenti S. Treatment of vitiligo with the 308 nm excimer laser. Clin Exp Dermatol 2004;29:133–7. Hong SB, Park HH, Lee MH. Short-term effects of 308nm xenon-chloride excimer laser and narrow-band ultraviolet B in the treatment of vitiligo: a comparative study. J Korean Med Sci 2005;20:273–8. Parsad D, Pandhi R, Dogra S, Kumar B. Clinical study of repigmentation patterns with different treatment modalities and their correlation with speed and stability of repigmentation in 352 vitiliginous patches. J Am Acad Dermatol 2004;50:63–7. Westerhof W, Nieuweboer-Krobotova L. Treatment of vitiligo with UV-B radiation vs topical psoralen plus UV-A. Arch Dermatol 1997;133:1525–8. Passeron T, Ostovari N, Zakaria W, Fontas E, Larrouy JC, Lacour JP, Ortonne JP. Topical tacrolimus and the 308nm excimer laser: a synergistic combination for the treatment of vitiligo. Arch Dermatol 2004;140:1065–9. Westerhof W, Nieuweboer-Krobotova L, Mulder PG, Glazenburg EJ. Left–right comparison study of the combination of fluticasone propionate and UV-A vs. either fluticasone propionate or UV-A alone for the long-term treatment of vitiligo. Arch Dermatol 1999;135:1061–6. Huai Q, Kim HY, Liu Y, et al. Crystal structure of calcineurin–cyclophilin–cyclosporin shows common but distinct recognition of immunophilin–drug complexes. Proc Natl Acad Sci USA 2002;99:12037–42. Smit N, Sellar K, Romijn F, et al. A study of calcineurin function in epidermal and melanoma cell cultures. Pigm Cell Res 2004;17:575 (abstract). Fisher GJ, Duell EA, Nickoloff BJ, et al. Levels of cyclosporin in epidermis of treated psoriasis patients differentially inhibit growth of keratinocytes cultured in serum free versus serum containing media. J Invest Dermatol 1986;91:142–6. Brady AJ, Wing AJ. Hyperpigmentation due to cyclosporin therapy. Nephrol Dial Transplant 1989;4: 309–10. Ozkaya-Bayazit E, Diz-Kucukkaya R, Akasya E, et al. Bullous acral erythema and concomitant pigmentation on the face and occluded skin. J Eur Acad Dermatol Venereol 2000;14:139–40. Rebora A, Delmonte S, Parodi A. Cyclosporin Ainduced hair darkening. Int J Dermatol 1999;38:229–30.

21 Asensio V, del Pozo LJ, Asensio M, Lerida MT. Megalotrichiasis and poliosis caused by cyclosporin A. Med Clin (Barc) 1991;97:39. 22 Lepe V, Moncada B, Castanedo-Cazares JP, et al. A double-blind randomized trial of 0.1% tacrolimus vs 0.05% clobetasol for the treatment of childhood vitiligo. Arch Dermatol 2003;139:581–5. 23 Grimes PE, Morris R, Avaniss-Aghajani E, et al. Topical tacrolimus therapy for vitiligo: therapeutic responses and skin messenger RNA expression of proinflammatory cytokines. J Am Acad Dermatol 2004;51:52–61. 24 Lee JY, Kang WH. Effect of cyclosporin A on melanogenesis in cultured human melanocytes. Pigment Cell Res 2003;16:504–8. 25 Lan CC, Chen GS, Chiou MH, et al. FK506 promotes melanocyte and melanoblast growth and creates a favourable milieu for cell migration via keratinocytes: possible mechanisms of how tacrolimus ointment induces repigmentation in patients with vitiligo. Br J Dermatol 2005;153:498–505. 26 van Geel N, Ongenae K, De Mil M, et al. Double-blind placebo-controlled study of autologous transplanted epidermal cell suspensions for repigmenting vitiligo. Arch Dermatol 2004;140:1203–8. 27 Barman KD, Khaitan BK, Verma KK. A comparative study of punch grafting followed by topical corticosteroid versus punch grafting followed by PUVA therapy in stable vitiligo. Dermatol Surg 2004;30:49–53. 28 Parsad D, Kanwar AJ, Kumar B. Psoralen-ultraviolet A vs. narrow-band ultraviolet B phototherapy for the treatment of vitiligo. J Eur Acad Dermatol Venereol 2006;20:175–7. 29 Savant SS. Autologous miniature punch skin grafting in stable vitiligo. Indian J Dermatol Venereol Leprol 1992; 58:310–4. 30 Singh KG, Bajaj AK. Autologous miniature skin punch grafting in vitiligo. Indian J Dermatol Venereol Leprol 1995;61:77–80. 31 Falabella R, Barona M, Escobar C, et al. Surgical combination therapy for vitiligo and piebaldism. Dermatol Surg 1995;21:852–7. 32 Olsson MJ, Juhlin L. Epidermal sheet grafts for repigmentation of vitiligo and piebaldism, with a review of surgical techniques. Acta Derm Venereol 1997;77:463–6. 33 Gauthier Y, Surleve-Bazeille JE. Autologous grafting with noncultured melanocytes: a simplified method for treatment of depigmented lesions. J Am Acad Dermatol 1992;26:191–4.

CHAPTER 36

Surgical depigmentation of vitiligo: bleaching cream, laser and cryosurgery Monique R.T.M. Thissen

Vitiligo, a common acquired skin disorder, is a serious problem because patients with this disease exhibit depigmented areas on their skin that are very sensitive to sunburns and otherwise may lead to tremendous cosmetic and psychosocial problems [1]. There are several treatment options, like UV therapy, topical steroids, and pigment cell transplantation, aimed at creating repigmentation [2]. In case of extensive vitiligo – the so-called vitiligo universalis which is not a very common type of vitiligo – the loss of pigment may be so extreme that restoring or stabilizing the depigmentation process with several therapies is not amenable [3,4]. In these patients, the remaining pigmented parts of the skin could be further treated with depigmentation therapies to remove the pigment remnants to obtain at least a uniform skin color [2]. In this chapter different techniques for depigmentation, like topical bleaching cream, laser therapy, and cryotherapy, will be described.

the rest of his life. The patient might not be able to repigment when a cure for vitiligo may become available in future. These considerations should be taken into account. In vitiligo the disappearance of melanocytes, and thus the appearance of depigmented patches is sometimes related to micro-traumata to the skin. This is called Köbner phenomenon. The presence of this Köbner phenomenon, which is more pronounced in progressive than in stable vitiligo, may be a positive predictive value for successfulness of laser and maybe also cryotherapy-induced depigmentation [5–8]. Patients also have to know that with some therapies it takes a long time to achieve depigmentation which may even not be permanent. After therapy repigmentation may occur, either spontaneously or sunlight/UV-induced leading to irregular leukoderma or follicular pigmentation [7].

Depigmentation techniques Depigmentation: the moment of the decision and indications When none of the therapy options for repigmentation is effective and the skin has already become depigmented over 80%, both dermatologist and patient may decide to attempt removing the remaining pigmentations. This may be seen especially in patients with a dark-skin type and when the residual pigmented areas are located on the face and hands. However, if the depigmentation therapy is successful, it will be a point of no return and the patient has to realize this well. The absence of pigmentation protection from the sun will be necessary for

Bleaching cream Destroying the melanocytes in the residual pigmentations can be realized by daily topical application of bleaching creams. This is the oldest method to obtain depigmentation. There is a significant relation between the duration of use of the creams and the effectiveness; longer usage leads to better results. The active substances in these creams are either monobenzylether of hydroquinone (MBEH) or 4-methoxyphenol (4-MP), the latter also called “p-hydroxyanisole,” “mequinol” or “monomethylether of hydroquinone” [7,9,10]. Both MBEH and 4-MP exhibit melanocytotoxic properties by a variety of actions [11,12,13]. Due to

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the deeper localization the melanocytes in the hair follicles seem to be more difficult to destroy compared to their epidermal equivalents, so this can be the explanation for the follicular repigmentation pattern sometimes observed after the treatment has been terminated [11]. There is a long-term, worldwide experience with these creams and although the use of it may frequently result in total depigmentation or otherwise a satisfying effect for the patient, there is a serious risk for cutaneous and ocular side effects [14,15]. A small fraction of these substances will reach the blood circulation after transcutaneous uptake and may affect other melanocyte-containing structures like eyes and neurocutaneous tissue resulting in local damage. Cutaneous side effects such as severe erythema, burning and itching sensations are seen frequently. However, hypothetically this inflammatory response might also contribute to further destruction of the melanocytes. To minimize these risks the cream should be applied only in small amounts on well-circumscribed parts of the body. This means that it often takes several months or even years before the skin will be totally depigmented. The final efficacy of both MBEH and 4-MP seems to be equal but visible depigmentation with 4-MP begins between 4 and 12 months after starting treatment whereas with MBEH the bleaching effect becomes evident after 1 month [9]. Adverse skin reactions on the other hand are less common and less severe with 4-MP compared to MBEH [14]. Both 4-MP and MBEH can result in incomplete depigmentations showing irregular leukodermas [16,17]. There are also advantages of using bleaching substances to depigment the skin. Besides the fact that the therapy is cheap and useful for all skin types at any age, the creams are easy to apply and treatment can be done at home.

Laser therapy In a certain number of patients the desired permanent depigmentation cannot be achieved at all with bleaching substances. Together with the other disadvantages of bleaching creams different options for removing the pigment remnants have been

investigated. One of these is the use of lasers, especially those capable of creating selective damage to melanocytes and melanin-containing tissue while leaving the rest of the skin unharmed. In general it is known that the higher the wavelength of the emitted laser light is, the deeper this light will penetrate in the tissue. In the skin there are several substances acting as chromophores picking up different wavelengths of laser light, sometimes competing with each other. For the chromophore melanin the absorption of the laser light is most optimal above 600 nm (lower wavelengths are better absorbed by blood and for wavelengths greater than 900 the absorption coefficient becomes to low) [18]. In the so-called Q-switched laser technology the laser light is emitted in small fractions with a pulse duration of several nanoseconds. The duration of the energy pulse is shorter than the natural thermal relaxation time of the melanosomes. This means that no energy (i.e. heat) is transduced from these melanosomes into surrounding tissues, thus destroying melanin-containing structures and sparing adjacent structures from cell damage and scar formation [18]. Clinically both the Q-switched ruby laser (694-nm wavelength) and the Q-switched alexandrite (755-nm wavelength) can be used for depigmentation therapy [6,7,19]. Theoretically the light of the Q-switched alexandrite laser can penetrate deeper into the skin because of the higher wavelength. Therefore, this equipment may be more effective for depigmentation because of reaching both the superficial and the deeper located melanocytes in the hair follicles [20]. The energy intensity which is necessary to destroy the pigment depends on the original skin type and on the type of laser equipment used [6,7,19]. The number of treatment sessions needed to achieve total depigmentation depends on the size of the areas to be treated. Generally the area becomes depigmented after a single treatment. Before treating large areas it is recommended to start with a test-spot (e.g. 5 cm2) and evaluate the effect after 2 months to check if the obtained depigmentation is permanent. Laser therapy is applicable for every skin type. Great advantages of laser treatment are the rapid onset of depigmentation within 4–7 days after

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treatment and the possibility to depigment relatively large areas at once. The treatment may be painful which could be a reason to minimize the size of the treated area. Otherwise, local anesthesia can be used to diminish the pain without interfering with the final effectiveness of the treatment [7]. Another limitation is that, unlike the use of bleaching creams, laser therapy is possible only in the hospital. In addition, laser treatment is much more expensive than bleaching creams.

which can be applied in an outpatient setting. Specific aftercare is not required except topical antiseptics in case of blistering and there is a minimal risk for infections or other complications. Only those persons with a history of cryoglobulinemia should not be treated in this way. It is recommended to start cryotherapy with a test-area.

Cryotherapy

1 Nordlund JJ, Ortonne JP. Vitiligo vulgaris. In: Nordlund JJ, Boissy RE, Hearing VJ, et al (eds.) The Pigmentary System: Physiology and Pathophysiology. New York: Oxford University Press, 1998;513–51. 2 Njoo MD, Westerhof W, Bos JD, Bossuyt PM. The development of guidelines for the treatment of vitiligo. Arch Dermatol 1999;135:1514–21. 3 Njoo MD, Spuls PI, Bos JD, et al. Nonsurgical repigmentation therapies in vitiligo: meta-analysis of the literature. Arch Dermatol 1998;134:1532–40. 4 Njoo MD, Westerhof W, Bos JD, Bossuyt PM. A systematic review of autologous transplantation methods in vitiligo. Arch Dermatol 1998;134:1543–9. 5 Njoo MD, Das PK, Bos JD, Westerhof W. Association of the Köebner phenomenon with disease activity and therapeutic responsiveness in vitiligo vulgaris. Arch Dermatol 1999;135:407–13. 6 Thissen MR, Westerhof W. Laser treatment for further depigmentation in vitiligo. Int J Dermatol 1997;36: 386–8. 7 Njoo MD, Vodegel RM, Westerhof W. Depigmentation therapy in vitiligo universalis with topical 4-methoxyphenol and the Q-switched ruby laser. J Am Acad Dermatol 2000;42:760–9. 8 Radmanesh M. Depigmentation of the normally pigmented patches in universal vitiligo patients by cryotherapy. J Eur Acad Dermatol Venereol 2000;14:149–52. 9 Mosher DB, Parish JA, Fitzpatrick TB. Monobenzylether of hydroquinone: a retrospective study of treatment of 18 vitiligo patients and a review of the literature. Br J Dermatol 1977;97:669–79. 10 Grimes PE. Vitiligo: an overview of therapeutic approaches. Dermatol Clin 1993;11:325–8. 11 Riley PA. Hydroxyanisole depigmentation: in vivo studies. J Pathol 1969;97:185–91. 12 Riley PA. Hydroxyanisole depigmentation: in vivo studies. J Pathol 1969;97:193–206. 13 Riley PA. Mechanism of pigment cell toxicity produced by hydroxyanisole. J Pathol 1970;101:163–9.

A third method to obtain a depigmented skin is cryotherapy. Application of liquid nitrogen is used for many indications in dermatology. Compared to other cellular components of the skin-like keratinocytes, fibroblasts and vascular structures especially the melanocytes can be easily and rather selectively destroyed by cryotherapy [21,22]. For this reason one of the adverse results after cryotherapy is a permanent leukoderma in the treated area [23]. In vitiligo skin the melanocytes are more prone to physical (thermal) damage than those in non-vitiligo normal skin. This might be partially due to Köbner phenomenon. The easiest way to treat is by using closed cryoprobes in different diameters according to the size and location of the area to be treated. The liquid nitrogen should be applied until a small white frosted rim is seen on the skin just outside the probe. For depigmentation only a single freezethaw cycle is advised. In cases of larger pigmented areas the treatment is best performed in several sessions with intervals of 1–3 weeks guided by the personal discomfort after treatment like edema, pain, blistering, and time needed to heal. Of course patients have to be informed about the healing process and the fact that the final cosmetic aspect will be obtained in 4 weeks after the therapy. In general, the depigmentation will be permanent although it might be necessary to repeat the treatment once or more with intervals of 4–6 weeks [8]. If the therapy is performed by experienced hands the results will be a smooth depigmentation without scarring or damage to the surrounding structures. Cryotherapy is a safe and cost-effective method

References

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14 Nordlund JJ, Forget B, Kirkwood J, Lerner AB. Dermatitis produced by applications of monobenzone in patients with active vitiligo. Arch Dermatol 1985; 121:1141–4. 15 Hedges TR, Kenyon KR, Hanninen LA, Mosher DB. Corneal and conjunctival effects of monobenzone in patients with vitiligo. Arch Ophthalmol 1983;101:64–8. 16 Colomb D. Depigmentation en confettis après application de Leucodinine B sur chloasma. Ann Dermatol Venereol 1982;109:899–900. 17 Boyle J, Kennedy CT. Leucoderma induced by monomethylether of hydroquinone. Clin Exp Dermatol 1985;10:154–8. 18 Goldman MP, Fitzpatrick RE. Laser-tissue interaction. Cutaneous Laser Surgery, St. Louis: Mosby Yearbook, 1994;1–18.

19 Kim YJ, Chung BS, Choi KC. Depigmentation therapy with Q-switched ruby laser after tanning in vitiligo universalis. Dermatol Surg 2001;27:969–70. 20 Rao J, Fitzpatrick RE. Use of the Q-switched 755-nm alexandrite laser to treat recalcitrant pigment after depigmentation therapy for vitiligo. Dermatol Surg 2004;30:1043–5. 21 Kuflik GE. Cryosurgery updated. J Am Acad Dermatol 1994;31:925–44. 22 Zouboulis CC. Cryosurgery in dermatology. Eur J Dermatol 1998;8:466–74. 23 Zabriskie NA, Nordlund JJ, Nerad JA. Unusual skin depigmentation following eyelid cryosurgery. Ophthal Plast Reconstr Surg 1996;12:296–8.

CHAPTER 37

Future directions in surgical management of vitiligo Yvon Gauthier

At present, we have a long experience of surgical management of vitiligo whatever technique used. We have a good knowledge of the limitations and adverse effects of melanocyte transplantation. However, it is always difficult to predict the future directions of a treatment destined to a disease with unclear pathogenesis. Nevertheless, we are beginning to try to analyze what are the current problems frequently encountered in surgical management of vitiligo and what would be the potential improvements in these techniques. Due to the time-consuming nature of surgical therapies these treatment regimens are most often limited to segmental or localized vitiligo although they can be successful in some cases of generalized vitiligo also. Surgical modalities may be considered in inactive, non-progressive disease only. Areas such as the ankles, dorsal fingers, and the dorsum of the feet tend not to repigment as well because of Köbnerization, which can occur on the grafted macules. So the ideal patient for melanocyte transplantation has localized, non-progressing patches of vitiligo in a non-acral location. In our opinion, the main future directions in surgical management could be: increasing the effectiveness, increasing the safety by minimizing the adverse effects, improvement of the outcome, and the permanence of the repigmentation.

explanation for some poor results. The aim of medical treatment might be to improve or to suppress the unfriendly melanocyte environment in the vitiligo skin, since transplantation of melanocytes does not alter the supposed underlying causes in vitiligo: autoimmune, neural, or cytotoxic processes. We hope that a true etiological medical treatment will be developed in the next few years.

Improvement of melanocyte proliferation and migration Halo phenomenon and also small white spots between the transplanted areas and the surrounding normal appearing skin are frequently observed after melanocyte transplantation [1]. This white border is often difficult to repigment by any means (Fig. 37.1). The coalescence phenomenon between the grafted melanocytes and also with the melanocytes of the border zone is a complex and still mysterious process. On piebald guinea pig skin we have previously

Increasing effectiveness Improvement of the melanocyte environment A better knowledge of vitiligo pathogenesis will allow us to understand why repigmentation failed to be produced in some cases and what was the

Fig. 37.1 White rim after basal cell layer suspension

transplant on achromic macules post-halo nevus.

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(A) (A)

(B) Fig. 37.2 After 4 months good coalescence and spread-

ing of grafted areas on piebald guinea pig skin after autologous basal cell layer suspension transplant (distance between the grafted areas not exceeding 8 mm).

observed that the spontaneous coalescence between some black grafted spots occurred only if the distance between them was not exceeding approximately 8 mm (Figs. 37.2 and 37.3). The induction of melanocyte migration may involve complex mechanisms including chemokinesis, chemotaxis, haplotaxis, cell-to-cell adhesion, and cell matrix adhesion [2]. Many investigators have reported that certain factors such as basic fibroblast growth factor, endothelin, stem cell factors, leukotriene, TGF
, and type IV collagen can induce melanocyte migration. Endothelin and basic fibroblast growth factor are usually produced by keratinocytes particularly after ultraviolet (UV) stimuli [3]. These considerations convinced us that the use of basal layer suspension including keratinocytes and melanocytes could be more appropriate than the use of pure cultured melanocytes. Keratinocytes could regulate melanocyte growth

(B) Fig. 37.3 After 4 months no coalescence of grafted areas

after autologous basal cell layer suspension transplant (distance between the grafted areas exceeding 8 mm).

and differentiation and as well give a good melanocyte–keratinocytes ratio. Guerra et al. [4] determined that appropriate melanocyte–keratinocyte ratio is maintained in cell culture when primary keratinocytes are seeded at a density of 4  104 cells/cm2. A good and appropriate ratio was maintained if keratinocytes were subcultivated 1–2 days after reaching confluence and reseeded at a density of at least 4  104 cells/cm2. These secondary cultures have been proven useful in treating large areas of vitiligo [4]. With basal cell layer suspension method the donor site can cover an achromic area 5–8 times larger. During repigmentation vitiligo macules usually retain a white border and UV can rarely induce slight pigmentation from the margin inside the white patch. This clinical observation led some authors to

Future directions in surgical management of vitiligo

279

Adverse effects are usually observed after skin transplant (minigrafting technique, suction blister epidermal grafting technique, and ultra-thin epidermal sheet technique) but not after cultured or non-cultured cell transplants [7]. The majority of these adverse effects might be avoided in the future. • At donor site: An appropriate dressing and a good stabilization of vitiligo by medical treatment could inhibit scar or keloid formation and also prevent the onset of hypopigmentation. • At recipient site: Cobblestone appearance of the graft was a specific adverse effect of minigrafting technique which can easily be avoided by making holes deeper than the thickness of the graft. Milia and partial loss of grafts were the most common adverse effects with ultra-thin epidermal sheet techniques. To prevent partial loss of grafts a good immobilization of mobile sites is needed during first 2 weeks. A case report has also documented transmission of human papillomavirus (HPV) infection (verruca) from operator’s finger to the vitiligo lesion treated with suction blister epidermal grafting [8].

In vitiligo vulgaris skin we have reported detachment from the basement membrane and transepidermal elimination of melanocytes following minor mechanical traumas in normally pigmented skin [9,10]. A similar phenomenon could occur on grafted areas located on the extremities, and submitted to repeated and strong frictions. We propose that this transepidermal elimination in vitiligo should be regarded as a possible mechanism of the chronic loss of pigment cells, as the melanocyte adhesion system is less well organized and far weaker than the system which firmly holds epidermal keratinocytes bounded to each other and to the basement membrane. Interactions between melanocytes and the basement membrane are mediated by integrins [11] and interactions between melanocytes and keratinocytes are mediated by cadherins [12] in association with -catenin which has a key role in the development of the melanocytes lineage and a further role in intracellular signaling and gene transcription [13]. To date, no marked differences have been found in the overall level of expression of integrins/cadherins between control, non-lesional, or lesional vitiligo vulgaris skin. However, the tissue level of divalent cations, which are supposed to play important roles, has, to our knowledge, still not been investigated. Further studies are needed to determine why melanocytes detach from the basement membrane and how the triggering or precipitating factors (immunological, neural, and impaired redox status) could act. So in the future, it would to be hoped that the adhesiveness of grafted melanocytes could be improved to obtain a permanent repigmentation in all cases.

Improvement of the outcome: permanence of the repigmentation

Future indications of surgical management of vitiligo

The question always arises whether the repigmentation induced by grafting methods is permanent or not. Some patients with segmental vitiligo macules have retained the repigmentation achieved at the end of a long-term follow-up period. On the contrary, in patients with vitiligo vulgaris, sometimes there is reactivation of the disease that may lead to a secondary failure of the treated skin due usually to the Köbner phenomenon.

Good indications of surgical therapies

hypothesize that there was some inhibitory mechanism for melanocyte migration from the margin [5]. But Le Poole [6] in a study performed with an organotypic culture of human skin has not found any inherent migration defect which would be responsible for impaired repigmentation of vitiligo macule. Further investigations are needed to elucidate the problem.

Increasing the safety by minimizing the adverse effects

Segmental vitiligo is the least common pattern and occurs in a dermatomal or quasi-dermatomal distribution. It will remain an excellent indication because it almost always responds with complete repigmentation in the transplanted area, regardless of the transplant method used. Moreover, segmental vitiligo retain all their repigmentation after long-term follow-up [1]. The full repigmentation of

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large segmental vitiligo areas will need several sessions of melanocyte transplantations. Ultra-thin epidermal sheet transplantation will be chosen for the treatment of areas easy to immobilize. Basal cell layer suspension transplant will be used on achromic lesions located on the periorificial areas of the face and in areas with greater movements.

Indications of surgical therapies In a lesion of vitiligo vulgaris, it is more difficult to achieve full repigmentation, but it may respond well if the disease is not progressive. Selection of patients in the group of vitiligo vulgaris is crucial. Good signs when decisions are to be made about transplant will be: spontaneous repigmentation of some areas, no appearance of new spots during the last few years, short duration time, and not very extensive total vitiligo area.

Contraindications of surgical therapies Patients with extensive vitiligo vulgaris and those who have not had completely stable, non-progressive vitiligo for at least 2 years should not be chosen for transplantation. Surgical therapies might be avoided on vitiligo areas subject to strong frictions after transplantation, for example, during professional activities because the risk of secondary depigmentation is very high.

Conclusion Finally, we think that the future advances in medical treatment could significantly facilitate the surgical management of vitiligo. The main objective of medical therapies could be the stabilization of the disease and the repigmentation of the macules where there were still remaining melanocytes located in the epidermal and follicular reservoirs. In any case, there will always be a “niche” for surgical management of vitiligo patches with total loss of melanocytes, affecting reasonably large areas.

References 1 Olsson MJ, Juhlin L. Long-term follow-up of leucoderma patients treated with transplants of autologous cultured melanocyte, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol 2002;147: 893–904. 2 Norris DA, Horikawa T, Morelli JG. Melanocyte destruction and repopulation in vitiligo. Pigm Cell Res 1994;7:193–203. 3 Yohn JJ, Morelli JG, Walchak JJ, et al. Cultured human keratinocytes synthesize and secrete endothelin 1. J Invest Dermatol 1993;100:23–6. 4 Guerra L, Capurro S, Melchi F, et al. Treatment of “stable” vitiligo by Timedsurgery and transplantation of cultured epidermal autografts. Arch Dermatol 2000; 136:1380–9. 5 O’Donnel DB, Saddler M, et al. Tenascin is abundant in the skin of vitiligo patients. J Invest Dermatol 1992;9:620 [Abstract]. 6 Le Poole C, Van den Wijngaard R, Westerhof W, et al. Organotypic culture of human skin to study melanocyte migration. Pigm Cell Res 1994;7:33–43. 7 Njo D, Westerhof W, Bos J, Bossuyt M. A systematic review of autologous melanocytes transplantation methods in vitiligo. Arch Dermatol 1998;134:1543–9. 8 Kang HY, Song J, Im S. Verruca vulgaris following treatment of vitiligo with epidermal grafting. Br J Dermatol 2000;143:645–6. 9 Gauthier Y, Cario-André M, Lepreux S, et al. Melanocyte detachment after skin friction in non lesional skin of patients with generalized vitiligo. Br J Dermatol 2003;148:95–101. 10 Gauthier Y, Cario-André M, Taïeb A. A critical appraisal of vitiligo etiologic theories. Is melanocyte loss a melanocytorragy? Pigm Cell Res 2003;16:322–3. 11 Hara M, Yaar TM, Tang A, et al. Rôle of integrins in melanocyte attachment and dendricity. J Cell Sci 1994;107:2739–48. 12 Tang A, Eller MS, Hara M, et al. E cadherin is the major mediator of human melanocyte adhesion to keratinocytes in vitro. J Cell Sci 1994;107:2739–48. 13 Hary L, Brault V, Kleber M, et al. Lineage-specific requirements of beta catenin in neural crest development. J Cell Biol 2002;159:867–80.

CHAPTER 38

Informed consent Mats J. Olsson

Introduction/background It is most important that the patients get sufficient information prior to surgery. This information should be given as early as possible in the process so the patients get a chance to take their time going through the material and ask questions if needed. It also gives the patients a base upon which to form their decisions. General information regarding the disease and the surgery (including pre-surgery requirement), both verbally and in writing should be given at the first consultation if you as a specialist consider a surgical treatment to be the best choice for the particular case and if the patient requests such treatment option. Pre-surgery information should describe the steps involved in the procedure and also mention some of the aftercare, so that the patient can plan in advance for the day of surgery and the first days thereafter. This might for the patient involve applying for a sick leave, arranging home help, and notifying their friends. It is also important that the patient is notified about the specific pre-surgical requirements regarding the surgical method to be used and the condition of the patient. Such requirements can be a test for blood-borne virus infections (e.g. hepatitis C and human immunodeficiency virus) if dermabrasion is to be used. Dermabrasion and laser ablation can create an aerosol and it is important for the operating personnel to take necessary protective precautions. Other requirements are to avoid medication with anti-coagulative properties, such as acetyl salicylic acid (i.e. Aspirin), 10 days before the surgical procedure and to pre-treat the patient with anti-viral tablets if a positive history of herpes simplex exists and the area to be treated is on face. To ensure that the patient has taken in and understood the preceding information and emphasize its

importance, an Informed Consent should be signed prior the surgery.

Clinical studies Informed Consent is also used when a surgical procedure is conducted for a research purpose (e.g. to be evaluated or compared with other methods and/or medications), but in that case the study requires a specific research plan, risk–benefit analysis, and patient information, which are all to be approved by the local/institutional ethics committee for human trials [1]. The layout and requirements in a clinical study are quite complex and specific for each different trial and individual country and institution, and I will therefore not elaborate on Informed Consent related to research trials in this chapter. The International Conference on Harmonisation (ICH) has worked out guidelines for Good Clinical Practice (GCP) which can be of help when setting up a study. These guidelines are updated and published on the homepages of the European Medicines Agency (EMEA) [2] and Food and Drug Administration (FDA) [3]. It is not always easy for the study patient or a patient receiving a medical treatment to fully understand written or verbally given information. Current data reveal that even subjects with no known cognitive impairments often fail to give valid consent [4]. We must in our own clinical studies do what we can to ensure that research subjects provide valid consent.

Layout of form The content and structure of the document depend on the method to be used and the type of leukoderma to be treated.

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An example of a simple Informed Consent for surgical treatment of vitiligo is given below, which can be modified to fit your personal need depending

on the transplantation method, type of patients, and the local policy at your hospital:

Uppsala, Sweden, 2005

Uppsala Vitiligo Clinic Address Postal code

Declaration I hereby certify that I have received oral and written information about pigment cell transplantation and aftercare in surgical treatment of vitiligo. I am aware that this is not a cure for the underlying cause of the disease itself, only a possibility to improve appearance of the depigmented areas. This treatment does not alter the immunological background cause of the disease and therefore does not change the likelihood for a future progression of the disease. It is not possible to know before transplant procedure how well the new pigment cells will be accepted in each individual patient’s skin, due to possible autoimmune activity or elevated activities of neuropeptides. Local injection of anesthesia will be used to numb the areas but some discomfort can still be experienced. Known earlier reactions against anesthesia, antibiotics or any other medication must be reported to the treating physician before the procedure. The laws and regulations for the treatment and hospital care are those of Sweden.

Signature

Place and Date

Name in full

References 1 Vetting the ethics of research involving humans. www.forskningsetikprovning.se/eng 2 EMEA-guidance. www.emea.eu.int

3 FDA-guidance. www.fda.gov/oc/gcp/guidance.html 4 Wendler D. Can we ensure that all research subjects give valid consent? Arch Intern Med 2004;164:2201–4.

Index

Note: Page numbers in italics refer to figures and tables. 308-nm excimer laser, 35, 255, 256, 257 632.8-nm helium-neon laser, 255 achromic area size limitations related to, 147 acral vitiligo, 225–8 guidelines for management, 228 surgical modalities autologous melanocyte transplantation, 225–7 camouflage tattooing, 225 acrofacial vitiligo, 22 see also lip-tip vitiligo acrylic acid, 193–4, 196, 197 Adaptic®, 217 albinism, 75, 243–4 allylamine, 193, 196, 197 Ampligreffe, 129, 129–30 anesthesia, of recipient site leukoderma treatment basal cell layer suspension transplantation, 156 cultured autologous melanocyte transplantation, 174 ultra-thin epidermal sheets transplantation, 117 antegrade migration of amelanotic melanocytes, 231 from melanocyte reservoir, in hair bulb, 231 areola, 27, 72, 104, 112 autoantibodies, 7, 21 autocytotoxic theory, 8–9

autograft repigmentation, 88 autologous cultured epidermal grafts in lip vitiligo, 217 autologous epidermal cell suspensions transplantation, 62–3, 263 autologous melanocyte transplantation, 177, 225–7 contraindications, 58 cultured melanocytes, 227 epidermal grafting, 226 miniature punch grafting, 227 selection criteria age, 58 disease stability, 56–7 Köbner phenomenon, 57 localization, of lesions, 57–8 motivation, of patient, 58 surface area, 57 vitiligo type, 56 split-thickness skin grafting, 226–7 see also cultured autologous melanocyte transplantation Band-Aid®, 135 basal cell layer suspension transplantation, 147, 159, 277, 280 adverse effects, 145–6 leukoderma treatment after care, 156–8 anesthesia, of recipient site, 156 dermabraded lesions, 152–3 documentation, 158

follow-up evaluation, 158 free cells, release and preparation, 154–6 premedication, 156 shave biopsies, donor tissue, 153–4 BB-UVB (broadband UVB), 32–3 Bcl-2, 9 beard area, 111 beta-carotene, 36 biopsy, risks, 198 biosafety cabinet, 164–5, 165–6 BL-3-type laboratory, 204–5 bleaching cream method, 273–4 bleeding pattern, on donor area in thin split-thickness graft, 110 blister-induced epidermal grafts, 263 see also suction blister epidermal grafts bupivacaine, 131 calcineurin inhibitors, 34, 268, 269 camouflage tattooing, 225 camouflaging, 36–7, 251 candidate genes, for vitiligo, 5–6, 6 canthaxanthin, 36 Cavitron Ultrasonic Surgical Aspirator (CUSA), 139, 141 cell carrier system, of melanocytes and keratinocytes development background, 191–2 for grafting, 192–3 issues, 197–8

283

284

Index

cell carrier system, of melanocytes and keratinocytes (cont’d) surface engineered carrier production plasma polymerization, 193–4 cell culture procedures, 170–4, 194–7, 205 quality controls, 182–3 cell storage area, 161–2 cell storage equipment, 162 for cleanness and hygiene, 164 centrifuge, 167 hemacytometer, 166–7 incubator, 165 laminar-flow workbench, 165–6 inverted microscope, 163–4 laboratory glass, 163 pipette, 163 waterbath, 163, 164 cell therapy, 180, 188, 204, 217 autologous, 203 SCT, 203 cell transfer assessment carrier dressing to wound bed model, 194–5 cellular grafts, 70, 72, 73 transplantation autologous epidermal cell suspensions, 62–3 cultured autologous epidermal grafts, 63–5 cultured autologous melanocytes, 63 cellular immunity, 6–7 centrifuge, 167 chemical epilation, 111, 236 before epidermal grafting on hair-bearing skin, 102 chemical leukoderma, 76, 152, 169, 240–1 classification non-segmental vitiligo, 20, 21–4 of repigmentation diffuse, 15 marginal, 15 perifollicular, 14–15 segmental vitiligo, 20, 24–9 of surgical therapies, 60 cellular grafts, 62–5, 64 tissue grafts, 59–62, 64

cleanness and personal hygiene, in tissue culture laboratory, 164–5 CO2 laser resurface, 111, 142, 233, 240, 259 resurface, 260–4 cobblestone, 72, 91, 92, 93, 104, 144, 227, 279 Code of Federal Regulations (CFR), 204 combination therapies, 34–5, 267–72 corticosteroids, 34 topical, 31, 268, 269, 270 cryopreservation, 167, 174, 181, 183 cryotherapy, 275 cultured autologous epidermal grafts transplantation, 63–5, 194–7 cultured autologous melanocyte transplantation, 63, 170–4, 194–7 adverse effects, 146 leukoderma treatment aftercare, 174–6 anesthesia, of recipient site, 174 cryopreservation, 174 culturing, 173 defrosting, 174 dermabraded lesions, 170 documentation, 177 follow-up evaluation, 176–7 foreskin samples, donor tissue, 171 free cells, release and preparation, 171–3 full thickness skin samples, donor tissue, 171 premedication, 174 shave biopsies, donor tissue, 170–1 see also autologous melanocyte transplantation cultured cellular graft transplantation, in vitro safety concerns GCP, 205–6 GMP, 204–5 SCT regulatory environment, 203–4 cultured epithelial autografts (CEA), 191–2 cultured melanocyte transplantation, 170–4, 227

see also cultured autologous melanocyte transplantation cultured melanocyte–keratinocyte, 199 cyanoacrylate, 101, 111, 135, 137, 228 cyclosporine A (CsA), 269 defrosting, of cells, 174 Delnet®, 156, 175 depigmentation, 3, 7, 8, 10, 41–2, 50, 57, 159, 178, 218, 240 therapies, 35–6 bleaching cream, 273–4 cryotherapy, 275 laser therapy, 274–5 dermabrasion, 15, 58, 63, 88, 101, 112, 145, 156, 175, 240 halo nevi, 241, 242 laser-assisted, 259 lesion cell suspension, 152–3 cultured melanocytes, 170 superficial, 214, 223 and thin split-thickness skin grafting, 232–3 dermatography, 249 Dermatological Global Assessment (DGA), 81 dermo-epidermal grafts, 42–3, 96, 212, 214, 216 diascopy, 119, 211, 243 “difficult to treat” sites, 70–2, 111–12 diffuse repigmentation, 15, 16 dihydroxyacetone (DHA), 36 dihydroxyphenylalanine (DOPA), 15, 183 Discard-A-Pad, 130, 131 discoid lupus erythematosus (DLE), 88, 243 leukoderma, 73 donor site dressing, 131 elbows and knees, 72, 159, 178 EMLA®, 109, 110, 117, 118, 156, 174, 221, 223 epidermal cell suspension, 44 transplantation, 62–3, 263 epidermal grafting, 17–18, 25, 43, 88, 180, 263 advantages and limitations, 104–6

Index chemical epilation, 102 cultured autologous grafts, 63–5, 217 efficacy and safety, 103–4 grafts harvesting, 100–1 transfer and dressing, 102–3 in vitro cultured, 234–5 suction blistering, 61, 70–2, 73, 75, 96–100, 213–15, 226 induction time, 98–100 systemic PUVA, 43, 233–4 epidermal suspensions, 44, 57 epidermis-bearing melanocytes, in vitro reconstituted, 44–5 culture conditions, for optimal M/K ratio, 181, 196 pigmentary disorders treatment, 181 epithelial grafts, in vitro cultured transplantation, 180 cultured epidermis, clinical applications, 182–4 epidermis-bearing melanocytes, 181 postoperative management, importance, 184 regenerated epidermis, histological examination, 184 success rate, 184–5, 186–7 Er:YAG laser, 59, 111, 158, 184, 259, 263, 264, 265 European Medicines Agency (EMEA), 203, 281 European Union SCT regulatory environment, 203–4 exchange grafts punch grafts, 42 excimer laser see Xenon chloride (Xecl) excimer laser excimer light see monochromatic excimer light eyebrows, 24–5, 111 eyelids, 72, 111, 117, 156, 220–1 transplantation procedures, 221 fingers, 58, 70, 112, 159, 178, 226, 227–8, 260, 264, 265 Fixomull®, 118, 154, 170–1 flip-top grafts, 62

flip-top pigment transplantation, 42–3, 134–7 cases, 135–6 indications, 134 procedure and materials, 134–5 results, 135 focal vitiligo, 21, 104, 241 Food and Drug Administration (FDA), 204, 256, 281 FOXD3 (“Forkhead box” D3), 5–6 full-thickness punch grafts, 59–61, 144 full thickness skin grafts, 41, 128 genetics, 4–6 epidemiological data, 4 FOXD3, 5–6 heterogeneity, 4–5 major histocompatibility complex (MHC), 5 susceptibility loci, 5 genitals, 72, 221–3 GluStitch™, 135 Good Clinical Practice (GCP), 203, 205–6, 281 Good Manufacturing Practice (GMP) BL-3-type laboratory, 204–5 cell culture procedures, 205 materials and reagents, source and characterization, 205 quality assurance system, 205 Goulian–Weck knife, 154, 170 graft harvesting anesthesia, 109 instruments, 109–10 methods, 110 site, 109 Greens Media, 192, 199 hair follicles, 120, 229–31 grafting, 62 melanocyte forms, 229 portions, 229–30 transplantation color match, 126–7 complications, 126 indications, 123 limitations, 126 Malakar’s method, 124–5

285

Na’s method, 123–4 pigment appearance and spread, 125–6 postoperative care, 125 preoperative worknote, 123 hairy area, 111 halo nevi, 73, 241–2 Hautschlitzapparat, 128 hemacytometer, 166–7 cell concentration, calculation, 166–7 Neubauer-type chamber, 166 herpes-labialis-induced lip leukoderma (HILL), 50, 73, 92 high-efficiency particulate air (HEPA), 165 human wound bed model, 194–5 Humby knife, 109, 110 humoral immunity, 7 hydroquinone derivatives, 152, 169, 240, 241, 263 4-hydroxyanisole, 36 hypopigmentation disorders, 70, 73–6 immunomodulators, 33–4 calcineurin inhibitors, 34 corticosteroids, 34 incubator, 165 Indermil™, 135 Informed Consent, 281 background, 281 clinical studies, 281 layout form, 281–2 intense pulse light (IPL) therapy, 152, 169, 240 intricities, 61, 129 inverted microscope see microscope iron oxide, 217, 249 Jelonet®, 90 keratin, 226 keratinocytes, 4, 180, 199, 268 basic fibroblast growth factor (b-FGF), 4 stem cell factors (SCF), 4, 9 Köbner’s phenomenon, 28, 36, 52, 57, 134, 144, 252, 273 experimentally induced (KP-e), 52

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Index

labia majora, 223 labial mucosa, 218 laboratory design, 161 laboratory glass, 163 laminar-flow workbench, 165–6 high-efficiency particulate air (HEPA), 165 Langerhans cells, 4 laser ablation, 102 for repigmenting vitiligo 308-nm excimer laser, 255 632.8-nm helium-neon laser, 255 therapy, 274–5 laser application, in transplantation procedure alternative treatment methods, comparison, 263–5 blister-induced epidermal graft, 263 minigraftng, 263 Silktouch Flashscanner, 264 split-thickness skin grafting method, 264 CO2 laser resurfacing, 259, 260–3 Er:YAG, 259 thin skin graft, 260–3 hair pattern, 263 laser induced leukoderma, 240 leukoderma treatment, 151, 238 basal cell layer suspension transplantation, 151 aftercare, 156–8 anesthesia, of recipient site, 156 dermabraded lesions, 152–3 documentation, 158 follow-up evaluation, 158 free cells, release and preparation, 154–6 premedication, 156 pretreatment issues, 151 shave biopsy, donor tissue, 153–4 surgical methods, 152 cultured autologous melanocyte transplantation, 168 aftercare, 174–6 anesthesia, of recipient site, 174 cryopreservation, 174 culturing, 173

defrosting, 174 dermabraded lesions, 170 documentation, 177 follow-up evaluation, 176–7 foreskin samples, donor tissue, 171 free cells, release and preparation, 171–3 full thickness skin samples, donor tissue, 171 premedication, 174 shave biopsies, donor tissue, 170–1 laser-assisted dermabrasion, 259 types albinism, 76, 243–4 by laser treatment, 240 chemical, 76, 240–1 discoid lupus erythematosus (DLE), 73, 243 focal vitiligo, 241 halo nevi, 73, 241–2 idiopathic guttate hypomelanosis, 76 herpes labialis lip leukoderma, 73, 92 nevus anemicus, 242–3 nevus depigmentosus, 242 piebaldism, 73, 239 post-burn, 75–6, 240 segmental vitiligo, 238–9 Waardenburg’s syndrome, 239–40 ultra-thin epidermal sheet transplantation, 115–22 leukotrichia, 25, 70, 72, 231–2 chemical epilation, 236 dermabrasion and thin splitthickness skin grafting, 232–3 epidemiology, 229 epidermal grafting in vitro cultured, 234–5 and systemic PUVA, 233–4 histological considerations, 229–31 minigrafting, 235–6 normal repigmentation process, 231 repigmentation, 45 hypotheses, 231 lip leukoderma, 211 due to herpes labialis, 73, 92

lip-tip vitiligo, 104, 225 see also acrofacial vitiligo lips, 72, 90, 111–12, 211 autologous cultured epidermal grafts, 217 micropigmentation or tattooing, 217–18 minigrafting, 215–16 suction blister epidermal grafts, 213–15 surface anatomy, 211, 212 thin and ultra-thin split thickness grafts, 216–17 liquid nitrogen-induced blisters, 101–2, 142 major histocompatibility complex (MHC), 5 Malakar’s method, 124–5 marginal inflammatory vitiligo, 23–4 marginal repigmentation, 15, 16, 267–8 Mastisol®, 118, 154, 156, 171, 175 matrix metalloproteinases (MMPs), 15 medical and surgical therapies, combining, 267 marginal repigmentation, 267–8 PUVA, 268 versus topical corticosteroids, 270–1 topical calcineurin inhibitor, 268–9 UVB TL01, 268, 269–70 medical treatment, 31, 144 camouflaging, 36–7 combination therapies, 34–5 depigmentation therapies, 35–6 immunomodulators, 33–4 calcineurin inhibitors, 34, 268–9 corticosteroids, 34, 270 photoprotection, 36 phototherapy UVA phototherapy, 31–2, 268 UVB phototherapy, 32–3, 268 prostaglandin, 35 psychological support, 37 systemic antioxidant therapy, 35 topical calcipotriol, 35 medically induced repigmentation mechanisms diffuse repigmentation, 15 marginal repigmentation, 15 perifollicular repigmentation, 14–15

Index Medicines and Healthcare products Regulatory Authority (MHRA), 198, 199, 200 melanocyte, 14, 269 culturing, 173–4 defective adhesion, 10 improvement adverse effect, avoiding, 279 environment, 277 indications, 279–80 proliferation and migration, 277–9 repigmentation, grafting method, 279 migration phototherapy induced stimulation, 126 transfer, 103 melanocyte-bearing epidermis, 225–7 palmoplantar surface proteins, 226 melanocyte disorder, 3–4 keratinocytes basic fibroblast growth factor (b-FGF), 4 stem cell factors (SCF), 4, 9 Langerhans cells, 4 MSC, transcription factors, 3 in vitiligo macules, 3–4 melanocyte growth factor deficient theory, 9–10 melanocyte growth medium M2®, 172–3, 194–8 melanocyte–keratinocyte culturing, delivery of for grafting cell carrier system, 191–4, 197–8 human wound bed model, 194–5 results, 195–7 risks, 198–200 on surfaces, 194 melanocyte–kekeratinocyte (M/K) ratio, 181, 182–3 melanocyte stem cells (MSC) transcription factors, 3 melanocyte suspensions de-epithelialized recipient area, 175 in vitro cultures, 44–5, 170–4 melanocyte transplantation complications after basal cell layer suspension transplant, 145–6

after cultured autologous epidermal transplant, 146 after skin transplant, 144–5 limitations achromic areas size, 147 transplantation technique, 147 vitiligo macule location, 146–7 vitiligo pathogenesis, 146 Mepitel®, 119, 156, 157, 175 mesh grafts advantages and limitations, 131–2 historical background, 128 instruments, 129 preoperative workup, 129 procedure, 129–31 donor site dressing, 131 harvesting, 129 meshing, 129–31 postoperative care, 131 recipient and donor area treatment before surgery, 129 4-methoxyphenol, 36 micro-phototherapy, focused, 33 micropigmentation, 73, 212, 217, 249 adverse effects and limitations, 252 chemical substance, 249 cosmetic results, factors affecting, 252 equipments and procedure, 250 preparations, 250 principles, 249 in vitiligo, 250–2 see also tattooing Micropore®, 51, 89, 90, 125, 157, 176, 210, 216 microscope see inverted microscope milia, 112, 121, 279 formation, 145, 216–17 miniature punch grafting, 89, 90, 227 minigrafting, 17, 43–4, 59–61, 72, 73, 87–94, 144–5, 235–6, 263 advantages, 92 complications donor site, 91–2 recipient site, 91 follow up, 90–1 in lip vitiligo, 215–16 method, 89–90 mini-punch grafting evolution, 87–8 punch instrument, 87

287

minigrafting test, 51, 57, 50, 134 see also test grafting mini-punch grafting (MPG) evolution, 87–8 autograft repigmentation, 88 monobenzylether of hydroquinone (MBEH), 36, 273, 274 monochromatic excimer light, 33, 268 monomer, 193–4 monomethylether of hydroquinone (MMEH), 36 mucosal vitiligo, 21–2, 222–3 Na’s method, 123–4 NB-UVB (narrowband UVB), 31, 33, 52, 93 “Neubauer”-type chamber, 166 nevus anemicus, 242–3 vascular malformation, 242, 243 nevus depigmentosus, 75, 242 melanocyte isolation, 242 nipples and areolas, 72 non-segmental vitiligo clinical characteristics acrofacial, 22 focal, 21 marginal inflammatory, 23–4 mucosal, 21–2 progression, affecting factors, 22 trichrome, 22–3 universal, 22 vulgaris, 22 and segmental vitiligo, comparison, 22 occiput, 123, 126 oculocutaneous albinism (OCA), 243 OP-SITE®, 216 oral PUVA photochemotherapy, 32 outcome adverse events, 73 assessment methods photographic image analysis, 81 transparent sheets, 81–2 treatment evaluation, from patients’ point of view, 82 visual assessment, 81 current evaluation methods, 80 “difficult to treat” areas, 70–2 guidelines, 76

288

Index

outcome (cont’d) leukodermas/hypopigmentation disorders, 73–6 leukotrichia, 72 parameters repigmentation, 80–1 patient population, description, 81 treated area, size of, 72–3 outer root sheath (ORS), 14, 229 oxidative stress, 8, 35 Padgett dermatome, 129 palmoplantar epidermis, 226 palms and soles, 70 pathogenesis, 6–10 classic hypotheses autocytotoxic theory, 8–9 autoimmune disease, 6 cellular immunity, 6–7 humoral immunity, 7 neural hypothesis, 7–8 genetics epidemiological data, 4 FOXD3, 5–6 heterogeneity, 5 limitations, 146 melanocyte disorder, 3–4 keratinocytes, 4 Langerhans cells, 4 new hypotheses melanocyte defective adhesion, 10 melanocyte growth factor deficient theory, 9–10 melanocyte survival, disorder of, 9 viral infections, 10 perifollicular repigmentation, 14–15, 16 personal hygiene, in tissue culture laboratory, 164–5 Petri dish, 154–5, 171, 172 phenol derivatives, 152, 169, 240 chemical structure, 241 phosphate buffered saline (PBS) solution, 117–18, 171 photographic image analysis, 81 photoprotection, 36 phototherapy, 31, 92, 126, 268 PUVA, 268 versus topical corticosteroids, 270–1

UVA phototherapy oral PUVA photochemotherapy, 32 topical PUVA photochemotherapy, 31–2 UVB phototherapy BB-UVB, 32–3 PUVB, 33 focused micro-phototherapy, 33 NB-UVB, 33 UVB TL01, 268, 269–70 phototoxic blisters, 102, 132 piebaldism, 29, 63, 73, 180, 239 pigment spread, 60, 88, 93, 270 and pigment appearance, 125–6 pigmentary disorders cultured epidermis, clinical applications cell cultures, quality controls, 182–3 patient, selection of, 183 receiving bed, preparation, 183–4 epidermis-bearing melanocytes, in vitro reconstituted, 181 pipette, 163 plasma polymerization, 193–4, 196 polymer carrier dressing, risks, 200 post-burn leukoderma, 75–6, 240 postoperative care, 184 hair follicles transplantation, 125 mesh grafts, 131 post-wound-care, 209–10 thin split-thickness skin grafts, 111 postoperative patient information, 209–10 povidone iodine, 129 premedication leukoderma treatment basal cell layer suspension transplantation, 156 cultured autologous melanocyte transplantation, 174 ultra-thin epidermal sheets transplantation, 116 preoperative workup for mesh graft, 129 preparatory area, 161 prostaglandin, 35 pseudocatalase, 33, 35

psoralen plus ultraviolet A (PUVA), 60, 220, 268 versus topical corticosteroids, 270–1 psychological support, 37 pteridines, 8 punch grafts, 42 punch instrument, 87 pure epidermal grafts, 214 PUVB (with BB-UVB), 33 quadrichrome vitiligo see trichrome vitiligo quality assurance system, 205 recipient area and donor area treatment before surgery, 129 for epidermal grafting chemical epilation, 102 dermabrasion, 101 laser ablation, 102 liquid nitrogen-induced blisters, 101–2 phototoxic blisters, 102 suction blistering, 101 preparation anesthesia, 110 epidermis removal, 110 methods, 111 regenerated epidermis histological examination, 184 repigmentation, 32, 34, 69, 70, 72 of leukotrichia, 45 hypotheses, 231 medically induced repigmentation diffuse, 15 marginal, 15 perifollicular, 14–15 surgical method development epidermal grafting, 43 epidermal suspensions, 44 epidermis with melanocyte, in vitro cultures, 44–5 melanocyte suspensions, in vitro cultures, 44–5 minigrafting, 43–4 thin dermo-epidermal grafts, 42–3 surgically induced repigmentation, 15–18

Index retrograde migration, of melanocytes, 231 roof epidermal blisters grafting, 145 seed grafts, 62, 139, 140–141 segmental vitiligo, 90, 238–9, 279–80 clinical features age of onset, 24 bilateral segmental vitiligo, 29 body and scalp hair, 24–5 classification, on face, 25–7 family history, 24 incidence, 24 Köbner’s phenomenon, 28 lesions distribution, 25 lesions progression, 27 progression, affecting factors, 27–8 site of involvement, 24 treatment response prognosis, 29 and non-segmental vitiligo, comparison, 22 semi-automatic color segmentation technique, 81 shave biopsies, donor tissue, 153–4, 170–1 sieve graft, 128 Silktouch Flashscanner, 264 silver knife, 109 skin grafting in ancient times, 41 principles and biology contracture, 108 graft revascularization, 108 graft take adherence, 108 renaissance, 41 skin resurface in laser-assisted dermabrasion CO2-laser, 259 Er:YAG laser, 259 skin transplant adverse effects of minigrafting technique, 144–5 of roof epidermal blister grafting, 145 of ultra-thin epidermal sheet transplantation, 145 SLEV1, 5 Sofratulle®, 102, 111, 130, 131

somatic cell therapy (SCT), 203 regulatory environment European Union, 203–4 United States, 204 split-thickness skin graft, 53, 61, 112, 128, 137, 226–7, 264 see also thin split-thickness skin grafts stability, 49 assessment, 50 establishing factors, 57 period, 49 status, in patches, 49–50 standard operating procedure (SOP), 198, 204 stem cell factors (SCF), 4, 15 sterile area, 161 suction blister epidermal grafts (SBEG), 61–2, 70, 226 advantages and limitations, 104–6 chemical epilation, 102 efficacy and safety, 103–4 grafts harvesting, 100–1 grafts transfer and dressing, 102–3 course, 103 in lip vitiligo, 213–15 recipient site preparation dermabrasion, 101 laser ablation, 102 liquid nitrogen-induced blisters, 101–2 phototoxic blisters, 102 suction blistering, 96–8, 101 induction time, 98–100 see also blister-induced epidermal grafts surface engineered carrier production plasma polymerization, 193–4 surgical depigmentation Köbner phenomenon, 273 techniques bleaching cream, 273–4 cryotherapy, 275 laser therapy, 274–5 surgical therapies classification, 60 cellular grafts, 62–5, 64 tissue grafts, 59–62, 64 development, history and chronology depigmentation, 41–2

289

epidermal grafting, 43–4 epidermal suspensions, 44 epidermis with melanocyte, in vitro cultures, 44–5 exchange grafts, 42 leukotrichia repigmentation, 45 melanocyte suspensions, in vitro cultures, 44–5 minigrafting, 43–4 skin grafts, 41 thin dermo-epidermal grafts, 42–3 patient selection and preoperative information autologous melanocyte transplantation, 56–8 surgically induced repigmentation mechanisms, 15–18 Surgipad®, 90, 216 susceptibility loci, for vitiligo, 5 synthetic melanins, 36 systemic antioxidant therapy, 35 systemic lupus erythematosus (SLE), 243 systemic PUVA and epidermal grafting, 233–4 systemic steroids, 34 T-cell, 7, 53–4, 268 tacrolimus, 34, 35, 269 tattooing, 73, 217, 249, 250 see also micropigmentation Tegaderm®, 110, 118, 135, 154, 156, 157, 170, 171, 175, 176 test grafting (TG), 51, 60, 89, 109 see also minigrafting test thermoepilation, 126 Thiersch–Ollier grafts see thin splitthickness skin grafts thin dermo-epidermal grafts, 42–3 thin split-thickness skin grafts, 108, 260–3, 264 advantages and limitations, 112–13 course and adverse effects, 112 and dermabrasion, 232–3 efficacy, 112 method “difficult to treat” sites, modifications at, 111–12 graft harvesting, 109–10

290

Index

thin split-thickness skin grafts (cont’d) optimum results, prerequisites, 112 postoperative care, 111 recipient area preparation, 110–11 patient selection, 108–9 post- and preoperative views, 261–2 and ultra-thin split thickness grafts in lip vitiligo, 216–17 see also split-thickness skin grafts thioglycolate depilatory, 102 tissue culture laboratory, setting, 161 cells and materials storage, equipment, 162 centrifuge, 167 cleanness and personal hygiene, 164–5 hemacytometer, 166–7 incubator, 165 laboratory design cell storage (room) area, 161–2 preparatory area, 161 sterile area, 161 laboratory glass, 163 laminar-flow workbench, 165–6 microscope, 163–4, 164 pipetting, 163 waterbath, 163, 164 tissue grafts, 70, 72, 73 flip-top grafts, 62 full-thickness punch grafts, 59–61 hair follicle grafts, 62 seed grafts, 62 split-thickness grafts, 61 suction blister grafts, 61–2 topical calcineurin inhibitors, 34, 268–9 cyclosporin, 269 signal transduction, 268

tacrolimus, 269 see also topical immunomodulators (TIM) topical calcipotriol, 35 topical corticosteroids (TCS), 268 versus PUVA, 270–1 topical immunomodulators (TIM), 269 topical PUVA photochemotherapy, 31–2 topical steroids, 34, 220 toes, 70, 227–8 transparent plastic film, 119 transparent sheets, 81–2 transplantation method, 70, 71, 116 treated area, size of, 72–3 treatment evaluation, from patients’ point of view, 82 Tribonate®, 117, 118, 153, 156, 170, 174 trichrome vitiligo, 22–3 trypan blue, 166 trypsination, 155, 172 trypsinization, 145 L-tyrosine, 8 ultrasonic abrasion and seed grafts, 139, 140–1 cases, 141–2 surgical aspirator, 139–40 ultrasonic surgical aspirator, 139–40 CUSA, 139, 141 sonopet, 141 structure and principle, 139 ultra-thin epidermal sheet transplantation, 115–22, 145, 216 leukoderma treatment anesthesia, of recipient site, 117 area calculation, 116 donor area, 118 evaluation and documentation, 119–20

follow-up inspection, 119 premedication, 116 recipient site preparation, 117–18 transplantation phase, 118–19 ultraviolet (UV) radiation, 268 United Kingdom MHRA, 198, 200 United States SCT regulatory environment, 204 United States Code (USC), 204 universal vitiligo, 22 UVA phototherapy, 31–2 oral PUVA photochemotherapy, 32 topical PUVA photochemotherapy, 31–2 UVB phototherapy BB-UVB, 32–3 micro-phototherapy, focused, 33 NB-UVB, 33 PUVB (with BB-UVB), 33 UVB TL01, 269–70 repigmentation pattern, 270 vermillion, 211 visual assessment, 81 vitiligo macule location limitations, 146–7 vitiligo vulgaris, 22, 279–80 Waardenburg’s syndrome, 239–40 watchmaker’s pin-vise, 217, 250 waterbath, 163, 164 Wood’s light, 51, 119, 211, 238 Xenon chloride (Xecl) excimer laser, 35, 255 Zimmer® air-driven ultra-dermatome, 118 motor-driven ultra-dermatome, 109–10