Clinical Applications of Bones: Allografts and Substitutes

Clinical Applications of Bone Allografts and Substitutes Biology and Clinical Applications

SERIES IN ALLOGRAFTS IN BONE HEALING: BIOLOGY AND CLINICAL APPLICATIONS Advances in Tissue Banking Specialist Publications

Editor-in-Chief: Glyn O. Phillips Published Vol. 1

Bone Biology and Healing edited by Glyn O. Phillips

Vol. 2

Bone Morphogenetic Protein and Collagen edited by Glyn O. Phillips

Vol. 3

Clinical Applications of Allografts and Substitutes edited by Glyn O. Phillips

Allografts in Bone Healing: Biology and Clinical Applications - Vol. 33

Clinical Applications of Bone (Nografts Substitutes

and

Biology and Clinical Applications

Editor

Glyn 0 Phillips Phillips Hydrocolloid Research, UK

> World Scientific NEW JERSEY • LONDON • SINGAPORE • BEIJING • SHANGHAI • HONGKONG • TAIPE, . CHENNA,

Published by World Scientific Publishing Co. Pte. Ltd. 5 Toh Tuck Link, Singapore 596224 USA office: 27 Warren Street, Suite 401-402, Hackensack, NJ 07601 UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.

CLINICAL APPLICATIONS OF ALLOGRAFTS AND SUBSTITUTES Copyright © 2005 by World Scientific Publishing Co. Pte. Ltd. All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.

For photocopying of material in this volume, please pay a copying fee through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to photocopy is not required from the publisher.

ISBN 981-256-343-1

Printed in Singapore by World Scientific Printers (S) Pte Ltd

ALLOGRAFTS IN BONE HEALING: BIOLOGY AND CLINICAL APPLICATIONS International Advisory Board H. Burchardt, USA A. Gross, Canada M. Itoman, Japan J. Kearney, UK J. Komender, Poland B. Loty, France P. Mericka, Czech Republic D.A.F. Morgan, Australia D. Pegg, UK M. Salai, Israel W.W. Tomford, USA Y. Vajaradul, Thailand H. Winkler, Austria N. Yusof, Malaysia N. Triantafyllou, Greece R. Capanna, Italy W.W. Boeckx, Belgium C.J. Yim, Korea

V

CONTENTS

Introduction to the Series

ix

Preface

xiii

List of Contributors

xvii

Chapter 1

The IAEA Code of Practice for the Radiation Sterilisation of Tissue Allografts for Validation and Routine Control Volume 7, Chapter 8

Chapter 2

Chapter 3

Chapter 4

Chapter 5

Chapter 6

Preserved Bone Allografts in Reconstructive Orthopaedics Volume 6, Paper 12

1

57

Clinical Strategy of Application of Deep Frozen Radiation Sterilised Bone Allografts Volume 6, Paper 6

67

Clinical Results and Organisational Aspects of Autogenous and Allogenous Bone Grafting in the Treatment of 226 Patients with Primary Osseous Neoplasms Volume 1, Chapter 3.6

83

New Approaches to Comparative Evaluation of Allogenic and Autologous Bone Transplants Procured in Various Ways Volume 7, Chapter 19

89

The Use of Freeze-dried Mineralised and Demineralised Bone Volume 3, Chapter 2.1

Vll

105

Vlll

Chapter 7

Chapter 8

Preserved Allogenic Rib Cartilage in Reconstructive Surgery Volume 6, Paper 12

127

Bone Substitutes and Related Materials in Clinical Orthopaedics Volume 1, Chapter 3.2

139

INTRODUCTION TO THE SERIES This series* is aimed directly at orthopaedic surgeons, who use or propose to use musculoskeletal allografts in their clinical practice. It is not a subject which comes naturally or easily to this group of clinicians, who seem to be always overloaded with the day-to-day calls of surgical practice. Often, they must rely on infrequent conference talks or specialist review articles for their information. Consequently, it is a field riddled with myths and inconsistencies. • • • • • • • • • •

How are these grafts prepared? Are they safe? Which are most effective in promoting bone healing? Does radiation used to sterilisation damage the bone or weaken the graft when used for structural purposes? Which graft should be used for which procedure? Are they free of viruses, particularly HIV? What does sterility mean in relation to an allograft? Do they retain any bone morphogenic protein after tissue bank processing? What about their immunogenicity? What are the growth factors which assist in the bone healing process?

These are only few of the questions, which have been posed to me during numerous training courses and workshops with orthopaedic surgeons. This series aims to answer these questions and more and do so in an accessible manner. It is a ready reference for any orthopaedic surgeon involved in this work and will point them to even more specialised papers for further detail. The difficulty in gaining access to authoritative information in this diverse subject is its inter-disciplinary character. At one end of *The papers in this series are collected from Advances in Tissue Banking and Radiation and Tissue Banking.

xx

X

the spectrum is the tissue banker, who is involved with screening potential donors, undertaking serological tests to eliminate potential harmful micro-organisms and procuring the tissues, in association with medical colleagues. Thereafter, there is a series of processing and sterilisation procedures, conducted within a total quality system which documents and ensures complete traceability, which ends with the allograft professionally packaged and ready for the surgeon. At the other end of the spectrum is the surgeon, facing a bewildering array of such grafts. In between there are so many specialities, such that currently the information flow is mainly based on chat and experience between surgeons. This series aims to bridge this great divide by describing what grafts should be used, what are the factors which influence their ability to promote bone healing and details about the clinical effectiveness of the work carried out up to this time. The subject is developed stepwise, but each contribution has been prepared by a specialist who has direct experience in practical aspects of the subject. Volume 1 deals with the biological aspects of bone healing and immunology, the growth factors which control bone repair and specialist factors associated with particular grafts such as demineralised bone. Volume 2 describes the influence of the components of bone, the biochemistry of collagen, the process of osteoinduction, and factors which might reduce the functioning of these important molecular triggers, and dispels some myths about the effects of radiation. Volume 3 describes the general clinical use of various allografts, a comparison between autografts and allografts, and an evaluation of the value of bone substitutes compared with human allografts. Volume 4 describes in more detail specific procedures for application of allografts in various reconstructions: in the knee, the spine, in neurosurgery, total hip and revision hip arthroplasty. Volume 5 deals with allografts in the treatment of bone tumours and prosthetic composites and evaluating long term results of allograft in the management of bone tumours.

XI

All the contributors have also been authors within the Advances in Tissue Banking series and received the accolade of their peers across the subject spectrum. They are, therefore, not narrow specialists and so can present a wide perspective which the series aims to do, and to do so with an authority based on achievement. It is a pleasure to recommend the series to all orthopaedic surgeons who have an open mind about the subject and are prepared to read and learn. Glyn O. Phillips Series Editor

PREFACE

The clinical practice of using bone grafts to repair, replace or supplement the bone stock has a long history, dating back to McEwen in 1881. When a group of surgeons, in which Geoffrey Burwell was a leading figure showed that frozen preserved allograft was superior in performance to fresh allogeneic bone, the road was pointed to the more extensive use of bone grafts. However, generally the practice remained a "cottage industry" well into the latter part of the 20th century. This involved orthopaedic surgeons keeping pieces of bone in individual hospital cold store, which had been rescued after surgery, usually femoral heads after hip replacement, and using these as required on an individual basis. There were many exceptions and these surgeons were usually associated with the pioneering tissue banks, which first emerged first in the 1950's. Notable among the early tissue banks was the Bethesda Naval Tissue Bank in the USA, the Wakefield Tissue Bank in the UK, the Bank at Hradecs Kralove in Czechoslovakia, the Charite Hospital Bank in Berlin, the Democritos Bank in Greece and bank in Warsaw which celebrated its 40th anniversary in 2004. The explosion came in the 1990's and onwards, with the result that more than one million bone grafts were used in the USA during 2004. This volume reflects the growth of the subject, giving a cross-section of specialised experience. Despite this remarkable growth the safety of allografts remains a major concern due to microbial and viral contamination of tissues. Existing methods and processing for sterilising tissues are proving, in many instances inadequate. Infections have been transmitted from the graft to the recipient and in the USA, the Centre for Disease Control and other regulatory bodies, have

xm

XIV

drawn attention to the need for a reliable end sterilisation method which does not damage the functionality of the final tissue. The International Atomic Energy Agency (IAEA) has given special attention to the widely used method of using ionising radiations for such sterilisation. There is a great deal of misunderstanding about this method and a rigorous approach is needed if the method is to be used to its full potential. Accordingly the IAEA have set out a Code of Practice for this application of radiation, which is described in the first contribution since it is fundamental to the whole field of surgical use of tissue allografts. The following two contributions document the Polish experience led by Janusz Komender. A tissue bank has been operating in Poland since 1963 and more than 100,000 grafts of bone, cartilage dura mater, skin and fascia have been prepared and used in the various branches of reconstructive surgery. Historically and scientifically this work is important, not the least because they have consistently used radiation sterilised bone grafts. As such they have the widest experience of this type of graft, and their contributions positively dispel the myth that radiation destroys the clinical value of the allograft. Satisfactory graft substitution was observed in 90.8% of all patients. Their second contribution concentrates on the use of deep frozen radiation sterilised bone allografts. They find that such allografts undergo "creeping substitution" (incorporation) in 3 to 6 months. Both contributions give a wealth of experience in the use of radiation sterilised grafts. There is no real conflict between the use of autografts and allografts, although this debate is still often perpetuated. Autografts are, of course, the gold standard. Shortage of autograft bone and the advisability of introducing a second lesion are factors which ultimately decide which should be used in particular circumstances. The contribution of Sarkar and colleagues from Germany compare the clinical results and organisational aspects of autogeneous and allogenous bone grafting. This contribution shows that using allogenous grafts does not increase the risk of post-operative infections. In contrast

XV

to the Polish experience these workers do not favour graft sterilisation. The Russian experience in this field has not been readily available and so the contribution by Professor Kalinin and his colleagues is important since it illustrates the approach in that great country. They have developed a model which contributes to the continuing discussion about allografts versus autografts. They find demineralised bone to be a highly promising transplantation material, a subject further considered in the next contribution. Demineralised bone is a specialist tissue graft which has mostly been used in maxillofacial surgery. Christian Delloye, from Belgium, however, compares the more general use of freeze-dried mineralised and demineralised bone. The used of freeze-dried bone has not been as popular in Europe as in the USA. As a structural material it is not appropriate since freeze drying significantly weakens the bone, much more so than the effects of radiation. As a leading member of the European Association for Musculoskeletal Tissue (EAMST) Dr Delloye appropriately draws attention to the need to keep strictly to the European Standards when processing his grafts. His conclusion is that freeze dried bone remains a reliable bone substitute. The orthopaedic surgeon needs to be supported with other grafts, apart from bone. Cartilage is one of the most important of these. Despite the advances in tissue engineering, allogenic rib cartilage offers excellent properties and enables the surgeon to shape the implant as required, particularly for reconstructions of the face. The contributions of Sladowski and colleagues demonstrate that cartilage offers long term support for soft tissues and degradation does not occur within the first four years. Experience of using more than 2500 such grafts is described, with positive results in 75% of cases. Despite the advances in using human bone allografts, it must often be conceded, either because lack of availability or shortage of these grafts at the desired time that bone substitutes must be considered. Professor Aho from Finland provides an excellent

XVI

survey of what is now available. Moreover, he evaluates their clinical effectiveness. He concludes that most of these substitutes can be used as fillers for reconstruction of moderately sized (1-4 cm in diameter) cystic lesion in the human skeleton. Only a few can be used as a replacement of a weight-bearing skeletal part. The volume, therefore, provides an international expert evaluation of the use of bone, bone substitutes and related allografts, and describes the practices and clinical results in particular procedures. It will provide a ready reference for anyone wishing to carry out a quick survey of the subject. Glyn O. Phillips Editor

LIST OF CONTRIBUTORS

J. KOMENDER Department of Transplantology Orthopaedics and Neurosurgery Central Clinical Hospital Military Medical University in Warsaw, Poland A. KOMENDER Department of Transplantology Orthopaedics and Neurosurgery Central Clinical Hospital Military Medical University in Warsaw, Poland H. MALCZEWSKA Department of Histology & Embryology Medical University in Warsaw Orthopaedics and Neurosurgery Central Clinical Hospital Military Medical University in Warsaw, Poland W. MARCZYNSKI Institute of Traumatology Orthopaedics and Neurosurgery Central Clinical Hospital Military Medical University in Warsaw, Poland WOJCIECH MARCZYNSKIJANUSZ KOMENDER Institute of Traumatology Orthopaedics and Neurosurgery of Central Clinical Hospital Military School of Medicine in Warsaw, Poland JANUSZ KOMENDER Bank of Human Tissues Medical Academy in Warsaw, Poland

xvn

XV111

M.R. SARKAR Klinik ftir Unfall-, Hand- und Wiederherstellungschirurgie Chirurgie III, Universitat Ulm, Germany M. SCHULTE Klinik ftir Unfall-, Hand- und Wiederherstellungschirurgie Chirurgie III, Universitat Ulm, Germany G. BAUER Klinik ftir Unfall-, Hand- und Wiederherstellungschirurgie Chirurgie III, Universitat Ulm, Germany E. HARTWIG Klinik ftir Unfall-, Hand- und Wiederherstellungschirurgie Chirurgie III, Universitat Ulm, Germany A.V. KALININ Russian Research Institute of Traumatology and Orthopaedics, named after R.R. Vreden Baikov Str. 8, 195427 St. Petersburg, Russia V.I. SAVELIEV Russian Research Institute of Traumatology and Orthopaedics, named after R.R. Vreden Baikov Str. 8, 195427 St. Petersburg, Russia A.A. BULATOV Russian Research Institute of Traumatology and Orthopaedics, named after R.R. Vreden Baikov Str. 8, 195427 St. Petersburg, Russia Ch. DELLOYE Catholic University of Louvain St-Luc University Clinics Brussels, Belgium

XIX

D. SLADOWSKI Department of Transplantology Warsaw Medical University, Poland A. KOMENDER Department of Transplantology Warsaw Medical University, Poland J. KOMENDER Department of Transplantology Warsaw Medical University, Poland H. MALCZEWSKA Department of Histology & Embryology Warsaw Medical University, Poland A.J. AHO Department of Surgery The Turku University Central Hospital The Biomaterial Project, University of Turku Turku, Finland J.T. HEIKKILA Department of Surgery The Turku University Central Hospital The Biomaterial Project, University of Turku Turku, Finland

1 IAEA CODE OF PRACTICE FOR THE RADIATION STERILISATION OF TISSUE ALLOGRAFTS: REQUIREMENTS FOR VALIDATION AND ROUTINE CONTROL

AN IAEA CONSULTATION DOCUMENT

1. Introduction This code of practice for the radiation sterilisation of tissue allografts adopts the principles which the International Standards Organisation (ISO) applied to the radiation sterilisation of health care products. The approach has been adapted to take into account the special features associated with human tissues, and the features which distinguish them from industrially produced sterile health care products. The code, as described here, is not applicable if viral contamination is identified. Thus, it is emphasised that the human donors of the tissues must be medically and serologically screened. To further support this screening, it is recommended that autopsy reports are also reviewed if available. This adaptation of established ISO methods can thus only be applied for sterilisation of tissue allografts if the radiation sterilisation described here is the terminal stage of a careful detailed, documented sequence of procedures, involving: • donor selection; • tissue retrieval;

1

2

• • • •

tissue banking general procedures; specific processing procedures; labelling; and distribution;

all of which are conducted according to the IAEA International Standards for Tissue Banks. It shall not be used outside this context. The methods proposed here for the establishment of a sterilisation dose are based on statistical approaches used for the sterilisation of health care products (ISO 11137:1995, ISO 13409:1996, ISO 15844:1998, AAMI TIR 27:2001) and modified appropriately for the low numbers of tissue allograft samples typically available. For a standard distribution of resistance (SDR), the tissue bank may elect to substantiate a sterilisation dose of 25 kGy for microbial levels up to 1,000 colony forming units (cfu) per allograft product. Alternatively, for the SDR and other microbial distribution, specific sterilisation doses may be validated depending on the bioburden levels and radiation resistances (Dio values) of the constituent microorganisms. International standards have been established for the radiation sterilisation of health care products which include medical devices, medicinal products (pharmaceuticals and biologies) and in vitro diagnostic products (ISO 11137:1995 (E); ISO 11737-1: 1995; ISO 11737-2:1998; ISO/TR 13409:1996, ISO/TR 15844:1998 and AAMI TIR 27:2001). Following intensive studies of the effects of ionising radiation on chemical, physical and biological properties of tissue allografts and their components, these are now radiation sterilised using a variety of methods and practices. Through its radiation and tissue banking programme, the International Atomic Energy Agency has sought during the period 2001-2002 to establish a code of practice for the radiation sterilisation of tissue allografts and its requirement for validation and routine control of the sterilisation of tissues.

3

Annex A describes the methods for selecting a sterilisation dose. Annex B provides three worked examples applying these methods. Annex C gives tables which contain microbial survival data relating to Standard Distribution of Resistances. Annex D gives a bibliography of key references for the sterilisation of tissues by ionising radiation. This code sets out the requirements of a process, in order to ensure that the radiation sterilisation of tissues produces standardized sterile tissue allografts suitable for safe clinical use. Although the principles adopted here are similar to those used for the sterilisation of health care products, there are substantial differences in practice arising from the physical and biological characteristics of tissues. For health care products, the items for sterilisation come usually from large production batches. For example, syringes are uniform in size and have bacterial contamination arising from the production process, usually at low levels. It is the reduction of the microbial bioburden to acceptable low levels which is the purpose of the sterilisation process, where such levels are defined by the sterility assurance level (SAL). The inactivation of microorganisms by physical and chemical means follows an exponential law and so the probability of a surviving microorganism can be calculated if the number and type of microorganisms are known and if the lethality of the sterilisation process is also known. Two methods are used in ISO 11137:1995 to establish the radiation doses required to achieve low SAL values. Method 1 of ISO 11137:1995 relies on knowing the bioburden (assuming a Standard Distribution of Resistances) before irradiation and uses this data to establish a verification dose, which will indicate the dose needed for a SAL of 10~2. The method involves a statistical approach to setting the dose based on three batches and hence relatively large numbers of samples are required for both establishing the initial bioburden and the verification dose, both per product batch. A further adaptation of method 1 for

4

a single production batch has also been developed (ISO/TR 15844-1998). In Method 2 of ISO 11137:1995, the bioburden levels are measured after giving a series of incremental doses to the samples, these doses being well below the dose required for a SAL of 10"6. In this method, 280 samples are required to determine the dose to produce a SAL value of 10"2, from which the dose needed to yield a SAL value of 10"6 may be extrapolated. No assumptions are made in method 2 about the distribution of microorganisms and their resistances. In a later ISO/TR 13409:1996, Method 1 was adapted to allow the use of as few as 10 samples to determine the verification dose. In this modification, the dose needed for a SAL value of lO^1 is used to establish the dose required for a SAL value of 10"6. The sole purpose, however, of this modification is to substantiate whether 25 kGy is an appropriate dose to achieve a SAL value of 10~6. In AAMI TIR 27:2001, another method to substantiate the sterilisation dose of 25 kGy was developed. 1.1. Sterilisation of tissue allografts Tissues used as allografts comprise a wide range of materials and bioburden levels such that the above quality assurance methods developed for health care products cannot be applied without careful and due consideration given to the differences between health care products and tissue allografts. Tissues which are sterilised currently include: bone, cartilage, ligaments, tendons, fascias, dura mater, heart valves, vessels, skin and amnion. Unlike health care products, the variability in types and levels of bioburden in tissues is much greater than that found for health care products where the levels of microbial contamination are usually low and relatively uniform in type and level. In addition, tissue allografts are not products of commercial production processes involving large numbers of samples. These

5

differences mean that extra attention must be given to the following: (a) uniformity of sample physical characteristics (shape and density); (b) uniformity of bioburden in sample; (c) donor screening for viral contamination; and (d) whether low numbers of samples can be used for sterilisation dose setting purposes. 2. Objective The objective of this code is to provide the necessary guidance in the use of ionising radiation to sterilise tissue allografts in order to ensure their safe clinical use. 3. Scope This code specifies requirements for validation, process control and routine monitoring of the selection of donors, tissue processing, preservation, storage and the radiation sterilisation of tissue allografts. They apply to continuous and batch type gamma irradiators using the radioisotopes 60Co and 137Cs, electron beam accelerators and X-rays. The principles adopted here are similar to those elucidated in ISO 11137:1995 in that statistical approaches to establishing doses to assure sterility of the tissue products are proposed.

4. References The following standards contain provisions which are relevant to this code: ISO 9001:2000 Quality management systems — Requirements. ISO 11137:1995 Sterilisation of health care products — Requirements for validation and routine control Radiation — sterilisation.

6

ISO 11737-1: 1995 Sterilisation of medical devices — Microbiological methods — Part 1. ISO 11737-2:1998 Sterilisation of medical devices — Microbiological methods — Part 2. ISO/TR 13409:1996 Sterilisation of health care products — Radiation sterilisation — Substantiation of 25 kGy as a sterilisation dose for small or infrequent production batches. ISO/TR 15844:1998 Sterilisation of health care products — Radiation sterilisation — Selection of sterilisation dose for a single production batch. AAMI Technical Information Report (TIR 27):2001 — Sterilisation of health care products — Radiation sterilisation-Substantiation of 25 kGy as sterilisation dose — Method VDmax. ISO/ASTM 51261 (2002) Guide for Selection and Calibration of Dosimetry Systems for Radiation Processing. IAEA (May, 2002) International Standards for Tissue Banks. 5. Definitions The majority of the definitions relating to the sterilisation process are given in ISO 11137:1995. The following definitions are particularly useful for this code and are given below. Allograft: A graft transplanted between two different individuals of the same species. Allograft product: An allograft or a collection of allografts within a primary package. Absorbed dose: The quantity of radiation energy imparted per unit mass of matter. The unit of absorbed dose is the gray (Gy), where 1 gray is equivalent to the absorption of 1 joule per kilogram (1 Gy = 100 rad). Batch (irradiation): Quantity of final product irradiated at the same cycle in a qualified facility.

7

Batch (production): Defined quantity of finished tissue product from a single donor that is intended to be uniform in character and quality, and which has been produced during a same single cycle of processing. Bioburden: Population of viable microorganisms on tissue allograft and package prior to the sterilisation process. Distribution: Transportation and delivery of tissues for storage or use in recipient. Dose mapping: An exercise conducted within an irradiation facility to determine the distribution of the radiation dose throughout a load of tissue allograft or simulated items of specified bulk density, arranged in irradiation containers in a defined configuration. Dosimeter: A device having a reproducible measurable response to radiation, which can be used to measure the absorbed dose in a given material. Dosimetry system: System used for determining absorbed dose, consisting of dosimeters, measuring instrumentation and procedures for the system's use. Dw: Radiation dose required to inactivate 90 per cent of the homogeneous microbial population where it is assumed that the death of microbes follows first-order kinetics. Good tissue banking practice (GTBP): Practice that meets accepted

standards as defined by relevant government or professional organisations. Irradiator: Assembly that permits safe and reliable sterilisation processing, including the source of radiation, conveyor and source mechanisms, safety devices and shield. Positive test of sterility: A test of sterility which exhibits detectable microbial growth after incubation in a suitable culture medium.

8

Qualification: Obtaining and documenting evidence concerning the processes and products involved in tissue donor selection, tissue retrieval, processing, preservation and radiation sterilisation that will produce acceptable tissue allografts. Recovery efficiency: Measure of the ability of a specified technique to remove microorganisms from a tissue allograft. Reference standard dosimeter: Dosimeter, of high metrological quality, used as standard to provide measurements traceable to and consistent with measurements made using primary standard dosimeters. Routine dosimeter: A dosimeter calibrated against a primary or reference dosimeter and used routinely to make dosimetric measurements. Sample item portion (SIP): Defined standardized portion of a tissue allograft that is tested. Sterile: Free of viable micro-organisms. Sterility assurance level (SAL): Probability of a viable microorganism being present on a tissue allograft after sterilisation. Sterilisation: A validated process to destroy, inactivate, or reduce microorganisms to a sterility assurance level (SAL) of 10~6. (Sterility is expressed by several national legislations and international standards as a SAL of 10~6.) Sterilisation dose: Minimum absorbed dose required to achieve the specified sterility assurance level (SAL). Test of sterility: Test performed to establish the presence or absence of viable microorganisms on tissue allograft, or portions thereof, when subjected to defined culture conditions. Tissue bank: An entity that provides or engages in one or more services involving tissue from living or cadaveric individuals for transplantation purposes. These services include assessing

9

donor suitability, tissue recovery, tissue processing, sterilisation, storage, labeling and distribution. Validation: Refers to establishing documented evidence that provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes. A process is validated to evaluate the performance of a system with regard to its effectiveness based on intended use. Verification dose: Dose of radiation to which tissue allograft, or portions thereof are nominally exposed in the verification dose experiment with the intention of achieving a predetermined sterility assurance level (SAL). 6. Personnel Responsibility for the validation and routine control for sterilisation by irradiation including tissue donor selection, tissue retrieval, processing, preservation, sterilisation and storage shall be assigned to qualified personnel in accordance with subclauses 6.2.1 and 6.2.2 of ISO 9001:2000, whichever is applicable. 7. Validation of Pre-sterilisation Processes 7.1. General An essential step in the overall radiation sterilisation of tissues is rigorous donor selection to eliminate specific contaminants. Full details about donor selection, tissue retrieval, tissue banking general procedures, specific processing procedures, labelling and distribution are given in IAEA international standards for tissue banks. Such tissue donor selection, retrieval, processing and preservation are processes which determine the characteristics of the tissue allograft prior to the radiation sterilisation process. The most important characteristics are those relating to use of

10

the tissues as allografts, namely, their physical, chemical and biological properties, the latter including the levels and types of microbial contamination. Validation of these processes shall include the following: (a) (b) (c) (d)

qualification of the tissue bank facilities; qualification of the tissue donors; qualification of the tissue processing and preservation; certification procedure to review and approve documentation of (a), (b) and (c); (e) maintenance of validation; and (f) process specification. 7.2. Qualification of the tissue bank facilities Tissue banks shall have facilities to receive procured tissues and to prepare tissue allograft material for sterilisation. Such facilities are expected to include laboratories for the processing, preservation and storage of tissues prior to sterilisation. These laboratories and the equipment contained therein shall meet international standards enunciated by the various tissue bank professional associations and now combined in the IAEA International standards for tissue banks. A regularly documented system should be established which demonstrates that these standards are maintained, with special emphasis on the minimisation of contamination by microorganisms throughout the tissue retrieval, transportation, processing, preservation and storage stages to bioburden levels which comply with the IAEA international standards for tissue banks. Tissue banks shall also have access to qualified microbiological laboratories to measure the levels of microorganisms on the tissue allografts at various stages in their preparation for the purposes of assessing both the levels of contamination at each stage and also typical bioburden levels of the pre-irradiated tissue allografts. The standards expected of such laboratories are specified in: ISO 11737-1:1995 and ISO 11737-2:1998.

11

The overall purpose of the above facilities contained within tissue banks is to demonstrate that they are capable of producing preserved tissue allografts which have acceptably low levels of microorganisms in the preserved product prior to their sterilisation by radiation.

7.3. Qualification of tissue donors The main aim of the tissue donor selection process carried out prior to processing, preservation, storage and sterilisation is to produce tissue allografts which are free from transmissible infectious diseases. Such a selection process in order to produce acceptable tissues shall include the following minimal information: (a) time of retrieval of tissue after death of donor, conditions of body storage; (b) age of donor; (c) medical, social and sexual history of donor; (d) physical examination of the body; (e) serological (including molecular biology) tests; and (f) analysis of autopsy as required by law. Such information shall be used to screen donors to minimise the risk of infectious disease transmission from tissue donors to the recipients of the allografts. The information so collected shall be comprehensive, verifiable and auditable following good practice on tissue banking, as specified in the IAEA international standards for tissue banks. The following serological tests shall be carried out as a minimum on each donor: (a) antibodies to human immunodeficiency virus 1 and 2 (HIV 1, 2); (b) antibodies to hepatitis C virus (HCV); (c) hepatitis B surface antigen (HBs-Ag); and (d) syphilis: non-specific (e.g. VDRL) or preferably specific (e.g. TPHA).

12

Other tests may be required by statutory regulations or when specific infections are indicated as specified in the IAEA international standard for tissue banks.

7.4. Qualification of tissue processing and preservation The processing of tissue allograft materials such as bone, cartilage, ligaments, fascias, tendons, dura mater, heart valves and vessels, skin and amnion comprises various stages such as removal of bone marrow, defatting, pasteurisation, antibiotic treatment, percolation and treatment with disinfectants such as hypochlorite, ethyl alcohol and glycerol. The inclusion of any or all of these stages will depend on a number of factors including: (a) the preferred practice of the tissue bank; (b) the nature of the tissue (and its anticipated use in the clinic); and (c) the degree of contamination of the procured tissue. The preservation of the processed tissue allografts may include: (a) (b) (c) (d) (e)

freeze drying; deep freezing; air drying; heat drying; and chemical treatment.

An important function of these processes in Sees. 7.2 to 7.4 is to produce tissue allografts which have low levels of microbial contamination and in particular less than 1,000 cfu per allograft product when it is desired to substantiate a sterilisation dose of 25 kGy. In the latter case, for a bioburden of 1,000 cfu per allograft product, a 25 kGy dose is sufficient to achieve a SAL of 10~6 for a standard distribution of resistances. The capacity of all of the tissue processing and preservation procedures

13

to remove microorganisms should be checked periodically and documented.

7.5. Maintenance of validation For each of the qualifications detailed above in Sees. 7.2-7.4, a validation process should be specified, which will demonstrate that the standards expected will be maintained. As a minimum, these validation processes shall include: (a) an audit of the origin and history of the procured tissues with reference to 7.3 (a) to (d); (b) a random, statistically significant sampling of procured tissues (that is, prior to processing and preservation) followed by a laboratory-based screening for viruses and infectious agents (see Sec. 7.3); (c) measures of particle count and microbial contamination in the environment of each of the separate facilities of the tissue bank; (d) random, statistically-significant sampling of tissue allografts prior to and after tissue processing and preservation for measurements of bioburden levels; and (e) determination of the ability of the tissue processing and preservation procedures to both reduce the levels of microorganisms and to produce the levels of bioburden required for the radiation sterilisation process. This should ensure a microbial contamination level of 1,000 cfu per allograft product or less when it is required to substantiate a sterilisation dose of 25 kGy. 7.6. Process specification A process specification shall be established for each tissue allograft type. The specification shall include: (a) the tissue allograft type covered by the specification; (b) the parameters covering the selection of tissue for processing;

14

(c) details of the tissue processing and preservation carried out prior to irradiation as appropriate to each tissue type; (d) details of the equipment, laboratory and storage facilities required for each of the processing and preservation stages, particularly with regard to acceptable contamination levels; (e) details of the routine preventative maintenance programme; and (f) process documentation identifying every processed tissue, including details of its origin (see Sec. 7.3), its processing and preservation, dates of performing all processes, details of process interruptions, details of any deviations from the adopted processing and preservation procedures.

8. Validation of the Serilisation Process 8.1. General The guidance given here is based on the procedures specified in previous documents (ISO 11137:1995, ISO/TR 13409:1996, ISO/ TR 15844:1998 and AAMI TIR 27:2001) for the sterilisation of health care products. More emphasis is given here, however, on the factors which affect the ability of the sterilisation process to demonstrate that an appropriate sterility assurance level (SAL) can be achieved with low numbers of tissue allografts, which may have more variability in the types and levels of microbial contamination than is found in health care products and which may also be more variable in size and shape. More specifically, several approaches to establishing a sterilisation dose are proposed for the small numbers of tissue allografts typically processed. Emphasis is placed on the need to take into account both the variability of bioburden from one tissue donor to another, as well as the variability of size and shape of tissue allografts, which can affect both the accuracy of product dose mapping (and hence the sterilisation dose itself) and also the applicability of using Sample Item Portions (SIP) of a tissue allograft product.

15

Validation of the sterilisation process shall include the following elements: (a) qualification of the tissue allografts and their packaging for sterilisation; (b) qualification of the irradiation facility; (c) process qualification using a specified tissue allografts or simulated products in qualified equipment; (d) a certification procedure to review and approve documentation of (a), (b) and (c); and (e) activities performed to support maintenance of validation. 8.2. Qualification of the tissue allografts for sterilisation 8.2.1. Evaluation of the tissue allograft and packaging Prior to using radiation sterilisation for a tissue allograft, the effect that radiation will have on the tissue allograft and its components shall be considered. The key references given in Annex D contain information on this aspect. Similarly, the effect of radiation on the packaging shall also be considered. Guidance on the latter is given in Annex A of ISO 11137:1995. Using such information, a maximum acceptable dose shall be established for each tissue allograft and its packaging. 8.2.2. Sterilisation dose selection A knowledge of the number and resistance to radiation of the microorganism population as it occurs on the tissue allografts shall be obtained and used for determination of the sterilisation dose. For the sterilisation of health care products, a reference microbial resistance distribution was adopted in ISO 111371:1995 for microorganisms found typically on medical devices. Studies should be carried out to establish the types of microorganisms that are normally found on the tissue types to be

16

sterilised as well as their numbers and resistance to radiation. Such studies should take account of the distribution of the microorganisms within the tissue allograft itself since this may not be uniform. This should be determined by taking sample item portions (SIP) of the tissue and demonstrating that there are no significant statistical variations in distribution from SIP to SIP. If such studies show a consistent distribution of microoranisms from one tissue allograft to another, and one which is less resistant than the standard distribution of resistances (SDR) (see Table 1), then a table similar to B24 in ISO 11137:1995 giving a distribution of resistances appropriate to the allografts may be constructed for the purpose of sterilisation dose setting. This would allow the use of appropriate and perhaps lower sterilisation doses than would be the case if method 1 in ISO 11137:1995, based on the SDR in Table 1, were used. In the absence of such studies, the SDR may be used to establish sterilisation doses. To establish a sterilisation dose which will give a sterility assurance level (SAL) of 10"6, the methods based on those in ISO 11137:1995, ISO/TR 15844:1998, ISO/TR 13409:1996 and AAMI TIR 27:2001 should be used. A summary of these approaches as they apply to tissue allografts is given in Annex A. 8.2.3. Technical requirements The technical requirements to generate the information required for selection of the sterilisation dose shall be: (a) access to qualified microbiological and dosimetric laboratory services; (b) Microbiological testing performed in accordance with ISO 11737-1:1995 and ISO 11737-2:1998; and (c) access to a 60Co or 137Cs radiation source, or electron beam or X-ray irradiators.

17

8.2.4. Transfer of sterilisation dose The conditions for transferring the sterilisation dose between two irradiation facilities are the same as those given in ISO 11137:1995 (Sec. 6.2.3) and apply equally to tissue allografts. 8.3. Qualification of the irradiation facility The principles covering the documentation of the irradiation system, its testing, calibration and dose mapping are covered in ISO 11137:1995 (Sec. 6.3) and apply equally to tissue allografts. 8.4. Qualification of the irradiation process 8.4.1. Determination of the product-loading pattern The principles given in ISO 11137:1995 (Sec. 6.4.1) covering this shall also apply for the sterilisation of tissue allografts. 8.4.2. Product dose mapping In general, the guidelines given in ISO 11137:1995 (Sec. 6.4.2) apply also to tissue allografts. However, it should be recognised that the product dose mapping of relatively uniform (i.e. in shape, size, composition and density) health care products is a more straight-forward task than the product dose mapping of tissue allografts, which by their nature are more variable in their physical characteristics. In particular, the density of tissue allografts may vary depending on their water content. In addition, some tissue allografts may be heterogeneous in their distribution of density within the product, requiring an appropriate number of dosimeters for the dose mapping exercise. A consideration of these factors affecting the actual absorbed dose in tissue allografts must be undertaken so that the level of accuracy in delivering a dose to a particular tissue can be determined.

18

The acceptability of the accuracy of delivering a dose to tissue allografts will depend on the dose delivered in the verification dose experiments. If, for example, the actual dose delivered at its lowest possible accuracy limit is less than 90% of the verification dose, then the verification test must be repeated at a higher dose. Similarly, the minimum absorbed dose administered for sterilisation should take into account the likely variation in dose delivered so that sterilisation can be assured. As a guideline, uncertainties in the delivered dose should be within ±10%. 8.5. Maintenance of validation The guidelines covering calibration of equipment and dosimetric systems, irradiator requalification and sterilisation dose auditing are the same as given in ISO 11137:1995 (Sec. 6.6) and apply equally to tissue allografts. 8.6. Routine sterilisation process control The guidelines covering process specification, tissue allograft handling and packing in the irradiation container, sterilisation process documentation are similar to those given in ISO 11137: 1995 (Sec. 7) and apply equally to tissue allografts. 9. Quality, Safety and Clinical Application of the Tissue Allograft A programme to demonstrate the quality, safety and clinical application of the tissue allograft throughout its shelf life shall be performed. Sampling procedures appropriate to the tissue type should be devised for this purpose. 10. Documentation and Certification Procedures Information gathered or produced while conducting the qualification and validation of the tissue allografts, tissue bank facilities

19

and tissue processing, preservation and radiation sterilisation procedures shall be documented and reviewed for acceptability by a designated individual or group and retained in accordance with ISO 9001:2000 and the IAEA international standard for tissue banks or revision thereof, whichever is applicable. 11. Management and Control Control of the procedures involved in the selection of tissue donors, tissue processing and preservation prior to sterilisation by radiation and the radiation sterilisation process itself, shall be fully documented and managed in accordance with ISO 9001:2000 and IAEA International Standard for Tissue Banks, whichever is applicable. Annex A. Establishing a Sterilisation Dose A.I. Scope This annex describes the practices and procedures for determining the bioburden levels of the tissue allografts and the application of this information to establish the radiation sterilisation dose. It must to be emphasised hat such samples must be the end results of the series of validated donor screening and subsequent procedures as are described in the IAEA international standards for tissue banks. A.2. Selection of tissue allograft products Tissue allografts can be prepared from a wide range of tissues such as skin, amnion, bone, cartilage tendons and ligaments. If samples can be prepared from these tissues, which are reasonably reproducible in shape, size and composition and also in sufficient numbers for statistical purposes, then the usual sampling procedures apply, as given, for example, in ISO 11137 and ISO/ TR 13409. However, if allograft products are both few in number

20

(less than 10) and cannot be considered as identical products then it may be necessary to take multiple sample item portions of a single tissue allograft product for both bioburden analysis prior to sterilisation and also for the purpose of establishing a sterilisation dose. In such instances, it is important to have confidence in the distribution of microorganisms throughout the sample, obtained, for example, by periodic monitoring of such products.

A.3. Sample item portion (SIP) The SIP shall validly represent the microbial challenge presented to the sterilisation process. SIPs may be used both to verify that microorganisms are distributed evenly, bioburden estimation and for establishing a sterilisation dose. It is important to ascertain that the SIPs are representative, not only in shape size and composition but also in bioburden. Statistical tests should be applied to establish this. At least 20 SIPs should be used (10 for bioburden testing and 10 for the verification dose experiments).

A.4. Bioburden determination Bioburden determination could include the count of aerobic bacteria, spores, yeasts, molds and anaerobic bacteria. Many factors determine the choice of the tests most appropriate for the tissue allograft. At a minimum, the aerobic bacteria and fungi should be counted. The objective of the bioburden determination is to: (a) determine the total number of viable microorganisms within or on a tissue allograft and the packaging after completion of all processing steps before sterilisation; (b) act as an early warning system for possible production problems; and (c) calculate the dose necessary for effective radiation sterilisation.

21

The validation of the bioburden estimation requires the determination of the effectiveness and reproducibility of the test method. The steps to estimate bioburden are the shown in the following flow chart and full details can be found in ISO 117371:1995. Sample collection

For large production batches, randomly select units or SIPs of tissue allografts. For small production batches, take either sample item portions (SIPs) or whole sample from tissues allografts. For a single large piece of allografts, collect the total volume of the eluent solution from the last washing of the tissue allograft processing. Transport of the sample to the laboratory

During transportation, tissue samples for bioburden estimation should be kept under the same conditions as for the whole production batch. Removal of micro-organisms from the sample

Stomaching: This method is particularly suitable for skin, amnion and other soft tissue-like films or in the form of a tube. The test item and a known volume of eluent should be enclosed in a sterile stomacher bag. Reciprocating paddles operate the bag and force the eluent through and around the item. The time of treatment should be recorded. Shaking with or without glass beads: The test item is immersed in a known volume of eluent within a suitable vessel and shaken using a mechanical shaker (reciprocating, orbital, vortex mixing or wrist action). Glass beads of a defined size may be added to increase surface abrasion and thereby recovery efficiency. The time and frequency of shaking should be recorded.

22

Ultrasonication: The test item is immersed in a known volume of eluent within a suitable vessel. The time and ultrasonic intensity of the treatment should be recorded. Flushing: The test item is flushed with a known volume of eluent and the resulting solution is collected. Transfer to culture medium and incubation

A number of transferring methods can be employed, including: membrane filtration, pour plating, spread plates, most probable number (MPN). Enumeration

For tissue bioburden determination, the total microbial count should be carried out. Characterization

For contaminants that are commonly found and those suspected to be most radiation resistant should be isolated and characterized. A.5. Verification dose experiments In ISO 11137, the concept of establishing a verification dose for a SAL value which is much higher than 10"6, for example, for a SAL value of 10~2 was proposed as an experimental method of establishing the sterilisation dose corresponding to a SAL of

io-6.

For such verification dose experiments, samples of tissue allografts should be taken from production batches and irradiated at the calculated verification dose. In these experiments it is assumed (and should be demonstrated statistically) that the tissue allograft products are reasonably uniform in shape, size, composition and bioburden distribution. For single batch sizes up to 999, the numbers of sample required may be obtained from

23

Table 1 of ISO/TR 13409. For minimum batch sizes of 20-79, for example, 10 samples are required for the bioburden determination and 10 for the verification dose experiment. In general, the number of samples required for the bioburden determination and verification dose experiments will depend on the number of batches and the number of samples in each batch. For each circumstance, the number of positive sterility tests allowed in the verification dose experiment should be calculated statistically using an acceptable range of values of probability for 0, 1, 2, 3 etc. positive tests of sterility. For the 100 samples used in method 1 of ISO 11137, for example, there is a 92% chance of there being 1% positives when up to 2 positives are detected and a 10% chance of accepting a batch with 5.23% positives (W.A. Taylor and J.M. Hansen, Alternative Sample Sizes for Verification Dose

Experiments and Dose Audits, Radiation Physics and Chemistry (1999) 54, 65-75). For the 10 samples taken in ISO/TR 13409:1996 from a batch of 20, up to one positive test of sterility is proposed. For 30 or more, up to 2 positive tests of sterility are proposed (ISO/TR 13409:1996). It should be noted here that these latter statistical tests do not offer the same degree of protection as obtained when accepting up to two positive tests of sterility for a sample size of 100. For example, when accepting up to one positive test of sterility in a sample size of ten, there is a 95% chance of accepting a batch with 3.68% positives and a 10% chance of accepting a batch with 33.6% positives. Alternative sampling strategies are now available [see Taylor and Hansen (1999) above] which include for example, double sampling plans which can minimise sample sizes and yet offer similar protection. For single batches of low sample sizes, protection levels similar to those of the 100 sample approach in ISO 11137 can only be obtained by accepting a small number (possibly even zero) of positive sterility tests. For example, accepting up to one positive for a sample size of 50 offers similar protection. Hence, in ISO/TR 13409:1996 the verification dose for 10 samples taken from a batch of 20 is that which is required to

24

produce a SAL of 10 1 (the reciprocal of the number of SIPs used) and is that dose which will yield not more than one positive test of sterility from the ten irradiated SIPs. In order to calculate the verification doses as well as the doses required to produce a SAL value of 10 ~6, one of several approaches may be taken to establish an appropriate verification dose for low sample numbers (up to 100 but typically much less). The methods proposed here for the establishment of a sterilisation dose are based on statistical approaches used previously for the sterilisation of health care products (ISO 11137: 1995, ISO 13409:1996, ISO 15844:1998, AAMI TIR 27:2001) and modified appropriately for the typical low numbers of tissue allografts samples available. For a standard distribution of resistance (SDR), the tissue bank may elect to substantiate a sterilisation dose of 25 kGy for microbial levels up to 1,000 cfu per unit. Alternatively, for the SDR and other microbial distribution, specific sterilisation doses may be validated depending on the bioburden levels and radiation resistances (Dw values) of the constituent microorganisms. (a) For establishing specific sterilisation doses for standard distribution of resistance and other microbial distribution for samples sizes between 10 and 100 an adaptation of method 1 of ISO 11137:1995 may be used. Method 1 of ISO 11137 is normally used for multiple batches containing a large number of samples per batch. For batches of 100 samples for example, verification dose experiments are carried out for a SAL of 10 ~2. A successful experiment (up to 2 positive tests of sterility) will then enable the dose required to achieve a SAL value of 10~6 to be calculated from the survival curve of a standard distribution of resistances (SDR). In this code, an extension of Table 1 of ISO 11137 is given so that verification doses for SAL values between 10"2 and 10"1 may be found for bioburden levels up to 1,000 cfu per allograft product. These SAL values correspond to relativelow sample sizes of 10-100. This allows method 1 to be used for typical tissue allografts where relatively low numbers of samples are available and also where the distribution of microbial radiation

25 resistances is known and different to the SDR. The worked example given later uses this approach and, in addition, applies it (with appropriate statistical sampling, see above) to a microbial population which has a different distribution of radiation resistances than the SDR. However, for low bioburden levels combined with low sample numbers, it may be anticipated that there is an increased probability using this adaptation of method 1 that the verification dose experiment may fail. In the case of failure, the methods outlined in (b) and/or (c) may be used. (b) For substantiation of a 25 kGy sterilisation dose, the method in ISO/TR 13409:1996 may be used to calculate the verification dose. This is an accredited method and is essentially a modification of the method in (a) above and applies only to a standard distribution of resistances. In this method, the verification dose for a given SAL is approximated to the initial bioburden by a series of linear relationships. Each linear equation is valid for a particular ten-fold domain of bioburden level, e.g., 1-10 cfu. The method in ISO/TR 13409:1996 can only be used to substantiate a dose of 25 kGy. It should be noted that the statistical approach allowing up to one positive test for sample sizes up to 30 and up to 2 positive tests for sample sizes above 30 does not offer the same level of protection as for the 100 samples in ISO 11137 until the sample size reaches 100. Alternative sampling strategies may be employed (Taylor and Hansen, 1999) for all the verification dose methods proposed here. (c) For substantiation of a 25 kGy sterilisation dose, an alternative and more recent method in AAMI TIR 27 may be used. The modification takes into account how the verification dose varies with bioburden level for a given SAL (and sample size) on the assumption that an SAL of 10~6 is to be achieved at 25 kGy. Depending on the actual bioburden levels to be used (1-50 or 51-1,000 cfu per allograft product), a linear extrapolation of the appropriate SDR survival curve is made from either (log No, 0 kGy) or (log 10"2) to (log 10"6, 25 kGy) for 1-50 cfu and 511,000 cfu, respectively. For bioburden levels less than 1,000 cfu per allograft unit, these constructed survival curves represent a

26

more radiation resistant bioburden than would otherwise be the case. The validity of this approach arises from the purpose of the method which is to validate a sterilisation dose of 25 kGy. For all bioburden levels below 1,000 cfu per allograft product, this means that for the reference microbial resistance distribution given in Table B24 of ISO 11137:1995 for medical devices, a more conservative approach to the calculation of a verification dose is taken. Hence, this modification allows the use of greater verification doses than would be allowed using the formula given in either method 1 of ISO 11137 or in ISO/TR 13409:1996. The result is that there are fewer unexpected and unwarranted failures relative to verification doses experiments carried out using the method in ISO/TR 13409:1996. At a bioburden level of exactly 1,000 cfu per allograft product (the maximum in both methods), there is no difference in the outcome of the methods, i.e., the calculated verification doses are identical. A.6. Procedures (a) Establish test sample sizes Select at least 10 allograft products or SIPs, as appropriate, for the determination of the initial bioburden. The number of allograft products or SIPs should be sufficient to represent validly the bioburden on the allograft product(s) to be sterilised. Select between 10 and 100 allograft products (or SIPs) for the verification dose experiments and record the corresponding verification dose SAL (= 1/n, where n is the number of allograft products or SIPs used). For 20-79 allograft products in a single batch, 10 allograft products may be used for both the bioburden determination and the verification dose experiment. (b) Determine the average bioburden Using methods such as those in ISO 11737-1:1995 and as described above (Bioburden estimation), determine the average

27

bioburden of at least 10 allograft products or SIPs (the number will depend on the number of batches and the number of samples in the batches). For SIP values less than unity, the bioburden level for the whole product should be calculated and should be less than 1,000 cfu per allograft product for verification dose experiments carried out to substantiate a 25 kGy sterilisation dose. (c) Establish the verification dose The appropriate verification dose depends on the number of samples (allograft products or SIPs) to be used in the experiment (= I/number of samples). The verification dose calculation depends on which of the three methods above is being used, as follows: (i) For establishing specific sterilisation doses for standard distribution of resistance and other microbial distribution for samples sizes between 10 and 100: an adaptation of method 1 of ISO 11137:1995. Calculate the dose required to achieve the required SAL from a knowledge of the initial bioburden level and from the microbial distribution and associated radiation resistances. This may be calculated from the equation, Ntot = N0(1)10-(D/Di) + N0(2)10-(D/D2) + - + N0(n)10-(D/D»), where Ntoi, represents the numbers of survivors; Afy,) represents the initial numbers of the various microbial strains i (where i = 1 - ri); and D\, D2, ..., D^ represent the Dw values of the various microbial strains. D represents the radiation dose and n the number of terms in the equation for a standard distribution of resistances (n = 10). For the reference standard distribution of resistances (Davis, K.W., Strawderman, W.E. and Whitby, J.L. (1984). /. Appl. Bacteriol. 57, 31-50) used in ISO 11137:1995 for medical devices (see Table 1), this equation will produce data similar to Table B.I of ISO 11137:1995 but for SAL values

28

between 10 2 and 10 r instead. By equating Ntot to the selected SAL value and by using the appropriate Dw values for each microbial type together with their numbers prior to irradiation, the verification dose, D, for SAL values between 10~2 and 10"1 can be calculated. These values are set out in Table 2(a). The same calculation can be used to find the sterilisation dose for the desired SAL of 10"6 or reference can be made to Table B.I of ISO 11137:1995. In this method, the sterilisation dose is calculated using the bioburden level of the whole product. Alternatively, approximate values of the verification doses to achieve the same SAL values may be calculated using the equation given in ISO/ TR 13409:1996 (see next paragraph). (ii) For substantiation of a 25 kGy sterilisation dose, method ISO/TR 13409:1996: From a knowledge of the average bioburden and the number of samples or SIPs to be used in the verification experiment, the verification dose for a standard distribution of resistances is approximated by the equation: Verification dose at a the selected SAL = I + [S x log (bioburden)] where I and S are given in Annex C, Table 3 of this code. (iii) For substantiation of a 25 kGy sterilisation dose, AAMI TIR 27:2001: The calculation of the verification dose follows the procedures by Kowalski and Tallentire, 1999 (Radiat. Phys. Chem. 54, 55-64) where the bioburden levels refer to either the SIP or whole product whichever is being used in the verification dose experiment: For bioburden levels of 1 to 50 cfu per allogmft product or SIPs

Step 1: DIin = 25 kGy/(6 + log NQ), Step 2: Verification dose = Dlin (log No - log SAL V D)/ where Diin represents the D\Q dose for a hypothetical survival curve which is linear between the coordinates (log No, 0 kGy) and (log 10 ^6, 25 kGy) for initial bioburden levels, No, up to 1,000 cfu per allograft product. This linear plot therefore represents a constructed survival curve in which there is 1 out of

29

106 probability of a survivor at 25 kGy. The method is valid therefore only for the substantiation of a 25 kGy sterilisation dose regardless of whether a lower dose could in fact be validated. For bioburden levels of 51 to 1,000 cfu per allograft product or SIPs

Step 1: For a particular value of bioburden, use Table B.I of ISO 11137:1995 to identify the doses (kGy) corresponding to SAL values of 10"2 [D(10"2)] and 10"6 [D(10"6)]. From these values, calculate TDW from the following equation: TDW = (Dose™6 kGy - Dose"2 kGy)/4, where TDW represents the hypothetical Dw value for a survival curve for a standard distribution of resistances which has been modified such that it is linear between log 10"2 and log 10"6 (log SAL values) when plotted against dose, with the log 10"6 value being set at 25 kGy. Essentially, this produces a survival curve which is more resistant to radiation than the SDR (for bioburden levels less than 1,000 cfu per allograft product) and one which is appropriate to substantiation of a 25 kGy sterilisation dose only. Step 2: Verification dose = 25 kGy - [TDW (log SALVD + 6)], where SAL V D is the sterility assurance level at which the verification dose experiment is to be performed. (d) Perform verification dose experiment Irradiate the tissue allografts or SIPs thereof at the verification dose. Irradiation conditions of the samples for verification of the substerilisation dose should be the same as the whole batch which is to be sterilised. For example, if the produced tissue batch is irradiated in frozen condition, the samples for the substerilisation dose verification studies should be irradiated in the same condition and the frozen condition should be kept during the whole irradiation process.

30

The defined test sample size (SIP < 1), according to the SAL and batch size, is exposed to radiation at the verification dose. The dose delivered should not be less than 90% of the calculated verification dose. Test the tissue allografts for sterility using the methods in ISO 11737-2:1998 and record the number of positive tests of sterility. The irradiated SIPs, of all types of tissue allografts, are transferred to a growth medium and incubated for at least 14 days at an appropriated temperatures. Positive and negative sterility tests results should be registered. For bone and skin allografts, an additional test is recommended to detect anaerobic bacteria. (e) Interpretation of results For a verification dose experiment performed with up to 30 allograft products or SIPs, statistical verification is accepted if there is no more than one positive test of sterility observed. For 30 to 100 products or SIPs, statistical verification is accepted if there are no more than two positive tests of sterility observed (ISO/TR 13409:1996). Where the verification dose experiment is successful, the dose required to produce a SAL of 10"6 for the whole allograft product should be calculated for procedure c(i) as indicated above and calculated in Annex C, Table 2(b). For procedures c(ii) and c(iii), a successful verification dose experiment substantiates the use of 25 kGy as a sterilisation dose.

A.7. Routine use of sterilisation doses The routine use of a sterilisation dose calculated in procedure c(i) or of 25 kGy as substantiated by either procedure c(ii) or c(iii) shall only be valid if the tissue selection and tissue processing procedures have been demonstrated to produce tissues allografts with consistent bioburden levels. It should be demonstrated that the level of variation in bioburden, is consistent with the

31

sterilisation dose to be used routinely. In such cases, sterilisation dose audits should be carried out at regular intervals, at least every three months. Annex B. Sterilisation of Tissue Allografts (Examples of Sterilisation Procedures) B.I. Limited number of amnion samples with low bioburden and low bacterial resistance using method 1 of ISO 11137:1995 to calculate the verification dose B.I.I. Introduction This method uses method 1 of ISO 11137:1995 but applies it to sample sizes of less than 100 in a single production batch. The example chosen consists of a single batch of 20 amnion membranes ( 5 x 5 cm) from which 10 are used for the bioburden determination and 10 are used for the verification dose experiment. The data used in the example are consistent with data on bioburden levels, bacterial types and distribution found in Hilmy et al. (2000). /. Cell Tissue Banking 1, 143-147. In that study, the most radiation resistant microbes were assumed to have a D10 value of 1.8 kGy, i.e., a distribution which differs from the reference microbial resistance distribution in that there are no microbes with a D10 value higher than 1.8 kGy. Furthermore, the tissue processing and preservation procedures have produced tissue allografts which are much lower than 1,000 cfu per allograft product. For such samples, a sterilisation dose which is significantly less than 25 kGy is confirmed from the verification dose experiment. B.I.2. Procured tissue qualification (a) Tissue type: ... Amnion samples of 5 x 5 cm (b) Screening of tissue for transmission of disease: ...

32

Age of donor: ... 25 ... Medical, social and sexual history: ... None to suggest risk of transmissible disease Serological tests: ... HIV (HIV-1,2 Ab) ... negative; Hepatitis C (HCV-Ab) ... negative; Hepatitis B (HBs-Ag) ... negative; Syphillis (VDRL) ... negative. B.I.3. Tissue processing and preservation qualification (a) Description of processing technique ... hypochlorite, (b) Description of preservation technique ... lyophilization (c) Typical microbial levels of procured tissue before processing ... in the range of 5,000-10,000 cfu per tissue ... (d) Typical bioburden levels of processed and preserved tissues ... 57 cfu per allograft product (Note 1) It is noted from the study of Hilmy et al. (see above) that the bioburden levels of the processed tissue (i.e. before sterilisation by irradiation) decreased from about 1,400 cfu to 120 cfu during the study period 1994 to 1997, with 1998 data showing an average of 57 cfu per allograft product (range 12-160 cfu). Clearly, good processing techniques can have a dramatic effect on the bioburden levels of the tissue being prepared for sterilisation by irradiation. The level of reduction used in this example is probably therefore a conservative estimate of the degree of elimination of bacteria B.1.4. Qualification of tissue allografts for sterilisation Typical bioburden distribution: The distribution of bacterial resistances given below is assumed to consist entirely of bacteria with a D10 value of 1.8 kGy and represents a distribution which is similar but not identical to the standard distribution of resistances, i.e.: Dio (kGy) 1.8; Frequency 1.0.

33

B.I.5. Calculation of the sterilisation dose Stage Stage 1 Production batch size

Value 40

Comments 5 x 5 cm amnion samples.

10

Test sample size for the verification dose experiment

10

Verification dose required for SAL 10"1 (= 1/10).

20

10 for bioburden; 10 for verification dose experiment.

Stage 2 Obtain samples Stage 3 SIP Average bioburden Stage 4 Verification dose calculation

i—i

Test sample size for bioburden determination

The whole allograft product is used.

57

Bioburden results of 15, 91, 99, 30, 30, 99, 8, 84, 91, 23.

3.2 k

Using the bacterial resistance distribution given above (and not the SDR), the survival equation is constructed (see Annex A) and used to calculate the verification dose (D) for a JV(tot) value of 0.1 (equivalent to a SAL value of 0.1, the reciprocal of the number of samples used) and where the total initial number of microorganisms (Continued)

34

(Continued)

Stage

Value

Comments per product (SIP = 1) is equal to 57. The survival equation is: Ntot = 57 x lO-P/1-8) From this data, the verification dose is calculated as 3.2 kGy.

Stage 5 Verification dose experiments

3.3 kGy (delivered dose) 1 positive/10 samples

The sterility test yielded one positive test out of ten and therefore the verification dose experiment was successful (but note that the level of protection is significantly less than allowing up to 2 positives for a sample size of 100, see Annex A) and the sterilisation dose for SAL = 10"6 can be calculated from the survival equation given above (= 14.0 kGy). Note: In the case that a SIP < 1 was taken instead, the bioburden for the whole product should be used to calculate the sterilisation dose.

B.I.6. Conclusion This example shows how the combination of good tissue processing and preservation and sterilisation by ionising radiation, for samples which are known to have bacterial contamination relatively susceptible to radiation, can allow the use of a sterilisation dose which is much less than 25 kGy.

35

B.2. Limited number of amnion samples requiring only substantiation of 25 kGy as a sterilisation dose B.2.1. Introduction In this example, it is assumed that there is a standard distribution of resistances which defines the bacterial contamination of the tissue allografts. The example chosen consists of a single batch of 40 amnion membranes ( 5 x 5 cm) from which 10 are used for the bioburden determination and 10 are used for the verification dose experiment. The data used in the example are consistent with data on bioburden levels, bacterial types and distribution found in Hilmy et al. (2000). /. Cell Tissue Banking 1, 143-147. Furthermore, for the limited number of samples to be tested, it is required only to establish that a 25 kGy dose may be used to achieve an SAL of 10~6. It is shown below that when the method in ISO 13409:1996 is applied for 20 samples (10 for the bioburden determination and 10 for the verification dose experiment), from a batch size of 40, the samples fail the verification dose experiment. To increase the probability of a successful verification dose experiment, whilst at the same time substantiating a sterilisation dose of 25 kGy, the method of Tallentire and Kowalski is applied (see Annex A). This allows the use of a higher verification dose and it is then found that the samples pass this test, substantiating the use of a 25 kGy sterilisation dose. B.2.2. Procured tissue qualification (a) Tissue type ... Amnion ( 5 x 5 cm) (b) Screening of tissue for transmission of disease Age of donor ... 25 Medical, social and sexual history ... None to suggest risk of transmissible disease Serological tests: HIV (HIV-1,2 Ab) ... negative; Hepatitis C (HCV-Ab) ... negative; Hepatitis B (HBs-Ag) ... negative; Syphillis (VDRL) ... negative.

36

B.2.3. Tissue processing and preservation qualification (a) Description of processing technique ... hypochlorite (b) Description of preservation technique ... ly optimization (c) Typical microbial levels of procured tissue before processing ... in the range of 5,000-10,000 cfu per tissue ... (d) Typical bioburden levels of processed and preserved tissues ... 57 cfu per allograft product (Note 1). B.2.4. Qualification of tissue allografts for sterilisation Typical bioburden distribution (it is assumed that the standard distribution of resistances, see Annex A, is valid). Stage Stage 1 Production batch size

Value 40

Comments 5 x 5 cm amnion samples.

Test sample size for bioburden determination

10

Test sample size for the verification dose experiment

10

Verification dose required for SAL 10-1 (= 1/10).

20

10 for bioburden; 10 for verification dose experiment.

Stage 2 Obtain samples Stage 3 SIP

1

The whole allograft product is used. (Continued)

37 (Continued)

Stage Average bioburden

Value

Comments

57

Bioburden results of 15, 91, 99, 30, 30, 99, 8, 84, 91, 23. Average bioburden for whole product 57 cfu. (This is less than 1,000 cfu and therefore the method may be used.) Note: If a SIP <1 was taken, then the bioburden of the whole product should be calculated and should be less than 1,000 cfu per allograft product for this method to be valid.

Stage 4 Verification dose 4.6 kGy The verification dose is calculated using the method in ISO/TR 13409: calculation (1) 1996. In this method (applicable to a standard distribution of resistances only), the verification dose for a given SAL is approximated to the initial bioburden by a series of linear relationships using the parameters I and S (see below). Each linear equation is valid for a particular ten-fold domain of bioburden level, e.g. 10-100 cfu. For a bioburden of 57 and sample size of 10, / and S values of 0.67 and 2.23 respectively are obtained from ISO/TR 13409:1996 and are given here in Annex C, Table 3. The verification dose is given by: Dose = I + [S x log (average SIP bioburden)] = 0.67 + (2.23 x log x 57) = 4.6 kGy. (Continued)

38 (Continued)

Stage Stage 5 Verification dose experiments (1)

Verification dose calculation (2)

Value

Comments

4.5 kGy (delivered dose) 2 positives/ 10 samples

The sterility test yielded two positive tests out of ten and therefore the verification dose experiment was not successful and a sterilisation dose of 25 kGy could not be substantiated.

8.6 kGy

A new verification dose was calculated using the method of Tallentire and Kowalski (see Annex A). This method takes into account how the verification dose for a standard distribution of resistances (reference microbial resistance distribution) varies with bioburden level for a given SAL (and sample size) on the assumption that an SAL of 10~6 is to be achieved at 25 kGy. Application of method 1 of ISO 11137:1995 for bioburden levels of less than 1,000 cfu would yield sterilisation doses of less than 25 kGy. The method of Tallentire and Kowalski assumes instead that only substantiation of a 25 kGy sterilisation dose is required regardless of the bioburden level. Extrapolation of the reference distribution to produce an SAL of 10"6 at 25 kGy for bioburden levels of less than 1,000 cfu allows the use of higher verification doses (Continued)

39 (Continued)

Stage

Value

Comments than would be predicted by method 1 of ISO 11137:1995 and hence a greater probability of a successful verification dose experiment. For a bioburden level of 120 (i.e. between 51 and 1,000), the doses corresponding to this bioburden for SAL values of 10"6 and 10"2 are found from Table 1 of ISO11137 and are designated Dose"6 and Dose"2 respectively, from which the TDio is calculated as follows: TDW = (Dose"6 kGy - Dose"2 kGy)/4 = (20.4 - 7.3)/4 = 3.27 kGy, where TDW represents the hypothetical Dio value for a survival curve for a standard distribution of resistances which has been modified such that it is linear between log 10~2 and log 10"6 (log SAL values) when plotted against dose, with the log 10~6 value being set at 25 kGy. Essentially, this produces a survival curve which is more resistant to radiation than the SDR (for bioburden levels less than 1,000 cfu per allograft product) and one which is appropriate to substantiation of a 25 kGy sterilisation dose only. Note: Table 1 of ISO 11137:1995 does not have a value corresponding to a (Continued)

40 (Continued)

Stage

Value

Comments bioburden of 57 and so the next highest value of 59.2 is used. The verification dose, VD, is then calculated, as follows: VD = 25 kGy - [TDW (log SALVD + 6)] = 25 - [3.27 (log 0.1 + 6)] = 8.6 kGy, where SAL V D is the sterility assurance level at which the verification dose experiment is to be performed, (= the reciprocal of the number of samples), in this case, 0.1.

8.5 kGy The 10 samples are irradiated at this Verification 1 positive/ verification dose and tested for dose experiments (2) 10 samples sterility. The sterility tests yielded one positive test out of ten and therefore the use of 25 kGy as a sterilisation dose (SAL = 10"6) could be substantiated (note however that this result does not offer the same level of protection when allowing up to 2 positives in a sample size of 100, see above).

B.2.5. Conclusion Although the tissue processing and preservation produced tissues with relatively low bioburden for which sterilisation doses substantially less than 25 kGy could have been used (see example above), the tissue bank required only a method to substantiate a sterilisation dose of 25 kGy. The application of the methods of ISO 13409:1996 and of Tallentire and Kowalski,

41

which are particularly suitable for bioburden levels much less than 1,000 cfu per allograft product, allowed the use of relatively high verification doses and, hence a greater probability of success. In the example chosen, the method in ISO 13409:1996 failed and hence the method of Tallentire and Kowalski was used as well. For tissue banks which prefer to use a standard 25 kGy sterilisation dose, this latter method will be more efficient in that fewer verification dose experiments will fail.

B.3. Limited number of bone samples with very low bioburden and SDR using ISO/TR 13409:1996 to calculate the verification dose (SIP < 1) B.3.1. Introduction This method uses ISO/TR 13409:1996 an applies it to a sample of 40 small pieces of bone. Typically, very low bioburden levels are found after processing. In this example, very low SIP values are used so that most of the allograft product can be retained for use. B.3.2. Procured tissue qualification (a) Tissue type: ... bone cut into 40 small pieces (chips) (b) Screening of tissues donor Age of donor ... 36 ... Medical, social and sexual history: None to suggest of transmissible disease Serological tests: HIV (HIV-1,2 Ab) ... negative; Hepatitis C (HCV-Ab) ... negative; Hepatitis B (HBs-Ag) ... negative; Syphillis (VDRL) ... negative. B.3.3. Tissue processing and preservation qualification (a) Description of processing technique ... cut into standardised small pieces.

42

(b) Description of preservation technique ... frozen. (c) Typical bioburden levels of processed and preserved tissues ... 40 cfu per allograft product. B.3.4. Qualification of tissue allografts for sterilisation Stage Stage 1 Production batch size

Value

Comments

5

Bone cut into 40 small pieces (1 cc each) packed in flask, produced from one donor in one processing batch.

Test sample size for bioburden determination

10

According ISO/TR 13409:1996, Table 1.

Test sample size for verification dose experiment

10

According ISO/TR 13409:1996, Table 1.

20

A random sample of 20 standardised product portions of 1 cc each was obtained from the production batch.

Stage 2 Obtained samples

Stage 3 SIP

0.025

Calculated from 1/40.

SIP bioburden

1

Bioburden results of 1, 0, 2, 0, 1, 2, 1, 1, 1, 1 were observed from the 10 SIP tested, for an average bioburden of 1.

Average bioburden

40

The average bioburden for the product tested was calculated as follow: 1/0.025 = 40. This is (Continued)

43

(Continued)

Stage

Value

Comments less than 1,000 cfu per allograft product and therefore this method is valid.

Stage 4 Verification dose calculation

1.3

Stage 5 Verification dose 1.3 kGy experiment (delivered dose) 0 positive/ 10 samples

Stage 6 Interpretation of results

Verification dose formula: I + (S x log (average SIP bioburden) kGy. According ISO/TR 13409: 1996, Table 2, the I and S values are 1.25 and 1.65 respectively: = 1.25 + (1.65 x log 1) = 1.25 kGy = 1.3 kGy The test sterility yielded 0 positive from the 10 SIPs tested. Therefore, the verification experiment was successful and no further action was necessary. The test of sterility result was acceptable, the sterilisation dose of 25 kGy was confirmed.

B.3.5. Conclusion Although a lower sterilisation dose could be justified if the adaptation of method 1 of ISO 11137:1995 was applied, the tissue bank elected to use ISO/TR 13409:1996 to substantiate a 25 kGy sterilisation dose only.

44

Annex C. Tables 1, 2 and 3 Table 1. Microbial standard distribution of resistance (SDR) (Davis, K.W., Strawderman, W.E. and Whitby, J.L. (1984). The rationale and computer evaluation of a gamma sterilisation dose determination method for medical devices using a substerilisation incremental dose sterility test protocol, /. Appl. Bad. 57, 31-50). D10 (kGy) 1.0 1.5 2.0 2.5 2.8 3.1 3.4 3.7 4.0 4.2 % 65.487 22.493 6.302 3.179 1.213 0.786 0.350 0.111 0.072 0.007 Table 2(a). Radiation dose (kGy) required to achieve given SAL for different bioburden (cfu) having standard distribution of resistances. Sample size in) 10 15 1/10 1/15 SAL (1/n) Bioburden 0.06 0.08 0.09 0.10 0.12 0.14 0.17 0.19 0.22 0.26 0.29 0.34 0.39 0.44 0.50 0.57 0.65 0.73 0.83 0.93 1.0 1.2 1.4 1.6 1.8 2.0

1.1

1.0 1.1 1.2 1.3 1.3 1.4

1.2 1.3 1.4 1.5 1.6 1.7 1.7

1.5 1.5 1.7 1.8 1.9 1.9 2.0

1.0 1.1

25 20 1/20 1/25

35 30 1/30 1/35

1.0 1.1 1.2 1.3 1.4 1.4 1.5

1.0 1.1 1.2 1.3 1.3 1.4 1.5 1.6 1.7

1.6

1.7

1.0 1.1 1.2 1.3 1.4 1.5 1.5 1.6 1.7 1.8 1.9

1.7 1.7 1.9 2.0 2.1 2.2 2.2

1.8 1.9 2.0 2.1 2.2 2.3 2.4

2.0 2.0 2.1 2.3 2.4 2.5 2.5

2.0

1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.6 1.7 1.8 1.9 2.0 2.1

1.0 1.1 1.2 1.3 1.4 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2

2.1 2.1 2.3 2.4 2.5 2.6 2.7

2.2 2.2 2.4 2.5 2.6 2.7 2.8

2.3 2.3 2.5 2.6 2.7 2.8 2.9

1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.6 1.7 1.8 1.9

60 1/60

80 70 90 1/70 1/80 1/90

2.2

1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4

1.0 1.1 1.1 1.2 1.3 1.5 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6

1.1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7

2.3 2.4 2.5 2.7 2.8 2.9 3.0

2.5 2.5 2.7 2.8 2.9 3.0 3.1

2.7 2.8 2.9 3.1 3.2 3.3 3.4

2.8 2.9 3.0 3.2 3.3 3.4 3.5

40 45 50 1/40 1/45 1/50

1.0 1.1 1.2 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.1

1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.2 3.3

100 1/100

1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.4 3.5 3.6

45 Table 2(a). (Continued) Sample size (n) 10 15 20 25 30 35 40 45 50 60 70 80 90 SAL(l/«) 1/10 1/15 1/20 1/25 1/30 1/35 1/40 1/45 1/50 1/60 1/70 1/80 1/90 2.2 2.6 3.0

1.8

2.1

2.3

2.5

2.8

2.9

3.0

3.0

3.2

3.3

3.5

1.9

2.2 2.3

2.4

2.6 2.7 2.9

3.0

2.5

3.2 4.0 4.4 5.0 5.4 6.0 7.0

2.1 2.2 2.3 2.4 2.5 2.5 2.7

3.2 3.3 3.4 3.6 3.7 3.8

3.4 3.5 3.5 3.8 3.9 4.0 4.1

3.5 3.6 3.7

2.5 2.6 2.7 2.8 2.9 3.0

2.7 2.8 3.0 3.0 3.2 3.2

3.1 3.2 3.3 3.5 3.6

3.6 3.8 3.8 4.0 4.1

8.0 9.0

2.8 2.9 3.0 3.0 3.1 3.2 3.3 3.3 3.4 3.4 3.5 3.5 3.6

3.1 3.2 3.4 3.5 3.6 3.7 3.7 3.8 3.8 3.9 4.0 4.0 4.1

10 11

12 13

2.0

2.4 2.6 2.8 2.9 3.0 3.0 3.1 3.3 3.4 3.5 3.6 3.7 3.7 3.8 3.9 4.0 4.0

3.3

3.5 3.6

3.7 3.8 3.9 4.0 4.0 4.1 4.2 4.2 4.3 4.3 4.4 4.5 4.7 4.9

2.6 2.9

3.0

3.1

2.9 3.1 3.2 3.3

3.1 3.3 3.4 3.5 3.6 3.6 3.8 3.9 4.0 4.1

3.2 3.4 3.5 3.6 3.7

3.4 3.5 3.6 3.8 3.9 4.1 4.1 4.2 4.3

3.8

4.3

4.5

30

4.0

4.5

4.6

4.6 4.8

4.4 4.4 4.5 4.6 4.6 4.7 4.7 5.0 5.2 5.0 5.3 5.2 5.5 5.3 5.6 5.4 5.7 5.5 5.8 5.6 5.9 5.7 6.0 5.8 6.1 5.9 5.9 6.0 6.1

14 15 16 17 18 19 20 25 35

4.1

40

4.3

45 50 55 60 65 70 75

4.4 4.5

80 85 90 95 100 150 200

250 300 350

4.1 4.1 4.2 4.2

4.6

4.9 5.0 5.1

4.7 4.8 4.8

5.2 5.3 5.4

4.8 5.0 5.1 5.2 5.3 5.4 5.5 5.6

4.9 5.0 5.0 5.1

5.4 5.5 5.6 5.6

5.6 5.7 5.8 5.8

5.2 5.2 5.7

5.7

5.9

5.8 6.2 6.6

5.9 6.4 6.8

6.0 6.2 6.5 6.6

6.8 7.0 7.2

6.2 6.2 6.3 6.4 6.4 6.5 7.0 7.3 7.6

6.1 6.2 6.7 7.0 7.0 7.3 7.2 7.5 7.8 7.4 7.7 8.0

4.2

4.3 4.4 4.4

4.5 4.6 4.6 4.7 4.7 4.8 5.0 5.2 5.4 5.6 5.7 5.9 6.0 6.1 6.2 6.2 6.3 6.4 6.5 6.5 6.6 6.7 7.1 7.5 7.8 8.0 8.2

3.8 3.9 4.0 4.2 4.3 4.4 4.4 4.5 4.6 4.7

4.7 4.8 4.9 4.9 5.0 5.2 5.4 5.6 5.7 5.9 6.0 6.1 6.2 6.3

3.7 3.8 3.9 4.0 4.2 4.3 4.4 4.5 4.6 4.7

4.7 4.8 4.9

4.9 5.0 5.1 5.1 5.4 5.6 5.7 5.9 6.0 6.1 6.3 6.4 6.4

6.4 6.5 6.6 6.6 6.7

6.5

6.8

6.9 7.0 7.5

6.8 7.3 7.7 7.9 8.2 8.4

6.6 6.7 6.8 6.8

7.8 8.1 8.3 8.5

3.9 4.0

4.1 4.3

4.4 4.4 4.5 4.6 4.7 4.8 4.9 4.9 5.0 5.0 5.1 5.1

5.4 5.6 5.7 5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.9 7.0

4.2 4.3 4.4

4.6 4.7 4.8 4.9 5.0 5.0 5.1 5.2 5.3 5.3 5.4 5.4

5.7 5.9 6.1 6.2 6.4 6.5 6.6 6.7 6.8

3.9 4.0 4.1 4.2 4.3 4.5 4.6

4.7 4.8 4.9 5.0 5.1

5.2 5.3 5.3 5.4 5.5 5.5 5.6 5.9 6.1 6.2 6.4 6.5 6.7 6.8 6.9

4.2 4.3 4.4 4.6 4.7 4.9 5.0 5.1 5.2 5.3 5.3 5.4

5.5 5.6 5.6 5.7 5.7 6.0 6.2 6.4 6.6 6.7 6.8 6.9 7.0

6.9

7.0 7.1

7.1 7.2

7.0 7.0 7.1 7.2

7.2 7.2 7.3 7.4

7.3 7.4 7.5 7.5

7.0 7.1

7.3 7.3

7.4 7.5

7.6 7.9 8.2 8.5 8.7

7.8 8.2 8.5 8.7 8.9

7.6 7.7 8.0 8.2 8.4 8.5 8.7 8.8 8.9 9.1 9.1 9.3

3.6 3.7 3.9 3.9 4.1 4.2 4.4 4.5 4.6 4.7 4.8 5.0 5.1 5.2 5.3 5.4

5.5 5.6 5.6 5.7 5.8 5.8 5.9 6.1 6.3 6.5 6.7 6.8 7.0 7.1 7.2

7.3 7.4 7.5 7.5 7.6 7.7 7.8 7.8 8.3 8.7 9.0 9.2 9.4

100 1/100 3.7 3.8 4.0 4.0 4.2 4.3 4.5 4.6 4.7 4.8 5.0 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.7 5.8 5.9 5.9 6.0 6.3 6.5 6.6 6.8 7.0 7.1 7.2

7.3 7.4 7.5 7.6 7.7 7.7 7.8 7.9

7.9 8.5 8.8 9.1 9.4 9.5

46 Table 2(a). (Continued) Sample size in) SAL (1/n) 400 450 500 550 600 650 700 750 800 850 900 950 1,000

10 1/10

15 1/15

20 25 1/20 1/25

30 1/30

35 1/35

40 45 1/40 1/45

6.7 6.9 7.1 7.2 7.3 7.4 7.5 7.6 7.6 7.7 7.8 7.9 7.9

7.4 7.5 7.7 7.8 7.9 8.0 8.1 8.2 8.2 8.3 8.4 8.5 8.5

7.6 7.7 7.8 8.0 8.1 8.2 8.3 8.4 8.5 8.5 8.6 8.7 8.7

8.2 8.3 8.5 8.6 8.7 8.8 8.9 9.0 9.0 9.1 9.2 9.3 9.3

8.4 8.5 8.7 8.8 8.9 9.0 9.1 9.2 9.3 9.3 9.4 9.5 9.6

8.5 8.7 8.8 9.0 9.1 9.2 9.3 9.4 9.4 9.5 9.6 9.7 9.7

7.9 8.0 8.1 8.2 8.4 8.5 8.5 8.6 8.7 8.8 8.9 8.9 9.0

8.7 8.9 9.0 9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.8 9.9

50 1/50

60 1/60

70 1/70

80 90 1/80 1/90

8.8 9.1 9.0 9.2 9.1 9.4 9.2 9.5 9.3 9.6 9.5 9.7 9.6 9.8 9.7 9.9 9.7 10.0 9.8 10.1 9.9 10.1 10.0 10.2 10.0 10.3

9.3 9.4 9.6 9.7 9.8 9.9 10.0 10.1 10.2 10.3 10.3 10.4 10.5

9.4 9.6 9.7 9.9 10.0 10.1 10.2 10.3 10.4 10.4 10.5 10.6 10.7

9.6 9.8

9.7 9.9

9.9 10.0 10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 10.8

10.0 10.2 10.3 10.4 10.5 10.6 10.6 10.7 10.8 10.9 11.0

Table 2(b). Radiation dose (kGy) required to achieve an SAL of 10 different bioburdens having standard distribution of resistances. Bioburden Dose 0.06 0.08 0.09 0.10 0.12 0.14 0.17 0.19 0.22 0.26 0.29 0.34 0.39 0.44 0.50 0.57 0.65 0.73 0.83

10.4 10.6 10.8 11.0 11.3 11.5 11.7 11.9 12.1 12.3 12.5 12.7 12.9 13.1 13.3 13.5 13.6 13.8 14.0

Bioburden Dose 0.93 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.6 3.0 3.2 4.0 4.4 5.0 5.4 6.0 7.0 8.0 8.8

14.2 14.2 14.3 14.6 14.8 14.9 15.2 15.3 15.5 15.8 16.0 16.2 16.3 16.5 16.6 16.8 17.0 17.2 17.3

Bioburden Dose 9.0 10 11 12 13 14 15 16 17 18 19

20 30 40 50 60 70 80 90

17.4 17.6 17.7 17.9 18.0 18.1 18.2 18.3 18.4 18.5 18.6 18.7 19.3 19.7 20.1 20.3 20.6 20.8 21.0

100 1/100

6

for

Bioburden Dose 100 150 200 250 300 350 400 450 500 550 600

650 700 750 800 850 900 950

1,000

21.1 21.8 22.2 22.6 22.9 23.1 23.3 23.5 23.7 23.8 24.0 24.1 24.2 24.3 24.4 24.5 24.6 24.7 24.8

47

Table 3. I and S for calculation of verification dose for test sample size and bioburden level (ISO/TR 13409:1996). Verification dose at a given SAL = I + (S x log (Avergare SIP bioburden)). I = intercept; S = slope. Test sample size

10 20 30 40

50 60 70 80 90

Bioburden 1 to 10

Bioburden 11 to 100

Bioburden 101 to 1,000

I

S

I

S

i

S

1.25 1.71 2.00 2.21 2.38 2.52 2.65 2.76 2.86

1.65 1.82 1.93 2.01 2.07 2.12 2.16 2.19 2.22

0.67 1.14 1.46 1.69 1.88 2.03 2.16 2.30 2.39

2.23 2.41 2.49 2.55 2.59 2.63 2.66 2.67 2.70

-0.26 0.35 0.71 1.00 1.21 1.40 1.55 1.67 1.80

2.71 2.81 2.87 2.90 2.93 2.95 2.97 2.99 3.00

Annex D. Key References for the Sterilisation of Tissues by Ionising Radiation D.I. Bone AKKUS, O. and RIMNAC, CM. (2001). Fracture resistance of gamma radiation sterilised cortical bone allografts, /. Orthop. Res. 19, 927-934. CORNU, O., BANSE, X., DOCQUIER, P.L., LUYCKX, S. and DELLOYE, C. (2000). Effect of freeze-drying and gamma irradiation on the mechanical properties of human cancellous bone, /. Orthop. Res. 18, 426-431. MOREAU, M.F., GALLOIS, Y., BASLE, M.F. and CHAPPARD, D. (2000). Gamma irradiation of human bone allografts alters medullary lipids and releases toxic compounds for osteoblastlike cells, Biomaterials 21, 369-376. SILBERMAN, F. and KAIRIYAMA, E. (2000). Radiation sterilisation and the surgical use of bone allografts in Argentina, Advances in Tissue Banking 4, 27-38.

48

ARAKI, N., MYOUI, A., KURATSU, S., HASHIMOTO, N., INOUE, T., KUDAWARA, I., UEDA, T., YOSHIKAWA, H., MASAKI, N. and UCHIDA, A. (1999). Intraoperative extracorporeal autogenous irradiated bone grafts in tumour surgery, Clin. Orthop. 368, 196-206. RUSSELL, J.L. and BLOCK, J.E. (1999). Clinical utility of demineralised bone matrix for osseous defects, arthrodesis and reconstruction: impact of processing techniques and stud methodology, Orthopedics 22, 524-531. MARCZYNSKI, W., TYLMAN, D. and KOMENDER, J. (1997). Long-term follow up after transplantation of frozen and radiation sterilise bone grafts, Ann. Transplant. 2, 64-66. RUSSELL, J., SCARBOROUGH, N. and CHESMEL, K. (1997). Re: Ability of commercial demineralised freeze-dried bone allograft to induce new bone formation, /. Peridontol. 68, 804-806. ZHANG, Q., CORNU, O. and DELLOYE, C. (1997). Ethylene oxide does not extinguish the osteoinductive capacity of demineralised bone. A reappraisal in rats, Ada Orthop. Scand. 68, 104-108. FIDELER, B.M., VANGSNESS, C.T. Jr., LU, B., ORLANDO, C. and MOORE, T. (1995). Gamma irradiation: effects on biomechanical properties of human bone-patellar tendon-bone allografts, Am. J. Sports Med. 23, 643-646. GOERTZEN, M.J., CLAHSEN, H., BURRIG, K.F. and SCHULITZ, K.P. (1995). Sterilisatation of canine anterior cruciate allografts by gamma irradiation in argon. Mechanical and neurohistological properties retained one year after transplantation, /. Bone Joint Surg. Br. 77, 205-212, retracted publication. WHITE, J.M., GOODIS, H.E., MARSHALL, S.J. and MARSHALL, G.W. (1994). Sterilisation of teeth by gamma radiation, /. Dent. Res. 73, 1560-1567.

49

LOTY, B., TOMENO, B., EVRARD, J. and POSTEL, M. (1994). Infection in massive bone allografts sterilised by radiation, Int. Orthop. 18, 164-171. YAHIA, L.H., DROUIIN, G. and ZUKOR, D. (1993). The irradiation effect on the initial mechanical properties of meniscal grafts, Biomed. Mater. Eng. 3, 211-221. ZASACKI, W. (1991). The efficacy of application of lyophilized, radiation-sterilised bone graft in orthopedic surgery, Clin. Orthop. 272, 82-87. KOMENDER, J., MALCZEWSKA, H. and KOMENDER, A. (1991). Therapeutic effects of transplantation of lyophilized and radiation-sterilised, allogeneic bone, Clin. Orthop. 272, 38-49. DZIEDZIC-GOCLAWSKA, A., OSTROWSKI, K., STACHOWICZ, W., MICHALIK, J. and GRZESIK, W. (1991). Effect of radiation sterilisation on the osteoinductive properties and the rate of remodeling of bone implants preserved by lyophilization and deep-freezing, Clin. Orthop. 272, 30-37. ANGERMANN, P. and JEPSEN, O.B. (1991). Procurement, banking and decontamination of bone and collagenous tissue allografts: guidelines for infection control, /. Hosp. Infect. 17, 159-169. LOTY, B., COURPIED, J.P., TOMENO, B., POSTEL, M., FOREST, M. and ABELANET, R. (1990). Bone allografts sterilised by irradiation. Biological properties, procurement and results of 150 massive allografts, Inst. Orthop. 14, 237-242. WEINTROUB, S. and REDDI, A.H. (1988). Influence of irradiation on the osteoinductive potential of demineralised bone matrix, Calcif. Tissue Int. 42, 255-260. MACDOWELL, S. (1988). Irradiated cartilage, Plast. Surg. Nurs. 8, 14-15.

50

WANGERIN, K., EWERS, R. and BUMANN, A. (1987). Behaviour of differently sterilised allogenic Iyophilized cartilage implants in dogs, /. Oral Maxillofac. Surg. 45, 236-242. LINBERG, J.V., ANDERSON, R.L., EDWARDS, J.J., PANJE, W.R. and BARDACH, J. (1980). Preserved irradiated homolgous cartilage for orbital reconstruction, Opthalmic Surg. 11, 457462. HOROWITZ, M. (1979). Sterilisation of homograft ossicles by gamma radiation, /. Laryngol. Otol. 93, 1087-1089. KOMENDER, J., MALCZEWSKA, H. and LESIAK-CYGANOWSKA, E. (1978). Preserved bone in clinical transplantation, Arch. Immunol. Ther. Exp. (Warz) 26, 1071-1073.

KOMENDER, J. (1978). Evaluation of radiation-sterilised bone and clinical use, Ada Med. Pol. 19, 277-281. BURWELL, R.G. (1976). The fate of freeze-dried bone allograft, Transplant. Proc. 8, 95-111.

DEXTER, F. (1976). Tissue banking in England, Transplant. Proc. 8, 43-48. KOMENDER, J., KOMENDER, A., DZIEDZIC-GOCLAWSKA, A. and OSTROWSKI, K. (1976). Radiation-sterilised bone grafts evaluated by electron spin resonance technique and mechanical tests, Transplant. Proc. 8, 25-37. URIST, M.R. and HERNANDEZ, A. (1974). Excitation transfer in bone. Deleterious effects of cobalt 60 radiation-sterilisation of bank bone, Arch. Surg. 109, 586-593. IMAMALIEV, A.S. and GASIMOV, R.R. (1974). Biological properties of bone tissue conserved in plastic material and sterilised with gamma rays (clinico-experimental study), Ada Chir. Plast. 16, 129-135.

51

OSTROWSKI, K., DZIEDZIC-GOCLAWSKA, A., STACHOWICZ, W., MICHALIK, J., TARSOLY, E. and KOMENDER, A. (1971). Application of the electron spin resonance technique for quantitative evaluation of the resorption rate of irradiated bone grafts, Calcif. Tissue Res. 7, 58-66. TARSOLY, E., OSTROWSKI, K., MOSKALEWSKI, S., LOJEK, T, KURNATOWSKI, W. and KROMPECHER, S. (1969). Incorporation of lyophilized and radiosterilised perforated and unperforated bone grafts in dogs, Ada Chir. Acad. Sci. Hung. 10, 55-63. OSTROWSKI, K., KECKL Z., DZIEDZIC-GOCLAWSKA, A., STACHOWICZ, W. and KOMENDER, A. (1969). Free radicals in bone grafts sterilised by ionizing radiation, Sb. Ved. Pr. Lek. Fak. Karlovy Univerzity Hradci Kralove Suppl.: 561-563. MARQUIT, B. (1967). Radiated homogenous cartilage in rhinoplasty, Arch. Otolaryngol. 85, 78-80.

D.2. HIV SMITH, R.A., INGELS, J., LOCHEMES, J.J., DUTKOWSKY, J.P. and PIFER, L.L. (2001). Gamma irradiation of HIV-1, /. Orthop. Res. 19, 815-819. HERNIGOU, P., GRAS, G., MARINELLO, G. and DORMONT, D. (2000). Inactivation of HIV by application of heat and radiation: implication in bone banking with irradiated allograft bone, Ada Orthop. Scand. 71, 508-512. CAMPBELL, D.G. and LI, P. (1999). Sterilisation of HIV with irradiation: relevance to infected bone allografts, Aust. N. Z. J. Surg. Jul 69, 517-521. SALAI, M., VONSOVER, A., PRITCH, M., VON VERSEN, R. and HOROSZOWSKI, H. (1997). Human immunodeficiency virus

52

(HIV) inactivation of banked bone by gamma irradiation, Ann. Transplant. 2, 55-56. FIDELER, B.M., VANGNESS, C.T. Jr., MOORE. T., LI, Z. and RASHEED, S. (1994). Effects of gamma irradiation on the human immunodeficiency virus. A study in frozen human bonepatelar ligament-bone grafts obtained from infected cadavera, /. Bone Joint Surg. Am. 76, 1032-1035. CAMPBELL, D.G., LI, P., STEPHENSON, A.J. and OAKESHOTT, R.D. (1994). Sterilisation of HIV by gamma irradiation. A bone allograft model, Int. Orthop. 18, 172-176. BEDROSSIAN, E.H. Jr. (1991). HIV and banked fascia lata, Ophthal. Plast. Reconstr. Surg. 7, 284-288.

D.3. Biomaterials HOLY, C.E., CHENG, C, DAVIES, J.E. and SHOICHET, M.S. (2001). Optimizing the sterilisation of PLGA scaffolds for use in tissue engineering, Biomaterials 22, 25-31. ANDRIANO, K.P., CHANDRASHEKAR, B., MCENERY, K., DUNN, R.L., MOYER, K., BALLIU, CM., HOLLAND, K.M., GARRETT, S. and HUFFER, W.E. (2000). Preliminary in vivo studies on the osteogenic potential of bone morphogenetic proteins delivered from an absorbable puttylike polymer matrix, /. Biomed. Mater. Res. 53, 36-43. CHEUNG, D.T., PERELMAN, N., TONG, D. and NIMNI, M.E. (1990). The effect of gamma-irradiation on collagen molecules, isolated alpha-chains and cross linked native fibers, /. Biomed Mater. Res. 24, 581-589. BRUCK, S.D. and MUELLER, E.P. (1988). Radiation sterilisation of polymeric implant materials, /. Biomed. Mater. Res. 22, 133144.

53

SCHWARZ, N., REDL, H., SCHIESSER, A., SCHLAG, G., THURNHER, M., LINTNER, F. and DINGES, H.P. (1988). Irradiation-sterilisation of rat bone matrix gelatin Ada Orthop. Scand. 59, 165-167. PHILLIPS, G.O. (1984). Chemical processes induced during radiation sterilisation of cellulose, Anselme Payen Award Symposium at American Chemical Society, 188th National Meeting (Philadel-

phia). NAKAMURA, Y., OGIWARA, Y. and PHILLIPS, G.O. (1985). Free Radical Formation and Degradation of Cellulose by Ionising Radiations, Polymer Photochemistry 6, 135-159. PHILLIPS, G.O. (1985). Radiation Degradation of Cellulosic Systems. In: Proc. Int. Symp. Fiber Science and Technology (Hakone,

Japan), 88-90. WOZNIAK-PARNOWSKA, W. and NAJER, A. (1978). Studies on the sterilisation of pharmaceutical base materials with ionizing radiation and ethylene oxide, Ada Microbiol. Pol. 27, 161-168.

B.4. Soft tissues TYSZKIEWICZ, J.T., UHRYNOWSKA-TYSZKIEWICZ, LA., KAMINSKI, A. and DZIEDZIC-GOCLAWSKA, A. (1999). Amnion allografts prepared in the Central Tissue Bank in Warsaw, Ann. Transplant. 4, 85-90. MARTINEZ PARDO, M.E., REYES FRIAS, M.L., RAMOS DURON, L.E., GUTIERREZ SALGADO, E., GOMEZ, J.C., MARIN, M.A. and LUNA ZARAGOZA, D. (1999). Clinical application of amniotic membranes on a patient with epidermolysis bullosa, Ann. Transplant. 4, 69-73. JOHNSON, K.A., ROGERS, G.J., ROE, S.C., HOWLETT, C.R., CLAYTON, M.K., MILTHORPE, B.K. and SCHINDHELM, K.

54

(1999). Nitrous acid pretreatment of tendon xenografts crosslinked with glutaraldehyde and sterilised with gamma irradiation, Biomaterials 20, 1003-1015. MAEDA, A., INOUE, M., SHINO, K., NAKATA, K., NAKAMURA, H., TANAKA, M, SEGUCHI, Y. and ONO, K. (1993). Effects of solvent preservation with or without gamma irradiation on the material properties of canine tendon allografts, /. Orthop. Res. 11, 181-189. HINTON, R., JINNAH, R.H., JOHNSON, C, WARDEN, K. and CLARKE, H.J. (1992). A biomechanical analysis of solventdehydrated and freeze-dried human fascia lata allografts. A preliminary report, Am. J. Sports. Med. 20, 607-612. BUMANN, A., KOPP, S., EICKBOHM, J.E. and EWERS, R. (1989). Rehydration of lyophilised cartilage grafts sterilised by different methods, Int. J. Oral. Maxillofacial Surg. 18, 370-372. CANTORE, G., GUIDETTI, B. and DELFINI, R. (1987). Neurosurgical use of human dura mater sterilised by y rays and stored in alcohol: long term results, /. Neurosurg. 66, 93-95. ARMAND, G., BAUGH, P.J., BALAZS, E.A. and PHILLIPS, G.O. (1975). Radiation protection of hyaluronic acid in the solid state, Radiat. Res. 64, 573-580. HALL, A.N., PHILLIPS, G.O. and RASSOL, S. (1978). Action of ionizing radiations on a hyaluronate tetrasaccharide, Carbohydrate Res. 62, 373-376. MOORE, J.S., PHILLIPS, G.O. and RHYS, D. (1973). Chemical effects of ?-irradiation of aqueous solutions of chondroitin-4sulphate, Int. J. Radiat. Biol. 23(2), 113-119. LITWIN, S.B., COHEN, J. and FINE, S. (1973). Effects of sterilisation and preservation on the rupture force and tensile strength of canine aortic tissue, /. Surg. Res. 15, 198-206.

55

DONNELLY, R.J., APARICIO, S.R., DEXTER, R, DEVERALL, P.B. and WATSON, D.A. (1973). Gamma-radiation of heart valves at 4 degrees C; a comparative study using techniques of histochemistry and electron and light microscopy, Thorax 28, 95-101. MANDELCORN, M.S. and CRAWFORD, J.S. (1972). Feasibility of a bank for storage of human fascia lata sutures, Arch. Opthalmol. 87, 535-537. KORLOF, B., SIMONI, E., BARYD, I., LAMKE, L.O. and ERIKSSON, G. (1972). Radiation-sterilisation split skin: a new type of biological wound dressing. Preliminary report, Scand. J. Plast. Reconstr. Surg. 6, 126-131. RITTENHOUSE, E.A., SANDS, M.P., MOHRI, H. and MEERENDINO, K.A. (1970). Sterilisation of aortic valve grafts for transplantation, Arch. Surg. 101, 1-5. WELCH, W. (1969). A comparative study of different methods of processing aortic homograft, Thorax 24, 746-749. MALM, J.R., BOWMAN, F.O. Jr., HARRIS. P.D., KAISER. G.A. and KOVALIK, A.T. (1969). Results of aortic valve replacement utilizing irradiated valve homografts, Ann. N.Y. Acad. Sci. 30, 740-747. BALAZS, E.A., DAVIES, J.V., PHILLIPS, G.O. and YOUNG, M. (1967). Transient intermediates in the radiolysis of hyaluronic acid, Radiat. Res. 31, 243-255.

2

PRESERVED BONE ALLOGRAFTS IN RECONSTRUCTIVE ORTHOPAEDICS

J. KOMENDER,1 A. KOMENDER,1 H. MALCZEWSKA2 and W. MARCZYNSKI3 department of Transplantology, 2Department of Histology & Embryology, Medical University in Warsaw, 3 Institute of Traumatology, Orthopaedics and Neurosurgery Central Clinical Hospital, Military Medical University in Warsaw, Poland

1. Introduction Tissue banking experience was achieved over a long period (1963-2001) since the creation of tissue banks in Poland. Thanks to initiative of two outstanding Polish professors: Adam Gruca, director of Orthopaedic Clinic in Warsaw and Kazimierz Ostrowski, director of the Department of Histology & Embryology in Warsaw, organisation of tissue processing and co-operation with orthopaedic clinics, radiation-chemistry laboratories and microbiological units was started. Since 1963, a tissue bank has been operating in Warsaw and around 100 000 grafts of bone, cartilage, dura mater, skin and fascia have been prepared and used in various branches of reconstructive surgery. In 1967, the tissue bank in Katowice, connected to the Blood Transfusion Centre, was created. In 1978, a similar unit was organised in Kielce as the Cryobiological Unit in the local blood transfusion centre. These three units created the first network of multi-tissues banks in Poland. In the 1980s, in several 57

58

medical institutions (Cardiosurgery Clinic in Zabrze, Institute — Centre of Health Child, Cardiosurgery Clinic in Krakow) the heart valves and blood vessels banks were organised. Later, two eye tissues banks (in Warsaw and in Lublin) were founded. All of these units are in contact, once a year within the frame of Polish Transplantation Society, when a session on tissue banks is organised. Most of the workers attend the meetings of EATB. Twice, in 1977 and in 1999, worldwide meetings of tissue bank specialists in Poland were organised. Implementation of radiation sterilisation as main sterilisation procedure has been adopted since the very beginning. Some studies performed in our tissue bank were fundamental for allografts radiation-sterilisation (DziedzicGoclawska, 1979; Dziedzic-Goclawska et al., 1979; Komender, 1976; Ostrowski et al, 1980; Ostrowski et al, 1996). The Central Tissue Bank established good links of collaboration with orthopaedic units, and that enable us to perform interesting analyses of use of preserved allografts in clinics. 2. Transplantation of Lyophilised and Radiation-Sterilised Bone Grafts In 1960s and 1970s, lyophilised bone most often was used for orthopaedic reconstructions. Thanks to the good co-operation with several orthopaedic units, records on the follow-up of patients after bone transplantation can be gathered. This analysis is based on 1014 cases of preserved (lyophilised and radiation-sterilised) allogenic bone transplantation, where 27 biological and clinical variables were taken into consideration and detailed results were published earlier (Komender et al, 1991). The following general conclusions may be drawn: more than 91% of operated patients reached full restoration or significant improvement of their condition; after surgery 80.3% of patients reached their full physical fitness; in 5.1% of the patients the results of treatment were unsatisfactory; in cases of benign tumours and congenital changes, the highest number of "very good" results of treatment was achieved; unsatisfactory results of treatment most often appeared in post-traumatic deformation and degenerative diseases (Table 1).

59 Table 1. Transplantation of lyophilised, radiation-sterilised bone allografts. Results of treatment in various diagnoses. Result of treatment

Diagnoses Very good

Satisfactory

Difficult to estimate

%

n

%

6

0.6

38

3.8

0.6

5

0.5

186

18.4

0

0

0

0

2

0.2

3.0

1

0.1

4

0.4

41

4.1

86

8.5

5

0.5

7

0.7

140

13.9

3.4

38

3.8

3

0.3

7

0.7

82

8.1

141

14.0

213

21.1

11

1.1

17

1.7

382

37.8

25

2.5

73

7.2

7

0.7

4

0.4

109

10.8

9

0.9

18

1.8

1

0.1

2

0.2

30

3.0

375

37.1

547

54.2

36

3.6

52

5.1

1010

100

n

%

n

%

n

%

11

1.1

19

1.9

2

0.2

107

10.6

68

6.7

6

Malignant tumours

0

0

2

0.2

Unspecific inflammations

6

0.6

30

Specific inflammations

42

4.2

Degenerative changes

34

Traumas Benign tumours

Congenital change Scolioses Others Total

Total

Unsatisfactory n

Chi2 = 75.479; degrees of freedom, 24; p < 0.01

A comparative analysis of the distribution of clinical results obtained in this group was carried out using the Chi2 test (Mainland, 1963). Some correlations and coincidences were presented. In order to find out the meaning of the correlations; the "indices of coincidence" were calculated according to Pearson (1904). Pearson's index permits a comparison of coincidence of different pairs of variables even with a various degree of freedoms, thus showing a strong relationship between the variables. As expected, numerous

60

variables showed significant correlation with the estimated final result of treatment (Komender et al., 2001). Three sets of indices of coincidence for the "final result of treatment", "fitness for work" and "role of the graft" are evaluated. A strong relationship was found between: "rebuilding of grafts", "early estimated result of treatment" and "re-operation" with the "final result of treatment". What concerns the rebuilding of transplanted allografts seems to be the most important and sensitive factor in the post-surgery observation. Less important factors seem to be "diagnosis" and "anatomical localisation". When the patients with unsatisfactory results of treatment were analysed, the most often localisation is related to the vertebral column or crus. No significant coincidence was found with "age", "handicap level" or other parameters. Numerous patients even in their 70s were operated with the use of preserved allografts and the age seemed having no influence on the final result of treatment. The sequence of variables is different when they are compared with the "fitness for work" after surgery. This coincides with "age", "diagnosis" and "physical efficiency". Such characteristics as: "complications intra- or post-surgery", "wound healing", etc. do not show any significant relationship with "fitness for work". The sequence of variables is different when they are compared with the "role of the graft". A very strong coincidence is expressed with "performed re-operation" and with the "rebuilding of graft". Reoperation is always an unfavourable incident in course of treatment and if surgeon is obliged to remove the allografts, a positive clinical effect can rarely be expected. Impaired graft substitution is also a rather bad prognostic factor. A comparison of coincidence indices shows how significant "rebuilding of the grafts" is in post-surgery estimation. Lyophilised bone allografts are not so readily used now. 3. Transplantation of Deep-Frozen and Radiation-Sterilised Allogenic Bone Grafts In 1980s and 1990s, deep-frozen bone allografts are most often use for transplantation in humans. In the Institute of Traumatology,

61

Orthopaedics and Neurosurgery of the Military Medical Academy in Warsaw, during 1981-1995, biostatic bone allografts, frozen and radiation-sterilised were transplanted into numerous patients (Kwiatkowski and Ratynski, 1999; Marczynski etal, 1999). The results achieved in group of 596 female (53%) and 529 male (47%) are presented here. The mean age of patients was 36 years and varied from 2 to 70 years. The grafts were most often used in the second decade of life for 336 patients (30%), or in the third decade for 247 (22%). The indications for bone transplantations varied, 32 diagnoses were categorised into five groups: bone union failure in 401 cases (36%), congenital anomalies in 307 cases (27%), benign tumours in 133 (12%), 68 (6%) of post inflammatory changes and arthroses, 216 (19%) of bone necrosis and others, with: hip joint hypoplasia in 168 cases (15%), scolioses in 146 cases (13%) and solitary cysts in 135 cases (12%). Various types of grafts were used, depending on indications: chips 663 (59%), bars 270 (24%), slices of bone 168 (15%) and solid, large grafts 22 (2%). The manner in which the graft was implanted depended on the diagnosis and location of the pathological change. The grafts were implanted as follows: without covering by periosteum in 517 cases (46%), in incomplete intraosseus apposition of grafts in 292 cases (26%), intraosseus in 213 cases (19%), in subperiosteal position 56 cases (5%) and by intramuscular implantation 45 grafts (4%). In spinal reconstructions, bars were often used —198 cases (63%), while in femur surgery, chips were mainly used (184 — 78%). In hip surgery, slices of bone were most frequently transplanted (136 — 58%), while chips dominated in reconstructions of the crural bone (148 — 78%). The result of treatment was analysed 2 to 11 years after surgery. The progress of rebuilding of bone grafts was evaluated in physical and X-ray examinations that were carried out at regular intervals after operation. Analysis shows that by the 3rd month, 517 (46%) grafts were completely rebuilt; the patients of this group were mainly in the second decade of life. Within 6 months after operation, a further 416 (37%) grafts were rebuilt, by the 9th month 101 (9%), and by the 12th month 22 (2%). Within next the 12 months, a further 54 (4.8%) were rebuilt. However, 10 grafts (0.8%) became sequestered with no sign of rebuilding. The number of grafts rebuilt

62

Table 2. Transplantation of frozen radiation-sterilised bone allografts. Rebuilding of grafts introduced in various position. Position of graft

No periosteal cover Incomplete intraosseus Intraosseus Subperiosteal Intramuscular*

Rebuilt grafts

Total

n

%

n

%

466 251 187 56 0

90.1 86.0 87.8

517 292 213 56 46

100 100 100 100 100

100 0

*all grafts were resorbed

and those in the process of being rebuilt was 1022 (90%). All grafts implanted in muscles were partially or completely reabsorbed (Table 2). The rebuilding of grafts varied, depending on numerous factors such as: diagnosis, site of grafting, age of the recipient, type, size and the way of the graft introduced. Grafts of spongy bone were rebuilt within 3 to 6 months, grafts of compact bone, in the form of bars were rebuilt within 6 to 24 months. A comparison of age and of graft substitution shows highly effective substitution in the first decade of life 96%. In the second decade, their effectiveness was also high (95%). In the subsequent decades of life, substitution of grafts diminishes. Satisfactory graft substitution was observed in 1022 cases (90.8%) of all patients. The number of transplantations that were estimated to be unsatisfactory was 103 (9.2%). 4. Conclusion The application of allogenic, biostatic frozen grafts reduce the extent and duration of operations, and the "creeping substitution" of implanted bone lasts 3 to 8 months, thus progress of the graft substitution depends on its structure and the position of transplants. Observation of results of bone allografts application is still progressing and now the group of patients under observation

63

is over two thousand. Development of surgical techniques, new generation of antibiotics and new technology of tissue banking certainly will influence the clinical results. 5. References BLOEM, R.M., TOMFORD, W.W. and MANKIN, HJ. (1996). Histological observations on retrieved human allografts. In: Orthopaedic Allografts Surgery, A.A. Czitrom and H. Winkler, eds., Springer, Wien-New York, pp. 61-66. BURCHARDT, H. and ENNENKING, W.P. (1978). Transplantation of bone, Surg. Clin. North Am. 58, 403. BURWELL, R.G. (1969). The fate of bone grafts. In: Recent Advances in Orthopeadic, A.G. Appley, ed., Churchill, London, p. 115. DZIEDZIC-GOCLAWSKA, A. (1976). Effect of Radiation sterilisation on biostatic tissue grafts and their constituents. In: Sterilisation by Ionizing Radiation, E.R.L. Gaughran and AJ. Goudie, eds., Montreal, Multiscience, Vol. 2, p. 156. DZIEDZIC-GOCLAWSKA, A., OSTROWSKI, K., STACHOWICZ, W. and MOUTIER, R. (1979). Decrease of crystallinity of bone mineral in osteopetrotic rats, Metab. Bone Dis. Relat. Dis. 2: 33-37. HEAD, W.C., MALININ, T.L. and BERKLACICH, F. (1987). Freezedried proximal femur allografts in revision total hip arthroplasty, Clin. Orthop. 215, 109.

KOMENDER, A. (1976). Influence of preservation procedures on some mechanical properties of human haversian bone, Mat. Med. Pol. 10, 13. KOMENDER, A. (1977). Metody konserwacji tkanek. In: Przeszczepy Biostatyczne, J. Komender, ed., PZWL, Warsaw, Vol. I, pp. 33-48. KOMENDER, J. and KOMENDER, A. (1977). Evaluation of radiationsterilised tissue in clinical use. In: Sterilisation of Medical Products

64

by Ionizing Radiation, E.R.L. Gaughran and A.J. Goudie, eds., Multisc Publ. Ltd., Montreal, p. 188. KOMENDER, J., MALCZEWSKA, H. and KOMENDER, A. (1991). Therapeutic effects of transplantation of lyophilised and radiation-sterilised, allogeneic bone, Clin. Orthop. 272, 38-49. KOMENDER, J., MARCZYNSKI, W., TYLMAN, MALCZEWSKA, H., KOMENDER, A. and SLADOWSKI, D. (2001). Preserved tissue allografts in reconstructive surgery, Cell. Tiss. Banking 21, 103-112. KRYST, L. (1981). Przeszczepianie tkanek w chirurgii szcz_kowotwarzowej. In: Przeszczepy Biostatyczne, J. Komender, ed., PZWL, Warsaw, Vol. II, pp. 151-161. KWIATKOWSKI, K. and RATYNSKI, G. (1999). The usage of frozen allografts of spongy bone filling the loss of bone after hip prosthesis, Ann Transpl. 4, 59-63. Mainland, O. (1963). Elementary Medical Statistics, WB Saunders, London, Philadelphia, p. 231. MARCZYNSKI, W., KOMENDER, J., BARANSKI, M. and KRAUZE, K. (1999). Frozen and radiation sterilised bone allografts in treatment of posttraumatic malformation of bone, Ann. Transpl. 4, 41-45. MARCZYNSKI, W., KOMENDER, J., STEPIEN, K. and BARANSKI, M. (1999). Fusion of spine in children scoliosis with frozen and radiation-sterilised bone allografts, Ann. Transpl. 4, 30-34. OSTROWSKI, K., DZIEDZIC GOCLAWSKA, A. and STACHOWICZ, W. (1980). Radiation induced paramagnetic entities in tissue mineral and their use in calcified tissue research. In: Free Radicals in Biology, W. Pryor, ed., Academic, New York, Vol. 4, p. 321. OSTROWSKI, K., WLODARSKI, K., DZIEDZIC-GOCLAWSKA, A., MICHALIK, J. and STACHOWICZ, W. (1996). Allogeneic Bone grafts: Study of radiation sterilised bone tissue by electron

65

paramagnetic resonance spectrometry and a new model of periosteal induction of osteogenesis. In: Orthopaedic Allograft Surgery, A.A. Czitrom and H. Winkler, eds., Springer, Wien-New York, pp. 45-52. PEARSON, K. (1904). On the theory of contingency. In: Memoirs, Biometric Series No. 1. Draper's Co., London, p. 119.

CLINICAL STRATEGY FOR APPLICATION OF DEEP FROZEN-RADIATION STERILISED BONE ALLOGRAFTS

WOJCIECH MARCZYNSKIJANUSZ KOMENDER Institute of Traumatology, Orthopaedics and Neurosurgery of Central Clinical Hospital Military School of Medicine in Warsaw, Poland JANUSZ KOMENDER Bank of Human Tissues Medical Academy in Warsaw, Poland

1. Introduction Orthopaedic diseases of osseous tissue and its post-traumatic damage usually require surgical treatment which often consists of reconstruction. In many cases the quantity of osseous tissue of the place of intervention is insufficient for adequate reconstruction. In these patients autogenic or allogenic bone grafts should be used (Dziedzic-Goclwska, 1977; Goldberg and Stevenson, 1987; Komender, 1977; Marczynski, 1991, Mazess, 1988; Ostrowski, 2000; Wlodarski, 1991). Obtaining autografts requires an additional surgery and it is often impossible to gain a sufficient quantity of tissue (Nusbickel et al., 1989). For these reasons, our attention has shifted towards frozen allografts which we have successfully used for many years. 67

68 The use of allogenic biostatic frozen and radiation sterilised bone grafts has become commonplace in orthopaedics and traumatology. Bone grafts stabilise bone structure in places where healing is disturbed or osteogenesis decreased. Grafts consist of either spongy or compact bone and have different forms. Bone allografts can be placed in many different ways. In some cases they are inserted intramuscularly and connected to the bone only at their ends. As a result an osseous bridge is formed (Alho et al., 1989; Anderson, 1989; Malinin et al, 1985; Marczynski and Tylman, 1991; Mazurkiewucz and Trelinska, 1985; Tylman, 1986). 2. Methods and Results Allogenic biostatic frozen and radiation sterilised bone grafts were transplanted to 1376 patients in the Institute of Traumatic Surgery, Orthopaedics and Neurosurgery in Warsaw between 1981 and 1998. 731 patients (53%) were female, 645 (47%) were male. There were 827 adults (60%) and 549 children (40%). The age of patients was 2 to 70 with a mean of 36 years (Fig. 1). Most grafts were used in patients aged 11 to 19 (426, 31%) and patients in their 20 (298, 21%). Bone transplantation was performed in patients with up to 32 different diagnoses. The most frequent indications were scoliosis (206 cases, 15%), hip joint hypoplasia Age Groups of Graft Recipients (No. of case analysed: 1,376)

Fig. 1. Age groups of graft recipients.

69

(177 cases, 13%), and solitary cysts (179 cases, 13%). All diagnoses were divided into six groups: bone union failure (489 cases, 35%), congenital anomalies (374, 27%), benign neoplasms (173, 12%), prosthesis reimplantation (43, 3%), arthrosis (80, 7%) and miscellaneous cases (91, 7%) (Fig. 2). Various types of grafts were used, depending on indications. Spongy bone grafts where used in 1085 (79%) and compact in 295 (21%) patients. Slivers were used in 839 cases (61%), bars in

Diagnoses (No. of cases analysed: 1,376)

Fig. 2. The indications for the bone transplantation

Forms of Bone Allografts (No. of cases analysed: 1,376)

Fig. 3. Forms of bone allografts.

70

295 cases (21%), plasters in 218 cases (16%) and solid grafts in 24 cases (2%) (Fig. 3). The way in which the graft was implanted depended on the diagnosis and the location of the affected osseous tissue. The grafts were placed periosteally in 1206 cases (88%) and subperiosteally in 170 cases (12%). Adhesion apposition was used in 661 cases (48%), incomplete intraosseus implantation in 335 cases (24%), intraosseus implantation in 316 cases (23%) and intramuscular implantation in 64 cases (5%). The grafts were implanted in the spine in 394 cases (28%), in the femur in 284 cases (21%), in the hip in 271 cases (20%), in the tibia in 229 cases (17%), in the arm in 126 cases (9%), in the forearm in 42 cases (3%), in the foot in 18 cases (1%) and in the hand in 12 cases (1%) (Fig. 4). In the spine slivers were used in 217 cases (55%) and bars in 177 (45%). In the femur slivers were used in 224 cases (79%), bars in 31 cases (11%) and plasters in 29 cases (10%). In the hip surgery plasters were used in 144 cases (53%), slivers in 123 cases (46%) and bars in 4 cases (1%). In crural bones slivers were used in 114 cases (50%), plasters in 98 cases (43%) and bars in 17 cases (7%). In the arm slivers were used in 83 cases (66%), plasters in 26 cases (21%) and bars in 17 cases (13%). In the forearm slivers were used in 37 cases (88%) and plasters in 5 cases (12%). The result of the treatment was analysed 2 to 15 years after the surgery.

Anatomical Location of Grafts in Sceleton (NO. of cases analysed: 1,376)

18, 1% Fig. 4. Anatomical location of grafts in the skeleton.

71

% of Remodelled Grafts at Various Time Intervals (No. of cases analysed: 1,376)

Fig. 5. Percentage of remodelled grafts at various time intervals.

The progress of the bone graft remodelling was evaluated on the basis of physical and X-ray examinations. The examinations were carried out at regular intervals. Three months after the surgery, 745 grafts (46%) were incorporated, 509 (37%) six months after the surgery, 123 (9%) nine months after the surgery and 28 (2%) twelve months after the surgery. After this period, 68 more grafts (5%) were incorporated and 17 grafts (1%) became sequesters (Fig. 5). Among our patients, there were six cases of infection and one case of congenital pseudoarthrosis. All 316 intraosseus grafts (100%) were incorporated. 661 adhesive insertion grafts were incorporated (90%). The grafts which were incompletely embedded in the bone were incorprated in 335 cases (86%). All grafts which had been placed subperiosteally were incorporated where 1085 of the grafts apposed outside periosteum (90%) were incorporated. All grafts placed in muscles were partially or completely resorbed. The total number of grafts which had either completely incorporated or were in the process of incorporation amounted to 1238 (90%). The time of graft incorporation depended on many factors such as diagnosis, site of implantation, patient's age, the type and size of the graft as well as the method of its implantation. Spongy bone grafts were incorporated after 3 to 6 months, compact bone grafts

72

% of Allografts Remodelled vs. Patients' Age (No. of cases analysed: 1,376)

Fig. 6. Percentage of allografts remodelled versus patient's age.

in the form of bone bars were incorporated after 6 to 24 months. By correlating patients' age with the efficacy of the procedure, we have shown that the treatment was highly effective in patients aged 2 to 10 (100%). In patients aged 11 to 19 the efficacy of the treatment was 95% and in patients in their 20s it was 88%. In older age groups the efficacy of bone graft implantation decreased (Fig. 6). High or satisfactory efficacy of the treatment was achieved in 1249 cases (90%). In 126 cases (9%), the procedure was not effective or it was not possible to assess the graft. We have achieved high overall efficacy of graft implantation in all six groups created on the basis of the diagnosis. 3. Examples of Graft Use in Specific Clinical Situations Prosthesis reimplantation of the hip joint is performed on a large scale all over the world. Aseptic loosening of the hip joint prosthesis often occurs many years after implantation. The process of loosening is caused by surgical errors as well as mechanical and biological factors. Free particles of polyethylene, metal and acrylic cement initiate the process. Loosening is often accompanied by bone destruction. When reconstruction of the damaged bone is attempted ways of reconstructing acetabulum and femur and achieving functional stability of the hip joint prosthesis must be determined. Surgical technique used during hip arthroplasty

73

Fig. 7. X-ray of the hip after total arthroplasty. Radiograph showing picture after surgery, after loosening prothesis, and after revision with used bone allografts.

enables precise reconstruction of bone stock both around the acetabulum and femur metaphysis. For this purpose, we used morselised, frozen allogenic spongy bone grafts and 43 patients (3%) underwent hip arthroplasty. Radiograms were taken immediately after the surgery and 3, 6 and 12 month later. The grafts were incorporated 3 to 6 months after the surgery. The patients were allowed to exert full body-weight on the joint 6 month after the surgery (Fig. 7). Post-traumatic spinal fractures require surgical treatment in 1520% of patients. Post-traumatic instability and spinal canal stenosis with or without neurological symptoms are indications for surgical treatment. Surgical methods include decompression of spinal canal structures, internal stabilisation along with immobilisation of the fewest possible number of segments, which enabled us to bring patients early into the erect position without the necessity of external immobilisation. For the surgery, we used frozen allografts of spongy morselised or compact bones in the form of bars. After the bone grafts had been incorporated the mechanical stabilisation was replaced by the biological stabilisation. We treated 68 patients (5% of all patients) with thoracic and lumbar spine fractures. Primary neurological complications of various degrees were diagnosed in 56 patients (82% of patients with spinal

74

fractures). We treated 8 patients with fractures in the thoracic section Th} to Thio (11%). The fractures occurred most commonly in the transitional section Thn to L2 (50 patients, 74% of patients with spinal fractures). 10 fractures occurred in the lumbar section L3 to L5 (15% of patients with spinal fractures) The bone grafts were incorporated 3 to 6 months after surgery (Fig. 8). Congenital and developmental scolioses were treated in 102 children between 1990 and 1997. Clinically and radiologically confirmed progression of the scoliosis was an indication for surgical treatment. Two surgical methods were used: a one-stage correction

Fig. 8. Radiograph showing instability fracture Li after injury and transpedicular stabilisation, with used frozen allografts.

Fig. 9. Telescope rod with dystraction instrument.

after

75

Fig. 10. Spondylodesis with use morsellised, frozen bone grafts during last dystraction of the rod.

and a multiple-stage correction. One-stage correction was achieved with the aid of one or two distraction rods. In scolioses which increased with the child's age, multiple-stage treatment was carried out with the use of a telescopic rod type RRC-2 (Fig. 9). Fifty three children, whose bone development had not been completed according the Risser test, were treated this way. Apart from mechanical correction and stabilisation, allogenic biostatic radiation sterilised frozen bone grafts were implanted in all operated patients to ensure additional biological stabilisation (Fig. 10). The choice of a particular type of graft depended on the clinical situation. If the graft was to be placed in the lamina and the spinosus process of vertebra, morsellised bone grafts were used. We recently used morsellised bone grafts which were mixed with crushed garamycin sponge before implantation. Among the multiple-stage treated scolioses the angle before the surgery was 40 to 100 degrees, average 70 degrees, and after the surgery 13 to 40 degrees, average 27 degrees. The percentage of post-surgical correction was 56 to 74%. In each child we treated the loss of the correction due to fast growth. On average we performed 5 correction surgeries on one child. For these surgeries, we used frozen bone grafts which were applied in two stages. During the implantation of the telescope rod the grafts were placed in the area

76

Fig. 11. Pre-operative and post-operative radiographs of the scoliosis. Example of multiple-stage treatment using a telescope rod in the scolioses increasing with the growth of the child.

where the hooks had been implanted. In children undergoing the final correction, morselised bone was implanted on the decorticated surface of the lamina and the spinoous process of the veretebra in the space between the telescope rod and the spine. The bone grafts were incorporated 3 to 6 months after the surgery (Fig. 11). Bone cysts cause pain and fractures but they have no specific symptoms. They are usually identified by a casual X-ray of a bone or because of a pathological fracture. Frozen and radiation-sterilised bone allografts were implanted in 179 patients (13%) with cysts resulting from benign tumors. Most of them were children (137 cases, 76% of all cyst cases). Adults constituted 24% (42 cases). The most common location of bone cysts was the humerus (53 patients, 30%), the femur (47 patients, 26%), the tibia and the fibula (47 cases, 26%). Other bone cysts were found in the hip (10 cases, 6%), hands and feet (9 cases, 5%) and forearm (4 cases, 2%). The most common type was a solitary bone cyst (127 cases, 71%). Other types of cysts were cartilaginous tumors (12 cases, 7%), fibrocellular tumors (9 cases, 5%), osteoid osteomas (6 cases, 4%) and aneurysmal bone cysts (9 cases, 5%). During surgical treatment the bone cysts were excised and examined, its sites electrocoagulated and mechanically and chemically cleaned. Then they were filled with morselised spongy or

77

Fig. 12. Radiograph showing multiloculare cyst of the femur pre-operative and 3 months post-surgery with use frozen bone grafts.

Fig. 13. Radiograph showing rebuilding bone allografts after 6 and 12 months postsurgery.

cortical bone grafts. The cyst tissue underwent histopathological examination. Physical examination and X-rays were evaluated every 6-8 weeks. Allograft incorporation was observed in 82 patients (46%) within three months and in 66 more patients (37%) six months after the surgery (Figs. 12 and 13). Frozen bone grafts were used in 489 patients (35% of all patients) with a post-traumatic non-union bone. The age of the patients was 12 to 69 with a mean of 34 years. Frozen bone grafts were used in 306 patients (62.5% of miscellaneous group patients) with a delayed-union bone, in 34 patients (5%) with primary posttraumatic bone defects, in 117 patients (23.9%) with pseudoarthrosis and in 42 patients (8.6%) with habitual dislocation of the

78

shoulder joint. The specific diagnosis and the indications for the surgery determined the method of graft implantation which was intraosseus, incompletely intraosseus, adhesive or intramuscular. In 459 cases (94%) spongy bone grafts were used. In 30 cases (4%) cortical bone grafts were used, in 288 cases (58.9%) morselised bone grafts, in 171 cases (35.1%) cancellous blocks and in 30 cases (6%) cortical struts. The process of the bone graft remodelling was evaluated on the basis of physical and X-ray examinations, which were carried out every 3 months after the surgery. In 225 patients (46%) the grafts were incorporated 3 months after the surgery, in 181 cases (37%) after six months, in 44 cases (9%) after nine months, in 10 cases (2%) after twelve months. In 24 cases (5%) the remodelling process lasted over a year. In 5 cases (1%) the grafts became sequesters due to infection. In 42 cases (8.6%) partial resorption of the grafts was observed. Seventeen patients required a second bone grafting (Fig. 14). Surgical filling of defects after intra-articular fracture of proximal tibia was performed in 15 patients between 1995 and 1998, In which 70% of fractures involved the lateral tibia condyle, 10% medial condyle and 20% both condyles. All patient were diagnosed with an X-ray and CT in order to establish the location and extent of the bone defect. For the tibia reconstruction we used morsellised bone grafts and screw or plate stabilisation. Clinical assessment of functions of the operated knee joints was carried out according to

Fig. 14. Bone defect of the tibia post inflammatory process. Osteosynthesis by bone bar and screw. Defect was fulfilled by frozen morsellised bone grafts.

Fig. 15. Anero-posterior X-ray and CT of fracture condylus lateralis of the tibia.

79

80

Fig. 16. Operational filling of defects after intra-articular fracture of proximal tibia with stabilisation by screws; immediately after surgery and 3 months late.

the Lysholm-Gillquist score and IKDS score. Frozen bone grafts were incorporated 3 to 6 months after the surgery (Figs. 15 and 16). 4. Conclusions 1. The application of allogenic, biostatic frozen grafts makes it possible to reduce the extent and duration of operations. 2. The allogenic, biostatic frozen and radiation sterilised bone grafts undergo the "creeping substitution" (incorporation) in 3 to 6 months. 3. The allogenic grafts in various forms (granulate, bone wreckage, plasters, solid grafts and bone bars) suit different surgical requirements. 4. The progress of the graft substitution (incorporation) depends on its structure and the site of implantation. It can be assessed by an X-ray examination. 5. Morselised frozen allografts of spongy bone enable the reconstruction of osseous tissue damaged in the process of hip joint prosthesis loosening. 6. Frozen bone grafts are advantageous osteogenic material. Bone graft transplantation enables spine stabilisation after the removal of instruments.

81

7. Application of frozen bone grafts to produce a biological spondylodesis in the surgical treatment of scolioses shortens the operation time, limits the blood loss and the risk of complications. Moreover, it does not cause any additional untoward effects and scars. 5. References ALHO, A., KARAHARJU, E.O., KORKALA, O., LAASONEN, E.M., HOLMSTROM, T. and MULLER, C. (1989). Allogenic grafts for bone tumor. 21 cases of osteoarticular and segmental grafts, Ada Orthop. Scand. 60, 143-153. ANDERSON, W.J. (1989). Allograft bone for arthrodesis and repair of skeletal hand problems, /. Hand Surg. 14, 332-335. DZIEDZIC-GOCLAWSKA, A. (1977). Radiation sterilisation of the tissue. In: Biostatic Grafts. cz.I, J. Komender, ed., PZWL, Warszawa, p. 50. GOLDBERG, V.M. and STEVENSON, S. (1987). Natural history of autografts and allografts, Clin. Orthop. 225, 7-16. KOMENDER, J. (1977). Biostatic Allografts. Publ., Co., PZWL, Warsaw, pp. 24-46. MALININ, T.I., MARTINEZ, O.V. and BROWN, M.D. (1985). Banking of massive osteoarticular and intercalary bone allografts12 years experience, Clin. Orthop. 197, 44-57. MARCZYNSKI, W. and TYLMAN, D. (1991). Operating treatment of idiopatic necrosis of the head of the femur with use allogenic bioststatic frozen bone grafts, Military Health Service Sci. ]. 11(12), 692-696. MARCZYNSKI, W, (1995). Treatment of Non-union and Defects of Bone.

Publ. Co., Bellona, Warsaw, pp. 10-28. MAZURKIEWICZ, H. and TRELINSKA, A. (1985). Role of bone allografts in reconstruction defects of acetabulum and femur

82

in replacement of the hip, Chir. Org. Motus Orthop. Polonica 50, 403-408. MAZESS, R.M. (1988). Bone densitometry (letter), Am. J. Roentgenol. 150, 207-208. NUSBICKEL, F.R., DELL, P.C, MCANDREW, M.P. and MOORE, M.M. (1989). Vascularized autografts for reconstruction of skeletal defects following lower extremity trauma. A review, Clin. Orthop. 243, 65-70. OSTROWSKI, K. (2000). Infections transferred by transpants. Section of Medical Scientes Polish Academy of Scientes, Publ. Co., Warsaw, pp. 5-15. TYLMAN, D. (1986). Bone union of fracture of bones — Biological aspect, and influence physical agent. Chir. Org. Motus Orthop. Polonica 51, 433-446. LODARSKI, K.H. (1991). Bone histogenesis mediated by nonosteogenic cells, Clin. Orthop. 272, 8-15.

4 CLINICAL RESULTS AND ORGANISATIONAL ASPECTS OF AUTOGENOUS AND ALLOGENOUS BONE GRAFTING IN THE TREATMENT OF 226 PATIENTS WITH PRIMARY OSSEOUS NEOPLASMS

M.R. SARKAR, M. SCHULTE, G. BAUER & E. HARTWIG Klinik fur Unfall-, Hand- und Wiederherstellungschirurgie Chirurgie III, Universitat Ulm, Germany

1. Introduction Operative therapy of osseous neoplasms consists of curettage or resection, and this inevitably leads to skeletal defects. They are frequently large, sometimes including major parts of a joint. Therefore, reconstruction of osseous defects is a prime condition for limb salvage. Which are the methods available for reconstructing a skeletal defect in orthopaedic tumour surgery? They comprise of bone grafting with autogenous or allogenous material, callus distraction, and implantation of synthetic materials like tricalcium phosphate or endoprosthetic devices.

83

84

Callus distraction seems to be an interesting alternative. However, for large defects the time required for transport and consolidation will be extremely long, easily more than one year. It is common knowledge that local problems during such a long time become a major challenge and the psychological demands on the patient are tremendous. Finally, the osteostimulative effects of callus distraction are a point of concern after operative treatment of osseous neoplastic disease. 2. Clinical Data It is beyond the scope of this contribution to comment on the controversy whether massive allografts or mega-prostheses should be preferred for reconstruction in specific situations. This report is focused on the outcome of bone grafting procedures in 226 patients who were operated on because of primary osseous neoplasms during the years 1980 to spring of 1996 at the University of Ulm. There were 101 tumour-like lesions, 61 benign and 64 malignant tumours. Autogenous grafts were used in 110 patients, allografts in 97 patients, and in 19 cases a combination of both, or with tricalcium phosphate, was implanted. 65% of the patients were adolescents or young adults below the age of 30 which is characteristic of primary osseous neoplasms in contrast to skeletal metastases, which predominantly appear at a more advanced age. The majority of tumours were located in the pelvic bones (32%) or in the lower extremities (44%). Upper extremity involvement was seen in 15% and spinal lesions in 9%. There was no statistical difference between the distribution of autogenous or allogenous grafts as to the location, the sex or the age of the patients. In bone tumour surgery outcome evaluation comprises three very different fields: early and late local complications of surgery, oncological results (local recurrence, metastatic spread, death) and functional aspects (pain, joint mobility, ambulation etc). The analysis presented here is focussed on clinical results and complications attributable to the method of reconstruction used in this series. This

85

study was devised because bone tumour surgery is different from trauma surgery for a variety of reasons: 1. No soft tissue damage due to blunt trauma or open wounds, 2. Aspects of radical surgery in malignant tumours (including untypical approaches), 3. Additional problems due to the underlying disease and concomitant therapy. The oncological outcome of every tumour patient treated at our institution is regularly and closely monitored with a follow-up programme. However, these data are not shown here because our series comprises too many histological entities with very different therapeutic and prognostic implications. This precludes an analysis of the oncological outcome with respect to the grafting procedure involved. Also, an evaluation of the functional outcome only makes sense for a given procedure (e.g. alloplastic joint replacement of the knee versus an osteocartilagineous allograft), but not for a multitude of different tumour locations and surgical interventions. Wound healing was not problematic and by primary intention in most cases. The infection rate was 2% (n = 4) for the entire series. No difference was observed between infection rates of patients having received autografts or allografts. Graft resorption without adequate formation of new bone occurred in 5% (n = 11) of the cases. This phenomenon was seen somewhat more frequently in the autogenous group than after allografting (6 vs 1; 4 cases in the group with combined implants). However, since our patients were not prospectively randomised to receive one kind of graft or another, we prefer to regard our results as indicative of certain trends. We do not consider statistical analysis of the data mentioned to be appropriate. The same is true for late implant failure resulting in graft fracture. This complication was seen in 3% (n = 6) and necessitated revision surgery. If possible, stable osteosynthesis was the preferred method of treatment like in a fracture of a long diaphyseal allograft. It has been shown by Enneking and others that the core of large allografts

86

remains vital even years after implantation, since revascularisation eventually ends before it comprises the entire graft. However, graft fractures have the propensity to heal provided sufficient stabilisation is achieved and the surrounding host soft tissues convey good vascular supply. In other cases a second grafting procedure was necessary. 3. Discussion Bone grafting for the reconstruction of skeletal defects in oncologic surgery has to respect the rules developed for post-traumatic defects: grafts may only be placed in a well vascularised site under conditions of absolute mechanical stability and sterility. Viable soft tissue coverage is a crucial factor for successful graft incorporation. Depending on location and load patterns, cancellous chips or cortical segments are used. Autogenous bone is generally considered the optimal graft because it integrates faster and with fewer complications than any other material. However, it requires a separate operative procedure in the same patient and the amount available is limited — especially in children and adolescents. Allogenous grafts carry the risk of viral infection for the recipient. The harvesting and storage of allografts creates additional costs, administrative, legal and ethical problems. Nevertheless, allografts are the only therapeutic option besides endoprosthetic devices for large-size reconstructions. Our series shows that the use of allografts does not increase the risk of post-op infections. Also, the rate of long term failures is low due to progressive incorporation. In order to protect bone graft recipients, it has been suggested that only sterilised bone grafts should be used and several methods were developed in recent years: autoclaving, high dose radiation and chemical impregnation. While the first two work reliably, chemical methods were shown to be less effective because permeation of the respective media is limited and difficult to control. The severe sideeffect of most sterilisation procedures is the loss of any osteoinductive

87

potential in the allograft. In addition, mechanical characteristics can also be altered (less by irradiation than by autoclaving). Many sterilised grafts will eventually fail both mechanically and biologically. For these reasons, we do not favour graft sterilisation. Rather, at our institution, a dual strategy for minimising the risks associated with allografts is followed. Femoral heads from patients undergoing total hip replacement surgery are subjected to thermodesinfection. This technique was developed by Knaepler who invented a device that heats the bone specimen in a sterile chamber to a temperature of 80°C for half an hour. At this temperature level, biological and mechanical qualities of the graft are only moderately altered, while most bacterial and viral transmitters of diseases are eliminated including HIV, but not hepatitis B and C virus. The donors are informed about this procedure and their consent (including permission to perform serological examinations for bone banking purposes) is documented in the written form. Also, negative bacteriological testing of the graft itself is required. Large allografts (diaphyseal segments of long bones, entire femora, tibiae, etc) are harvested in co-operation with the Organ Transplantation Unit (OTU) of the University of Ulm. The necessary medical and legal procedures required by German law are taken care of by the doctors of the ICU. When, during their talks with the relatives of the patient, they raise the issue of organ transplantation, they will also seek permission for bone or joint explantation. If this is consented, the trauma surgeon joins the explantation team and removes the bones or joints after the internal organs, but still under conditions of uncompromised sterility. Depending on what has been agreed with the donor's family, two different kinds of procedures for bone graft retrieval are possible: via a limited approach only vertebral bodies or ciorticocancellous segments of the pelvic bones can be harvested because the laparotomy necessary for liver or kidney explantation is used exclusively. Separate incisions permit an extended procedure for the retrieval of whole femora, tibiae, humeri, knee joints, or an entire hemipelvis.

88

The bone grafts are packed in three sterile plastic bags and stored at -80°C. The recipients of the same donor's kidneys are routinely entered into a clinical follow-up programme co-ordinated by the OTU. During the regular check-ups of these patients a serological re-testing is done after six months. These test results are handed to the Bone bank Working Group and only when they show that the kidney recipient remained sero-negative will the corresponding bone grafts be authorised for implantation. At our institution, the various organisational and legal issues involved in bone banking are dealt with by an interdisciplinary Bone Bank Working Group led by a senior trauma surgeon. Two younger trauma surgeons share organisational tasks; other members of the group are two transplantation surgeons, a representative of the kidney transplantation curatorium and a senior theatre nurse who is in charge of the thermodesinfection machine and also regularly checks the deep freezers which are installed right in the theatre area. This structure has proven so effective that so far we have always been able to graft patients with material from our own bank, which renders our department independent of external providers.

5

NEW APPROACHES TO COMPARATIVE EVALUATION OF ALLOGENIC AND AUTOLOGOUS BONE TRANSPLANTS PROCURED IN VARIOUS WAYS

A.V. KALININ, V.I. SAVELIEV and A.A. BULATOV Russian Research Institute of Traumatology and Orthopaedics, named after R.R. Vreden Baikov Str. 8, 195427 St. Petersburg, Russia

1. Introduction Any research work dealing with comparative evaluations of different ways of bone tissue conservation, starts with the choice of an experimental model. The latter should correspond to the aims and the goals of the research, as well as provide for standardisation of conditions in the process of experiment conduction. These conditions are rather difficult to achieve due to both external and internal factors. The external factors include peculiarities of operative technique with various degrees of tissue trauma, and security of graft fixation, or purulent complications. With acquired experience and model improvement the external factors may be under control. But it is more difficult to overcome the undesirable influence of intrinsic factors such as age, gender, individual and constitutional peculiarities of donors and hosts. The most reasonable decision under these conditions is to conduct such kinds of research on genetically similar lines of

89

90

experimental animals. But inbred animals, especially large ones, are still too expensive to be used in everyday practice of the majority of experimental laboratories. It should be emphasised that experimental models used at present for the sake of studying bioplastic characteristics of bone transplants, do not allow us to take into consideration the influence of all, or at least the majority of the enumerated factors. As a rule they are based on procurement of transplantation material from several animals-donors and the transplantation to other animal-recipients, into osseous defects created in various skeletal sites (Einhorn et a\., 1984). The transplanted grafts usually differ in their size, form and composition, which in combination with the absence of a standard method of graft fixation, has certain influences over the processes of bone regeneration, their speed and quality. These very reasons may explain existing disagreement about the term of consolidation and remodelling of osseous grafts (including demineralised bone) preserved in various ways. Besides that, the known experimental models are labour consuming, and need for their implementation plenty of animals — a number of them being killed without surgery due to the need of a great amount of transplantation material. In 1967 Saveliev devised an original model for comparative studies of bone transplant evolution. According his suggestion several grafts (from two to six) received from one animal-donor were transplanted to another animal-host (both of them being dogs). Rib fragments were used as transplantation material; they were placed in rib defects created in experimental animals by rib resection, both on the left and the right sides of the chest (Fig. 1). Thus the suggested model allowed us to standardise the transplantation material, and eliminate most of the unfavourable factors described above. The expenditure of animals decreased to a considerable degree because several operations were done on one and the same dog. In contrast to other models all manipulations on one animal, including its euthanasia, were performed at the same time — which excluded the influence of time factor.

91

DOG «A»

DOG «B>;

Fig. 1. A schematic drawing of the operations. Allotransplantation — transplantation of rib fragments from Dog "A" to Dog "B" after their sterilisation and conservation in different ways.

And finally, this model allowed one-stage orthotopic transplantation of bone grafts identical in their form, size and structure, to that of an animal-recipient. The following operation technique was used. Under intravenous thiopental anaesthesia, hair was cut in the area of the greatest convexity of the rib cage on the left and right sides in a strip 10-15 cm long. Due to the danger of injuring the pleura in the process of rib excision with subsequent pneumothorax development, dogs were usually intubated so that lung ventilation could be started if necessary. The skin was incised parallel to the spine. Then the soft tissues were cut transversely to the ribs. The periosteum was incised along the rib at the necessary length, and detached from it with a raspatory. After that a rib fragment was resected with cutting forceps. The intramedullary canals of the remaining parts of the ribs were widened with an awl. One end of the pin with the graft mounted on it was inserted into the vertebral end of the rib at a depth of 1-1.5 cm. After that, with the help of strong clamps the stumps of the rib were separated as far as possible to allow us to insert the other

92

end of the pin into the sternal rib stump, and the stumps were approximated to be in contact with the ends of the graft. The intercostal muscles were stitched with continuous sutures, followed by stitching of m. latissimus dorsi. The dog was extubated. The animals did not need any special care after the operation. They were sacrificed under general anaesthesia in due time, and macrospecimens were removed. The latter were studied macroscopically, roentgenographically (in two views), histologically, and in other ways. Later Saveliev (1978) devised a new version of the experimental model, according to which rib grafts removed from one side of the chest after their treatment with sterilising or preserving agents, were transplanted into rib defects created on the other side of the chest of the same animal (Fig. 2). Thus the first version was concerned with allogenic, and the second one — with autologous bone plasty. The application of the second version considerably increased the reliability of the achieved results, because the transplantation material was

DOG «C>

Fig. 2. A schematic drawing of the operations. Autotransplantation — transplantation on the left side of the chest of the Dog "C" of rib fragments excised earlier on the right side, sterilised and preserved in different ways.

93

in complete conformance with individual characteristics of the host's body. In other words, in contrast to the first variant there was no disagreement between the antigenic uniform transplantation material and the non-uniform host bed. Alongside this, the follow-up period became considerably shorter: the longest follow-up time not exceeding six months in contrast to one to two years in the transplantation of allogenic bone. Elimination of difficulties caused by the necessity to reproduce similar experimental conditions, and possibilities of adequate control provided by a fresh (not preserved) rib fragment, resulted in the fact that all peculiarities of reparative osteogenesis found in experiments on transplantation of osseous tissue (preserved in different ways and for various periods of time), could be interpreted solely with respect to their influence. It is also worth mentioning that not a single test model used nowadays in experiments on bone transplantation, allows us to use the same amount of bone with uniform structure, volume and form as the one which is being described. In attempts to improve the second version of the discussed model, various ways of graft fixation were studied, which resulted in elaboration of the original method consisting in securing grafts with flat spear-like intramedullary rods made out of pins usually used in trauma and orthopaedic surgery. This rod securely joined the ends of the resected ribs, obliterated motion at their junction with the graft, and prevented graft displacement ("twisting") around the rib axis. In addition to that, it decreased the force acting at the central part of the graft, especially at the height of its remodelling which helped to prevent fractures and pseudarthroses. This spear-like rod appeared especially useful for holding demineralised bone grafts, which are difficult to stabilise with other means. In addition to fixation method improvements it was suggested that we dissect a rib fragment 2-3 mm smaller than the graft; this allowing us to achieve some compression along the line of bone junction. All these suggestions helped to improve conditions for grafts and favoured their substitution with new bone.

94

The described versions of the model were used by the authors of this article in the study of bioplastic characteristics of allogenic and autologous bone grafts preserved in various ways. 2. Material and Methods The first experimental series (allotransplantation) included 45 operated dogs; the second one (autotransplantation) consisted of 38 animals. In the first series, rib fragments up to 35 mm in length were received from mature dogs-donors. Into the defects created on both sides of the rib cage of dogs-recipients, six grafts were placed: three on each side. Five of them represented allogenic material: the first graft was procured under sterile conditions and preserved by freezing at -20°C; the second one was preserved in 0.5% formalin solution; the third and the fourth were demineralised in 2.4 N and 1.2 N HCl solution respectively;

t t / 1 I // a

b e

d

e

f

g

Fig. 3. Rib X-rays after allotransplantation of the following fragments: (a) intact rib; (b) demineralised in 1.2 N HCl solution; (c) procured under sterile conditions and preserved by freezing at -20°C; (d) demineralised in 2.4 N HCl solution; (e) preserved in 0.5% formalin solution; (f) autograft (reference); (g) sterilised with gaseous ethylene oxide, and preserved by freezing.

95

the fifth graft was sterilised with gaseous ethylene oxide and preserved by freezing (Saveliev, 1971). The sixth (control or reference) graft was autologous; it was taken out during the operation and positioned into the defect. Fixation was achieved with metal pins. The time of graft preservation before their transplantation didn't exceed one month. All in all, 270 grafts were transplanted. The animals were sacrificed under general anaesthesia at one, three, six, nine and 12 months after the operation, and macrospecimens were removed. The latter were examined with macroscopic, roentgenographic (Fig. 3) and histologic (Fig. 4) methods.

i'V.

2.

3.

Fig. 4. Three months after the operation. Histotopogramms of ribs with transplanted allografts. Grafts: 1. procured under sterile conditions and preserved by freezing at -20°C; 2. preserved in 0.5% formalin solution; 3. demineralised in 2.4 N HC1 solution; 4. sterilised with gaseous ethylene oxide and preserved by freezing; 5. autograft removed during the operation (reference); 6. demineralised in 1.2 N HC1 solution.

96

Histologic specimens were stained with hematoxylin-eosin and according to van Gieson. The second experimental series was aimed at studying the bioplastic characteristics of osseous autografts preserved by freezing, with formalin and by demineralisation. On the left side of the dog's chest, fragments of three ribs were resected subperiosteally, and autografts 35 mm in length were prepared out of them. The defects formed in this way were left unfilled. The wound was completely closed in layers. The rib fragments were thoroughly freed of periosteal remnants and preserved by freezing under the temperature of -20°C, in 0.5% neutral formalin and by demineralisation in 2.4 N HC1 solution with subsequent washing in sterile saline solution, and keeping in 70° alcohol under +4°C. The length of graft preservation depending upon the experimental conditions, amounted to one, three and six months. After that period of time another operation was performed on the contralateral (right) side of the chest. It consisted in rib resection (leaving an intact rib in-between) and substitution of these defects with preserved grafts. Four rib grafts were transplanted to each dog; one of them — "fresh" (removed during the surgery) — served as the control (reference). It should be pointed out that the most crucial moment of the operation consisted in detachment of the pleural sheet of the periosteum from the internal side of the rib with a raspatory. While doing this it is possible to injure the pleura. Pneumothorax developing under these circumstances is dealt with in a usual way: the pleura is sutured, and air is pumped out of the pleural cavity. The dog is immediately intubated, and artificial lung ventilation is started. Intubation may be performed before the operation. Our experience shows that this complication is rare and doesn't affect the transplantation outcome. No animals died during the surgery. The dogs were sacrificed in 15, 30, 90, 180 and 270 days. The removed macro-specimens were examined with macroscopic, roentgenographic (Fig. 5) and histologic (Fig. 6) methods. Histologic specimens were stained with hematoxylin-eosin and according to van Gieson.

97

Fig. 5. X-rays of the ribs after autotransplantation. Autografts: (a) intact rib, (b) preserved in 0.5% formalin solution; (c) sterilised with gaseous ethylene oxide and preserved by freezing; (d) demineralised in 2.4 N HC1 solution; (e) "fresh", removed and transplanted during the operation.

'I • n

'••""

V; a .

b.

'-' ;'c.

"

d.

Fig. 6. Nine months after the operation. Histotopogramms of ribs with transplanted autografts: (a) sterilised with gaseous ethylene oxide and preserved by freezing at -20°C; (b) preserved in 0.5% formalin solution; (c) demineralised in 2.4 N HC1 solution; (d) "fresh", excised and transplanted during the operation.

98

3. Results Our experiments on allotransplantation showed that in one month after the surgery, washed, de-proteinised allografts and autografts were seen in X-rays as shadows with distinct outlines, with the density approaching that of the host's ribs. The space between the ends of the grafts and the rib stumps was clearly identified. The demineralised graft could not be visualised at this time, but the osseous bed of the host demonstrated a marked periosteal reaction. Periosteal growth was also seen at the ends of the ribs with unsubstituted defects. In three months the washed graft looked less dense; its contours became irregular. The ends of the graft and the rib stumps were connected by periosteal bone callus. The shadow of the autograft (reference) was as dense as the host's rib. The ends of transplanted autologous bone were smooth; periosteal bone growth was clearly seen. In the place of the demineralised graft an osseous regenerate could be defined, the form and the structure of its shadow resembling the roentgenographic shadow of an intact rib. In the area of unfilled defects, end plates of sclerotic osseous tissue appeared on the rib stumps. Six months after the transplantation of demineralised bone and autologous tissue (reference) good osseous regenerates with an organotypic structure were formed. Roentgenographic and histologic studies showed active periosteal and endosteal bone formation, especially marked on the side of the pleural sheet of the periosteum. The demineralised graft was more intensely replaced than the autologous one (reference), the latter still demonstrating on histological specimens the presence of particles of old bone deprived of osteocytes in the bulk of the osseous regenerate. Bone formation, in response to washed grafts sterilised with ethylene oxide, was less marked. The weakest osteogenic reaction accompanied transplantation of deproteinised bone. The shadow of the graft endured resorption and fragmentation nearly along its whole length. No worthy osseous regenerate was formed. At the site of the defect left unfilled after the operation, there appeared a scar band joining the sclerotic rib stumps.

99

In the second experimental series, the following stages of evolution of grafts and receiving beds were noted. On the 15th day, besides inflammatory changes, mild periosteal and endosteal reactions were observed — mainly on the side of the terminal areas of the receiving bed, as well as phenomena of osteoclastic graft resorption. Narrow fissures were seen between the grafts and the rib stumps. The demineralised graft was not visible in X-rays. By the 30th day primary osseous union appeared between the osseous bed and "fresh" (i.e., removed during the operation), frozen and formalinised grafts. Periosteal reparation was more marked than two weeks earlier, especially at the internal (pleural) surface of the grafts. The outline of their ends was not clearly seen in X-rays; histologically, newly formed osseous tissue of cancellous or lamellar character was defined in the area of the union. After 90 days, at the site of transplantation — predominantly from the side of the pleural surface — one could find organised osseous regenerates joining the ends of the rib stumps and incorporating remodelling grafts (Fig. 2). Autologous bone preserved in formalin needed more time for remodelling in comparison with frozen and "fresh" tissue. Demineralised bone demonstrated the most complete substitution by this time. At this term on histologic specimens of each regenerate, one could distinctly visualise a well pronounced cortical layer and an intramedullary cavity filled with a dense net of newly formed trabeculae with the presence of myeloid and fatty bone marrow. Fragments of old bone were found within the bulk of trabeculae. After 180 days roentgenographic and histologic examinations demonstrated further resorption of graft particles embedded in osseous tissue conjoining the ends of the resected ribs. By the 270th day practically complete anatomical restitution of rib integrity was achieved; their structure did not differ from intact ribs. The site of transplantation could be defined only by the presence of metallic rods. Histologically, in the depth of the new bone formed after formalinised graft transplantation, its small fragments enduring remodelling were still seen.

100 4. Discussion The experiments have shown that demineralised rib fragments possess high bioplasic activity. In most of the animals, after a short time (3-6 months) after alloplasty there appeared new bone with an organotypic structure. After transplantation of frozen and formalinised grafts, nine to 12 or even more months were needed for complete restoration of rib integrity. Judging by the speed of new bone formation and remodelling after transplantation of both allogenic and autologous osseous tissue, demineralised grafts were the best, followed by frozen and formalinised material. But around frozen and formalinised grafts, as compared with demineralised ones, denser osseous tissue was always formed, although it happened much later. The results achieved in the present study allow us to evaluate the role of various components of osseous tissue in the processes of reparation. First of all, they show that the ground substance in autografts has a positive influence over transplantation outcomes in contrast to mineral elements, which, being present in transplants, hinder their assimilation. Resorption and utilisation of the mineral basis of the bone demand additional energetic and temporal expenditures on the part of the host's body. Comparing the results of morphologic examination of autologous and allogenic grafts preserved in one and the same way, one can note similarities as well as differences. Similarities consist in necrobiosis of the grafts; their infiltration with cellular elements; and resorption and synchronous (at best) substitution with newly formed osseous tissue. Differences are concerned both with reparation tempo, and quality of reparation. Studying morphologic remodelling of bone auto- and allotransplants, we came to the persuasion that the peculiarities of reparative osteogenesis depend in many respects upon the biological type of osseous tissue. Our findings showed that the reasons why reparation processes didn't proceed at a similar speed lay in the fact that they had important qualitative manifestations in their essence. Thus, a characteristic histologic

101

feature of the early period following bone allotransplantation consisted in formation around the graft of fibrillar connective tissue without any participation of osteogenic cells of the receiving bed, which usually didn't occur in cases of autotransplantation. Besides that, one saw lesser activity of osteogenic elements of the osseous bed; weak endosteal bone formation; and prevalence of resorption processes over restorative activity, especially in the middle part of allografts. Allogenic and autologous transplantations differed also in such aspects as an increase in new osseous tissue amount, resorption speed of fatty bone marrow, mineral substances and collagen. The causes of these and many other differences are not yet known. It is interesting to point out that transplantation of demineralised autologous bone gave better results in comparison with allografts treated in the same way, although in both cases the transplantation material consisted mainly of the ground substance. Hence, it may be concluded that the host's body accumulates its own proteins at a greater speed, and that autologous protein possesses greater osteoinductive abilities in comparison with foreign matter. To our mind, these differences might be explained on the basis of either dissimilar antigen activity of allogenic and autologous collagen, or its structural (molecular) dissimilarity. Interesting findings were received in the process of comparative study of reparation processes after transplantation of autologous bone preserved by freezing, in weak solutions of formalin and by means of demineralisation. Before the start of the experiments it was supposed that these factors would cause certain biochemical and morphological changes in the graft, due to which the latter might be deprived of its advantages. But in reality the situation was different. An autologous osseous graft preserved by freezing at -20°C after being transplanted to its host, gave practically the same results as the one freshly produced during the operation. Better bioplastic qualities in comparison with allotransplantation were demonstrated by autologous bone kept in 0.5% formalin solution for three months, or treated

102

with HC1. Truly, here as in earlier described cases, remodelling of formalinised grafts was slightly slower in comparison with frozen ones, and that of demineralised grafts, slightly faster. The experiments on autotransplantation lend another confirmation to the fact that viability of transplantation material was not the only, and moreover not the principal, prerequisite of success of grafting in clinical practice. Thus, with the help of the improved experimental model the following clinically important findings have been received: • Demineralised bone is a highly promising transplantation material suitable for clinical application. • The methods of biologic tissue preservation in 0.5% formalin solution and by freezing do not exclude each other from the point of view of their clinical application, but remodelling of formalinised bone is more slow. • Viability of isolated bone grafts has no decisive influence over transplantation outcomes. In conclusion it should be stated that the comparative evaluation of bioplastic characteristics of bone grafts based on the original experimental model has confirmed its definite usefulness and informational value. It allows us to eliminate the influence of immune, species-linked and other factors, to standardise in this way, experimental conditions, and to receive reproducible data. 5. References EINHORN, T.A., LANE, J.M., BURSTEIN, A.H., KOPMAN, C.R. and VIGORITA, V.J. (1984). The healing of segmental bone defects induced by demineralised bone matrix, /. Bone Joint Surg. 66-A, 274-279. SAVELIEV, V.I. (1967). Chemical sterilisation of tissue grafts and their usage in plastic surgery. Auto-abstract of M.D. Doctor Dissertation. Omsk, 30 p.

103

SAVELIEV, V.I. (1971). Gaseous sterilisation of tissue grafts and a stationary appliance for this purpose, Ortopedia, Travmatologia i Protezirovanie 2, 76-78. SAVELIEV, V.I. (1978). An experimental model for bone graft comparative study. In: New Methods of Prevention, Diagnosis and Treatment in Orthopedic Diseases. Leningrad, pp. 136-141.

6

THE USE OF FREEZE-DRIED MINERALISED AND DEMINERALISED BONE

Ch. DELLOYE Catholic University of Louvain St-Luc University Clinics Brussels, Belgium

1. Introduction The use of freeze dried bone is not as popular in Europe as in the USA where the pioneer work of freeze drying was performed by Flosdorf and Hyatt (1952). The Korean war prompted the use of freeze dried bone — the first human tissue after blood components to be preserved in this way. The main advantage of lyophilisation or freeze drying is storage at room temperature and this remains true till today. The strategic aspect of the storage was not neglected by the US Navy Tissue Bank where the method was set up for bone. Kreuz et al (1951) and Carr and Hyatt (1955) later reported good clinical results with freeze dried bone. From a recent survey of tissue banking activities in the American Association of Tissue Banks (AATB), it appeared that 302 542 bone allografts have been distributed in 1992 alone by accredited or similar bone banks in the USA. Freeze drying was by far the number one preservation means as 83.5% of bone allografts were stored in this way (Strong et al, 1996). This impressive number of

105

106

bone allografts also included about 130 000 vials of cortical bone powder. The freeze drying technique has made the bone allograft very popular in the USA where it is largely available. 2. Aims and Equipment for Freeze Drying* Freeze drying may be defined as a technique to achieve the dry state by freezing a wet matter and sublimating the resulting ice. The aim of freeze drying is to obtain a chemically stable product at room temperature without alteration of the original properties of the product. Stability is achieved by removing the water via application of the triple point of water. At this particular point, all three phases of water coexists. In vacuum and under cold conditions, it is then possible to directly pass from the solid state to the gas phase. Practically, water in the substance is first solidified by freezing. Then, the frozen water is removed by sublimation, which means the transformation from ice to the vapour state without passing through the liquid phase or, in other words, without melting. This is particularly advantageous in the sense that water is removed without chemical or physical denaturation. The final product is stable as there is no water left for chemical reactions to occur. The procedure of freeze drying is usually divided into three stages: freezing the product, primary drying by sublimation of the ice, and finally when no ice is visible anymore, a secondary drying where the residual water is removed by an application of heat. The final product must not have a residual moisture content of more than 5% of the dry weight. The most common method of determining the residual moisture is by gravimetry: after freeze drying, the dried substance is weighed on an analytical balance then placed at 90°C and weighed daily until no further changes in weight is detected. The difference must not exceeded 5% of the dry weight (Malinin et al, 1984). *See also Advances in Tissues Banking Vol 1, Chap. 6

107

The basic requirements for freeze drying is to have a door-fitted chamber where the substance is placed. A vacuum is obtained using a mechanical pump and the latter is protected from humidity by placing a refrigerated water vapour trap between the pump and the chamber. The speed of the procedure is dependent on the temperature (vapour pressure) difference between the chamber and the trap. In our laboratory, the prepared bones are frozen at about -30°C during the primary drying phase. Both a powerful pumping system that is able to reach about 1.10~5rnmHg and a trap that is cooled with liquid nitrogen are required to work at these low temperatures. In our experience when working at 1.10~5mmHg vacuum, cancellous bones are freeze dried in about two days with a residual moisture of 1.1. ± 0.8% of dry weight (unpublished data). 3. Properties of Freeze Dried Bone 3.1. Mechanical properties The sterile freeze dried bone which is ready for use results either from a procedure carried out under constant sterile conditions or from a final sterilisation. It is currently accepted that the compressive strength of the bone is not modified after being freeze dried (Bright and Burchardt, 1983; Pelker et al, 1983). However, freeze drying of cortical bone produces a significant deleterious reduction in the torsional strength of the long bone (Pelker et al, 1983) as well as in bending (Triantaphyllou et al, 1976). Considering only the mechanical influence of irradiation alone, it is agreed that the threshold dose limit is 30 kGy above which there is a significant decrease in the torsional strength (Komander, 1976; Bright and Burchardt, 1983). In compression, the dose of 60 kGy appears as the threshold for both cortical (Komender,

108

1976; Lory et al, 1990) and cancellous bone (Anderson et al, 1992). But the association of freeze drying and irradiation will combine their effects and again causes more pronounced effect and, in particular, in flexion and torsion. Although there is no unanimous agreement on the exact influence depending on how the tests are carrying out, the decrease varies from 10-70% of the original properties with a more pronounced effect on torsional properties (Triantaphyllou et al, 1975; Pelker et al, 1983). Another issue is the choice of the optimal sequence for preservation and irradiation. According to data published by Pelker et al (1983), irradiation of a dried substance would be more harmful than that of a wet substance. However, this aspect remains, conflicting (Hault and Powlison, 1989; Randall et al, 1991; Strong and MacKenzie, 1993) and requires additional studies. Another matter of debate is the influence of rehydration on the mechanical recovery. Bright and Burchardt (1983) contended that there is a progressive return to normal values within 24 hours while Conrad et al (1993) observed a decrease in strength after 24-hour rehydration. The most important aspect for the surgeon is to know that the bone will be brittle during implantation and that it will return progressively to more normal mechanical resistance in the days after surgery. 3.2. Biological properties The changes induced by freeze drying are first produced by the necessary freezing of the tissue to convert water into ice. It is during this stage that cells are killed. However, it is possible to maintain a cellular survival. To achieve cell survival after freeze drying, the use of cryoprotective additives is mandatory (Greaves, 1966). This has been applied successfully for viruses by the pharmaceutical industry (Strong and MacKenzie, 1993). In a conventionally freeze dried tissue, there is no cell survival and consequently, a freeze dried bone is not osteogenic by itself. In

109

other words, a freeze dried bone is not able to produce new bone by itself. Freeze drying is an appropriate technique to preserve osteoinductive (if any) and osteoconductive properties of bone. The osteoinductive capacity of a hydrochloric acid (HCl)-mineralised bone has been shown to still induce new bone 11 years after being dried (Delloye et al, 1986). Extensive experience with non-demineralised bones shows that the osteoconductive capacity of the preserved bone is fully retained with freeze drying (Delloye et al, 1987, 1991).

4. Preparation of Bone to be Freeze Dried Bones that will be processed are usually procured at the mortuary room according to the common European standards (EATB and EAMST, 1997) and according to Belgian law. Pieces of bones are cut from the explanted epiphyses around the knee only. Bones are freed from any residual soft tissues, washed with a jet, and treated with various chemical agents to remove the bone marrow and to obtain a virucidal effect against the viruses of HIV, Hepatitis B and C and prion diseases. Lipid extraction of bones has been shown to result in a faster and more complete cellular invasion of the bone implant (Aspenberg and Thoren, 1990). The bones are rinsed, freeze dried for two days, packaged under vacuum, and finally freeze dried at 25 kGy (Delloye et al, 1987). A freeze dried bone is usually packaged in a glass jar or plastic envelope. Plastic wrapping and conventional sterilisation packaging system were found to be convenient when combined, as the outer air-sealed plastic provides chemical and mechanical protection of the bone while the inner peel-off package offers an easily usable form to unpack the content in an operating theatre under strike conditions (Fig. 1).

110

**:?

Fig. 1. Aspect of the ready-for-use freeze dried bone implant that has been packaged under vacuum. This allows easy detection of any air leakage.

Ill

5. Clinical Indications of Freeze Dried Bones 5.1. Non-demineralised bone 5.1.1. Locations In the majority of the cases, cancellous bone or cortico-cancellous bone are used. The main indication is a local loss of bone. Such a small skeletal defect is currently observed in some orthopaedic operations like osteotomies (Figs. 2 and 3), in benign tumours (Figs. 4-6), in revision arthroplasties (Fig. 7), and in spine arthrodesis (Figs. 8 and 9).

Fig. 2. Bone lengthening in a bone with Ollier's disease: (a) Immediate post-operative view of the lengthening achieved with two large freeze dried bone blocks and plating. The patient is six years old; (b) aspect at two months with an apparent union; (c) three years after, the patient underwent a second lengthening with an Ilizarov's procedure. To be successful, the bone ends (where previously the freeze dried bone was) must be vascularised. Aspect of the elongated bone with new bone arising from either end of the osteotomy.

112

Fig. 3. Failure of union with a Maquet's procedure using freeze dried bone. There is a fibrous layer interposition due to inadequate fixation.

Fig. 4. Pre-operative aspect and final aspect (at four years post-operation) of a simple bone cyst that has been curetted and packed with freeze dried bones. Uneventful course.

113

Fig. 5. Progressive remodelling of a freeze dried bone block that was implanted into a cavity after removal of recurrent benign tumour in the calcaneum. Gradual restoration of the original trabecular network.

VI '.88

Fig. 6. Reconstruction of a tibial plateau after a recurrent chondroblastoma. Progressive incorporation of the graft at five years. The patient is asymptomatic.

However, all the locations for bone grafting are not equal in terms of osteoconduction. Cellular invasion of an acetabular bone graft will be more difficult than for a graft implanted in a tibial plateau because in the first location, the graft has been placed most often close to hardware (cup, screws, cement) and because the

114

Fig. 7. Reshaping of a tibial plateau for arthroplasty in a 75-year-old woman. Aspect at seven months after surgery.

Fig. 8. Excision of a vertebral body for tumour. Reconstruction carried on with a diaphyseal freeze dried ring fixed with one unicortical screw and a plate. A portion of a rib was used as autograft to fill the inner part of the ring. Aspect at four years after procedure with an uneventful course.

115

Fig. 9. Very long-term (12 years) reconstruction of a L4 vertebra that was completely removed because of Ewing's sarcoma. Reconstruction of the body using the proximal end of a tibia that has been freeze dried and combined to a posterior fusion. The patient is completely asymptomatic.

adjacent bone environment is presumably poorer in osteogenic elements as a consequence of revision surgery of the acetabulum. Consequently, the observation of Hooten et al (1996) that allografts from the acetabulum were poorly invaded is not surprising. Whether they are morcellised or in bulk will not change the environment. If they are in bulk, any fracture will result in an overall collapse. In contrast, if they are morcellised and protected mechanically (e.g. by a plate), a full collapse is not expected as the fracture will be limited to only one component of the graft. A progressive failure still remains possible as the loading on the remaining part of the graft becomes higher. Within a long bone, the metaphysis and epiphysis are preferred locations for bone grafting because they contain more osteoblasts than the diaphysis. In our opinion, treating a non-union at the

116

mid-diaphysis of an adult long bone with only a bone allograft has some risk of failure because this material is not osteogenic by itself. The only exception to that is the growing bone which has more osteogenic capacity than a fully-grown bone (Fig. 2). The spine is becoming another suitable location where a freeze dried bone allograft may serve as a good substitute. The diaphyseal segment of freeze dried bone is quite useful as a substitute for a vertebral body at the cervical (fibular ring) or the lumbar body (tibial or femoral ring). Salib et al (1997) recently reported very favourable results from using material and we have the same experience at our institution (Fig. 7). A freeze dried bone that is placed in compression is able to sustain a high load at long-term (Fig. 8). In contrast to the acetabulum, the location at the spine is more favourable: the allograft is placed in compression and abuted in cancellous bone with an osteoblast-rich environment. Here, an expected good incorporation is realistic. Even more so, the allograft used as a ring can serve as a container for bone autograft, like a segment of rib for instance. The clinical results are excellent and reproducible provided the graft has been placed correctly. In any case, it is mandatory that the recipient bone of the graft must be prepared with a resulting bleeding and appearance of cancellous rather than cortical bone. The implant must preferably be impacted or press-fitted into the bed. It should not be fixed with screws alone, if possible, because freeze dried bone is brittle and consequently may crack. If these guidelines are followed, a freeze dried bone will give a high level of satisfaction to the implanting surgeon. 5.1.2. Reliability of freeze dried bone in a clinical setting We conducted three studies to assess the value of the freeze dried bone. In our first study (Delloye et al, 1987), 72 consecutive cases where the graft was of sufficient size and at a distance from the surgical hardware, if present, to be clearly visible on the antero-

117

posterior and lateral views. In majority, most grafts were at the lower limbs. The majority which had been used in revision arthroplasty could not be included in the study, since they were not clearly visible on X-rays. Few grafts at the spine could be analysed for the same reasons. The grafts were followed for at least one year; 78% produced very good results (as defined as an obvious union of the freeze dried bone to the recipient cancellous bed within six months and a preserved original bone volume); in 11% of the cases, the rate was good (defined as a more irregular but still achieved union at six months or a decreased bone volume of less than 25% from the original volume); finally, 11% of the cases that were considered as failures (defined as non-union at six months or a resorption, including a collapse (if present), of more than 25% of the starting volume). Interestingly, amongst the eight failed cases, six were due to inappropriate preparation of the bed, poor spacefilling, or to inadequate graft fixation (Fig. 3). Only two cases could be attributed to the graft itself — one with resorption and one with a collapse. In a second study (Cornu et al, 1995), 64 consecutive patients undergoing a tibial tubercle elevation with a bone graft (Maquet's procedure) were studied. This operation allows a good lateral view of the bone graft. Autograft was compared to freeze dried bone allograft in a matched population for sex, weight and age. Amongst the clinical data analysed, only the mean hospital stay was significantly lower in the allograft group (p < 0.03). The patient satisfaction index and the amount of blood received, when applicable, were not significant variables. A similar radiological score when applied in both groups did not reveal a statistical difference. Fixation with two screws was found to be associated with less resorption than with one-screw fixation in both auto- and allografts. It was concluded from the study that freeze dried bone allografts can be used in that operation. Finally, the efficacy of freeze dried, non-demineralised bone was assessed in spinal fusion for scoliosis (Recht et al, 1993). Again, two

118

groups of patients — one receiving exclusively bone autografts and the other receiving autograft supplemented with freeze dried bones — were analysed by the loss of angular correction on X-rays. In boths goups, the loss was not statistically different at one year, so that the authors concluded that freeze dried bone could be recommended in spine surgery for scoliosis. A similar conclusion was muscle by Fabry (1992). From these clinical studies, it appears that provided basic surgical technique for a sound graft preparation and fixation is applied, a freeze dried irradiated bone can be a reliable bone substitute to an autograft. 5.1.3. Freeze dried bone allograft as an antibiotic carrier, or can a bone allograft be used in a septic environment? Another interesting aspect of a bone allograft is its potential use as a vehicule for antibiotic. Hernigou et al (1992) was the first to adress this specific issue. He showed that fresh and preserved cancellous human bone was able to release antibiotic for several days at a level of 1000 times above the minimal bactericidal concentration and this was observed after only 30 minutes of exposure. These observations were confirmed in vivo as a preserved femoral head soaked for 30 minutes in 500 mg of vancomycin released the antibiotic, as measured in urines for at least three weeks. Winkler and Georgopoulos (1997) observed similar findings with vancomycin. The release was found to be effective for several weeks in patients. Most surgeons consider the use of an allograft inappropriate in septic conditions. Having regard to the above-mentioned experiments, and that the pharmaceutical industry provides bovine collagen as an antibiotic carrier, and considering also the clinical experience of vascular surgeons using preserved arterial allografts as a salvage procedure for life-threatening massive infection of Dacron arterial implants, it is believed that a preserved bone allograft, provided it has been soaked in an appropriate antibiotic solution, may be used in septic conditions.

119

5.2. Demineralised bone It took about 30 years from Urist's discovery in 1965 to accept that hydrochloric acid demineralisation of bone was able to induce new bone formation in a muscle of rat (Urist, 1994). This observation caused a tremendous impetus to the isolation and characterisation of several non-collagenous proteins from the bone matrix called the bone morphogenetic proteins. These are now produced by genetic engineering and are available for clinical studies (Wozney et al, 1988). In the meantime, several tissue banks, especially in the USA, have promoted the use of demineralised bone powder. Its use has been popular especially in the maxillo-facial area. In this regard, the paper reported by Glowacki et al (1981) had a sharp positive influence. However, its use as a powder in orthopaedic surgery has not been so popular because expected results were probably too high (Kakiuchi et al, 1985). In Europe, the use of demineralised bone powder remains very limited because there are many causes for failure such as the quality of the starting material (age of the donor), the influence of sterilants such gamma-iradiation and ethylene oxide, and because alternative methods to powder use such as bone autograft or the mechanical stimulation of bone growth, as promoted by Ilizarov (1989), remain popular in Europe. However, demineralised bone powder should be used in nonunion or other conditions where new bone formation would be desirable. We have experimented in an unusual indication like a benign tumour called an aneurysmal bone cyst (Delloye et al, 1996). This pathologic process removes normal bone by osteoclastic resorption and may cause extensive bone loss. However, in very rare instances, this resorption trend may stop spontaneously. This fact indicates that reversal of the mechanism is possible. We hypothesised that the bone powder could provoke the reversion or at least stop the resorption. In two patients where bone had disappeared, new bone was largely produced and could reshape new bone while in three others, resorptive activities were successfully halted (Fig. 10).

Fig. 10. Use of HCl-partially demineralised bone powder to induce healing of a devastating aneurysmal bone cyst in a calcaneum of a 16-year-old girl, (a) Pre-operative aspect with disappearance of almost all the calcaneum; (b) six weeks after surgery, there is, to some extent, a restitution of the calcaneum; (c) at four months, slow reconstruction of the bone. No recurence; (d) aspect at two-and-a-half years after surgery. Weight bearing was resumed at eight months.

120

121

The future is governed by the more liberal use of recombinant bone morphogenetic factors with however an appropriate carrier to the intended use. Even more so, we expected to use such proteins to heal large massive bone allografts that fracture as a result of cyclic loading without repair capacity. 6. Conclusions Freeze dried bone remains a reliable bone substitute for the orthopaedic surgeon. The fate of the graft is directly dependent on the manner in which the recipient bone has been prepared and the mechanical stability of the graft. Provided these requirement are observed, a freeze dried bone is a very useful material for the surgeon. 7. References ANDERSON, M., KEYAK, J. and SKINNER, H. (1992). Compressive mechanical properties of human cancellous bone after gamma irradiation, /. Bone Joint Surg. 74A, 747-752. ASPENBERG, P. and THOREN, K. (1990). Lipid extraction enhances bank bone incorporation, Ada Orthop. Scand. 61, 546-548. BRIGHT, R. and BURCHARDT, H. (1983). The biomechanical properties of preserved bone grafts. In: Osteochondral Allografts. Biology, Banking and Clinical Applications. G. Friedlaender, H. Mankin and K. Sell, eds., Little, Brown and Company, Boston, pp 223-232. CARR, C. and HYATT, G. (1955). Clinical evaluation of freeze-dried bone grafts, /. Bone Joint Surg. 37A, 549-566. CONRAD, E. ERICKSEN, D., TENCER, A., STRONG, D. and MacKENZIE, A. (1993). The effects of freeze-drying and rehydration on cancellous bone, Clin. Orthop. 290, 279-284.

122

CORNU, O., De HALLEUX, J., BANSE, X. and DELLOYE, Ch. (1995). Tibial tubercle elevation with bone grafts. A comparative study of autograft and allograft, Arch. Orthop. Trauma. Surg. 114, 324-329. DELLOYE, Ch., HEBRANT, A. and COUTELIER, L. (1986). Osteoinduction in twelve year-preserved decalcified alloimplants in rats, Clin. Orthop. 205, 309-310. DELLOYE, Ch., ALLINGTON, N., MUNTING, E. and VINCENT, A. (1987). L'os de banque lyophilise. Technique et resultats apres 3 annees d'utilisation, Ada Orthop. Belg. 53, 1-11. DELLOYE, Ch., De HALLEUX, J., CORNU, J., WEGMANN, E., BUCCAFUSCA, G.C. and GIGI, J. (1991). Organizational aspects of bone banking in Belgium, Ada Orthop. Belg. 57, 26-34. DELLOYE, Ch., De NAYER, P., MALGHEM, J. and NOEL, H. (1996). Induced healing of aneurysmal bone cysts by demineralized bone particles, Arch. Orthop. Trauma. Surg. 115, 141-145. EUROPEAN ASSOCIATION OF MUSCULOSKELETAL TRANSPLANTATION (EAMST) and EUROPEAN ASSOCIATION OF TISSUE BANKS (EATB). (1997). Common standards for musculo skeletal tissue banking. Osterreichisches Bundesinstitut fur Gesundheitswesen, Vienna, Austria. FABRY, G. (1991). Allograft versus autograft bone in idiopathic scoliosis surgery: A multivariate statistical analysis, /. Pediatr. Orthop. 4, 465-467. FLOSDORF, E. and HYATT, G. (1952). The preservation of bone grafts by freeze-drying, Surgery 31, 716-722. GLOWACKI, J., KABAN, L.B., MURRAY, J., FOLKMAN, J., and MULLIKEN, J.B. (1981). Application of the biological principle of induced osteogenesis for craniofacial defects, Lancet 1, 959-963.

123

GREAVES, R. (1966). The importance of the prefreezing stage on the viability of freeze-dried organisms. In: Advances in Freeze-Drying, L. Rey, ed., Hermann, Paris, pp 95-102. HAUT, R. and POWLISON, A. (1989). Order of irradiation and lyophilisation effects of the strength of patellar tendon allografts, Trans. Orthop. Res. Soc. 14, p 514.

HERNIGOU, Ph., GLORION, Ch v GIRARD-PIPAU, F., DERIOT, H. and GOUTALLIER, D. (1992). Liberation in vitro et in vivo des antibiotiques a partir des greffes ossseuses, Rev. Chir. Orthop. 78 (Suppl I), 216-217. HOOTEN, J., ENGH, Ch., HEEKIN, R. and VINH, T. (1996). Structural bulk allografts in acetabular reconstruction, /. Bone Joint Surg. 78B, 270-275. ILIZAROV, G. (1989). The tension-stress effect on the genesis and growth of tissues, Clin. Orthop. 238, 249-263. KAKIUCHI, M., HOSOYA, T., TAKAOKA, T., AMITANI, K. and ONO, K. (1985). Human bone matrix gelatin as a clinical alloimplant. A retrospective review of 160 cases, Int. Orthop. 9, 181-185. KOMENDER, A. (1976). Influence of preservation on some mechanical properties of human haversian bone, Mater. Med. Pol. 8, 13-17. KREUZ, F.P., HYATT, G. and TURNER, T. (1951). The preservation and clinical use of freeze-dried bone, /. Bone Joint Surg. 33, 863-872. LOTY, B., COURPIED, J.P., TOMENO, B., POSTEL, M , FOREST, M. and ABELANET, R. (1990). Radiation sterilized bone allografts, Int. Orthop. 14, 237-242. MALININ, T., MA W.N. and FLORES, A. (1984). Freeze-drying of bone by allotransplantation. In: Osteochondral Allografts; Biology,

124

Banking and Clinical Applications, G. Friedlaender, H. Mankin and K. Sell, eds., Little, Brown and Company, Boston, pp 181-192. PELKER, R., FRIEDLAENDER, G. and MARKHAM, T. (1983). Biomechanical properties of bone allografts, Clin. Orthop. 174, 54-57. RANDALL, R., PELKER, R., FRIEDLAENDER, G. and PANJABI, M. (1991). The sequence dependency of freeze-drying and irradiation on the biomechanical properties of rat bone, Trans. Orthop. Res. Soc. 16, p 480. RECHT, J., BAYARD, F., DELLOYE, Ch. and VINCENT, A. (1993). Freeze-dried allograft versus autograft bone in scoliosis surgery. A retrospective comparative study, Eur. Spine }. 2, 235-238. SALIB, R., GRABER, J. and EASTLUND, T. (1997). Fusion rate and operative technique using an ethylene oxide-sterilized freeze-dried composite bone allograft in anterior lumbar interbody fusion, Tissue and Cell Report 4, 23-28. STRONG, M. and MacKENZIE, A. (1993). Freeze-drying of tisssues. In: Musculoskeletal Tissue Banking. W. Tomford, ed., Raven Press, New York, pp 181-208. STRONG, M , EASTLUND, T. and MOWE, J. (1996). Tissue banking activities in the United States: 1992 survey of AATB-inspected tissue banks, Tissue and Cell Report 3, 15-18. TRIANTAPHYLLOU, N., SOTIOPOULOS, E. and TRIANTAPHYLLOU, J. (1975). The mechanical properties of the lyophilised and irradiated bone grafts, Ada Orthop. Belg. 41(Suppl I), 35^4. URIST, M.R. (1994). The search for and the discovery of bone morphognetic protein (BMP). In: Bone Grafts, Derivatives and Substitutes, M.R. Urist, B. O'Connor, R. Burwell, eds., ButterworthHeinzmann, Oxford, England, pp 315-362.

125

WINKLER, H. and GEORGOPOULOS, A. (1997). Bone grafts as a delivery system for antibiotics. First experience with vancomycin chips. 6th Annual Meeting of EAMST, Barcelona, pp 31-32. WOZNEY, J.M., ROSEN, V. and CELESTE, A.J. (1988). Novel regulators of bone formation: Moleculor clones and activities, Science, 242, 1528-1532.

7 PRESERVED ALLOGENIC RIB CARTILAGE IN RECONSTRUCTIVE SURGERY

D. SLADOWSKI, A. KOMENDER and J. KOMENDER Department of Transplantology Warsaw Medical University, Poland H. MALCZEWSKA Department of Histology & Embryology Warsaw Medical University

1. Introduction Despite the impressive developments in tissue engineering, rib cartilage is still frequently used for reconstructions in a face region (Komender et ah, 1986; Kryst, 1981; Meeuwsen and De Vries, 1996; Sailer, 1983; Thomassin, 2001) and for other clinical procedures (Tomford et al., 1996). Transplants of allogenic cartilage are prepared in tissue banks from sterilised rib cartilage obtained from cadaveric donors (Komender and Komender, 1977). This material offers excellent physical properties enabling to obtain desired shape of implant in a relatively easy way. Graft might be carved from single cartilage, or might be composed from several elements glued together. In the opposite to living cartilage, preserved cartilage do not distort so often after transplantation. It offers long term support for soft tissues with slow degradation rate which usually does not occur within first four years after

127

128

transplantation. The other advantage of preserved grafts is avoidance of additional trauma inflicted during autologus tissue retrieval. Since 1963 (establishment of the Central Tissue Bank in Warsaw) more than 2500 grafts prepared in the Central Tissue Bank in Warsaw have been used in the reconstructive surgery. This vast clinical material allows clinical evaluation of the material in long time period after surgery. Our assessment was based on 440 complete, validated reports of reconstructive surgery performed in 11 surgical wards with the use of radiation-sterilised cartilage material. The average duration of illness before transplantation was 107.3 months with SD = 102.3. Time of hospitalisation was 23.5 days (SD = 15.2). Interval of time between surgery and estimation of the result of treatment was 98.2 months with SD = 46.0. The result of the treatment was estimated according to a four-grade scale: very good, satisfactory, difficult to estimate and unsatisfactory. It was found that 33.5% of all analysed patients achieved very good results of treatment, in 41.8% of the patients the result was satisfactory and in 19.9% failures were found. In approximately 4.1% of the patients, the result could not be classified in a conclusive way. From the vast array of factors which might affect the results of the reconstructive surgery conducted with the use of cartilage material, several most important have been identified for analysis. The following variables were analysed: age, sex, diagnosis, location, duration of illness, the time of hospitalisation, general complications, local complications, reoperations, use of antibiotics, form of grafts (single fragment, several fragments, with patients own tissue), early estimated result after hospitalisation, interval between surgery and control examination, concomitant diseases, result of treatment, estimation of graft resorption and estimation of the role of the graft. Evaluation of the results was performed after one-year observation period. Obtained results indicate that in more than 75% of the cases, positive results of treatment may be achieved, however it must be remembered that the final outcome of the treatment depends on several factors which should be taken into consideration by the surgeon performing reconstructive surgery.

129

2. Material The material prepared in The Central Tissue Bank in Warsaw is obtained from 18-55 years old donors, both sexes, excised 24 hours after death and preserved using the standard operating procedure. After removal of perichondrium, tissue is stabilised in 70% ethanol for 4 hours, then washed in 0.9% NaCl for 24 hours and immersed in a 0.9% NaCl solution. Closed vials with grafts are radiationsterilised with a dose of 33 kGy, in gamma source. After sterility control, grafts are registered and sent to the surgical wards together with the appropriate questionnaire which should be returned back to the Tissue Bank for the final evaluation of graft performance. Cartilage is usually transplanted mainly as a single fragment (56.4%), or two or three fragments (41.4%). Only occasionally preserved material is combined with autologus tissue (2.0%). Number of fragments has no effect on the final outcome of the surgery, but it must be remembered that there is a need for graft prepared and packed in a way facilitating the use of multiple fragments. Our data indicate that cartilage is usually used in the posttraumatic surgery (47.5% of cases in our material) in the group of patients in their second (26.9%) and third (39.4%) decade of their life. Age distribution of patients from the other age groups are similar up to 50 years of age (7.7% of the patients were in the first decade, 10.9% in the fourth, 9.8% in the fifth, 5.3% of the patients were over fifty years of age) (Table 1). The sex distribution is similar in all age groups. The other areas of the use of cartilage are congenital deformations (28.9%), unspecific inflammations (16.8%), postoperative malformations (3.0%), specific inflammations (1.8%), malignant tumours (1.4%) and benign tumours (0.5%). One of the most important factors affecting final results of the treatment is the age of the recipient. Successful outcome of the surgery increases with age, ranging from 70% in case of young patients (0-20 years) to more than 90% in case of patients older than 50 years (Fig. 1). This reflects age dependent decrease in resorption rate. In the most frequently reported age group (1030 years) successful treatment can be expected in more than 75% of

130

Table 1. Results of cartilag e transplantatio n in various age groups. Age m

years

Result of treatment Very good

Satisfactory

Doubtful

Unsatisfactory

Total

n

%

n

%

n

%

n

%

n

27.3 30.5 33.5 35.4 54.8 21.7

15 46 77 19 12 15

45.5 39.0 44.5 39.6 28.6 65.2

0 4 7 4 1 2

0.0 3.4 4.0 8.3 2.4 8.7

9 32 31 8 6 1

27.3 27.1 17.9 16.7 14.3

33 118 173 48 42

>5 1

9 36 58 17 23 5

4.3

23

10 5

Total

148

33.9

184

42.1

18

4.1

87

19.9

437

100

0-10 11-20 21-30 31-40 41-50

% 8

27 40

11

Fig. 1. Relation between age and success of the treatment with cartilag e graft.

131

Fig. 2. Age dependent distribution of results.

cases. The fourth and fifth decades of life seem to be preferable for cartilage transplantation when unsatisfactory results are almost not existing (Fig. 2).

3. Observation After surgery some local changes can be observed. The most common-oedema occurs in more than half cases (65.8%) and is not associated with the results of the surgery (Fig. 3). Other local changes are less frequent (purulence 5.9%, infiltration 2.5%, accelerated resorption of grafts 0.5%). It is interesting that in one fourth of all cases no oedema or other local changes after surgery can be observed (25.3%). Preserved cartilage is predominantly used in nose surgery (more than a half of all reported cases 58.4%, reconstruction of ear (16.6%), and correction of mandible (11.1%) (Table 2). Unfortunately, in a long term observation (more than 7 years), the results of the nose reconstruction are usually hampered by the young age of the patients which usually undergo this kind of surgery. In this age

132

Fig. 3. Local changes observed after transplantation are not prognostic (except accelerated resorption). Table 2. Location of transplanted cartilage and results of reconstructions. Result o f Treatment

Localisation Very good

Satisfactory

Doubtful

Unsatisfactory

Total

n

%

n

%

n

%

n

%

n

%

Nose Ear concha Mandible Maxilla Orbit Front

75 3 30 7 8 5

29.2 17.8 63.8 56.7 50.0 38.5

119 33 10 11 6 4

46.3 45.2 21.3 36.7 37.5 30.8

10 1 5 0 1 1

3.9 1.4

20.6 35.6

0.0 6.3 7.7

53 26 2 2 1 3

23.1

257 73 47 30 16 13

59 17 11 7 4 3

Total

48

33.9

183

42.0

18

4.1

87

20.0

436

100

10.6

4.3 6.7 6.3

133

Fig. 4. Nose, age dependent distribution of results.

group (10-30 years) complete resorption of the transplanted material occurs in almost 30% of cases. There are only around 50% chances of obtaining positive results (58% in our material) (Fig. 4). The final result of the treatment indicates that cartilage grafts are the most suitable for mandible corrections, where unsatisfactory results are observed very seldom (4.3%). The use of autologus bone obtained from iliac crest seems less attractive as failure can reach 24% (Bahat O, Fontanessi, 2001). In our material we observed over 63% of all "very good" results in case of patients with mandible reconstructions. Cartilage is transplanted to mandible not only to improve the shape of the bone but also to enhance the regeneration of the bone in the alveolar process. Similar distribution of the results of treatment was found in the group of patients with cartilage transplanted into maxilla. In case of nose surgery, the unsuccessful outcome of the treatment can be expected more frequently. Unsatisfactory results can be expected in almost 20% of all cases. In most cases, failure is caused by degradation and resorption of transplanted material. It must be stated however, that up to now, no other satisfactory treatment has been developed and

134

even in case of graft resorption, the surgery can be performed once again. (In control examinations, full resorption of grafts was seen in 23.0% of the patients and limited resorption in 9.3% of the patients). It must be also remembered that resorption usually does not occur during first four years after transplantation. Our research conducted on the problem of cartilage graft resorption resulted in creation of a new preservation procedure which slows down cartilage degradation (Pawlowski et al., 1986) and we hope to observe more favourable results in the future. The most complicated surgery conducted with the use of preserved cartilage is reconstruction of the ear concha. Despite intensive resorption at this location, cartilage is quite frequently used (16.6% of all cases). Most of the "unsatisfactory results" can be expected after reconstruction in this location (35.6%) but still 63% of the results of treatment of patients are successful ("very good" and "satisfactory" together). The use of this material for ear

Fig. 5. Age and results of auricular concha reconstruction.

135

reconstruction seems to be very promising in case of older patients (> 60 years) when no unsatisfactory results have been observed (Fig. 5). From other locations less frequently reported, it must be stated that the use of preserved cartilage can be advocated as a material of choice in case of reconstructive surgery of maxilla and orbit where unsatisfactory results are very infrequent (6.7% and 6.3% respectively). The incidence of "unsatisfactory results" of treatment in children and young patients exceeded 27%. The tissues of children and young patients seem to react strongly to cartilage grafts, which is not observed in older age groups. It is interesting that over 50% of all the "unsatisfactory results" can be expected in the group of patients that were operated for posttraumatic malformations. The results of treatment in this group were found significantly worse than that of the others. Table 3. Results of cartilage transplantation in various diagnoses. Diagnoses

Result of treatment Very good

Satisfactory

Doubtful

Unsatisfactory

Total

n

%

n

%

n

%

n

%

n

%

Traumas

65

31.1

91

43.5

8

30.8

45

21.5

209

62

Congenital changes

36

28.3

52

40.9

4

30.1

35

27.6

127

8

Unspecific inflammations

34

46.6

29

39.7

5

60.8

5

6.8

73

22

Postoperative deformations

8

1.5

4

0.8

0

0.0

1

7.7

13

4

Specific inflammations

2

5.0

5

5.2

0

0.0

1

12.5

8

2

Tumours

3

2.9

2

8 .6

1

4.3

1

14.3

7

2

48

3.9

83

1 .9

18

0.1

88

20.1

437

100

Total

136

Transplantation of cartilage in "congenital malformations" gives even less favourable results: "very good" and "satisfactory" together 69.2% and "unsatisfactory" 27.6%. Transplantation of preserved cartilage in "unspecific inflammations" and "postoperative malformations" seems to be very effective (nearly 90% of positive results of surgery). The groups of "specific inflammations" and "tumours", however, not so numerous, presented a high percentage of positive results of treatment which indicates that the use of preserved cartilage should be advocated in this kind of treatment (Table 3). Preserved costal, allogenic cartilage is the proper material for use in reconstructive surgery of the face. More than 33% of all operations were completed with full clinical success. In 42.0% of all operations, the results were found to be "satisfactory", which means that positive results should be expected in 75% of treated patients. It is also true that in nearly 20% of the patients, the result of treatment was "unsatisfactory" which means that the facial reconstructions were unsuccessful. This requires some additional explanation. If the successful transplantation of bone depends on the process of grafts substitution by regenerating the patient's own bone (creeping substitution), then the clinical of cartilage transplantation depends on a stable state of transplant in years (Komender, 1986; Kryst, 1981, Pawlowski, 1986). It often happens that for some unknown reasons, cartilage grafts are resorbed quickly. In our material the rapid resorption was found in 23% of all cases, while accelerated resorption was seen several days after transplantation in two patients (0.5% of the cases). 4. Conclusion Costal, allogenic, preserved cartilage is often used for reconstruction of malformations in the region of the face. The examination of patients between 1 to 17 years after surgery, reveals positive results of treatment in 75% of cases. Unsatisfactory results of transplantation (19.9% in the whole group) are correlated mainly with younger patients, congenital or post-traumatic malformations and location in ear concha.

137

5. References BAHAT, O. and FONTANESSI, R.V. (2001). Efficacy of implant placement after bone grafting for three-dimensional reconstruction of the posterior jaw, Int. }. Periodontics Restorative Dent. 21, 220-231. KOMENDER, J. and KOMENDER, A. (1977). Evaluation of radiationsterilized tissue in clinical use. In: Sterilization of Medical Products by Ionising Radiation, E.R.L. Gaughran and A.J. Goudie, eds., Multisc Publ. Ltd., Montreal, p. 188. KOMENDER, J., MALCZEWSKA, H. and KOMENDER, A. (1991). Therapeutic effects of transplantation of lyophilised and radiation-sterilised, allogeneic bone, Clin. Orthop. 272, 38-49. KOMENDER, J., MALCZEWSKA, H. and PAWLOWSKI, A. (1986). Preserved allogenic cartilage in reconstructive surgery, Probl. Haematol. Transfusiol. Transpl. 13, 288-293. KRYST, L. (1981). Przeszczepianie tkanek w chirurgii szczekowotwarzowej. In: Przeszczepy Biostatyczne, J. Komender, ed., PZWL, Warsaw, Vol. II, pp. 151-161. MEEUWSEN, F. and DE VRIES, PH.A. (1996). Preservation of human costal cartilage for transplants in nasal surgery. In: 4th International Conference European Association of Tissue Banks, Byk Jr. Chr, A. Lechat and R. von Versen, eds., Monduzzi Editore Bologna, pp. 79-82. PAWLOWSKI, A., MALEJCZYK, J., SLUBOWSKI, T., SLADOWSKI, D. and MOSKALEWSKI, S. (1986). Arrested resorption of costal cartilage grafts subjected to hydrochloric acid in rats, Otolaryng. Pol. 40, 25. SAILER, H.F. (1983). Transplantation of lyophilized cartilage in maxillo-facial surgery. In: Experimental Foundations and Clinical Success, Karger, Basel-New York, p. 178. THOMASSIN, J.M., PARIS, J. and RICHARD-VITTON, T. (2001). Management and aesthetic results of support grafts in saddle nose surgery, Aesthetic. Plast. Surg. 25, 332-337.

138

TOMFORD, W.W., OHLENDORF, C. and MANKIN, H.J. (1996). Articular cartilage cryopreservation and transplantation. In: Orthopaedic Allograft Surgery, A. Czitrom and H. Winkler, eds., Springer, Wien-New York, pp. 269-274.

8

BONE SUBSTITUTES AND RELATED MATERIALS IN CLINICAL ORTHOPAEDICS

A.J. AHO & J.T. HEIKKILA Department of Surgery The Turku University Central Hospital The Biomaterial Project, University of Turku Turku, Finland

1. Introduction Bone substitutes have been studied for more than 100 years, but the clinical need for them has rapidly increased during the last 30 years due to revision surgery after total hip replacements (THR, Charnley, 1960) and limb salvage surgery for bone tumours (Imamaliev, 1969; Ottolenghi, 1982; Parrish, 1966). In these operations, large quantities of bone is needed, exceeding the amount of autogenous bone available. The developments within anesthesiology has also made large, more demanding reconstructive orthopaedic operations possible. A bone substitute material, bovine bone, decalcified by muriatic acid treatment, has already been used to fill small bone defects 100 years ago (Senn, 1889). At about the same time, Macewen (1881) performed the first massive bone allograft operation using another kind of bone substitute material, allogenic bone, for the treatment of osteomyelitic bone defect in the humerus.

139

140

The original approach was to select materials which are as inert as possible, but later bioactive materials with controlled reactivity were tailored to be used as bone substitutes. The original approach has been transferred from stainless steel towards materials such as hydroxyapatite and bioactive glass. The ideal bone substitute should be: (1) non-toxic; (2) biocompatible; (3) able to support the loads subjected on the original bone; (4) bioactive; (5) osteoinductive-osteoconductive; (6) allow new bone ingrowth or ongrowth; (7) disappear with the same speed as the new bone growth occurs; (8) close to biomechanical values of the natural bones; (9) easy to handle; and (10) moldable or shapeable preoperatively. The bone substitutes can be grouped according to various methods, but the main groups are: (1) calcium phosphates; (2) calcium Table 1. List of bone substitutes. 1. Calcium phosphates Hydroxyapatites, HA • Bone (bovine)-derived • Synthetic ceramics • Coralline HA — Porites, Goniopora • HA-composites • Tricalcium phosphates, TCP 2. Calcium carbonates Natural coral 3. Calcium sulphate — Plaster of Paris 4. Glass and glass-ceramics 5. Polymers 6. Metals 7. Bone and bone-derived materials • Autograft, allograft (bank bone), xenograft • Demineralised bone matrix (DBM) 8. Osteoinductive growth factors • BMPs • TGFP-family

141

carbonates; (3) calcium sulphate; (4) glass and glass-ceramics; (5-6) artificial materials, such as metals and polymers; (7) bone and bonederived materials; and (8) osteoinductive growth factors (Table 1). Hydroxyapatites (HA) are the main constituents of bone (65%). Two basic approaches exist in the development of HA-materials to be used instead of bone: first, to remove organic phases from the bone by different chemical and physical methods, and second, to sinter inorganic materials into calcium ceramics. 2. Calcium Phosphates 2.1. Calcium phosphates of biologic origin, bone-derived, bone apatite Deproteinised bovine bone After the original experiments with decalcifying effect by Senn, various methods have been used to deproteinise bone. Before the 1st World War, Orell (1937) already produced a bone substitute, Os purum, by soaking bovine bone in warm potassium hydroxide to remove antigenic proteins and fats. Other deproteinised bone substitutes were Kiel bone, anorganic bone, Oswestry bone, (Table 2) marketed today as macroporous Bio-Oss® and Endobone®. In general, they all possessed some beneficial properties, such as low inflammatory reaction and normally good appositional bone formation. The disadvantages included slow and inconstant resorption and osteogenic properties (Burwell, 1969), and they could not be used to bridge defects. Also, their manufacturing was troublesome. However, modern technical sintered modifications of these kind of bone-derived substitutes are presently in clinical use mainly in German-speaking Europe as Pyrost® (Mittelmaier and Katthagen, 1983), Osteograf® (Coramed) and Bon Ap. A certain kind of inorganic bone, Ossar®, was used in our institution during the 1960s. Good biocompatibility and new bone incorporation was observed both in experimental and clinical studies (Viikari and Aho, 1963; Fig. 1).

142 Table 2. Bone substitutes prepared by removing proteins and other components from bovine bone; calcium phosphates of biological origin.* Author

Preparation

Os purum®

Orell (1937) Orell (1953)

Soaking in warm KOH, acetone

Some residual collagen

Cavity filling in Sweden in the 1930s-1940s

Kiel bone®

Maatz and Baumeister (1957) Hallen (1966) Salama (1983)

H2O2 maceration, acetone

Deproteinisation partial

Cavity filling, non-union, good results in 62-84%

Anorganic bone (Ossar®)

Williams and EthyleneIrvine (1954) diamine Hurley (1958) extraction Viikari and Aho (1963) Kramer (1964)

Deproteinisation partial

Cavity filling; good results in over 80%

Oswestry bone

Roaf and Hancet (1963) Kramer et al (1966)

H2O2 ethylenediamine extraction

Fully deproteinisation, bone conducting

Cavity filling, spinal fusion, expander of autograph

Pyrost®

Mittelmaier and Katthagen (1983)

Gentle burning, sintering

Totally deproteinised, ceramic like, sintered, crystalline structure

Pathological fractures, operative bone defects

Name of bone substitute

Properties

Clinical use

*Other materials are marketed as Bio-Oss®, Endobone®, Osteograf®, BonAp (HiMed)

143

Fig. 1. (A) Cavity bone defect in the proximal tibia (dog) filled with particulate anorganic bone (Ossar®, Turku, Finland). Good incorporation and new bone format.on by trabeculous bone (B) at three, and by lamellar bone at six months van Gieson stain (magnified: 330x).

Synthetic ceramic calcium phosphates/hydroxyapatites (HA) Albee and Morrison (1920) were the first to report good clinical results with regard to the use of a synthetic calcium phosphate salt, triple calcium phosphate (TCP). In the 1950s it was revealed that the main component of bone resembled hydroxyapatite. But only in the mid70s Jarcho (1976), Denissen (1979), Aoki (Aoki et al, 1977) and deGroot (1980), at about the same time but independently, were able to produce synthetic hydroxyapatite. Jarcho (1976) was first to show chemical bonding of bone with hydroxyapatite (Fig. 2). It has since been used as dense and porous implants. The team apatite includes a family of

144

;

<

•

*

.

•

.

-

.

,-.'-•

Fig. 2. SEM picture illustrating bone bonding of hydroxyapatite cone (HA) and host bone (arrow) without intervening fibrous tissue. Bone trabeculae BT.

compounds having similar structure but not identical compositions. Calcium hydroxyapatite has a definite composition Ca10(PO4)6(OH2)2 with a stoichiometric Ca/P ratio of 1.67 corresponding bone tissue and belongs to the hexagonal system (LeGeros and LeGeros, 1993). Biological apatites are usually carbonate substituted. Coralline HA and bone-derived CaP apatites differ from biological apatites due to their crystallinity, composition and reactivity. In the biological surroundings on the surface of HA as well as on bioactive glass and glass-ceramics, a carbonated hydroxyapatite layer is formed, promoting the adhesion of matrix-producing cells and organic molecules as a result of surface charges (LeGeros and LeGeros, 1993). These reactions led to the bone-bonding of the materials (Jarcho et al, 1977; Daculsi, 1990a). They are osteoconductive, able to guide bone formation on their surface when implanted in a bony environment. These materials have also shown good biocompatibility. Their disadvantages are brittleness and low resistance against fatigue fractures (deGroot et al, 1987), and particle migration. The increased porosity and proportion of TCP decreases the compressive strength. Thus, calcium phosphates are not suitable for mechanical

145

Table 3. Calcium phosphate hydroxyapatite, HA, calcium carbonate and calcium sulphate used as clinical bone substitutes in the 1970s-1980s. Filler effect

Spinal Local Coating for Segment fusion fracture prostheses replacement injections (THR*)

TCP with bone marrow

++

+

Deproteinised bone

++

+

Bone-derived calcium phosphates

++

+

Synthetic calcium phosphates, HA

++

+

HA + TCP

++

++

Natural coral

++

Calcium sulphate

++

+

+ +

+ +

+

promising in experimental studies, clinical data needed "'"'"reliable clinical results *THR = total hip replacement

loading. Good results, on the other hand, have been reported of their use as filling for bone defects. Clinical use. In clinical applications, the dental and craniomaxillofacial applications were reported first (Boretos, 1987; Kent et al, 1986) (Table 3). Ceros 80®, Calcitec® (dense) and Bioroc® (microporous) are some of the commercial materials on the market at the moment. In orthopaedics, good results have been reported when porous HA was used to fill moderate-sized defects in long bones after tumour excision (Uchida et al, 1990; Inoue et al, 1992). HA has also been used in spinal fusions (Koyama and Handa, 1986).

146

Fig. 3. A diagram illustrating the structural parts of a metal (Cobalt-Cram, Titanium, stainless steel) prosthesis for total hip replacement. The proximal part of the stem can be coated with hydroxyapatite or bioactive glass. The prosthesis can be applied with or without bone cement (methylmetacrylate) fixation between metal and bone. Socket is used for acetabular fixation.

HA-coating. The beneficial role of hydroxyapatite as coating on porous metal implants (Fig. 3) was shown by Ducheyne et al (1980). The HA on the surface of the implant was found to be incorporated with the host bone without fibrous tissue interposition (Cook et al, 1988). Early clinical results — one two-year follow-up — have indicated less subsidence of the HA-coated prosthesis (Karrholm et al, 1994; Kroon and Freeman, 1992). The clinical trials using THR's coated with 50 \x of HA indicated at six years a 100% survival rate with HA-coated prosthesis (Geesink, 1990; Geesink and Hoefnagels, 1995). However, a randomised control study in patients with primary THR's did not show an advantage of HA-coated prostheses with a two-year follow-up (Rothman et al, 1996). This might be due to delamination, solubility and resorption of HA in the biological surroundings and by the osteoclasts. On the other hand, canine experiments indicated enhanced bone ingrowth with HA-coated

147

implants even in the presence of osteopenic knee bone (S0balle/1993). Coating methods utilising other materials such as glass, glass ceramics, and metals are discussed in the chapters related to the matter. HA-composites. Because the use of particulate HA-material has some drawbacks, such as difficult handling properties, migration of the particles, brittleness, and resorption of the material, composite material has been created using modern technology. A multitude of experimental studies on HA-composites has been made, combining HA with collagen (Collagraft, USA; Collapat®, Mittelmeier and Katthagen, 1983), fibrin, polymethylmetacrylate and polylactic acid. In these composite materials the non-resorbable matrices may decrease the area of ceramics available to bone contact, cause toxic effects (PMMA) on bone healing (Heikkila et al, 1996) and obstruct pores of the material, e.g. when using corals (Tencer et al, 1987). However, potential for biomedical application of these composite materials exists, particularly with regard to their biomedical properties, e.g. apatite-wollastonite glass ceramics — polyethylene composites which have shown higher microhardness and Young's modulus (Wang et al, 1996; Bonfield, 1996). Also, a composite material formed of sintered HA-particles and PLLA showed a significantly increased mechanical strength up to 120 MPa (Shikinami et al, 1996). These composite materials seem to reduce the mechanical weakness of Ca-P biomaterials on an experimental level. A good clinical example of composite material application was a successful treatment of a large bone defect in the human tibia with a composition of HA with bioactive glass, resulting in a pronounced remodelling of the cortical bone during the seven-year follow-up (Aho et al, in press). 2.2. Tricalcium phosphates, TCP Macroporous biphasic calcium phosphate is a material which combines the osteoconduction and the resorption property of HA with the more rapid resorption of TCP. The combination (60% HA, 40% TCP) has been applied to scoliotic patients during spinal fusion operations

148

(Passuti et al, 1989), as filler material after bone tumour resection (Daculsi et al, 1990b), and for the treatment of tibial fractures (Suzuki et al, 1994). A tight bone contact and good stabilisation of the implant material was observed. BCP is a synthetic composition of HA and (3-TCP. Mixing autogenous bone with the biphasic calcium phosphate seems to increase the beneficial effect of the implant material (Moore et al, 1987). In general, the use of calcium phosphates has recently been at a low level clinically. 2.3. Coralline HA One of the interesting materials that has been developed during recent years is the coral-derived HA. The preparation was published by Roy and Linnehan (1974), who developed a method for the processing of hydroxyapatite in the skeletons of Porites and Goniopora corals. Coralline calcium carbonate is transformed into hydroxyapatite using hydrothermal reaction in elevated pressure and aqueous NH 4 phosphate solution. The three-dimensional structure of the resulting HA resembles that of cortical or cancellous bone with pore sites ranging from 230-600 \i (Bucholz et al, 1987). This material has been used to reconstruct traumatic bone defects (Bucholz et al, 1987; Holmes et al, 1986) and in plastic surgery. The composition of coralline HA and fJ-TCP is marketed as macroporous Interpore. 2.4. Natural coral — calcium carbonate The other coral-based bone substitute material is calcium carbonate of the natural coral (Biocoral®, aragonite, macroporous). It has been reported to be enzymatrically changed into calcium phosphate of bone. The biological function of carboanhydrase changes the coral calcium carbonate into hydroxyapatite in the surrounding bone. Good experimental and clinical results have been reported. Direct osteoblastic new bone apposition has been observed with corals. Due to its good osteoconductive properties, the natural coral cylinder has been successfully studied in experimental bone substitute in the

149

healing of segmental defects in weight-bearing bone in sheep (Gao et al, 1995; Gao, 1996). However, its suitability to bridge larger defects and its resorption rate need more studies because its compressive strength is low compared to cortical bone, 4.3-9.3 MPa versus 131 MPa (Piecuch et al, 1984). Clinically, Biocoral® has been proven to be a suitable filling material for small defects in craniofacial surgery (Roux et al, 1988), spinal fusion (Pouliquen et al, 1989), and more widely in orthopaedics (Loty et al, 1990). Clinical trials are needed for more accurate evaluations. Biocoral® has been experimentally used as carrier for bone morphogenetic protein to enhance the repair of an experimental segmental tibial defect (Gao, 1996). 2.5. Injectable materials Injectable TCP was already tested in experimental fractures in the 1930s (Murray, 1931; Haldeman and Moore, 1934). A control investigation was performed by Niwa et al (1980), they showed superior results while using hydroxyapatite in comparison to TCP. Recently, an injectable coral-derived calcium phosphate material (Norian SRS) has also been introduced (Constanz et al, 1995). This material resembles the chemical structure of coral and is resorbable. It has been experimentally used to treat metaphyseal fractures, and this technique can also be applied for osteoporotic Colles fracture. Clinical tests are now being made in USA and Europe. Injectable bioactive glass has also been developed by the Biomaterial Project of Turku (Brink et al, 1996). Regarding fracture treatment also composite glass biomaterials are in progress. 2.6. Calcium sulphate, plaster of Paris The hemihydrate form of calcium sulphate (CaSO4H2O) with water results in irregularly-shaped crystals. In the beginning of this century, bone defects (Dreesman, 1893; Peltier et al, 1957) were treated with acceptable results, Peltier et al (1957) even observed clinically better

150

bone regeneration than seen with autografts. However, calcium sulfate does not give structural support, it is biocompatible, brittle and resorbable, but not osteoinductive. It might be used as a carrier for antiseptic materials and antibiotics and perhaps also for growth factors, BMPs. A novel modification of calcium sulphate used as a bone filler and "manufactured by proprietary process with purity and consistency in mind", Osteoset® is marketed in the USA. 3. Bioactive Glass and Glass Ceramics Boactive glass is composed mainly of Na2O, CaO, SiO2 and P2O5. Bioactive glass was first introduced by Hench in 1967 (Hench et al, 1971). The first bioactive glass, Bioglass®, has a silica content of 46% by weight. Several glass ceramic materials were developed later in the 1980s in Japan (A-WGC, Cerabone®), Germany (Ceravital®) and

Fig. 4. SEM picture illustrating bone bonding at the interface (IF) between bioactive glass (BG; S53P4) and host bone (B).

151

Finland (e.g. S53P4). These materials are biocompatible, bioactive and have a direct chemical and tissue contact between the glass surface and host bone without an intermediate layer of connective tissue (Fig. 4). They are produced by melting the constituent oxides at 1300-1400°C and then by controlled cooling. After implantation, a complex series of biological and physiochemical reactions occur at the interphase. Based on pH changes on the glass surface, leaching and dissolution occurs and a Si-rich layer is formed at the glass surface. A carbonated calcium phosphate layer forms on it by precipitation and supersaturation (Karlsson et al, 1989; Ducheyne et al, 1992; Andersson, 1990). The carbonated calcium phosphate on the surface of the glass is responsible for bone bonding (Fig. 5). The ultrastructure of the interphase consists morphologically of a laminar structure of fine granular material with several sublayers indicating the presence of proteins, calcium salts and acid proteoglycans (Aho et al, 1993, 1996). Compositionally, the silica content of the glass should range between 45% and 53% for

Fig. 5. Histological picture illustrating bone contact and bone bonding (arrows) between bioactive glass granule (S53P4) and new bone (NB). Z = reaction zone between glass (BG) and bone.

152

the glass to be bioactive. Glass with a Si content of more than 60% does not form a Ca-P layer, and is not bioactive. The advantage of bioactive glass is the adjustability of its surface reaction when changing the composition. Layered composites can be produced by special techniques. The CaP-surface layer is formed in situ and closely resembles that of bone. The disadvantages include mechanical weakness and low fracture toughness. However, the modulus of elasticity of 30-55 MPa is similar to that of the cortical bone's 30-85 MPa. Clinical praxis. Clinical use of bioactive glass was started in the 1980s, first in dental applications as alveolar ridge maintenance devices and middle ear implants in otorhinolaryngology, and later in a particulate form (Perioglass®) in paradontology (Wilson et al, 1994) (Table 4). Glass ceramics (Ceravital®, Gross et al, 1993; Bioverit®, Vogel et al, 1990) have also been used as middle ear implants. In Japan, apatite woUastonite glass ceramics (Cerabone®) was developed for orthopaedic purposes (Kokubo et al, 1985; Yamamuro et al, 1988). It Table 4. Clinical studies of bioactive glass and glass-ceramics in clinical maxillofacial, dental and orthopaedic surgery. Author Hench (1996) Wilson

Trademarks Bioglass® 45S5 Perioglass® Biogran®

Maxillofacial, otorhinological, dental surgery, ear ossicles, alveolar ridge, periodontal defect, pulp capping, sinus lift Coating for artificial dental root

Kudo et al (1990) Yamamuro et al (1990)

Speciality, anatomic site

Cerabone® A-WGC

Filling of bone tumour cyst cavities, iliac crest prostheses, spine intervertebral prostheses

153

Table 4. (Cont'd) Trademarks

Author

Speciality, anatomic site Spine anterior vertebra reconstruction with Kaneda device

Asano et al (1992) Kawanabe et al (1993)

BIS-GMA (Resin + CaO-SiO2-P2O5 glass powder)

bone cement in clinical (?) use in Japan

Heikkila et al (1995, 1996)

S53P4 Bioactive glass

Cavity filling of tumour surgery. Tibial condyle fractures — substitution of depressed bone fragment

Aho et al (1997)

bioactive glass-Ha composite mixture

Large cavity substitution of the tibia due to fibrous dysplasia

Aitasalo et al (1994)

S53P4

Filler, obliteration of frontal sinus

Suominen and Kinnunen (1996)

S53P4

Plastic surgical reconstruction of facial and orbital bones

Turunen et al (1996)

S53P4

Sinus lift operations — alveolar ridge

has been used as a filler in cavity bone defects (Yamamuro et al, 1990), as vertebral prostheses because of its high strength properties (Yamamuro et al, 1990; Asano et al, 1992; Shimizu et al, 1992), and as block implants for glenoplasty (Sedel et al, 1992). Bioactive glass has also been used as coating material for dental root implant (Kudo et al, 1990). AW glass ceramic coated hip prostheses for dogs have been developed in Japan; however, reports on its clinical application

154

Fig. 6. Curves indicating new bone growth at the interface between lines HA and bioactive glass in the distal subchondral bone of rabbit. No significant difference was found at six and twelve months.

are not yet available. The incorporation with surrounding bone contacts of the bioactive glass and HA are comparable (Fig. 6). In Turku, S53P4, the bioactive glass used recently has been developed and tested by the Turku Biomaterial Project (Andersson and Karlsson, 1988; Andersson, 1990) in the 1980s, and is in clinical use. The first randomised prospective series using S53P4 granules with autogenous bone as cavity filler was started in 1993 (Fig. 5). Bone formation and fibrovascular tissue growth between the granules has been observed (Heikkiia et al, 1995; Suominen, 1996). The same bioactive glass has also been applied in the treatment of depressed tibial condyle fractures (Fig. 7), and clinical series for spinal posterior fusion (Fig. 8) is under way in patients with intervertebral instability and unstable vertebral fractures. In otorhinolaryngology, chronic frontal sinusitis have also been treated successfully with S53P4 granules (Aitasalo et al, 1994, 1997). In plastic surgery it has been used to reconstruct, e.g. orbital floor after blow-out fractures (Suominen and Kinnunen, 1996).

155

Fig. 7. Radiographs illustrating a depressed condylar fracture of the proximal tibia in a 76-year old patient. The condyle was elevated and bioactive glass was used as bone filler support. (A) Before operation, (B) postoperative with 1.5 years followup. In the prospective clinical series, autografted patients serve as the controls.

In the near future, composite materials with bioactive glass will be developed and tested to increase the handling properties and biomechanical strength of S53P4.

4. Polymers Degradable polyglycol acid (PGA) and polylactic acid (PLLA) have been developed for bone fixation purposes (Rokkanen, 1991). Good clinical results have been obtained using these rods and screws, as published in many papers. These pin-like shaped devices have been used for fixation of small bone fragments in human beings (Bdstman et al, 1989; Partio, 1992), and they are biocompatible thus allowing the bone healing. The degradation of these implants occurs from three months to several years, depending on the polymer. Material-related complications are relatively few, they are limited to

156

Fig. 8. Diagram and clinical model of posterolateral fusion illustration applying bioactive glass as bone substitute.

infections to a low degree and sinus formation, resulting from too rapid degradation. It has been suggested and recently experimentally tested that a suitable bone substitute might be PGA/PLLA coated with HA or bioactive glass. The idea of the function strategy is that the HA or bioactive glass will give the immediate bone contact and the polymer will resorb with time, thus allowing physiological bone healing. Polyethylene (PE), polyacetal (PCM) and polysulfon (PS) have been used in connection with metal prosthesis for friction decrease, e.g. as acetabular prosthesis socket material (Fig. 3) and polypropan (PP) and silicon for finger prosthesis for over twenty years. Polyacetal hip prosthesis (Mathys) stem with a steel core has not been found to

157

present a more acceptable loosening rate than achieved by other prostheses (Niinimaki et al, 199A). A composite of PE with calcium phosphate ceramics (Hapex®) has been developed at the beginning of the 1980s. Recently, a composite material combining PE and bioactive A-W GC (Bonfield, 1996) has been introduced and suggested for clinical use. As can be seen, a very active world-wide research is focussed particularly on polymer investigation. Some synthetic bioactive polymers with bone bonding capacity have also been developed. The best documented one is a copolymer named Polyactive® comprising of polyethylene oxide and polybutylene trephthalate (PEO/BTF). These polymers presumably allow calcification inside the hydrogel resulting in bone bonding (Ikada, 1996).

4.1. Polymethylmetacrylates (PMMA) and cements The medical use of polymethylmetacrylates, methylesters of the methacrylate acid, as so-called bone cements was started in the 1950s after the development of self-curing or cold-curing polymethylmetacrylate. Because it is not biocompatible, has toxic effects during the implantation procedure and is not bone conducting, several experiments to develop bioactive cements have been made during recent years processing glass ceramics (Kokubo et al, 1991) and glass ionomers (Hatton and Brook, 1992). Ca-P cements have a compressive strength between 10 and 40 MPa and are thus indicated only for replacement of cancellous bone defects and are not useful for implant fixation because of the poor mechanical strength. A promising bone cement combining polymethylmetacrylate with hydroxyapatite powders has been developed (Oonishi et al, 1989). Also, a combination of Bis-GMA and A-W glass (Nakamura, 1996) ceramics powder is in experimental use in Japan. This cement is bone bonding and has higher mechanical properties than PMMA cement. At present, other modifications of bioresorbable calcium phosphate together with polymers are at an experimental level.

158

5. Metals Many metals (stainless steel, Cr, Co, Ni, Al, Mo, V, Ti) and their alloys, such as Co-Cr (Vitallium), have been used for joint prostheses and fixation plates in orthopaedics (Fig. 3). The advantages of metals include strong mechanical and fatigue properties. However, they are related to many disadvantages, such as inerty, corrosion, modulus differences compared to bone, and fixation problems at the interface between the metal and bone cement. The metal prostheses have been applied most for the treatment of arthrotic large weight-bearing joints of the lower extremity, and as tumour megaprostheses (Chao, 1983; Kotz et al, 1986; Choong et al, 1996). The favourable tribologic properties of aluminium and zirconium as hard material for femoral head balls are worth mentioning. The important drawbacks related to metal prostheses are the unphysiological tribochemical abrasion, fatigue wear production (Collier et al, 1991) between metal surface and bone with stress protection phenomenon and micromovements, bone resorption and granulomatous lesions resulting in loosening of the prosthesis in long-term follow-up (Appel et al, 1990; Santavirta et al, 1990). The changes occur both in connection with methylmetacrylate fixation and without it. However, the final clinical results using HAcoating to enhance fixation of hip prosthesis seem uncertain for the present (see p 80). A report by Miyaji et al (1994) indicating a method for the development of the surface of the metal into bioactive surface itself, is interesting. A beneficial effect to avoid stress shielding bone resorption has been found in the metaphyseal and diaphyseal areas using isoelastic femoral stem with a follow-up of nine years (Niinimaki and Jalovaara, 1995). Osteolysis caused by the wear debris of polyethylene (PE, UHMWPE, ultra high molecular weight polyethylene) with metal surface femoral head balls is a well-known disadvantage (e.g. Kabo et al, 1993; Nakamura, 1996). There is evidence that by decreasing the radius size of the head, the wear rate can be diminished (Kesteris et al, 1996). Some progress is, however, evident because the use of metals such as aluminium and zirconium as bioinert ceramics in arthroplasty

159

have shown a diminished friction against PE socket, and are therefore superior to other metals. The osteolysis which always develops with PE socket was significantly lower using aluminium head and socket in a total hip arthroplasty (Sedel et al, 1990). Recently, clinical series using metal to metal cup-ball application are in progress in USA. 6. Bone and Bone-Derived Materials 6.1. Bone tissue banking Bone banking is at present one of the best and most common methods to procure allogenic bone for bone substitution procedures (Mankin et al, 1987; Aro and Aho, 1993; Delloye, 1990). Bone banking methods are described elsewhere in this volume (Papers 3.1 and 3.6). Transmission of transmittable virus infections is a theoretical risk related to frozen bone allografts (Sanzen and Carlsson, 1997; Tomford, 1995). In recent years, processed decalcified allogenic human bone products have been marketed (Tutoplast®, Dembone®, Perfobone®, Gendler, 1986). 6.2. Bone-derived biologically active substances The subject is presented here only to illustrate its progress. Demineralised bone matrix and morphogenic proteins (BMPs) produced by decalcifying techniques (bovine, reindeer) (Urist, 1965; Urist et al, 1992) as well as the large amount of growth factors such as TGF-(3 family have been used to induce bone formation in connection of carrier materials (Lindholm et al, 1993; Lind et al, 1996). The medical application of growth factors are at present being transferred to prospective clinical series. Regarding processed-related materials, e.g. autoclaved bone, an interesting observation has been made: there was no significant difference concerning the incorporation of an experimental 1/3 ulna defect between frozen allograft and autoclaved autograft. However, supplementation with allogenic bone matrix was needed to improve the incorporation (Kohler et al, 1986).

160

7. Conclusions Referring to orthopaedic clinical praxis, most of the substitutes presented above can be used for filler-reconstruction of moderatesized (1-4 cm of diameter) cystic lesions in human skeleton. The basic demand is bioactivity with bone bonding capacity and biocompatibility of the material used. Many synthetic calcium phosphates, hydroxyapatites, glass materials and glass ceramics, coral-derived products, and tricalcium phosphates exhibit these properties. The most important disadvantage is brittleness and low compression tolerance (bending compression and biomechanic properties). Only a few can be used as a replacement of a weightbearing skeletal part. The modern approach is to develop bioactive bone bonding materials to replace previous biomaterials simply adopted from other fields of high technology. At the moment, tailor-made materials for different applications are being developed. Many polymers are at an experimental state to produce new composites with bioactive materials. The requirements for the ideal bone substitute material are so demanding that not a single material is able to fulfill them all. Metals (aluminium, zirconium, titanium) and metal alloys (Co-Cr, for example) will, for the present, remain the main components of metal prostheses after tumour resections, or simply because of athrosis. The future aim will be to combine the strength of metals and polymers with the osteoconductivity, or preferably osteoinductivity, and bioactivity of other types of materials resulting in an ideal bioactive composite implant with a good bioactivity, osteoconductivity and possible osteoinductivity, suitable hardness, strength and modules corresponding to biomechanical properties of bone. However, bone tissue as allografts (bank bone) and autografts will further be needed as a replacement alternative of total bone ends in tumour surgery and revision arthroplasty.

161

9. References AHO, A.J., HEIKKILA, J.T., AHO, H.J. and YLI-URPO, A. (1996). Transmission electron microscopy at the interface between S53P4 and bone. In: Biocemmics Vol 9, T. Kokubo, T. Nakamura and F. Miyaji, eds., Elsevier Science Ltd, Oxford, pp 41-44. AHO, A.J., HEIKKILA, J.T., ANDERSSON, O.H. and YLI-URPO, A. (1993). Morphology of osteogenesis in bioactive glass interface, Ann. Chirurg. Gynaecol. 82, 145-153. AHO, A.J., SUOMINEN, E., ALANEN, A., YLI-URPO, A., KNUUTI, J., ANDERSSON, O.H. and AHO, H.J. Cortical remodelling of the tibia after filling a large lesion due to fibrous dysplasia by compatible glass-hydroxyapatite mixture, Arch. Orthop. Trauma Surg. (in press). AITASALO, K., PELTOLA, M., SUONPAA, J., YLI-URPO, A., ANDERSSON, O.H., VARPULA, M. and HAPPONEN, R.-P. (1994). Obliteration of frontal sinuses with bioactive glass after chronic suppurative sinusitis. One-year follow-up. In: Bioceramics Vol 7, O.H. Andersson, R.P. Happonen and A. Yli-Urpo, eds., Butterworth-Heinemann Ltd, Oxford, pp 409-414. AITASALO, K., SUONPAA, J., PELTOLA, M. and YLI-URPO, A. Obliteration of frontal sinuses with bioactive glass after chronic suppurative sinusitis. Four-year follow-up. In: Bioceramics Vol 10, R. Sedel, ed., Paris (in press). ALBEE, EH. and MORRISON, H.F. (1920). Studies in bone growth. Triple calcium phsophate as a stimulus to osteogenesis, Ann. Surg. 71, 32-36. ANDERSSON, O.H. and KARLSSON, K.H. (1988). Models for physical properties and bioactivity of phosphate opal glasses, Glastechnische Berichte 61, 300-305.

162

ANDERSSON, O.H. (1990). The bioactivity of silicate glasses (Thesis). Abo Akademi University, Turku, Finland. AOKI, H., KATO, K., OGISO, M. and TABATA, T. (1977). Sintered hydroxyapatite as a new dental implant material, /. Dental Outlook 49, 567-575. APPEL, A.M., SOWDER, W.G., SIVERHUS, S.W., et al (1990). Prosthesis associated pseudomembrane-induced bone resorption, Br. f. Rheumatol. 29, 32-36.

ARO, H.T. and AHO, A.J. (1993). Clinical use of bone allografts, Ann. Med. 25, 403^12. ASANO, S., KANEDA, K. SATOH, S. and TAKEDA, N. (1992). Anterior spinal reconstruction with apatite- and wollastonitecontaining glass-ceramic vertebral prosthesis and kaneda device. In: Bioceramics Vol 5, T. Yamamuro, T. Kokubo and T. Nakamura, eds., Kobunshi Kankokai Inc, Kyoto, pp 443-450. BONFIELD, W. (1996). Composite biomaterials. In: Bioceramics Vol 9, T. Kokubo, T. Nakamura and F. Miyaji, eds., Pergamon, Oxford, pp 11-13. BORETOS, J.W. (1987) Advances in bioceramics, Advanced Ceramic Materials 2, 15-30. BOSTMAN, O., HIRVENSALO, E., VAINIONPAA, S., MAKELA, E.A., VIHTONEN, K., TORMALA, P. and ROKKANEN, P. (1989) Ankle fractures treated using biodegradable internal fixation, Clin. Orthop. 238, 195-203. BRINK, M , PITKANEN, V, TIKKANEN, J., PAAJANEN, M. and GRAEFFE, G. (1996). Spherical particles of a bioactive glass — manufacturing and reaction in vitro. In: Bioceramics Vol 9, T. Kokubo, T. Nakamura and F. Miyaji, eds., Pergamon, Oxford, pp 127-130.

163

BUCHOLZ, R.W., CARLTON, A. and HOLMES, R.E. (1987). Hydroxyapatite and tricalcium phosphate bone graft substitutes, Orthop. Clin. N. Amer. 18, 323-334. BURWELL, R.G. (1969). The fate of bone grafts. In: Recent Advances in Orthopaedics, A.G. Apley, ed., J & A Churchill Ltd, London, pp 115-207. CHAO, E.Y.S. and IVINS, J.C. (eds.) (1983). Tumor prostheses for bone and joint reconstruction. The design and application, ThiemeStratton Inc, New York. CHARNLEY, J. (1960). Anchorage of the femoral head prosthesis to the shaft of the femur, /. Bone Joint Surg. (Br) 42B, 28-30. CHOONG, P.F.M., SIM, EHV PRITCHARD, D.J., ROCK M.G. and CHAO, E.Y.S. (1996). Megaprostheses after resection of distal femoral tumors, Ada Orthop. Scand. 67(4), 345-351. COLLIER, J.P., SURPRENANT, V.A. JENSEN, R.E. and MAYOR, M.B. (1991). Corrosion at the interface of cobalt-alloy heads on titaniumalloy stems, Clin. Orthop. 271, 305-312. CONSTANZ, B.R. ISON, I.C., FULMER, M.T., POSER, R.D., SMITH, S.T., VAN WAGONER, M., ROSS, J., GOLDSTEIN, S.A., JUPITER, J.B. and ROSENTHAL, D.I. (1995). Skeletal repair by in situ formation of the mineral phase of bone, Science 267, 1796-1799. COOK, S.D., THOMAS, M.K.A., KAY, J.F. and JARCHO, M. (1988). Hydroxylapatite-coated porous titanium for use as orthopaedic biologic attachment system, Clin. Orthop. 230, 303-312. Daculsi, G., LeGeros, R.Z. and Deudon, C. (1990a). Scanning and transmission electron microscopy, and electron probe analysis of the interface between implants and host bone. Osseo-coalescence versus osseo-integration, Scanning Microsc. 4, 309-314.

164

DACULSI, G., PASSUTI, N., MARTIN, S., DEUDON, C, LEGEROS, R.Z. and RAHER, S. (1990b). Macroporous calcium phosphate ceramic for long bone surgery in humans and dogs. Clinical and histological study, /. Biomed. Mater. Res. 24, 379-396. DELLOYE, C. (1990). The bridging capacity of a cortical bone defect by different bone grafting materials and diaphyseal distraction lengthening. An experimental study (Thesis). Catholic University of Louvain, Louvain. DENISSEN, H. (1979). Dental root implants of apatite ceramics. Experimental investigations and clinical use of dental root implants made of apatite ceramics (Thesis). Vrije Universiteit te Amsterdam, Amsterdam. DREESMAN, H. (1893). Read before the surgical section. The New York Academy of Medicine, March 13. DUCHEYNE, P., BIANCO, P., RADIN, S. and SCHEPERS, E. (1992). Bioactive materials: Mechanical and bioengineering conditions. In: Bone-Bonding Biomaterials, P. Ducheyne, C. van Blitterwijk and T. Kokubo, eds., Reed Healthcare Communications, Leiderdorp, The Netherlands, pp 1-12. DUCHEYNE, P., HENCH, L.L., KAGAN, A., MARTENS, M., BURSEN, A. and MULIER, J.C. (1980). Effect of hydroxylapatite impregnation on skeletal bonding of porous and coated implants, /. Biomed. Mater. Res. 14, 225-237.

GAO, T.J. (1996). Bioactive delivery system for extracted bone morphogenic protein (Thesis). Acta Univ. Tamperensis, Ser A, Vol 511, Tampere. GAO, T.J., LINDHOLM, T.S., KOMMONEN, B., RAGNI, P., PARONZINI, A., LINDHOLM, T.C. and PAJAMAKI, J. (1995). Comparative study on potential of natural coral and tricalcium phosphate cyliners on healing a segmental diaphyseal defect of sheep, Bioceramics 8, 199-222.

165

GEESINK, R.G. (1990). Hydroxyapatite-coated total hip prostheses. Two-year clinical and roentgenographic results of 100 cases, Clin. Orthop. 261, 39-58. GEESINK, R.G. AND HOEFNAGELS, N.H. (1995). Six year results of hydroxyapatite-coated total hip, J. Bone Joint Surg. 77, 534-547. GENDLER, E. (1986). Perforated demineralized bone matrix: A new form of osteoinductive biomaterial, /. Biomed. Mater. Res. 20(6), 687-697. DE GROOT, K. (1980). Bioceramics consisting of calcium phosphate salts, Biomaterials 1, 47-50. DE GROOT, K., GEESINK, R., KLEIN, C.P. and SEREKIAN, P. (1987). Plasma sprayed coatings of hydroxylapatite, /. Biomed. Mater. Res. 21, 1375-1381. GROSS, U., MULLER-MAI, C. and VOIGT, C. (1993). Ceravital bioactive glass-ceramics. In: An Introduction to Bioceramics, L.L. Hench and J. Wilson, eds., World Scientific, Singapore, pp 105123. HALDEMAN, K.O. and MOORE, J.M. (1934). Influence of a local excess of calcium and phosphorus on the healing of fractures, Arch. Surg. 29, 385.

HATTON, P.V. and BROOK, I.M. (1992). Characterisation of the ultrastructure of glass-ionomer cement, Brit. Dent. J. 173, 275-277. HEIKKILA, J.T., AHO, A.J., AHO, H.J., YLI-URPO, A. and HAPPONEN, R.-P. (1995). Bone formation in rabbit cancellous bone defects filled with bioactive glass granules, Ada Orthop. Scand. 66(5), 463^67. HEIKKILA, J.T., AHO, A.J., KANGASNIEMI, I. and YLI-URPO, A. (1996). Polymethylmetacrylate composites: Disturbed bone formation at the surface of bioactive glass and hydroxylapatite, Biomaterials 17(18), 1755-1760.

166

HEIKKILA, J.T., MATTILA, K.T. ANDERSSON, O.H., KNUUTI, J., YLI-URPO, A. and AHO, A.J. (1996). Behaviour of bioactive glass in human bone. In: Bioactive Glass as a Bone Substitute in Experimental and Clinical Bone Defects, J.T. Heikkila Thesis. Annales

Universitatis Turkuensis Tom 240. HENCH, L.L., SPLINTER, R.J., ALLEN, W.C. and GREENLEE, T.K. (1971). Bonding mechanism at the interface of ceramic prosthetic materials, /. Biomed. Mat. Res. Symp. 2(1), 117-143. HOLMES, R.E., BUCHOLZ, R.W. and MOONEY, V. (1986). Porous hydroxyapatite as a bone-graft substitute in metaphyseal defects, /. Bone Joint Surg. 68A, 904-911. IKADA, Y (1996). Bone-related polymeric biomaterials. In: Bioceramics Vol 9, T. Kokubo, T. Nakamura and F. Miyaji, eds., Pergamon, Oxford, pp 15-18. IMAMALIEV, A.S. (1969). The preparation and preservation and transplantation of articular bone ends. In: Recent Advances in Orthopaedics, A.G. Apley, ed., J & A Churchill Ltd, London, pp 209-263. INOUE, O., SHIMABUKURO, H., SHINGAKI, Y. and IBARAKI, K. (1992). Our application of high porosity hydroxyapatite cubes for the treatment of non-cystic benign bone tumors. In: Bioceramics Vol 5, T. Yamamuro, T. Kokubo and T. Nakamura, eds., Kobunshi Kankokai Inc, Kyoto, pp 411-418. JARCHO, M. (1976). Hydroxylapatite synthesis and characterization in sense of polycrystalline forms, /. Mater. Sci. 11, 2027-2035. JARCHO, M., KAY, J.R, GUMAER, K.I., DOREMUS, R.H. and DROBECK, H.P. (1977). Tissue cellular and subcellular events at a bone-ceramic hydroxyapatite interface, /. Bioeng. 1, 79-92. KABO, J.M., GEBHARD, J.S., LOREN, G. and ARMSTUTZ, H.G. (1993). In vivo wear of polyethylene acetabular components, /. Bone Joint Surg. 75B, 254-258.

167

KARLSSON, K. H., FROBERG, K. and RINGBOM, T. (1989). A structural approach to bone adhering of bioactive glasses, /. NonCryst. Solids 112, 69-72. KARRHOLM, J., MALCHAU, H., SNORRASON, F. and HERBERTS, P. (1994). Micromotion of femoral stems in total hip arthroplasty. A randomized study of cemented, hydroxyapatite-coated, and porous-coated stems with roentgen stereophotogrammetric analysis. /. Bone joint Surg. 76A, 1692-1705. KENT, J.N., FINGER, I.M., QUINN, J.H. and GUERRA, L.R. (1986). Hydroxylapatite alveolar ridge construction: Clinical experiences, complications, and modifications, J. Oral Maxillofac. Surg. 44, 3749. KESTERIS, U., ILCHMANN, T., WINGSTRAND, H. and ONNERFALT, R. (1996). Polyethylene wear in Scanhip® arthroplasty with a 22 or 32 mm head, Ada Orthop. Scand. 67, 125-127. KOKUBO, T., ITO, S., SHIGEMATSU, M., SAKKA, S. and YAMAMURO, T. (1985). Mechanical properties of a new type of apatite-containing glass-ceramic for prosthetic application, /. Mater. Set. 20, 2001-2004. KOKUBO, T., YOSHIHARA, T., NISHIMURA, N. and YAMAMURO, T. (1991). Bioactive bone cement based on CaO-SiO2-P2O5 glass. In: AW Glass-Ceramic — Development, Characterisation, Modification,

and Clinical Application, T. Yamamuro, ed., The Fellow Club of the Department of Orthopaedic Surgery, Kyoto University, Kyoto, pp 678-680. KOTZ, R., RITSCHL, P. and TRACHTENBRODT, J. (1986). A modular femur-tibia reconstruction system, Orthopaedics 9, 1639-1652. KOYAMA, T. and HANDA, J. (1986). Porous hydroxyapatite ceramics for use in neurosurgical practice, Surg. Neurol. 25, 71-73.

168

KROON, P.O. and FREEMAN, M.A.R. (1992). Hydroxyapatite coating of hip prostheses. Effect on migration into the femur, /. Bone Joint Surg. 74B, 518-522. KUDO, K., MTYASHAVA, M., FUJIOKA, Y, KAMEGAI, T., NAKANO, H., SEINO, Y, ISHIKAWA, F., SHIOYAMA, T. and ISHIBASHI, K. (1990). Clinical application of dental implant with root of coated bioglass: Short-time results, Oral Surg. Oral Med. Oral Pathol. 70, 18-23. KOHLER, P. (1986). Reimplantation of bone after autoclaving. Reconstruction of large diaphyseal defects in the rabbit. Thesis. Stockholm. LEGEROS, R.Z. and LEGEROS, J.P. (1993). Dense hydroxyapatite. In: An Introduction to Biocerantics, L.L. Hench and J. Wilson, eds., World Scientific, Singapore, pp 139-180. LIND, M, OVERGAARD, S., NGUYEN, T., ONGPIPATTANAKUL, B., BUNGER, C. and S0BALLE, K. (1996). Transforming growth factor-P stimulates bone ongrowth. Hydroxyapatite-coated implants studied in dogs. Ada Orthop. Scand. 67, 611-616. LINDHOLM, T.C., GAO, T.J. and LINDHOLM, T.S. (1993). Granular hydroxyapatite and allogenic demineralized bone matrix in rabbit skull defect augmentation, Ann. Chir. Gynaecol. 82, 91-98. LOTY, B., ROUX, F.X., GEORGE, B., COURPIED, J.P. and POSTEL, M. (1990). Utilisation du corail en chirurgie osseuse, Int. J. Orthop. (SICOT) 14, 255-259. MACEWEN, W. (1881). Observations concerning transplantation on bone, Proc. R. Soc. Lond. 32, 232-247. MANKIN, H.J., GEBHARDT, M.C. and TOMFORD, W.W. (1987). The use of frozen cadaveric allografts in the management of patients with bone tumours of the extremities, Orthop. Clin. North Am. 18, 275-289.

169

MITTELMAIER, H. and KATTHAGEN, B.D. (1983). Klinische Erfahrungen mit Collagen-Apatit-Implantation zur lokalen Knochenregeneration, Z. Orthop. 121, 115-123. MIYAJI, E, ZHANG, X., YAO, T., KOKUBO, T. OHTSUKI, C , KITSUGI, T., YAMAMURO, T. and NAKAMURA, T. (1994). Chemical treatment of Ti metal to induce its bioactivity. In: Bioceramics Vol 7, O.H. Andersson, R.P. Happonen and A. YliUrpo, eds., Butterworth-Heinemann Ltd, Oxford, pp 119-124. MOORE, D.C., CHAPMAN, M.W. and MANSKE, D.J. (1987). The evaluation of a biphasic calcium phosphate ceramic for use in grafting long-bone diaphyseal defects, /. Orthop. Res. 5, 356-365. MURRAY, J.E., REF. HALDEMAN, K.O. and MOORE, J.M. (1934). Influence of a local excess of calcium and phosphorus on the healing of fractures, Arch. Surg. 29, 385-396. NAKAMURA, T. (1996). Bioceramics in orthopaedic surgery. In: Bioceramics Vol 9, T. Kokubo, T. Nakamura and F. Miyaji, eds., Pergamon, Oxford, pp 31-34. NIINIMAKI, T. and JALOVAARA, P. (1995). Bone loss from the proximal femur after arthroplasty with an isoelastic femoral stem, Ada Orthop. Scand. 66, 347-351. NIINIMAKI, T., PURANEN, J. AND JALOVAARA, P. (1994). Total hip arthroplasty using isoelastic femoral stems, /. Bone Joint Surg. 76B, 413-418. NIWA, S., SAWAI, K., TAKAHASHI, S., TAGAI, H., ONO, M. and FUKUDA, Y. (1980). Experimental studies on the implantation of hydroxylapatite in the medullary canal of rabbits, First World Biomaterials Congress, Baden near Vienna, Austria. OONISHI, H., KUSHITANI, S., AONO, M., MAEDA, E., TSUJI, E. and ISHIMARU, H. (1989). Interface bioactive bone cement by using PMMA and hydroxyapatite granules. In: Bioceramics Vol 1, Ishiaku-Euro-America, pp 102-107.

170

ORELL, S. (1937). Surgical bone grafting with "os purum", "os novum" and "boiled bone", /. Bone Joint Surg. 19, 873-885. OTTOLENGHI, C.E., MUSCOLO, D.L. and MAENZA, R. (1982). Bone defect reconstruction by massive allograft: Technique and results of 51 cases followed for 5 to 32 years. In: Clinical Trends in Orthopaedics, L.R. Starub and P.D. Wilson, eds., Thieme-Stratton Inc, New York, pp 171-183. PARRISH, EF. (1966). Treatment of bone tumours by total excision and replacement with massive autologous and homologous grafts, /. Bone Joint Surg. 48A, 968-990. PARTIO, E.K. (1992). Absorbable screws in the fixation of cancellous bone fractures and arthrodeses. A clinical study of 318 patients, University of Helsinki, Helsinki. PASSUTI, N., DACULSI, G., ROGEZ, J.M., MARTIN, S. and BAINVEL, J.V. (1989). Macroporous calcium phosphate ceramic performance in human spine fusion, Clin. Orthop. 248, 169-176. PELTIER, L.F., BICKEL, E.Y., LILLO, R. and THEIN, M.S. (1957). The use of plaster of Paris to fill defects in bone, Ann. Surg. 146, 6169. PIECUCH, J.F. GOLDBERG, A.J., SHASTRY, C.V. and CHRZANOWSKI, R.B. (1984). Compressive strength of implanted porous replamineform hydroxyapatite, /. Biomed. Mater. Res. 18, 39-45. POULIQUEN, J.C., NOAT, M., VERNERET, C, GUILLEMIN, G. and PATAT, J.L. (1989). Coral as a substitute for bone graft in posterior spine fusion in childhood — preliminary results. French J. Orthop. Surg. 3, 272-280. ROKKANEN, P. (1991). Absorbable materials in orthopedic surgery, Ann. Med. 23, 109-115.

171

ROTHMAN, R.H., HOZACK, W.J., RANAWAT, A. and MORIARTY L. (1996). Hydroxyapatite-coated femoral stems. A matched-pair analysis of coated and uncoated implants, /. Bone Joint Surg. 78A, 319-324. ROUX, EX., BRASNU, D., LOTY, B., GEORGE, B. and GUILLEMIN, G. (1988). Madroporic coral: A new bone graft substitute for cranial surgery, /. Neurosurg. 68, 510-513. ROY, D.M. and LINNEHAN, S.K. (1974). Hydroxyapatite formed from coral skeleton carbonate by hydrothermal exchange, Nature 247, 220-222. SANTAVIRTA, S., KONTTINEN, Y.T., BERGROTH, V., ESKOLA, A., TALLROTH, K. and LINDHOLM, T.S. (1990). Aggressive granulomatous lesions associated with hip arthroplasty, /. Bone Joint Surg. {Am.) 72-A, 252—258.

SANZEN L. and CARLSSON, A. (1997). Transmission of human Tcell lymphotrophic virus type 1 by a deep-frozen bone allograft, Ada Orthop. Scand. 68, 72-74. SEDEL, L., FUMER, P., SHIBUYA, T. and YAMAMURO, T. (1992). Apatite-wollastonite glass-ceramics (AW G-C) used as self procedure for recurrent shoulder dislocation stabilization (a preliminary clinical trial). In: Bioceramics Vol 5, T. Yamamuro, T. Kokubo and T. Nakamura, eds., Kobunshi Kankokai Inc, Kyoto, pp 427^34. SEDEL, L., KERBOULL, L., CHRISTEL, P., MEUNIER, A. and WITVOET, J. (1990). Alumina — on alumina hip replacement. Results and survivorship in young patients, /. Bone Joint Surg. 72B, 658-663. SENN, N. (1889). On the healing of aseptic bone cavities by implantation of antiseptic decalcified bone, /. Med. Sci. (Am.) 98(3), 219-243.

172

SHIKINAMI, Y., HATA, K. and OKUNO, M. (1996). Ultra-highstrength resorbable implants made from bioactive ceramic particles /polyactide composites. In: Bioceramics Vol 9, T. Kokubo, T. Nakamura and F. Miyaji, eds., Pergamon, Oxford, pp 391-394. SHIMIZU, K., IWASAKI, R., MATSUSHITA, M. and YAMAMURO, T. (1992). Posterior lumbar interbody fusion using AW-GC vertebral spacer. In: Bioceramics Vol 5, T. Yamamuro, T. Kokubo and T. Nakamura, eds., Kobunshi Kankokai Inc, Kyoto, pp 435442. S0BALLE, K. (1993). Hydroxyapatite ceramic coating for bone implant fixation. Mechanical and histological studies in dogs, Ada Orthop. Scand. 64 (Suppl No 255). SUOMINEN, E. (1996). Bioactive ceramics in reconstruction of bone defects. Studies with bioactive glasses, glass-ceramics, hydroxyapatite and their composites (Thesis). Ann. Univ. Turkuensis, Ser D, Tom 231, Turku. SUOMINEN, E.A. and KINNUNEN, J. (1996). Bioactive glass granules and plates in the reconstruction of defects of the facial bones, Scand. J. Plast. Reconstr. Surg. Hand Surg. 30, 281-289.

SUZUKI, K. and KURABAYASHI, H. (1994). Efficiency of hydroxyapatite-tricalcium phosphate-composite (HAP-TCP) for bone defect of tibia fracture — comparison between HAP-TCP and autogenous iliac bone. In: Bioceramics Vol 7, O.H. Andersson, R.P. Happonen and A. Yli-Urpo, eds., Butterworth-Heinemann Ltd, Oxford, pp 435-440. TENCER, A.F., WOODARD, P. L. SWENSON, J. and BROWN, K. L. (1987). Bone ingrowth into polymer coated porous synthetic coralline hydroxyapatite, /. Orthop. Res. 5, 275-282. TOMFORD, W.N. (1995). Current concept review. Transmission of disease through transplantation of musculoskeletal allografts, /. Bone Joint Surg. 77, 1742-1754.

173

TURUNEN, T., PELTOLA, J., KANGASNIEMI, I., JUSSILA, J., UUSIPAIKKA, E. and YLI-URPO, A. (1995). Augmentation of the maxillary sinus wall using bioactive glass and autologous bone. In: Bioceramics Vol 8, J. Wilson and L.L. Hench, eds., pp 259-264. UCHIDA, A., ARAKI, N., SHINTO, Y, YOSHIKAWA, H., KURISAKI, E. and ONO, K. (1990). The use of calcium hydroxyapatite ceramic in bone tumour surgery, /. Bone Joint Surg. (Br.) 72B, 288-302. URIST, M.R. (1965). Bone formation by autoinduction, Science 150, 893-899. URIST, M.R., CHANG, J.J., BROWNELL, A.G., HUO, YK. and LINDHOLM, T.S. (1992). Native bone morphogenetic protein. In: New Trends in Bone Grafting, T.S. Lindholm, ed., Acta Univ. Tamperensis, Ser B, Vol 40, University of Tampere, Tampere, pp 29-39. VIIKARI, S.J. and AHO, A.J. (1963). The use of heterogenous, anorganic bone as grafting material for osseous defects, Ann. Chir. Gyn. 52, 665-678. VOGEL, W., HOLAND, W., NAUMAN, K., VOGEL, J., CARL, G., GOTZ, W. and WANGE, P. (1990). Addendum glass-ceramics for medicine and dentistry. In: Handbook of Bioceramics Vol 1, Bioactive Glasses and Glass-Ceramics, T. Yamamuro, L.L. Hench and J. Wilson, eds., CRC Press, Boca Raton, FL, pp 353-355. WANG, M., KOKUBO, T. and BONFIELD, W. (1996). A-W glass ceramic reinforced polyethylene for medical applications. In: Bioceramics Vol 9, T. Kokubo, T. Nakamura and F. Miyaji, eds., Pergamon, Oxford, pp 387-390. WILSON, J., CLARK, A.E., DOUEK, E., KRIEGER, J., KING, S.W. and SAVILLE, Z.J. (1994). Clinical applications of bioglass implants. In: Bioceramics Vol 7, O.H. Andersson, R.P. Happonen and A. Yli-Urpo, eds., Butterworth-Heinemann Ltd, Oxford, pp 415-422.

174

YAMAMURO, Tv SHIKATA,J., KAKUTANI, Y, YOSHII, S., KTTSUGI, T. and KOZO, O. (1988). Novel methods for clinical applications of bioactive ceramics. Bioceramics: Material Characteristic versus in vivo behavior, Ann. N.Y. Acad. Sci. 523, 107-114. YAMAMURO, T. SHIKATA, J., OKUMURA, H., SOSHII, S., KOTANI S. and KOKUBO, T. (1990). Preclinical and clinical applications of A-W glass ceramic to various orthopaedic conditions. Bioceramics 2, 361—366. YAMAMURO, T. SHIKATA, J., KAKUTANI, Y, YOSHII, S., KITSUGI, T. and ONO K. (1994). Novel methods for clinical applications of bioactive ceramics. In: AW Glass-Ceramics — Development, Characterisation, Modification, and Clinical Application, J. Yamamuro, ed., The Fellow Club of the Department of Orthopaedic Surgery, Kyoto University, Kyoto, pp 914-921.