Table Of ContentHandbook of Histology Methods
for Bone and Cartilage
Handbook of Histology Methods
for Bone and Cartilage
Edited by
Yuehuei H. An,
MD
Department of Orthopaedic Surgery,
Medical University of South Carolina, Charleston, SC;
and Department of Bioengineering, Clemson University, Clemson, SC
and
Kylie L. Martin,
BSc
Department of Orthopaedic Surgery,
Medical University of South Carolina, Charleston, SC
Springer Science+Business Media, LLC
© 2003 Springer Science+Business Media New York
Originally published by Humana Press Inc. in 2003
Softcover reprint of the hardcover 1st edition 2003
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Due diligence has been taken by the publishers, editors, and authors of this book to assure the accuracy of the information
published and to describe generally accepted practices. The contributors herein have carefully checked to ensure that the drug
selections and dosages set forth in this text are accurate and in accord with the standards accepted at the time of publication.
Notwithstanding, as new research, changes in government regulations, and knowledge from clinical experience relating to
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This publication is printed on acid-free paper. ∞
ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials.
Production Editor: Kim Hoather-Potter.
Cover design by Yuehuei H. An and Patricia F. Cleary.
Cover art: Background image: multiple fluorochrome labeling of bone (see Chapter 5). From left to right: root with
periodontal ligament, alveolar bone, fatty bone marrow of the mandible (see Chapter 16); micrograph of a pediatric distal
femoral physis (see Chapter 21); osteoblasts and woven bone areas stained with toluidine blue and acid fuchsin (see
Chapter 23); scanning electron micrograph of a bone specimen following removal of marrow and bone cells (see Chapter
34); and growth plate cartilage and bone of rat distal femur (contributed by Dr. Q. Kay Kang).
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10 9 8 7 6 5 4 3 2 1
Library of Congress Cataloging in Publication Data
Handbook of histology methods for bone and cartilage/edited by Yuehuei H. An and
Kylie L. Martin.
p.;cm
Includes bibliographical references and index.
ISBN 978-1-61737-277-3 ISBN 978-1-59259-417-7 (eBook)
DOI 10.1007/978-1-59259-417-7
1. Bones--Histology--Handbooks, manuals, etc. 2. Cartilage--Histology--Handbooks,
manuals, etc. I. An, Yuehuei H. II. Martin, Kylie L.
[DNLM: 1. Bone and Bones--anatomy & histology--Handbooks. 2. Bone and
Bones--physiology--Handbooks. 3. Cartilage--anatomy & histology--Handbooks. 4.
Cartilage--physiology--Handbooks. 5. Histological Techniques--Handbooks. 6. Tissue
Harvesting--Handbooks. WE 39 H23613 2003]
QM101.H33 2003
611'.0184--dc21
2002035286
To Q. Kay Kang, MD
Without her love, inspiration, and support,
this book would not have been possible.
Yuehuei H. An,MD
To my husband Charles, and my daughters Lisa and Heather,
for their patience, understanding, love, and support.
Kylie L. Martin,BSC
v
PREFACE
Histotechnology and histomorphometry are the major methodologies in bone and cartilage-
related research. Handbook of Histology Methods for Bone and Cartilage is an outgrowth of
the editors’ own quest for information on bone and cartilage histology and histomorphometry.
It is designed to be an experimental guide for personnel who work in the areas of basic and
clinical bone and cartilage, orthopedic, or dental research. It is the first inclusive and organized
reference book on histological and histomorphometrical techniques on bone and cartilage
specimens. The topic has not previouslybeen covered adequately by any existing books in the
field.
Handbook of Histology Methods for Bone and Cartilage has six major parts and is designed
to be concise as well as inclusive, and more practical than theoretical. The text is simple and
straightforward. Large numbers of tables, line drawings, and micro- or macro-photographs,
are used to help readers better understand the content. Full bibliographies at the end of each
chapter guide readers to more detailed information. A book of this length cannot discuss every
method for bone and cartilage histology that has been used over the years, but it is hoped that
major methods and their applications have been included.
Yuehuei H. An,MD
Kylie L. Martin, BSc
vii
TABLE OF CONTENTS
Preface...........................................................................................................................................vii
About the Editors.........................................................................................................................xii
Contributors................................................................................................................................xiii
List of Color Plates ....................................................................................................................xvii
PART I – INTRODUCTION
1. Introduction to Experimental Bone and Cartilage Histology
Yuehuei H. An and Helen E. Gruber .......................................................................................3
PART II – STRUCTURE AND FUNCTION OF BONE AND CARTILAGE
2. Cell Structure and Biology of Bone and Cartilage
William R. Walsh, Mark Walton, Warwick Bruce, Yan Yu,
Ronald M. Gillies, and Martin Svehla .................................................................................. 35
3. Normal Structure and Function of Bone
Torben Steiniche and Ellen M. Hauge.................................................................................. 59
4. Structure and Function of Articular Cartilage
Jerry C. Y. Hu and Kyriacos A. Athanasiou......................................................................... 73
PART III – TISSUE HARVESTING, FIXATION, AND PREPARATION
5. Bone-Labeling Techniques
Reinhold G. Erben ................................................................................................................. 99
6. Human Bone Biopsy
Robert S. Weinstein............................................................................................................. 119
7. Biopsy Issues for Bone and Cartilage Tumors
John L. Eady ........................................................................................................................ 129
8. Tissue Harvesting and Fixation
Linda L. Jenkins and Karen J. L. Burg ............................................................................... 143
9. Common Fixatives in Hard-Tissue Histology
Antonio Scarano, Giovanna Iezzi, and Adriano Piattelli ................................................... 159
10. Decalcification of Bone Tissue
Robert A. Skinner................................................................................................................ 167
11. Principles of Embedding and Common Protocols
Yuehuei H. An, Patricia L. Moreira, Qian K. Kang, and Helen E. Gruber....................... 185
12. Infiltration Techniques and Results in Different Types of Resin
Antonio Scarano, Giovanna Orsini, and Adriano Piattelli................................................. 199
PART IV – SECTIONING TECHNIQUES
13. Histological Techniques for Decalcified Bone and Cartilage
Qian K. Kang, Jackie C. LaBreck, Helen E. Gruber, and Yuehuei H. An........................ 209
ix
x Contents
14. Techniques for Sectioning Undecalcified Bone Tissue Using Microtomes
William L. Ries.................................................................................................................... 221
15. Cutting and Grinding Methods for Hard-Tissue Histology
Thomas W. Bauer and Diane Mahovlic.............................................................................. 233
16. Bone Sectioning Using the Exakt System
Karl Donath and Michael Rohrer........................................................................................ 243
17. Bone Sectioning Using a Modified Inner Diamond Saw
Joop G. C. Wolke, Jan-Paul C. M. van der Waerden,
Christel P. A. T. Klein, and John A. Jansen ....................................................................... 253
18. Bone Sectioning Using the Precise 1 Automated Cutting System
Antonio Scarano, Manlio Quaranta, and Adriano Piattelli ................................................ 265
19. Bone, Cement, and Metal Implant Interface Preparation
with a High-Pressure Water Cutter
Jian-Sheng Wang and Lars Lidgren.................................................................................... 271
PART V – STAINING TECHNIQUES
20. Basic Staining and Histochemical Techniques and Immunohistochemical
Localizations Using Bone Sections
Helen E. Gruber and Jane A. Ingram.................................................................................. 281
21. Basic Staining Techniques for Cartilage Sections
Helen E. Gruber and Jane A. Ingram.................................................................................. 287
22. Histochemical and Immunohistochemical Staining of Cartilage Sections
Andreas G. Nerlich.............................................................................................................. 295
23. Staining Techniques for Plastic-Embedded Specimens
Antonio Scarano, Giovanna Petrone, and Adriano Piattelli............................................... 315
24. In Situ Hybridization of Bone and Cartilage
Shintaro Nomura and Seiichi Hirota................................................................................... 321
PART VI – ANALYSIS TECHNIQUES
25. Bone Histomorphometry: Concepts and Common Techniques
Jean E. Aaron and Patricia A. Shore................................................................................... 331
26. Histological Analysis of Bone-Implant Interface
John A. Jansen ..................................................................................................................... 353
27. Histomorphometric Analysis of Bone–Cement and Cement–Metal Interface
Jian-Sheng Wang, Gonzalo G. Valdivia, Michael J. Dunbar, Cecil H. Rorabeck,
Robert B. Bourne, and Suzanne Maher............................................................................... 361
28. Histologic Analysis of Bone Healing
Ryland B. Edwards, III, Mandi J. Lopez, and Mark D. Markel......................................... 375
29. Histomorphometry of Metabolic Bone Conditions
Ellen M. Hauge, Torben Steiniche, and Troels T. Andreassen.......................................... 391
30. Histology of Articular Cartilage Repair
Stefan Nehrer and Myron Spector....................................................................................... 411
Contents xi
31. Histological Analysis of Cartilage Conditions
Theodore R. Oegema, Jr., Cathy S. Carlson, and Ada A. Cole ......................................... 423
32. Histological Analysis of Soft-Tissues Biomaterial Interface:
Relevance to Dental Implants
Antonio Scarano, Gian Antonio Favero, Elisabetta Fiera,
and Adriano Piattelli............................................................................................................ 439
33. Pathological Diagnosis of Common Tumors of Bone and Cartilage
Jasvir S. Khurana and Krishnan K. Unni............................................................................ 447
PART VII – ELECTRON MICROSCOPY AND RADIOGRAPHY
34. Methods for Transmission and Scanning Electron Microscopy
of Bone and Cartilage
Helen E. Gruber and Winston W. Wiggins......................................................................... 497
35. SEM Methods for Observation of the Bone-Implant Interface
Masashi Neo and Takashi Nakamura.................................................................................. 505
36. TEM Methods for Observation of the Bone-Implant Interface
Joseph Hemmerlé................................................................................................................. 513
37. Radiological Examination of Calcified Tissues with Emphasis on Bone
Victor Fornasier and Charles L. Ho.................................................................................... 531
38. Microangiography, Macrosectioning, and Preparation
for Contact Radiography
Yebin Jiang, Yunzhao Wang, Dianmin Xue, and Yuming Yin......................................... 549
PART VIII – RECOGNIZING AND AVOIDING ARTIFACTS
39. Recognizing and Interpreting Histology Artifacts in Hard-Tissue Research
Thomas W. Bauer ................................................................................................................ 561
Index .......................................................................................................................................571
xii About the Editors
ABOUT THE EDITORS
Yuehuei H. (Huey) An, MD, graduated from the Harbin Medical University,
Harbin, China in 1983 and was trained in orthopedic surgery at the Beijing Ji
Shui Tan Hospital, and in hand surgery at Sydney Hospital, Sydney, Aus-
tralia. In 1991, Dr. An joined Dr. Richard J. Friedman in the Department
of Orthopaedic Surgery at the Medical University of South Carolina to
establish the Orthopaedic Research Laboratory, which is now a multifunc-
tional orthopedic research center. Dr. An has published more than 100
scientific papers and book chapters, more than 100 research
abstracts, and has also edited 5 books, including Animal Models in Ortho-
paedic Research (CRC Press, 1999), Mechanical Testing of Bone and the
Bone-Implant Interface (CRC Press, 2000), Handbook of Bacterial Adhe-
sion (Humana Press, 2000), Internal Fixation in Osteoporotic Bone
(Thieme, 2002), and Orthopaedic Issues in Osteoporosis (CRC Press,
2002). He is an active member of eight academic societies in the fields of
orthopedic surgery, biomaterials, biomechanics, and tissue engineering.
Kylie L. Martin, BSc, is an histological technician and laboratory manager
of the Orthopaedic Research Laboratory for the Department of Orthopaedic
Surgery, Medical University of South Carolina, Charleston, SC, USA. Ms.
Martin received her baccalaureate degree from the Western Australian
Institute of Technology (now Curtin University of Technology). She has
been at the Medical University of South Carolina since 1979, working in a
number of research laboratories, and has extensive experience in experi-
mental histology and cell biology. Ms. Martin has published several journal
articles and book chapters.
