Bone Research Protocols

443 Pages·2003·5.822 MB·English

by  James A. Gallagher (auth.), Miep H. Helfrich PhD, Stuart H. Ralston MD (eds.)

Bone Research Protocols
M E T H O D S I N M O L E C U L A R M E D I C I N ETM
John M. Walker, SERIES EDITOR
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M E T H O D S I N M O L E C U L A R M E D I C I N ETM
Bone Research
Protocols
Edited by
Miep H. Helfrich,
PhD
University of Aberdeen Medical School
Aberdeen, UK
Stuart H. Ralston,
MD
University of Aberdeen Medical School
Aberdeen, UK
Humana Press Totowa, New Jersey
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Library of Congress Cataloging-in-Publication Data
Bone research protocols / edited by Miep H. Helfrich, Stuart H. Ralston
p. ; cm. -- (Methods in molecular medicine ; 80)
Includes bibliographical references and index.
ISBN 1-58829-044-1 (alk. paper); E-ISBN 1-59259-366-6
1. Bones--cultures and culture media--Laboratory manuals. 2. Bone cells--Laboratory manuals I.
Helfrich, Miep H. II. Ralston, Stuart. III. Series.
[DNLM: 1. Bone and Bones--cytology. 2. Cell Culture. 3. Histological Techniques. 4. Osteoblasts. 5.
Osteoclasts. WE 200 B7129 2003]
QP88.2.B5898 2003
612.7'5'072--dc21
2003044967
Preface
The last decade has seen a tremendous advance in our understanding of
bone biology. The genes responsible for the majority of rare inherited bone
disorders have been identified and much progress has been made in the
identification of genes in polygenic disorders such as Paget’s disease and
complex multigene diseases such as osteoporosis. Transgenic technology has
identified further genes, sometimes unexpectedly, with profound effects on
bone. This wealth of new genetic information will undoubtedly lead to extensive
cell biological studies to understand the mechanisms by which these gene
products affect bone mass and bone strength.
InBone Research Protocolsa catalogue of protocols has been assembled
to perform such mechanistic studies. In the tradition of the Methods in
Molecular Medicineseries, the chapters are practical laboratory protocols that
should enable the reader to carry out the techniques from scratch. To our
knowledge this is the first time such a truly practical manual on well-established
bone methods has been assembled, and this volume aims to be complementary
to and follow on from the more theoretical Methods in Bone Biology, edited
by Arnett and Henderson (1).
We have concentrated on laboratory, as opposed to clinical, protocols
and on those that are specific to bone and bone tissue only. For example, there
are no differences in analysis of DNA and RNA from bone or other tissues,
but special considerations apply to isolation of DNA and RNA from bone and
these are described. Equally, histological and histochemical procedures for
soft tissues are often easily adapted to bone and are not specifically covered,
apart from those included as analysis tools in various chapters. Tissue fixation,
embedding and sectioning of bone, however, present unique problems and
such methods are described as part of the chapters dealing with electron
microscopy and immunostaining. Much progress has been made in digital image
analysis recently and several chapters (dealing with confocal microscopy, bone
resorption assays and histomorphometry) include a detailed description on
how to make best use of this powerful technology.
We have given much attention to in vitro methods, but have also
included a number of protocols for in vivo studies of bone resorption and bone
formation and their in vivo analysis. Generally, one method is given for each
v
vi Contents
technique, with the exception of in vitro osteoclast formation studies, for which
several protocols are described, illustrating that many methods, often only
subtly different, are described in the literature for different species and sources
of growth factors. Those interested in this field are encouraged to read all
methods first before deciding which one is most appropriate for their particular
application.
The osteoblast protocols concentrate on primary cultures, rather than
cell lines. For a thorough discussion of osteoblast cell lines and their specific
features we recommend the comprehensive review by Hughes and Aubin in
Methods in Bone Biology. We have included a chapter on measurement of
bone strength ex vivo, since this parameter is critical in analysis of bone
pathology. Two chapters on methods for mechanical studies on bone are
included, concentrating on in vitro studies. This area of bone research is
expanding rapidly and we expect that further additions to this section, such as
methods for in vivo loading of bone, currently not covered, may be necessary
with time.
We hope that Bone Research Protocols will help those entering the
bone field to establish new techniques in their laboratories. For those already
experienced in bone research, we hope that they will benefit from the detailed
description of the methods, in particular the many pointers and pitfalls, which
the authors were specifically asked to discuss. We certainly learned a lot!
Finally, we are grateful to Leica, Zeiss Buehler, ADCIS, Amersham, and
Bilplane for financial support with color reproductions.
Many thanks are owed to Nicki Duncan for her excellent assistance with
the editorial work, to all authors for their willingness to share their trade secrets,
and to Prof. John Walker and Craig Adams at Humana Press for giving us the
opportunity to add a volume on bone research methods to the Methods in
Molecular Medicine series.
Stuart H. Ralston, MD
Miep H. Helfrich, PhD
1.Arnett, T. R. and Henderson, B. (1998) Methods in Bone Biology. Chapman Hall,
London, UK, p. 314.
Contents
Preface .................................................................................................v
Contributors.........................................................................................ix
PART I CULTURE OF OSTEOBLASTS AND OSTEOCYTES
1 Human Osteoblast Culture
James A. Gallagher.....................................................................3
2 Osteoblast Isolation from Murine Calvariae and Long Bones
Astrid Bakker and Jenneke Klein-Nulend..............................19
3 Mineralizing Fibroblast-Colony-Forming Assays
Andrew Scutt, Lynsey Reading, Nanette Scutt,
and Karen Still.......................................................................29
4 Osteocyte Isolation and Culture
Peter J. Nijweide, Arie van der Plas, Marcel J. Alblas,
andJenneke Klein-Nulend....................................................41
PART II CULTURE OF OSTEOCLASTS
5 Isolated Osteoclast Cultures
Astrid Hoebertz and Timothy R. Arnett..................................53
6 Primary Isolation and Culture of Chicken Osteoclasts
Patricia Collin-Osdoby, Fred Anderson,
and Philip Osdoby.................................................................65
7 Isolation and Purification of Rabbit Osteoclasts
Fraser P. Coxon, Julie C. Frith, Helena L. Benford,
and Michael J. Rogers..........................................................89
8 Generating Human Osteoclasts from Peripheral Blood
Afsie Sabokbar and Nicholas A. Athanasou........................101
9 Generating Human Osteoclasts from Bone Marrow and
Peripheral Blood
Adrienne M. Flanagan and Helen M. Massey.......................113
10 Generating Murine Osteoclasts from Bone Marrow
Naoyuki Takahashi, Nobuyuki Udagawa, Sakae Tanaka,
and Tatsuo Suda..................................................................129
vii
viii Contents
11 Osteoclast Formation in the Mouse Coculture Assay
Robert J. van 't Hof..................................................................145
12 RANKL-Mediated Osteoclast Formation
from Murine RAW 264.7 Cells
Patricia Collin-Osdoby, Xuefeng Yu, Hong Zheng,
and Philip Osdoby...............................................................153
PART III BONE ORGAN CULTURE
13 Analysis of Osteoclast Function in Mouse Calvarial Cultures
Michael J. Marshall and Marit N. Rowlands.........................169
14 Assessing Bone Formation
Using Mouse Calvarial Organ Culture
I. Ross Garrett..........................................................................183
PART IV HISTOLOGICAL AND ULTRASTRUCTURAL TECHNIQUES
15 In Situ Hybridization and In Situ Reverse Transcription
Polymerase Chain Reaction in Human Bone Sections
Andrew P. Mee and Judith A. Hoyland.................................201
16 Techniques for the Study of Apoptosis in Bone
Brendon S. Noble and Hazel Y. Stevens..............................225
17 Protein Localization in Wax-Embedded and Frozen Sections
of Bone Using Immunohistochemistry
Sharyn Bord.............................................................................237
18 Detection of Noncollagenous Bone Proteins
in Methylmethacrylate-Embedded Human Bone Sections
Johannes P. T. M. van Leeuwen and Pieter Derkx..............249
19 Fluorescence Imaging of Bone-Resorbing Osteoclasts
by Confocal Microscopy
Stephen A. Nesbitt and Michael A. Horton..........................259
20 Bone Histomorphometry
Shobna Vedi and Juliet Compston.......................................283
21 Transmission Electron Microscopy of Bone
Vincent Everts, Anneke Niehof, and Wouter Beertsen......299
22 Scanning Electron Microscopy of Bone
Deborah Marshall, Miep H. Helfrich,
and Richard M. Aspden.....................................................311
Contents ix
PART V IMAGING TECHNIQUES
23 Bone Measurements by Peripheral Quantitative
Computed Tomography in Rodents
Jürg A. Gasser.........................................................................323
PART VI IN VIVO TECHNIQUES
24 Studies of Local Bone Remodeling:
The Calvarial Injection Assay
Robert J. van ’t Hof, Claire E. Clarkin,
and Kenneth J. Armour.......................................................345
25 Inflammation-Induced Osteoporosis: The IMO Model
Kenneth J. Armour and Katharine E. Armour......................353
26 Ovariectomy and Estrogen Replacement in Rodents
Jade W. M. Chow.....................................................................361
PART VII MECHANICAL LOADING TECHNIQUES
27 Mechanical Testing of Bone Ex Vivo
Richard M. Aspden..................................................................369
28 Bone Cell Responses to Fluid Flow
Hazel Y. Stevens and John A. Frangos................................381
29 Methods for Analyzing Bone Cell Responses to Mechanical
Loading Using In Vitro Monolayer and Organ Culture Models
Andrew A. Pitsillides, Victoria Das-Gupta,
Dominic Simon, and Simon C. F. Rawlinson...................399
PART VIII MOLECULAR ANALYSIS
30 Extraction of Nucleic Acids from Bone
Tracy L. Stewart and Val Mann..............................................425
31 Analysis of Gene Expression in Bone by Quantitative RT-PCR
Deborah Ireland.......................................................................433
Index.................................................................................................441
Contributors
MARCEL J. ALBLAS • Department of Molecular Cell Biology, Leiden Univer-
sity Medical Center, Leiden, The Netherlands
FRED ANDERSON • Department of Biology, Washington University,
St. Louis, MO
KATHARINE E. ARMOUR • Bone Research Group, Department of Medicine
and Therapeutics, University of Aberdeen Medical School, Aberdeen,
UK
KENNETH J. ARMOUR • Bone Research Group, Department of Medicine and
Therapeutics, University of Aberdeen Medical School, Aberdeen, UK
TIMOTHY R. ARNETT • Department of Anatomy and Developmental
Biology, University College London, London, UK
RICHARD M. ASPDEN • Department of Orthopaedics, University of Aberdeen,
Aberdeen, UK
NICOLAS A. ATHANASOU • Department of Pathology, Nuffield Orthopaedic
Centre, Headington, Oxford, UK
ASTRID BAKKER • Department of Oral Cell Biology, ACTA-Vrije
Universiteit, Amsterdam, The Netherlands
WOUTER BEERTSEN • Department of Periodontology, Academic Centre for
Dentistry Amsterdam, The University of Amsterdam, Amsterdam, The
Netherlands
HELENA L. BENFORD • Bone Research Group, Department of Medicine and
Therapeutics, University of Aberdeen, Aberdeen, UK
SHARYN BORD • Bone Research Group, University of Cambridge School of
Clinical Medicine, Cambridge, UK
JADE W. M. CHOW • Department of Cellular Pathology, St. George’s
Hospital Medical School, London, UK
CLAIRE E. CLARKIN • Bone Research Group, Department of Medicine and
Therapeutics, University of Aberdeen Medical School, Aberdeen, UK
PATRICIA COLLIN-OSDOBY • Department of Biology, Washington University;
Division of Bone and Mineral Metabolism, Washington University
Medical School, St. Louis, MO
JULIET COMPSTON • Bone Research Group, Department of Medicine, Uni-
versity of Cambridge School of Clinical Medicine, Cambridge, UK
FRASER P. COXON • Bone Research Group, Department of Medicine
and Therapeutics, University of Aberdeen, Aberdeen, UK
xi