CELLULAR AND MOLECULAR BIOLOGY OF BONE Edited by Masaki Noda Department of Molecular Pharmacology Division of Functional Disorder Research Medical Research Institute Tokyo Medical and Dental University Tokyo, Japan ACADEMIC PRESS, INC. A Division of Harcourt Brace & Company San Diego New York Boston London Sydney Tokyo Toronto Cover photo: Longitudinal section through neonatal rat femur stained with Masson trichrome. Photo courtesy of R. Tracy Ballock, M.D. This book is printed on acid-free paper. @ Copyright © 1993 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press, Inc. 1250 Sixth Avenue, San Diego, California 92101-4311 United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW1 7DX Library of Congress Cataloging-in-Publication Data Cellular and molecular biology of bone / edited by Masaki Noda, p. cm. Includes bibliographical references and index. ISBN 0-12-520225-3 (hardcover) 1. Bone cells. 2. Bones—Molecular aspects. I. Noda, Masaki. QP88.2.C46 1993 599' .087~dc20 93-14783 CIP PRINTED IN THE UNITED STATES OF AMERICA 93 94 95 96 97 98 QW 9 8 7 6 5 4 3 2 1 CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors' contributions begin. Abdul-Badi Abou- Samra (321), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114 Jane E. Aubin (1), Medical Research Council Group in Periodontal Physiology, University of Toronto, Toronto, Ontario M5S 1A8 R. Tracy Ballock (97), Laboratory of Chemoprevention, National In­ stitute of Health, National Cancer Institute, Bethesda, Maryland 20892 Roland Baron (445), Departments of Orthopedics, Cell Biology, and Cell and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510 Jeffrey Bonadio (169), Department of Pathology, Howard Hughes Med­ ical Institute, University of Michigan, Ann Arbor, Michigan 48109 Myles A. Brown (257), Departments of Medicine, Dana Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115 Munmun Chakraborty (445), Departments of Orthopedics, Cell Biol­ ogy, and Cell and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510 Diptendu Chatterjee (445), Departments of Orthopedics, Cell Biolo­ gy, and Cell and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510 Gilbert J. Cote (343), Departments of Medicine and Cell Biology, Bay­ lor College of Medicine and VA Medical Center and Section of Endocrinology, M.D. Anderson Cancer Center, University of Texas, Houston, Texas 77030 Marie Demay (321), Massachusetts General Hospital, and Harvard Medical School, Boston, Massachusetts 02114 xvii • xviii Contributors Randall L. Duncan (413), Renal Division, Jewish Hospital/Washington University, St. Louis, Missouri 63110 Gregor Eichele (287), V. and M. McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030 Robert F. Gagel (343), Departments of Medicine and Cell Biology, Baylor College of Medicine and VA Medical Center and Section of Endo­ crinology, M.D. Anderson Cancer Center, University of Texas, Hous­ ton, Texas 77030 Christopher K. Glass (257), Division of Cellular and Molecular Medicine and, Center for Molecular Genetics, University of California, San Di­ ego, La Jolla, California 92093 Steven A. Goldstein (169), Orthopedic Research Laboratories, Section of Orthopedic Surgery, University of Michigan, Ann Arbor, Michigan 48109 Agamemnon E. Grigoriadis (497), Research Institute of Molecular Pathol­ ogy, A-1030, Vienna, Austria Anne-Marie Heegaard (191), Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892 Johan N.M. Heersche (1), Medical Research Council Group in Periodon­ tal Physiology, University of Toronto, Toronto, Ontario M5S 1A8 William Home (445), Departments of Orthopedics, Cell Biology, and Cell and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510 Keith A. Hruska (413), Renal Division, Jewish Hospital of St. Louis, St. Louis, Missouri 63110 Kyomi Ibaraki (191), Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892 Harald Jiippner (321), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114 Sandra A. Kerner (235), Departments of Pediatrics and Cell Biology, Baylor College of Medicine, Houston, Texas 77030 and Ligand Phar­ maceuticals, Inc., San Diego, California 92121 Janet M. Kerr (191), Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892 Robert A. Kesterson (235), Departments of Pediatrics and Cell Biology, Baylor College of Medicine, Houston, Texas 77030 and Ligand Phar­ maceuticals, Inc., San Diego, California 92121 Seong-Jin Kim (97), Laboratory of Chemoprevention, National Institutes of Health, National Cancer Institute, Bethesda, Maryland 20892 Henry Kronenberg (321), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114 Contributors xix • Jane B. Lian (47), Department of Cell Biology, University of Massa­ chusetts M1edical Center, Worcester, Massachusetts 01655 Sergio Line (539), National Institute of Dental Research, National Insti­ tutes of Health, Bethesda, Maryland 20892 Abderrahim Lomri (445), Departments of Orthopedics, Cell Biology, and Cell and Molecular Physiology, Yale University School of Medi­ cine, New Haven, Connecticut 06510 Meetha Medhora (413), Renal Division, Jewish Hospital/Washington University, St. Louis, Missouri 63110 Lynn Neff (445), Departments of Orthopedics, Cell Biology, and Cell and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510 Keiichi Ozono (235), Departments of Pediatrics and Cell Biology, Baylor College of Medicine, Houston, Texas 77030 and Ligand Pharmaceuti­ cals, Inc., San Diego, California 92121 Sara Peleg (343), Department of Medical Specialities, Section of Endo­ crinology, M. D. Anderson Cancer Center, University of Texas, Hous­ ton, Texas 77030 J. Wesley Pike (235), Departments of Pediatrics and Cell Biology, Baylor College of Medicine, Houston, Texas 77030 and Ligand Pharmaceuti­ cals, Inc., San Diego, California 92121 Jan-Hindrik Ravesloot (445), Departments of Orthopedics, Cell Biology, and Cell and Molecular Physiology, Yale University School of Medi­ cine, New Haven, Connecticut 06510 Craig Rhodes (539), National Institute of Dental Research, National In­ stitutes of Health, Bethesda, Maryland 20892 Felice Rolnick (413), Renal Division, Jewish Hospital/Washington Uni­ versity, St. Louis, Missouri 63110 Gino Segre (321), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114 Susan M. Smith (287), Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706 Teruki Sone (235), Departments of Pediatrics and Cell Biology, Baylor College of Medicine, Houston, Texas 77030 and Ligand Pharmaceuti­ cals, Inc., San Diego, California 92121 Gary S. Stein (47), Department of Cell Biology, University of Massa­ chusetts Medical Center, Worcester, Massachusetts 01655 Christina Thaller (287), V. and M. McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030 1 Present address: Faculdade de Odontalgia de Piracicabe-UNICAMP, Av. Limeira s/n, Caixa Postal 52, 13400 Piracicaba-Sao Paulo, Brazil. • xx Contributors 2 Kursad Turksen (1), Medical Research Council Group in Periodontal Physiology, University of Toronto, Toronto, Ontario M5S 1A8 Erwin F. Wagner (497), Research Institute of Molecular Pathology, A-1030 Vienna, Austria Zhao-Qi Wang (497), Research Institute of Molecular Pathology, A-1030 Vienna, Austria John M. Wozney (131), Genetics Institute, Cambridge, Massachusetts 02140 Yoshihiko Yamada (539), National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892 Kensuke Yamakawa (413), Renal Division, Jewish Hospital/Washington University, St. Lo3uis, Missouri 63110 Toshiyuki Yoneda (375), Department of Medicine, University of Texas Health Science Center, Division of Endocrinology and Metabolism, San Antonio, Texas 78284 Marian F. Young (191), Bone Research Branch, National Institute of Den­ tal Research, National Institutes of Health, Bethesda, Maryland 20892 2Present address: Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637. ^Present address: Division of Molecular Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan 101. PREFACE The study of bone cell biology has been undertaken by multidisciplin- ary groups whose fields cover basic sciences such as developmental biology, molecular endocrinology, genetics, physiology, pharmacol­ ogy, and biochemistry, as well as clinical sciences such as endocrinol­ ogy, orthopedics, and dental medicine. Bone biology has been re­ garded as one of the applied sciences; however, recent progress in bone biology suggests that it could serve as a leading model in each of the above-mentioned disciplines. In the past few years, research in this field has advanced rapidly because of the availability of new technologies and recent developments in biology. Molecular and cel­ lular biological techniques have been the most successful in bone biology research. This rapid progress, however, increases the knowl­ edge gap between researchers—even between those working within the same field of bone biology. To accomplish the goals of bone biology research most efficiently, it is necessary to compile ground-breaking information into a concise format. The purpose of this book is to introduce forefront research in bone biology, where powerful molecular and cellular biological tech­ niques are successfully utilized, as well as to give a comprehensive picture of the most up-to-date information in bone biology. This book offers a concise state-of-the-art view of bone biology and will provide a resource not only for experts in the field, but also for undergraduate students, newcomers, and practitioners. Masaki Noda xxi 1 OSTEOBLASTIC CELL LINEAGE JANE E. AUBIN, KURSAD TURKSEN, and JOHAN N. M. HEERSCHE I. Introduction II. Cells of the Osteoblast Lineage A. General Morphological and Histological Definition B. Osteoblast Phenotypic Expression and Osteoblast Markers HI. Origin and Lineage of the Osteoblast A. Mesenchymal Stem Cells and Multipotential and Restricted Progenitors B. Osteo-Chondroprogenitors IV. Osteoblast Heterogeneity: Subpopulations, Stages of Differentiation, or Aberrant Expression in Vitro? V. Indirect Identification of the Osteoprogenitor Cell A. Bone Formation in Vitro B. A Colony Assay for the Quantification of Osteoprogenitor Cells and Assessment of Their Proliferative and Self-Renewal Capacity C. Regulation of the Osteoprogenitors VI. Monoclonal Antibodies for Identification of Cells in the Osteoblast Lineage VII. Concluding Remarks References Cellular and Molecular Biology of Bone Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved. 1 • 2 Jane E. Aubin et al I. INTRODUCTION Bone formation takes place in the organism during embryonic develop­ ment, growth, remodeling, and fracture repair and when induced experimentally—for example, by the implantation of decalcified bone matrix or purified or recombinant members of the bone morphogenetic protein family (Reddi, 1985; Urist, 1989; Wozney et al, 1990; Wozney, 1992). There is clearly a large reservoir of cells in the body capable of osteogenesis throughout life. During the past decade, new methods have been developed to study the cell biology of bone and gain insight into the various cell types important in bone function (for reviews, see Rodan and Rodan, 1984; Nijweide et al, 1986,1988; Heersche and Aubin, 1990; Aubin et al, 1990b). It has also become increasingly clear that the metabolic activities of bone are under the control of a large number of systemic and local factors (Martin et al, 1987; Stern, 1988; Marcus, 1988; Martin, 1989; Mundy, 1989). Despite these advances, many questions remain. For example, detailed knowledge of the lineage of the osteo­ blast, including identification of transitional steps from stem cell to com­ mitted osteoprogenitor to osteoblast, interactions of cells within the lin­ eage, and identification and regulation of stem cells and different levels of committed progenitors, is largely lacking. This chapter provides a review of the osteoblast lineage and possibilities for recognizing stages of differentiation or maturity. As such, it reviews current concepts of the origin, lineage, and differentiation of osteoblasts and currently available tools to study them, with emphasis on in vitro model systems. II. CELLS OF THE OSTEOBLAST LINEAGE A. General Morphological and Histological Definition Based on morphological and histological studies, osteoblastic cells are categorized in a presumed linear sequence progressing from osteo­ progenitor to preosteoblasts, to osteoblasts, and then to lining cells or osteocytes (Nijweide et al, 1986; Martin et al, 1987; Marks and Popoff, 1988; Bonucci, 1990; Wlodarski, 1990). Earlier morphological definitions of the active osteoblast as a cuboidal, polar, basophilic cell lining the bone matrix at sites of active matrix formation (Cameron, 1968; Holtrop, 1975) have been supplemented more recently by elucidation of their specific products—for example, type I collagen (Leblond, 1989), osteo­ calcin (Hauschka et al, 1989), osteopontin (SPP1) (Butler, 1989), and bone sialoprotein (Sodek et al, 1992a,b). Active osteoblasts give a strong histo- chemical reaction for alkaline phosphatase (APase) that disappears when cells cease their synthetic activity (Doty and Schofield, 1976) or become embedded in matrix as osteocytes (Holtrop, 1975). However, a number of criteria, such as morphology (Marotti, 1976; Villaneuva et al, 1 Osteoblastic Cell Lineage 3 • 1981), biosynthetic activity detected by biochemical analysis (Otawara and Price, 1986), immunohistochemistry (Mark et al., 1988) or in situ hybridization (Heersche et al, 1992), suggest that newly differentiated osteoblasts (cuboidal, osteocalcin low or negative) differ from more ma­ ture osteoblasts later in their secretory lifetime (more flattened osteo­ calcin high). Thus, maturational stage—not only stage of differentia­ tion—ultimately will have to be elucidated with these and other markers (see later). In a region where osteoblasts are laying down bone matrix, the cuboidal cells directly behind them have been called preosteoblasts (Pritchard, 1952; Luk et al., 1974). Based on kinetic studies, it has been suggested that preosteoblasts are the precursors of the osteoblast in the regions of growing bone (Owen, 1963, 1967; Kember, 1971). Pre­ osteoblasts morphologically resemble the osteoblast and show some markers of the osteoblast (e.g., APase activity [Doty and Schofield, 1976]), but they are clearly recognizably different from osteoblasts in not expressing others (see later). Other possibly earlier precursor cells may reside in the heterogeneous layer of proliferating cells behind the osteo- blast/preosteoblast layer (Pritchard, 1972a,b). These earlier cells may be osteoprogenitor cells (Young, 1962). In addition to their position in the tissue near bone surfaces, osteoprogenitors are fibroblastic or spindle- shaped with oval or elongated nuclei and notable glycogen content (Scott, 1967). Their appearance and location in the tissues are the main criteria to define their presence. It seems likely, but there is no proof, that the farther away from the bone surface the osteogenic cell is the less differentiated it will be (Scott, 1967). The osteocyte is considered the most mature or terminally differenti­ ated cell of the osteoblast lineage (Jande and Belanger, 1973; Holtrop, 1975). Osteocytes are embedded in bone matrix occupying spaces (la- cuanae) in the interior of bone and are connected to adjacent cells by cytoplasmic projections within channels (canaliculi) through the miner­ alized matrix (Menton et al., 1984). The presence of gap junctions be­ tween the cytoplasmic projections is thought to allow these cells to communicate. Some, but not all, of the biochemical features of the osteo­ blast are expressed in the osteocyte (see also later). In the adult, the majority of bone surfaces are occupied by another cell type with a distinct phenotype, the bone lining cell, which displays a flat and highly elongated cell shape with a spindle-shaped nucleus (Menton etal., 1984). Bone lining cells are usually designated as part of the osteoblast lineage because they are believed to be derived from osteo­ blasts that have ceased their activity and flattened out on bone surfaces that are undergoing neither formation nor resorption (Luk et al., 1974; Miller and Jee, 1987). They have fewer organelles than the active osteo­ blast (Cameron, 1968), further suggesting that they may be largely inac-