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Bone Regeneration and Repair: Biology and Clinical Applications provides current information regarding the biology of bone formation and repair, reviews the basic ence of autologous bone

Bone Regeneration

and Repair

Bone Regeneration

Contents i

Bone Regeneration and Repair

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ii Contents

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iv Contents

© 2005 Humana Press Inc.

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Cover illustrations: Figure 5, Chapter 14, “Bone Grafting for Total Joint Arthroplasty: Biology and Clinical tions,” by M Hamadouche et al Figure 2B, Chapter 8, "Grafts and Bone Substitutes," by D Sutherland and

Applica-M Bostrom.

Cover design by Patricia F Cleary.

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Library of Congress Cataloging in Publication Data

Bone regeneration and repair : biology and clinical applications / edited by Jay R Lieberman and Gary E Friedlander.

p ; cm.

Includes bibliographical references and index.

ISBN 0-89603-847-5 (alk paper) e-ISBN 1-59259-863-3

1 Bone regeneration 2 Regeneration (Biology) 3 Transplantation of organs, tissues, etc.

[DNLM: 1 Bone Regeneration—physiology 2 Bone Regeneration—genetics 3 Bone and Bones—physiology.

WE 200 B71285 2005] I Lieberman, Jay R II Friedlander, Gary E.

as bone graft incorporation However, in some circumstances the regenerative capacity

of bone is altered or damaged in a manner that precludes such a special pattern of repair.Fracture nonunions, lost bone stock supporting total joint arthroplasties, and periodontaldefects are frustrating examples of these difficult clinical challenges Allogeneic boneand even autogenous bone grafts have not provided solutions for all these problems, attimes related to limitations of their regenerative capacities and also when not used in amanner that respects their biological or biomechanical needs

Over the past few decades, scientists and clinicians have been exploring the use ofgrowth factors and bone graft substitutes to stimulate and augment the body’s innateregenerative capabilities The development of recombinant proteins and the applica-tion of gene therapy techniques could dramatically improve treatment for disorders ofbone, cartilage and other skeletal tissues

Bone Regeneration and Repair: Biology and Clinical Applications provides current

information regarding the biology of bone formation and repair, reviews the basic ence of autologous bone graft, skeletal allografts, bone graft substitutes, and growthfactors, and explores the clinical applications of these exciting new technologies Anoutstanding group of contributors has thoughtfully and skillfully provided currentknowledge in this exciting area This book should be of value to those in training,clinicians, and basic scientists interested in regeneration and repair of the musculoskel-etal system

sci-Jay R Lieberman, MD

Gary E Friedlaender, MD

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Contents

vii

Preface vColor Illustrations ixContributors xi

1 Bone Dynamics: Morphogenesis, Growth Modeling, and Remodeling

Jeffrey O Hollinger 1

2 Fracture Repair

Charles Sfeir, Lawrence Ho, Bruce A Doll, Kodi Azari,

and Jeffrey O Hollinger 21

3 Common Molecular Mechanisms Regulating Fetal Bone Formation

and Adult Fracture Repair

Theodore Miclau, Richard A Schneider, B Frank Eames,

and Jill A Helms 45

4 Biology of Bone Grafts

Victor M Goldberg and Sam Akhavan 57

5 Cell-Based Strategies for Bone Regeneration:

From Developmental Biology to Clinical Therapy

Scott P Bruder and Tony Scaduto 67

6 Biology of the Vascularized Fibular Graft

Elizabeth Joneschild and James R Urbaniak 93

7 Growth Factor Regulation of Osteogenesis

Stephen B Trippel 113

8 Grafts and Bone Graft Substitutes

Doug Sutherland and Mathias Bostrom 133

9 Gene Transfer Approaches to Enhancing Bone Healing

Oliver Betz, Mark Vrahas, Axel Baltzer, Jay R Lieberman,

Paul D Robbins, and Christopher H Evans 157

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viii Contents

10 Bone Morphogenic Proteins and Other Growth Factors to Enhance

Fracture Healing and Treatment of Nonunions

Calin S Moucha and Thomas A Einhorn 169

11 The Ilizarov Technique for Bone Regeneration and Repair

James Aronson 195

12 Biology of Spinal Fusion: Biology and Clinical Applications

K Craig Boatright and Scott D Boden 225

13 Bone Allograft Transplantation: Theory and Practice

Henry J Mankin, Francis J Hornicek, Mark C Gebhardt,

and William W Tomford 241

14 Bone Grafting for Total Joint Arthroplasty:

Biology and Clinical Applications

Moussa Hamadouche, Daniel A Oakes, and Daniel J Berry 263

15 Biophysical Stimulation Using Electrical, Electromagnetic,

and Ultrasonic Fields: Effects on Fracture Healing and Spinal Fusion

James T Ryaby 291

16 Vascularized Fibula Grafts: Clinical Applications

Richard S Gilbert and Scott W Wolfe 311

17 Craniofacial Repair

Bruce A Doll, Charles Sfeir, Kodi Azari, Sarah Holland,

and Jeffrey O Hollinger 337

18 Bone Regeneration Techniques in the Orofacial Region

Samuel E Lynch 359

Index 391

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Contents ix

Color Illustrations

The following color illustrations are printed in the insert that follows page 212:

Chapter 1, Figure 3B, p 10: Cutting cone in BMU

Chapter 3, Figure 1, p 47: Gene expression during mesenchymal cell condensation

and cartilage development

Chapter 3, Figure 2, p 49: Gene expression during cartilage maturation, vascular

invasion, and ossification

Chapter 3, Figure 3, p 52: Gene expression during early, intermediate, and late

stages of nonstabilized fracture healing

Chapter 5, Figure 2, p 69: Mid-diaphysis of a stage 35 embryonic chick tibia

Chapter 5, Figure 7, p 78: Reactivity of antibodies SB-10 and SB-20 in longitudinal

sections of developing human limbs

Chapter 15, Figure 3, p 296: Three-dimensional reconstructions of rat trabecular

x Contents

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JAMES ARONSON, MD • Chief of Pediatric Orthopaedics, Arkansas Children’s Hospital,

Professor, Departments of Orthopaedics and Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR

KODI AZARI, MD • University of Pittsburgh School of Medicine, Pittsburgh, PA

AXEL BALTZER, MD • Praxis und Klink für Orthopaedie, Dusseldorf, Germany

DANIEL J BERRY, MD • Orthopaedic Department, Mayo Clinic, Rochester, MN

OLIVER BETZ, P h D • Center for Molecular Orthopaedics, Harvard Medical School, Boston,

MA

K CRAIG BOATRIGHT, MD • The Emory Spine Center, Department of Orthopaedic Surgery,

Emory University School of Medicine, Atlanta, GA

SCOTT D BODEN, MD • The Emory Spine Center, Department of Orthopaedic Surgery,

Emory University School of Medicine, Atlanta, GA

MATHIAS BOSTROM, MD • Hospital for Special Surgery, New York, NY

SCOTT P BRUDER, MD, P h D • Department of Orthopaedics, Case Western Reserve University,

Cleveland, OH; and DePuy Biologics, Raynham, MA

BRUCE A DOLL, DDS, P h D • Department of Periodontics, University of Pittsburgh

School of Dental Medicine, Pittsburgh, PA

B FRANK EAMES • Department of Orthopaedic Surgery, University of California, San

Francisco, CA

THOMAS A EINHORN, MD • Professor and Chairman, Department of Orthopaedic Surgery,

Boston Medical Center, Boson University School of Medicine, Boston, MA

CHRISTOPHER H EVANS, P h D • Center for Molecular Orthopaedics, Harvard Medical

School, Boston, MA

GARY E FRIEDLAENDER, MD • Department of Orthopaedics and Rehabilitation, Yale

University School of Medicine, New Haven, CT

MARK C GEBHARDT, MD • Chief of Orthopaedics, Beth Israel Deaconess Hospital and

Othopaedic Oncology Service, Children’s Hospital, Boston, MA

RICHARD S GILBERT, MD • Mount Sinai School of Medicine, New York, NY

VICTOR M GOLDBERG, MD • Department of Orthopaedic Surgery, Case Western Reserve

University, Cleveland, OH

MOUSSA HAMADOUCHE, MD, P h D • Department of Orthopaedic and Reconstructive Surgery

(Service A), Centre Hospitalo-Universitaire Cochin-Port Royal, Paris, France

JILL A HELMS, DDS, P h D • Department of Orthopaedic Surgery, University of California,

San Francisco, CA

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xii Contents

SARAH HOLLAND, MD • University of Pittsburgh School of Medicine, Pittsburgh, PA

JEFFREY O HOLLINGER, DDS, P h D • Bone Tissue Engineering Center, Departments of

Biomedi-cal Engineering and BiologiBiomedi-cal Sciences, Carnegie Mellon University, Pittsburgh, PA

LAWRENCE HO • University of Pittsburgh School of Medicine, Pittsburgh, PA

FRANCIS J HORNICEK, MD, P h D • Orthopedic Oncology Service, Massachusetts General

Hospital and Children’s Hospital, Harvard Medical School, Boston, MA

ELIZABETH JONESCHILD, MD • Seattle Hand Surgery Group, Seattle, WA

JAY R LIEBERMAN, MD • Department of Orthopaedic Surgery, David Geffen School of

Medicine, University of California, Los Angeles, CA

SAMUEL E LYNCH, DMD, DMS c • BioMimetic Pharmaceuticals Inc., Franklin, TN

HENRY J MANKIN, MD • Orthopedic Oncology Service, Massachusetts General Hospital

and Children’s Hospital, Harvard Medical School, Boston, MA

THEODORE MICLAU, MD • Department of Orthopaedic Surgery, University of California,

San Francisco, CA

CALIN S MOUCHA, MD • Division of Adult Joint Replacement & Reconstruction, Department

of Orthopedics, New Jersey Medical School, University of Medicine & Dentistry of New Jersey (UMDMJ), Newark, NJ

DANIEL A OAKES, MD • Orthopaedic Department, Mayo Clinic, Rochester, MN

PAUL D ROBBINS, P h D • Department of Molecular Genetics and Biochemistry, University

of Pittsburgh School of Medicine, Pittsburgh, PA

JAMES T RYABY, P h D • Senior Vice President, Research and Development,

OrthoLogic Corp., Tempe, AZ

RICHARD A SCHNEIDER, P h D • Department of Orthopaedic Surgery, University of California,

San Francisco, CA

TONY SCADUTO, MD • Shriners Hospitals for Children, Los Angeles, CA

CHARLES SFEIR, DDS, P h D • University of Pittsburgh School of Dental Medicine, and

Bone Tissue Engineering Center, Carnegie Mellon University, Pittsburgh, PA

DOUG SUTHERLAND, MD • Hospital for Special Surgery, New York, NY

WILLIAM W TOMFORD, MD • Orthopedic Oncology Service, Massachusetts General

Hospital and Children’s Hospital, Harvard Medical School, Boston, MA

STEPHEN B TRIPPEL, MD • Department of Orthopaedic Surgery, Indiana University

School of Medicine, Indianapolis, IN

JAMES R URBANIAK, MD • Division of Orthopaedic Surgery, Duke University Medical

Center, Durham, NC

MARK VRAHAS, MD • Center for Molecular Orthopaedics, Harvard Medical School, Boston,

MA

SCOTT W WOLFE, MD • Professor of Orthopedic Surgery, Weill Medical College of

Cornell University, and Attending Orthopedic Surgeon, Hospital for Special Surgery, New York, NY

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Bone Dynamics 1

1

From: Bone Regeneration and Repair: Biology and Clinical Applications

Edited by: J R Lieberman and G E Friedlaender © Humana Press Inc., Totowa, NJ

skel-Considerable information is available in the literature on bone morphogenesis, growth, modeling,and remodeling However, in preparing this review, it struck me that the line distinguishing growth,modeling, and remodeling, curiously, was sometimes gossamery Fundamental and guiding buildingblocks from seminal publications of several distinguished workers helped focus my attention on keyelements embodying definitions and principles necessary for a review chapter

This chapter will provide a landscape of events embracing morphogenesis, growth, modeling, andremodeling The benefits enjoyed by this author during the writing of this chapter are the sinew andpower to inspire admiration and respect for the complexities and unity of form and function of the

206 bones of the skeleton (1) I share this with you.

WORKING DEFINITIONS AND FOUNDATIONAL PRINCIPLES

Consensus definitions for knotty physiological processes can provide a sturdy platform for dialog.The underpinning for the chapter definitions was scoured from several sources, timeless epistles, con-solidated, and reduced by the author The curious reader can seek additional enlightenment and moredetail in references provided

Morphogenesis begets growth Morphogenesis is a consummate series of events during genesis, bringing cells together to permit inductive opportunities; the outcome is a three-dimensional

embryo-structure, such as a bone (2) The term growth embraces processes in endochondrally derived, lar bones that increase length and girth prior to epiphyseal plate closure (3) Intramembranous bone,

tubu-not tubular in general form, but curved and platelike, without physes, enlarges in size under the aegis

of a genetic script and then stops In the cranium, the physis analog is the fontanelle Fontanelles such

as the bregmatic, frontal, occipital, mastoid, and sphenoidal provide linear space for growth (i.e.,enlargement, increase in size) Heuristically, bone growth presupposes genetic controls prompting cellmitogenesis, differentiation, quantitative amplification, and enlargement (increase in cell mass andsize)

Nononcologic cells have a built-in “governor” for cell divisions For example, human fetal blasts can undergo 80 cycles of cell divisions, whereas fibroblasts from an adult stop after about 40divisions, and interestingly, embryonic mice fibroblasts stop at 30 divisions Mechanisms controlling

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2 Hollinger

cell divisions are generally unknown; however, cyclin-dependent kinase inhibitor proteins, decrement

in cyclin-dependent kinases, and cell contact-dependent cell–cell interactions have been implicated (4,

5) From an embryological perspective, morphogenetic codes directing cell populations prompt

induc-tive interactions for building three-dimensional structures (2) A morphogenetic code could provide the

guidelines ruling cell numbers, size, and growth Therefore, growth may be perceived as dynamic eventsmentored by molecular cues

The process that permits bone growth is modeling, an active pageantry of cells embraced in ious partnerships Cells eagerly craft the growing 206 (1) bones using a three-dimensional blueprint

myster-that permits clinical recognition of a bone, whether it is the femur of the 6-month-old infant, a old toddler, a 14-year old teenager, a 30-year-old surgery resident, or a 70-year-old professor emer-itus The preprogrammed architectural mold is a translation from an as yet to be deciphered genetictome, hormonal directives (e.g., growth hormone), and mechanical cues: “modeling must alter both

3-year-the size and architecture” (6,7).

The final product of growth and modeling is a skeletal complex of 206 adult bones demanding

continuous maintenance, which is accomplished by remodeling Remodeling sustains structure and

patches blemishes in the adult skeleton, while responding to homeostatic demands to ensure calciumand phosphate balance: “remodeling… [is] replacement of older by newer tissue in a way that need

not alter its gross architecture or size” (6,7).

In summary, as described in several recent reports (8–14) and stated succinctly by Frost; “Growth determines size Modeling molds the growing shape Remodeling then maintains functional compe- tence” (6,7).

MORPHOGENESIS AND GROWTH

For modeling to occur, there must be a structure to model Fundamental questions need to be posed:(1) Why (and how) does a congregation of cells occur in a designated positional address? (2) Why(and how) do cells of that congregation produce a structure recognized as “a bone”? Molecular cues

is the obvious answer They drive cells, cells interact with other cells, and a structure, bone, takesshape However, the response “molecular cues” spawns another query: Why are certain molecular cues

expressed? Morphogenesis is the consummate porridge of molecular cues, and the inspiration for the cues is tangled in the genetic code Morphogenesis begets growth, which begets modeling.

Morphogenesis is an epochal series of events during embryogenesis that brings cells together for

inductive opportunities; the outcome is the skeletal system Morphogenesis and bone are linked to a

powerful family of cell morphogens: bone morphogenetic protein (15,16) There are other key tive morphogens that will be noted (17).

induc-Morphogenesis involves control centers with positional addresses in the developing embryo, wherecells of that center regulate other cells through signaling factors The signaling factors are proteins en-

coded by conserved multigene families; some multigene examples include bone morphogenetic proteins

(bmp), epidermal growth factors (egf), fibroblast growth factors (fgf), hedgehogs, and Wnts (2,17–26).

The hedgehog family in vertebrates consists of three homologs of the Drosophila melanogaster

hedgehog gene: desert hedgehog, Indian hedgehog, and sonic hedgehog (shh) Shh may be the most

important for the skeletal system, in that it mediates formation of the right–left axis (chicks) and

initiates the anterior–posterior axis in limbs Shh in limb bud formation induces fgf4 expression, which acts with Wnt7 The name Wnt comes from fusing the D melanogaster segment polarity gene wing-

less with the name of its vertebrate homolog integrated.

Signaling centers destined to be limb buds consist of aggregations of mesenchymal and epithelial

cells and may be under the control of fgf8, fgf10, and shh (27) (reviewed in ref 2) There are four axial levels where mesenchymal–ectodermal aggregates interact, called the apical ectodermal ridge (AER) (28) Here, four limb buds form, and in the posterior zone of the AER, at the zone of polarizing

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Bone Dynamics 3

activity (ZPA), shh acts as a mitogen for mesenchymal cells Wisps of mesenchymal tissue stream in

a centrifugal direction from the midline, and a further consolidation of cell phenotypes occurs, givingshape and form to a chondrogenic anlagen, where chondrocytes predominate and types II, IX, and XI

collagens prevail (reviewed in ref 13).

Clusters of genes, homeobox genes (Hox genes), ensure limb bud location and limb constituents (reviewed in refs 18 and 29) In mice with abnormalities in expression of Hox genes, loss of digits can occur (associated with Hoxa, Hoxd (30), and Hoxd-13 may cause syndactyly in humans (31) The shh mediates anteroposterior patterning for metatarsal and metacarpals, as well as orchestrat- ing expression of bmps, fgfs, and Sox9 (the cartilage gene regulator for endochondral bone formation) (reviewed in ref 17) Shh prompts fgf4 expression in ectoderm, bmp2 expression in mesoderm (32), and regulates anterior–posterior positioning and distal limb growth (33).

With these cues flying around during morphogenesis, there is a potential for cells to get “confused.”Through unidentified mechanisms, recklessness is not the rule, but rather, coordination and harmony

among cells and cueing molecules propel growth The process of growth and the dynamics of

model-ing (i.e., shapmodel-ing growmodel-ing bones) produce delicate digits, lovely shaped incus, maleus, and stapes,and the hulky femur In addition to the signals for mitogenesis and differentiation, there are signals

for programmed cell death: apoptotic signals.

As a symphony of life and death events, embryogenesis is a marvelous consortium of movementshoned by a molecular tool kit that determines where congregations of cells will occur, the interactionsamong the cells, and the shape, size, and position of structures derived from that congregation, as well

as the death of cells Bundling of molecules in selectively positioned batches direct body position, form,cell, tissue, and organ development This concept is underscored by the work reported by Storm and

colleagues (21,34) on brachyopodism in mice (caused by a mutation in growth differentiation factors

5, 6, and 7) and by evidence from Kingsley on the short-eared mouse (associated with a corruption in the genetic coding for bmp5) (19) The short-ear null mutation causes alterations in the size and shape

of ears, sternum, and vertebrae that do not affect size and shape of limbs In contrast, brachypodismnull mutations reduce the length of limb bones and the number of segments in the digits but do notaffect ears, sternum, ribs, or vertebrae Explanation for the two phenotypes is that a mosaic for signal-ing centers exists, and during embryogenesis, some of the tiles in the mosaic become corrupted Theoutcome is determined by the tiles corrupted

During embryogenesis, controlling gates must be invoked to either stop or redirect events; cellular stopping mechanisms broadly may include cell contact and extracellular inhibitory signals

extra-Bmps are powerful, proactive inducers of events and must be tempered A family of anti-bmps has been

identified, and includes noggin, fetuin, chordin, cerberus, and DAN (reviewed in ref 35) Noggin onizes bmp-induced chondrocyte apoptosis (36) (Chondrocyte apoptosis is required for joint forma- tion [37]) When noggin expression is disrupted in mice, multiple skeletal defects occur, including

antag-short vertebrae, malformed ribs and limbs, and the absence of articulating joints In terms of cartilagedevelopment and the growth of bone, growth differentiating factor-5 appears to be required in mice

for joint cartilage, as long as cartilage-inducing signals from bmp-7 are absent (34).

Intracellular stopping mechanisms exist as well, and for bone may include the intracellular

signal-ing molecules known as smads (the mammalian homolog to the D melanogaster gene Mothers against

decapentaplegic) (38,39) Smad is a contraction of D melanogaster Mothers against decapentaplegic

—dpp—and Caenorhabditis elegans Sma Bmps bind to serine–threonine transmembrane receptors, causing receptor phosphorylation, which activates a smad complex that transduces a signal to the cell

nucleus and transcription ensues (Fig 1) (35) (More will be said about this in the section on blasts.) Other smad complexes abrogate the process (reviewed in ref 35).

osteo-To this point, considerable information has been mentioned about cues, and nothing yet on thecellular craftsmen executing functions that result in growth and modeling Pluripotential cells thatcan become chondrogenic, osteoblastic, and osteoclastic lineage cells will be mentioned next

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4 Hollinger

Chondrocytes

Limb buds containing pluripotential mesenchymal cells destined to develop through endochondral

bone formation express type IIb collagen, a chondrocyte-unique transcript of the alpha1(II) gene, type

IX and type XI collagens, and matrix glutamic acid (gla) protein (reviewed in ref 25) Implicated in transcriptional control of chondrocyte differentiation has been Sox9 (17,40) Sox9 and type II colla-

Fig 1 BMP receptor binding and intracellular signal transduction BMPs bind types I and II

serine/threo-nine kinase receptors (BMPR-1 A/B and BMPR-II) to form a heterodimer Following binding, the type II tors phosphorylate (P) the glycine/serine-rich domain of the type I receptor The type I receptor phosphorylates the MH2 domain (Smad homology domain) of Smads 1, 5, and possibly 8 (Smad 6 may block the phosphory- lation cascade by binding the type I receptor.) Following phosphorylation, the Smad1,5,8 complex either may bind to Smad 4 and translocate to the nucleus or may bind to Smad 6 and the signal is terminated The Smad1, 5,8–Smad 4 complex translocated across the nuclear membrane can activate gene transcription either directly or indirectly through activation of the osteoblast-specific factor-2 (Osf2) (With permission from Schmitt, J M., Hwang, K., Winn, S R., and Hollinger, J O [1999] Bone morphogenetic proteins: An update on basic biology

recep-and clinical relevance J Orthoped Res 17, 269–278.

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Bone Dynamics 5

gen are chondrocyte-specific genes coexpressed by chondrogenic lineage cells Chondrocyte

differ-entiation, maturation, and hypertrophy appear to be controlled by fibroblast growth factors, fibroblast

growth factor receptors (41), parathyroid hormone-related peptide (PTHrP) (42,43), and the teinase gelatinase B (44).

metallopro-PTHrP controls the rate of differentiation of chondrocytes into hypertrophic chondrocytes Forexample, bone explants exposed to elevated PTHrP have a delayed differentiation of hypertrophiedchondrocytes; in PTHrP-deficient mice, there is premature differentiation of chondrocytes into hyper-

trophic chondrocytes (reviewed in ref 25) The upstream regulator for PTHrP is modulated by Indian

hedge hog (Ihh), a gene product localized to the cartilage anlagen in endochondral bone (45,46).

Until closure of the physes, long bones lengthen and increase in girth Physeal energies for

elon-gation are stimulated by growth hormone (GH), inspiring chondrocytes to express insulin-like growth factor-I (IGH-I) Acting in an autocrine manner, IGH-I “self-inspires” chondrocytes to express more IGH-I, proliferate, and, in a paracrine mode, incite other chondrocytes in a likewise fashion Osteo- blasts secrete IGF-I in response to PTH and GH; these factors are osteoanabolic, thus adding in expan- sion of girth (reviewed in ref 47).

Evidence suggests that fibroblast growth factor receptor 3 (fgfr3) negatively controls growth by limiting chondrocyte proliferation: absence of fgfr3 results in prolonged skeletal overgrowth (in mice)

(48) The metalloproteinase gelatinase B, a catalytic enzyme that is present in the extracellular matrix

of cartilage, appears to control the final component of chondrocyte maturation, apoptosis, and

vascu-larization (44).

Vascularization of hypertrophic cartilage heralds calcification of the chondrocytes followed byprogrammed cell death (i.e., apoptosis) Streaming toward the calcified chondrocyte Cathedral are pluri-potential mesenchymal cells destined to become chondroclasts, osteoblasts, and myeloid-derived cells,the osteoclast precursors

Osteoblasts and Osteocyctes

During the complicated processes of embryogenesis, dorsal–ventral orientation, and limb bud

devel-opment, a symphony of signaling cues (bmps, bmp-like molecules, fgf, homeobox gene products,

Ihh, shh, TGF- β, and Wnt) weave a tapestry providing positional addresses for groups of

pluripoten-tial cells as well as fate-determining cues (2,15,16,19,20,28,32,49–52) Cues for osteoblast lineage cell progression strongly suggest that the initiator is certain bmps, members of the TGF- β clan and bmp-

like gene expression products (growth differentiation factor-5, gdf-5) (53–59) (Certain bmps—except

bmp-1—cause osteoblast differentiation; TGF-β stimulates proliferation and can inhibit differentiation

[60]) The differentiation tempo is sustained through mediation with anti-bmps (e.g., noggin, chordin, fetuin, DAN, cerberus, reviewed in refs 35, 61, and 62) that can short-circuit binding to cognate recep-

tors, serine–threonine transmembrane receptor–ligand binding (20,63–65), and transmembrane signal transduction through smads The Smads shuttle signals received from receptor interaction with TGF-β

and BMPs (i.e., the ligands) to the nucleus, where another set of signals begins

Within the cell nucleus, an activated Smad complex can usher in the nuclear activities encoded by

DNA (reviewed in ref 35) (Fig 2) The process includes the nuclear transcriptional factor Runx-2 (a.k.a.core binding factor A: cbfa-1), which can stimulate expression of specific genes leading to differen-

tiation of the osteoblast phenotype (66–68) Runx-2 is a unique nuclear transcription factor for blast differentiation (reviewed in refs 35, 69, and 70) It is hypothesized that activation of Runx-2

osteo-results from a chain of events beginning with BMP-initiated receptor interaction, followed by lular Smad signaling

intracel-The smad-activated complex transits the cell cytoplasm, crosses the nuclear membrane, and binds

to DNA, where it induces a transcriptional response for Runx-2 Runx-2 gene activation initiates

expres-sion of Runx-2 protein, which binds to the osteocalcin transcription promoter, heralding osteoblast

differentiation (23) Osteocalcin and Runx-2 are osteoblast icons.

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6 Hollinger

In homozygous deficient Runx-2 mice, no osteoblasts form, and mice die postpartum due to costal muscle incompetency (66) Heterozygously mutated mice for Runx-2 have a phenotype con-

inter-sistent with cleidocranial dysostoses, the autosomal dominant disease characterized by hypoplastic

clavicles, open fontanelles, supernumerary teeth, and short stature (71).

The fate of the hard-working osteoblast can follow three pathways: programmed cell death

(apop-tosis), lining cells, and osteocytes Apoptosis is the pathway most frequently taken, followed in order

by osteocytes and lining cells Osteocytes and lining cells are required to sustain bone viability and to

respond to biomechanical signals These two phenotypes will be addressed in more detail in the tion on remodeling

sec-Osteoclasts

Balancing bone formation in the developing embryo and through the maturational period is the

osteoclast, which is derived from the monocyte (reviewed in refs 72 and 73) It is generally concluded

the osteoclast resorbs bone during growth, modeling, and remodeling

Several factors have been associated with osteoclast formation, including PTH, PTHrP, vitamin

D3, interleukins-1, -6, and -11, tumor necrosis factor (TNF), leukemia inhibitory factor, ciliary

neuro-tropic factor, prostaglandins, macrophage colony-stimulating factor (M-CSF), c-fms, c-fos,

granulo-Fig 2 The osteocalcin gene regulation is controlled by a promoter region where several specific nuclear

proteins can activate gene transcription Osteoblast-specific factor-2 (OSF-2) binds to the osteoblast-specific element-2 (OSE-2) by its runt domain Following this action the TATA box, a nucleotide sequence with T– thymine nucleotide–and A–adenine nucleotide, binds RNA polymerase II (Pol II) This complex transcribes the osteocalcin genetic sequence into mRNA (messenger ribonucleic acid) The mRNA is translated into the osteo- calcin protein on ribosomes The illustration also shows that within the osteocalcin promoter region is the gene- tic sequences for mouse osteocalcin E-box sequence-1 (mOSE1) and osteoblast specific elelment-1 (OSE1) (bp stands for base pairs.) (Modified and with permission from Schmitt, J M., Hwang, K., Winn, S R., and Hollinger,

J O [1999] Bone morphogenetic proteins: an update on basic biology and clinical relevance J Orthoped Res.

17, 269–278.

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line-Just as the osteoblast has a specific differentiation transcription factor (i.e., Runx-2), the factor for

the osteoclast is PU1 (78) PU1-deficient mice are osteopetrotic, lacking osteoclasts and macrophages

(78) Another transcription factor whose omission leads to osteopetrosis in mice is c-fos (79).

Osteoclasts anchor to the surface of bone previously occupied by osteoblasts, and they do so throughintegrin extracellular matrix receptors: αvβ3 (a vitronectin-type receptor), α2β1 (a collagen receptor),

and αvβ1 (80) In addition, osteopontin helps osteoclasts, as well as osteoblasts, stick to bone (12).

Skeletal growth is a multidimensional, genetically coded process that destines size A community

of cells with a determined social hierarchy, bonded by signaling cues, sculpt growing bones, a

pro-cess called modeling.

MODELING

Cells alter the shape and size of bone Is this growth or modeling? Appendicular bones grow inlength and girth Physeal growth centers permit elongation, whereas the periosteal surface moves cen-trifugally, powered by osteoblastic deposition Concurrently, endosteal growth proceeds centripetally,with a quanta of osteoclastic activity slowly enlarging the zone of bone marrow The growth of appen-dicular bones maintains a gross morphology so the appearance of the pediatric “little” femur looksremarkably like the “adult” femur In contrast, the axial and craniofacial skeletons do not possess physealgrowth centers Therefore, the axial growth for the vertebral bodies proceeds through a periosteal sur-face deposition titrated precisely with an endosteal deposition–resorption component The adjective

“drifts” (6,7,81,82) describe the waves of osteoblastic formation and osteoclastic resorption that move

and mold bone in four dimensions: volume and time This movement during growth is accomplished

by the process of modeling

The U-shaped mandible, mid-, and upper facial and cranial complexes may be viewed as plates ofbones mortised together, with fontanelles in the cranial complex and formation and resorption driftsenabling expansion for brain growth The skeletal complex of the cranium and upper face are oftenincorrectly described as “flat” bones Studying a skull and midface, average freshman predental andpremedical students would agree there is nothing “flat” in that area Rather, gentle curves prevail anddefine the format Therefore, bones of the craniofacial complex are correctly and accurately described

as “curved” bones Over 30 years ago, Enlow noted the intricate patterns of shaping, reshaping,

resorp-tion, and formation drifts of the growth of curved bones (81,82).

Instructional guidance for growth (which includes shape and size) of osseous skeletal elements is

controlled hormonally, and at pubescence the hormonal spigot is turned off, where GH, for example,

is quenched—growth ceases

Sustaining shape and size of bone in the adult skeleton is accomplished by the process of

remod-eling, where damaged bone is ceaselessly replaced by a tireless workforce of cells (6,7,13) But does

modeling really cease in the adult skeleton? The complexity of physiological issues and definitionsoften mire down rational dialog about the modeling and remodeling activities Are the differences

relative to timing? Relative to differences among processes? Frost proposes (6,7), and Kimmel scores (8), that processes of macromodeling and minimodeling continue in the adult skeleton, where

under-macromodeling increases the ability of bone to resist bending (by expanding periosteal and endostealcortices) and minimodeling rearranges trabeculae to best adapt to functional challenges

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