Physiology and Pharmacology of Bone - GBV · Physiology and Pharmacology of Bone Contributors A.-B. Abou-Samra, D.C. Anderson, H.C. Anderson, R. Baron, W. Born, F.R. Bringhurst, E.

Physiology andPharmacology of BoneContributorsA.-B. Abou-Samra, D.C. Anderson, H.C. Anderson, R. Baron,W. Born, F.R. Bringhurst, E. Canalis, M. Centrella,M. Chakraborty, D. Chatterjee, P.D. Delmas, J.A. Eisman,E.F. Eriksen, D.M. Findlay, J.A. Fischer, H. Fleisch, J.K. Heath,W. Home, H. Jiippner, M. Kassem, H.M. Kronenberg,L.E. Lanyon, A. Lomri, L. Malaval, T.J. Martin, T.L. McCarthy,F. Melsen, D.C. Morris, L. Mosekilde, G.R. Mundy, K.W. Ng,S. Nussbaum, A.M. Parfitt, M. Peacock, J.T. Potts, Jr., L.G. Raisz,J.-H. Ravesloot, V. Rosen, G. Segre, K.C. Shoukri, A. Vesterby,J.M. Wozney

Editors

Gregory R. Mundy and T. John Martin

Springer-VerlagBerlin Heidelberg New York London ParisTokyo Hong Kong Barcelona Budapest

Contents

CHAPTER 1

Calcium HomeostasisA.M. PARFITT. With 12 Figures 1

A. Introduction and Scope 1B. Concepts of Homeostasis 3C. Extracellular Fluid Free Calcium: The Controlled Variable 5D. Parathyroid Hormone: The Controlling Variable 8

I. Afferent Loop: Effects of Calcium on PTH Secretion 10II. Efferent Loop: Effects of PTH on Free Calcium 14

III. Short-Term Regulation as a Function of ParathyroidStatus 18

E. Other Calciotropic Hormones 20I. Calcitriol 20

II. Calcitonin - 22F. External Balance and/Turnover of Calcium 23

I. Dietary Intake and Intestinal Absorption 23II. Renal Excretion 24

III. Role of Intestine and Kidney in Calcium Homeostasis 27G. Bone and Bone Mineral in Relation to Calcium Homeostasis . . . . 29

I. Aspects of Bone Remodeling 30II. Role of Bone Remodeling in Calcium Homeostasis 32

III. Quiescent Bone Surfaces 34IV. Circulation of Bone, Macro and Micro 36V. Bone Mineral: Composition and Structure 37

VI. Movement of Calcium Ions in and out of Bone 40VII. Blood-Bone Equilibrium and Its Homeostatic Function . . . 44

VIII. Blood-Bone Equilibrium: Physiochemical andCellular Mechanism 47

H. Integration of Skeletal and Mineral Homeostasis 51References 54

XII Contents

CHAPTER 2

Bone Remodeling and Bone StructureE.F. ERIKSEN, A. VESTERBY, M. KASSEM, F. MELSEN, andL. MOSEKILDE. With 13 Figures 67

A. Introduction 67B. Bone Macro- and Microanatomy 68

I. Cortical Bone 68II. Cancellous Bone 71

C. Bone Remodeling 71I. Quantum Concept of Bone Remodeling 72

II. Coupling Phenomenon 73D. Evaluation of Bone Remodeling and Structure 73

I. Surface Area Estimates 74E. Evaluation of Bone Resorption 75

I. Cortical Bone 75II. Cancellous Bone ; 75

F. Evaluation of Bone Formation 76I. Cortical Bone 76

II. Cancellous Bone 76G. Bone Balance 77

I. Cortical Bone 77II. Cancellous Bone 77

III. Calculation of Activation Frequency 77IV. Calculation of Tissue Level Indices of Turnover 78

H. Indices Pertaining to Cancellous Bone Structure 78I. Trabecular Bone Volume 78

II. Marrow Space Star Volume 78I. The Bone Resorption Sequence 80

I. Cancellous Bone 80II. Cortical Bone 81

J. The Bone Formation Sequence 82I. Cancellous Bone 82

II. Cortical Bone 83III. The Bone Structural Unit 83

K. Bone Remodeling and Bone Loss 84L. Reversible Bone Loss 84

I. Cancellous Bone 85II. Cortical Bone 86

M. Irreversible Bone Loss 86I. Cortical Bone 86

II. Cancellous Bone 86III. Bone Turnover and Bone Loss 88

N. Implications for Bone Mass Measurements 89

Contents XIII

O. Physiological Ageing Processes in Bone: Differences BetweenFemales and Males 90

I. Bone Loss in Cortical Bone 90II. Bone Loss in Cancellous Bone 91

P. Relationship Between Bone Structure and Bone Strength 93I. Cancellous Bone :. 94

Q. Bone Remodeling in Metabolic Bone Disease 95I. High and Low Turnover Bone Disease 97

II. Bone Remodeling in Osteoporosis 99R. Final Remarks and Future Perspectives 99References 101

CHAPTER 3

Biology of the OsteoclastR. BARON, M. CHAKRABORTY, D. CHATTERJEE, W. HORNE, A. LOMRI,

and J.-H. RAVESLOOT. With 7 Figures I l l

A. Introduction I l lB. Main Morphological Features of the Osteoclast 112C. Structure-Function Relationship 115D. Motility, Attachment, and Establishment of

the Bone-Resorbing Compartment 116I. Cytoskeletal Organization 116

II. Attachment Apparatus 1171. Clear Zone 1172. Podosomes and the Sealing Zone 1183. Role of Integrins 1194. Regulation of Bone Resorption and

the Attachment Apparatus 121E. Proteins Destined for Export: Biosynthetic and Secretory

Functions of the Osteoclast 122I. Lysosomal Enzymes '. 122

II. Nature and Specificity of the Secreted Enzymes 124III. Generation of Oxygen-Derived Free Radicals and

Synthesis and Secretion of Other Proteins by the Osteoclast 127F. Cytosolic and Membrane Proteins: Membrane Composition and

Ion Transport 128I. Apical Membrane and the Process of Acidification 129

II. Role of the Basolateral Membrane and Ion Channels inAcidification, Intracellular pH, and MembranePotential Regulation 133

III. Handling and Regulatory Role of Calcium 135G. Conclusion 137References 140

XIV Contents

CHAPTER 4

Osteoblasts: Differentiation and FunctionT.J. MARTIN, D.M. FINDLAY, J.K. HEATH, and K.W. NG.With 3 Figures 149

A. Introduction 149B. Mature Members of the Osteoblast Lineage: Definitions 149C. Origin of Osteoblasts 151D. Osteoblast Lineage 153E. Protein Products of Osteoblasts 155F. Factors That Influence Osteoblast Differentiation 156G. Model of Osteoblast Differentiation 161H. Osteoblast. Proliferation 162I. Hormone Receptors and Responses of Osteoblasts 162J. Role of Osteoblasts in Intercellular Communication 165

I. Osteoclast Activation 165II. Osteoclast Formation 166

HI. Coupling of Resorption to Formation 168K. Proteinase Production by Osteoblasts 170L. Conclusion 173References 173

CHAPTER 5

Cytokines of BoneG.R. MUNDY. With 4 Figures 185

A. Introduction 185B. Nature of the Osteotropic Cytokines 185C. Cell Source of the Osteotropic Cytokines 186D. Interactions Between Systemic Factors and Cytokines 186E. Interactions Between Cytokines 187F. Diseases Associated with Abnormal Cytokine Production 187G. Interleukin-1 188H. Interleukin-1 Receptor Antagonist 194I. Tumor Necrosis Factor and Lymphotoxin 195J. Interleukin-6 197K. Gamma-Interferon 199L. /?2-Microglobulin 199M. Osteoclastpoietic Factor 200N. Colony-Stimulating Factors 201O. Leukemia-Inhibitory Factor (Differentiation-Inducing Factor) . . . 202P. Prostaglandins and Other Arachidonic Acid Metabolites 203Q. Transforming Growth Factor /? 206R. Bone Morphogenetic Proteins 206

Contents XV

S. Other Bone-Derived Growth Factors 206References 207

CHAPTER 6

Hormonal Factors Which Regulate Bone ResorptionG.R. MUNDY. With 5 Figures 215

A. Introduction 215B. Parathyroid Hormone 215

I. Effects of PTH on Bone Resorption 215II. Effects of PTH on Bone Formation 221

III. Signal Transduction Mechanisms for PTH in Bone Cells 221IV. Effects of PTH on Bone Turnover 222V. Effects of PTH on Calcium Homeostasis 224

C. 1,25-Dihydroxyvitamin D 225I. Vitamin D Receptor 226

II. Effects of 1,25-Dihydroxyvitamin D on Osteoclasts 228III. Effects of Vitamin D Metabolites on Cells of

the Osteoblast Lineage 230D. Calcitonin 230E. Amylin 233F. Cortisol 233G. Thyroid Hormones 234H. Estrogens 235References 238

CHAPTER 7

Factors That Regulate Bone FormationE. CANALIS, T.L. MCCARTHY, and M. CENTRELLA. With 1 Figure . . . . 249

A. Introduction 249B. Platelet-Derived Growth Factor 251C. Heparin-Binding Growth Factors 253D. Insulin-Like Growth Factors and Their Binding Proteins 255E. Transforming Growth Factor Beta 257References 261

CHAPTER 8

MineralizationH.C. ANDERSON and D.C. MORRIS. With 4 Figures 267

A. Introduction 267

XVI Contents

B. Direct Cellular Control of Mineralization ThroughMatrix Vesicles 267

C. Mechanism of Matrix Vesicle Calcification 271I. Role of Alkaline Phosphatase 271

II. Role of Lipids 272III. Constitutive Proteins of Matrix Vesicles 273IV. Biphasic Hypothesis of Mineralization-Crystal

Initiation Phase 274V. Biphasic Hypothesis of Mineralization-Crystal

Growth Phase 276D. Indirect Cellular Control of Mineralization 278

I. Effect of Bone Morphogens on the Calcification Mechanism 278II. Role of Growth Factors in Mineralization 279

III. Hormones That Affect Calcification 279IV. Pathological Calcification 280V. Vitamin D 281

E. Cellular Regulation of the Composition andMineralizing Potential of Matrix 281

I. Collagen 281II. Proteoglycans and Noncollagenous Proteins of Matrix 283

III. Control of Angiogenesis 284IV. Cellular Regulation of the Ionic Milieu at Calcification Sites 285V. Control of pH 287

References 288

CHAPTER 9

Pathogenesis of OsteoporosisL.G. RAISZ and K..C. SHOUKRI. With 1 Figure 299

A. Introduction 299B. Primary Osteoporosis 299

I. Calcium-Regulating Hormones 3021. Parathyroid Hormone 3022. 1,25-Dihydroxyvitamin D 3033. Calcitonin 303

II. Systemic Hormones 3041. Sex Hormones 3042. Other Systemic Hormones 305

III. Local Factors 3061. Interleukins 3072. Prostaglandins 3073. Growth Factors 308

IV. Calcium and Other Nutrients 309V. Physical Activity and Life-style 310

Contents XVII

VI. Alternative Hypotheses 311C. Secondary Osteoporosis 311

I. Glucocorticoid-Induced Osteoporosis 312II. Hyperparathyroidism 316

III. Hyperthyroidism 316IV. Hypogonadism 317V. Nutritional and Gastrointestinal Disorders 317

VI. Renal Disease 318VII. Multiple Myeloma and Other Hematologic Disorders 319

VIII. Mast Cells and Heparin 320IX. Osteogenesis Imperfecta 320X. Miscellaneous Therapeutic Agents 321

D. Conclusion 322References 323

CHAPTER 10

Vitamin D MetabolismJ.A. EISMAN. With 4 Figures 333

A. Overview 333B. Dermal Production of Vitamin D3 and Dietary Sources of

Vitamin D 335I. Substrate and Chemistry 335

II. Ultraviolet Light and Skin Pigmentation 336HI. Dietary Sources of Vitamin D 337

C. Serum Vitamin D Binding Protein 338I. Ethnic Differences 338

II. Binding Affinities and Function 338D. Hepatic 25-Hydroxylation 339

I. Microsomal 25-Hydroxylase 339II. Vitamin D Catabolism 339

III. 25-Hydroxyvitamin D Bioactivity 340E. Renal 1-Hydroxylation 340

I. Proximal Tubule Mitochondrial P450 340II. Regulation of Renal 1-Hydroxylation 342

HI. Pathophysiology of Renal 1-Hydroxylation 343F. Extrarenal 1-Hydroxylation 344

I. Physiological Extrarenal 1-Hydroxylation 345II. Pathological Extrarenal 1-Hydroxylation 345

G. Renal and Target Tissue 24-Hydroxylation 346I. Renal 24-Hydroxylation 346

II. Target Tissue 24-Hydroxylation 347III. Catabolic Versus Unique Functions of

24,25-Dihydroxyvitamin D 347

XVIII Contents

H. Vitamin D Catabolism 348I. Target Tissues 349

I. Bone and Calcium Related 349II. Hematopoietic Cells 351

III. Skin and Skin Appendages 352IV. Reproduction and Endocrine Glands 353V. Other Tissues 354

J. Vitamin D Receptor 354I. Functional Role 354

II. End Organ Resistance 356III. Molecular Biology 356IV. Vitamin D Response Element in Responsive Genes 357

K. Genomic and Nongenomic Effects 358L. Summary 361References 362

CHAPTER 11

Bisphosphonates: Mechanisms of Action and Clinical UseH. FLEISCH 377

A. Introduction 377B. Chemistry and General Characteristics 377C. Synthesis 379D. Methods of Determination 379E. History of the Development for Use in Bone Disease 380F. Mode of Action ! 380

I. Physicochemical Effects 380II. Effect on Calcification In Vivo 381

III. Inhibition of Bone Resorption 3821. Assessment of Activity 3822. Activity of Various Bisphosphonates 3843. Mechanisms of Action of Bone Resorption Inhibition.... 3844. Other Effects In Vivo 387

G. Pharmacokinetics 387H. Animal Toxicology 389I. Drug Interactions 390J. Clinical Use 390

I. Ectopic Calcification and Ossification 3901. Soft Tissue Calcification 3902. Urolithiasis 3913. Dental Calculus 3914. Fibrodysplasia Ossificans Progressiva 3915. Other Heterotopic Ossifications 391

II. Diseases with Increased Bone Resorption 3921. Paget's Disease 392

Contents XIX

2. Hypercalcemia of Malignancy andTumoral Bone Destruction 394

3. Hyperparathyroidism and Other Causes ofHypercalcemia , 396

4. Osteoporosis 397III. Other Indications 398

K. Adverse Events 398L. Contraindications 400M. Future Prospects 400References 401

CHAPTER 12

Paget's DiseaseD.C. ANDERSON. With 20 Figures 419

A. Introduction 419B. Nature of the Underlying Disease Process 419C. Epidemiology 420D. Direct Studies on the Etiology of Paget's Disease 423E. Possible Role of Canine Distemper Virus 423F. Properties of the Osteoclast That Might Make It Particularly

Susceptible to Persistent Infection with an RNA Virus 425G. Clinical Features 427

I. Bones Affected and Extent 427II. Symptoms and Signs 428

1. Features Due to Long-standing Excessive BonyRemodelling^ 429

2. Features Due to Secondary Arthritis 4303. Features Due to Pressure on Surrounding Structures . . . . . 4304. Neoplasia 431

H. Histopathology 431I. Clinical Assessment and Investigation 431J. Treatment of Paget's Disease 435

I. Bisphosphonates (Diphosphonates) 435II. Probable Mode of Action of Bisphosphonates in

Paget's Disease 439III. Future Work 439

References 440

CHAPTER 13

Hyperparathyroid and Hypoparathyroid Bone DiseaseM. PEACOCK. With 15 Figures 443

A. Introduction 443B. Parathyroid Hormone Function: Calcium Homeostasis 443

XX Contents

C. Classification of Parathyroid Disease 444D. Primary Hyperparathyroidism 446

I. Incidence 446II. Diagnosis 446

III. Pathogenesis 449IV. Nonskeletal Signs 450V. Skeletal Signs 450

1. Radiology 452, 2. Histology 455

3. Bone Mass and Fracture 455VI. Human Parathyroid Hormone 456

E. Secondary Hyperparathyroidism 457I. Renal Failure 457

1. Diagnosis 4572. Pathogenesis 4583. Skeletal Signs 459

II. Vitamin D Deficient Osteomalacia/Rickets 4601. Pathogenesis and Diagnosis 4602. Skeletal Signs 462

III. Ageing 4631. Diagnosis and Pathogenesis 463

IV. Others 467F. Tertiary Hyperparathyroidism 467G. Hypoparathyroidism 468

I. Diagnosis and Pathogenesis 4681. Nonskeletal Signs 4692. Skeletal Signs 469

References 471

CHAPTER 14

Skeletal Responses to Physical LoadingL.E. LANYON. With 4 Figures 485

A. Introduction 485B. Influences on Bone Form 485C. Hierarchy of Functional Control 487D. Nature of the Loading-Related Stimulus 490E. Experimental Studies: In Vivo 490F. Experimental Studies: In Vitro 494G. Loading of Bone Cell Cultures 496H. Implications of Experiments In Vivo 496I. Implications of Experiments In Vitro 497

I. Organ Culture 497J. Discussion 499K. Summary 502References 503

Contents XXI

CHAPTER 15

Parathyroid Hormone: Biosynthesis, Secretion, Chemistry, and ActionH.M. KRONENBERG, F.R. BRINGHURST, S. NUSSBAUM, H. JUPPNER,A.-B. ABOU-SAMRA, G. SEGRE, and J.T. POTTS, JR. With 5 Figures . . . 507

A. Physiologic Actions 508I. Actions in Bone 508

1. Effects upon Osteoclasts 5092. Effects upon Osteoblasts 510

II. Actions in Kidney 5121. Calcium Reabsorption 5132. Phosphate Reabsorption 5143. Other Renal Effects 516

B. Biosynthesis and Secretion of PTH 517I. Parathyroid Hormone Biosynthesis 517

II. Parathyroid Hormone Gene 520III. Parathyroid Hormone Secretion 522

1. Physiology 5222. Cellular Mechanisms 523

C. Structural Basis of PTH Function 525I. Parathyroid Hormone Binding and Activation Domains 526

II. Evolutionary Lessons: Rat and Chicken PTH 529III. Three-Dimensional Structure of PTH 530IV. Lessons from the Structure and Activity of PTHrP 532V. Structure-Activity Relationships for Middle and

Carboxyl Regions of PTH 534D. Parathyroid Hormone Receptors 535E. Second Messengers in PTH Action 540

I. Cyclic AMP 540II. Other Second Messengers 541

HI. Physiologic Roles of Different Second Messengers inPTH Action 543

IV. Second Messengers in PTH Regulation ofRenal Phosphate Transport 544

V. Modulation of PTH Signal Transduction 546F. Conclusion 549References 549

CHAPTER 16

Calcitonin Gene Products: Molecular Biology, Chemistry, and ActionsW. BORN and J.A. FISCHER. With 3 Figures 569

A. Introduction 569B. Calcitonin/CGRP Genes 570

I. Structure of DNA 570

XXII Contents

II. Regulation of Transcription 574III. Tissue-Specific Expression of Messenger RNA 575

C. Calcitonin Gene Products 577I. Biosynthesis 577

II. Structure and Tissue Distribution 578III. Regulation of Release 580IV. Metabolism 582

D. Biological Action 582I. Calcitonin Receptor and Targets 583

1. Receptors 5832. Biological Targets 586

II. Calcitonin Gene-Related Peptide Receptors and Targets . . . . 5881. Receptors 5882. Biological Targets 589

III. Other Gene Products 594E. Clinical Implications 595

I. Calcitonin 595II. Calcitonin Gene-Related Peptide 596

F. Conclusions 596References 598

CHAPTER 17

Parathyroid Hormone-Related Protein: Molecular Biology, Chemistry,and ActionsT.J. MARTIN. With 3 Figures 617

A. Introduction 617B. Molecular Biology 618

I. Isolation and Cloning 618II. Chromosomal Localization 618

III. Genomic Structure 619IV. Regulation of Gene 620V. Recombinant PTHrP 621

C. Chemistry 621I. Structure-Activity Relationships 621

II. Tertiary Structure 622III. Immunology 623IV. Molecular Processing 624V. Structural Conservation 624

D. Actions 625I. Second Messengers and Receptors 626

II. Postreceptor Events 626III. Actions on Bone 627IV. Actions on Kidney 629

Contents XXIII

E. Physiological Functions 630I. Parathyroid Hormone Related Protein as

an Oncofetal Hormone 630II. Paracrine Agent in Smooth Muscle Relaxation 632

III. Parathyroid Hormone Related Protein in Lactating Breast .. 632IV. Location in Epithelia 632V. Endocrine and Paracrine Roles 632

References 633

CHAPTER 18

Pathophysiology of Skeletal Complications of CancerG.R. MUNDY and T.J. MARTIN. With 1 Figure 641

A. Introduction 641B. Frequency of the Skeleton as a Site for Malignant Disease 641C. Cancers Which Involve the Skeleton 643D. Favored Skeletal Sites of Malignancy 644E. Complications of the Metastatic Process 644F. Pathophysiology of the Metastatic Process 647

I. Properties of Tumor Cells Which Favor Metastasis 648II. Tumor Cell Invasion at the Primary Site 650

1. Adhesion 6512. Secretion of Enzymes 6513. Cell Motility 652

III. Tumor Cells in the Bloodstream 652IV. Tumor Cell Arrest at the Metastatic Site 653V. Growth Regulatory Factors at Metastatic Sites 653

G. Potential Mechanisms for the Metastatic Process in Bone 654I. Chemotactic Factors 654

II. Growth Regulatory Factors 654III. Calcium 655IV. Proteolytic Enzymes 655

H. Factors Which May Be Involved in Osteolysis 656I. Parathyroid Hormone Related Protein 656

II. Transforming Growth Factor a 656III. Transforming Growth Factor /? 657IV. Prostaglandins of the E Series 658V. Parathyroid Hormone 659

VI. Vitamin D Sterols 659VII. Platelet-Derived Growth Factor 660

VIII. Procathepsin D : 660IX. Bone-Resorbing Cytokines 661

I. Factors Involved in Osteoblastic Effects 662I. Transforming Growth Factor /?2 663

XXIV Contents

II. Fibroblast Growth Factor 663III. Plasminogen Activator Sequence 663

References 663

CHAPTER 19

The Proteins of BoneP.D. DELMAS and L. MALAVAL. With 7 Figures 673

A. Introduction 673B. Collagen 676

I. Structure and Synthesis of Bone Collagen 6771. Structure of Collagens 6772. Type I Collagen Synthesis and Secretion 677

II. Disorders of Collagen Synthesis: Osteogenesis Imperfecta... 681III. Markers of Collagen Metabolism 682

1. Markers of Type I Collagen Synthesis: Propeptides 6822. Markers of Collagen Degradation 683

C. Gamma-carboxyglutamic Acid Containing Proteins of Bone 685I. Gamma-Carboxyglutamic Acid (GLA) 685

II. Osteocalcin 6851. Structure, Biosynthesis, and Tissular Distribution 6852. Functional Role 6863. Circulating Osteocalcin 688

III. Matrix GLA-Protein 689D. Proteoglycans ....._ 690

I. Biglycan and Decorin 6901. Structure 6902. Distribution 6923. Properties and Potential Role 692

II. CS-PGIII 693HI. Other Proteoglycans 693

E. SPARC/Osteonectin 694I. Structure 694

II. Binding to Hydroxyapatite and to Other Proteins 696III. Tissular Distribution 697IV. Biological Properties and Potential Role 698V. Circulating SPARC/Osteonectin 699

F. Bone RGD-Containing Proteins 699I. Bone Sialoproteins 700

1. Osteopontin 7012. Bone Sialoprotein II 702

II. Thrombospondin 704HI. Fibronectin 706

G. Other Proteins in Bone 707

Contents XXV

I. Proteases and Protease Inhibitors 707II. Plasma Proteins 709

H. Conclusion 710References 710

CHAPTER 20

Bone Morphogenetic ProteinsJ.M. WOZNEY and V. ROSEN. With 4 Figures 725

A. Introduction 725B. Biochemistry and Molecular Biology of the BMPs 726

I. In Vivo Assay System 726II. Discovery of Multiple Related Proteins 729

1. Purification from Bone 7292. Bone Morphogenetic Protein Family 7293. Recombinant Expression 7334. Expression of BMPs in Other Tissues 7345. Chromosomal Localization of the BMP Genes 735

C. Activities of Individual BMP Molecules 736I. In Vivo Activities 736

II. In Vitro Activities 737III. Bone Morphogenetic Proteins in Embryogenesis 739

D. Clinical Utility of BMPs 740I. Clinical Indications 740

II. Animal Studies 7411. Bone-Derived Extracts 7412. Recombinant Human BMPs 742

III. Human Studies with Bone Extracts 742E. Summary 743References 743

Subject Index 749