Basic and Applied Bone Biology [2 ed.] 0128132590, 9780128132593

Table of contents :
Cover
BASIC AND APPLIED BONE BIOLOGY
Copyright
Dedication
List of Contributors
Biographies
Preface (First Edition)
Preface (Second Edition)
Part I: Basic Bone Biology and Physiology
1 - Bone Morphology and Organization
The Functions of Bone
Bone Is Organized as a Multiscale Material
Bone Composition
The Nanoscale Organization of Bone
Collagen
Enzymatically Mediated Collagen Cross-Linking
Nonenzymatically Mediated Collagen Cross-Linking
Collagen Orientation
Bone Mineral
Noncollagenous Extracellular Matrix Proteins
Proteoglycans and Glycosaminoglycans
Glycoproteins
SIBLING Proteins
Osteocalcin
Osteonectin
The Microstructural Organization of Bone
Woven Bone
Primary Bone
Primary Lamellar Bone
Plexiform Bone
Primary Osteons
Secondary Bone
Interstitial Bone
The Macroscopic Organization of Bone
Cortical Bone
Cancellous Bone1
Skeletal Envelopes
The Blood Supply and Innervation of Bone
Blood Supply
Innervation of Bone
Bone Fluid Compartments
Bone Mass and Bone Quality
Rate of Bone Turnover
Bone Trabecular Architecture
Tissue Material (Intrinsic) Properties
Microdamage Accumulation
Suggested Readings
2 - Bone Marrow and the Stem Cell Niche
Niches in Development
Imaging of Niches
Bone Marrow Niche Cells
Nonhematopoietic
Osteoblastic Cells
Mesenchymal Stromal/Stem Cell Populations
Adipocytes
Endothelial Cells
Hematopoietic
Megakaryocytes
Macrophages
Neutrophils
Osteoclasts
Neuronal Regulation of the Hematopoietic Stem Cell Niche
Hormonal Regulation of the Hematopoietic Stem Cell Niche
Niches and Malignancies
Aging and the Bone Marrow Microenvironment
The Future
Suggested Readings
3 - Bone Cells
Osteoclasts
Osteoclast Morphology and Function
Osteoclast Differentiation and Fusion
Osteoclast Apoptosis
Regulation of Osteoclast Generation and Survival
Osteoblasts
Osteoblast Morphology and Function
Osteoblast Formation and Differentiation
Osteoblast Apoptosis
Regulation of Osteoblast Generation and Survival
Bone Lining Cells
Osteocytes
Osteocyte Morphology and Functions
Osteocytogenesis and Osteocyte Maturation
Osteocyte Apoptosis: Consequences and Regulation
Preservation of Osteocyte Viability by Mechanical Stimuli
Aging and Osteocyte Apoptosis
Hormonal Regulation of Osteocyte Life Span
Regulation of Bone Formation by Osteocytes: The Sclerostin Paradigm
Regulation of Bone Resorption by Osteocytes: RANKL and OPG
Regulation of Bone Mineralization by Osteocytes
Suggested Readings
4 - Local Regulation of Bone Cell Function
Overview of Cytokines and Growth Factors and Their Receptors
Definition: Cytokines Versus Growth Factors
Soluble Ligands
Membrane-Bound Ligands
Receptor Classification
Signal Transduction Cascades
Local Factors That Regulate Osteoclast and Osteoblast Differentiation and Function
Factors That Regulate Osteoclast Differentiation and Function
The RANKL–RANK–OPG System
Macrophage Colony-Stimulating Factor
Factors That Affect Osteoblast Differentiation and Function
Wnts
Canonical Wnt Signaling
Noncanonical Wnt Signaling
Inhibitors of Wnt Signaling
Dickkopf
Kremen
Sclerostin
Secreted Frizzled-Related Proteins
Wise
Insulin-Like Growth Factor
Parathyroid Hormone–Related Peptide
Fibroblast Growth Factors
Local Factors That Affect Both Osteoclasts and Osteoblasts
Interleukins
Tumor Necrosis Factor
Bone Morphogenetic Proteins
Transforming Growth Factor Beta
Regulation of Transforming Growth Factor Beta Bioavailability
Prostaglandins
Regulation of Bone Cell Differentiation and Function Through Cell–Cell Contact
Notch
Ephrin–Ephrin Receptor
Connexins
Local Angiogenic Factors That Regulate Bone Cell Activity
Cell Surface Attachment Molecules
Suggested Readings
5 - Bone Growth, Modeling, and Remodeling
Skeletal Development
Intramembranous Ossification
Endochondral Ossification
Bone Modeling
Events That Signal Modeling
Cellular Processes
Modeling During the Life Cycle
Longitudinal Growth
Radial Growth
Bone Drift
Bone Remodeling
Events That Signal Remodeling
Remodeling Cycle
Activation
Resorption
Reversal
Formation
Quiescence (Resting)
Bone Remodeling Cycle Duration
Bone Remodeling Rate
Bone Remodeling Balance
Bone Repair
Effects of Modeling and Remodeling on Bone Structure and Material Properties
Bone Structure Effects of Modeling and Remodeling
Bone Material Property Effects of Remodeling
Mineralization
Collagen Cross-Links
Microdamage
Laboratory Assessment of Modeling and Remodeling
Suggested Readings
Part II: Assessment of Bone Structure and Function
6 - Skeletal Imaging
X-ray
Conventional Radiography
Dual-Energy X-ray Absorptiometry
Computed Tomography
Clinical Diagnostic Computed Tomography
Dual Energy or Spectral Imaging Scanning
High-Resolution Peripheral Quantitative Computed Tomography
Microcomputed Tomography
Quantitative Computed Tomography (Densitometry)
Modeling Analysis
Magnetic Resonance Imaging
Ultrashort Echo-Time (TE) Sequences
Radionuclide Imaging
Positron Emission Tomography and Positron Emission Tomography–Computed Tomography
Ultrasound
Summary
Suggested Readings
Computed Tomography
Dual Energy X-ray Absorptiometry
Magnetic Resonance Imaging
Finite Element Analysis
Radionuclide Imaging
Ultrasound
7 - Skeletal Hard Tissue Biomechanics
Introduction to Basic Bone Structure and Function
Main Functions of Bone in the Body
Bone Is a Hierarchical Structure
Fundamentals of Solid Mechanics
The Force–Displacement Curve
Stress and Strain in Axial Loading
Modulus of Elasticity and Poisson’s Ratio
Methods of testing mechanical properties of whole bone
Axial Loading in Tension and Compression
Torsion
Bending
Factors Contributing to Whole Bone Mechanical Properties
Bone Mass: Cancellous Bone
Cancellous Architecture and Anisotropy
Cortical Bone Size (Mass) and Architecture
Bone Tissue Material Properties
The Bone Extracellular Matrix: Type I Collagen
The Bone Extracellular Matrix: Inorganic Hydroxyapatite
Methods of Testing the Mechanical Properties of Bone Tissue
Other Measures of Bone “Strength”
Suggested Readings
8 - Techniques in Histomorphometry
Histologic Specimens
Specimen Collection
In Vivo Labeling
Specimen Processing
Histomorphometric Analysis
Static versus Dynamic Measurements
Primary Versus Derived Variables
Referents
Bone Architecture and Geometry
Tissue Types
Cell Number and Activity
Dynamic Histomorphometry
Osteocytes
Microdamage
Marrow
Assumptions and Technical Aspects
Histologic Features of Disease and Treatment
Osteoporosis
Osteomalacia
Hyperparathyroidism
Primary Hyperparathyroidism
Secondary Hyperparathyroidism
Paget’s Disease of Bone (Osteitis Deformans)
Antiresorptive Therapy
Anabolic Therapy
Suggested Readings
9 - Skeletal Genetics: From Gene Identification to Murine Models of Disease
Genetic Analysis of Bone Phenotypes
Linkage Analysis
Genome-Wide Association Studies
Whole Exome and Whole Genome Analysis
Functional Analysis
Omics Analysis of Bone Phenotypes
Transgenic Technology and the Creation of Novel Research Models
Production of Transgenic Rodents
Animals that Arise From Pronuclear Injection
Animals Derived From Homologous Recombination in ES Cells
Pronuclear Microinjection
Isolation of Embryos for Microinjection
Superovulation
Collection of Single-cell Embryos
DNA Preparation for Microinjection
Pronuclear Microinjection of Embryos Microinjection
Transfer of Injected Embryos to Recipient Females
Analysis of Potential Transgenic Founders
Microinjection of Genetically Modified Embryonic Stem Cells
Generation of ES cell Lines
Homologous Recombination
Collection of Blastocysts
Injection of ES cells Into Blastocysts
Transfer of Injected Blastocysts to Recipient Females
Identification of Chimeric Animals
Designer DNA Nucleases: The CRISPR Revolution
Transgenic Animals and Bone Biology
Bone Cell–Specific Promoters
Conditional Models Using Cre-LoxP Technology
Bridging Human Disorders and Mouse Models of Bone Diseases
Secondary Challenges and Genetic Background Impacts on Skeletal Phenotypes
Summary
Suggested Readings
Part III: Skeletal Adaptation
10 - Skeletal Changes Across the Life Span
Prenatal Growth
Development and Maturation
Calcification and Mineral Homeostasis
Postnatal Growth
Newborn Mineral Homeostasis
Infancy and Childhood Skeletal Growth
Puberty and Adolescence
Race and Ethnic Differences
Endocrine Regulators
Genetic Regulation
Timing of Growth Rates
Development of Peak Bone Mass
Fracture Risk in Children
Skeletal Changes With Reproduction
Pregnancy
Lactation
Skeletal Changes With Aging
Skeletal Expansion and Age
Skeletal Mass and Age
Suggested Readings
Peak Bone Mass
Fetal Programming
Other reading
11 - Mechanical Adaptation
Wolff’s Law—A Historical Perspective
Components of Strain That May Be Osteogenic
Strain Magnitude
Strain Distribution
Strain Duration
Strain Rate
Strain Frequency
Polarity
Strain Energy
Animal Models of Skeletal Mechanotransduction
Intrinsic Loading Models
Extrinsic Loading Models
Invasive (Surgical) Models
Noninvasive Models
Rules for Bone Adaptation
Bone Responds Only Above (or Below) a Threshold of Strain or Strain Rate
Bone Responds Only to Dynamic Loads
The Loading Period Can Be Short
Rate-Related Phenomena are Critical to Response
Strain Rate is a Function of Strain Magnitude and Loading Frequency
The Mechanostat
Bone Adaptation is Error Driven
Set Points Define Four Different Mechanical Usage Windows
Modeling and Remodeling are Antagonistic Processes
Bone Formation on the Different Envelopes is Controlled by Local Conditions
Cell Sensitivity and Refractory Periods
A Test of Wolff’s Law
Mechanotransduction on a Smaller Scale: Cell Types and Their Environment
Fluid Flow in Bone Tissue Has Manifold Cellular Effects
Cellular Deformation and Stimulation
What Is the Mechanoreceptor?
Ion Channels
Cell Adhesion and the Cytoskeleton
G Protein Signaling
Coordinating the Biochemical Response to Mechanical Stimulation
Clinical Implications: Exercise Prescription for Optimizing Skeletal Health
Skeletal Benefits of Physical Activity and Exercise During Growth
Infancy
Childhood and Adolescence
Skeletal Responses to Physical Activity and Exercise in Adulthood
Principles of Training and Exercise Recommendations for Optimal Bone Health
Principle of Specificity
Principle of Progressive Overload
Principle of Reversibility
Principle of Initial Values
Principle of Diminished Returns
Summary
Suggested Readings
12 - Fracture Healing
Types of Bone Fracture
Primary and Secondary Repair Mechanisms
Stages of Fracture Repair
Inflammatory Response
Soft Callus Formation (Cartilage Formation)
Hard Callus Formation (Endochondral Ossification)
Bone Remodeling
Assessment of Fracture Healing
Methods of Evaluation
Biomechanical Stages of Fracture Healing
Cellular Events of Fracture Repair
Molecular Regulation of Chondrogenesis and Osteogenesis During Fracture Repair
Local Regulation of Fracture Repair
Prostaglandins
Bone Morphogenetic Proteins
Platelet-Derived Growth Factor
Vascular Endothelial Growth Factor
Endothelial Progenitor Cells
Skeletal Muscle Interactions
Systemic Factors in Fracture Repair
Aging
Diabetes Mellitus
Glucocorticoids
Effects of Osteoporosis Drugs on Fracture Healing
Parathyroid Hormone
Bisphosphonates
Denosumab
Effects of Mechanical and Electrical Stimulation on Fracture Repair
Electromagnetic Therapy
Low-Intensity Pulsed Ultrasound
Summary
Suggested Readings
Part IV: Hormonal and Metabolic Effects on Bone
13 - Calcium and Phosphate: Hormonal Regulation and Metabolism
Organ System Interplay Regulates Calcium and Phosphorus Metabolism
Distribution of Calcium and Phosphorus in the Body
Mineral Accrual and Loss Over the Life Span
Regulation of Whole Body Calcium Metabolism
Intestinal Calcium Absorption
Renal Calcium Reabsorption
Hormones Controlling Calcium Metabolism
Vitamin D and Its Metabolites
25-Hydroxyvitamin D Metabolism
1,25-Dihydroxyvitamin D Regulation and Metabolism
Vitamin D Actions
Parathyroid Hormone and Parathyroid Hormone-Related Peptide
Control of PTH Production and Release
Estrogen
Calcitonin
Somatotropin/Growth Hormone
Hormones of Pregnancy and Lactation
Regulation of Whole Body Phosphate Metabolism
Intestinal Phosphate Absorption
Renal Phosphate Reabsorption
Parathyroid Hormone Effects on Phosphate Reabsorption
Fibroblast Growth Factor 23 is an Important Hormone in Phosphate Reabsorption and Vitamin D Regulation
Changes in Dietary Phosphate Intake Alter Fibroblast Growth Factor 23 Concentration
Influence of Iron on FGF23 Metabolism
Summary
Suggested Readings
14 - Nutrition
Macro- and Micronutrient Roles in Bone Health
When and How Nutrients Influence Bone Mass
Perturbing Calcium Metabolism
Whole Diet Matters
Diet Versus Fortified Foods Versus Supplements
Acid–Base Balance, Fruit and Vegetable Intakes, and Bone Health
Dietary Bioactives and Their Mechanisms
Effects of Obesity and Weight Loss on Bone
Obesity, Osteoporosis, and Fracture Risk
Effects of Weight Loss on Bone
Correcting Nutrient Deficiency Versus Optimizing Intake
Challenges for Studying Effects of Nutrient Interventions on Bone Outcomes
Suggested Readings
15 - Hormonal Effects on Bone Cells
Direct Versus Indirect Effects of Hormones on Bone Cells
Parathyroid Hormone
Actions of PTH on Bone
PTH Receptors and Downstream Signaling
Effects of PTH on Osteoblasts and Bone Formation
Inhibition of Osteoblast Apoptosis by PTH
Downregulation of Sost/Sclerostin by PTH
Reactivation of Lining Cells by PTH
Effects of PTH on Osteoclastogenesis and Bone Resorption
Sex Steroids
Sex Steroid Production
Sex Steroid Receptor Signaling
Sex Steroids During Growth
Loss of Sex Steroids in the Adult Skeleton
Changes in Bone Cells Induced by Estrogen Deficiency
Effects of Estrogens and Androgens on Osteoclasts
Effects of Estrogens and Androgens on Osteoblasts and Osteocytes
Glucocorticoids
Epidemiology and Progression of Glucocorticoid-Induced Bone Disease
Glucocorticoids and Bone Cells
Glucocorticoid Receptors and Downstream Signaling
Thyroid Hormone
Growth Hormone
Insulin
1,25-Dihydroxyvitamin D3
Leptin
Suggested Readings
PTH
Sex Steroids
Glucocorticoids
Other Hormones
Part V: The Interaction of Bone With Other Organ Systems
16 - Bone and Muscle
Development and Structure of Muscle
Embryogenesis and Development
Muscle Structure
Regulation of Skeletal Muscle
The Mechanical Interaction of Muscle and Bone
Endocrine Cross Talk Between Muscle and Bone
Myostatin
Insulin-Like Growth Factor (IGF)-1
Cytokines
Brain-Derived Neurotrophic Factor
Irisin
Musclin
Beta-Aminoisobutyric Acid
Bone’s Endocrine Effects on Muscle
Sclerostin, Dkk1, and Wnts
RANKL
Osteocalcin
Fibroblast Growth Factor 23
Prostaglandin E2
Unknown Factors
Linked Diseases Between Bone and Muscle
Pleiotropic Genes for Bone and Muscle and Their Potential Role in Musculoskeletal Disease
Osteogenesis Imperfecta
Cachexia
Effects of Aging on the Musculoskeletal System
Sarcopenia
Summary
Suggested Readings
17 - Bone and the Immune System (Osteoimmunology)
Innate Immunity
Adaptive Immunity
B Cells and Bone Remodeling
T Cells and Bone Remodeling
Myeloid Cells and Bone Remodeling
Infections and Bone Cells
Endocrine Activation of Immune-Mediated Bone Loss
Role of Immune Activation in Estrogen Deficiency
Role of T Cells in Parathyroid Hormone Responses in Bone
Autoimmune Diseases Can Drive Local or Systemic Bone Loss
Rheumatoid Arthritis
Suggested Readings
18 - Bone and the Brain
Introduction to the Nervous System
Innervation of Bone
Neural Connections Between Bone and Brain
Functional Role of Central and Peripheral Efferent Pathways
The Sympathetic Nervous System
Leptin
Serotonin
Neuropeptide Y
Endocannabinoids
Cocaine- and Amphetamine-Regulated Transcript
Dopamine
Neuromedin U
The Parasympathetic Nervous System
Acetylcholine
Pituitary Adenylate Cyclase–Activating Polypeptide and Vasoactive Intestinal Peptide
Other Central Neurotransmitters That Impact Bone
Orexin
Oxytocin
Pro-opiomelanocortin and Melanocortin System
Functional Role of Sensory Neurotransmitters in Bone
Calcitonin Gene–Related Peptide
Substance P
VIP and PACAP
Nucleotides
Glutamate
Bone Feedback to the Brain
Bone Impact of Neurological Disorders
Conclusion
Suggested Readings
19 - The Microbiome and Bone
Introduction to the Microbiome
Effects of the Microbiome on Bone
Preclinical Manipulations of the Microbiome
The Effects of the Microbiome on Bone Mass, Remodeling, and Strength
Mechanisms Linking the Microbiome to Bone
Summary
Suggested Readings
20 - Bone and Kidney
CKD-MBD—Bone Abnormalities
Renal Osteodystrophy
Bone Imaging
Bone Biomarkers
Pathogenesis of Abnormal Bone in Patients With CKD
Abnormal Hormones
Disordered Osteoblast Function or Differentiation
Abnormalities of Bone Collagen
CKD-MBD—Biochemical Abnormalities
CKD-MBD—Vascular Calcification
Summary
Suggested Readings
Part VI: Skeletal Disease and Treatment
21 - Pharmaceutical Treatments of Osteoporosis
Osteoporosis
Definition
Risk Factors
Diagnosis
Pathogenesis
Osteoporosis Therapies
Calcium and Vitamin D
Antiremodeling Therapies
Estrogen
Selective Estrogen Receptor Modulators
Calcitonin
Bisphosphonates
Denosumab
Anabolic Therapies
Teriparatide
Abaloparatide
Antisclerostin Antibody
Treatment Guidelines and Decisions
Sequential Treatments
Antiremodeling Agent Followed by an Anabolic
Anabolic Followed by an Antiremodeling Agent
Antiremodeling Agent Followed by an Antiremodeling Agent
Combination (Concurrent) Treatments
Concurrent Antiremodeling Agent and Anabolic
Concurrent Antiremodeling Agents
Conclusion
Suggested Readings
22 - Bone and Cancer
Abnormalities of Bone Remodeling Induced by Cancer in Bone
Mechanisms Responsible for Bone Metastasis
Intrinsic Properties of Tumor Cells That Enhance Their Bone Metastatic Potential
Changes in the Phenotype of Cancer Cells Induced by the Bone Microenvironment
Generation of the Premetastatic Niche in Bone
Tumor Cell Homing to Bone
Immune Suppression in Bone Metastasis
Physical Characteristics of Bone Contribute to Bone Metastasis
Contributions of Bone Cells to Tumor Cell Dormancy, Reactivation, Chemoresistance, and Tumor Progression in Bone
Role of Osteoclasts in the Development and Progression of Bone Metastasis
Role of Osteoblasts in the Development and Growth of Bone Metastasis
Imaging of Bone Metastases
Bone Turnover Markers and Other Biomarkers of Cancer in Bone
Bone-Targeted Therapies for Bone Metastasis
Summary
Suggested Readings
23 - Sweet Bones: Diabetes Effects on Bone
Introduction
Fractures in Diabetes
Fracture Risk in Type 1 Diabetes
Fracture Risk in Type 2 Diabetes
Fracture Morbidity and Mortality in Diabetes
Fracture Healing in Diabetes
Skeletal Changes in Diabetes
Bone Mineral Density in Type 1 Diabetes
Bone Mineral Density in Type 2 Diabetes
Bone Geometry and Architecture in Type 1 Diabetes
Bone Geometry and Architecture in Type 2 Diabetes
Mineral Homeostasis and Bone Turnover in Diabetes
Other Hormonal/Cellular Level Changes in Diabetes
Osteocalcin
Adipocytokines
Oxidative Stress
Bone Tissue Material Properties
Bone Collagen Glycation
Management and Treatment Approaches
Effects of Diabetes Management on Bone Health
Insulin
Metformin
Sulfonylureas
Thiazolidinedione
Incretin-Based Treatments
GLP-1 Receptor Agonists
DPP-4 Inhibitors
Sodium-Glucose Cotransporter 2 Inhibitors
Amylin Analogs
Bariatric Surgery
Bone Health Management in Diabetes
Conclusions
Suggested Readings
Index
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
Back Cover

Citation preview

BASIC AND APPLIED BONE BIOLOGY SECOND EDITION Edited by

David B. Burr Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, United States; Department of Biomedical Engineering, Indiana University-Purdue University-Indianapolis, Indianapolis, IN, United States

Matthew R. Allen Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, United States; Department of Biomedical Engineering, Indiana University-Purdue University-Indianapolis, Indianapolis, IN, United States; Department of Medicine-Nephrology, Indiana University School of Medicine, Indianapolis, IN, United States; Roudebush Veterans Administration Medical Center, Indianapolis, IN, United States

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2019 Elsevier 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 photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-813259-3 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Stacy Masucci Acquisition Editor: Tari Broderick Editorial Project Manager: Karen Miller Production Project Manager: Kiruthika Govindaraju Cover Designer: Miles Hitchen Typeset by TNQ Technologies

This book is dedicated to the wonderful teachers I have had over many years—Denny, Bruce, Harold, Eric, Mitch, and Charles, among many others—who have so patiently taught me about the beauties and intricacies of our skeleton. And to my wife, Lisa, and son, Erik, who have tolerated and supported my obsession with bone. David B. Burr This book is dedicated to Kristine, Sophie, Gus, and Faye who provide me with a daily reminder that there is so much more to life than bone biology. And to my mom who has influenced my life more than she will ever know. Matthew R. Allen

v

List of Contributors

*Alexandra Aguilar-Pérez  Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, United States

Julia F. Charles  Department of Orthopaedics, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, United States

*Matthew R. Allen  Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, United States; Department of MedicineNephrology, Indiana University School of Medicine, Indianapolis, IN, United States; Department of Biomedical Engineering, Indiana University-Purdue UniversityIndianapolis, Indianapolis, IN, United States; Roudebush Veterans Administration Medical Center, Indianapolis, IN, United States

*Robert H. Choplin  Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN, United States Timothy J. Corbin  Department of Transgenics and Reproductive Technologies, Stowers Institute for Medical Research, Kansas City, MO, United States Robin Daly  Institute for Physical Activity and Nutrition, Deakin University, Melbourne, VIC, Australia *Linda A. DiMeglio  Department of Pediatrics, Division of Pediatric Endocrinology and Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, United States

Mary F. Barbe  Department of Anatomy and Cell Biology, School of Medicine, Temple University, Philadelphia, PA, United States *Teresita Bellido  Roudebush Veterans Administration Medical Center, Indianapolis, IN, United States; Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, United States

*Robyn K. Fuchs  Department of Health and Rehabilitation Sciences, Indiana University School of Medicine, Indianapolis, IN, United States *Theresa A. Guise  Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, United States

Nicoletta Bivi  Director-Assay Development, Laboratory of Experimental Medicine, Eli LIlly and Company, Indianapolis, IN, United States

Christopher J. Hernandez  Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY, United States; Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, United States; Hospital for Special Surgery, New York, NY, United States

*Andrea Bonetto  Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, United States *Lynda F. Bonewald  Department of Anatomy and Cell Biology and Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN, United States

*Kathleen M. Hill Gallant  Department of Nutrition Science, Purdue University, West Lafayette, IN, United States; Indiana University School of Medicine, Indianapolis, IN, United States

*Angela Bruzzaniti  Department of Oral Biology, Indiana University School of Dentistry and Department of Anatomy & Cell Biology, Indiana University School of Medicine, Indianapolis, IN, United States

Mary Beth Humphrey  Department of Medicine, University of Oklahoma Health Sciences Center and the Oklahoma City Veterans Administration, Oklahoma City, OK, United States

*David B. Burr  Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, United States; Department of Biomedical Engineering, Indiana University-Purdue University-Indianapolis, Indianapolis, IN, United States

*Erik A. Imel  Department of Pediatrics, Division of Pediatric Endocrinology and Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, United States; Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, United States

Laura M. Calvi  Department of Medicine-Endocrinology, University of Rochester Medical Center, Rochester, NY, United States

*Melissa A. Kacena  Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN, United States

*Are

members of the Indiana Center for Musculoskeletal Health, Indiana University School of Medicine, Indianapolis, IN, United States

xi

xii

List of Contributors

Dongbing Lai  Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, United States

*Lilian I. Plotkin  Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, United States

*Jiliang Li  Department of Biology and Center for Developmental and Regenerative Biology, Indiana University-Purdue University, Indianapolis, IN, United States

*Alexander G. Robling  Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, United States

Bruce H. Mitlak  Radius Health, Waltham, MA, United States *Sharon M. Moe  Division of Nephrology, Indiana University School of Medicine, Rodebush Veterans Administration Medical Center, Indianapolis, IN, United States

*G. David Roodman  Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, United States; Richard L. Roudebush VA Medical Center, Indianapolis, IN, United States Tae-Hwi Schwantes-An  Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, United States

Katherine J. Motyl  Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, ME, United States

Viral N. Shah  Barbara Davis Center for Diabetes, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, United States

Mary C. Nakamura  Department of Medicine, University of California at San Francisco, and San Francisco Veterans Administration Health Care System, San Francisco, CA, United States

David L. Stocum  Department of Biology and Center for Developmental and Regenerative Biology, Indiana University-Purdue University, Indianapolis, IN, United States

Thomas L. Nickolas  Department of Medicine-Nephrology, Columbia University Medical Center, New York, NY, United States *Munro Peacock  Department of Medicine, Indiana School of Medicine, Indianapolis, IN, United States Roger Phipps  Department of Pharmacology, Husson University School of Pharmacy, Bangor, ME, United States

*Joseph M. Wallace  Department of Biomedical Engineering, Indiana University-Purdue University, Indianapolis, IN, United States *Connie M. Weaver  Department of Nutrition Science, Purdue University, West Lafayette, IN, United States *Kenneth E. White  Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, United States

Biographies Dr. Burr is a University-Distinguished Professor of Anatomy and Cell Biology at Indiana University School of Medicine, and Professor of Biomedical Engineering at IUPUI. He joined the Indiana University School of Medicine faculty in 1990 as Chair of the Department of Anatomy (1990–2010), following faculty positions at the University of Kansas and West Virginia University Medical Schools. He served as President of the American Association of Anatomists (2007–09) and the Orthopaedic Research Society (2008–09), and was the Director of the Sun Valley Workshop on Musculoskeletal Biology for nearly 15 years (2004–17). He is a Fellow of both the American Association of Anatomists (AAA) and of the Orthopaedic Research Society (ORS). He has been the recipient of the Borelli Award from the American Society of Biomechanics (2008), the Gideon A. Rodan Award for Excellence in Mentorship from the American Society for Bone and Mineral Research (ASBMR), and the Henry Gray Scientific Achievement Award from the AAA. He serves as Editor-in-Chief for Current Osteoporosis Reports, Editor for Bone, and Associate Editor of the Journal of Musculoskeletal and Neuronal Interactions. He is the author of more than 250 research articles in the peer-reviewed literature; 56 book chapters and reviews; and 5 books on the structure, function, and mechanics of bone. Dr. Allen is a Professor of Anatomy and Cell Biology, Orthopaedic Surgery, and Medicine-Nephrology at Indiana University School of Medicine (ISUM), Biomedical Engineering at Indiana University-Purdue University-Indianapolis, and a nonclinical scientist at Roudebush VA Medical Center. He also holds an Assistant Dean position in Faculty Affairs | Professional Development | Diversity at IUSM. His research career, and interest in bone biology, began at Alma College during a summer research fellowship and continued during his years as a PhD student at Texas A&M (in Kinesiology) and postdoctoral fellow at IU School of Medicine. His research focuses on understanding how interventions can be maximized to strengthen the skeleton. He serves as Editor-in-Chief for Clinical Reviews in Bone and Mineral Metabolism and is on the Editorial Board for the Journal of Bone and Mineral Research, BONE, Osteoporosis International, Journal of Orthopaedic Research, JBMR-Plus and Bone Reports. He has authored more than 130 original research articles and 25 book chapters and reviews.

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Preface (First Edition) More than 10 years ago when we began to teach our graduate-level Basic Bone Biology course at Indiana University (IU), there were several excellent reference works available, primarily targeted to researchers working in a wide range of areas in skeletal biology. These included (and still include) Principles of Bone Biology, edited by John Bilezikian, Lawrence Raisz, and John Martin, which has since been expanded to two volumes; “Big Red” (Osteoporosis), the excellent and very complete reference edited by Bob Marcus, David Feldman, and Jennifer Kelsey; and the more succinct Primer of Metabolic Bone Diseases, updated and republished every few years by the American Society of Bone and Mineral Research. These are still available, and still excellent, but they do not serve well as textbooks for a bone biology course either because they are too extensive, too expensive, or do not cover relevant topics in sufficient depth. Therefore, we have chosen over the years to use primary reference materials—mostly, recent papers published in the peer-reviewed literature—for our course. From a didactic standpoint, this is an acceptable approach, and even a desirable one, especially for a graduate course in which the goal is to teach the student how to read and evaluate the literature. However, it became clear over time that this was not a sufficient surrogate for a true textbook. As the skeletal biology group at IU grew over the years, we incorporated topic experts to deliver lectures in their area of expertise. We soon realized that the course and content experts provided the foundation for building a textbook on basic and applied skeletal biology. As we discussed this idea with our colleagues here at IU and up the road at Purdue University, there was universal support and enthusiasm. Discussion with colleagues outside of our group made it clear that, beyond our own requirements, there was a need and a desire by the academic community for such a text. Writing this textbook began as something of a selfish idea—we needed it for our course—but we truly hope that it will be welcomed and used by others who find it appropriate for their own courses, or as a more modest reference than existing books on a wide range of topics in skeletal biology. Basic and Applied Bone Biology covers those topics that we feel are relevant to a modern course in skeletal biology. The book is organized, like bone, in a somewhat hierarchical manner. The first section begins with the

basic construction of bone, including its cellular structure and dynamics and the basic physiological processes that bone uses to grow and adapt itself over a lifetime. This is succeeded by several chapters related to the technical aspects used to assess bone in health and disease—various imaging modalities; biomechanical measurements useful for assessing bone properties; histomorphometric techniques to evaluate the dynamics of bone modeling and remodeling; and genetic approaches used to tease out the roles of specific genes, proteins, and epigenetic influences in the basic metabolic functions of bone. These early chapters provide the foundation for the next several chapters on skeletal adaptation, highlighting mechanically induced adaption of bone, fracture healing, and adaptation of the oral cavity associated with orthodontics and implants. Following this, the text transitions (gradually we hope) into areas that are more clinically related, the applied aspects referred to in the title. These chapters address growth and development, metabolic and hormonal processes, and how these are related to health and disease. The text ends with a chapter on pharmaceutical treatments for osteoporosis, which we hope incorporates both the clinical elements of treatment and the biological reasons for, and effects of, these treatments. Skeletal biology is, by its nature, interdisciplinary. The course that we teach at IU typically includes students in the basic medical sciences, general biology, the dental sciences, several engineering subspecialties, foods and nutrition, kinesiology, and rehabilitation sciences. We have written this textbook to cover a range of topics that we feel would be relevant to these groups and have attempted to write various chapters in a way that will be understandable to those students whose particular expertise and interest may not be in the area covered by a given chapter. We have also attempted to write the chapters so that they will be suitable for students at various levels of study, including undergraduate, graduate, and even postgraduate. We realize that the danger of this is that some chapters may be too superficial for students who are more expert in the area covered by that chapter. However, the textbook is meant to be supplemented by additional readings that delve into specific topics in greater depth for those who wish to specialize in that area. To this end, we have included a list of 10–15 suggested readings at the end of each chapter that can serve as a starting point for supplementary reading

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and discussion. Further, we have incorporated study questions at the end of each chapter. We have resisted the temptation to include answers to these questions. They are intended to be used for discussion (although they could also be used for testing), and there may not be a single “correct” answer. We hope that they will permit further exploration of the chapter topic, at the level appropriate for the student.

Finally, we have not only had a lot of fun putting this text together but have also learned a lot in areas that are not within our own expertise. We sincerely hope that it serves the same purpose for you. David B. Burr, PhD Matthew R. Allen, PhD February 16, 2013

Preface (Second Edition) Our understanding of the musculoskeletal system continues to expand. Since conceptualizing the first edition of Basic and Applied Bone Biology several years ago, there has been a rapid growth in knowledge about how bone interacts with other organ systems. When presented with the opportunity to revise this textbook in 2017, we saw an opportunity to tap into the comprehensive expertise of the expanding collection of investigators in the Indiana Center for Musculoskeletal Health at Indiana University School of Medicine, as well as that of our close colleagues from other institutions, to cover these emerging areas in musculoskeletal biology. This second edition of Basic and Applied Bone Biology takes the same fundamental approach to organization as did the previous edition, moving from basic cell biology to clinically focused topics. In addition to updates in all chapters and some reorganization of content between chapters, this new edition is highlighted by the addition of seven new chapters. In the opening section, Bone biology and physiology, we have added a chapter on bone

marrow and the stem cell niche to overview the complex interplay of cells that are generated and housed within bone. An entire new section, The Interaction of bone with other organ systems, covers bones’ connection to muscle, the immune system, the nervous system, the microbiome, and the kidney. Finally, the Skeletal disease and treatment section has been expanded to cover topics of skeletal interplay with cancer and diabetes. Our guiding principle for this revision, as with the original edition, was to provide a resource for the next generation musculoskeletal researcher. Whether you engage with the text as part of a structured course or less formally on your own, we hope the text gives you a framework of musculoskeletal biology and provides you with a foundation on which to make the next innovative leap in the field.

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David B. Burr, PhD Matthew R. Allen, PhD July 10, 2018

C H A P T E R

1 Bone Morphology and Organization David B. Burr1,2 1Department

of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, United States; 2Department of Biomedical Engineering, Indiana University-Purdue University-Indianapolis, Indianapolis, IN, United States

THE FUNCTIONS OF BONE

through endochondral ossification (see Chapter 5) and are part of the axial skeleton. It is not widely realized that bone is a blood-forming (hematopoietic) organ, but regions composed largely of spongy bone such as the iliac crest, vertebrae, and proximal femur are good sources of red blood cells throughout life. The marrow cavity within the bone is an important site of red marrow, indicative of hematopoiesis, during growth and development, but it is largely composed of yellow fat in adults. White fat and brown fat are also found in the human body, and while these are acted on by osteocalcin, which is produced by osteoblasts, these types of fat are not actually found within the bone marrow itself. White fat stores energy and secretes adipokines, and too much of it is associated with diabetes and other metabolic conditions that affect bone. Brown fat, on the other hand, burns lipid droplets and creates heat. Brown fat cells have large amounts of energy-producing mitochondria, which store iron. Young children have large amounts of brown fat, especially around the upper spine and across the shoulders. This keeps them warm, but it also provide a large iron reservoir that is important for their rapid metabolism and skeletal development. It is now known that brown fat is present in adults as well, but the amount of it declines with aging and may decline more in those who are prone to obesity. Yellow marrow fat originates from the same precursor cells that differentiate to become bone-forming osteoblasts. It provides an energy store and may contribute to lipid metabolism by regulating triglycerides. Because of the large surface area, regions with a lot of cancellous bone are also responsible for rapid turnover of bone tissue and play an important role in the long-term control of calcium balance. Bone turnover can be sensitive to changes in energy metabolism that occur as a function of aging, hormone deficiency, or the production of skeletal hormones, and this provides the means for long-term exchange of calcium

Bone is multifunctional, playing roles in mechanical support and protection, mineral homeostasis, and hematopoiesis. In recent years, it has become clear that bone also serves an important endocrine function. The mechanical functions of bone are by far the most widely recognized and studied. Both trabecular and cortical bones serve this function, although the nature of this function is partly specific to each. The dense cortical bone comprises most of the bone mass and takes on most of the role for load bearing. Although the more porous cancellous bone also supports load, one of its important functions is to redirect stresses to the stronger cortical shell. The mechanical function of bone extends beyond simple load bearing, which requires a certain degree of strength and stiffness. Because of its organization as a multiscale material, it is also highly adapted to avoid fractures caused by repetitive loading at physiologic levels, i.e., failure in fatigue. Bone also serves a protective function, especially in those vital areas such as the torso and head where injury can be fatal. In these locations, the bone microstructure is not different from that of bone in other locations, but it is organized in a manner that can absorb maximum energy with minimum trauma to the bone itself. For instance, the cranial vault is constructed of two thin plates of dense bone that sandwich porous cancellous bone (the porous appearance of this bone is why it is sometimes called spongy bone). Ribs are also constructed in this way, but with less dense cancellous bone. In the case of the ribs, the inherent curvature of the bone also increases its ability to absorb impact energy. Developmentally, bones that serve a protective function (e.g., calvarium and ribs) are formed, at least in part, through intramembranous ossification rather than

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and phosphate (as well as other minerals such as iron and magnesium). Although calcium provides bone with its stiffness and much of its strength, calcium ions in the mineral phase are also important for enzyme reactions, blood clotting, muscle contraction, and the transmission of nerve impulses. Both cortical and cancellous bones are sites of long-term storage and of the rapid exchange of ions within the mineral phase. The release of minerals through bone remodeling is a relatively lengthy process requiring timescales of days to weeks, and replacing these minerals completely takes even longer. However, the extensive surface area represented by the many osteocyte lacunae and canalicular channels provides a significant opportunity for short-term exchange to meet immediate demands. By way of example, the surface area of osteocyte canaliculi is estimated to be about 120 times greater than the surface area of all the cancellous bone in the body. The exchange of mineral that occurs at these surfaces is the source of the halo that can sometimes be observed around osteocyte lacunae, and which can appear either hypermineralized or hypomineralized depending on the content of labile calcium at this surface. Bone has been identified as an endocrine organ that helps to mediate phosphate metabolism and energy metabolism by secreting two hormones: fibroblast growth factor 23 (FGF23) and osteocalcin. Most of the body’s FGF23 is produced by osteocytes, the most abundant cell type in bone.

Trabecular bone

Trabecular packet and lamella

It causes a reduction in renal reabsorption of phosphate and a decrease in serum levels of 1,25-dihydroxyvitamin D3. Working with other hormones, bone helps to coordinate processes in the kidney and intestine that regulate its own mineralization. There is now also some evidence that the undercarboxylated form of osteocalcin, which is released from the bone matrix during resorption, helps to regulate pancreatic beta-cell proliferation and enhance insulin secretion. It also acts on adipocytes outside of bone to produce adiponectin, which can reduce insulin resistance. This has the dual effect of increasing glucose utilization and reducing fat in the body cavities. Regulation of the body’s energy stores by bone may also be mediated by leptin, acting through the autonomic nervous system and hypothalamus. Thus bone, through its several hormones, helps to coordinate processes in the bone marrow, brain, kidney, and pancreas that affect skeletal tissue mineralization, fat deposition, and glucose metabolism.

BONE IS ORGANIZED AS A MULTISCALE MATERIAL To achieve these functional goals, bone is organized in a hierarchical, fractal-like manner, from nanometerto millimeter-sized structures (Fig. 1.1). This contributes to an unusual combination of both high stiffness and

Mineralized collagen fibers

Collagen / Mineral composite Alpha chains

Osteon and lamella

Crystal lattice

Cortical bone

Microstructure and sub-microstructure

Organ and tissue 10–1m

10–4m

Nanostructure and ultrastructure 10–7m

Molecular structure 10–9m

10–10m

FIGURE 1.1  The hierarchical organization of bone. At the macroscopic level, bone is seen as a composite, with dense cortical bone forming an outside shell and cancellous (spongy, trabecular) bone within the marrow cavity. The cancellous bone serves to attenuate loads and to direct forces to the stronger cortical bone. At the microscopic level, cortical bone is composed of many secondary haversian systems, or osteons, that are the product of bone resorption and replacement with human new bone. These osteons are composed of a central canal carrying a blood vessel, nerves, and lymphatics surrounded by layers of concentric lamellae. Trabecular bone is also lamellar, but its structure comprises a combination of lamellae that run approximately parallel to the trabecular surface and the remnants of older remodeled bone that can appear osteonal in some cases. At the ultra- and nanostructural levels, bone is a composite of collagen fibers with plates of mineral interspersed within the collagen fibrils (intrafibrillar) and between the collagen fibers themselves (interfibrillar). Together, these can form a cross-fibrillar phase in which the crystals can expand beyond the dimensions of a single collagen fibril. The collagen fibrils are composed of molecules forming a triple helix composed of two α1 chains and a single α2 chain. Part of figure courtesy of Beck, et al. J. Struct. Biol. 1998;122:17–29.

I.  BASIC BONE BIOLOGY AND PHYSIOLOGY

The Nanoscale Organization of Bone

great toughness (these are often inversely related, see Chapter 7), as well as to its mechanical role in support and movement of the body. At the nanostructural level, bone is composed of organic and mineral components, mainly consisting of a matrix of cross-linked type I collagen mineralized with nanocrystalline carbonated apatite. The collagen and mineral combine to form a composite material, with mineral providing stiffness to the structure and collagen providing resilience and ductility. This composite not only protects the brittle mineral (hydroxyapatite) from breaking but also doubles its load carrying capacity while distributing the forces around discontinuities in bone and reducing stress in the bone matrix. At a microscopic (micrometer [10−6 m]) level, the individual collagen fibers with interspersed mineral are organized in different ways, depending specifically on the rate, location, and substrate (if any) on which it is formed. At this microstructural level, the organization of bone tissue is very much related to its functional needs: rapid formation for stabilization (in fracture healing) or rapid growth during development; or slower formation to adapt to changing mechanical needs or to replace preexisting bone to provide repair of damaged regions and maintain their unique mechanical properties. At this level, bone can be denser (cortical or compact bone) or rather porous (cancellous, trabecular, or spongy bone), depending on the specific mechanical or biological needs and its location.

Approximately 65% of bone by weight is composed of mineral (primarily carbonated apatite), but as a living tissue, its organic component, mostly type I collagen, contributes about 20%–25% to its composition (Fig. 1.2).

Organic (25%)

NCPs (10%)

The remainder (approximately 10%) is composed of water that is bound to the collagen–mineral composite and unbound water that is free to flow through canalicular and vascular channels in bone. Unbound water can be redistributed as the bone undergoes loading and probably contributes to the signals detected by cells, informing them of loading conditions (see Chapter 11). Water is exchanged on a nearly 1:1 basis with mineral, so as bone becomes more mineralized, water content declines, and vice versa. This is important to its mechanical behavior; more highly mineralized bone is stiffer because it has more mineral, and also because it has less water. In addition, although it is stiffer, drier bone tends to be more brittle, and therefore break more easily. About 90% of the organic portion of bone is type I collagen, with smaller amounts of types III and V collagen also found in the zone surrounding the bone cells. The remaining 10% is made up of noncollagenous proteins (NCPs) which play a vital role in regulating collagen formation and fibril size, mineralization, cell attachment, and microcrack resistance. Of this small amount of NCPs, about 85% is extracellular and the remainder is found within bone cells.

THE NANOSCALE ORGANIZATION OF BONE Collagen

BONE COMPOSITION

Water (10%)

5

Cell protein (15%) Extra cellular (85%)

Type 1 Collagen (90%)

Mineral (65%)

FIGURE 1.2  Bone is composed of an organic matrix, mineral, and water. Most of the organic matrix is type I collagen, but noncollagenous proteins (NCPs) that contribute to mineralization and adhesion are also present. Most of this is extracellular, although a small amount of protein is also found within the cells.

At a fundamental level, bone is composed of collagen fibers interspersed with plates of mineral both within and between the fibrils. Individual collagen molecules are formed from two α1 chains and a single α2 chain that assemble into a triple helix (Fig. 1.3A). Each chain is about 1000 amino acids in length, and the helical center portion of collagen molecules comprise repeating units of a Gly-X-Y triplet. The periodic repetition of glycine residues is essential to the formation of the triple helix structure. While almost all amino acids are present in collagen, X and Y groups are often occupied by proline and hydroxyproline residues. Both these amino acids form a ring with the main backbone of the chain, resulting in improved helical rigidity (Box 1.1). Hydroxyproline is particularly critical, and unique, to collagen as its hydroxyl group is essential for hydrogen bonding with water molecules. This interaction is so critical because the stability of the triple helix is maintained by a sheath of water molecules attracted by hydroxyproline. During the intracellular production stage, the nonhelical registration peptides (N-propeptide and C-propeptide) at both ends of the molecule secure the chains together by sulfur cross-links. The triple helix with its terminal propeptides is known as the procollagen molecule. Following the exocytosis of molecules, these propeptide

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(A)

(B)

(C)

FIGURE 1.3  Collagen fibrils are constructed from many collagen molecules. (A and B) Each collagen molecule is approximately 300 nm long and 1.5 nm thick. The molecules are stacked in a quarter-staggered array such that there are 67 nm hole zones between the ends of the molecules and spaces between the laterally contiguous molecules known as pores. (C) The hole and overlap zones give the collagen fibril its characteristic banded appearance when viewed with atomic force microscopy. Both holes and pores enable the deposition of mineral during primary and secondary mineralization processes. The diameter of the entire collagen fiber can vary; its thickness is regulated by the action of noncollagenous proteins such as decorin. Part of figure courtesy of Beck et al. 1998. J. Struct. Biol. 122:17–29.

BOX 1.1

O S T E O G E N E S I S I M P E R F E C TA Osteogenesis imperfecta (OI) is a set of heritable conditions of variable severity usually caused by mutations in the collagen I genes, but in some types, other genes such as CRTAP or P3H1 are involved. More than 200 mutations have been described affecting various aspects of collagen formation and cross-linking. In severe cases, an increase in the hydroxylation of lysine leads to a more gradual formation of the collagen triple helix. OI leads to skeletal fragility, and it is sometimes termed “brittle bone disease” because of the multiple fractures (sometimes prior to birth) that can occur. Eight known forms of OI have been described, with type I, the most mild, and type II, the most severe. The incidence of the condition is about 5–7 out of 100,000 people, with two-thirds of these cases afflicted with the milder type I and type IV forms. OI is sometimes characterized as an osteoporosis because it is associated with low bone mass, thin cortices, and architectural deterioration of trabeculae, but the changes to the bone matrix caused by the collagen mutations exacerbates the fragility that accompanies the low bone mass and architectural deficits. Because it is a collagen mutation, OI also affects nonmineralized tissues that harbor the COL1A1 or COL1A2 genes (such as the eyes and skin) and is associated with short stature, hearing loss, respiratory problems, and a disorder of tooth development.

There are several mouse models of OI, each with a slightly different genotype and phenotype. The most common of these are the oim/oim and Brittle IV (Brtl) mice. The oim mouse is characterized by a mutation in the COL1A2 gene associated with α1(I) homotrimers and nonfunctional proα2(I) chains. This leads to reduced collagen content and fiber diameter, and fewer divalent collagen cross-links, either because of the reduced collagen content or because of a more rapid transformation to more mature trivalent cross-links. The Brtl IV mouse is a model of type IV OI characterized by a substitution of a cysteine for a glycine in the triple helix of one of the Col1α1 alleles. Like the oim mouse, it is associated with reduced collagen fiber diameter, but unlike the oim, it also affects mineral crystalline organization and is not associated with alteration in collagen cross-linking. Both oim and Brtl animals have significantly lower mechanical properties, even after accounting for bone mass, illustrating the role collagen plays in mechanical integrity. Because of the heterogeneity of causes for OI, any animal model is not likely to recapitulate the human condition entirely. Choice of an appropriate animal model should also consider what type of OI is under study.

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The Nanoscale Organization of Bone

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(A)

(B)

Mineral particles

Collagen molecule

Mineral particles

40 nm

FIGURE 1.4  (A) Collagen molecules (green helix) are cross-linked within the fibril by bonds that are formed through enzymatic processes, and by those formed without the need for an enzymatic reaction. Enzymatically formed cross-links (black bars), such as pyridinoline or deoxypyridinoline, form near the ends of the molecules, the C- and N-termini. Nonenzymatically formed bonds (red bars), such as pentosidine, are randomly located between the molecules. Mineral (gray blocks) is deposited in the hole and pore zones between the collagen fibrils. Water (blue lines) and hydrogen bonds contribute to the bonding of mineral and collagen within the fibril. (B) Hole and pore zones between the molecules contain plates of bone mineral (hydroxyapatite). Water is bound to collagen in these spaces, and this alters the distribution of load sharing between the collagen and bone mineral deposited in this location.

regions are cleaved enzymatically, leaving nonhelical domains at both ends of the molecule, termed the N- or C-telopeptides (at the N-terminus or C-terminus, respectively). Cleavage of the registration peptides forms the mature collagen molecule, composed of the helical triple helix region and the nonhelical terminal N- and C-telopeptides. Lateral and longitudinal aggregation of collagen molecules is essential to extend from the nanoscale to the microscale. In this assembly, five molecules form a microfibril in a semihexagonal arrangement. Microfibrils aggregate laterally and longitudinally to make up fibers that are eventually about 150 nm in diameter and 10 μm in length. Electron microscopy images of collagen fibers reveal an approximately 67 nm banding pattern, called the D-banding pattern, which represents the space between the ends of contiguous collagen molecules, and the overlap between the end regions of laterally contiguous molecules (Fig. 1.3B). The mean diameter of collagen fibrils and their spacing is less in osteoporotic bone than in healthy bone, which may increase the bone’s fragility. Collagen fibrils are connected by different kinds of cross-links that can have profound effects on the material properties of the tissue and ultimately on the

mechanical behavior of the whole bone (Fig. 1.4). These can be broadly grouped into those formed through enzymatic processes and those formed through processes of nonenzymatic glycation, which create advanced glycation end products (AGEs). Enzymatically Mediated Collagen Cross-Linking Pyridinoline and deoxypyridinoline are two mature cross-links of collagen that are derived from an enzymatic pathway initiated by the enzyme lysyl oxidase. Pyridinoline is the maturation product of two hydroxylysyl (Hyl) residues from the telopeptide with a Hyl from the α-helix, whereas the deoxypyridinoline analog contains a lysine residue from the α-helix. These trivalent cross-links are very stable. The content of these mature cross-links in human bone collagen increases sharply up to the age of about 10–15 years and thereafter remains constant or possibly declines slightly, although the number of pyridinoline and deoxypyridinoline cross-links can change with treatments that alter bone turnover. An increased pyridinoline:deoxypyridinoline ratio has been related to increased compressive strength and stiffness in bone but probably has no effect on toughness or ductility.

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Nonenzymatically Mediated Collagen Cross-Linking There are several cross-links of collagen that result from the nonenzymatic condensation of arginine, lysine, and ribose. Pentosidine, a fluorescent AGE, constitutes the smallest fraction of these nonenzymatically glycated cross-links, but it is often used as a marker for their total content because it is one of the only AGEs that can be accurately quantified. Other AGEs include Nεcarboxymethyllysine (CML), furosine, imidazolone, and vesperlysine. Recently, CML, which is nonfluorescent, has been shown to be present in bones at levels 40–100 times greater than pentosidine. As AGE formation occurs over a period of years, proteins with long half-lives, such as collagen, can accumulate substantial amounts of AGEs with age; for example, pentosidine accumulation triples over the last three decades of life; CML accumulation may increase by 5–10 fold over the same period. In addition, AGEs have been shown to reduce collagen fibril diameter. Because they can be formed in the presence of sugars (such as glucose or ribose), they are accumulated by individuals with diabetes mellitus (Chapter 23); this is one contributor to the increased bone fragility found in these individuals. Accumulation of AGEs in the extracellular matrix of bone also regulates the proliferation and differentiation of bone-forming cells, the osteoblasts, through interaction with the AGE-specific receptor (RAGE). Binding to RAGE activates NF-κB in osteoblasts and stimulates the production of cytokines. The AGE–RAGE binding interaction also upregulates the production of reactive oxygen species, which elevates inflammation in the bone microenvironment and leads to bone loss. Accumulation of AGEs in collagen can impair osteoblast proliferation and differentiation, reduce osteocalcin secretion, and cause disruptions in cell–matrix interaction and cell adhesion that ultimately affect bone formation. AGEs also may regulate both osteoclastogenesis and osteoclast activity. Osteoclastic resorption is slowed in the presence of AGEs, in part, perhaps, because the solubility of collagen is reduced. These pathways—AGE regulation of osteoclast differentiation and activity, as well as effects on matrix solubility—may contribute to normal or even elevated bone mass that is found in people with type 2 diabetes who have high concentrations of AGEs in their bone. However, the presence of AGEs makes the bone material (tissue) brittle and thus more susceptible to fracture even though there is more bone mass. Collagen Orientation Historically, collagen in bone has been reported to be regularly organized, with collagen fiber bundles arranged parallel to each other in adjacent sheets (lamellae), either perpendicular to each other or arranged

alternately in adjacent lamellae. This stemmed from the different microscopic appearance of bone under polarized light (Fig. 1.5). It is likely that this is partly a function of the optics and plane of section, rather than the way that collagen bundles are arranged. Under cross-polarized light, collagen fiber bundles that are oriented transverse to the plane of viewing appear light, or birefringent, whereas those that are oriented parallel to the plane (i.e., longitudinally) are dark. This is because transversely oriented fiber bundles rotate the plane of polarized light with respect to the viewing plane, whereas those that run longitudinally do not. Alternately arranged, or intermediate, collagen fiber bundles are interpreted as representing a combination of fiber arrangements in successive lamellae. In reality, there are many variations on this theme, and it is likely that collagen fiber bundles even within a lamella are arranged in many different orientations, with the predominant orientation being responsible for what is observed either microscopically or by X-ray diffraction. The collagen in these layers has been shown to be preferentially oriented with respect to the predominant stress in the bone. Longitudinally oriented fibers are predominantly found in portions of bone that are under tension (i.e., being pulled further apart), whereas transversely oriented fibers are more abundant in regions that are usually under compression (i.e., being pushed closer together). This has been shown by mapping the numbers of light or dark osteons across sections of bone in which the primary loading directions are known. This has also been shown experimentally, by altering the direction of loading and observing the collagen fiber direction in the newly formed bone. Both approaches suggest that there is a relationship between collagen fiber orientation and the predominant direction of loading. For reasons to do with optics and light transmission, bone collagen may only appear in polarized light to be oriented preferentially in these directions. In reality, bone collagen may be organized in a twisted plywood configuration that is continuously rotated through 180 degrees cycles (Fig. 1.5B). In this scheme, the collagen fibers gradually, rather than abruptly, change orientation from one successive lamella to another. Under polarized light, this would make the bone appear lamellar, with differing light and dark areas. It is as if one took a piece of plywood in which the fibers in successive plies were perpendicular to each other, twisted it, and then examined the orientation of the individual plies end on. The fibers would then appear as arches, rather than as discrete and oriented fibers. Because the fiber orientation repeats in this model, the tissue-level structure appears lamellar, thus giving bone its characteristic laminar appearance under the microscope. Whether the collagen is twisted or not, how it becomes oriented in the directions it does is something of a mystery. It has been suggested that the orientation of

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The Nanoscale Organization of Bone

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FIGURE 1.5  (A) Variations in collagen orientation can be viewed using polarized light. Fibers that are oriented transversely to the direction of light appear bright, whereas those oriented along the path of the light are dark. In a cross section of cortical bone, dark osteons are composed of longitudinally oriented fibers. Some osteons on the right side of this image would also be characterized as alternately fibered. (B) Electron microscopy images of parallel and alternately fibered collagen bundles in bone are shown along with a schematic representation (top two images). Collagen in bone may in reality be organized in a helicoidal arrangement in which collagen orientation changes only slightly from lamella to lamella. The bottom image shows this effect in three dimensions. Panel (B) reproduced with permission from Ruggeri et al. In: Motta PM, editor. Recent advances in microscopy of cells, tissue, and organs. Rome: Antonio Delfino Editore, 1997.

the osteoblast depositing it determines the collagen orientation and that the mineral is simply deposited in the spaces between collagen fibrils. An alternative suggestion is that collagen is deposited without any preferred orientation and that the deposition of the mineral, which is charged, causes both the collagen and the mineral to become oriented in directions that are dependent on the mechanical environment. Which of these ideas is correct is still under debate.

Bone Mineral Bone mineral is composed of highly substituted, poorly crystalline carbonated apatite mineral, which nucleates within the gap regions between the ends of the collagen fibrils, also known as hole zones, as well as along the pores that run longitudinally between the fibrils (Fig. 1.4).

Mineral is initially deposited as an amorphous calcium phosphate, along with large amounts of calcium carbonate. As bone tissue matures, the carbonate content is reduced, and mineral crystals grow laterally, becoming more platelike, and orient themselves parallel to one another and to the collagen fibrils. The long axis of the mineral plate, or c-axis, aligns with the longitudinal axis of the bone. The average size of the mineral crystals in bone tends to span a wide range, but the majority (98%) has a thickness less than 10 nm. Eventually, the plates coalesce with other crystals to become larger polycrystalline aggregates that can become greater than the width of the collagen fibrils. As bone ages, the mineral crystals also can enlarge due to ion substitutions and changes in mineral stoichiometry. Therefore, the measured average size of mineral crystals is highly dependent on tissue age. However, it can be difficult to distinguish between smaller crystals with many

I.  BASIC BONE BIOLOGY AND PHYSIOLOGY

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1.  BONE MORPHOLOGY AND ORGANIZATION

Rate of new bone mineralization (as a % of interstitial bone mineralization)

imperfections and larger crystals with a few imperfections; both may demonstrate similar crystalline properties. More soluble carbonate can exist in a labile form on the surface of the crystallites, but it can also substitute for the phosphate and hydroxyl groups in carbonated apatite. These substitutions within the mineral allow it to be more easily resorbed. Such substitutions distort the shape and size of the crystals and reduce the stability of the mineral lattice. During episodes of acid load, sys− temic bicarbonate (HCO3 ) is consumed to buffer the − blood pH. The HCO3 deficiency is offset by carbonate and phosphate ions present in bone’s mineral reservoir. Thus, in chronic acidosis, the bone’s mineral reservoir not only helps to maintain acid–base homeostasis but also results in bone loss. In addition, different cations (e.g., magnesium, sodium, and strontium) can substitute for the calcium ions, and fluoride can substitute for the hydroxyl group in the apatite lattice; in some cases, the mechanical properties of bone are altered and in others the activity of osteoblasts and osteoclasts is affected. At one time, sodium fluoride (NaF) was considered a promising anabolic therapy for osteoporosis. There is evidence that NaF stimulates osteoprogenitor cells and preosteoblasts, promoting direct bone formation without the need for prior resorption. Moreover, fluoroapatite (the mineral with fluoride substituted for the hydroxyl groups) is more resistant to resorption than is the carbonated apatite. However, the substitution of fluoride into the mineral crystal increases the brittleness of bone and may therefore hasten rather than delay fracture. Although this may or may not occur with other ion substitutions in the mineral crystal, it is instructive in showing that hydroxyapatite is finely adapted to the bone’s specific mechanical needs.

100

80

There are two sequential and continuous phases to the deposition of mineral in bone: an initial and rapid increase in the number of mineral crystals due to heterogeneous nucleation (primary mineralization) and a slower growth and maturation of those crystals to an eventual size of about 40 nm × 3 nm × 7.5 nm. During primary mineralization, mineral is rapidly deposited within the collagen framework, achieving 65%–70% of its total mineralization within about 3 weeks after the initial deposition of collagen. During the secondary phase of mineralization, the bone matrix continues to accumulate mineral at a slower, more progressive rate until the amount of mineral reaches a physiologic limit (Fig. 1.6). Estimates for the completion of secondary mineralization range from a few months to many years.

Noncollagenous Extracellular Matrix Proteins There are numerous NCPs in bone that regulate and direct the construction and maintenance of the extracellular matrix. Although these proteins only account for about 2% of the bone by weight, they play vital roles in embryogenesis and development, regulate the formation and size of collagen fibrils, control mineralization, and provide conduits for cellular signaling and attachment. They can be divided into several large classes (Table 1.1), including   

1. P  roteoglycans (heparin sulfate, hyaluronan, small leucine-rich proteoglycans [SLRPs], and versican); 2. Glycoproteins (alkaline phosphatase [ALP], fibronectin, thrombospondin [TSP1 and 2], and vitronectin); 3. Proteins of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family that are associated with bone mineralization (dentin matrix acidic phosphoprotein 1 [DMP-1], matrix extracellular phosphoglycoprotein [MEPE], osteopontin, sialoproteins); 4. Osteocalcin (or bone Gla protein); and 5. Osteonectin (also known as secreted protein acidic and rich in cysteine [SPARC]).

Proteoglycans and Glycosaminoglycans 60

40

1 18 35

70

105 140 175 210 245 280 315 350 Days

FIGURE 1.6  Primary mineralization of bone occurs within the first 3 weeks after the osteoid is deposited (black colored region). Secondary mineralization occurs in part through a slower growth and maturation of the crystals and can require a year or more to complete (gray colored region).

Proteoglycans are a broad class of molecules defined by a core protein covalently bonded to a variable number of sulfated glycosaminoglycan side chains. Proteoglycans range widely in size, although those in bone tend to be in the smaller range. Proteoglycans in bone help to regulate mineralization by affecting apatite nucleation and growth. Hyaluronan and its receptor, CD44, work together to direct skeletal development. Hyaluronan is a long chain of nonsulfated glycosaminoglycan. It is found mostly in the periosteum and along the endocortical surfaces of bone, but it is also present around all major cell types, including

I.  BASIC BONE BIOLOGY AND PHYSIOLOGY

The Nanoscale Organization of Bone

11

TABLE 1.1  Noncollagenous Proteins in Bone PROTEOGLYCANS AND GLYCOSAMINOGLYCANS Heparan sulfate

Produced by osteoclasts and osteoblasts Plays important roles in cell–cell interactions

Hyaluronan

Nonsulfated glycosaminoglycan Hyaluronan in periosteum, endosteum, and around cells CD44 is the cell surface hyaluronan receptor and plays a role in development

Small leucine-rich proteoglycans

Provide structural organization in bone

Biglycan

Found in pericellular location undergoing morphological delineation Upregulated in osteoblasts and may act as shear sensors when found in osteocytes Binds collagen and TGF-β

Decorin

First appears in preosteoblasts and is downregulated in more terminal osteoblastic cells Binds to collagen and TGF-β and may regulate fibril diameter Inhibits cell attachment to fibronectin

Fibromodulin

Binds to distinct regions of collagen fibers Binds TGF-β

Osteoadherin

Contains RGD sequence Function unknown

Versican

CS-containing PG found in osteoid May capture space destined to become bone

GLYCOPROTEINS Alkaline phosphatase

Potential Ca2+ carrier Hydrolyzes inhibitors of mineral deposition such as pyrophosphates Loss of function leads to hypophosphatasia Bone formation marker Nonspecific and bone-specific forms (bone-specific alkaline phosphatase [BSAP])

Fibronectin

Produced during early stages of bone formation Binds cells in an RGD-independent manner May be involved in proliferation

Thrombospondin

Role in development—found in early stages of bone formation (MSCs and chondrocytes during cartilage development) Antiangiogenic

Vitronectin

Involved in cell attachment and spreading; shows specificity for osteopontin

SIBLING FAMILY OF GLYCOPROTEINS Bone sialoprotein

Limited pattern of expression Marks late stage of differentiation and early stage of mineralization

Dentin matrix acidic phosphoprotein 1 (DMP-1)

Expressed by osteocytes and osteoblasts Has affinity for hydroxyapatite and the N-terminus of type I collagen Regulates mineralization

Matrix extracellular phosphoglycoprotein

Expressed by osteocytes and osteoblasts Regulates mineralization Negative regulator of osteoblast activity

Osteopontin

Secreted by bone cells in early stages of osteogenesis Promotes adhesion of different tissues (cement line and periodontal ligament) Inhibits mineral formation and crystal growth Continued I.  BASIC BONE BIOLOGY AND PHYSIOLOGY

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1.  BONE MORPHOLOGY AND ORGANIZATION

TABLE 1.1  Noncollagenous Proteins in Bone—cont’d OTHER IMPORTANT NONCOLLAGENOUS PROTEINS Osteocalcin

Enhances calcium binding, controls mineral deposition Expressed by osteoblasts and osteocytes Bone remodeling marker Overexpressed in cancer and some autoimmune diseases

Osteonectin

Binds to collagen, HA, and vitronectin Located at sites of mineral deposition (possible nucleator) May play a role in osteoblast proliferation

Ca2+, calcium ion; CS, chondroitin sulfate; HA, hyaluronic acid; MSC, mesenchymal stem cell; OPG, osteoprotegerin; PG, proteoglycan; RGD, Arg-Gly-Asp.

osteocytes, within the bone matrix. Versican is a chondroitin sulfate–containing proteoglycan that is important for cartilage formation and is therefore found in the developing skeleton. However, it is also found in osteoid in adult bone, where it may inhibit or regulate mineralization. Heparan sulfate is produced by osteoblasts and osteoclasts and plays a role in cell–cell communication. It binds FGF and can act as a coreceptor on this protein. It also binds and modulates the activities of transforming growth factor beta (TGF-β) and osteoprotegerin/tumor necrosis factor receptor superfamily member 11B (OPG), both important signaling molecules during the process of bone remodeling and repair. SLRPs are small proteoglycan molecules that are involved in constructing the collagenous matrix, controlling the aggregation and size of collagen fibrils, and possibly assisting in collagen–mineral interactions. Perhaps the most important SLRPs are decorin and biglycan: both maintain osteoblast numbers, but they perform that function at different stages of osteoblast development. Decorin is expressed early during cell differentiation, by preosteoblasts, and is downregulated during terminal differentiation of osteoblasts. Biglycan, on the other hand, can induce apoptosis in osteoblast progenitor cells and is upregulated in mature osteoblasts. It is also found in osteocytes and in the pericellular regions of the matrix and may act as a sensor of shear stress in this location. Both decorin and biglycan function in a complementary manner to maintain osteoblast number. In addition, both bind to collagen and to TGF-β and can therefore modulate growth factor activity. Glycoproteins There are a number of glycoproteins in bone, and some of their functions are not completely understood. However, several are critical to the regulation of bone mineralization. ALP is used as a biomarker for bone formation because it hydrolyzes pyrophosphates, which inhibit mineral deposition by binding to mineral crystals. Neutralizing the pyrophosphates in bone allows normal crystal growth and leads to normal mineralization. ALP is produced by many different organs in addition

to bone (e.g., kidney and liver). Therefore, alterations in ALP levels are not necessarily an accurate indicator of the activity of mineralization processes in bone. However, bone-specific alkaline phosphatase can also be measured and represents a widely used and beneficial marker of bone formation and mineralization. Low levels or loss of function of ALP results in a condition known as hypophosphatasia, which causes hypercalcemia and can lead to death in children. TSP1 and 2 are antiangiogenic NCPs that are important during the early stages of bone formation and are found in mesenchymal stem cells and chondrocytes during cartilage development. TSP2 is a promoter of the mineralization process and increases in osteoid undergoing mineralization. Fibronectin and vitronectin are two other glycoproteins that bind to cells. The former may be important in the early stages of bone formation and cell proliferation. In contrast, vitronectin regulates cell attachment and spreading. It is found in the osteoclast plasma membrane and may collaborate with osteopontin in attaching osteoclasts to the mineral matrix. SIBLING Proteins The SIBLING family of phosphoproteins includes bone sialoprotein (BSP), DMP-1, MEPE, and osteopontin. All of these phosphoproteins play a role in bone mineralization. Osteopontin is secreted by osteoblasts in the early stages of osteogenesis. It inhibits mineral formation and crystal growth, and it is found locally in regions of lower mineralization, such as the cement line in bone and the periodontal ligament surrounding the teeth. It may also act to provide a scaffold between tissues with different matrix composition and to provide cohesion between them. It has been suggested that this is the bone glue that provides fiber matrix bonding, as well as crack bridging in the case of microcrack formation. Osteopontin also binds to osteoclasts and promotes the adherence of the osteoclast to the mineral in bone during the resorption process. DMP-1 is expressed by osteocytes and osteoblasts. It has a high affinity for hydroxyapatite and the N-telopeptide region of type I collagen and functions to locally regulate the mineralization process.

I.  BASIC BONE BIOLOGY AND PHYSIOLOGY

The Microstructural Organization of Bone

It has been implicated in DNA binding, gene regulation, and integrin binding. The absence of DMP-1 causes elevated FGF23 and results in hypophosphatemic rickets. It is unknown whether DMP-1 plays a role in the differentiation of osteoblasts to osteocytes. MEPE is another protein of the SIBLING family that regulates bone mineralization locally. It is found predominantly in odontoblasts and osteocytes, where it is highly expressed during the mineralization process. MEPE is highly expressed in tissues undergoing rapid mineralization, for example in the woven bone of a fracture callus, as well as in endochondral and intramembranous ossification. Animal studies suggest it is a negative regulator of osteoblast activity; the absence of MEPE results in a high bone mass and resistance to bone loss. Osteocalcin Osteocalcin enhances calcium binding and controls mineral deposition. It is expressed by osteoblasts and osteocytes. For this reason, it is used as a marker of bone formation, although it may also function to regulate osteoclasts and their precursors. Mice in which osteocalcin is absent have severe osteopetrosis. Therefore, osteocalcin can be more accurately viewed as a marker of bone remodeling, and its level increases with the remodeling rate even in those cases, such as postmenopausal osteoporosis, in which there is a severe imbalance between formation and resorption. Osteonectin Osteonectin is located at sites of mineral deposition, where it binds to hydroxyapatite, collagen, and

vitronectin, and may promote nucleation of new mineral crystals. It may also play a role in osteoblast proliferation and its absence results in osteopenia or low bone mass. It binds to several different growth factors (FGF2, plateletderived growth factor [PDGF], and vascular endothelial growth factor [VEGF]) and may regulate their activity.

THE MICROSTRUCTURAL ORGANIZATION OF BONE At the microstructural level, bone can be organized in a variety of different ways, determined by its function and the manner in which it is deposited. Most, but not all, bone is to some degree lamellar, meaning that collagen and mineral exist in discrete sheets that can be visualized under the microscope. The lamellae create circumferential bands of bone, each 3–7 μm thick, which give the appearance of tree rings, each separated by an interlamellar layer approximately 1 μm thick. The lamellae may be arranged around the endocortical (wall of the marrow cavity) or the periosteal (outer border of the bone) circumference of the bone (circumferential lamellae), within individual trabeculae, or concentrically around individual vascular channels (concentric lamellae; Fig. 1.7).

Woven Bone Woven bone is rapidly formed and highly disorganized and is therefore not arranged in a lamellar pattern (Fig. 1.8). Its rapid formation is the result of a large cell:bone (B)

(A)

13

Trabecular lamellae (primary and hemiosteon)

Cancellous envelope

(C)

Intracortical envelope

Periosteal envelope Endocortical envelope

FIGURE 1.7  (A) Macroscopically, bone appears as either porous cancellous bone or denser cortical bone. This structure creates four different kinds of surfaces, called envelopes, on which bone cells can act. (B) Trabeculae in the cancellous bone compartment consist mostly of primary lamellae. However, remodeled areas (areas in which bone has been resorbed and reformed) can also form hemiosteons, similar to half osteons. (C) The intracortical envelope in humans is packed with secondary osteons, or haversian systems.

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1.  BONE MORPHOLOGY AND ORGANIZATION

FIGURE 1.8  (A) Woven bone is laid down rapidly, and the collagen fibers within it lack any preferred organization. This is demonstrated by the diffuse tetracycline labeling (yellow) found among the pores within it. (B) Lamellar bone is deposited in sheets in a more organized fashion than woven bone. (C) The lamellar structure can be seen using polarized light microscopy.

volume ratio. It is usually, but not always, deposited de novo without any previous hard tissue or cartilage model (anlage). It is composed of small and randomly arranged type I collagen fibers that are rapidly mineralized, probably resulting in a tissue that is more highly mineralized than lamellar bone. Because it forms so quickly, it initially presents as a lattice structure, with large pores present within the mineralized structure. This is primarily a repair tissue, forming the callus that bridges the gap during fracture healing to provide stability for the bone during the healing process. It also occurs in response to inflammation, such as in osteomyelitis. However, woven bone is also formed in nonpathologic situations when mechanical loads are much higher than usual or are presented in a way to which the bone is not fully adapted and is found in the region of the growth plate during endochondral ossification during normal skeletal development.

Primary Bone There are three types of primary bone that are differentiated by their microscopic organization: primary lamellar bone, plexiform (or laminar) bone, and primary osteons. They are morphologically distinct and impart different mechanical and physiologic properties to satisfy their different functions. They are united by the commonality that they must be deposited directly onto a substrate of either bone or cartilage (or calcified cartilage), without resorption of preexisting bone.

Primary Lamellar Bone Primary lamellar bone (Fig. 1.8) is the principal type of bone formed on the periosteal surface. It is characterized by a series of parallel laminar sheets. It can become quite dense and has few vascular canals. Therefore, it is very strong and provides a primarily mechanical function. However, it is also deposited on the surfaces of the marrow cavity and on trabeculae within the marrow, where it can be quite labile. It may turn over rapidly and be replaced and may therefore serve to support calcium metabolism. Plexiform Bone Plexiform bone (Fig. 1.9) sometimes called fibrolamellar bone, is generally not found in humans (although it has been reported to occur around the time of the major growth spurts) but is found in many animals, especially those that grow rapidly (e.g., cows and sheep). It is a combination of nonlamellar bone, which forms a core substrate, and primary lamellar bone, which is deposited on the surface of the substrate. The nonlamellar portion forms de novo within the fibrous periosteum as buds of fine-fibered bone composed of small and randomly oriented collagen fibrils (Fig. 1.10). These buds of bone unite with adjacent buds to form a bridge of bone separated from the surface of the preexisting bone by a space that includes vascular elements. Plexiform bone derives its name from this interconnecting vascular plexus. The

I.  BASIC BONE BIOLOGY AND PHYSIOLOGY

The Microstructural Organization of Bone

15

FIGURE 1.9  Plexiform bone is composed of lamellar bone laid down on a woven bone core. (A) This gives it its bricks and mortar appearance which can be best visualized using reflected light microscopy. (B) Backscattered electron microscopy. Courtesy Dr. Mitchell Schaffler.

De novo bone formation

Lamellar bone formation

Periosteum Periosteum

Vascular spaces

FIGURE 1.10  The formation of plexiform bone. Bone formation begins with intramembranous ossification within the periosteal membrane. Vascular spaces surrounding these bone cores are filled by lamellar apposition.

initial bridging also provides a way to rapidly increase bone strength, as small amounts of bone on the outer surface will contribute significantly to its strength (see Chapter 7). The bridges of bone provide several surfaces on which lamellae can be deposited and are one of the reasons that the bone can form so rapidly. As the lamellae form on the surface of the nonlamellar bridges, they gradually fill in these vascular spaces, while retaining

smaller spaces for the vessels of approximately 25–50 μm in diameter. Primary Osteons Primary osteons are formed by infilling of enlarged vascular channels, usually found within well-organized lamellar bone. The osteonal lamellae are concentrically deposited on the surface of the canal until only a

I.  BASIC BONE BIOLOGY AND PHYSIOLOGY

16

1.  BONE MORPHOLOGY AND ORGANIZATION

small vascular canal remains. Bone cells are arranged in several circular layers (resembling a solar system) around the vascular canal. Primary osteons are only about 50–100 μm in diameter or smaller, typically have fewer than 10 lamellae, and do not have a well-defined boundary separating them from the rest of the existing matrix. It has been suggested that the existence of primary osteons is related to body size and rapid growth; the presence of primary osteons in rapidly growing deer antler supports this notion.

Secondary Bone Primary bone is new bone made in a space where bone has not previously existed, although it may be formed on an existing bone surface. When bone is the product of resorption of previously deposited bone followed by deposition of new bone in its place, it is called secondary bone. This distinction is important because the apposition of primary bone, which requires only formation, is different from that of secondary bone, which requires a coordinated (coupled) series of processes to resorb and replace the bone that was already there. This is one way of repairing microscopic damage, which is constantly being created. The result of this process of resorption and replacement is a secondary osteon or haversian system (Fig. 1.11). Secondary osteons form longitudinally arrayed fibers embedded in a matrix composed of interstitial lamellae but separated from the matrix by a ductile interface, the cement line. The secondary osteon is distinguished from a primary osteon in being larger (100–250 μm in diameter), having more concentric lamellae (approximately 20–25 lamellae), and having a cement line at its outer boundary. The number and size of the osteons varies with age in

(A)

Interstitial bone

predictable ways, becoming more numerous but smaller as we grow older. As with primary osteons, the lamellae surround a central haversian canal (approximately 50 μm in diameter) that carries a neurovascular bundle. Secondary osteons are about 1–10 mm long, running at an average angle of 11–17 degrees with respect to the long axis of the bone. However, the orientation of any individual osteon may be quite variable, as the vascular spaces they contain branch extensively. Moreover, haversian vessels are connected in a vascular plexus by other vessels that run between them in a more or less transverse direction. The canals in which these vessels run are called Volkmann’s canals. The vessels in these canals also connect the haversian capillaries with the marrow vasculature, and with the vascular plexus in the periosteal membrane. The cement line, or reversal line, represents a remnant of the reversal phase of bone remodeling, i.e., the point at which osteoclastic bone resorption stops and bone formation begins. It clearly demarcates the secondary osteon from its surrounding bone matrix (Fig. 1.11). Cement lines are mechanically important structures that serve as fiber reinforcements to the bone tissue. It is well established using histologic, birefringence, and electron microscopic techniques that the cement line is collagen deficient. There is some debate about whether cement lines are highly mineralized or deficient in mineral, but in either case, their mechanical function in preventing or deflecting crack growth would be the same. In addition to mineral and collagen, the cement line also contains high levels of certain kinds of NCPs, such as glycosaminoglycans and osteopontin. This makes a great deal of sense, as osteopontin plays a role in osteoclast adhesion during resorption and the cement line is where the osteoclasts stop resorbing.

(B)

Concentric lamellae

FIGURE 1.11  Secondary bone is formed by removal of preexisting bone and replacement with new bone. (A) In the cortex, this results in a secondary osteon, or haversian system, that surrounds a canal transmitting vessels and nerves associated with the vessels. (B) The osteon is bounded by a cement line that separates it from interstitial bone. Courtesy Dr. Mitchell Schaffler.

I.  BASIC BONE BIOLOGY AND PHYSIOLOGY

The Macroscopic Organization of Bone

Both proteoglycan and osteopontin inhibit mineralization, and it also makes sense that the cement line would be mineral deficient. Osteons have poor shear strength and a weak fiber–matrix interface at the cement line. The functional importance of cement lines lies in their ability to control fatigue and fracture processes, absorb energy by stopping crack propagation, and provide viscous damping in compact bone. They create significant point-specific stiffness variations in bone and increase the static toughness of bone by preventing deleterious crack growth, thereby improving fatigue properties. A viscous cement line may relieve locally high shear stresses by allowing deformation at this interface; the low shear stiffness makes it difficult to transmit energy to a growing crack.

Interstitial Bone Inevitably, bone that is remodeled will leave behind traces of old bone that is preexisting but not remodeled. This bone may appear to be lamellar but disorganized because its lamellae, in cross section, are incomplete. This interstitial bone represents the remains of either primary or secondary lamellar bone and fills the gaps between adjacent secondary osteons (Fig. 1.11). Because it has not been recently remodeled, its mean tissue age is older than osteonal bone (although it may formerly have been a complete osteon), and it is more highly mineralized and more susceptible to accumulation of microcracks.

THE MACROSCOPIC ORGANIZATION OF BONE At the macroscopic level, bone can be divided into dense cortical (or compact) bone and more porous cancellous bone, which is composed of trabecular struts. These types of bone are distinguished not only by their porosity but also by their location and function.

Cortical Bone Cortical bone is found as the primary component of the shafts or diaphyses of the long and short bones of the extremities (Fig. 1.7). The haversian canals in cortical bone create a porosity of about 3%–5%, although this increases with age and with osteoporotic changes to the skeleton. Compact bone is also found surrounding the cancellous bone of the vertebral body, at the ends (or metaphyses) of the long bones, in the iliac crest, and in the skull. It provides both support and protection.

17

Cancellous Bone1 Cancellous bone is found primarily in the metaphyses of the long bones, as well as in the vertebrae, ribs, and iliac crest. It is composed of plates and rods of bone, each about 200 μm thick, but comprising only about 25%–30% of the total tissue volume, with the remainder being marrow space (Fig. 1.7). In cancellous bone, the lamellae are arranged more or less parallel to the trabecular surface, but one finds what appear to be half osteons, or hemiosteons, that represent previous periods of surface resorption and subsequent formation (i.e., remodeling events) (Fig. 1.7). One surface of the hemiosteon borders the marrow cavity, rather than a canal, and is separated from the rest of the trabecula by a cement line. These hemiosteons are the product of the same type of bone remodeling found in cortical bone, but, because they start on a longer surface rather than on the surface of a haversian canal, they do not have the circular appearance of whole secondary osteons. As they are adjacent to the marrow cavity and can derive their blood supply from it, they neither need nor contain a central vascular channel. Occasionally, complete osteons can be found within a trabecula, although because osteonal diameter is similar to or larger than the thickness of a trabecula (approximately 150–180 μm), this is somewhat rare; the osteons found in trabeculae tend to be smaller than those found in cortical bone. If the trabecular strut becomes very thick, bone resorption can occur parallel to the primary orientation of the trabecula and through its approximate center. This initially creates a tunnel in the trabecula (trabecular tunneling), which is essentially a longitudinal section of an entire bone remodeling unit. This process eventually divides the single trabecula into two trabecular plates (Figure 8.21). Cancellous bone derives its primary mechanical benefit from its architecture, which provides structural support without increasing the weight of the entire bone. Because of its location within the marrow cavity, by itself it is not an efficient weight-bearing structure. One important mechanical function is to provide a means for the bone to funnel the stresses imposed on it to the stronger, more massive cortical bone. Because it is highly interconnected, it provides a series of struts that can strengthen bone, similar to the way that the struts in the Eiffel Tower make it a strong structure (Fig. 1.12). The importance of a connected architecture can be demonstrated by a simple 1  Cancellous bone is sometimes also called spongy bone or trabecular bone. Cancellous is preferred over trabecular when referring to this bone as a structure because cancellous imparts the idea of a porous structure. When referring to individual struts, the term trabeculae can be used. Spongy bone is not preferred because this bone is not spongy from a mechanical standpoint, even though its appearance resembles the structure of a sponge.

I.  BASIC BONE BIOLOGY AND PHYSIOLOGY

18

1.  BONE MORPHOLOGY AND ORGANIZATION

(A)

(C)

(B)

FIGURE 1.12  Architecturally, cancellous bone is composed of plates and rods running at various angles that are thought to reflect the orientation of the major stresses within the bone (A and B). These trabeculae act as struts to support the outer cortical structure, much as the struts of the Eiffel Tower act to support its outer framework (C). (C) Reproduced with permission from Östberg G. Mater. Des. 2002;23(7):633–640.

(A)

(B) 100 lbs.

(C) 50 lbs.

25 lbs.

FIGURE 1.13  The importance of bone architecture, independent of the amount of material (i.e., mass). Cancellous bone in which the cross struts are disconnected (C) is not very capable of supporting a load, even though there is as much mass as in (A). A smaller mass that is buttressed properly (B) may be able to support more load than a greater mass in which connectivity is compromised. Figures indicate maximum load.

analogy to a bench with cross struts between the primary load-bearing legs (Fig. 1.13). When these struts are connected, the stool can hold weight effectively. However, when those struts are not connected or not connected efficiently, even if they are still present, the stool will not serve as well as a load-bearing structure. The architecture of cancellous bone can be characterized by the number of trabeculae (Tb.N), how thick they are (Tb.Th), and how far apart they are (trabecular separation, Tb.Sp). Each of these factors contributes to the overall cancellous bone volume, but the same bone

volume can contain trabeculae that are organized in different ways, i.e., more trabeculae that are thinner or fewer, thicker trabeculae. There is likely to be an ideal relationship between Tb.Th and Tb.N, dependent on the location and primary direction of loading. However, it has been shown several times that the loss of complete trabeculae (reducing Tb.N) reduces the strength and stiffness of bone by two to three times more than does losing the same amount of bone via trabecular thinning (Fig. 1.14). The reason for this is that the loss of whole trabeculae reduces connectivity within the entire structure, which makes the structure much less capable of bearing weight and less able to direct stresses to the cortex than does maintaining the connections but making them thinner. In healthy human bone, the trabeculae tend to be shaped as plates of bone rather than as circular or elliptical rods (the extent to which the trabeculae are platelike or rodlike is sometimes called the structure model index). This provides greater strength in the loadbearing direction. It is more difficult to bend a plate in the direction of its greatest width than it is to bend a rod. When people begin to lose bone, as in osteoporosis, their bone plates become more rodlike and lose their connectivity (Fig. 1.15). In some locations, such as the vertebrae, trabeculae will be preferentially lost off-axis from the primary direction of loading so that the remaining struts run in a preferred direction aligned to the loading direction. This anisotropy (directional dependence) enables the structure to maintain its strength in the primary

I.  BASIC BONE BIOLOGY AND PHYSIOLOGY

19

The Macroscopic Organization of Bone

Estimated loss of strength, %

0

Trabecular Plates

–20

–40

–60

Trabecular number Trabecular thickness

–80

–100

0

5 10 Loss of BV/TV, %

15 Trabecular Rods

FIGURE 1.14  The thickness of the trabecular plates and their number do not contribute equally to the mechanical strength of the structure. A decrease in trabecular number has a much greater negative impact on bone strength than does the loss of an equal amount of bone through trabecular thinning. This again demonstrates the importance of connectivity in cancellous architecture. BV/TV = bone volume fraction, or the proportion of the tissue area that contains bone (rather than marrow or other spaces). BV, bone volume; TV, trabecular volume.

direction of loading, but puts it at risk if the axis of loading is changed. Cancellous bone beneath cartilage of the articular surface of the joints may function to cushion the joint by attenuating forces generated during movement. Immediately beneath the joint, the cancellous bone condenses to form a thin plate of compact bone, called the subchondral plate (Fig. 1.16). It has been suggested that the composite of subchondral plate and cancellous bone beneath it plays a role in the development of age-related arthritis, or osteoarthritis. The precise role of subchondral cortical and cancellous bone in the joint is still under debate. The spaces between the trabecular struts are regions in which blood cells are formed (i.e., red marrow). The differentiation of cells in the bone lineage can be partly diverted toward forming adipocytes, and the marrow within the diaphyses then becomes more fatty (i.e., yellow marrow) with age. However, some red marrow is present throughout life in the ends of the bones, as well as in the vertebrae, iliac crests, and ribs, thus making bone a primary blood-forming organ.

Skeletal Envelopes At the macroscopic level, there are four distinct surfaces in bone and these are defined by their location (Fig. 1.7): periosteal, endocortical, cancellous, and intracortical. It is convenient to divide the bone into these skeletal

FIGURE 1.15  Cancellous bone is composed of broader plates and thinner rods. When bone is lost, as in osteoporosis, more of the trabecular plates convert to a rodlike configuration. Reproduced with permission from Mosekilde L. Bone Mineral. 1990:13–35.

Cartilage Subchondral cortical bone

Cancellous bone

FIGURE 1.16  Beneath the cartilage in joints, there is a dense cortical bone plate called subchondral bone. This may serve to support the joint and regulate stresses. This becomes more porous cancellous bone as one moves distally toward the bony diaphysis.

envelopes because the different surfaces play different roles in the health of the bone and differ in the manner in which they respond to mechanical loads (see Chapter 11). They also are morphologically distinct, although several of them are interconnected, which provides pathways for communication among them. The outer surface of the diaphyses is called the periosteal surface, and it is covered by a thin fibrocellular membrane (periosteum) that contributes to bone formation along this surface. It is composed of two parts: an outer fibrous portion with a few fibroblast-like cells and a deep, or cambium, layer that is populated with highly osteogenic cells (Fig. 1.17). The cambium layer is the

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1.  BONE MORPHOLOGY AND ORGANIZATION

Bone Marrow

Lining Cells

FIGURE 1.17  The periosteal sheath covers the outer surface of bone. It is composed of two layers: an outer fibrous layer and an inner layer that is more highly cellular. These cells become active osteoblasts and contribute to bone apposition along this surface.

source of cells responsible for the growth, development, modeling/remodeling, and fracture repair of our bones. This accounts for the highly sensitive response of the periosteum to mechanical stimulation, infection, tumors, or mild injury. The cells of the periosteum are proliferative and capable of forming either highly organized lamellar bone under these conditions or disorganized woven bone in pathologic situations. The periosteum is well vascularized and innervated by both sympathetic and pain-sensitive fibers. Because mesenchymal cells are also present, cells in the deep layer of the periosteum can also differentiate into chondrocytes and form cartilage, most notably in adults during the fracture healing process. The surface surrounding the marrow cavity is the endocortical (or endosteal) surface. This surface is not covered by a membrane, but it is instead lined with a discontinuous (fenestrated) layer of osteoprogenitor cells (lining cells; Fig. 1.18). A space containing extracellular fluid exists between these cells and the surface of the bone matrix and may provide a barrier between the bone fluid within the canalicular and lacunar spaces and the extracellular fluid found in the marrow cavity and vessels. These lining cells are associated with capillaries near the bone surface and sinusoids in the marrow. Because diffusion is the principal mechanism for exchange between the extravascular fluid compartment and the endothelial layer of bone capillaries, the fenestrated nature of the cells lining this surface may act as a membrane, thus controlling fluxes of hydrophilic ions between the vascular and extravascular fluids. This membrane is important for the regulation of rapid calcium exchange between bone and the extracellular fluid compartment. Trabecular surfaces are morphologically and functionally similar to the endocortical envelope: cells line both surfaces and they are exposed to all the elements of the

FIGURE 1.18  Quiescent osteoblasts, or lining cells, cover the endocortical surface of bone. Notice that there is a space between the cells and the bone surface. This space contains fluid and may provide a barrier between the marrow cavity and the bone.

marrow cavity. Because vessels within the trabeculae are relatively uncommon, this fenestrated layer of cells may regulate nutrients that enter or leave the cellular canalicular system. Bone formation and resorption on trabecular surfaces respond to mechanical and biological signals in a manner parallel to that of endocortical surfaces. The surfaces of haversian canals represent a fourth skeletal envelope, the intracortical envelope. These surfaces are also covered by a fenestrated layer of resting osteoprogenitor cells, and because there is a neurovascular bundle that includes one or two vessels in the canal, they can likewise serve to regulate nutrient fluxes between the vascular system and the extracellular fluid compartments that provide nutrients for tissue within the dense cortical bone.

THE BLOOD SUPPLY AND INNERVATION OF BONE Blood Supply Calcified tissues present special problems of organization because material cannot diffuse rapidly through the dense tissue to nourish the cells; bone cells must therefore be within about 250 μm of their blood supply. This may be one reason that human bone (and that of other larger mammals, e.g., rabbits, dogs, and horses) is internally structured with primary and secondary osteons, as otherwise bone with a cortex thicker than 0.5 mm would not be able to support healthy osteocytes in the center of the cortex. It may also be the reason why the average diameter of a trabecula is about 200 μm. Moreover, the

I.  BASIC BONE BIOLOGY AND PHYSIOLOGY

The Blood Supply and Innervation of Bone

circular arrangement of cells around a central haversian canal in compact bone provides a geometrically efficient system for supplying the maximum amount of bone tissue from the minimum number of vessels. This means that secondary osteonal bone has a smaller surface area of canals per unit volume of bone than found in primary bone, and the connections between the vessels, the Volkmann’s canals, are further apart. Vessels in bone are formed primarily through angiogenesis, which can be defined as an expansion of the vascular network that occurs via endothelial cell processes (sprouting, migration, proliferation) as well as by anastomosis of vessels and subsequent “pruning.” Angiogenesis occurs in direct association with osteogenesis—in bone formation, fracture healing, and bone multicellular unit– based remodeling. There is a close relationship between angiogenesis and osteogenesis, partly driven by molecular signals that are common to both processes, and there is some evidence that the process of osteoblast differentiation itself will attract blood vessels. Osteoblasts are known to secrete VEGF, which stimulates angiogenesis and triggers other signaling responses in endothelial cells. Moreover, Notch is also known to be important to endothelial cell regulation. Together, VEGF and Notch help to control the development of anastomoses between vessels, a key component of circulatory abundance that is required of a metabolically active tissue like bone. Overexpression of Notch reduces vessel sprouting but increases perfusion (as does FGF1/2), whereas reduced Notch signaling promotes sprouting but limits perfusion. Notch regulates Noggin, an angiocrine factor (i.e., a molecule found in endothelial cells that can stimulate repair in damaged bone or other organs) but also an osteogenic factor that stimulates the differentiation of osteoprogenitor cells. This VEGF–Notch–Noggin signaling pathway highlights the interrelationship between osteogenesis and angiogenesis. One can imagine how important this becomes not only in normal bone remodeling but also in fracture repair (See Chapter 12). There are two types of capillaries in bone, termed H-type and L-type. H-type capillaries are found near the metaphysis, but they are connected through other vessels to the L-type sinusoidal capillaries found primarily in the diaphysis. These latter capillaries are surrounded by quiescent hematopoietic stem cells. H-type capillaries that are found endosteally have a blood–bone marrow barrier, but L-type capillaries are fenestrated, and drain blood into the venous sinuses which then lead out of the marrow cavity. These differences between the two types of capillaries are important in regulating hematopoietic stem cells (H-type) and leucocytes (L-type). As another indication of the close relationship between osteogenesis and angiogenesis, Notch is known to stimulate growth of H-type capillaries and expand the pool of vessel-associated osteoprogenitor cells. With aging, there are fewer

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H-type vessels, which is associated with a reduction in osteoprogenitor cells. Vessels in haversian canals have the structural characteristics of capillaries and are often paired within a canal. Unlike in the marrow cavity, venous sinusoids and lymphatic vessels are not found in haversian canals, although prelymphatic vessels may be present. The absence of venous sinusoids is one of the characteristics of a true capillary bed. The vessels are about 10–15 μm in diameter, but they tend to be wider near to the endosteal surface than the periosteal surface. The vessel walls contain no smooth muscle but are fenestrated capillaries lined by an incomplete layer of endothelial cells. They are similar in this way to vessels in other blood-forming organs such as the spleen and bone marrow. The endothelial cells of an osteonal vessel contain numerous pinocytic vesicles that may facilitate transport of water and nutrients across the capillary wall. A continuous 40–60 nm basement membrane surrounds the vessel and limits the rate of ion transport across the capillary wall. Endothelial extensions into the lumen of the capillary may be present and perhaps provide a greater surface area for exchange. In long bones, blood enters the marrow cavity through the nutrient artery, which penetrates the cortex and divides into ascending and descending branches running longitudinally within the medullary cavity. These ascending and descending arteries will supply medullary sinuses in the marrow that drain into the central venous sinus, along with cortical branches directed back toward the endosteum. These cortical branches pass through endosteal canals and feed arterial blood to fenestrated intracortical capillaries in the Haversian system. Intracortical capillaries anastomose with periosteal plexuses formed by arteries from neighboring connective tissue. Thus, perfusion of cortical bone in long bones is centrifugal, originating from the ascending and descending arteries in the marrow cavity and anastomosing with periosteal plexuses. Medullary sinuses drain centripetally into the central sinus which is drained by veins often paired with nutrient arteries, but can exit the cortex as independent emissary veins. The vessels in the bone cortex and in the marrow are sensitive to increased medullary hemostatic pressure, and alterations in pressure can cause pain. Blood flow in the medullary cavity is regulated by neuronal mechanisms via sympathetic nerve fibers. Metabolic mechanisms, such as acid metabolites, low pH, high carbon dioxide, or low oxygen tension will increase bone blood flow. Skeletal perfusion is essential to bone health and plays a crucial role in bone growth, fracture repair, and bone homeostasis. Disturbances to bone perfusion have been shown to have deleterious effects on bone health and function. Reduced bone perfusion has been associated with bone loss in a number of conditions,

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1.  BONE MORPHOLOGY AND ORGANIZATION

including aging, inflammation, infection, fractures, unloading, tumors, diabetes, smoking, and glucocorticoids. Impairments to bone perfusion have also been shown to reduce growth and repair. Additionally, conditions that alter bone remodeling (diabetes, disuse, aging, estrogen withdrawal, anabolic drug treatment) have each been associated to changes in bone blood flow caused by alterations in dilation and constriction of the principal nutrient artery, which regulates the resistance to flow in each long bone.

Innervation of Bone Very little is known about the innervation of the bone, or about the receptors and neurotransmitters that are expressed by nerves in bone. Although at one time it was thought that the bone itself did not contain sensory nerve fibers, it is now known that the marrow, periosteum, and even mineralized bone are supplied by primary afferent nerves as well as by postganglionic autonomic fibers from the sympathetic nervous system (See Chapter 18). The periosteum is the most richly innervated, with somewhat lower innervation in the bone marrow, and very little innervation in the mineralized bone. The density of sensory fibers in the periosteum may be higher as a mechanism to detect skeletal injury. Although bone mass declines with age, the density of sensory nerve fibers in bone is not known to decline, which may be one reason that musculoskeletal pain increases with age. The fibers of the primary sensory nerves are thin (although they can assemble to form bundles), poorly myelinated or unmyelinated, and have low conduction velocities. Sensory fibers express calcitonin generelated peptide (CGRP) in peptide-rich C- or Aδ fibers, as well as substance P, which is predominantly associated with unmyelinated nerve fibers. These peptides are most highly expressed in the periosteum, which is why periosteal tissues are so sensitive to pain. Neurofilament heavy polypeptide (NF200 or NF–H) is expressed by thinly myelinated primary afferent nerves (Aδ fibers) and at concentrations about equal to those of CGRP and substance P within bone. Because these fiber types are poorly myelinated, or unmyelinated, they have low conduction velocities: 70 years) has three times more nonenzymatic cross-links than bone from those younger than 50 years. The interaction of collagen and mineral adds another level of complexity. Although collagen supports most of the matrix strain in tension, coupled deformation between collagen and mineral allows collagen to transfer load to the mineral component. This evenly distributes forces and reduces stresses throughout the matrix. Smaller collagen fibers oriented around osteocytes alter local strains at the nano- and micrometer level, thus diverting cracks away from and preserving living cells. The orientation of mineral crystallites also changes the strain environment at the nanoscale. None of these effects are dependent on bone mass; they therefore represent a component of bone quality.

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25

Bone Mass and Bone Quality

FIGURE 1.21  Skeletal microdamage is traditionally visualized microscopically following en bloc staining with basic fuchsin. Microcracks can be observed using brightfield (A) or ultraviolet fluorescence (B) microscopy, the latter taking advantage of the natural fluorescent properties of fuchsin. Alternatively microcracks can be stained en bloc with fluorochromes such as calcein and alizarin (C and D). These stains cannot be visualized using brightfield (C) but can with appropriate filter sets and UV fluorescence (D).

Microdamage Accumulation

STUDY QUESTIONS

Microscopic damage occurs in bone as a consequence of daily activities that repeatedly load the bone (Fig. 1.21). The amount of damage present in bone is a function both of the amount of damage that is produced and the amount that is repaired through normal physiologic processes. Either increased production of damage or suppressed repair can elevate the level of microdamage in bone. The accumulation of microdamage may increase the fragility of bone, although under most conditions it is unlikely that sufficient damage can be accumulated in the tissue to cause a measurable change in properties. However, the accumulation of damage that occurs with age, in conjunction with the loss of bone mass, may contribute to the increased bone fragility that occurs in postmenopausal women. Microdamage accumulation in bone can not only reduce the stiffness of the tissue and decrease its strength but can also have a positive effect in releasing energy and delaying bone fracture.

  

1. D  efine the three main classes of noncollagenous extracellular matrix proteins, and give examples and functions of proteins from each class. 2. Explain how microcracks are kept from becoming larger at the nanoscalar and microstructural levels. 3. Explain how cancellous and cortical bone work together to provide mechanical support and protection to bone. 4. Compare and contrast woven bone, lamellar bone, primary bone, secondary bone, and interstitial bone. 5. Compare and contrast the four skeletal envelopes: periosteal, endocortical, trabecular, and intracortical. 6. Describe the compartments of water in bone and the roles played by each. 7. Describe the autonomic innervation of bone. 8. Define the four characteristics of bone quality.   

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Suggested Readings 1. Bonnucci E, Motta PM. Ultrastructure of Skeletal Tissues. Bone and Cartilage in Health and Disease. Boston: Kluwer Academic Publishers; 1990. 2. Brookes M, Revell WJ. Blood Supply of Bone: Scientific Aspects. London: Springer-Verlag; 1998. 3. Burr DB, Allen MR. Calcified tissue international, special issue: bone material properties and and skeletal fragility. Calcif. Tissue Int. 2015;97:199–241. 4. Castañeda-Corral G, Jimenez-Andrade JM, Blook AP, Taylor RN, Mantyh WG, Kaczmarska MJ. The majority of myelinated and unmyelinated sensory nerve fibers that innervate bone express the tropomyosin receptor kinase A. Neuroscience. 2011;178:196–207. 5. Dempster D, Felsednberg D, van der Geest S. The Bone Quality Book. Amsterdam: Elsevier; 2006. 6.  Enlow DH, Brown SO. A comparative histological study of fossil and recent bone tissues. Part III. Mammalian bone tissues. Tex. J. Sci. 1957;10:187–230. 7. Fonseca H, Moreira-Gonçalves D, Appell Coriolano H-J, Duarte JA. Bone quality: the determinants of bone strength and fragility. Sports Med. 2014;44:37–53. 8. Foote JS. A contribution to the comparative histology of the femur. Smithsonian Contrib. Knowl. 1916;35:1–242. 9. Fuchs RK, Allen MR, Ruppel ME, Diab T, Phipps RJ, Miller LM. In situ examination of the time-course for secondary mineralization of Haversian bone using synchrotron Fourier transform infrared microspectroscopy. Matrix Biol. 2008;27:34–41.

10. Fukumoto TJ. Bone as an endocrine organ. Trends Endocrinol. Metab. 2009;20:230–236. 11. Gurkan UA, Akkus O. The mechanical environment of bone marrow: a review. Ann. Biomed. Eng. 2008;36:1978–1991. 12. Jee WSS, Weiss L. The skeletal tissues. In: Weiss L, ed. Histology: Cell and Tissue Biology. New York: Elsevier Biomedical; 1983. 13. Kaplan FS, Hayes WC, Keaveny TM, et al. Form and function of bone. In: Simon SR, ed. Orthopaedic Basic Science. Chicago: American Academy of Orthopaedic Surgeons; 1994. 14. Karsenty G, MacDougald O, Rosen CJ. Interactions between bone, adipose tissue and metabolism. Bone. 2012;50(Special Issue):429–579. 15. Martin RB, Burr DB, Sharkey NA, Fyhrie DP. Skeletal Tissue Mechanics. second ed. New York: Springer-Verlag; 2015. 16. Reznikov N, Bilton M, Lari L, Stevens MM, Kröger R. Fractual-like hierarchical organization of bone begins at the nanoscale. Science. 2018;360:507–517. 17. Ruppel ME, Miller LM, Burr DB. The effect of the microscopic and nanoscale structure on bone fragility. Osteoporos. Int. 2008;19:1251–1265. 18. Sivaraj KK, Adams RH. Blood vessel formation and function in bone. Development. 2016;143:2706–2715.

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C H A P T E R

2 Bone Marrow and the Stem Cell Niche Laura M. Calvi Department of Medicine-Endocrinology, University of Rochester Medical Center, Rochester, NY, United States

Hematopoiesis is required throughout the life of an organism for the daily maintenance of blood products, necessary for oxygenation (erythropoiesis), clotting (megakaryopoiesis/thrombopoiesis), and maintenance of innate (myelopoiesis) and adaptive (lymphopoiesis) immunity. This highly hierarchical system is well characterized, thanks to fidelity of a large array of cell surface markers (many identified as a numerical cluster of differentiation, or CD) that have been used extensively to quantify and isolate specific cell populations using flow cytometry. Differentiated cells have varying life spans, from hours (in the case of neutrophils) to years (in the case of memory T cells and hematopoietic stem cells), and thus a large component of the hematopoietic system needs to be continually replaced in an astounding synthetic process that at homeostasis is carried on primarily by intermediate committed precursors. These committed precursors are derived from progenitors that may give rise to more than one differentiated lineage. Progenitors and precursors have limited self-renewal capacity, derive from hematopoietic stem cells, tissuespecific stem cells that persist throughout the life of an organism, and are critical to replenish the precursor populations, especially when there is increased demand for blood products or in the case of damage to the marrow. Hematopoietic stem cells are commonly used clinically in the setting of stem cell transplantation for the treatment of hematologic malignancies and other disorders. Stem cell transplantation requires treatment of the recipient with conditioning regimens (chemotherapy and/or radiation) that destroys the recipient’s native bone marrow and prepares it for the donor stem cells. This process is limited by the availability of donor stem cells, the morbidity of the conditioning regimens, the rate at which the donor stem cells regenerate the entire hematopoietic system (responsible for variable periods

Basic and Applied Bone Biology, Second Edition https://doi.org/10.1016/B978-0-12-813259-3.00002-6

of susceptibility to infection and reliance on transfused blood products), and, in cases in which the donor marrow is different from the recipients, by graft versus host disease, where the donor marrow immune cells attack the host. Therefore, extensive research has focused on understanding hematopoietic stem cells to expand their numbers while maintaining their multipotentiality and extensive capacity for self-renewal. Scientist working in the late 19th century and earlier 20th centuries first hypothesized that populations of nonhematopoietic stem cells may exist in the bone marrow and that these cells may participate in injury repair. With the advent of bone marrow transplantation in the laboratory and later in the clinic, observations were made to suggest that there may be bone of donor origin, supporting the idea of the mesenchymal stem cell (MSC) population. At the same time, investigators were noticing that populations enriched for hematopoietic stem cell capacity appeared to behave differently depending on whether they were grown in vitro or found within certain microenvironment (marrow or spleen). Moreover, initial studies suggested that the distribution of hematopoietic stem cells followed a hierarchical organization, with the most primitive cell populations being found at the endosteal surface of bone. In addition, it was noted early that when injected in the peripheral blood, hematopoietic stem cells would spontaneously home to the bone marrow, the organ where hematopoietic cells are naturally found, suggesting that supportive conditions were present specifically in the bone marrow to support hematopoiesis. Based on these observations, Schofield initially proposed the concept of the niche. Akin to the niche in ecology, niches conceptually represent the microenvironmental conditions that support a certain stem cell population. Schofield proposed that niche cells would be found in close proximity to supported stem

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© 2019 Elsevier Inc. All rights reserved.

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2.  BONE MARROW AND THE STEM CELL NICHE

6& $62–4 times body weight) and applied rapidly and/or incorporate odd or unusual loading patterns are more osteogenic than static or low-impact activities. For resistance training, the greatest benefits are achieved with high-intensity (70%–85% of maximum) programs that involve two to three sets of 8–12 repetitions performed at least 2 days/ week and which target the major muscle groups crossing the hip and spine. Although these findings form the basis for current exercise recommendations for bone and fracture prevention, it is important to recognize that any exercise prescription must take into account each individual’s clinical risk factors, BMD, and functional status.

Principle of Reversibility Reversibility relates to how bone responds once a given stimulus (e.g., exercise) is discontinued. The benefits of exercise on bone mass typically diminish progressively over time following discontinuation of training. While this supports the “use it or lose it” concept, bone structural adaptations in response to exercise during growth may persist into adulthood, despite decreased activity. For example, former young female gymnasts have significantly greater total and cortical bone area and estimated bone strength at the tibia and radius 10 years after retirement from gymnastics compared to healthy controls. The clinical significance of these structural adaptations is supported by data showing that small improvements in bone geometry (e.g., periosteal apposition) can lead to large increases in bone strength, independent of changes in BMD, because the resistance of bone to bending or torsional forces is related exponentially (to the fourth power) to its diameter. These findings also reinforce the importance of engaging in regular weight-bearing exercise during growth when the skeletal is undergoing significant modeling and thus has a greater capacity to alter its structure favorably in response to loading to maximize bone strength. Given the challenges associated with maintaining long-term participation in exercise, two other clinically important issues to consider are: (1) how quickly do any exercise-induced gains in bone revert to baseline values;

and (2) is there a minimal dose of loading that can maintain any initial exercise-induced skeletal benefits? In premenopausal women, the skeletal benefits of exercise are lost after 3–6 months of detraining. However, there is some evidence that the benefits of exercise on BMD and bone structure during growth can, at least partly, be retained with a lower level of exercise. Whether there is a minimal dose of exercise needed to retain any initial skeletal gains remains to be determined.

Principle of Initial Values When considering skeletal adaptations to exercise it is important to account for initial bone density and structure, as the greatest changes will typically occur in those with the lowest initial values. This observation was highlighted in a study involving resistance and jump training in premenopausal women. Those with the lowest initial bone mass experienced the greatest changes in response to training; for each unit lower BMD (0.01 g/cm2) at the greater trochanter, the training response was 12% greater. The initial values effect also closely reflects the principle of progressive overload, such that smaller bones will experience greater strain than larger bones exposed to the same load. Therefore, if the intensity or pattern of loading is of a sufficient magnitude or rate, or differs substantially from habitual activity, then bones should adapt accordingly even if the initial values are high. This was highlighted in a study of young female gymnasts who experienced significant gains (2.3%–5.0%) in lumbar spine and femoral neck BMD with continued training over 12 months, despite high initial BMD values.

Principle of Diminished Returns The principle of diminished returns relates to the concept that following any initial exercise-induced skeletal adaptations, subsequent gains are likely to be small with a similar loading regimen. Bone cells initially respond strongly to mechanical loading, but this response will eventually phase out as the cells adapt or accommodate to the new loads (signals). Although these principles have not been specifically tested in human clinical trials, the findings from several exercise interventions in older adults over 12–18 months indicate that the greatest changes in bone density occur during the initial 5–6 months. However, this phenomenon could be related to the principles of initial values and progressive overload, that is, following any initial skeletal adaptations bone may experience less strain if the loads remain unchanged. Indeed, there are studies that have demonstrated a linear increase in BMD with continued exercise training, which could be related to the fact that a progressive exercise program was implemented that resulted in sustained overload and thus ongoing skeletal adaptations.

III.  SKELETAL ADAPTATION

Suggested Readings

SUMMARY

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Suggested Readings

Exercise is a vital strategy to increase peak bone mass, structure, and strength during growth. It can also maintain or increase bone mass throughout the mid adult years and slow bone loss and reduce the risk and incidence of falls in the elderly, all of which are important to reduce the risk of fracture. Not all forms of exercise are equally effective, and a generic exercise prescription for bone is not appropriate because the exercise goals and guidelines vary across the lifespan according to the level of risk for each individual. At present, the optimal program to increase peak bone strength and improve or preserve skeletal integrity to reduce the risk of fractures remains uncertain. However, there is strong evidence to support the prescription of high-impact weight bearing during growth as an effective strategy to improve the mass, structure, and strength of bone. During adulthood and old age, multimodal exercise programs including moderate to high-intensity PRT (or power training) and short bouts of weight-bearing impact activities (as tolerated), and challenging balance, mobility, and step training activities are most effective for improving hip and spine BMD as well as muscle mass, strength, power, and function. However, further dose–response studies are needed with clearly defined and quantified intensity variables to better determine the minimum dose of exercise required to improve or maintain healthy bones at different stages of the lifespan.

STUDY QUESTIONS   

1. D  efine strain rate and its relationship to strain magnitude and loading frequency. How can strain rate and frequency be dissociated experimentally? 2. What are the four different mechanical usage windows and the minimum effective strains needed to stimulate each window? Describe the cellular activity and tissue outcomes of these windows. 3. What elements unique to osteocytes support the hypothesis that they act as mechanical sensors? 4. Describe how ion channels, adhesion/cytoskeletal molecules, and G protein-related molecules are involved in mechanical sensing. 5. What components (e.g., strain magnitude, distribution, loading rate, and refractory periods) would you include in an in vivo or clinical experimental designed to optimize the amount of bone formation that occurs in response to mechanical stimulation?  

1. Batra N, Burra S, Siller-Jackson AJ, Gu S, Xia X, Weber GF. Mechanical stress-activated integrin α5β1 induces opening of connexin 43 hemichannels. Proc. Natl. Acad. Sci. U.S.A. 2012;109:3359–3364. 2. Bidwell JP, Pavalko FM. The load-bearing mechanosome revisited. J. Bone Miner. Metab. 2010;8:213–223. 3. Fuchs RK, Bauer JJ, Snow CM. Jumping improves hip and lumbar spine bone mass in prebuescent children: a randomized controlled trial. J. Bone Miner. Res. 2001;16:148–156. 4. Han Y, Cowin SC, Schaffler MB, Weinbaum S. Mechanotransduction and strain amplification in osteocyte cell processes. Proc. Natl. Acad. Sci. U.S.A. 2004;101:16689–16694. 5. Hung CT, Allen FD, Pollack SR, Brighton CT. What is the role of the convective current density in the real-time calcium response of cultured bone cells to fluid flow? J. Biomech. 1996;29:1403–1409. 6. Koch JC. The laws of bone architecture. Am. J. Anat. 1917;21:179–293. 7. Kennedy OD, Herman BC, Laudier DM, Majeska RJ, Sun HB, Schaffler MB. Activation of resorption in fatigue-loaded bone involves both apoptosis and active pro-osteoclastogenic signaling by distinct osteocyte populations. Bone. 2012;50:1115–1122. 8. Martin RB, Burr DB, Sharkey NA, Fyhrie DP. Skeletal Tissue Mechanics. New York: Springer; 2015. 9. Robling AG, Niziolek PJ, Baldridge LA, et al. Mechanical stimulation of bone in vivo reduces osteocyte expression of Sost/ Sclerostin. J. Biol. Chem. 2008;283:5866–5875. 10. Rubin CT, Lanyon LE. Limb mechanics as a function of speed and gait: a study of functional strains in the radius and tibia of horse and dog. J. Exp. Biol. 1982;101:187–211. 11. Rubin CT, Lanyon LE. Regulation of bone formation by applied dynamic loads. J. Bone Jt. Surg. 1984;66 A:397–402. 12. Smalt R, Mitchell FT, Howard RL, Chambers TJ. Induction of NO and prostaglandin E2 in osteoblasts by wall-shear stress but not mechanical strain. Am. J. Physiol. 1997;273(4 Pt. 1):E751–E758. 13. Snow CM, Shaw JM, Winters KM, Witzke KA. Long-term exercise using weighted vests prevents hip bone loss in postmenopausal women. J. Gerontol. A Biol. Sci. Med. Sci. 2000;55:M489–M491. 14. Turner CH. Three rules for bone adaptation to mechanical stimuli. Bone. 1998;23:399–407. 15. Tveit M, Rosengren BE, Nilsson JA, Ahlborg HG, Karlsson MK. Bone mass following physical activity in young years: a mean 39-year prospective controlled study in men. Osteoporos. Int. 2013;24:1389–1397. 16. Warden SJ, Fuchs RK, Castillo AB, Nelson IR, Turner CH. Exercise when young provides lifelong benefits to bone structure and strength. J. Bone Miner. Res. 2007;22:251–259. 17. Winters KM, Snow CM. Detraining reverses positive effects of exercise on the musculoskeletal system in premenopausal women. J. Bone Miner. Res. 2000;15:2495–2503. 18. Wolff J. The Law of Bone Remodeling. Trans. by P.G.J. Maquet, R. Furlong. Berlin: Springer-Verlag; 1986. 19. Bass SL, Saxon L, Daly RM, et al. The effect of mechanical loading on the size and shape of bone in pre-, peri-, and postpubertal girls: a study in tennis players. J. Bone Miner. Res. 2002;17(12):2274–2280. 20. Gianoudis J, Bailey CA, Ebeling PR, et al. Effects of a targeted multimodal exercise program incorporating high-speed power training on falls and fracture risk factors in older adults: a community-based randomized controlled trial. J. Bone Miner. Res. 2014;29(1):182–191. 21. Allison SJ, Poole KE, Treece GM, et al. The influence of highimpact exercise on cortical and trabecular bone mineral content and 3D distribution across the proximal femur in older men: a randomized controlled unilateral intervention. J. Bone Miner. Res. 2015;30(9):1709–1716. 22. Kerr D, Morton A, Dick I, Prince R. Exercise effects on bone mass in postmenopausal women are site-specific and load-dependent. J. Bone Miner. Res. 1996;11(2):218–225.

III.  SKELETAL ADAPTATION

C H A P T E R

12 Fracture Healing Jiliang Li1, Melissa A. Kacena2, David L. Stocum1 1Department

of Biology and Center for Developmental and Regenerative Biology, Indiana University-Purdue University, Indianapolis, IN, United States; 2Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN, United States

A bone fracture or an osteotomy causes a break in the bone, which leads to the loss of anatomic continuity and/ or to mechanical instability of the bone. Fractures commonly happen because of falls, car accidents, or sports injuries. Fractures are often associated with penetration injuries on the battlefield. Other factors such as lower bone density and osteoporosis increase the incidence of fracture. Bone fractures are common and costly to the public due to high health-care expenditures. According to the 2004 Surgeon General’s Report, about 1.5 million Americans suffer a fracture because of osteoporosis each year. Hip fractures are the most devastating type of bone fracture and account for almost 300,000 hospitalizations per year. Among patients with osteoporotic fractures, 20% die and another 20% end up in a nursing home within a year of the fracture. Many become isolated, depressed, or afraid to leave home because they fear falling. Care for bone fractures from osteoporosis costs nearly US$18 billion each year. To find ways to treat bone fractures, it is essential to understand how fractures normally heal and what factors interfere with fracture healing. This chapter provides an overview of the fracture healing process at the cellular and molecular levels and a discussion of several key situations that complicate healing. Current therapeutic strategies that are aimed at accelerating fracture repair are also discussed.

TYPES OF BONE FRACTURE All fractures can be broadly described as closed (no skin break) or open (skin break). Open fractures are always associated with more damage to the surrounding soft tissue, including the periosteum, have a higher

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risk of infection, and often have a higher incidence of nonunion than closed fractures. Fractures of long bones, such as the femur, humerus, tibia, and other long bones, can be classified according to the characteristics of the force that causes them. Simple and comminuted fractures in which the bones are broken into two or several pieces, respectively, are caused by a single injury. Stress fracture, which is an overuse injury, results from repetitive loading. Simple fracture occurs when a bending force or twisting force is applied to a bone, resulting in two fragments with transverse, oblique, or long curved (spiral) edges of the broken bones. This type of fracture heals through the spontaneous repair processes we will discuss later. Comminuted fracture is characterized by the breaking of a bone into several small pieces and is the result of high velocity injuries, such as car accidents, falls from a height, or high-energy injuries with tissue loss caused by fragments from explosive devices on the battlefields. Repair of comminuted fractures follows a healing pattern similar to that of simple fractures, but on a larger scale. Such fractures generally are very difficult to treat and may result in a deformity of the injured part even after treatment. Stress fractures result when low-magnitude cyclically repeated force is applied over a long period, causing progressive accumulation of microdamage. Unlike simple and comminuted fractures, stress fractures and their associated fatigue damage heal via normal bone remodeling. This process involves the sequential and coordinated activity of osteoclasts and osteoblasts that remove and replace the damaged bone, respectively. If the repetitive loading is prolonged and/or microdamage cannot be repaired, the bone may eventually fail through propagation of the microdamage.

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PRIMARY AND SECONDARY REPAIR MECHANISMS Repair of long bones after fracture is a unique process that results in the restoration of normal bone anatomy and function after injuries. This repair can be divided into primary healing and secondary healing based on differences in the local motion between the fracture fragments. Primary healing involves a direct attempt by the cortex to reestablish continuity between the fracture fragments (Fig. 12.1). This process seems to occur only when the alignment stability and decrease in interfragmentary motion of the fracture fragments are established by rigid internal fixation. Osteoblasts derived from mesenchymal stem cells (MSCs) lay down osteoid on the exposed bone surfaces. New haversian systems will be reestablished across the original fracture line through intracortical remodeling. Secondary (spontaneous) healing involves a response of the periosteum and surrounding soft tissues at the fracture site (Fig. 12.2). The response from the periosteum is a fundamental reaction to bone injury; it is enhanced by limited fragment motion and inhibited by rigid fixation. Mesenchymal cells and osteoprogenitor cells contribute to the process of repair by a recapitulation of embryonic (A)

intramembranous ossification and endochondral bone formation. The new bone formed by intramembranous ossification is found peripheral to the site of the fracture. Osteoblast progenitor cells in the inner layer of the periosteum differentiate into osteoblasts in response to molecular signals produced during fracture and directly synthesize new bone matrix on the bone surface without first forming cartilage. This process does not contribute to directly bridging the fracture. Callus that forms by endochondral ossification is formed within the fracture site and involves the development of cartilage in response to hypoxia caused by the lack of blood supply. The chondrocytes are derived from MSCs in the periosteum and endosteum. They proceed through a state of hypertrophy and the cartilage matrix becomes calcified. The hypertrophic chondrocytes undergo apoptosis, and the calcified matrix is removed by invasion of osteoclasts and blood vessels, followed by osteoblast-induced bone formation. Fracture repair is clearly related to external factors, including the mechanical environment at the fracture site. Motion at the fracture site results in healing primarily through cartilage formation (endochondral ossification), and stability favors the direct formation of bone (intramembranous ossification). Most long bone fractures heal by a combination of intramembranous and endochondral ossification. Both intramembranous and endochondral ossification produce woven bone with poorly organized hydroxyapatite matrix. This is extremely important during fracture healing, as rapid new bone formation is required to quickly consolidate fracture fragments to restore the mechanical stability of bone. The mineral appositional rate of woven bone formation can be 2–4 times greater than the lamellar bone formation. The woven bone will be later remodeled by osteoclasts to achieve lamellar bone.

STAGES OF FRACTURE REPAIR Secondary fracture healing of long bones can be considered as a series of four discrete stages occurring in sequence and overlapping to a certain extent (Fig. 12.3).

(B)

Inflammatory Response

FIGURE 12.1  Directing healing of a transverse osteotomy in dog radius. (A) In the radiographs, there is no external callus formation. The fracture line disappears 5–6 weeks after production of osteotomy. (B) The longitudinal section at 10 weeks shows minimal callus formation around the fracture site. Reproduced with permission from Schenk R, et al. Experientia. 1963;19:593–595.

When trauma occurs, the continuity and vascular supply of bone is disrupted and a hematoma forms at the site of the injury. This leads to a loss of mechanical stability, a lack of local oxygen and nutrients, and a release of various factors from platelets. Macrophages, leukocytes, and other inflammatory cells then invade the area. The damage also sensitizes the surviving local cells and enables them to respond better to local and systemic messages. This inflammatory response peaks within the first 24 h and lasts for about 7 days.

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Stages of Fracture Repair

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(A)

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(B)

FIGURE 12.2  Secondary (spontaneous) fracture healing in dog radius after transverse defect-osteotomy. (A) Longitudinal section 14-week postfracture shows extensive bony callus formation along the periosteal surface and within the bone marrow cavity. (B) The area indicated by the black arrow in image A contains fibrous tissue that is sufficiently vascularized to permit intramembranous ossification. (C) The area indicated by the white arrow in image A shows that fibrocartilage has to mineralize (1) before undergoing resorption and vascular invasion (2). At other sites of the endochondral ossification, bone is deposited on persistent calcified cartilage or bone. (3). Reproduced with permission from Schenk R, et al. Experientia. 1963;19:593–595.

Soft Callus Formation (Cartilage Formation) The cells that are stimulated and sensitized during the inflammatory stage begin producing new vessels, fibroblasts, intracellular material, and supporting cells. The hematoma is replaced with fibrovascular tissue, a fibrin-rich granulation tissue. Fibrocartilage then develops and stabilizes the bone ends (Fig. 12.4). In mouse and rat models, the peak of soft callus formation occurs 7–10 days postfracture with a peak in both type II collagen and proteoglycan production.

Hard Callus Formation (Endochondral Ossification) After the soft callus forms, replacement of the cartilage and fibrovascular tissue occurs via vessel invasion and endochondral ossification (Fig. 12.4). Periosteal bone apposition also occurs, contributing to formation of the hard callus. The calcified cartilage is replaced with woven bone. The peak of hard callus formation usually occurs around 14 days postfracture in animal models, as indicated by callus volume as well as osteoblast markers,

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FIGURE 12.3  Stages of fracture repair. Stage 1: Following fracture or osteotomy, blood supply is disrupted and a blood clot (hematoma) forms. Stage 2: Progenitor cells in the periosteum and marrow differentiate into osteoblasts to facilitate intramembranous bone formation where an intact blood supply is preserved. In the fracture space where the tissue is hypoxic, the progenitor cells undergo chondrogenesis. The chondrocytes hypertrophy and their matrix becomes calcified, leading to chondrocyte apoptosis and invasion of the matrix by periosteal and marrow blood vessels. These vessels are accompanied by perivascular mesenchymal stem cells that differentiate into osteoblasts. Stage 3: The osteoblasts form woven bone on the calcified matrix. Stage 4: The remodeling process proceeds with osteoclasts and osteoblasts facilitating the conversion of woven bone into lamellar bone and eventually recreating the appropriate anatomical shape.

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FIGURE 12.4  Fracture repair of the closed femoral fracture in female Sprague–Dawley rats at 10 weeks postfracture. (A) Cartilage (star) is detected by the red stain with safranin O staining. (B) Endochondral ossification demonstrated by von Kossa staining shows cartilage (blue) and bone (black). (C) Tartrate-resistant acid phosphate staining reveals osteoclasts stained red with multiple nuclei, as seen in the enlarged insert.

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Stages of Fracture Repair

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FIGURE 12.5  (A) Contact microradiographs of cross sections at the fracture sites in a rat open femoral osteotomy model show formation of an outer cortical shell and resorption of the original fracture cortex (star) from 2 to 49 weeks following surgery in Sprague–Dawley rats. (B) Cross sections from rats continuously treated with bisphosphonate (incadronate, 100 μg/kg daily) show a larger callus area compared with the control animals. There is a highly porous shell in which the endocortical border is unclear at 25 weeks. The remnant of the original cortex is still observed at 49 weeks. Adapted from Li J, et al. J. Bone. Miner. Res. 1999;14:969–979; Li J, et al. J. Bone Miner. Res. 2000;15:2042–2051; Li C, et al. J. Bone Miner. Res. 2001;16:429–436 with permission of the American Society for Bone and Mineral Research.

such as type I collagen, alkaline phosphatase (ALP), and osteocalcin. Intramembranous ossification peripheral to the site of the fracture also contributes to the hard callus.

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Bone Remodeling In this last stage of fracture repair, woven bone is gradually replaced by lamellar bone via bone remodeling (see Chapter 5). Hard callus resorption by osteoclasts is followed by lamellar bone formation by osteoblasts to restore the anatomical structure of the preinjured bone and support mechanical loads. This process, also sometimes referred to as secondary bone formation, starts after 3–4 weeks and may take years to be completed before the original anatomic structure is restored. In adults, the original shape and cavity of new bone may never be fully restored. The new outer cortical shell develops over the cartilage core. The ischemic bone tissue (the original cortex) at the fracture site is resorbed as the outer cortical shell remodels inward to become the new diaphyseal bone (Fig. 12.5A). Osteoclasts identified by tartrate-resistant acid phosphatase staining carry out two tasks during fracture healing: removal of endochondral matrix (Fig. 12.4) and remodeling of woven bone (Fig. 12.6). Inhibition of bone resorption, for example, with bisphosphonate treatment, prevents the removal of the ischemic bone tissue and restoration of diaphyseal bone (Fig. 12.5B).

BM

FIGURE 12.6  Remodeling of woven bone in the outer cortical shell after fracture. (A) Tartrate-resistant acid phosphatase staining shows osteoclasts (arrows) with multiple nuclei on the periosteal surface of the bone, indicating ongoing bone resorption. (B) On the endosteal surface of the outer cortical shell, a group of active osteoblasts (arrows) are observed, indicating ongoing new bone formation toward bone marrow (BM). The outer cortical shell remodels inward to become the new diaphyseal bone, while the original fracture cortex is resorbed.

Fractures are usually considered healed when the bone stability has been restored by the formation of new bone that bridges the area of fracture even before the final shaping of the bone is achieved. An adequate blood supply and a gradual increase in mechanical stability

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are crucial for successful fracture healing. However, the interruption of normal healing processes results in fracture nonunion. Nonunion fractures, which are defined as the cessation of all reparative processes of healing without bone union, are traditionally classified as atrophic or hypertrophic. Atrophic nonunion, which typically shows little callus formation, results from poor vascularization at the fracture site. Hypertrophic nonunion is linked to inadequate immobilization (unstable fixation) and appears to have adequate blood supply and cartilage formation that leads to pseudarthrosis, a false joint associated with abnormal movement at the unhealed site of bone.

ASSESSMENT OF FRACTURE HEALING Methods of Evaluation Fracture healing involves a dynamic interplay of biological processes to restore the original anatomic structure and mechanical function of bone. Therefore, both structural and biomechanical evaluations are used to assess fracture repair. The extent and quality of structural repair can be evaluated using radiographic and histologic methods. The mechanical properties of a healing bone are assessed by mechanical tests. There is a consensus that mechanical tests provide the gold standard measures of healing in a laboratory setting. Clinical assessment of healing requires noninvasive methods, including clinical symptoms (pain or tenderness when bearing weight) and radiographic indicators.

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Plain radiography is a ubiquitous method used to evaluate fracture healing in both laboratory and clinical settings, due to its noninvasive nature. The most common radiographic definitions of fracture healing involve the bridging of fracture site by callus, obliteration of the fracture line, and continuity. Recently, computed tomography has been used to define union as bridging of >25% of the cross-sectional area at the fracture site. Callus volume can also be calculated using three-dimensional (3D) reconstructed images. Bone histology is an invasive method used to study the bone structure during fracture healing in the laboratory. Conventional bone histomorphometry can be applied in fracture studies. Compared with single longitudinal sections of a healing fracture, which do not capture all of the tissue heterogeneity within callus, transverse sections at the fracture line level can provide the more accurate measurement of cross-sectional area, as well as an estimation of tissue heterogeneity. On cross sections of callus, fibrous cartilage and bone tissues can be measured and calculated as the percentage of the total crosssectional callus area. Osteoblasts and osteoclasts can be specifically stained and their activities can be quantified, as shown in Fig. 12.6. In addition, fluorochromes, used widely for the measurement of bone formation–related parameters to estimate bone metabolism, can also be used to examine callus remodeling, in particular, during the middle and late stages of fracture healing (Fig. 12.7). To test the mechanical function of a healing bone, torsion and four-point bending tests are logical choices when studying fracture healing in long bones (See Chapter 7). The choice of the type of test is mainly determined by technical considerations. In general, torsion

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FIGURE 12.7  Fluorochromes can be used to examine callus remodeling during fracture repair. Calcein is injected before rats are sacrificed 2, 4, 6, and 16 weeks after femoral osteotomy. (A) Diffuse calcein labeling (green) suggests woven bone formation at 2 weeks. (B–D) Linear calcein labeling indicates lamellar bone formation during callus remodeling at 4, 6, and 16 weeks, respectively. These data suggest that measurement of fluorochrome labeling is a useful tool to estimate the rate of bone formation during normal fracture repair and repair occurring after various therapies.

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Cellular Events of Fracture Repair

is a better choice than four-point bending because torsion tests subject every cross section of the callus to the same torque, whereas four-point bending might create a nonuniform bending moment throughout the callus. As a result, failure of the callus during a four-point bending test does not necessarily occur at the weakest cross section of the callus. It is important to remember that threepoint bending should not be recommended to estimate the mechanical properties of a healing bone, especially during early stages of the healing process, because the site to which the force is applied is located at the original fracture line, which is composed primarily of cartilage, calcified cartilage, or less mature bone tissue depending on the healing stages. The outcome measures that can be obtained from mechanical tests, such as ultimate strength, stiffness, energy to failure, and torque in the torsion test, are structural rather than material properties. These structural properties of a fracture callus depend collectively on the individual tissues, including cartilage, calcified cartilage, and woven bone, and the spatial distribution of these tissues, as well as the overall geometry of the callus. As the callus geometry can now be easily measured using computed tomography, it is possible to roughly estimate the overall callus tissue material properties by normalizing the structural properties with the callus geometric parameters. However, the true measurement of the material properties of the callus requires direct testing of the individual tissues in the callus. For instance, nanoindentation can be used to measure the elastic properties of the individual tissues of callus.

Biomechanical Stages of Fracture Healing It is apparent that mechanical properties improve with the progress of fracture healing. During the ossification process of external callus, a fourfold increase in the total amount of calcium per unit volume and a twofold increase in hydroxyproline (an indicator of total collagen content) lead to a threefold increase in the breaking strength of the callus in a tensile test. Studies also suggest a high correlation between the hardness of fracture callus and its mineral content per tissue volume. In 1977, White et al. divided the whole period of secondary fracture healing into four biomechanical stages, based on the results of torsion tests performed on healing rabbit tibiae at multiple time points (Table 12.1). In stage 1, the bone fails through the original fracture with a low stiffness. In stage 2, the bone fails through the original fracture site with a high stiffness. In stage 3, the bone fails partially through the original fracture site and partially through the previously intact bone with a high stiffness.

TABLE 12.1  Biomechanical Stages of Fracture Repair Stages

Site of Failure

Stiffness

1

Original fracture

Low

2

Original fracture

High

3

Original fracture and previously intact bone

High

4

Previously intact bone

High

Adapted from Einhorn TA. Bone remodeling in fracture repair. 1992.

In stage 4, the bone fails though the previously intact bone with a high stiffness. These stages correlate with progressive increases in average torque and energy absorption to failure as healing progresses, but they do not map onto the four biological stages in a one-to-one manner. It is important to note that assessment of fracture healing must be based on both bone structure and mechanical properties. In some cases, especially with the treatment of antiresorptive agents, the recovery of mechanical properties of a healing fracture alone does not represent the restoration of original bone structure. Bisphosphonate treatment has been shown to enhance callus strength though inhibition of callus remodeling, resulting in a larger callus and larger proportion of mineralizing cartilage and woven bone (Fig. 12.5). In terms of material properties, callus tissues are not as strong as the well-organized lamellar bone tissues. However, the larger cross-sectional area and moments of inertia of the callus resulting from bisphosphonate treatment compared with intact bone can compensate for the inferior material properties. When being tested, the healing bone may fail at least partially through the previously intact bone. Although the histologic progress is delayed due to the suppression of callus remodeling by bisphosphonates, the recovery of biomechanical properties may not be affected, suggesting an inconsistency between restoration of anatomic structure and recovery of strength of healing bone caused by bisphosphonate treatment.

CELLULAR EVENTS OF FRACTURE REPAIR Bone formation during fracture healing is accomplished primarily by MSCs in the periosteum, with lesser contributions from the endosteum and marrow stroma. The first event in fracture repair is hemostasis to stop bleeding from damaged blood vessels in the bone and periosteum, resulting in the formation of a hematoma (clot) within the break. Platelets trapped in the hematoma degranulate, releasing platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and transforming growth factor beta (TGF-β), which

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chemoattract neutrophils and macrophages into the fracture zone to initiate an inflammatory phase. Osteocytes die back for a short distance on both sides of the fracture, leaving dead bone matrix that is degraded by osteoclasts derived from the macrophages. The fracture is then repaired by both intramembranous and endochondral ossification. Periosteal MSCs proliferate and differentiate directly into osteoblasts to form collars of intramembranous bone on either side of the fracture space (hard callus). The osteoblasts secrete a bone matrix rich in type I collagen and containing osteocalcin, the mineralization-associated glycoproteins osteonectin, osteopontin, and bone sialoprotein 2, and numerous proteoglycans. Within the fracture space, a cartilage template is formed by the proliferation of periosteal MSCs and their differentiation into chondrocytes (soft callus). The chondrocytes secrete a cartilage-specific matrix composed of aggrecan, types II and XI collagens, fibronectin, and hyaluronic acid and then undergo hypertrophy characterized by the upregulation of type X collagen and downregulation of other types of collagen. Next, the cartilage template is replaced by bone, a process that requires neoangiogenesis. The cartilage matrix calcifies and the hypertrophied chondrocytes release angiogenic signals that trigger the sprouting of capillaries in the periosteum before undergoing apoptosis. As osteoclasts degrade the calcified matrix, periosteal capillaries accompanied by perivascular MSCs invade the template. Some of these MSCs differentiate into osteoblasts, while others become residents of the reforming bone marrow. The osteoblasts differentiate into osteocytes of the cortical and trabecular bone. In this way, new bone is formed in the fracture space, while at the same time intramembranous bone forms directly from MSCs on either side of the fracture space. At first, the new bone is higher than the preexisting bone, but it is subsequently remodeled by osteoclasts to restore the normal shape of the bone. Besides the MSCs within the local environment adjacent to a bone fracture, systemic recruitment of skeletal progenitors has also been proposed. The presence and increase in circulating osteoblast precursors in response to bone injury suggests a recruitment of these progenitor cells from nonfracture sites to the fracture area. These circulating progenitor cells express the osteoblast marker ALP and are able to home to the bone-lining cells at the fracture sites. However, these cells might not integrate within new bone as osteocytes. It is unclear whether these circulating cells are directly involved in fracture repair by producing new bone matrix or indirectly by secreting osteoinductive factors. To discover the origin of the skeletal progenitors during fracture repair, the green fluorescent protein (GFP)– reporter mouse model has become a useful tool to track cell differentiation during fracture repair. For instance,

two GFP reporters, Col3.6GFPcyan and OcGFPtpz, driven by Cola1 (which encodes type I collagen) and BGLAP (which encodes osteocalcin; Oc) promoter fragments, respectively, are bred into the same mice to visualize both early and late stages of osteoblast differentiation. Following fracture, a histologic method that can preserve fluorescent signals in undecalcified bone sections can be used to observe osteoblasts. Osteoprogenitor cells arise from the flanking periosteum proliferate and migrate to fill the fracture zone by day 6. These cells differentiate to osteoblasts and chondrocytes, to form a new outer cortical shell. The hypertrophic chondrocytes are dispersed and the cartilage matrix is mineralized by young osteoblasts between days 7 and 14. The original fracture cortex is resorbed as the outer cortical shell remodels inward to become the new diaphyseal bone after 35 days. In addition, a variety of different GFP-reporter mouse models can be used to track the origin and fate of stem cells that contribute to fracture repair as well as track chondrocyte differentiation.

MOLECULAR REGULATION OF CHONDROGENESIS AND OSTEOGENESIS DURING FRACTURE REPAIR Formation of a hematoma after fracture is regulated as in other wounds by the production of tissue factor (TF) by the nonendothelial cells of damaged blood vessels. TF is the first element in the clotting cascade that ends in the production of thrombin, the molecule that induces platelet degranulation. Degranulation involves the release of α-granules and dense bodies. Molecules such as serotonin and thromboxane A2 that contribute to the vasoconstriction of hemostasis are released by the α-granules. The dense bodies contain fibrinogen, which along with plasma fibrinogen is converted to fibrin of the clot by thrombin. In addition, the dense bodies release the PDGF and TGF-β that initiates the inflammatory phase of fracture repair. During the inflammatory phase, a number of growth factors are released from sequestration in the bone matrix as it is degraded by osteoclasts and also from macrophages. These factors act as signals to activate the transcription factors that commit MSCs to differentiate along chondrogenic and osteogenic pathways. RUNX2 is a key transcription factor that commits MSCs to skeletogenic differentiation. SOX-9 is the key transcription factor that commits skeletogenic precursors to chondrogenesis, and transcription factor Sp7/osterix is the key transcription factor that determines differentiation into osteoblasts. Table 12.2 lists the molecular factors that regulate formation of the cartilage template during fracture repair. These factors are quite similar to those involved in skeletogenesis of endochondral bones during embryonic

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TABLE 12.2  Signaling Molecules, Transcription Factors, and Differentiation Markers Expressed in the Periosteum and Regenerating Tissues of a Fractured Endochondral Bone Tissues

Signals

Transcription Factors

Differentiation Markers

Periosteum

BMPs 2, 4, and 7

RUNX2

–

Soft callus

BMPs 2, 4, 5, and 7

SOX-9

–

SOX-9

Aggrecan, types II, IX, X, and XI collagen

Osterix

Osteocalcin, type I collagen

PDGF FGF2 IGF-I Chondro-callus

IHH BMPs 2, 4, 6, and 7 TGF-β FGFs 1 and 2

Ossification

BMP, bone morphogenic protein; FGF, fibroblast growth factor; IGF-I, insulin-like growth factor I; IHH, Indian hedgehog protein; PDGF, platelet-derived growth factor; TGF-β, transforming growth factor beta. Adapted from Stocum DL. Regenerative biology and medicine. 2012.

development. The expression of RUNX2 is activated by bone morphogenetic proteins (BMPs) 2, 4, 5, and 7. BMP receptor types IA and IB are expressed in the periosteal cells of uninjured bone and both they and BMPs 2, 4, and 7 are strongly expressed in periosteal mesenchymal cells in both the hard callus and soft callus. The expression of SOX-9 and soft callus formation is regulated primarily by fibroblast growth factor 2 (FGF2) in conjunction with the BMPs, PDGF, and insulin-like growth factor I (IGFI). Differentiation of the chondroblasts of the soft callus is regulated by BMPs, FGF1 and 2, and TGF-β. SOX-9 induces the expression of genes for cartilage markers such as types II, X, IX, and XI collagens and aggrecan. IHH transcripts (which encode Indian hedgehog protein [IHH]) are detected in chondrocytes and GLI1 transcripts (which encode Zinc finger protein GLI1) are expressed in a population of cells on the periphery of the callus that will reform the periosteum. In the embryonic development of endochondral bone, IHH, parathyroid hormone (PTH), and parathyroid hormone-related peptide (PTHrP) form part of a feedback loop that controls the rate at which chondrocytes mature. During hard callus formation and replacement of the cartilage template with bone, osteoblast differentiation by perivascular MSCs is regulated by RUNX2 and osterix in response to BMPs. Whether the same upregulation of hyaluronidase and adhesion proteins (neural cell adhesion molecule, fibronectin, and CYR61/IGF-binding protein 10) observed during the condensation of skeletal cells is required for MSC condensation within the soft callus is unknown. It is unlikely that molecules such as the Hox-A, HoxD, T-box transcription factors, sonic hedgehog, FGF4, FGF8, and LIM homeobox transcription factor 1, which

are involved in axial patterning of the skeletal condensations of the limb bud, play a similar role in repair of an endochondral fracture. This is because condensation and chondrocyte differentiation of the soft callus is taking place within a small gap in an already established pattern. Transcriptional profiling of intact versus fractured rat femur by subtractive hybridization and microarray analysis reveals that gene expression patterns change dramatically during fracture repair. Sixty-six percent of the total number of genes are homologous to multiple families of genes known to be involved in the cell cycle, cell adhesion, extracellular matrix, cytoskeleton, inflammation, general metabolism, molecular processing, transcriptional activation, and cell signaling, including components of the Wnt pathway. Thirty-four percent represent genes with unknown functions. The majority of these are grouped in two clusters marked by a sharp increase in activity at 3 days postfracture, which peak at day 14 and then decrease. This pattern suggests that these genes are involved in the proliferation and differentiation of chondrocytes. Wnt-β-catenin signaling plays a very important role in fracture healing. Treatment with lithium, an agonist of the Wnt-β-catenin signaling pathway, improves fracture healing; whereas treatment with Dickkopf-1, an antagonist of this pathway, suppresses fracture repair. Sclerostin, another antagonist of Wnt-β-catenin signaling, is also involved in fracture repair. Fractures in mice with a null mutation of Sost (the gene that encodes the sclerostin protein) show accelerated callus bridging, greater callus maturation, and significantly improved recovery of mechanical strength of repair bone. Similarly,

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systemic inhibition of sclerostin by administration of a sclerostin-neutralizing antibody greatly enhances new bone formation and the strength of the fracture callus in rats and nonhuman primates. The role of sclerostin in fracture repair suggests the involvement of osteocytes in fracture healing because sclerostin is primarily made by osteocytes. Furthermore, attention has also been paid to other osteocyte-specific proteins, such as dentin matrix acidic phosphoprotein 1 (DMP-1; encoded by the DMP1 gene) and FGF23. DMP-1 is one of the acidic phosphorylated extracellular matrix proteins of the small integrin-binding ligand N-linked glycoprotein family. DMP-1 is expressed in the mineralized tissues and is involved in the mineralization. In situ hybridization demonstrates that DMP1 mRNA is strongly expressed in preosteocytes and osteocytes in the bony callus during intramembranous and endochondral ossification until 14 days postfracture. FGF23 is expressed mainly by osteocytes. However, transcript and immunohistochemical analysis have shown a marked increase in FGF23 production in osteoblasts in the fracture callus during fracture repair. In addition, elevated FGF23 (C-terminal fragment) in serum is detected in patients following hip arthroplasty surgery. These data suggest that FGF23 may serve as an indicator of osteoblast differentiation in the early phase of bone healing.

LOCAL REGULATION OF FRACTURE REPAIR Prostaglandins The critical role of prostaglandins, in particular, the prostaglandin E2 (PGE2) signaling pathway in normal bone repair, was well documented in the first decade of the 21st century using genetically modified animal models. Prostaglandins are a family of lipid mediators that coordinate cell–cell communication through interaction with specific cell membrane receptors. These have effects on both bone formation and resorption that are mediated through the proliferation and differentiation of osteoblasts and the regulation of differentiation of osteoclasts. The prostaglandin G/H synthase 2/cyclooxygenase (COX) isozymes catalyze the rate-limiting step in the formation of prostaglandins from arachidonic acid. Two distinct PTGS/COX genes (encoding isozymes COX-1 and COX-2) have been cloned and characterized. The production of prostaglandins under physiologic conditions from the constitutive expression of COX-1 plays important roles in the cytoprotection of the gastric mucosa and kidney function. In comparison, prostaglandins derived from induced COX-2 expression are present in pathologic conditions such as cancer and during inflammation following acute injury. COX-2 is not normally detectable but

is rapidly induced through multiple signaling pathways in various cell types that participate in the inflammatory response. PGE2 is a major COX-2 product at inflammatory sites, where it causes vasodilation and increases local vascular permeability. The critical role of PGE2 induced by COX-2 during fracture healing has been demonstrated using a mouse model containing a null mutation of the Ptgs2/Cox2 gene (Cox2−/− mice). In Ptgs2-null mice, skeletal healing is significantly delayed compared with Ptgs1/ Cox1-null mice and wild-type controls. Thus, COX-2 has an essential role during normal fracture healing. This was found to be mediated through effects on osteoblastogenesis in both intramembranous and endochondral ossification. At fracture sites, COX-2 is expressed primarily in early stem cell precursors of cartilage that also express the COL2A1 gene that encodes a key part of type II collagen in humans and mice. COX-2 has been shown to regulate RUNX2 and osterix expression, which are key factors in chondrogenesis and osteoblastogenesis. Nonsteroidal antiinflammatory drugs (NSAIDs) have been widely used to suppress inflammation and reduce pain following bone injuries. NSAIDs exert their predominant effects by inhibiting COX enzyme activity. The newer COX-2-selective agents act to specifically inhibit COX-2. During the repair of complete fractures, inhibition of COX-2-dependent prostaglandin production results in significant healing defects. In animal studies, indomethacin, a commonly used prescription NSAID, delays endochondral ossification. COX-2-selective NSAID treatment can stop normal fracture healing and induces the formation of delayed- and nonunions. Clinical studies show that NSAIDs decrease bone repair, resulting in a higher nonunion incidence of long bone fractures and delayed spinal fusion. PGE2 is known to bind four different G proteincoupled receptors (GPCRs): EP1, EP2, EP3, and EP4. Interestingly, manipulation of different EP receptor subtypes has shown different effects on fracture healing. The EP1 receptor is involved in regulating intracellular calcium levels. EP2 and EP4 activation stimulates cyclic AMP (cAMP) through the Gs subunit of GPCRs. In contrast, EP3 activation results in a decrease in cAMP levels through the Gi, Gq, or Gs subunits of GPCRs, depending on the EP3 isoform. EP1−/− mice exhibit accelerated fracture healing. Selective agonists for both EP2 and EP4 have positive effects on bone healing, and EP2 and EP4 receptor knockout mice have impaired fracture healing and bone resorption. Treatment of COX-2−/− mice with an EP4 agonist rescues the impaired fracture healing. EP4-selective agonists also accelerated the delayed fracture healing of aged mice. The EP3 receptor, which negatively regulates cAMP levels, is suggested to have negative effects on bone formation. Therefore, the different EP receptors appear to mediate unique effects on the cells and tissues involved in fracture repair.

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Local Regulation of Fracture Repair

Bone Morphogenetic Proteins The activity of BMPs was first discovered by Marshall R. Urist in 1965 because of their capacity to induce ectopic bone formation at extraskeletal sites. BMPs belong to the TGF-β superfamily, which includes TGF-βs, activins/inhibins, nodal, myostatin, and Mullerian-inhibiting factor (or anti-Mullerian hormone). TGF-β superfamily proteins bind to serine/threonine kinase receptors, and transduce signals predominantly through Smaddependent mechanisms. To date, over 20 BMPs have been identified and characterized. Members of BMP family bind to dimers of types I and II serine/threonine kinase receptors. There are three distinct BMP type I receptors, called activin receptor–like kinase (ALK)-2, ALK-3 (BMPR-IA), and ALK-6 (BMPR-IB), and three distinct BMP type II receptors, BMP type II receptor (BMPR-II), activin type II receptor (ACTR-II), and activin type IIB receptors (ACTR-IIB). Type II receptors possess constitutively active kinase activity that phosphorylates type I receptors on ligand-receptor complex formation. Phosphorylated type I receptors transduce the signal to downstream target proteins. A major family of downstream targets of BMPs is the Smad proteins. Of the eight Smad proteins identified in mammals, Smad1, Smad5, and Smad8 are receptor-regulated Smads that are phosphorylated by the BMP type I receptor. Smad2 and Smad3 are activated by activin and TGF-β type I receptors. Smad4 is the only common-partner Smad (coSmad) in mammals, which is shared by both BMP and TGF-β/activin signaling pathways. Smad6 and Smad7 negatively regulate signaling by the other six Smads. However, the detailed downstream signaling molecules of BMPs have not been elucidated. BMPs and several Smads have been detected during fracture healing. In a rodent fracture model, overexpression of BMPs 2, 4, and 7, common-mediator Smad (Smad4), and receptor-regulated Smads (Smads 1 and 5) versus lower levels of inhibitory Smad (Smad6) can be detected at day 3 in osteogenic cells in the thickened periosteum and bone marrow at the fracture sites. At day 10, Smad6 increases dramatically and Smad4 remains elevated, while Smad1 and Smad5 decrease in the fracture callus. Smad7 is expressed only in vascular endothelial cells. By day 28, when new bone has replaced the fracture callus, the expression of all BMPs and Smads decreases, approaching control levels. During fracture healing, the expression patterns of Smads 1 and 5 are similar to that of BMPs 2 and 7. These data suggest that BMPs and downstream Smad family members play an important role in the early stage of fracture healing. Among BMPs, BMP-2 particularly plays a critical role in fracture healing. MSCs, osteoblasts, and chondrocytes all produce BMP-2 at the inflammatory stage of fracture healing. BMP-2 may initiate bone formation, including

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initiating the production of other BMPs involved in bone formation. Mice lacking the ability to produce BMP-2 in limb bones also lack the ability to heal fractures. Furthermore, BMP-2 is extremely important for intramembranous bone formation because BMP-2 is highly expressed in the periosteal MSCs. Animals lacking BMP-2 in a limb-specific manner show an almost complete lack of initial periosteal activation and an apparent failure of the bone to heal (Fig. 12.8). BMPs induce cartilage and bone formation during fracture repair by stimulating the proliferation and differentiation of stem cells into chondrocytes and osteoblasts, a process called osteoinduction. Osteoinduction is one of three requirements for bone regeneration, in addition to osteoconduction and osteogenesis. Osteoconduction is a process that supports the ingrowth of capillaries, perivascular tissues, and osteoprogenitor cells into the 3D structure of an implant or bone graft. Osteoconductive properties are determined by the material architecture, chemical structure, and surface charge. Autologous bone grafts have substantial osteoconductive properties. BMPs and bone grafts are usually needed in the treatment of delayed union or nonunion fracture. Because of the cartilage- and/or bone-inductive activities of BMPs, extensive research on BMPs have been conducted, with the aim of developing therapeutic strategies for the restoration and treatment of skeletal conditions resulting from trauma and degenerative bone diseases. Thus far, two BMPs have been used clinically: recombinant human (rh)BMP-2 and rhBMP-7 (also known as osteogenic protein-1 [OP-1]). Currently, both rhBMP-2 and rhBMP-7 are delivered on an absorbable collagen sponge during surgery. However, clinical outcomes are not as impressive as those seen in animal studies, in which more robust bone formation has been observed. The reasons for this are unclear. One possibility is a lack of sufficient numbers of responding cells at the site of implantation in the host. Another possibility is that BMPs may need to be delivered in combination with other growth factors as “cocktails.” A recent study showed that BMPs may be introduced in a single percutaneous injectable manner to accelerate fracture repair without direct exposure of the fracture site. An injectable strategy may be used in conjunction with the use of other growth factors, injectable grafting materials, and/ or stem cells to make the use of BMPs more effective and affordable.

Platelet-Derived Growth Factor PDGF are polypeptide growth factors secreted by platelets, macrophages, and osteoblasts. PDGF is a major mitogen for osteoblasts, fibroblasts, and smooth muscle cells. It promotes organogenesis, angiogenesis, and wound healing. PDGF has various isoforms (AA, AB,

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(A)

(B)

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FIGURE 12.8  Mice lacking BMP-2 (−/−) have no observable callus formation, and the space between the severed bone surfaces remains open at day 20. Histology was performed on the fractures at day 3 to look at initiation of the repair response (A–D). Control mice (+/+) show an expanded periosteal layer (box in A; magnified in B) containing actively proliferating periosteal cells. Black arrow in (A) indicates the fracture site and the red double arrow in (B) indicates periosteal expansion. BMP-2 mutant mice (−/−) lack any evidence of periosteal activation near the fracture (C; magnified in D). The black arrow in (C) indicates the fracture site and the red double arrow in (D) indicates the minimal extent of proliferation and expansion of the periosteum. Reproduced with permission from Tsuji K, et al. Nat. Genet. 2006;38:1424–1429.

BB, CC, and DD) that signal through two distinct dimerized receptors (α and β) with different affinities. Among these isoforms, PDGF-BB is recognized as the universal PDGF because of its ability to bind to all known receptor isotypes and due to its physiological functions. Following a fracture, platelets aggregate around the fracture site, releasing several growth factors, including PDGF, VEGF, and TGF-β, into the developing hematoma. PDGF attracts neutrophils, macrophages, and progenitor cells, as well as stimulates production of VEGF and interleukin-6 (IL-6) to the fracture site to regulate angiogenesis and promote bone healing. A new hypothetical paradigm has been proposed recently in which PDGF-BB could function at injury sites to mobilize the pericytes and stimulate pericytes to transit into MSCs. In this way, PDGF-BB both contributes to the osteogenic lineage and helps to stabilize newly formed blood vessels, accelerating bone healing. In clinical settings, PDGF is typically delivered locally to the fracture site in a solution of recombinant human PDGF-BB mixed with a beta-tricalcium phosphate scaffold. The US Food and Drug Administration (FDA) has granted premarket approval for the use of PDGF-BB with a beta-tricalcium phosphate scaffold for ankle and hindfoot fusion. The similar formulation of PDGF-BB with beta-tricalcium phosphate has also been approved by the PDA for the treatment of periodontally related bone defects.

Vascular Endothelial Growth Factor Vascularization is essential for bone formation. The importance of blood vessel formation in bone repair can be observed during endochondral ossification, where the cartilage template is replaced by primary bone formation. Chondrocytes at the growth plate undergo hypertrophy and then apoptosis, leaving cavities in a calcified matrix for new bone formation. Endothelial cells play a critical role by being part of the blood vessels that grow into the template, allowing MSCs to enter and form osteoblasts that begin to lay down osteoid matrix. Histologic evidence suggests that chondrocyte apoptosis occurs readily after the invasion of endothelial cells, despite the fact that the factors that induce chondrocyte apoptosis are unknown. The intimate relationship between the ingrowth of vasculature and formation of skeletal tissues suggests an important role of angiogenic mediators in fracture repair. One of the most important growth factors in stimulating vascularization is VEGF, which mediates vascular invasion that is requisite for ossification of the cartilage template. Besides new blood vessels needed for endochondral ossification during fracture repair, fracture healing results in increased blood flow to the surrounding tissue. The relevance of vessel formation is further demonstrated by the use of inhibitors of angiogenesis, which completely prevent healing of fractures and lead to nonunions. In contrast, factors

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such as VEGF delivered as a recombinant protein or via gene therapy have been shown to improve angiogenesis and bone healing by promoting a more rapid recovery of mechanical strength and mineralization of the callus. VEGF acts synergistically with BMP-4 to form cartilage by amplifying recruitment of MSCs and enhancing cell survival. Interestingly, the best ratio of VEGF:BMP-4 for cartilage formation is 1:5. Thus, VEGF along with BMP-4 is one combination of growth factors that might be useful for the enhancement of bone regeneration and fracture healing. While investigations of angiogenic factors and fracture repair have focused on VEGF and its three receptors, VEGFR-1 (FLT-1), VEGFR-2 (KDR/FLK-1), and VEGFR-3 (FLT-4), angiopoietin-1 (ANG-1), a ligand for the endothelium-specific receptor tyrosine kinase angiopoietin-1 receptor/tyrosine kinase with Ig and EGFhomology domains-2 (TIE-2), which has been identified as a novel angiogenic factor, has been localized to bone cells during fracture healing. Unlike the VEGF family that promotes angiogenesis via stimulation of endothelial cell division, ANG-1 has both angiogenic and antiapoptotic effects as well as enhances endothelial cell survival. VEGF often results in leaky, inflamed, and malformed vessels, whereas ANG-1 stimulates angiogenesis with nonleaky neovessel formation. Rats treated with cartilage oligomeric matrix protein (COMP)-ANG-1, a soluble and stable ANG-1 recombinant chimera, exhibit enhanced vascularity and larger callus volumes, which accelerate healing processes during distraction osteogenesis. COMP-ANG-1 also increases vascularity and osteogenesis in osteonecrotic femoral heads as well as the healing of bone allografts for spinal fusion in animal studies. Therefore, ANG-1 and its receptor are potential targets for stimulation of angiogenesis and bone regeneration. Despite the fact that VEGF protein and gene therapy have been shown to increase angiogenesis in rodents and rabbits, these positive proangiogenic data have unfortunately not been successfully reproduced in the clinic. Direct delivery of VEGF protein and gene into patients has failed to promote angiogenesis. It is still questionable whether VEGF therapy can be used to enhance fracture repair and bone regeneration in humans.

Endothelial Progenitor Cells Endothelial progenitor cell (EPC) therapy is a unique cell therapy for fracture healing. EPCs have been identified in both human and animals; they can form vessellike structures in vitro and new vessels in vivo. There are two types of blood vessel formation: vasculogenesis, the de novo formation of a blood vessel without existing vessels; and angiogenesis, the formation of blood vessels from preexisting vessels. It was originally thought

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that vasculogenesis was restricted to embryonic growth, while angiogenesis occurred during postnatal vessel formation. However, recent studies suggest that vasculogenesis also occurs in adults due to the presence of EPCs in the blood circulation. EPCs, under appropriate mobilization stimuli and permissive tissue-homing conditions, undergo neovascularization at the target site. In addition, endothelial cells have been shown to secrete BMP-2, which may help recruit osteoprogenitor cells to promote new bone formation at the fracture site. Furthermore, in the presence of BMP-4, endothelial cells may undergo an endothelial to mesenchymal transition to form MSCs. These endothelial cell–derived MSCs can differentiate into chondrocytes and osteoblasts. Therefore, the potential capacity of EPCs to form new vessels and bone tissues simultaneously makes EPCs an excellent candidate cell population to be tested for enhancement of fracture repair. The discovery that endothelial colony forming cells (ECFCs) are the vessel wall–derived EPCs that participate in angiogenesis has led to the use of ECFCs as a means to improve vascularization and callus formation after fracture in the rat femur. When ECFCs are implanted surgically or injected to the fracture area, more vascularization and callus formation can be seen, suggesting an anabolic effect of ECFCs on fracture repair.

Skeletal Muscle Interactions It is generally accepted that the rate and strength of fracture healing is intimately linked to the integrity of surrounding soft tissues, in particular skeletal muscle. The concomitant loss of surrounding tissue has been associated with poor healing outcomes in patients with open fractures and has been experimentally confirmed in animal studies. In the orthopedic field, the muscle– bone relationship is of utmost importance as surgeons must often battle increased complications, morbidity, and delayed fracture healing in cases with extensive soft tissue damage resulting from high energy trauma. Because of its proximity to bone and abundant vascularity, muscle is a potential source of stem cells and molecular signals for fracture healing. Muscle serves as a vascular supply for oxygen and nutrient exchange during fracture repair, promoting revascularization. The granulation tissue formed as one of the first steps in fracture healing is highly vascularized with small blood vessels. Muscle also is an important source of progenitor cells for fracture healing. Cells derived from muscle are capable of differentiating into cells expressing bone markers. Muscle-derived stem cells (MDSCs) are recruited and driven to osteogenic differentiation by BMPs. Lineage-traced MDSCs at fracture sites have been found to alter gene expression to give rise

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to chondrocytes with increased expression of chondrogenic marker, Sox9, and decreased expression of muscle marker, Pax3. In addition, muscle satellite cells (MyoD-lineage cells) that are the stem cells that regenerate muscle tissue have been found to have osteogenic potential under skeletal injury conditions. Satellite cells have been observed to express both myoblastic (Pax7 and MyoD) and osteoblastic (APL and Runx2) markers and are capable of differentiating into osteoblasts spontaneously. In cases where the bone marrow or periosteum is compromised, myogenic cells of the MyoD lineage contributed to fracture repair and served as a secondary supply of cells. Muscle is capable of supplying osteoprogenitor cells, and the muscular osteoprogenitors possess similar osteogenic potential to those progenitors derived from the periosteum. Muscle is also capable of providing osteogenic growth factors and myokines. Fracture healing is initiated by an inflammatory cascade, which is mediated in part by muscle-derived inflammatory cytokines (i.e., IL-1, IL-6, TNFα). These muscle-derived cytokines play an important role in mounting and maintaining an appropriate inflammatory response in early fracture healing, critical for adequate fracture repair. As discussed earlier, BMP-2 and BMP-7 are FDA approved for use in bone healing due to their role in osteoblast differentiation and fracture repair. Unfortunately, concerns have arisen regarding the multiple side effects and off-label usage of BMPs including a recent link to oncogenic side effects with use of BMP-2. More novel approaches to utilization of BMP-2 in fracture healing include modified muscle cells that secrete BMP-2. Critical size rat femoral defects underwent quicker bridging and restored mechanical strength when receiving activated muscle-secreted BMP-2. Though not a member of the TGFβ superfamily, and not used in the clinical setting currently, BMP-1 is secreted by muscle and may play a role in fracture healing. BMP-1 is a protease secreted by muscle that cleaves procollagen. In patients with traumatic blast injuries, both BMP-1 protein and mRNA levels were elevated, suggesting a significant role for BMP-1 in musculoskeletal repair. Therefore, better understanding of the roles of muscle-derived BMPs in skeletal tissue regeneration is warranted to improve musculoskeletal repair in patients who suffer extensive traumatic injuries. Myostatin is the most well-known muscle-derived protein (see Chapter 16). It has been implicated to play a significant inhibitory role in fracture repair. Small molecule inhibition of myostatin following orthopaedic trauma improves muscle regeneration and fracture healing. It is reasonable to postulate that inhibition of myostatin may be a plausible intervention to improve fracture healing outcomes in patients with significant musculoskeletal injuries.

IGF-1 is recognized as a key myokine that may affect fracture healing. IGF-1 plays a role in muscle fiber repair and regenerative processes by activating and promoting proliferation of satellite cells. Delivery of IGF-1 to ovine bone defects promotes accelerated bone formation. Given the fact that skeletal muscle upregulates expression of IGF-1 in response to injury, local production of IGF-1 by nearby skeletal muscle tissue may support bone healing.

SYSTEMIC FACTORS IN FRACTURE REPAIR Aging Children have the highest potential for healing broken bones. Elderly people are prone to develop several complications, such as nonunion, delayed union, and so on, after a bone fracture. Despite the fact that nonunions in the aging population are a significant clinical problem, little progress has been made in understanding the mechanisms involved in the healing potential decline resulting from aging (see Chapter 10). The decreased healing potential associated with aging is closely related to the decline of cellular activities of stem cells, chondrocytes, and osteoblasts. Previous studies have defined cellular and molecular changes that occur during fracture healing as animals age. Compared with juvenile mice (4 weeks old), chondrocyte maturation and onset of endochondral ossification are delayed by 5–7 days, as evidenced by the reduced expression of collagen type II and type X as well as delayed invasion of blood vessels in adult (6 months old) and elderly mice (18 months old). The completion of endochondral ossification is delayed by 7 days in adult mice and by 14 days in elderly mice, compared with juvenile mice, in which nearly all of the cartilage has been replaced by day 14 following the creation of fracture. Total bone volume in the external callus of the adult and elderly mice is also significantly less than in juvenile mice. These data suggest that both chondrogenesis and osteoblastogenesis decrease with aging. The reduced chondrocyte and osteoblast differentiation in elderly mice suggests that changes in progenitor cell population during human aging may be important. A reduced total number of MSCs, a decrease in responsiveness of MSCs, and an enhanced adipogenic potential at the expense of chondrogenesis and osteogenesis may be involved. In addition, endothelial cell activity and the factors that regulate endothelial cells decrease with aging, suggesting that the decline in angiogenesis potential in elderly patients may negatively affect bone healing.

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Systemic Factors in Fracture Repair

Current research suggests at least two molecular mechanisms are involved in the fracture healing associated with aging: accumulation of oxidative stress and reduction of COX-2 activity. It is known that oxidative stress resulting from an increase in intracellular reactive oxygen species (ROS) is a major determinant of aging and life span. Accumulation of ROS and limited capacity to neutralize ROS result in impaired fracture healing in the elderly. Oxidative stress can attenuate the activity of the Wnt signaling pathway, which has been shown to have an anabolic effect on bone formation, and play a critical role during fracture repair. Thus, antioxidative stress treatment may improve bone healing. In addition, expression of Cox2 mRNA is reduced by 75% in fractures between aged mice and young mice during the early healing phase 5 days after a fracture. COX-2 expression peaks at the exact time when stem cells are changing into cartilage within the fracture callus of young mice and is significantly reduced during that period in older mice. The aged mice show a dramatic decrease in the expression of other genes known to contribute to bone formation as well (e.g., those encoding osteocalcin and type X collagen). These data indicate that in aging animals gene expression is altered early in fracture repair, with consequences for the entire healing cascade. Local injection of an EP4 agonist, which directly activates the EP4 receptor in place of the missing COX-2, to the fracture site of aged mice compensated for the reduced fracture repair observed with aging, leading to a significant reduction in immature cartilage and more efficient formation of mature bone.

Diabetes Mellitus Diabetes mellitus (DM), or simply diabetes, is defined as a group of metabolic diseases characterized by high glucose levels that result from defects in the body’s ability to produce and/or use insulin (See Chapter 23). There are two types of diabetes: type 1 DM (T1DM; also called insulin-dependent DM) and type 2 DM (T2DM; also called noninsulin-dependent DM). Increased fracture risk and low bone mineral density (BMD) have both been linked to T1DM, but in T2DM, BMD is usually normal or elevated, even though the bone is fragile and at risk of fracture, suggesting that factors other than BMD affect bone fragility. Fracture healing is impaired in patients with diabetes. In the well-established diabetic animal models, fracture healing is also impaired, as evidenced by the reduced external callus area, reduced collagen matrix secretion, and reduced recovery of biomechanical properties. Impaired chondrogenesis and osteogenesis during the healing process results from the combined effects of hypoinsulinemia and hyperglycemia. It is not a surprise that systemic insulin treatment, used to normalize blood glucose,

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in a diabetic rat has been shown to reverse the deficit in bone healing. Local intramedullary delivery of insulin to the fracture site, which does not provide systemic management of glucose, can also reverse the healing deficit in mesenchymal cell proliferation and chondrogenesis at the early healing stage, as well as in bone mineralization and biomechanical properties at the late healing stage in a diabetic rat fracture model. Several popular growth factors, including rhBMP-2, rhBMP-7, and recombinant human PDGFBB (rhPDGF-BB) have been shown to positively affect fracture healing in diabetic animal models.

Glucocorticoids Glucocorticoid therapy has negative effects on the skeleton and is the most common cause of secondary osteoporosis (See Chapter 15). The inhibitory effects of glucocorticoid on fracture healing have been known since the 1950s. Formation and calcification of the external fracture callus are significantly reduced, as evidenced by the smaller callus area and lower bone mineral and BMD in glucocorticoid-treated animals. Glucocorticoid treatment results in a significant delay in endochondral ossification and bone remodeling in callus (Fig. 12.9). Glucocorticoid-treated animals exhibit less extensive bridging of the fracture and osteotomy and a less mature callus. When evaluated biomechanically, both torsional and four-point bending tests show a similar 50%–70% reduction in ultimate strength, stiffness, and energy to failure of glucocorticoid-treated animals compared with vehicle-treated controls. The detrimental effects of glucocorticoid on fracture healing may result from its direct actions on osteoblasts and chondrocytes. Glucocorticoid has been shown to impair osteoblast activity and promote osteoblasts apoptosis. Glucocorticoid administration suppresses osteoblast proliferation and differentiation via inhibition of BMPs and RUNX2 expression. Regarding chondrocytes, glucocorticoid decreases chondrocyte proliferation and secretion of cartilage matrix. In addition, studies have shown that glucocorticoid inhibits angiogenesis, which is critical for successful fracture repair. Taken together, delayed or nonunion fractures would occur under longterm glucocorticoid treatment. The current strategy used to treat glucocorticoidinduced skeletal disorders is to prevent glucocorticoidinduced osteoporosis and decrease the incidence of fractures. Few studies have investigated how the repair of fractures that occur during glucocorticoid treatment can be improved. The PTHrP analog has been shown to be an effective therapy for impaired bone healing in rabbits on corticosteroid therapy. Local delivery of BMP-2 and BMP-7/OP-1 may enhance osteotomy healing in glucocorticoid-treated animals. An ideal strategy would be a systemic treatment that can enhance fracture healing as well as improve bone quality.

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FIGURE 12.9  Glucocorticoid inhibits callus formation and delays endochondral ossification during fracture repair. Images show representative histologic images stained with von Kossa and enlarged histologic images from the corresponding squares. (A) Mice implanted with placebo pellets. (B) Mice implanted with slow release prednisolone pellets (1.5 mg/kg/day). No cartilage remains in the large callus of mice implanted with placebo pellets (A), whereas the callus of the prednisolone-treated mice is smaller and contains residual cartilage (B). Reproduced with permission from Doyon AR, et al. Calcif. Tissue Int. 2010;87:68–76.

EFFECTS OF OSTEOPOROSIS DRUGS ON FRACTURE HEALING

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Parathyroid Hormone PTH has a positive effect on BMD and bone formation rate in humans and other animals with fractures and primary and secondary osteoporosis (Chapter 21). PTH is a polypeptide consisting of 84 amino acids. Its bioactive 34-amino acid N-terminal fragment, PTH(1–34), exhibits potent anabolic skeletal effects through its anabolic and antiapoptotic effects on osteoblasts when given by intermittent injection. Systemic administration of PTH(1–34) accelerates the restoration of bone mechanical properties following fracture by augmenting chondrogenesis and osteogenesis at fracture sites. A study using the Pth-null mouse model showed that endogenous PTH also plays an essential role in fracture repair. Without endogenous PTH, fracture repair is markedly inhibited. Furthermore, without endogenous PTH, the effect of exogenous PTH is also attenuated. However, compared with endogenous PTH, exogenous PTH is more efficacious in enhancing fracture healing. PTH accelerates the normal fracture healing processes. Consistent among many animal models, PTH enhances callus formation in the early stage of healing, as evidenced by a larger external callus volume and greater BMD at the fracture sites. The increased callus formation results from the enhancement of chondrogenesis and osteogenesis (Fig. 12.10). The larger cartilage volume in callus is consistent with the upregulation of the chondrocyte transcription factor, SOX-9, as well as the increase in expression of mRNA for types II and X collagen. The mRNA levels of PTHrP and IHH are enhanced by PTH treatment at the fracture sites. PTHrP and IHH proteins may in turn mediate effects of PTH on chondrogenesis. Furthermore, PTH stimulates the differentiation and proliferation of progenitor cells into osteoblasts, as evidenced by the enhanced expression levels

7

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FIGURE 12.10  Parathyroid hormone treatment (PTH) enhances chondrogenesis during fracture repair. Images show histologic features in the fracture calluses of control and PTH-treated groups. Daily subcutaneous injections of 10 μg/kg of rhPTH(1–34)/teriparatide (PTH-treated group) or vehicle solution alone (control group) were administered during the entire healing period. Midsagittal sections from the fracture calluses on days 7, 14, 21, and 28 were stained with toluidine blue (pH 4.1). Areas showing metachromasia represent the cartilage area. Fracture calluses of the control group (A–D) and those of the PTH-treated group (E–H). Asterisks show the fracture site. Scale bars, 500 μm. Reproduced with permission from Nakazawa T, et al. Bone. 2005;37:711–719.

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Effects of Osteoporosis Drugs on Fracture Healing

of the transcription factors RUNX2 and osterix, and the osteoblast markers, type I collagen, ALP, and osteocalcin at the fracture sites. IGF-I expression is enhanced at the fracture sites under PTH treatment. IGF-I may mediate effects of PTH on osteogenesis in addition to chondrogenesis. The enhanced callus formation at the early healing stage under intermittent PTH treatment leads to a larger callus size that results in the early recovery of callus strength. In contrast to many other bone anabolic agents, PTH may accelerate endochondral ossification and bone remodeling during fracture healing. The increase in osteoclast number and activity under PTH treatment contributes to the much faster replacement of cartilage by woven bone and the conversion of woven bone into lamellar bone in callus. These positive PTH effects lead to a much faster recovery of mechanical properties and significantly shorten the fracture healing time. Studies using aging, estrogen-deficient, and glucocorticoid-treated animal models have demonstrated that these pathologic conditions significantly attenuate the effects of PTH on fracture repair. Callus formation and endochondral ossification are somehow delayed compared with those under normal circumstances. One explanation could be that the pathologic conditions impair the responsiveness of osteoprogenitor cells to PTH treatment. It remains to be investigated whether a higher dose and longer treatment time is necessary to effectively treat osteoporotic fractures. Clinical studies have shown the beneficial effects of PTH on fracture healing. Teriparatide, i.e., recombinant human PTH(1–34) (rhPTH[1–34]) at 20 or 40 μg/day accelerates fracture healing in postmenopausal women who have sustained a distal radial fracture that is in need of closed reduction but not operative treatment. A oncedaily injection of 100 μg of PTH(1–84) can reduce the healing time by 38% (7.8 weeks in PTH-treated patients versus 12.6 weeks in placebo control) in osteoporotic women who have fractures of the pubic and/or ischial rami of the pelvis. Due to the complicated role of PTH in vivo, more clinical trials are necessary to further elucidate the role of PTH in fracture healing in patients.

Bisphosphonates Bisphosphonates are used worldwide as a successful and usual first-response treatment for patients suffering from osteoporosis (see Chapter 21). These agents are usually successful at increasing bone mass and trabecular bone thickness, decreasing the risk of fracture, and decreasing bone pain, thus enabling individuals to have a better quality of life. Bisphosphonates bind tightly to calcium at normal pH, but in the sealed zone of an osteoclast (pH 3.5), they are quickly released.

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After being endocytosed, they quickly disrupt osteoclast function and usually lead to osteoclast apoptosis. Bisphosphonates may stay in bone tissues for over 10 years. The long half-life of bisphosphonates raises questions about how they affect fracture repair. Bisphosphonates have an inhibitory effect on callus remodeling without interfering with chondrogenesis and osteogenesis in the process of fracture healing. A lack of osteoclast activity due to bisphosphonate treatment results in prolonged endochondral ossification and a significant delay in bone remodeling during fracture healing. Therefore, a larger callus is seen during the late healing stage that lasts for a very long time in bisphosphonate-treated subjects (Fig. 12.5B). The larger callus, containing a larger proportion of mineralized but unremodeled tissue, contributes to the significant enhancement of callus strength at the late healing stage. Although more osteoclasts may be present inside the callus in those treated with bisphosphonates, most of the osteoclasts become apoptotic and cannot function. Callus remodeling may catch up with the normal control after withdrawal of bisphosphonates when fractures occur. With the development of a new generation of bisphosphonates with more powerful antiresorptive ability, the dosing regimens of bisphosphonates used to treat osteoporosis have evolved from the use of alendronate and risedronate daily and weekly to the use of ibandronate monthly or quarterly and even zoledronic acid at a once-yearly infusion. However, all bisphosphonates have shown similar effects on fracture healing.

Denosumab Denosumab, a human monoclonal antibody, a specific inhibitor of RANK ligand, is also a very effective antiresorptive compound (see Chapter 21). Denosumab has been approved by FDA and used to treat postmenopausal osteoporosis and related disorders. Denosumab and bisphosphonates reduce osteoclast-mediated bone resorption in fundamentally different ways. But, unlike bisphosphonates, the effects of denosumab on fracture repair have not been extensively studied despite the fact that denosumab treatment significantly reduces fracture risk in osteoporotic patients. In the process of fracture healing, RANK ligand expression reaches a peak level during the period of cartilage resorption and stays at an elevated level thereafter until woven bone has been remodeled. Similar to bisphosphonates, denosumab delays callus remodeling but does not affect the recovery of mechanical strength in mice. In clinical trials, healing of nonvertebral fractures can proceed without an increased risk of delayed union in patients treated with denosumab every 6 months. Different from bisphosphonates that tend

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to accumulate more at the fracture sites, denosumab does not accumulate at the fracture site, and its effects on bone may be fully reversible on discontinuation.

EFFECTS OF MECHANICAL AND ELECTRICAL STIMULATION ON FRACTURE REPAIR Electromagnetic Therapy Areas of active bone growth and regeneration or areas of bone deposition during physiologic bone remodeling are electronegative with respect to less active areas; thus, electric fields may be part of the normal process of bone development and regeneration. Direct electrical currents have been used to treat nonunion fractures and osteotomies, as first described in 1971, which are reported to respond well to applications of either cathodal current or electromagnetic fields. Bone regeneration can be accelerated in osteotomies made 3.5–4.5 cm distal to the head of the fibula in dogs. A voltage field was induced in the fibula by inductively coupling pulsed electromagnetic fields of low frequency and strength directly to the bone across the skin. Not only did the stimulated osteotomies heal faster than control osteotomies but also the regenerated bone was more highly organized and stronger than control regenerated bone, even though the mass of callus formed was less than in controls. This method has been used to successfully treat tibial pseudoarthroses in young patients. Pseudoarthrosis is a rare, local bone dysplasia that has a very low probability of correction by conventional techniques. Pulsed electromagnetic fields have also been used as a noninvasive postoperative treatment for lumbar vertebral fusion. The use of electromagnetic fields has been advocated to promote the synthesis of extracellular matrix proteins of bone cells and the secretion of growth factors from osteoblasts to stimulate angiogenesis and new bone formation. Pulsed electromagnetic field therapy may enhance angiopoietin-2 expression. It may also affect several membrane receptors and stimulate osteoblasts to secrete several growth factors such as BMPs 2 and 4, TGF-β, and FGF2. These anabolic effects of electromagnetic fields on bone formation contribute to the enhancement of fracture repair.

Low-Intensity Pulsed Ultrasound Low-intensity pulsed ultrasound (LIPUS) has been shown to have significant beneficial skeletal effects. Ultrasound refers to a high-frequency nonaudible acoustic energy that travels in the form of longitudinal mechanical waves. Traditionally used by physical therapists to intervene in injuries to soft tissues, it is most commonly

used with intensity in the range of 0.5–2.0 W/cm2. In comparison, to intervene in injuries to hard tissues (such as bone) pulsed-wave ultrasound with a spatially averaged, temporally averaged intensity (ISATA) of below 0.1 W/cm2 is preferred. ISATA refers to the average ultrasound power over the area of the ultrasound beam (spatial average) and the average of this intensity over a complete pulse cycle (ultrasound “on” and “off” period; temporal average). Pulsed-wave ultrasound with an ISATA below 0.1 W/ cm2 is termed LIPUS and is preferred in the intervention of fracture healing, as its low ISATA significantly reduces the risk of any thermal or cavitational tissue damage— LIPUS has US FDA approval to be applied to bone. A number of in vitro studies have shown LIPUS to have direct effects on osteoblasts, including alteration of transmembrane ion transfer, stimulation of immediate-early response genes, elevation of mRNA levels for bone matrix proteins, such as osteocalcin and BSP, and increased synthesis of cytokines and growth factors, including c-Fos, COX-2, IGF-I, nitric oxide, p38/MAPK, PGE2, PI3-K, and VEGF. These changes are consistent with a bone-forming response. This bone-forming response is supported by studies using bone rudiments. In 17-day-old fetal mouse metatarsal bone rudiments, LIPUS treatment for 21 min/day over a period of 7 days was found to stimulate a threefold increase in the average length of the calcified diaphysis, when compared with control rudiments. LIPUS stimulates bone union. The initial benefit of LIPUS on the skeleton in vivo is the induction of bone repair in fractures displaying either delayed union or nonunion. In a fracture nonunion model in rodents, 6 weeks of LIPUS treatment stimulated union in 50% of fractures. This is compared with a 0% union rate in contralateral fractures treated with inactive-LIPUS (placebo). Clinically, LIPUS stimulates union in more than 85% of fractures that have otherwise failed to heal. In addition to its benefits on fractures displaying a failed healing response, LIPUS can substantially accelerate the rate of repair of fresh fractures. LIPUS also promotes greater bone content in fracture callus, more rapid endochondral ossification, and quicker recovery of stiffness in ovariectomy-induced osteoporotic, as well as diabetic, rats. In humans, LIPUS can reduce the time for recovery of clinical and radiographic union by 30%–38%. This represents a reduction in healing time of 58, 37, and 19 days in tibial diaphyseal, distal radius, and scaphoid fractures, respectively.

SUMMARY Fractures are the most common traumatic injuries in humans. Successful fracture healing depends on a series of complex biological processes that result in bony

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Suggested Readings

union. Delayed union or nonunion, commonly seen in an aging population, is a devastating clinical complication of fractures that has been a persistent problem despite progress in surgical procedures, hardware, and physical therapy modalities. Although there have been a number of advances in both physical and biological therapies aimed at enhancing fracture healing at both the local and systemic levels, current treatments used to tackle delayed- and nonunion are still limited. There is a great need for new candidate molecules that might provide treatment results equivalent to or better than now achieved, with fewer side effects. Stem cell therapy is an emerging area in the field of fracture repair and bone regeneration. Scientists and surgeons must continue to optimize conditions for harvest, selection, expansion, and formulation of osteogenic cell populations. The targeted delivery of these cells with relevant osteoinductive substances is critical to successful bone healing. Current and novel therapeutic strategies for fracture healing must not only show efficacy (bone healing) but must also limit negative side effects, thereby improving patient outcomes.

STUDY QUESTIONS   

1. D  escribe how periosteum, soft tissue, bone marrow, and cortical bone respond differently to fracture. 2. Explain how NSAIDs have a negative effect on fracture healing. 3. What individual characteristics have a negative impact on fracture healing? What are the mechanisms underlying the negative impact? 4. Briefly describe the four stages of fracture repair and the four biomechanical stages of fracture healing. Describe why both anatomy and mechanics must be taken into account when analyzing fracture healing. 5. Although bisphosphonates have a positive effect on BMD, they have a negative impact of fracture healing. Explain how these drugs interfere with the normal healing process. 6. Describe muscle–bone interactions during fracture healing.   

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Suggested Readings 1. Adami S, Libanati C, Boonen S, et al. Denosumab treatment in postmenopausal women with osteoporosis does not interfere with fracture-healing: results from the FREEDOM trial. J. Bone Jt. Surg. Am. 2012;94(23):2113–2119. 2. Chao EYS, Aro HT, Mow VC, Hayes WC. Biomechanics of fracture fixation basic orthopaedic biomechanis. In: Mow VC, Hayes WC, eds. Basic Orthopaedic Biomechanis. Philadelphia: Lippincott-Raven Publishers; 1997:317–351. 3. Davis KM, Griffin KS, Chu TG, et al. Muscle-bone interactions during fracture healing. J. Musculoskelet. Neuronal Interact. 2015;15(1):1–9. 4. Einhorn TA. The cell and molecular biology of fracture healing. Clin. Orthop. Relat. Res. 1998;355:S7–S21. 5. Einhorn TA, Gerstenfeld LC. Fracture healing: mechanisms and interventions. Nat. Rev. Rheumatol. 2015;11(1):45–54. 6. Ferguson C, Alpern E, Miclau T, Helms JA. Does adult fracture repair recapitulate embryonic skeletal formation? Mech. Dev. 1999;87(1–2):57–66. 7. Ferguson CM, Miclau T, Hu D, Alpern E, Helms JA. Common molecular pathways in skeletal morphogenesis and repair. Ann. N.Y. Acad. Sci. 1998;857:33–42. 8. Gerstenfeld LC, Wronski TJ, Hollinger JO, Einhorn TA. Application of histomorphometric methods to the study of bone repair. J. Bone Miner. Res. 2005;20(10):1715–1722. 9. Hankenson KD, Dishowitz M, Gray C, Schenker M. Angiogenesis in bone regeneration. Injury. 2011;42(6):556–561. 10. Kayal RA, Alblowi J, McKenzie E, Krothapalli N, Silkman L, Gerstenfeld L. Diabetes causes the accelerated loss of cartilage during fracture repair which is reversed by insulin treatment. Bone. 2009;44(2):357–363. 11. Lu C, Miclau T, Hu D, Hansen E, Tsui K, Puttlitz C. Cellular basis for age-related changes in fracture repair. J. Orthop. Res. 2005;23(6):1300–1307. 12. Ominsky MS, Li C, Li X, Tan HL, Lee E, Barrero M. Inhibition of sclerostin by monoclonal antibody enhances bone healing and improves bone density and strength of nonfractured bones. J. Bone Miner. Res. 2011;26(5):1012–1021. 13. Reddi AH. Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nat. Biotechnol. 1998;16(3):247–252. 14. Ren Y, Liu B, Feng Y, Shu L, Cao X, Karaplis A. Endogenous PTH deficiency impairs fracture healing and impedes the fracture healing efficacy of exogenous PTH(1–34). PloS One. 2011;6(7):E23060. 15. Rosen CJ, Compston JE, Lian JB. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. seventh ed. Washington: American Society for Bone and Mineral Research; 2008. 16. Stocum DL. Regenerative Biology and Medicine. second ed. Academic Press; 2012. 17. Ushiku C, Adams DJ, Jiang X, Wang L, Rowe DW. Long bone fracture repair in mice harboring GFP reporters for cells within the osteoblastic lineage. J. Orthop. Res. 2010;28(10):1338–1347. 18. Zhang X, Schwarz EM, Young DA, Puzas JE, Rosier RN, O’Keefe RJ. Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair. J. Clin. Investig. 2002;109(11):1405–1415.

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C H A P T E R

13 Calcium and Phosphate: Hormonal Regulation and Metabolism Linda A. DiMeglio1, Erik A. Imel1,2 1Department

of Pediatrics, Division of Pediatric Endocrinology and Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, United States; 2Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, United States

ORGAN SYSTEM INTERPLAY REGULATES CALCIUM AND PHOSPHORUS METABOLISM Mineral homeostasis involves complex interactions between organ systems, primarily involving the skeleton, intestine, and kidneys. This interplay is regulated by hormones: calciotropic hormones (parathyroid hormone [PTH], parathyroid hormone-related peptide [PTHrP], calcitonin, and vitamin D metabolites), phosphate-regulating hormones (fibroblast growth factor 23 [FGF23], PTH, and vitamin D metabolites), sex steroids (see Chapter 15), glucocorticoids, growth factors, and thyroid hormone. Mineral homeostasis can also be influenced by cytokines and other inflammatory mediators (see Chapter 4). As bone is negatively affected by chronic inadequate dietary intake of calcium or phosphate and by inherited or acquired defects in calcium or phosphate homeostasis, one might reasonably conclude that the primary function of calcium and phosphate homeostasis is to maintain bone integrity. However, processes in the bone, intestine, and kidney work in concert to achieve the primary goal of maintaining plasma calcium within a relatively narrow range. Appropriate adaptations occur in response to abnormalities in one system to combat calcium or phosphorus dysregulation. Kinetic studies show that when plasma calcium concentrations decrease, the body compensates by increasing calcium resorption from the bone, increasing the efficiency of calcium reabsorption in the kidney, and improving the efficiency of dietary calcium absorption from the intestine (Fig. 13.1).

Basic and Applied Bone Biology, Second Edition https://doi.org/10.1016/B978-0-12-813259-3.00013-0

When renal calcium excretion is excessive (e.g., due to high dietary salt intake), compensatory bone and intestinal mechanisms work to maintain plasma calcium. When bone resorption is elevated (during menopause or other disease states), plasma calcium increases with resulting suppression of both intestinal calcium absorption and renal calcium reabsorption. Similarly, when plasma phosphate concentrations decrease, both intestinal absorption and renal phosphate reabsorption increase to compensate; phosphate can also be mobilized from skeletal stores (Fig. 13.1). Conversely, when plasma phosphate concentrations increase, intestinal absorption is downregulated and renal phosphate reabsorption decreases to allow net phosphate excretion. Homeostatic mechanisms for calcium react rapidly within minutes to maintain plasma calcium within a narrow range. However, normal ranges of plasma phosphate are wider, and homeostatic responses are slower, gradually compensating for changes over hours to days. Consequently, plasma phosphate concentration varies more throughout the day in an individual than does plasma calcium. Abnormalities in these homeostatic mechanisms lead to important disease states with effects on muscle and bone.

Distribution of Calcium and Phosphorus in the Body The adult human body contains about 1000  g of calcium and 700  g of phosphorus. Most (>99%) of the calcium is stored in the skeleton (Table 13.1); only approximately 7 g of calcium is found within cells,

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Ca absorption Pi absorption

Ca absorption Pi absorption

High serum Ca/Pi

Low serum Ca/Pi

Ca excretion Pi excretion

Ca deposition Pi deposition

Ca resorption Pi resorption

Ca reabsorbtion Pi reabsorption

FIGURE 13.1  Plasma calcium and phosphate concentrations are regulated through interactions of intestinal absorption, bone mineral deposition and resorption, and kidney mineral excretion via regulation of reabsorption. Under normal homeostatic mechanisms, when plasma calcium or phosphate is high, intestinal absorption becomes downregulated, and the mineral balance is shifted toward bone deposition or kidney excretion of calcium or phosphate. When plasma calcium or phosphate is low, mechanisms are engaged that increase intestinal absorption, skeletal resorption, and/or renal reabsorption. Dysregulation of these systems results in abnormal plasma levels of calcium or phosphate. Ca, calcium; Pi, inorganic phosphate.

TABLE 13.1  Approximate Distribution of Calcium and Phosphorus in Adult Humans Total Body Content (g)

% in Skeleton

% in Soft Tissue

% in Plasma

TABLE 13.2  Approximate Normal Human Serum Concentrations of Minerals, Regulating Hormones, and Vitamins Mass Units

SI Units

8.5–10.5 mg/dL

2.1–2.6 mM

4.4–5.2 mg/dL

1.1–1.3 mM

 Adults

2.5–4.5 mg/dL

0.8–1.5 mM

  Children (newborn)

4.3–9.4 mg/dL

1.4–3.1 mM

  Children (1–5 months)

4.8–8.1 mg/dL

1.6–2.6 mM

  Children (6–24 months)

4.0–6.8 mg/dL

1.3–2.2 mM

  Children (2–3 years)

3.6–6.5 mg/dL

1.16–2.1 mM

  Children (3 years to puberty)

3.6–5.6 mg/dL

1.16–1.8 mM

  Children (pubertal)

3.3–6.0 mg/dL

1.07–1.95 mM

Serum PTH

10–60 pg/mL

1.1–6.3 pM

Serum 25-hydroxyvitamin D

25–80 ng/mL

62–200 nM

Serum 1,25-dihydroxyvitamin D

21–65 pg/mL

50–156 pM

Intact FGF23

etidronate) (Fig 21.14A)

  

In addition to binding to the bone surface, it has been shown that bisphosphonates enter the bone through canaliculi and can be found on many internal surfaces (Fig. 21.14B). Bisphosphonates are released from the bone during the process of bone resorption and are endocytosed by osteoclasts. The non–nitrogen-containing bisphosphonates form nonhydrolyzable ATP analogs that cannot be activated as a source of energy and so result in cell dysfunction and ultimately cell apoptosis. The nitrogencontaining bisphosphonates decrease osteoclast activity by inhibiting the farnesyl diphosphate synthase (FPPS) enzyme in the mevalonate (cholesterol biosynthesis) pathway. The FPPS enzyme is responsible for formation of prenyl chains (15-carbon farnesyl and 20-carbon geranylgeranyl) that attach to small GTPase proteins (Rab, Rac, Ras, Rho) and are required for the correct intracellular localization and functioning of these proteins. Loss of GTPase function results in decreased osteoclast

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FIGURE 21.12  The bisphosphonate drug class includes several different drugs, all with a similar core structure. There is a phosphonate– carbon–phosphonate (P–C–P) backbone with two important side chain attachments, R1 and R2. The first-generation drugs include etidronate, clodronate, and tiludronate. The second- and third-generation drugs included a nitrogen-containing alkyl chain or heterocyclic ring as the R2 side chain, and this greatly increased bone mineral binding and potency for osteoclast inhibition. R1 is important for bone mineral binding. For the nitrogen-containing bisphosphonates, R2 is also involved in bone mineral binding and in addition is responsible for binding to the active site of the farnesyl diphosphate synthase (FPPS) enzyme. Inhibition of FPPS decreases osteoclast activity and function. The nitrogen-containing bisphosphonates are the most prevalent in clinical use.

activity and potentially osteoclast apoptosis (Fig 21.15). Consequently there may not be fewer osteoclasts, just poorly functioning osteoclasts with reduced resorptive capability. Indeed, this has been observed histologically (compare this to the effects of another antiremodeling agent, denosumab, below). In addition, because their life span is prolonged, some of the osteoclasts become

enlarged and multinucleated. Again, these have reduced resorptive capability. Similar to the binding to bone, in the FPPS active site the phosphonate groups bind to divalent cations (magnesium) and the nitrogen-hydrogen group forms hydrogen bonds with adjacent lysine and threonine molecules. The length of this N–O bond determines the enzyme

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FIGURE 21.13  The two phosphonate groups (P) and the R1 (OH in etidronate and nitrogen bisphosphonates) are responsible for bone mineral binding (binding to calcium in hydroxyapatite or HAP). In addition, in nitrogen-containing bisphosphonates the nitrogen in the R2 side chain forms a hydrogen bond with an OH group in HAP. The angle (X°) of the N–H–OH bond helps determine bone binding affinity differences among the drugs (≥125 degrees strong bond;  risedronate > ibandronate >  alendronate (Fig 21.16)

  

Numerous clinical trials have shown that all the nitrogen-containing bisphosphonates reduce bone remodeling compared with placebo (Fig. 21.17). Alendronate, risedronate, and zoledronic acid increase BMD at the spine and hip in all osteoporotic conditions and significantly reduce vertebral and nonvertebral (including hip) fracture risk in PMO. There are some differences among drugs. For example, ibandronate has not been shown to reduce nonvertebral fracture risk and so is only indicated for use in PMO. There are also differences in the rate of onset of effect and in the rate at which bone metabolism returns to pretreatment levels when the drug is discontinued. Bisphosphonates have very low oral bioavailability (5 years). These fractures are nontraumatic and occur in the femoral shaft below the

FIGURE 21.18  Zoledronic acid has the highest bone-binding affinity and highest osteoclast inhibitory potency. It has been shown to have long-lasting efficacy following a single dose in both Paget’s Disease and osteoporosis (A). Five years following a single dose in postmenopausal osteoporotic women, P1NP levels (a biomarker of bone formation) remain significantly lower than baseline (A) and bone mineral density (BMD) gains in the spine, hip, and total body were still present (B). Sustained suppression of remodeling has also been shown 6.5 years following a single dose in Paget’s disease patients, who have very high baseline turnover rates (A). Data from Reid IR, et al. J. Bone. Miner. Res. 2011;26:2261–2270; Grey A, et al. Bone. 2012;50:1389–1393.

lower trochanter. They can be preceded by pain and a visible change in the femoral cortex on radiographs. Radiographically they appear similar to stress fractures. Recent data from bone biopsies of proximal femoral cortical bone next to the fracture site suggest that the cortical bone becomes hypermineralized, potentially leading to decreased fracture toughness and increased crack propagation. This supports the role of remodeling suppression in the pathophysiology, although a causal relationship is still to be established. The condition is seen in 1–10 in 10,000, so there are clearly other factors (genetic, anatomical) that increase risk of it occurring. One likely contributor is femoral bowing, which increases the tensile stresses on the lateral femoral cortex where the fracture begins. However, despite the severity of these adverse effects, they are sufficiently rare that the benefit–risk is

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still strongly in favor of treating with a bisphosphonate. It is estimated that for every 100 hip fractures prevented, there is one atypical femoral fracture. The long bone residence time for bisphosphonates results in maintenance of efficacy on discontinuation of dosing for a period of time that is likely influenced by disease severity and the drug being used. Largely to minimize severe adverse effects such as atypical femoral fractures described above, consideration of a drug holiday has been suggested by the FDA on a case-by-case basis. While bisphosphonate drug holidays have become commonplace, there is little prospective placebo-controlled data to support when to start a drug holiday and for how long to discontinue treatment. In the Fracture Intervention Trial Long-Term Extension (FLEX), women who had been treated with alendronate for 5 years were treated with alendronate for an additional 5 years or switched to placebo for 5 years. Vertebral BMD was maintained over 5 years even without treatment, although hip BMD began to decline by the end of the 5-year period. There was no difference in nonvertebral fractures, but there were more clinical vertebral fractures in the placebo group. In the Health Outcomes and Reduced Incidence with Zoledronic Acid Once Yearly-Pivotal Fracture Trial (HORIZON-PFT), women were treated with zoledronic acid for 6 years (6 IV infusions) or for 3 years followed by 3 years placebo. There were significant decreases in BMD at spine and femoral neck in the placebo group. There was no difference in nonvertebral fractures, but there were significantly more morphometric vertebral fractures in the placebo group. Other studies provide conflicting data, and effects of bisphosphonate discontinuation may depend on factors such as current BMD, age, other medications, co-morbidities, and lifestyle. More data are needed to clarify these issues. Recommendations proposed by a recent ASBMR Task Force (2016) suggested that for women at low to moderate fracture risk, a drug holiday (of 2–3 years) after 5 years oral or 3 years IV bisphosphonate treatment should be considered. For women at high risk (older, low hip T-score, high fracture risk assessment, previous major osteoporotic fracture, or fracture on therapy), treatment should be continued for up to 10 years (oral) or 6 years (IV), with periodic evaluation. Clinical judgment is required on a case-by-case basis. The recommendations may be applicable to men and patients with glucocorticoid-induced osteoporosis. Denosumab Receptor activator of NF-κB ligand (tumor necrosis factor ligand superfamily member 11 or RANKL) is produced by cells of the osteoblast lineage, and through binding to the RANK receptor on osteoclast precursors stimulates differentiation and activation of mature osteoclasts (Chapter 3). OPG (tumor necrosis factor receptor superfamily member 11B) also produced by osteoblasts

403

acts as a decoy receptor for RANKL, downregulating osteoclast development and activation and causing osteoclast apoptosis. Denosumab is a fully human IgG2 RANKL monoclonal antibody. It binds to RANKL and essentially mimics the actions of OPG, the natural inhibitor of RANKL. Although both denosumab and bisphosphonates inhibit osteoclast-mediated bone resorption, they have different effects on osteoclasts. By blocking differentiation and inducing apoptosis, denosumab reduces the number of osteoclasts. Bisphosphonates, on the other hand, decrease osteoclast activity by blocking the FPPS enzyme, but the inactive osteoclasts may persist in the tissue. Histologically, therefore, fewer osteoclasts would be seen with denosumab treatment but not necessarily with bisphosphonate treatment. In the Phase III placebo-controlled 3-year fracture trial (The Fracture Reduction Evaluation of Denosumab in Osteoporosis or FREEDOM trial), denosumab (60 mg subcutaneously every 6 months) resulted in a relative risk reduction in new vertebral fractures (RR = 0.32), nonvertebral fractures (RR  =  0.80), and hip fractures (RR = 0.6) compared with placebo. Bone turnover markers were reduced by 86%, and bone formation (bone formation rate and activation frequency) assessed histomorphometrically in iliac crest biopsies was decreased more than 95%. Based on the clinical trial data, denosumab is indicated for treatment of PMO in women at high risk of fracture, of osteoporotic men at high risk of fracture, and of women and men on cancer therapies (for breast and prostate cancer) at high risk of fracture. Common adverse effects include back and musculoskeletal pain, hypercholesterolemia, and cystitis. Major adverse effects from the HORIZON trial, extension trials, and postapproval experience include hypocalcemia, low bone turnover (adynamic bone disease), osteonecrosis of the jaw, atypical femoral fractures, and serious infections (especially skin and cellulitis). The bone adverse effects are the same as those seen with bisphosphonates and are clearly related to suppression of remodeling. Although RANKL is a member of the TNF-α family, denosumab does not appear to block the TNF-α receptor. However, increased risk of infections is a major adverse effect with TNF-α inhibitors and results from T cell suppression leading to decreased priming of neutrophils in response to infectious agents. Whether this is the cause of infections seen with denosumab is unclear. In a 12-month head-to-head trial with clinical doses of alendronate (70 mg weekly) in PMO, denosumab produced significantly greater increases in BMD at all skeletal sites (total hip, femoral neck, lumbar spine, distal 1/3-radius) and a significantly greater decrease in bone turnover markers (P1NP and sCTX1). Interestingly, the differences in sCTX1 were seen at 1 and 3 months postdenosumab injection but disappeared 6 months postinjection (i.e., just before the next 6-monthly injection),

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suggesting partial recovery of remodeling with denosumab. Despite greater suppression of remodeling leading to higher BMD with denosumab (Fig. 21.17), there are no data to suggest this results in greater fracture risk reduction. The non–head-to-head data suggest that bisphosphonates and denosumab produce similar fracture risk reduction. The very high suppression of remodeling with denosumab results in the same problems seen with bisphosphonates (hypocalcemia, adynamic bone disease, accumulation of unrepaired microdamage). The partial recovery of remodeling biomarkers seen in some but not all subjects suggests that remodeling begins to increase 1–2 months prior to the next injection. There may be sufficient remodeling during this time to prevent long-term problems such as accrual of microdamage. It’s important to note, however, that in the comparative trial with alendronate, remodeling was still suppressed about 70% 6 months following a dose of denosumab, i.e., about the same as with oral alendronate. Denosumab binds to RANKL, and while this may be closely associated with the bone surface, denosumab does not bind to or get incorporated into bone. Once the antibody levels decline, bone remodeling increases and BMD starts to decline and is close to pretreatment levels within 12 months (Fig. 21.19). Because of this, if denosumab treatment is discontinued, treatment with another agent (likely a bisphosphonate or an anabolic agent) needs to be initiated, at least in high-risk subjects. An interesting observation on discontinuation of denosumab is an overshoot or rebound effect in which remodeling markers (especially the resorption markers) increase above pretreatment levels, and BMD declines rapidly toward or sometimes below pretreatment levels. The cause of this overshoot is unclear. One suggestion is that in an attempt to maintain homeostasis, during denosumab treatment there is an increase in the RANKL/ OPG ratio so that on denosumab discontinuation there is increased osteoclastogenesis. Similar effects are seen on stopping estrogen therapy. Whether this rebound effect increases fracture risk compared with untreated subjects is unclear. What is clear is that this decline in BMD is, over time, associated with increased fracture risk, and so on discontinuation of denosumab, treatment with another agent is needed. The decline in BMD can be attenuated with a bisphosphonate, though in one study a single dose of zoledronic acid only partially prevented the BMD decline. It is possible that this reflects the degree of remodeling suppression with denosumab. If denosumab suppresses remodeling >90%, then any subsequent agent that suppresses remodeling less than this (even at 70%) may result in increased remodeling and some loss of BMD. However, based on indirect comparisons from the Phase III trial data, it seems unlikely that such differences in bone remodeling result in differences in fracture risk (Fig. 21.17; Table 21.4).

FIGURE 21.19  In women with postmenopausal osteoporosis denosumab produces large significant increases in bone mineral density (BMD) at both the spine and hip. In contrast to bisphosphonates, which bind to the bone and have long-lasting remodeling suppression effects even after treatment withdrawal, the withdrawal of denosumab treatment leads to reversal of effect. Just 1 year after cessation of treatment, the gains in BMD over the previous 2 years are lost at both the hip and spine. Other studies have even shown an overshoot or rebound effect where on discontinuation of denosumab treatment BMD was lost to below baseline levels. Data from Miller PD, et al. Bone. 2008;43:222–229; Miller PD, et al. J. Clin. Endocrinol. Metab. 2011;96:394–402.

Anabolic Therapies There are currently two approved anabolic therapies, teriparatide and abaloparatide. These are PTH or PTHrP analogs and act through the PTH1 receptor (PTHR1) signaling pathway. An antisclerostin antibody, romosozumab, has completed Phase III trials and is awaiting regulatory approval. PTH 1–84 is the primary endogenous regulator of extracellular calcium and phosphate levels in the body (Chapters 13 and 15). Low serum calcium increases PTH output from the parathyroid gland. In turn, PTH has the following physiologic actions to increase extracellular calcium levels: increased renal reabsorption of calcium, decreased renal reabsorption of phosphate, increased vitamin D (via upregulation of renal 1α-hydroxylase), which in turn increases intestinal absorption of calcium, and increased bone remodeling. Therefore, continuous PTH has a catabolic effect on bone. The observation that intermittent PTH has an opposite and favorable effect on bone (decreased remodeling and an anabolic action) was first reported by Fuller Albright in the 1920s. This observation was largely ignored until small clinical trials were initiated with PTH formulations in the 1970s. The precise mechanism for the differential effects of intermittently administered PTH and endogenous PTH is unknown, although the brief elevation of PTH levels may modulate the osteoblast cell cycle in different

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Osteoporosis Therapies

ways (Chapter 15 for more details). A continuous high level of PTH, as seen in hyperparathyroidism, acts on osteoblasts to upregulate RANKL, downregulate OPG, and increase osteoclastogenesis. This stimulates bone resorption more than bone formation resulting in bone loss. Intermittently administered PTH decreases osteoblast apoptosis and increases cell survival. It decreases sclerostin output from osteocytes, which results in a disinhibition of the Wnt pathway and increases osteoblast activity. It also downregulates RANKL and upregulates OPG in lining cells and osteoblasts, which decreases osteoclastogenesis. If this occurs it is short-lived because clinical data show that after a lag, bone resorption is increased along with bone formation due to the coupling of the two processes. However, the two processes remain imbalanced, and new erosion cavities are overfilled with new bone, leading to net bone formation. Two PTH drugs that have bone anabolic activity (teriparatide and abaloparatide) are approved in the United States for treatment of osteoporosis. While both bind to and signal through the PTH1 receptor (PTH1R) and have similar mechanisms of action, it is worth considering them separately for reasons that are apparent from the discussion. PTH (1–84) was approved in Europe for treatment of osteoporosis but is no longer available. Its effects are similar to PTH (1–34), and clinical trials were unable to demonstrate superiority to PTH (1–34). PTH (1–84) is approved in the United States and Europe for treatment of hypoparathyroidism. Teriparatide Teriparatide is a recombinant human protein of the 1–34 N-terminal amino acids of endogenous PTH (rhPTH1-34). Both full-length PTH and teriparatide bind to PTH receptor types 1 and 2 (PTHR1 and PTHR2) with the same affinity and have the same physiologic actions on bone and kidney. Teriparatide given daily by SC injection has two important actions on bone. Initially it appears to stimulate apposition of bone directly on trabecular surfaces, possibly through a modeling process that does not require prior bone resorption. It subsequently stimulates bone remodeling, like endogenous PTH, but with a greater effect on osteoblasts compared with osteoclasts. The initial effect, i.e., formation without an increase in prior resorption, creates an “anabolic window” in which bone volume increases rapidly. The subsequent increase in bone remodeling continues to increase bone volume by allowing osteoblasts to “overfill” resorption spaces, resulting in a positive BMU balance (Chapter 5, Fig. 5.12). Together these effects result in an increase in bone mass, improved microarchitecture (seen as increased trabecular thickness and connectivity), and reduced fracture risk. The increase in trabecular connectivity is more often a division of thick trabeculae into two trabeculae via remodeling but rejoining of

405

FIGURE 21.20  Two years treatment with teriparatide (parathyroid hormone [PTH]) in postmenopausal women produced significant increases in hip and spine bone mineral density (BMD) compared with placebo-treated controls. This was associated with significant reduction in vertebral and nonvertebral fractures. Data from Neer, et al. NEJM. 2001;334:1434–1441.

broken trabeculae has been observed (Chapter 15 for more details). In the pivotal Phase III placebo-controlled fracture trial, teriparatide (20 μg/day subcutaneously) increased bone density at the spine and hip but not at the distal radius. Interestingly there is a loss of bone at the femoral neck for the first 6 months of treatment caused by increased remodeling. Eventually, however, bone formation “catches up” with the resorption rate, and net bone formation is evident by 12–18 months of treatment. Teriparatide decreased the risk of vertebral fractures by 65% and nonvertebral fractures by 35% after 20 months (Fig. 21.20). The study was terminated early because of the finding of osteosarcoma in the rat carcinogenicity study (see below). Based on these data, teriparatide is approved for treatment of women with PMO at high risk of fracture. It is also indicated for treatment of men with osteoporosis at high risk of fracture and subjects with glucocorticoid-induced osteoporosis at high risk of fracture. In all indications, teriparatide is limited to 2-year lifetime use, although this is being reevaluated. Other clinical trials and analyses illustrate bone effects of teriparatide. In a head-to-head trial with alendronate, teriparatide produced greater increases in BMD at trabecular bone sites (e.g., spine and femoral neck). However, there was significant loss of BMD at the distal radius with teriparatide, whereas there was no loss with alendronate. Teriparatide increased bone formation and resorption markers, and the effect was sustained for 12 months. Increased bone formation and remodeling have been shown with histomorphometry of iliac crest bone biopsies. The histomorphometric data and other analyses also show increased bone mass after teriparatide treatment. Overall, compared with bisphosphonates and denosumab,

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21.  PHARMACEUTICAL TREATMENTS OF OSTEOPOROSIS

teriparatide produced similar fracture risk reductions in vertebral and nonvertebral fractures. Some bisphosphonates and denosumab separately decrease hip fracture risk, but teriparatide did not. In a recent 2-year head-to-head trial with risedronate in postmenopausal women with severe osteoporosis, there were significantly fewer vertebral and clinical fractures in the teriparatide-treated group, but there was no significant difference in nonvertebral fractures. Teriparatide has a box warning for osteosarcoma. Because of this, teriparatide should not be used in Paget’s disease, in pediatric or young adults with open epiphyses, or in subjects with a history of skeletal malignancies. Lifetime use for more than 2 years is not recommended. However, the clinical relevance of osteosarcoma to the population treated with teriparatide is unclear, and after 16 years of clinical use, no significant increase over the background incidence for osteosarcoma has been detected. Other major adverse effects with teriparatide are hypercalcemia, calciuria, and orthostatic hypotension. Abaloparatide Abaloparatide is a 34-amino acid peptide with 76% homology with parathyroid hormone–related protein (PTHrP) (1–34) and 41% homology to PTH (1–34). Abaloparatide is a potent and selective activator of the PTHR1 signaling pathway. Abaloparatide is differentiated from PTH and PTHrP ligands based on its affinity and greater selectivity for the G protein–dependent (RG) (versus the G-independent (R0)) receptor conformation of PTHR1. This selectivity potentially produces a more transient stimulation of osteoblast c-AMP compared with PTH, resulting in less of an effect on bone resorption and less hypercalcemia. A phase III study conducted in postmenopausal women with low BMD with or without prevalent fracture (ACTIVE trial) demonstrated that abaloparatide, 80 μg/day, subcutaneously reduced the incidence of new vertebral, nonvertebral, major osteoporotic, and clinical fractures by 86%, 43%, 70%, and 43%, respectively, compared with placebo. Abaloparatide produced a smaller increase in the bone resorption marker CTX and a lower incidence of hypercalcemia compared with teriparatide. Adverse events that most often led to study drug discontinuation were nausea, dizziness, headaches, and palpitations, which were generally mild to moderate in severity. Serious adverse events leading to discontinuation occurred at similar rates in the abaloparatide and teriparatide groups. Use of abaloparatide for 18 months followed by alendronate for 24 months improved spine, total hip, and femoral neck BMD and reduced vertebral, nonvertebral, and major and clinical fractures compared with that observed after 18 months of placebo followed by 24 months of alendronate. Abaloparatide is approved for treatment of postmenopausal women with osteoporosis at high risk for fracture.

In labeling it has the same statements and warnings as teriparatide. It is approved for 2 years lifetime use, and this is in conjunction with teriparatide, i.e., treatment with teriparatide and/or abaloparatide should be for a maximum of 2 years. Abaloparatide has a box warning for osteosarcoma and warnings for hypercalcemia, hypercalciuria, and orthostatic hypotension. Once treatment is stopped with teriparatide, and presumably also abaloparatide, bone formation decreases more quickly than bone resorption and net bone loss resumes until baseline BMD is reestablished (i.e., all new bone formed during treatment will be lost). Because of this, treatment with an antiremodeling needs to be initiated once anabolic therapy is stopped (see Sequential Treatments section). Antisclerostin Antibody Sclerosteosis and van Buchem disease are two rare sclerosing bone disorders manifesting as high bone mass that is most pronounced in the mandible and skull, and which results from endosteal hyperostosis. Sclerosteosis is associated with mutations in the SOST gene (encodes the sclerostin protein), while van Buchem disease is associated with deletions in a downstream region of the gene that contains regulatory elements for SOST transcription. Sclerostin is secreted by osteocytes and regulates bone mass by inhibiting osteoblast activity. Specifically, sclerostin has been shown to antagonize canonical Wnt signaling by binding to LRP4/5/6, thereby decreasing Wnt-β-catenin signaling through frizzled receptors and downregulating gene transcription for bone formation (Chapters 3 and 4). Blocking sclerostin produces a disinhibition of osteoblasts in part by converting lining cells into active osteoblasts and upregulating bone formation. One sclerostin antibody, romosozumab, is in late-stage clinical development. Another, blosozumab, is no longer being considered as a treatment agent. Like the other anabolic therapies, antisclerostin antibodies do not bind to or accumulate in bone. In the initial Phase III trial (Fracture Study in Postmenopausal women with Osteoporosis or FRAME), 12 months treatment with romosozumab (210 mg subcutaneously once monthly) was compared with placebo (double-blinded). After 12  months, all subjects were switched to open-label denosumab. Romosozumab increased bone density at the spine, hip, and femoral neck (Fig 21.21A). It decreased the risk of new vertebral fractures by 73% and clinical fractures by 36% compared with placebo. It had no significant effect on nonvertebral fractures. The bone turnover markers paint an interesting picture. After the first injection, there was a rapid (within 14 days) ∼150% increase in P1NP. This was accompanied by a similarly rapid decrease (∼50%) in CTX. This suggests uncoupling of formation and resorption and the potential for a strong bone anabolic effect, and confirms preliminary Phase II data. However, the

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Treatment Guidelines and Decisions

407

FIGURE 21.21  In the Phase III clinical trial in postmenopausal women, 12 months treatment with the antisclerostin antibody romosozumab produced substantial increases in bone mineral density (BMD) at spine, total hip, and femoral neck. This was associated with a significant decrease in vertebral but not nonvertebral fracture risk compared with placebo treatment (A). Romosozumab was administered once monthly, subcutaneously. An increase in the bone formation marker, P1NP, was seen with injection over the first 6 months, but the effect diminished and no increase was seen for months 9 through 12 (B). A consistent decrease in the bone resorption marker β-CTX was seen throughout the 12 months (B). Data from Cosman F, et al. NEJM. 2016;375:1532–1543.

P1NP effect was not sustained. There were progressively smaller transient increases after each injection, and the P1NP levels returned to baseline and placebo group levels between 6 and 9 months. On the other hand, the CTX levels were more constant and remained below baseline levels throughout the 12 months (Fig 21.21B). Thus an initial anabolic action transitioned to a more antiremodeling action. Similar effects of romosozumab on BMD and bone biomarkers were seen in other Phase III trials. These effects on remodeling were confirmed by histomorphometry of iliac crest bone biopsies taken after 2 and 12 months of treatment with romosozumab. Like the other anabolics, antisclerostin antibody has a more profound effect on bone volume and biomechanical properties at sites with more cancellous bone (vertebra, femoral neck) than at sites that are predominantly cortical (femoral and humeral diaphysis, distal radius). Moreover, bone quality is maintained as material properties are either preserved or improved. The bone that is formed is lamellar, without fibrosis or evidence of woven bone. In two other Phase III trials, 12 months treatment with romosozumab was compared with alendronate (before all subjects converted to open-label alendronate for months 12–24; the ARCH trial) and with teriparatide (open label) in women who were discontinuing oral bisphosphonate therapy (the STRUCTURE trial). In both studies, romosozumab produced greater increases in BMD at spine and total hip. Romosozumab appeared to have a beneficial effect on hip cortical bone, whereas teriparatide did not. Compared with alendronate at 12 months, romosozumab produced a significantly greater decrease in vertebral fracture risk (by 37%) but not in nonvertebral fracture risk. However, after 24 months there was lower vertebral, nonvertebral and hip fracture risk in subjects treated with romosozumab (12 months) followed by

alendronate (12 months) compared with those treated for 24 months with alendronate. Overall the safety profile of romosozumab was good, but there was a higher incidence of serious cardiovascular events (cardiac ischemic and cerebrovascular events) compared with alendronate in the Phase III trial. Romosozumab was not associated with a higher incidence of cardiovascular events in the placebo-controlled Phase III trial. A Phase III clinical trial with blosozumab showed doserelated increases in spine, hip, and femoral neck BMD over 12 months similar to those seen with romosozumab. When treatment was discontinued, spine BMD declined and ∼50% of the BMD gain was lost within 12 months. The rate of loss was similar in total hip but appeared slower in the femoral neck especially with the high-dose treatment. On discontinuation there were no consistent differences in P1NP or CTX between blosozumab groups and placebo (i.e., no rebound effect as seen with denosumab).

TREATMENT GUIDELINES AND DECISIONS Unlike many other chronic diseases, there is no generally accepted stepwise treatment paradigm for osteoporosis. One reason for this is that treatment is almost always with a single agent. Antiremodeling agents are not given in combination (with the exception of DUAVEE, the combination of estrogen and a SERM), and the data for added benefit of an antiremodeling-anabolic combination over the single agents alone are inconclusive. Table 21.3 is a compilation of the treatment guidelines provided by the National Osteoporosis Foundation, the American Association of Clinical Endocrinologists and American College of Endocrinology, the Endocrine

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Society, and the American College of Physicians. Typically first-line therapy is one of the more potent bisphosphonates, followed by denosumab or occasionally PTH for those at high risk of fracture. The bisphosphonate ibandronate and the SERM raloxifene have not shown efficacy against nonvertebral fractures, and so they are used TABLE 21.3  Treatment Guidelines (USA) (a Compilation Based on Professional Society Guidelines) FIRST-LINE THERAPY • Alendronate, risedronate (oral bisphosphonates) • Zoledronic acid (IV bisphosphonate) • Denosumab (RANKL monoclonal Ab; for those at high risk of fracture) • Teriparatide (PTH) (possibly first line for those unable to use oral therapy and at high risk of fracture) ALTERNATE THERAPY IN PMO FOR THOSE NEEDING JUST SPINAL EFFICACY • Ibandronate (oral and IV bisphosphonate) • Raloxifene (SERM; especially in women at high risk for invasive breast cancer) OTHER THERAPIES • Abaloparatide (PTHrP) (too new for recommendation but like teriparatide) • Calcitonin (last-line therapy for osteoporosis) • Bazedoxifene/conjugated estrogen (DUAVEE; for short-term prevention of PMO) • Estrogen (no longer indicated for prevention or treatment of osteoporosis) • Other SERMs are approved in other geographies but not in the United States PMO, postmenopausal osteoporosis; PTH, parathyroid hormone; PTHrP, parathyroid hormone–related peptide; RANKL, receptor activator of NF-κB ligand; SERM, selective estrogen receptor modulator.

in PMO subjects at lower risk of fracture. However, raloxifene is particularly useful in women at high risk of ER+ invasive breast cancer. The rationale for these recommendations is seen from the clinical data presented in the preceding sections. A comparison of the efficacy of different treatments would be helpful in decision-making, but there are few direct head-to-head comparisons. The relative risks for vertebral, nonvertebral, and hip fractures from the Phase III clinical trials are shown in Table 21.4. In addition, a metaanalysis was performed to compare results across studies. Data from the metaanalysis are included in Table 21.4 where the relevant information is not reported in the Phase III trial. The choice of therapy for an individual patient should include consideration of the patient’s clinical history, risk of fracture and preferences, as well as the risks and benefits of a particular therapy. Age can be a factor because younger patients are at higher risk for vertebral fracture and older patients at higher risk for hip fracture. Another important question is how long to treat a patient. With bisphosphonates there is now consideration for a drug holiday depending on fracture risk. With other drugs, treatment needs to be continued for maintenance of efficacy unless treatment is switched to another agent (see section below).

Sequential Treatments Sequential (sometimes called follow-on) treatment is used to improve efficacy in subjects who have an inadequate response to a therapy and also when a treatment other than a bisphosphonate is discontinued. Sequential treatment is a necessity when treatment with denosumab,

TABLE 21.4  Relative Risk of Vertebral, Nonvertebral, and Hip Fractures From Phase III Clinical Trials Drug

Duration (months)

Vertebral

Nonvertebral

Hip

Alendronate

36

0.52 (0.42–0.66)b

0.68 (0.49–0.92)b

0.47 (0.26–0.79)b

Risedronate

36

0.59 (0.43–0.82)b

0.60 (0.39–0.94)b

0.70 (0.60–0.90)b

Ibandronate

36

0.49 (0.32–0.73)b,a

1.11 (0.65–1.22)a

Not reported

Zoledronic acid

36

0.30 (0.24–0.38)b

0.75 (0.64–0.87)b

0.59 (0.42–0.83)b

Denosumab

36

0.32 (0.26–0.41)b

0.80 (0.67–0.95)b

0.60 (0.37–0.97)b

Raloxifene

36

0.70 (0.50–0.80)b

0.90 (0.80–1.10)

1.10 (0.60–1.90)

Teriparatide

24

0.35

(0.22–0.55)b

Abaloparatide

18

0.14 (0.05–0.39)b

12

(0.16–0.47)b

Romosozumab

0.27

0.62

(0.40–0.97)b,a

0.50 (0.09–2.75)a

0.57 (0.32–1.00)b

Not reported

0.75 (0.53–1.05)

Not reported

Relative risk (95% confidence intervals) of vertebral, nonvertebral, and hip fractures from Phase III clinical trials. aA few values are not presented as relative risk in the Phase III study publications, so these values are odds ratios as presented in Hopkins et al. bSignificant reduction in fracture risk with treatment versus placebo. Alendronate: Black DM, et al. J. Clin. Endo. Metab. 2000;85:4118–4124; Risedronate: Harris ST, et al. JAMA. 1999;282:1344–1352; McClung MR, et al. NEJM, 2001;344:333– 340; Ibandronate: Chestnut CH, et al. J. Bone Miner. Res. 2004;19:1241–1249; Zoledronic acid: Black DM, et al. NEJM. 2007;356:1809–1822; Denosumab: Cummings SR, et al. NEJM. 2009;361:756–765; Raloxifene: Ettinger B, et al. JAMA. 1999;282:637–645; Teriparatide: Neer RM, et al. NEJM. 2001;344:1434–1441; Abaloparatide: Miller, PD et al. JAMA. 2016;316:722–733; Romosozumab: Cosman F, et al. NEJM. 2016;375:1532–1543; Meta-analysis: Hopkins, RB et al. BMC Musculoskel Disord. 2011;12:209

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Treatment Guidelines and Decisions

raloxifene, teriparatide, or abaloparatide (or romosozumab) is discontinued (Table 21.5). Unlike bisphosphonates, these agents do not bind to or accumulate in bone, and so effects on BMD and fracture risk are not maintained when treatment is discontinued. In addition, use of the anabolic agents is currently limited to 2 years lifetime. Antiremodeling Agent Followed by an Anabolic Most studies with this sequence have examined the efficacy of teriparatide following a period of bisphosphonate administration to determine if the anabolic efficacy is reduced by prior antiremodeling treatment. Overall the studies suggest that there is a transient delay or blunting of the expected BMD increase with teriparatide. Thus teriparatide still has bone anabolic activity, but effects may vary depending on the bisphosphonate used for the initial treatment and the region of the skeleton measured. Postmenopausal women treated with denosumab for 2 years and then switched to teriparatide for 2 years experienced an immediate anabolic response shown by serum biomarkers yet experienced substantial and significant loss of BMD over the first 1 year at the hip and femoral neck and over the whole 2 years at the distal radius. This may be linked to the overshoot effect seen with discontinuation of denosumab. It does suggest that 2 years treatment with an anabolic agent will be less effective against nonvertebral fracture risk when given immediately after cessation of denosumab treatment. Anabolic Followed by an Antiremodeling Agent The 2-year limit on anabolic therapy, combined with data demonstrating loss of BMD after discontinuation, means that when stopping treatment with an anabolic a switch to an antiremodeling is necessary. Clinical studies have examined the effects of giving teriparatide for 2 years and then switching to a bisphosphonate or placebo. Bisphosphonate treatment produced a further increase in

TABLE 21.5  Sequential Treatments First Treatment

Second Treatment

Third Treatment (if Needed)

Bisphosphonate Denosumab

Bisphosphonate

Bisphosphonate Denosumab

PTH/PTHrP

Bisphosphonate Denosumab

Raloxifene

Bisphosphonate Denosumab PTH/PTHrP

Bisphosphonate Denosumab

PTH/PTHrP

Bisphosphonate Denosumab

PTH, parathyroid hormone; PTHrP, parathyroid hormone–related peptide.

409

BMD at spine and hip, while those treated with placebo lost significant BMD. As might be expected, switching to raloxifene after 2 years teriparatide increased BMD at the spine but not the hip. Switching from abaloparatide after 18 months to alendronate for up to 24 months led to a further increase in BMD at the spine and hip, and in addition maintained the antifracture efficacy seen with abaloparatide treatment alone. Antiremodeling Agent Followed by an Antiremodeling Agent It does appear that because of the overshoot effect seen after discontinuing denosumab, maintaining the BMD gains on discontinuation is a problem. The decline in BMD can be attenuated with a bisphosphonate depending on dosing regimen. In an extension of the FREEDOM trial, a single dose of zoledronic acid was given to a small number of patients immediately after cessation of denosumab (7 years). This partially prevented the BMD decline at the spine but not at the hip. In an extension of the FRAME trial in a small number of subjects, a single dose of zoledronic acid was delayed until ∼2 months after cessation of denosumab (2 years) to try and increase bone uptake. In this study 70%–85% of the BMD gains at the spine and hip were maintained after 12 months.

Combination (Concurrent) Treatments Because the different classes of therapies have different mechanisms of action, one hypothesis that has been tested is that combination treatments (two drugs given concurrently) provide better efficacy than either agent alone. From a mechanistic perspective, the most interesting combination is that of an antiremodeling with a bone anabolic agent. The rationale is that the anabolic agent will increase bone mass by stimulating formation, and the antiremodeling agent will reduce loss by decreasing remodeling rate and the imbalance of resorption over formation, thus giving benefits to both the formation and resorption sides of the remodeling process. However, it is possible that because the anabolic agents also increase bone remodeling, the two opposite effects on bone remodeling will cancel each other and result in decreased efficacy with combined anabolic/antiremodeling treatment. Concurrent Antiremodeling Agent and Anabolic Several studies have been performed with bisphosphonates (alendronate, risedronate, and zoledronic acid) and denosumab combined with teriparatide. All studies have small sample sizes and different study durations and drug doses making general conclusions challenging. The effects appear to be bone site-specific, time-dependent, and different in treatment-naive versus previously treated subjects. A systematic review/ metaanalysis of seven randomized controlled trials with

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a bisphosphonate–anabolic (PTH or teriparatide) combination concluded that compared with the anabolic agent alone, the combination produced a greater increase in hip and femoral neck BMD over the short term (6–12 months) that was maintained over 18–24 months. A recent study in treatment-naive postmenopausal women showed that a denosumab–teriparatide combination produced significantly larger increases in BMD at all sites except the distal radius compared with either agent alone over 24 months. In the combination treatment group, the changes in bone turnover markers resembled more an antiremodeling action (decreased remodeling) than an anabolic action. Dose-ranging studies may be needed to find the optimal balance of antiremodeling and anabolic effects on remodeling. Importantly for informed clinical decision-making, the effects of combination treatment on fracture risk need to be assessed in large-scale, well-controlled clinical trials. Whether such trials will be performed is unclear. Concurrent Antiremodeling Agents There are data that support the idea that combination treatment with two antiremodeling agents produces greater increases in BMD than either agent alone. This is to be expected and likely results from a larger decrease in bone remodeling. These data were generated some years ago with HRT and a bisphosphonate (alendronate and risedronate) and raloxifene with alendronate. None of the studies looked at fracture risk, and with the recognition that high suppression of bone remodeling results in serious adverse effects with the potent antiremodeling agents (bisphosphonates and denosumab), the risk–benefit of such treatment needs to be considered. Combined antiremodeling treatment is typically not used or recommended.

CONCLUSION Tremendous progress has been made since the 1990s in developing new treatments for osteoporosis. Not that long ago patients at high risk for fracture could only receive estrogen, calcitonin, or calcium plus vitamin D supplement. Now there is a range of agents that have demonstrated substantial efficacy based on large-scale controlled trials, which can be used under different patient circumstances.

STUDY QUESTIONS   

1. D  efine osteoporosis. How is it diagnosed? What are the modifiable and nonmodifiable risk factors for osteoporosis? 2. Describe the mechanism of action of nitrogencontaining bisphosphonates.

3. D  iscuss the differences between denosumab and zoledronic acid. 4. Describe the mechanism of action of the SERM raloxifene and how this results in its clinical efficacy and adverse effects. 5. Compare and contrast the actions and clinical effects of teriparatide, abaloparatide, and romosozumab. 6. What is the potential advantage of combination therapy over monotherapy for treating osteoporosis? What are the current limitations of combination therapy?

Suggested Readings 1.  Austin M, Yang Y-C, Vittinghoff E, Adami S, Boonen S, et al. Relationship between bone mineral density changes with denosumab treatment and risk reduction for vertebral and nonvertebral fractures. for the FREEDOM Trial J. Bone Miner. Res. 2011;27:687–693. 2. Bauer DC, Black DM, Bouxsein ML, et al. Treatment-related changes in bone turnover and fracture risk reduction in clinical trials of anti-resorptive drugs: a meta-regression. J. Bone Miner. Res. 2017;33:634–642. 3. Black DM, Reid IR, Cauley JA, et al. The effect of 6 versus 9 years of zoledronic acid treatment in osteoporosis: a randomized second extension for the HORIZON-Pivotal Fracture Trial (PFT). J. Bone Miner. Res. 2015;30:934–944. 4. Black DM, Rosen CJ. Postmenopausal osteoporosis. N. Engl. J. Med. 2016;374:254–262. 5. Bone HG, Wagman RB, Brandi ML, et al. 10 years of denosumab treatment in postmenopausal women with osteoporosis: results from the phase 3 randomised FREEDOM trial and open-label extension. Lancet. 2017;5:513–523. 6. Burr DB, Russell RGG, eds. Burr DB, Russell RGG, eds. BONE: Special Issue on Bisphosphonates; Vol. 49. ; 2011. 7. Camacho PM, et al. American Association of Clinical Endocrinologists and American College of Endocrinology clinical practice guidelines for the diagnosis and treatment of postmenopausal osteoporosis - 2016. Endocr. Pract. 2016;22(Suppl. 4):1–42. 8. Cosman F. Combination therapy for osteoporosis: a reappraisal. Bonekey Rep. 2014;3:518. PMCID: PMC4007166. 9. Cosman F, et al. Clinician’s guide to prevention and treatment of osteoporosis (National Osteoporosis Foundation). Osteoporos. Int. 2014;25:2359–2381. 10. Ke HZ, Richards WG, Li X, Ominksy MS. Sclerostin and Dickkopf-1 as therapeutic targets in bone diseases. Endocr. Rev. 2012;33:747–783. 11. Marcus R, Feldman D, Kelsey J. Osteoporosis. fourth ed. San Diego: Academic Press; 2013 (multiple relevant chapters). 12. Orwoll ES, Bilezikian JP, Vanderschueren D. Osteoporosis in Men. second ed. Amsterdam: Academic Press; 2010 (multiple relevant chapters). 13. Russell RG, Watts NB, Ebetino FH, Rogers MJ. Mechanisms of action of bisphosphonates: similarities and differences and their potential influence on clinical efficacy. Osteoporos. Int. 2008;19:733759. 14. Saito T, Sterbenz JM, Malay S, Zhong L, MacEachern MP, Chung KC. Effectiveness of anti-osteoporotic drugs to prevent secondary fragility fractures: systematic review and meta-analysis. Osteoporos. Int. 2017;28:3289–3300. 15.  The Womens’ Health Initiative Steering Committee. Effects of cojnugated equine estrogen in postmenopausal women with hysterectomy: the Womens’ Health Initiative randomized controlled trial. J. Am. Med. Assoc. 2004;291:1701–1712.

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C H A P T E R

22 Bone and Cancer G. David Roodman1,2, Theresa A. Guise1 1Department

of Medicine, Indiana University School of Medicine, Indianapolis, IN, United States; L. Roudebush VA Medical Center, Indianapolis, IN, United States

2Richard

Bone involvement with cancer frequently occurs and is the third most common site for cancer metastasis. Bone is only exceeded by lung, liver, and lymph nodes as a metastatic site for solid tumor metastasis (Fig. 22.1). For example, up to 90% of patients with advanced prostate cancer have bone metastasis, and 60%–70% of breast cancer (BC) patients have bone involvement with advanced disease. Bone involvement also occurs with many other cancers, including lung, kidney, and melanomas. In contrast, bone disease is rare in most hematological malignancies except for multiple myeloma (MM), which is the most frequent cancer to involve bone. Among newly diagnosed MM patients, 65% to 70% are present with bone involvement 25

Percent

20 15 10 5

and >90% have bone involvement with advanced disease. Unfortunately, once cancers metastasize to bone, the vast majority of patients are currently incurable and suffer the adverse effects of cancer in bone. These effects include severe bone pain, pathologic fractures, spinal cord and nerve compression syndromes, and derangements of calcium and phosphate homeostasis, which have major consequences for patient survival and quality of life (Table 22.1). Cancer in bone is the leading cause of severe pain for patients with advanced malignancies and is the most frequent cause of cancerrelated pain. Further, cancer-related bone destruction can cause systemic muscle weakness that increases the risk of falls and can result in fractures that negatively impact performance status, survival, and quality of life. These severe effects of cancer in bone result from the marked dysregulation of the normal bone remodeling process that is induced by cancer cells and results in severe imbalances in bone formation and resorption. This chapter will provide an overview of the mechanisms by which bone metastases develop and affect normal bone homeostasis, present a brief summary of current imaging techniques to evaluate the presence of cancer in bone, discuss the utility of bone resorption markers to detect and follow patients with bone metastasis, and review current treatments for and prevention of bone metastasis.

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TABLE 22.1  Consequences of Cancer in Bone

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FIGURE 22.1  Relative proportion of metastasis by site. Bone is a very frequent site for cancer metastasis and is only exceeded by lymph nodes, liver, and lung.

Basic and Applied Bone Biology, Second Edition https://doi.org/10.1016/B978-0-12-813259-3.00022-1

• Severe bone pain that is frequently undertreated. • Frequent pathologic fractures. • Decreased performance status. • Increased mortality. • Systemic muscle dysfunction.

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© 2019 Elsevier Inc. All rights reserved.

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ABNORMALITIES OF BONE REMODELING INDUCED BY CANCER IN BONE The normal skeleton is constantly being remodeled to repair accumulated microdamage and transform woven bone into lamellar bone during the fracture repair (See Chapters 5 and 12). Bone formation and resorption are tightly coupled processes and are controlled by hormones such as PTH, 1,25 vitamin D3, and prostaglandins; cytokines produced by cells in the microenvironment; and cell-to-cell interactions between osteoclasts and osteoblasts in bone. Osteoblasts and osteoclasts communicate through bidirectional signaling between the cell surface receptor EphB4 on osteoblasts and its membrane-bound ligand Ephrin B2 on osteoclasts. Ephrin B2 on osteoclasts binds EphB4 on osteoblasts, which promotes osteoblast differentiation and simultaneously suppresses osteoclast differentiation by reverse signaling through Ephrin B2. Cancer in bone severely disturbs the balanced coupling between osteoclasts and osteoblasts and induces imbalances in bone destruction and/or formation (Fig. 22.2). These abnormalities in bone remodeling result in severe bone pain, increased fracture risk, enhanced tumor growth, and increased resistance of the tumor cells to chemotherapy. The imbalance in bone remodeling can result in metastases that are either osteolytic or osteoblastic (Fig. 22.3). This classification of metastases is based on the radiologic appearance of the involved bone. Osteolytic metastases occur when the imbalance in bone remodeling is predominantly bone destructive

while osteoblastic metastases result when increased new bone formation predominates. However, this distinction is relative and represents the extremes of a continuum. Many patients have a mixture of both osteolytic and osteoblastic metastases. Osteolytic metastases are the most common type of bone metastasis and have major effects on the survival and quality of life of patients. For example, pathologic fractures that occur frequently in MM and BC patients with osteolytic lesions increase their risk of death by >20% and 32% respectively, compared to patients without fractures. Further, many patients with osteolytic metastases suffer severe bone pain and have systemic muscle dysfunction that results from the increased bone destruction. Unfortunately, current treatments do not eradicate cancer in bone in the vast majority of patients, inadequately control bone pain in roughly 40% of patients, and incompletely block bone loss. These severe consequences of osteolytic cancer in bone represent major unmet medical needs that require novel mechanism-based therapeutic approaches. The abnormalities in bone remodeling present in osteolytic and osteoblastic metastases are best exemplified by MM (purely lytic) and prostate cancer (purely osteoblastic on radiographs). Bone lesions in MM are purely lytic, because of increased local osteoclast (OCL) activity adjacent to MM cells accompanied by severely suppressed osteoblast (OB) activity (Fig. 22.2). This imbalance of the normal bone remodeling process results in little or no new bone formation despite increased bone resorption. This lack of bone formation explains why bone scans,

Myeloma

Normal

FIGURE 22.2  Bone remodeling is uncoupled in myeloma. The fundamental concept of bone biology that of bone remodeling where bone is removed by the activity of osteoclasts and then replaced by the activity of osteoblasts is imbalanced in myeloma, where osteoclast activity is greatly increased while osteoblast activity is markedly suppressed.

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Mechanisms Responsible for Bone Metastasis

413

Osteolytic

• Breast, myeloma, lung • Bone destruction mediated by osteoclasts

Osteoblastic

• Prostate & breast • Bone formation mediated by osteoblasts

FIGURE 22.3  Types of tumor metastases in bone. Solid tumors that have a propensity to metastasize to bone to cause either bone destruction, osteolytic metastases, as in this X-ray of a patient with breast cancer; or new bone formation, osteoblastic metastases, as in this patient with prostate cancer. These respective phenotypes are due to tumor stimulation of either the bone resorbing osteoclast or the bone-forming osteoblast.

Radiograph

Bone scan

FIGURE 22.4  Bone scans can underestimate bone involvement in myeloma. Bone scans measure reactive bone formation that is severely suppressed in the osteolytic lesion of a myeloma patient shown on the radiograph. The bone scan of this lesion is negative even though there is a large lytic lesion present.

which measure reactive new bone formation, underestimate the extent of bone lesions in many MM patients (Fig. 22.4). Moreover, suppression of osteoblast activity can persist even when patients are in long-term remission, so that the majority of bone lesions caused by MM do not heal. In contrast, men with bone metastases from prostate cancer predominantly have osteoblastic lesions with increased numbers of irregular bone trabeculae (Fig. 22.3). Although these metastases are osteoblastic, bone resorption is also increased and may exceed the levels seen in patients with myeloma. This finding explains why antiresorptive drugs such as bisphosphonates or Denosumab (discussed below and in Chapter 21) also decrease bone pain and pathologic fractures in patients with metastatic prostate cancer. Similarly, although most bone metastases caused by BC are osteolytic, osteoblastic areas are detectable in these patients, and 15% to 20% of patients with BC have predominantly osteoblastic metastases.

MECHANISMS RESPONSIBLE FOR BONE METASTASIS Stephen Paget, a British surgeon, reported in 1889 his autopsy studies of women who died of metastatic BC. He found that bone was a preferred site for BC metastasis. To explain his results, he advanced the “seed and soil” hypothesis that proposed that cancer cells only grow in “congenial” secondary sites. This hypothesis went against the prevailing dogma that cancer cells spread in a stochastic manner from the primary site and would grow in any tissue that they “seeded.” Since Paget’s original studies, numerous contributing factors have been identified that explain why bone is a preferential site for tumor metastasis. These factors include intrinsic properties of the tumor cells that enhance their bone metastatic potential, changes induced by tumor cells in bone that create the premetastatic niche that supports tumor

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cell homing and growth in bone, changes that occur in tumor cells once they are in the bone microenvironment that enhance their survival in bone, the bone cell effects on tumor cell homing and dormancy in bone, the role of bone cells in suppressing antitumor immune responses in bone, and the physical properties of bone itself that promote the preferential colonization and growth of cancer cells in bone. Some examples of the contributions of these factors to bone metastasis are discussed below.

INTRINSIC PROPERTIES OF TUMOR CELLS THAT ENHANCE THEIR BONE METASTATIC POTENTIAL Kang and coworkers were one of the first groups to identify the specific gene expression signatures in cancer cells that increased their bone metastatic potential. These investigators isolated human MDA-231 BC cells from bone metastasis in mice, expanded these cells, and reinoculated them into mice in vivo to confirm that these BC clones had a high propensity to metastasize to bone rather than other metastatic sites. They found that these bone-trophic clones expressed a specific four-gene signature that increased their bone-metastatic potential. The four genes included the chemokine receptor CXCR4 that binds CXCL12 (the product of the chemokine, CXC motif, ligand 12 [CXCL12] gene) that is expressed on bone cells and increases cancer cell homing to homing;

fibroblast growth factor 5 (FGF5, an angiogenic factor); interleukin 11 (IL-11, an osteoclast stimulatory factor); and osteopontin. BC cells that express at least three of these four genes have an increased propensity to metastasize to bone. BC cells that also express increased levels of both matrix metalloproteinase 1 (MMP1) and ADAMTS1 (a disintegrin-like and metalloproteinase with thrombospondin type 1 motif, 1) have enhanced bone metastatic potential. Kang found that the combination of MMP1 and ADAMTS1 cooperated to cleave amphiregulin (AREG), an epidermal growth factor family member, from the BC cell membrane. AREG in turn increased bone resorption by suppressing osteoprotegerin (OPG) expression by osteoblasts. OPG is a decoy receptor for receptor activator of nuclear factor kappa-B ligand (RANKL) that blocks its capacity to induce osteoclast formation and activity (Fig. 22.5). Increasing bone resorption enhances BC metastasis to bone.

CHANGES IN THE PHENOTYPE OF CANCER CELLS INDUCED BY THE BONE MICROENVIRONMENT Circulating prostate and BC cells that colonize bone can acquire an osteoblast-like phenotype. These cancer cells express bone-related genes that facilitate their survival and growth in bone and escape antitumor cell immune responses in bone. This process of “osteomimicry” can

FIGURE 22.5  RANK ligand and osteoclast formation. Multiple cytokines and hormones induce expression of RANK Ligand (RANKL) on bone marrow stromal cells (BMSCs) and osteocytes. RANK Ligand binds its receptor RANK on osteoclast precursors to induce osteoclast differentiation and enhance osteoclast activity and survival. Osteoprotegerin, a member of the TNF receptor superfamily, is a soluble decoy receptor for RANKL that is produced by BMSCs, mature osteoblasts, and osteocytes and blocks the interaction between RANKL and its receptor on osteoclasts. Reproduced with permission from Roodman, GD. Mechanisms of bone metastasis. N. Engl. J. Med. April 15, 2004;350(16):1655–1664.

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415

Generation of the Premetastatic Niche in Bone

Tumor Cells AXII/ CXCL12 AXIIR/ CXCR4

ADHESION

Stromal Cells/OBL FIGURE 22.7  Homing of tumor cells to bone. Multiple adhesion FIGURE 22.6  Epithelial mesenchymal transition is induced in cancer cells in bone. Epithelial cancer cells can undergo epithelial mesenchymal transition (EMT) in bone that increases their migration and invasion potential. This process results in downregulation of epithelial markers on the tumor cells; loss of their cell polarity and cell-to-cell adhesion due to downregulation of the expression of cadherins and other cell-to-cell adhesion proteins, causing loss of polarity in the tumor cells and tight binding to adjacent cells. This allows the cancer cells to become more like mesenchymal stem cells that can differentiate to multiple types of cells as well as metastasize to other sites. Increased TGFbeta signaling in tumor cells as well as other cytokines in the bone microenvironment can drive EMT through upregulation of Snail, a transcription factor that plays a fundamental role during EMT.

result from increased expression of microRNA (miR)218 in metastatic BC cells, which directs a Wnt signaling circuit that promotes osteoblast-like cell differentiation. Further, epithelial cancer cells can undergo epithelial mesenchymal transition (EMT) in bone that increases their migration and invasion potential (Fig. 22.6). EMT results in downregulation of epithelial markers on the tumor cells and loss of cell polarity and cell-to-cell adhesion due to downregulation of the expression of cadherins (i.e., E-cadherin) and other cell-to-cell adhesion proteins. This results in loss of cell polarity in the tumor cells and tight binding to adjacent cells. This allows the cancer cells to become more like mesenchymal stem cells that can differentiate to multiple types of cells as well as metastasize to other sites. Increased TGFβ signaling in tumor cells as well as other cytokines in the bone microenvironment can induce EMT. Increased TGFβ signaling in tumor cells upregulates expression of Snail, a transcription factor that plays a fundamental role during EMT (Fig. 22.7). Increased transforming growth factor beta (TGFβ) signaling in the tumor cells also plays a crucial role in prostate cancer and BC bone metastasis. TGFβ

molecules and their ligands are expressed by tumor cells and cells in the bone microenvironment. For example, Annexin II receptor and CXCR4 expressed on tumor cells facilitate tumor cell homing to bone through binding of Annexin II and CXCL12 expressed on marrow stromal cells and osteoblasts.

controls expression of multiple genes in breast and prostate cancer cells that promote bone metastasis, including CXCR4, MMP1, IL-11, Jagged 1 (JAG1), and parathyroid hormone-like hormone (PTHrP). These genes can increase tumor cell homing to bone, increase osteoclast activity, and alter the bone matrix to increase tumor cell growth in bone.

GENERATION OF THE PREMETASTATIC NICHE IN BONE Multiple elements present at the primary tumor site can influence the development of bone metastasis. For example, tumor-associated stromal cells at the primary tumor site can increase the bone metastatic potential of BC cells. Stromal cells such as cancer-associated fibroblasts (CAFs) can preferentially increase the growth of specific tumor clones that express SRC (the product of the SRC [V-SRC avian sarcoma viral] oncogene) that are present in the heterogeneous mixture of BC cells present in the primary tumor. SRC expression allows these cancer cells to respond to the CAFderived factors, CXC motif chemokine 12 (CXCL12), and insulin-like growth factor (IGF1). The limited amounts of these CAF-derived factors in the primary tumor site preferentially increase the growth of cancer cells with high SRC activity to metastasize to the CXCL12-rich bone microenvironment. SRC activity in turn promotes BC cell growth and survival in bone marrow. Tumor cells also release factors that prepare the premetastatic niche in bone for tumor cell colonization. These factors include lysyl oxidase, which increases osteoclast activity, extracellular vesicles that

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TABLE 22.2 Tumor-Derived Osteoblastic Factors Endothelin-1

sERB3

Parathyroid hormone–related protein fragments

Platelet-derived growth factor BB

Adrenomedullin

Vascular endothelial growth factor

Insulin-like growth factors

Fibroblast growth factors

Bone morphogenic proteins

CCNs (CTGF, Cyr61)

UPA

Interleukin 18

Transforming growth factor beta UPA, urokinase-type plasminogen activator.

contain micro-RNAs that induce angiogenesis or increase osteoclast activity, and factors that blunt the antitumor cell immune response in the bone microenvironment. These immunosuppressive factors increase regulatory T cells and myeloid suppressor cells that further enhance tumor cell colonization of bone (see below). In addition, cytokines produced by the primary tumor or in the bone microenvironment, such as TGFβ, also increase expression of adhesion molecules on bone marrow stromal cells (BMSC) and osteoblasts and/or interact with hypoxiainducible factor 1 alpha (HIF1α) to increase expression of vascular endothelial growth factor (VEGF) and CXCR4 in the bone microenvironment. All these changes in turn augment bone metastasis by increasing angiogenesis and tumor cell homing to bone (Table 22.2).

TUMOR CELL HOMING TO BONE As noted above, increased expression of adhesive molecules, cytokine receptors, and receptor ligands on the surface of tumor cells also plays an important role in bone metastasis. CXCR4, which binds to CXCL12 (SDF1) expressed on the surface of pericytes and marrow stromal cells in bone, acts as a homing receptor for BC cells, prostate cancer cells, and MM cells to bone (Fig. 22.7). CXCL12 regulates, at least in part, the expression of the vitronectin receptor, integrin alpha (v) beta 3, expression in cancer cells, which plays an important role in the homing to bone and bone marrow as well as tumor cell interactions with endothelial cells and induction of angiogenesis. Tumor cells expressing alpha v beta 3 integrin or E-cadherin also home to the bone marrow by binding osteopontin, bone sialoprotein, vitronectin, or N-cadherin, respectively. Similarly, MM and other cancer cells that home to bone express alpha 4 beta 1 integrin that binds vascular cell adhesion molecule 1 on marrow stromal cells. Additionally, BC and prostate cancer cells express RANK, the receptor for RANK ligand, which further promotes their homing to the marrow.

IMMUNE SUPPRESSION IN BONE METASTASIS Tumor cells can activate macrophages in the bone marrow and at the primary tumor site to change them from M1 (antitumorigenic) to M2 (protumorigenic) macrophages that in turn enhance metastasis. This increase in M2 type macrophages in bone increases the growth of prostate cancer bone metastasis in several ways. Tumorassociated macrophages in the marrow produce cytokines and angiogenic factors that suppress T cell-mediated antitumor responses. In addition, activated T-regulatory cells and Th17 cells also are increased in cancer-involved bone and cause immunosuppression as well as express RANKL and IL-17. IL-17 in turn enhances RANKL induction and augments the effects of RANKL to increase osteoclast activity, which stimulates cancer cell growth in bone. Taken together, these results demonstrate that multiple mechanisms are employed by cancer cells in bone to suppress the antitumor immune response in bone. Further, TGFβ can induce expression of the Notch receptor ligand, Jagged 1, on BC cells to activate Notch signaling in osteoblasts that increases production of interleukin 6 (IL-6). IL-6 can enhance tumor growth in bone and increases the size of lytic lesions by stimulating osteoclast activity. In support of the importance of Notch signaling in BC bone metastasis, a recent study showed that a therapeutic antibody targeting Jagged 1 on tumor cells blocked BC bone metastasis in a preclinical model and sensitized the bone metastasis to chemotherapy.

PHYSICAL CHARACTERISTICS OF BONE CONTRIBUTE TO BONE METASTASIS The physical characteristics of bone itself may also contribute to tumor cell colonization. The bone extracellular matrix is extremely rigid with a high tissue modulus. Exposure of BC cells to the high matrix rigidity in bone increases levels of the GLI2 (Gli-Kruppel family member 2) gene transcription factor in BC cells. GLI2 in turn increases parathyroid hormone-related protein (PTHrP) promoter activity and TGFβ signaling, to further promote BC growth in bone.

CONTRIBUTIONS OF BONE CELLS TO TUMOR CELL DORMANCY, REACTIVATION, CHEMORESISTANCE, AND TUMOR PROGRESSION IN BONE As noted above, bone metastases result from a multistep process that is initiated when circulating tumor cells colonize bone. Tumor cells enter the bone marrow

VI.  SKELETAL DISEASE AND TREATMENT

Role of Osteoclasts in the Development and Progression of Bone Metastasis

compartment and do not spread throughout the bone but rather home to specialized microenvironments or niches, termed osteoblastic or vascular niches. These niches are the same areas in bone where hematopoietic stem cells (HSCs) reside. The osteoblastic niche regulates and supports HSC self-renewal, quiescence, and differentiation. Shiozawa and colleagues showed that prostate cancer cells directly compete with HSCs for this niche and that they can displace HSCs from the osteoblastic niche, which releases HSCs into the circulation. Similarly, when myeloma cells home to bone, they also interact with the osteoblastic niche and are retained there in a dormant state. Dormancy protects the myeloma cells from the effects of chemotherapy and allows them to remain quiescent in bone for long periods of time. The molecular mechanisms responsible for tumor cell dormancy are not well defined but may involve binding of the Annexin II receptor on myeloma cells and prostate cancer cells to Annexin II expressed on osteoblastic lineage cells in bone. Annexin II binding to myeloma and prostate cancer cells can regulate tumor cell growth (Fig. 22.7) and also increases AXL expression in prostate cancer cells, which induces TGFβ-2 signaling to further enhance tumor cell dormancy. Further, cells of the osteoblast lineage also contribute to maintaining tumor cells in the dormant state within HSC niches. Bone matrix proteins produced by marrow stromal cells as well as tumor cells such as osteopontin, bone sialoprotein, and decorin also affect bone metastasis. High levels of osteopontin increase bone metastasis, whereas overexpression of decorin inhibits bone metastasis. These results suggest that blocking the interactions of BC cells with osteopontin or overexpressing decorin may provide new approaches for treating bone metastasis. Cancer cells in bone also contribute to tumor cell dormancy. Sharma and coworkers found that dormant prostate cancer cells increased BMP7 expression by BMSCs, which can maintain prostate cancer cells in a dormant state. In contrast, other researchers reported that dormant BC cells in bone reside on the vasculature of metastatic sites, and that tumor cell dormancy in this case is mediated by thrombospondin-1 produced by endothelial cells. How dormant cells are activated in bone to eventually become overt metastasis remains a topic of intensive investigation. Osteoclastic bone resorption can release and activate dormant myeloma cells and BC cells from their niches, analogous to release of HSCs from their niche. Other reports suggest that reactivation of dormant tumor cells may be under tumor cell control, through factors that may be intrinsic to the colonizing tumor cells or acquired once the cells enter the niche.

417

ROLE OF OSTEOCLASTS IN THE DEVELOPMENT AND PROGRESSION OF BONE METASTASIS Preclinical studies in mouse models of bone metastasis found that increased osteoclast activity promotes tumor metastasis and tumor growth in bone, and that blocking bone resorption decreased osteolytic metastasis. Similarly, clinical studies (discussed below) found that blocking bone resorption with osteoclast inhibitors reduced skeletal related events (SREs) and increased overall and progression free survival of patients with bone metastasis. Further, adjuvant use of osteoclast inhibitors also decreased development of bone metastasis in prostate cancer patients and postmenopausal women with BC at high risk to develop bone metastasis. Multiple factors induced or produced by tumor cells in the bone microenvironment enhance osteoclastic bone resorption. The increased bone destruction in turn releases growth factors such as transforming growth factor beta (TGFβ), IGF1, and others to increase tumor cell growth. This results in a “vicious cycle” (Fig. 22.8) whereby tumor cells in bone induce osteoclastic bone resorption that further releases growth factors present in bone matrix that further increase tumor growth. The receptor activator of nuclear factor kappa-B (RANK)/ RANK ligand (RANKL) signaling pathway is a major regulator of both normal and pathologic bone remodeling (Fig. 22.5). RANKL is a type II homotrimeric (a trimer derived from three identical monomers) transmembrane protein that exists as a membrane-bound or secreted soluble protein, that is produced by cleavage of the full-length form on the cell surface. RANKL is mainly expressed in bone by BMSC, osteoblasts, and osteocytes and is also secreted by activated T lymphocytes. Several studies suggest that osteocytes are the major source of RANKL production in normal bone and produce 10-fold higher levels of RANKL than do osteoblasts. RANKL binds its receptor RANK, a member of the tumor necrosis factor (TNF) receptor superfamily, expressed on the surface of osteoclast precursors and mature osteoclasts. The binding of RANKL to RANK stimulates multiple signaling cascades that are vital for osteoclast differentiation, survival, and activity. OPG, a member of the TNF receptor superfamily, is a soluble decoy receptor for RANKL that is produced by BMSCs, mature osteoblasts, and osteocytes and normally blocks the interaction between RANKL and its receptor on osteoclasts. The RANKL/OPG ratio is crucial for regulating osteoclast formation and activity. Many of the cytokines, chemokines, and hormones produced by tumor cells that induce osteolytic metastasis, e.g., parathyroid hormone-related protein (PTHrp), 1,25-OH2VitD3, prostaglandins, interleukin 1 beta (IL1β),

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22.  BONE AND CANCER

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FIGURE 22.8  The vicious cycle of osteolytic bone metastasis. When cancer cells metastasize to bone, bone resorption is increased by osteoclast activating factors (OAFs) produced by the tumor cells. These (OAFs) include PTHrP, IL-6, and M-CSF that increase osteoclast formation. In addition, tumor cells induce cells in the bone environment, such as marrow stromal cells and osteocytes, to produce RANKL and decrease production of osteoprotegerin (OPG). Tumor cells also induce adipocytes to produce inflammatory cytokines that can increase osteoclast formation and enhance tumor growth. The increased bone resorption releases growth factors from the bone matrix, which in turn increase tumor growth. This creates a vicious cycle of tumor cells increasing bone resorption that in turn increases tumor growth in bone.

macrophage inflammatory protein 1 alpha (MIP1α), and TNF alpha, do so by increasing RANKL expression. It is unclear if tumor cells in bone produce significant levels of RANKL or only induce its production by acting on cells (predominantly osteoblasts and osteocytes) in the bone microenvironment. Several studies reported that RANKL was expressed by MM cells in bone marrow biopsies and by human and murine MM cell lines and that RANKL produced by MM cells and chronic lymphocyte leukemia cells induced the release of tumor necrosis factor alpha (TNFα), IL-6, and IL-8 by the malignant cells via an autocrine/paracrine mechanism. These cytokines increased the survival and growth of malignant cells and exacerbated bone destruction. However, other investigators found that (CD138+) MM cells and MM cell lines do not express RANKL, and that increased expression of RANKL occurred only when human MM cells were cocultured with BMSCs. Regardless of the source of RANKL, the increased RANKL/OPG ratio in MM patients correlates with poor prognosis and reduced survival. Importantly, preclinical models of MM bone disease demonstrate that blocking RANKL-induced osteoclast formation, via administration of recombinant OPG or RANK-Fc, significantly decreased osteolytic lesions and tumor growth in mice. In addition to inducing RANKL or decreasing OPG, products of tumor cells can activate osteoclastic bone resorption in other ways. Jagged 1 expressed on BC cells can activate Notch signaling in osteoclast precursors

to induce osteoclast formation. Further, other factors such as matrix metalloproteinase 7, produced by osteoclasts in response to prostate cancer cells, can cleave ­membrane-bound RANKL to release a soluble protein that increases bone destruction. In addition, matrix metalloproteinase 13 (MMP13) produced by myeloma cells can increase osteoclast precursor fusion to enhance osteolysis. This effect of MMP13 was independent of its proteolytic activity.

ROLE OF OSTEOBLASTS IN THE DEVELOPMENT AND GROWTH OF BONE METASTASIS Osteoblasts also contribute to the bone metastatic process. Osteoblasts produce receptor activator of RANKL and OPG as well as release angiogenic factors and enhance chemoresistance of tumor cells in bone. Immature osteoblasts (OBs), osteocytes, and BMSCs produce RANKL, whereas more mature osteoblasts and osteocytes produce OPG. As noted above, multiple tumor-derived factors can induce RANKL expression by immature osteoblast or block OPG expression by mature osteoblasts or osteocytes. For example, direct interactions between myeloma cells and osteocytes increase RANKL expression and decrease OPG expression by osteocytes. In addition, myeloma cells inhibit osteoblast precursor differentiation, thereby increasing the RANKL/OPG ratio and osteoclast

VI.  SKELETAL DISEASE AND TREATMENT

Role of Osteoblasts in the Development and Growth of Bone Metastasis

Osteoblast Wnt activity

DKK1 expression

Osteoclast Osteoblast Blastic

Lytic

FIGURE 22.9  Expression levels of DKK1 contribute to the type of bone metastasis. Tumor cell-derived factors regulate expression of DKK1, a suppressor of osteoblast differentiation in bone. Low DKK1 levels result in osteoblastic metastasis, whereas high DKK1 levels are seen with osteolytic metastasis.

formation, activity, and survival. Hepatocyte growth factor (HGF) and vascular endothelial growth factor alpha that are present in the tumor microenvironment also induce expression of RANKL and macrophage colony– stimulating factor by mature osteoblasts through transactivation of c-Met, the receptor for HGF, further increasing osteolysis. Blocking both c-Met and VEGF receptor 2 (VEGFR2) with a dual kinase inhibitor of c-Met and the VEGFR2 present on osteoblasts could significantly reduce the growth of prostate cancer in bone. Thus, targeting these pathways in osteoblasts could represent a new approach for treating prostate cancer metastatic to bone. Tumor cell regulation of osteoblast differentiation also contributes to development, type, and progression of bone metastasis. Tumor cells can enhance or suppress osteoblast differentiation. In osteoblastic metastasis, tumor cells produce osteoblast differentiation factors such as endothelin-1, bone morphogenic proteins (BMPs), insulin-like growth factors, platelet-derived growth factors, and fibroblast growth factors and/or suppress production of Wnt signaling antagonists such Dickkopf-1 (DKK1) and increase tumor cell growth (Fig. 22.9). In one study, administration of a selective endothelin-1 receptor antagonist to mice injected with prostate cancer cells decreased both osteoblastic metastases and tumor burden, although it had no effect on tumor growth at orthotopic sites. BMPs, including BMP-6 and BMP-2, also increase osteoblast differentiation in osteoblastic metastasis independently of endothelin-1. Both of these proteins also increase local invasiveness of prostate cancer cells in the bone microenvironment. Overproduction of urokinase-type plasminogen activator (uPA) by prostate cancer cells also increases bone metastasis. Cells transfected with an antisense DNA to uPA had a threefold decrease in bone metastases compared with empty vector transfected cells. An antiurokinase receptor antibody also blocked bone metastasis by prostate cancer cells. These observations suggest that blocking the production of osteoblast-inducing activity by tumors may decrease tumor growth in bone.

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Conversely, tumor cells that induce osteolytic lesions can also inhibit osteoblast differentiation. MM causes the most profound inhibition of osteoblast differentiation (Fig. 22.4). Multiple factors produced by MM cells or induced by their interaction with osteocytes or BMSCs act to suppress osteoblast differentiation. These inhibitors include soluble factors such as interleukin 7 (IL-7), TNFα, and the Wnt signaling antagonists DKK1 and sclerostin. Adhesive interactions between myeloma cells and BMSCs also suppress osteoblast differentiation. Several studies reported that DKK1 levels are also increased in osteolytic BC bone metastasis as well as early in the course of prostate cancer bone metastasis. However, with tumor progress DKK1 levels decrease in response to parathyroid hormone-related protein (PTHrp) produced by the prostate cancer cells. This results in formation of the osteoblastic metastasis that are characteristic of prostate cancer. Importantly, suppression of bone formation in MM persists even after MM cells are eradicated, but the basis for the protracted blockade of bone formation in MM is unclear. Recent studies found that epigenetic changes occur in the primary genes controlling osteoblast differentiation, RUNX2 (runt-related transcription factor 2) and SP7 (transcription factor Sp7, also called Osterix), master genes required for osteoblast differentiation, when BMSCs are exposed to myeloma cells. These investigators showed that the transcriptional repressor Gfi-1 (growth factor-independent 1) is upregulated in BMSCs of myeloma patients and can induce long-term suppression of osteoblast differentiation. Gfi-1 binding to the RUNX2 promoter 1 (RUNX2-P1) locus converts it from a poised bivalent state to a repressed state by recruiting histone modifiers (histone deacetylase 1 [HDAC1], lysine-specific histone demethylase 1, and enhancer of zeste homolog 2 [EZH2]) to induce epigenetic changes at the RUNX2 locus. This decreases H93K9ac and H3K4me3 stimulatory chromatin marks and increases H3K27me3 repressive modifications on the RUNX2 promoter that are essential for repression of RUNX2 gene transcriptions. These repressive H3K27me3 chromatin changes in RUNX2 persist even after removal of MM cells. Chromatin immunoprecipitation analysis also showed that BMSCs from MM patients have decreased H93K9ac on RUNX2, but showed no significant difference in H3K27me3 when compared with normal stromal cells. Importantly, knockdown of Gfi-1 or selective pharmacological inhibition of HDAC1 and EZH2 activity prevented repression of Runx2 and other osteoblast differentiation markers induced by MM cell exposure, and restored osteoblast differentiation. These data suggest that treatment of MM patients with clinically available HDAC1 and EZH2 corepressor inhibitors may reverse the profound osteoblast suppression that is associated with MM, allowing the repair of osteolytic lesions.

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The important contributions of osteocytes to the progression of cancer in bone are just becoming appreciated. Osteocytes comprise more than 95% of all bone cells (compared with 1%–2% for osteoclasts, and 3%–4% for osteoblasts) and are central regulators of physiologic bone remodeling (See Chapter 3). The osteocyte dendritic network allows direct cell-to-cell contact between osteocytes and with cells on the bone surface and in the bone marrow. This network also distributes osteocytesecreted molecules within the bone/marrow microenvironment and into blood vessels, so that they can enter the general circulation. Osteocytes produce RANKL, OPG, Sost/sclerostin, and DKK1, which regulate differentiation and activity of osteoclasts and osteoblasts. In addition, apoptotic osteocytes target bone remodeling to specific areas in bone. Induction of osteocyte apoptosis is sufficient to increase resorption and bone loss, suggesting that osteocyte apoptosis underlies pathological conditions involving enhanced bone resorption. The mechanisms underlying osteoclast recruitment and differentiation driven by osteocyte apoptosis are not fully understood. What is clear is that osteocyte apoptosis is increased in bone lesions of patients with MM, and the number of apoptotic osteocytes positively correlates with the number of osteoclasts on bone. Myeloma cells interact directly with osteocytes, and these interactions activate bidirectional notch signaling between MM cells and osteocytes. Notch signaling increases

MM cell proliferation and induces osteocyte apoptosis (Fig. 22.10). Osteocyte apoptosis is further increased by TNFα secreted by the MM cells. Osteocyte apoptosis induced by MM cells in turn increases RANKL expression in osteocytes, which stimulates osteoclast recruitment. In addition, MM cells increase the expression of Sost/sclerostin in osteocytes, which inhibits osteoblast differentiation. Deletion of the Sost gene or neutralization of its product sclerostin with an antisclerostin antibody stimulates bone formation and reduces bone resorption induced by MM cells in vivo. Some investigators have reported a role for osteocytes in prostate cancer bone metastasis, finding that tumor-generated pressure in bone activates mechanosensing osteocytes, which promotes prostate cancer bone metastasis. Recently, Bortezomib, a proteosome inhibitor with antitumor activity in myeloma has been shown to decrease the numbers of apoptotic osteocytes in vitro and the number of dead osteocytes in the bones, possibly contributing to its therapeutic effects in MM. Recently, several studies have reported a role for bone marrow adipocytes in bone metastasis. Bone marrow adipocytes are one of the most abundant cells in adult bone marrow and increase with age. By age 65, adipocytes comprise 60% of marrow volume. Adipocytes are derived from the same common mesenchymal stromal precursor as osteoblasts, which are induced to differentiate to specific lineage by differentiation factors in the

FIGURE 22.10  Myeloma osteocyte interactions regulate bidirectional notch signaling that increases tumor growth and osteoclast activity. Multiple myeloma (MM) cells interact directly with osteocytes and activate bidirectional notch signaling between MM cells and osteocytes, which increases MM cell proliferation and induces osteocyte apoptosis. Osteocyte apoptosis is further increased by tumor necrosis factor alpha (TNFα), secreted by the MM cells. Osteocyte apoptosis in turn increases RANKL and sclerostin expression in osteocytes that stimulates osteoclast recruitment and blocks osteoblast differentiation.

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Bone Turnover Markers and Other Biomarkers of Cancer in Bone

bone marrow microenvironment. Marrow adipocytes share properties of both brown and white fat cells but have unique functions that differ from both. They secrete fatty acids, cytokines, and adipokines, e.g., leptin and adiponectin that control calorie intake and insulin sensitivity. Cell-to-cell interactions between cancer cells and adipocytes induce morphologic and phenotypic changes in the adipocytes that decrease adiponectin expression, a suppressor of tumor growth, decrease their lipid content, and decrease expression of adipocytic genes. These cell-cell interactions also increase expression of inflammatory cytokines and chemokines such as IL-6, TNFα, CXC motif chemokine ligand 12 (CXCL12), and leptin, which can increase myeloma cell growth and migration and prevent myeloma cell apoptosis. Adipocyte-derived CXCR1 and CXCL12 also increase osteoclast activity in solid tumor metastasis, which enhances the growth and survival of prostate cancer bone metastasis in preclinical models. Further, adipocyte secretion of IL-1β and leptin attracts BC cells to bone. Platelets and megakaryocytes also play an important role in bone metastasis. Alpha-2 beta-3 integrin on platelets can influence melanoma cell homing to bone. Further, lysophosphatidic acid, a platelet-derived lipid, increases the growth of BC cells in bone and production of the osteoclastogenic factors, IL-6, IL-8, and monocyte chemoattractant protein 1. Consistent with these findings, antiplatelet therapy decreased breast and melanoma bone metastasis in mice. In contrast, megakaryocytes (the hematopoietic cells that produce platelets) decrease bone metastasis by suppressing osteoclast formation through production of OPG and increase bone formation. Similarly, increasing megakaryocyte numbers in the marrow blocks prostate cancer bone metastasis.

IMAGING OF BONE METASTASES Multiple imaging modalities are used to diagnose, follow, and determine the response of bone metastasis to treatment. These modalities include skeletal radiographs, 99m Technetium-labeled bisphosphonate bone scans, whole body computerized tomography (WBCT), magnetic resonance imaging (MRI), fluorodeoxyglucose positron emission tomography (FDG-PET) scans, and PET-CT (see Chapter 6). All of these imaging techniques have strengths and limitations for identifying the presence of bone metastasis. Whole body skeletal surveys (WBSS) are the traditional gold standard for identification of lytic bone lesions in MM. However, WBSS are not very sensitive for detecting early lesions caused by bone metastasis because a minimum of 30% of trabecular bone must be removed for a lytic lesion to be detectable by skeletal surveys. WBSS can identify both osteoblastic and lytic metastasis. However, they cannot be used to

421

assess spatial or structural details of the tumor and cannot be used to determine responses to therapy in MM patients because bone lesions in MM rarely heal. These issues have led to the use of WBCT, WBMRI, or PET-CT to evaluate MM patients. Low-dose WBCT, MRI, and PET-CT were found to be superior to WBSS for the detection of MM bone disease, with MRI and low-dose WBCT having equivalent sensitivity, specificity, and detection rates. Bone scans should not be used to evaluate MM patients because they can underestimate bone involvement in myeloma. This is because bone scans measure reactive new bone formation rather than bone destruction (Fig. 22.4). Bone scans with 99m Technetium-labeled diphosphonates can be used to detect bone lesions in BC and prostate cancer patients and are used for staging patients with these diseases. Osteoblastic lesions accumulate more of the radioactive bisphosphonate, as bone scans measure reactive bone formation. Bone scans are low cost and have high sensitivity but relatively low specificity. Further, the anatomic localization of small lesions is limited and false positive results can occur. Finally, positron emission tomography (PET) is better for detecting small lesions and has the advantage that it detects extramedullary disease. PET-CT is highly sensitive for detecting early bone marrow involvement and for diagnosing lytic bone metastases and can be used to monitor therapeutic responses.

BONE TURNOVER MARKERS AND OTHER BIOMARKERS OF CANCER IN BONE Bone turnover markers have been used to measure bone resorption and formation in patients with bone metastasis and MM, for detection of bone metastasis, as prognostic biomarkers and to assess treatment response in patients. When osteoclasts resorb bone, they release protons and proteolytic enzymes that degrade type I collagen in the bone matrix (Chapter 5). This results in release of both N- and C-terminal fragments of collagen (NTX and CTX), which are detectable in the blood or urine. These markers have been used to measure osteoclastic bone resorption in patients with bone metastasis and myeloma. In addition, osteoclasts release tartrate resistant acid phosphatase 5b, a marker protease of osteoclasts, which has also been used to measure bone resorption in patients with bone metastasis and in preclinical models of cancer in bone. Bone formation markers used clinically include procollagen type I propeptides, P1NP, and P1CP, which are removed from procollagen before formation of mature collagen 1 in bone matrix. Procollagen 1 is synthesized by osteoblasts and then cleaved by specific proteases to form type I collagen. P1NP is the most sensitive marker

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of bone formation and reflects the rate of new bone formation. Bone-specific alkaline phosphatase tests have been used as a marker of bone formation, but they are not very sensitive and can cross-react with the liver isoform of alkaline phosphatase. Multiple studies have compared levels of bone turnover markers and clinical outcomes in patients with bone metastasis who are treated with bone-targeted therapies. These studies show that patients with very high levels of NTX or bone-specific alkaline phosphatase are at increased risk for skeletal-related events compared with patients with normal levels of these markers. Further, failure to normalize elevated bone resorption markers on treatment with bone-targeted therapies (zoledronate or denosumab) is associated with disease progression and significantly reduces overall survival. Finally, bone turnover markers have also been used to predict the efficacy of bone-targeted therapy. These studies show that patients with very high NTX levels prior to starting treatment receive the greatest benefit from treatment and have increased overall survival. However, although bone turnover markers are useful in studies of large groups of patients, they cannot be used to diagnose bone metastasis in individual patients or predict treatment responses in individuals. This is because of differences among patient characteristics, the effects of treatment on bone turnover, and inherent variability of the assays.

BONE-TARGETED THERAPIES FOR BONE METASTASIS Current bone-targeted therapies focus on blocking osteoclast activity because safe, effective bone anabolic agents are still not approved for patients with cancer in bone. Bisphosphonates and Denosumab are the primary agents approved for the treatment and prevention of bone metastasis (Chapter 21). Bisphosphonates approved for treating myeloma in the United States are zoledronic acid given at 4 mg IV over 15–30 min and pamidronate given at 90 mg IV over 120 min every 3–4 weeks. All these agents decrease osteoclast activity and reduce development of new osteolytic lesions, pathological fractures, and hypercalcemia in patients with bone metastasis. Zoledronate can decrease bone and solid organ metastasis when used as an adjuvant treatment in postmenopausal women with BC. Bisphosphonate treatment also improves bone pain through inhibition of osteoclast-mediated proton release. Renal insufficiency and osteonecrosis of the jaw (ONJ) are uncommon but major complications associated with bisphosphonate therapy. Although bisphosphonates are very effective for preventing SREs (pathologic fractures, surgery to bone, radiation to bone, and spinal cord compression) in patients with bone metastasis or

at high risk of developing bone metastasis, major questions exist about when to initiate bisphosphonate treatment in patients, how frequently to treat patients, and the duration of treatment. Because the incidence of ONJ is related to duration of bisphosphonate treatment of >2 years, tooth extraction, and surgery to the jaw and is more frequent with zoledronate-based regimens, several studies have assessed if extending the dosing interval for zoledronate from monthly to every 3 months still maintains its efficacy. The Z-Mark study examined the efficacy of 3 monthly vs. monthly zoledronate therapy in MM patients who had received 1–2 years of monthly zoledronate therapy and had urinary N-telopeptide of type I collagen (uNTX) levels of 50 nmol/mmol creatinine, those who developed an SRE on the study, or those who had disease progression were treated with monthly zoledronate. Of the 121 patients, 79 received the 3 monthly schedules with only 12 patients developing an SRE over the 2 years of the study. This low SRE rate (8.9%) supported less frequent dosing of zoledronate in patients who had received 1–2 years of monthly treatment and have stable disease. It also reflected recent results that show very low rates of SREs in MM patients receiving more effective modern therapies. A second larger randomized trial compared every 12–4-week zoledronate therapy in 1544 patients with bone metastasis and 278 patients with myeloma. This study found no differences in SREs, incidence of ONJ or renal dysfunction between the treatment groups. However, only 795 of the 1822 patients randomized initially completed the 2 year study, with a similar high dropout rate for the MM patients. Taken together, these studies are not definitive but suggest that less frequent zoledronate dosing may be feasible in cancer patients with bone metastasis. Denosumab, a monoclonal antibody that binds RANKL, is approved for the treatment of bone metastasis in patients with solid tumors and just recently for MM. Its use is also approved for the prevention of bone metastasis in men with castrate-resistant prostate cancer at high risk for progression. Denosumab treatment was found to be superior to zoledronate for preventing SREs in patients with BC and conferred a survival advantage for patients with BC and lung cancer bone metastasis. Further, Denosumab can be given to patients with renal impairment, as it is an antibody. However, dental monitoring and vitamin D and calcium supplementation must be used for patients receiving Denosumab as is done for patients receiving bisphosphonates, because of the risk of ONJ and hypocalcemia. Importantly, there are no data on extending the dosing interval for Denosumab in patients with bone metastasis. This should be considered carefully because several studies have shown that patients with osteoporosis receiving Denosumab can have accelerated bone loss after stopping Denosumab.

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Suggested Readings

SUMMARY

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Suggested Readings

Bone is a frequent site of metastasis, and bone metastasis causes significant morbidity and increased mortality for patients, which result from a marked dysregulation of the normal bone remodeling process. Recent research has focused on identifying the molecular mechanisms responsible for osteolytic and osteoblastic metastasis, tumor cell homing to bone, the factors involved in the preparation of the premetastatic niche and that block the immune responses to cancer cells in bone. Identification of the molecular mechanisms responsible for tumor cell dormancy and reactivation should allow for the development of novel mechanism-based therapies to combat and prevent the colonization and progression of bone metastasis, and the devastating sequelae associated with cancer in bone.

STUDY QUESTIONS   

1. H  ow does the primary tumor site contribute to bone metastasis? 2. What is the contribution of increased osteoclast activity to bone metastasis? 3. What factors determine if a bone metastasis is osteolytic or osteoblastic? 4. What new therapeutic targets are emerging to treat/prevent bone metastasis? 5. What determines tumor dormancy in bone?  

1. Coleman RE. Skeletal complications of malignancy. Cancer. 1997;80(8 Suppl.):1588–1594. 2. Waning DL, Mohammad KS, Reiken S, et al. Excess TGF-beta mediates muscle weakness associated with bone metastases in mice. Nat. Med. 2015;21(11):1262–1271. 3. Roodman GD. Mechanisms of bone metastasis. N. Engl. J. Med. 2004;350(16):1655–1664. 4. Roodman GD. Osteoblast function in myeloma. Bone. 2011;48(1):135–140. 5. Coleman RE, Major P, Lipton A, et al. Predictive value of bone resorption and formation markers in cancer patients with bone metastases receiving the bisphosphonate zoledronic acid. J. Clin. Oncol. 2005;23(22):4925–4935. 6. Pickup M, Novitskiy S, Moses HL. The roles of TGFbeta in the tumour microenvironment. Nat. Rev. Cancer. 2013;13(11):788–799. 7. Zhang XH, Jin X, Malladi S, et al. Selection of bone metastasis seeds by mesenchymal signals in the primary tumor stroma. Cell. 2013;154(5):1060–1073. 8. Weilbaecher KN, Guise TA, McCauley LK. Cancer to bone: a fatal attraction. Nat. Rev. Cancer. 2011;11(6):411–425. 9. Croucher PI, McDonald MM, Martin TJ. Bone metastasis: the importance of the neighbourhood. Nat. Rev. Cancer. 2016;16(6):373–386. 10. Delgado-Calle J, Anderson J, Cregor MD, et al. Bidirectional notch signaling and osteocyte-derived factors in the bone marrow microenvironment promote tumor cell proliferation and bone destruction in multiple myeloma. Cancer Res. 2016;76(5):1089–1100.

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C H A P T E R

23 Sweet Bones: Diabetes Effects on Bone Viral N. Shah1, Linda A. DiMeglio2 1Barbara

Davis Center for Diabetes, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, United States; 2Department of Pediatrics, Division of Pediatric Endocrinology and Wells Center for Pediatric Resarch, Indiana University School of Medicine, Indianapolis, IN, United States

INTRODUCTION Diabetes is a disease of chronically high circulating blood glucose concentrations (Table 23.1). It affects nearly 1 in 11 people worldwide. There are two clinical types of diabetes: type 1 diabetes (T1D) and type 2 diabetes (T2D) (Fig. 23.1). The degree of hyperglycemia in persons with diabetes is assessed clinically using a laboratory measure known as hemoglobin A1c (Box 23.1) The incidence of T1D is about 23 new cases per 100,000 persons per year; approximately 1.25 million American children and adults have T1D. Due to care advances in therapeutics and technology, most persons with T1D now live to later adulthood, and the prevalence of T1D in the population has increased over time. Persons who develop T1D inherit a genetic predisposition to the disease. Over time, potentially triggered by environmental exposures (several viral infections are associated with T1D), they experience innate and adaptive immune activation, which results in destruction of pancreatic insulin-producing β cells. Treatment for T1D is insulin replacement by injection or continuous infusion (using a pump device worn on the body that delivers insulin through a catheter placed under the skin). Nearly 1.5 million Americans receive a T2D diagnosis each year; around 30 million Americans have this disease. T2D initially primarily features insulin resistance and worsens over time due to evolving insulin deficiency from a pancreatic β cell failure. Treatment for T2D usually begins with insulin sensitizers (which improve insulin action) and secretagogues (which enhance insulin secretion by β cells); eventually insulin therapy is often required. Both T1D and T2D are well known to be associated with long-term disease complications, including microvascular

Basic and Applied Bone Biology, Second Edition https://doi.org/10.1016/B978-0-12-813259-3.00023-3

disease (retinopathy, neuropathy, nephropathy), macrovascular complications (cardiovascular disease and stroke), and neurocognitive dysfunction. Complication risk increases with longer duration of disease and with chronically higher blood glucose concentrations. Since the early 1990s, therapeutic approaches have attempted to reduce diabetes complications by lowering average blood sugars. With improved diabetes care, there have been substantial reductions in diabetes complications and improved longevity. These changes have led to an aging population with diabetes facing additional ageassociated diabetes complications such as osteoporosis, falls/fractures, and cognitive dysfunction. Bone disease was first reported as an associated complication of diabetes nearly 100 years ago. The first published report, from 1927, noted osteopenia in hand X-rays of children with T1D. Nine years later, William Riely Jordon established the association between peripheral diabetic neuropathy and the chronic painless ankle and foot joint destruction known as Charcot foot (Box 23.2). Diabetes affects aspects of bone turnover, bone morphology, and bone mineral density (BMD) (Fig. 23.3). These changes ultimately result in increased fracture. As outlined below, diabetes affects bone health through multiple mechanisms including hyperglycemia, side effects from diabetes medications, and concurrent vascular complications. Although the most severe acute bone phenotypes were ameliorated with the advent of widely available insulin therapy in the 1920s, now with higher incidences of both types of diabetes and longer life expectancies for those affected, bone disease is emerging as a major health concern.

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© 2019 Elsevier Inc. All rights reserved.

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23.  SWEET BONES: DIABETES EFFECTS ON BONE

TABLE 23.1  Diabetes Overview Nondiabetic individuals

• Fasting plasma glucose ŽǁĂůĐŝƵŵ ĂŶĚsŝƚĂŵŝŶ

/ŶƐƵůŝŶ ƌĞƐŝƐƚĂŶĐĞ͕ ŚŝŐŚĞƌD/͕ ĂďĚŽŵŝŶĂů ĂĚŝƉŽƐŝƚLJ

/'&Ͳϭ

,ŝŐŚĞƌďŽŶĞŵŝŶĞƌĂůĚĞŶƐŝƚLJ ŚƌŽŶŝĐ ŝŶĨůĂŵŵĂƚŝŽŶ

/ŶĐƌĞĂƐĞĚĨĂůůƌŝƐŬ

>ŽǁĞƌďŽŶĞŵŝŶĞƌĂůĚĞŶƐŝƚLJ

%

KƌĂůŵĞĚŝĐĂƚŝŽŶƐ͕ ^'>dͲϮŝŶŚŝďŝƚŽƌƐ ĂŶĚWWZͲJ ĂŐŽŶŝƐƚƐ

/ŶĐƌĞĂƐĞĚĨƌĂĐƚƵƌĞƌŝƐŬ

,LJƉĞƌŐůLJĐĞŵŝĂ ,LJƉĞƌĐĂůĐŝƵƌŝĂ

ŚƌŽŶŝĐ ĚŝĂďĞƚĞƐ ĐŽŵƉůŝĐĂƚŝŽŶƐ

ZK^

' KƐƚĞŽĐLJƚĞĂŶĚ ŽƐƚĞŽďůĂƐƚ ĚLJƐĨƵŶĐƚŝŽŶ

ŚƌŽŶŝĐ ŝŶĨůĂŵŵĂƚŝŽŶ

>ŽǁďŽŶĞƚƵƌŶŽǀĞƌ >ŽǁďŽŶĞ ŵŝŶĞƌĂůĚĞŶƐŝƚLJ

ůƚĞƌĞĚďŽŶĞ ŐĞŽŵĞƚƌLJ ĂŶĚƐƚƌƵĐƚƵƌĞ

ůƚĞƌĞĚďŽŶĞ ŵĂƚĞƌŝĂů ƉƌŽƉĞƌƚŝĞƐ

/ŶĐƌĞĂƐĞĚ ĨĂůůƌŝƐŬ

/ŶĐƌĞĂƐĞĚĨƌĂĐƚƵƌĞƌŝƐŬ

FIGURE 23.3  (A) Potential mechanisms for diabetes-induced bone fragility. Type 1 diabetes (T1D) (left panel) is associated with higher peripheral tissue insulin levels and insulin resistance in the liver, leading to low insulin-like growth factor (IGF-1) levels. Persons with disease onset prior to completing linear growth can have smaller bones and abnormal bone structure. Those with celiac disease may have malabsorption with lower calcium absorption and poorer vitamin D stores. These factors all can lead to lower bone mineral density (BMD) in T1D. People with type 2 diabetes (T2D) (right panel) have hyperinsulinemia and obesity, leading to increased IGF-1 and increased BMD. Hyperglycemia is common to both T1D and T2D; decreased levels of amylin, GIP, and GLP-1 lead to lower low bone turnover. Chronic inflammation and use of SGLT-2 inhibitors and PPAR-γ agonists also increases fracture risk. In addition, hypoglycemia due to insulin treatment and/or diabetes therapies can increase the risk for falls leading to increased risk of fractures. (B) Effects of hyperglycemia on bone. Increased glucose excretion as a result of hyperglycemia causes increased calcium excretion due to osmotic diuresis which may be lead to reduced BMD. Excess glucose results in advanced glycation end product (AGE) accumulation. AGEs can change bone material properties. Reactive oxygen species (ROS) can produce osteocyte and osteoblast dysfunction leading to low bone turnover. ROS are also associated with chronic inflammation that can change bone geometry and structure. Diabetes complications, particularly peripheral neuropathy and retinopathy, not only change bone material properties and bone structure but also increase risk for falls. All these factors result in a net higher risk for fractures. Effects detrimental to bone health are indicated in red. GIP, glucose-dependent insulinotropic peptide; GLP-1, glucagon-like peptide; PPAR-γ, peroxisome proliferator-activated receptor gamma; ROS, reactive oxygen species; SGLT-2, sodium-glucose cotransporter 2.

risk 4.5; range 1.3–14.9) for any fracture in persons with T1D (Fig. 23.5A). Other studies have focused on risk of specific fracture types. Hip fractures are simpler to document than many other types of fracture as almost all hip fractures are symptomatic and require hospitalization for surgical

treatment. Hip fractures are also associated with high morbidity and mortality and therefore are very clinically important. Hip fractures in people without diabetes are more common in postmenopausal women than in similar-aged men, yet, interestingly, hip fracture risk in both men and women with T1D appears similar. Observed hip

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Fractures in Diabetes

(A)

(B)

Fracture Incidence Females

0

10 20 30 40 50 60 70 80 Age (years)

─────

T1D

─ ─ ─ ─

No Diabetes

Fracture Incidence Males

10 20 30 40 50 60 70 80 Age (years) ─────

T1D

─ ─ ─ ─

No Diabetes

FIGURE 23.4  Generalized representation of fracture incidence by age in females (A) and males (B) with type 1 diabetes (T1D, solid lines) compared with nondiabetic peers (dashed lines). For both women and men, the risk of fracture is higher for persons with T1D than peers with the increases becoming most evident around age 40 for females and age 50 in male. Adapted from Diabetes Care (2015) 38:1913–1920.

Any Fracture Hip Fracture

7 ─ 6 ─ 5 ─ 4 ─ 3 ─ 2 ─

(B) Spine Bone Mineral Density Z-score

Estimated Relative Risk of Fracture

(A)

+1 ─

-1 ─

1 ─ T1D

T2D

T1D

T2D

FIGURE 23.5  (A) Estimated relative risk of fracture in persons with diabetes compared with nondiabetic peers. People with type 1 diabetes (T1D) have a four- to fivefold greater risk of any fracture and a nearly sevenfold increased risk of hip fracture compared with peers. For persons with type 2 diabetes (T2D) the relative risk of fracture is greater compared with the general population with a ∼10% increased risk of any fracture and a ∼twofold increased risk of hip fracture in T2D. The risk of fracture in T2D is lower than in T1D. (B) Persons with T1D have lower bone mineral density (BMD) than nondiabetic peers (decrement at the spine of ∼0.2 SDS). Persons with T2D have higher BMD than nondiabetic peers. Higher BMDs in a person with T2D do not appear to have a protective effect on fracture risk. Data adapted from Osteoporos Int (2007) 18:427–444.

fracture rates compared with nondiabetic peers range widely with reported increases of 6- to 18-fold, depending on gender(s) and ascertainment method. A Danish National Database study reported the risk of hip fracture in men and women with T1D to be 70% higher than the general population. This greater risk appears to extend across the life span (starting around the age of 40) and persists as persons with T1D age. In general, hip fractures seem to occur about 10–15 years earlier in persons with T1D compared with those without diabetes, and people with diabetes have an approximately sevenfold higher risk of fracture than nondiabetic peers (Fig. 23.5A).

Unlike hip fractures, vertebral fractures are generally asymptomatic and undiagnosed unless assessed with imaging. Very few studies have reported risk for vertebral fractures in persons with T1D. Metaanalyses have shown a lower risk for vertebral fracture compared with hip fracture, which may be in part due to underreporting of vertebral fractures in many studies. A recent metaanalysis reported almost 2.88 times higher risk for vertebral fracture in subjects with T1D compared with controls. Of note, there also have been some case reports of persons with T1D sustaining multilevel vertebral compression fractures in association with nocturnal seizures.

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There are few data on fractures at sites other than hip and spine. A health improvement database network study from the United Kingdom showed a higher rate of lower extremity fractures in subjects with T1D, with risk consistently higher across all ages. Similarly, a recent study surveyed 756 adults with T1D from the Type 1 Diabetes Exchange clinic registry. Forty-eight percent of adults with T1D reported at least one fracture since their diagnosis. Of 659 reported fractures, 45% involved peripheral bones such as the metatarsals or metacarpals. These data suggest that fragility fractures of non-axial bones are common among people with T1D.

Fracture Risk in Type 2 Diabetes Most epidemiologic studies of people with T2D show an increased risk of fracture compared with the general population, although relative risks are lower than for those with T1D (Fig. 23.5A). Although absolute fracture rates are higher in women with and without T2D than men, most studies report higher relative risks for men with T2D than for women with T2D. This increased risk appears to start early during the disease, as T2D increases the likelihood of low-energy fracture for both middle-aged women and men (relative risks 1.87 and 2.38, respectively). Metaanalyses have generally reported relative risks of hip fracture between 1.2 and 2.8 for persons with T2D compared with controls. Very few studies report on vertebral or extremity fractures in persons with T2D, although a metaanalysis of 30 studies described positive associations between T2D and hip, vertebral, and foot fractures, but no association between T2D and wrist, proximal humerus, or ankle fractures. The relative risks for vertebral facture and any fracture were 1.16 and 1.05, respectively.

Fracture Morbidity and Mortality in Diabetes Fractures, particularly hip fractures, are frequently associated with complications that can lead to loss of independence, financial hardship, and increased risk of death. These risks appear to be even greater for persons with diabetes. Given the much higher prevalence of T2D compared with T1D, particularly in aging segments of the population, most of the epidemiologic data on morbidity and mortality after fracture in diabetes are from studies of persons with T2D. These findings do not necessarily extrapolate to persons with T1D. When compared with persons without diabetes, persons with diabetes have a 1.7-fold greater fracturerelated hospitalization risk. This risk increases with concomitant insulin therapy. People with diabetes also are at increased risk for post-fracture wound infections, other wound-related complications, and septicemia. One-year

survival rates after hip fracture are lower for older adults with T2D than those without diabetes (68% compared to 87%). Advanced age, male gender, uncontrolled diabetes, retinopathy, heart failure, glucocorticoid use, and postoperative complications are predictors of excess mortality after fracture with most observed deaths occurring within 2 years of fracture.

Fracture Healing in Diabetes Fracture repair involves recruitment of mesenchymal stem cells and then differentiation to chondrocytes that form cartilage (see Chapter 12). The transition from cartilage to bone requires angiogenesis. Fracture healing time in patients with diabetes is prolonged by 40%–80% with a three- to fourfold increase in risk for delayed union or nonunion. Data from animal studies suggest that diabetes-related delayed fracture healing is due to a variety of factors including reduced growth factors concentrations at the site of injury, microvascular disease, and advanced glycation end product (AGE) accumulation. Poor diabetes control and concomitant diabetes complications increase the risk of impaired healing. Amputation is another concern among patients with diabetes, particularly after ankle fractures. Persons with T2D have a four- to fivefold increased rate of amputation after ankle fracture compared with controls without diabetes.

SKELETAL CHANGES IN DIABETES Persons with diabetes are subject to the same general determinants of bone health as persons without diabetes. These include nonmodifiable risk factors such as genetics, age, gender, and race as well as controllable lifestyle factors such as diet, physical activity, and smoking (see Chapter 21). Additional diabetes-specific determinants include age at diagnosis (higher rates of fracture with younger age at diagnosis) and microvascular complications (higher fracture rates with prevalent complications) (Fig. 23.8). Sensory neuropathy, visual impairments, and hypoglycemia increase fall risk and thereby increase fracture risk. Additionally, chronic kidney disease (see Chapter 20) and neuropathy likely affect regional bone mineralization and quality, increasing fragility. Both insulin deficiency (particularly for persons with T1D) and resistance (particularly for persons with T2D) also have adverse effects on bone health (Fig. 23.3). Relative insulin deficiency and lower insulin-like growth factor 1 (IGF-1) concentrations may affect bone accrual in children and lead to bone loss in older persons with diabetes.

VI.  SKELETAL DISEASE AND TREATMENT

Skeletal Changes in Diabetes

Falls are a major risk factor for fracture. Persons with diabetes may have episodes of severe hypoglycemia (with loss of consciousness and/or seizure), diabetic neuropathy, poor vision due to retinopathy, and postural hypotension, all of which predispose to falls. Some of the adjunctive medications used commonly as therapy for people with diabetes, such as proton pump inhibitors to reduce stomach inflammation, diuretics (particularly angiotensin converting enzyme inhibitors), and psychotropic medications can also increase fall risk. Given the observed increases in fracture, many investigators have studied the skeletal properties of persons with diabetes. Bone density by dual-energy X-ray absorptiometry (DXA, see Chapter 6) is the most commonly reported measure, although many studies have done macroscale geometric bone measurements, including assessments of cross-sectional area and cortical thickness. The most recent reports incorporate threedimensional imaging techniques including quantitative computed tomography (QCT) of axial bone and peripheral bone (pQCT), micro–magnetic resonance imaging (micro-MRI), and high-resolution pQCT (HR-pQCT) to assess effects of diabetes on three-dimensional structures of cortical and trabecular bone.

Bone Mineral Density in Type 1 Diabetes Studies to date suggest that some (but not all) of the increased fracture risk observed in persons with T1D is attributable to moderate decreases in bone density compared with controls. Bone density decreases were first reported during the 1970s and 80s using X-ray and single photon absorptiometry assessments. Initially it was believed that bone mineral loss begins with the onset of clinical diabetes. However, subsequent studies using more sensitive absorptiometry methods, including DXA, confirmed lower BMD from even the time of diagnosis of T1D in adults suggesting that BMD and/or bone mineral content (BMC) is affected earlier at the clinical onset of diabetes. Most, but not all pediatric studies, have reported lower BMD and/or BMC in children and adolescents with T1D compared with controls. Lower BMC is associated with lower IGF-1 concentrations and higher exogenous-administered insulin dose (suggestive of insulin resistance) regardless of age and sex. In contrast to BMD studies in pediatric populations, studies in adults with T1D report only modest or no difference in BMD at lumbar spine and femoral neck compared with nondiabetic peers, suggesting that over time the deficit observed during childhood/adolescence is entirely or almost entirely overcome. Some data suggest a decrement in spine z-score of −0.2 compared with controls (Fig. 23.5B). A few small longitudinal studies have also reported normalization of BMD or bone size over time in persons with established

431

T1D. However, given the observed increases in fracture compared with peers, bone accrual and maintenance in persons diagnosed with T1D warrant further study.

Bone Mineral Density in Type 2 Diabetes The effect of T2D on BMD is different from that of T1D. Most studies from Western countries report higher axial BMD in persons with T2D compared with population controls without diabetes. Studies showed higher BMD at the spine, with z-scores on average 0.4 SD higher than controls (Fig. 23.5B). Increases are also observed at the hip. The higher BMD observed in T2D is similar in men and women and across racial and ethnic groups including Mexican American, White, and Black. Of note, studies from East Asian countries, where there is a lower prevalence and magnitude of obesity in persons with T2D, have revealed more inconsistent results, reporting similar or higher BMD at the hip and/or spine in those with T2D. As in nondiabetic populations, BMD is greater in those with higher body mass index (BMI), suggesting that the higher body weight is in part responsible for the higher BMD. A few studies have shown higher BMD in T2D even after adjusting for BMI. This suggests that additional factors such as hyperinsulinemia and higher circulating IGF-1 concentrations contribute to the observed increase in BMD.

Bone Geometry and Architecture in Type 1 Diabetes The structure of bone in diabetes is affected at all levels (Fig. 23.6). Although early studies suggested that children and adolescents with T1D have smaller distal radial and tibial cross-sectional area and that the bones fail to grow appropriately over 1 year, subsequent studies with longer follow-up demonstrated that bone size normalized over a subsequent 5 years of treatment, suggesting that the impairment in bone growth is transient in childhood-onset T1D. QCT data also suggest cortical bone deficits in adults with T1D. pQCT data suggest that in adult men with T1D, the distal radius cortex is thinned but that this is not due to overall smaller bone size, as these men have larger overall cross-sectional area and larger trabecular cross-sectional area compared with controls. At the intertrochanteric proximal femur, adult men have lower cortical thickness and cortical cross-sectional area. The femoral neck cortex is thinner, and the femoral diaphysis has a smaller outer perimeter and lower cross-sectional cortical area. HR-pQCT studies do not suggest large differences in trabecular microarchitecture at the distal radius or tibia between persons with T1D and controls. However,

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23.  SWEET BONES: DIABETES EFFECTS ON BONE

Effects of diabetes at different levels of bone organization

•

Modest BMD reduction, altered geometry (smaller cross-sectional area) in T1D • Increased BMD in T2D

• •

Cortical porosity Altered trabecular architecture

• Non enzymatic glycation of collagen • Alteration in mineral: matrix ratio

• Low bone turnover • Fewer osteoblasts, dysfunctional osteocytes

FIGURE 23.6  Effect of diabetes at different levels of bone organization. At an organ/tissue macroscale, bone mineral density is lower with smaller cross-sectional area in persons with type 1 diabetes (T1D) and higher in type 2 diabetes (T2D). At the microstructural level, using highresolution quantitative computed tomography, studies have reported reduced volumetric bone density and increased cortical porosity, increased trabecular separation, and thinning among patients with diabetes compared with peers. At nanoscale, diabetes affects bone tissue due to glycation of collagenous and noncollagenous proteins resulting in compromised tissue material properties. At the cellular level, hyperglycemia results in low bone turnover due to dysfunctional osteoblasts, osteocytes, and osteoclasts. Additionally, due to low bone turnover, microcracks accumulate causing brittle bone.

persons with T1D and confirmed microvascular disease tend to have thinner trabeculae and lower trabecular number. Yet, using microfinite element analysis, neither estimated radial bone strength nor stiffness is compromised. Using micro-MRI, women with T1D have lower trabecular number per unit volume (Tb.N/BV) and higher trabecular spacing in the proximal tibia, suggesting compromised trabecular architecture. Overall, these studies suggest that T1D affects cortical bone structure with more minor changes to the trabecular compartment. QCT (pQCT, HR-pQCT) and micro-MRI assessments permit precise characterization of 3D cortical and trabecular microarchitecture (Fig. 23.7) but are not used clinically for fracture prediction due to prohibitive costs and (for QCT measures) relatively high radiation exposure depending on site assessed. An alternate way to estimate fracture risk is to calculate a trabecular bone score (TBS). TBS is a two-dimensional textural index of pixel gray-level variations in lumbar spine DXA images, providing an indirect index of trabecular microarchitecture. TBS (TBS insight) has distinct advantages clinically, in that it integrates with existing DXA scanners, requires no additional scans or radiation exposure, and allows for retrospective analysis of

past DXA. Though TBS does not provide three-dimensional trabecular architecture information, ex vivo and in vivo studies have shown good correlations between TBS and trabecular bone volume to tissue volume ratio, number of trabeculae, and their connectivity and negative correlations with the space between trabeculae. TBS improves fracture risk prediction in adjunct to BMD and it has been incorporated into Fracture Risk Assessment Tool (FRAX). Two studies have used TBS to evaluate trabecular architecture in adults with T1D. One found no overall TBS differences between persons with T1D and controls, although TBS was lower in persons with T1D and history of fracture. A second study showed lower TBS compared with controls (1.42 ± 0.12 vs. 1.44 ± 0.08, P = .02) after adjusting for age, sex, smoking, and lumbar spine BMD. However, TBS was within the normal range in both groups. In addition, insulin resistance and other metabolic syndrome components such as diastolic blood pressure, BMI, and triglycerides were inversely associated with TBS. Interestingly, in both studies, men with T1D had lower TBS compared with women with T1D. The utility of TBS in fracture prediction is unknown in T1D.

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433

Mineral Homeostasis and Bone Turnover in Diabetes

(A)

(B)

(C)

1 mm

1 mm

(D)

(E)

(F)

(G)

(H)

(I)

FIGURE 23.7  High-resolution peripheral quantitative computed tomography images of ultradistal radius. Representative of median cortical porosity in (A–C) a nondiabetic person, (D–F) a person with type 2 diabetes (T2D) without microvascular complications, and (G–I) a person with T2D and microvascular disease. Images are of (A, D, G) midstack tomograms and three-dimensional visualization of (B, E, H) trabecular and (C, F, I) cortical bone (gray) and cortical porosity (green). Reproduced with permission from Shanbhogue VV, Hansen S, Frost M, Brixen K, Hermann AP. Bone disease in diabetes: another manifestation of microvascular disease? Lancet Diabetes Endocrinol. 2017;5:827–838.

Bone Geometry and Architecture in Type 2 Diabetes Most studies of bone geometry/architecture in T2D have been performed in postmenopausal women using HR-pQCT. These have generally reported significantly compromised cortical microarchitecture (i.e., higher cortical porosity) at the distal radius and tibia compared with nondiabetic controls. Similar differences are seen in males and of persons from different ethnic groups. Distal radius and tibial trabecular microarchitecture are generally normal, although with greater heterogeneity than seen in nondiabetic populations. The observed changes are more evident in persons with T2D who have had fracture, require insulin therapy, and/or have microvascular complications (Fig. 23.8). Some T2D studies have evaluated the utility of TBS for fracture prediction. A retrospective study of 29,407

women with T2D >50 years of age reported lower lumbar spine TBS despite higher BMD and found TBS to be a strong BMD-independent predictor of fracture in women. Similar data exist for men with T2D. It has been reported that TBS is inversely associated with HbA1c, fasting plasma glucose, fasting insulin, and insulin resistance even after adjusting for age and BMI. These data suggest that TBS is a useful fracture prediction tool in T2D.

MINERAL HOMEOSTASIS AND BONE TURNOVER IN DIABETES Diabetic effects on mineral homeostasis are modest. Hyperglycemia can promote hypercalciuria (Fig. 23.3B) but does not seem to have a significant effect on overall calcium homeostasis. Studies have consistently reported no significant differences in circulating concentrations of calcium,

VI.  SKELETAL DISEASE AND TREATMENT

434 (A)

23.  SWEET BONES: DIABETES EFFECTS ON BONE

Onset of diabetes

120

Onset of MVD

Healthy Tb Healthy Ct MVD-Tb

Annual change (%)

100

MVD-Ct MVD+Tb MVD+Ct

80 60 40 20 0

(B)

Insulin Onset of resistance MVD

120

Annual change (%)

100 80 60 40 20 0

0

10

20

30

40 Age

50

60

70

80

FIGURE 23.8  Model of effects of diabetes on bone throughout life, displayed as bone mass in the trabecular and cortical compartments, as a function of age. In healthy individuals, trabecular bone accumulates until approximately age 20–25 years, followed by a plateau until midlife and minimal loss thereafter. Cortical bone accretion continues until age 40–45 years, followed by bone loss that is initially slow and accelerates with progressive aging. (A) Model of type 1 diabetes (T1D). In T1D, significant bone loss might be present at the time of diagnosis and is relatively rapid in the first few years of clinical diabetes, declining gradually or normalizing thereafter to a steady-state value with increasing disease duration. Additional insults, such as the onset of microvascular disease, either at a young age before the accretion of peak bone mass or at a time when the initial bone loss has not yet stabilized, could present as late-onset bone loss in both the trabecular and cortical compartments. (B) Model of type 2 diabetes. Insulin resistance before and after the diagnosis of diabetes results in superior trabecular and cortical bone mass compared with nondiabetic peers. Additional insults, such as the onset of microvascular disease (MVD), exaggerate typical age-related bone loss most evidently in the cortical compartment. Healthy Tb = trabecular compartment of healthy individuals. Healthy Ct = cortical compartment of healthy individuals. MVD − Tb = trabecular compartment of patients with diabetes without microvascular disease. MVD − Ct = cortical compartment of patients with diabetes without microvascular disease. MVD + Tb = trabecular compartment of patients with diabetes and microvascular disease. MVD + Ct = cortical compartment of patients with diabetes and microvascular disease. Reproduced with permission from Shanbhogue VV, Hansen S, Frost M, Brixen K, Hermann AP. Bone disease in diabetes: another manifestation of microvascular disease? Lancet Diabetes Endocrinol. 2017;5:827–838.

phosphorus, and parathyroid hormone (PTH) between persons with T1D and controls. Serum 25-hydroxyvitamin D concentrations are lower in T1D than in controls, although the effect of this on bone outcomes is uncertain. Diabetes is also commonly associated with extracellular and/or intracellular magnesium deficiency. Magnesium deficiency can affect bone metabolism, particularly PTH secretion and activity, and has been linked to osteoporosis in animal models and human epidemiologic studies. However, not a conclusive link between magnesium deficiency and compromised bone health in diabetes has been established. T1D and T2D are generally characterized as low bone turnover diseases with lower bone formation and bone resorption rates than is observed in individuals without diabetes. Of note, these data primarily come from measurements of circulating bone turnover markers, as there are very few studies using bone histomorphometry in humans with diabetes, and those that are available have inconsistent results. There is also very little known about effects of overall glycemic control, duration of diabetes, and diabetes complications on bone turnover. Although alkaline phosphatase does not seem to vary between persons with diabetes and controls, the bone formation marker procollagen 1 intact n-terminal propeptide (P1NP) is decreased (to a lesser extent in T1D than T2D). Additionally, the bone resorption markers c-terminal telopeptide crosslinks (CTX) and n-terminal telopeptide (NTX) are lower in persons with T1D and T2D compared with nondiabetic controls. Tartrate-resistant acid phosphatase (TRAP) is also lower in T2D compared with controls. Circulating sclerostin concentrations, a potent inhibitor of bone formation and to a lesser extent bone resorption, are higher in persons with diabetes (more so in T2D than T1D) and may be responsible for the low bone turnover in patients with diabetes. Osteoprotegerin, which inhibits bone resorption, is also greater in persons with diabetes compared with controls. Low bone turnover results in an accumulation of aged bone mineral and increases the likelihood of change in bone mineral composition resulting in bones that are more prone to fracture. In postmenopausal women without diabetes, bone turnover markers are associated with increased fracture risk. However, there are no reports evaluating the predictive value of bone turnover markers on fracture risk in patients with diabetes.

OTHER HORMONAL/CELLULAR LEVEL CHANGES IN DIABETES Osteocalcin Osteocalcin (OC) is produced by osteoblasts and osteocytes and interacts with hydroxyapatite. Sympathetic

VI.  SKELETAL DISEASE AND TREATMENT

Bone Tissue Material Properties

nervous system signaling may increase its secretion (see Chapter 18). It is used as a marker of bone formation, although it fluctuates with food intake and therefore needs to be consistently measured in the same fasting/ fed state. OC can be fully ɣ-carboxylated at three glutamate terminals or undercarboxylated (uOC) if fewer than three terminals are carboxylated. Both OC and uOC have been associated with β cell function and insulin sensitivity, primarily based on T2D mouse data. It is not clear that this same pathway is as important in humans. In murine models, OC absence increases bone mass and peripheral fat and is associated with β cell deficiency, hypoinsulinemia, and hyperglycemia. In humans greater OC may be associated with improved glucose tolerance and insulin secretion. OC injections improve blood glucose control and protect against T2D. uOC may stimulate adiponectin secretion from adipocytes (see below). Circulating OC and uOC concentrations are lower in persons with T1D and T2D compared with controls, although the observed decreases are more modest in T2D. Overall OC appears to be negatively correlated to HbA1c.

Adipocytokines Adipose tissue secretes many hormones, collectively called adipocytokines. These hormones regulate important biological processes such as appetite, fat distribution, insulin sensitivity, energy balance, and inflammation. Although the full set of human adipocytokines is still not entirely characterized, it has become clear that adipose tissue is a source of more than 600 potentially secretory proteins. Several of these adipocytokines, including leptin, adiponectin, fibroblast growth factor 21 (FGF21), inflammatory markers such as TNF-alpha and IL-6, and bone morphogenic proteins, are involved in bone metabolism and glucose homeostasis. Though animal and human studies suggest links between bone metabolism and diabetes, the role of adipocytokines in bone metabolism in patients with diabetes is unclear. A few suggestive associations are outlined below. Studies have shown low circulating serum leptin concentrations in T1D and near-normal leptin levels in T2D compared with people without diabetes. Leptin’s effect on bone mass is twofold and complex. Through the central pathway, leptin appears to increase bone loss while through the peripheral pathway, leptin seems to increase bone formation. Most studies show either weak or no association between leptin levels and bone mineral changes in patients with T1D or T2D. Adiponectin is known to improve insulin sensitivity and reduce inflammation. It stimulates bone turnover, by stimulating both osteoblast and osteoclast induction. Overall higher adiponectin levels are associated with

435

worse bone phenotypes. Most, but not all, clinical studies have shown that serum adiponectin is negatively associated with BMD and positively with biochemical markers of bone turnover. Persons with T1D and T2D have higher levels of inflammatory markers such as TNF-alpha and IL-6. Studies have shown an inverse association of TNF-alpha and IL-6 with BMD in patients with diabetes. Many diabetes treatments, including insulin, are also associated with changes in body weight. As body weight is a major determinant of BMD, these treatments may also confound any observed associations between adipocytokines and bone metabolism in this population.

Oxidative Stress Mitochondrial production of reactive oxygen species (ROS) is high in persons with diabetes, with higher levels in persons with greater hyperglycemia. High circulating ROS overwhelm cellular antioxidant capacity. This results in oxidative tissue damage, which is a mechanism for the development of complications in diabetes. This tissue damage may be accentuated by acute blood sugar fluctuations. Oxidative stress decreases bone formation by decreasing osteoblast function. It does this by inhibiting osteoblast differentiation, increasing osteoblast apoptosis, and antagonizing osteoblastogenesis by decreasing Wnt-pathway signaling. Oxidative stress also stimulates osteoclast formation and enhances osteoclast activity.

BONE TISSUE MATERIAL PROPERTIES As diabetes-associated fracture risks are greater than expected based on observed BMD and bone morphology, deficits in bone quality are assumed to exist in persons with diabetes. Bone quality is a broad term that includes structural characteristics such as bone geometry and microstructure and bone tissue material composition and properties (see Chapter 1). Impaired bone material properties can arise from altered mineral to matrix ratios, changes in mineral composition, and/or compromised collagen quality. The effect of diabetes on bone tissue composition and material properties is generally unknown. Some animal studies and a few human studies have reported advanced glycation end product (AGE) accumulation (see below) and altered collagen maturation in the context of diabetes. Noncollagenous protein expression may also be changed in diabetes and influence bone fragility. Understanding bone tissue level changes requires bone biopsy, which is invasive, costly, and time-consuming.

VI.  SKELETAL DISEASE AND TREATMENT

436

23.  SWEET BONES: DIABETES EFFECTS ON BONE

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FIGURE 23.9  Advanced glycation end product (AGE) accumulation in collagen. Hyperglycemia, carbonyl stress, and oxidative stress induce excess AGE formation. The first step for forming AGEs is the nonenzymatic glycation, oxidation, or glycoxidation between helical amino groups and sugar to form a Schiff base. The compound is then stabilized by spontaneous Amadori rearrangement. The Amadori adduct undergoes further reactions to form permanent AGEs cross-links, such as pentosidine. AGE accumulation on collagen reduces bone quality, strength, and postyield properties. Additionally, AGEs interact with the receptor for AGEs (RAGE) expressed by bone cells, inhibiting their functionality and decreasing bone turnover. This induces an even greater accumulation of AGEs in bone with development of diabetic osteopathy, thus increasing fracture risk. Reproduced with permission from Merlotti D, Gennari L, Dotta F, Lauro D, Nuti R. Mechanisms of impaired bone strength in type 1 and 2 diabetes. Nutr. Metab. Cardiovasc. Dis. 2010;20:683; AGEs and bone ageing in diabetes mellitus. J. Diabetes Metab. July 2013;4(6).

A recently developed microindentation tool (commercially known as an OsteoProbe™) can be used to measure bone material strength (known as a bone material strength index [BMSi]) in vivo. BMSi by microindentation is reported to be lower in T2D suggesting deterioration of tissue material properties as a possible reason for increased fracture risk in this population. Bone material properties are regulated by factors, including bone turnover, glycation, and oxidative stress as outlined below.

Bone Collagen Glycation Advanced glycosylation end products (AGEs) are compounds generated from Amadori compounds through nonenzymatic Maillard condensation reactions between reducing sugars and amino acids of proteins, lipids, and amino acids (Fig. 23.9A and B). Carboxymethyl-lysine and pentosidine are well-recognized AGEs. Higher glucose concentrations increase the likelihood of AGE formation. AGEs accumulate in the organic matrix of bone in part because bone-related proteins, such as type 1 collagen, have a long half-life and therefore have many opportunities to be glycated. While enzymatic cross-linking improves bone strength, AGEs increase nonenzymatic collagen crosslinking across and within fibers (see Chapter 1) which

decreases bone strength. AGE accumulation is associated with changes in micro- and macroscopic bone mechanical indices that reduce energy absorption and bone toughness. Higher levels of serum and urine pentosidine concentrations increase the likelihood of fracture. AGEs also act as agonists for receptors for AGEs (RAGEs), found on cell surfaces. Greater RAGE expression is related to the progression of diabetes-related complications. Increases in RAGE expression are associated with inhibited osteoblast differentiation and may increase osteoblast apoptosis.

MANAGEMENT AND TREATMENT APPROACHES Effects of Diabetes Management on Bone Health Some of the commonly used therapies for diabetes management and their potential impacts on bone health are outlined below (Table 23.2). Insulin Animal studies demonstrate anabolic effects of insulin on bone health. However, in persons with T2D insulin

VI.  SKELETAL DISEASE AND TREATMENT

Management and Treatment Approaches

TABLE 23.2  Effect on Bone and Fracture Risk With Commonly Used Antihyperglycemic Treatment for Type 2 Diabetes Antihyperglycemic Drugs

Effect on Bone

Fracture Risk with Use

Insulin

Anabolic

↑

Metformin

±

↔

Sulfonylureas

±

↔

Thiazolidinediones

++

↑↑

DPP-4 inhibitors

+

↔

GLP-1R analogs

+

↔

SGLT-2 inhibitors

−

↑a

Amylin analogs

±

NA

Bromocriptine, alpha-glucosidase inhibitors, and meglitinides

NA

NA

NA, no information available. aFracture risk is increased with canagliflozin. Limited information is available on fracture risk with other SGLT-2i.

treatment is associated with an increase in fracture risk. This may be due to insulin being used in T2D generally in persons with longer-standing, more severe disease, many of whom have diabetes complications. Insulin-treated persons with T2D are also at an increased risk for hypoglycemia and falls. Persons with T1D are uniformly treated with insulin, so dissecting out the effect of insulin therapies on bone in this population clinically is not readily feasible. Metformin Metformin is a biguanide insulin sensitizer. It serves as a first-line therapy for most persons with T2D and is, rarely, used as adjunctive therapy in persons with T1D. It works by inducing AMP-activated protein kinase (AMPK). AMPK stimulation promotes osteoblast differentiation and mineralization in cell culture. It also increases bone marrow progenitor cell induction toward osteogenic lines and may protect osteoblasts from adverse effects of hyperglycemia. It enhances fracture repair in rats and prevents bone loss in the ovariectomized rat. Clinical studies suggest that metformin has a modest positive or neutral effect on bone turnover, BMD, and fracture risk in persons with T2D. Sulfonylureas Sulfonylureas are also commonly prescribed for persons with T2D. They work by stimulating pancreatic β cells to release insulin. In bone they activate PI3-K/Akt pathways and may stimulate osteoblast proliferation and differentiation. They also may increase alkaline phosphatase and OC. There are limited clinical data on their effects on bone health. Some studies have found that sulfonylurea use is associated with significant reductions in risk for any fracture, whereas others have not found

437

this benefit. Overall, the effects of sulfonylureas on bone metabolism are neutral to positive. Thiazolidinedione The thiazolidinedione (TZD) class of drugs includes the drugs rosiglitazone and pioglitazone. TZDs work as treatments for T2D by acting on the nuclear hormone receptor peroxisome proliferator-activated receptor gamma to improve insulin sensitivity, particularly in adipocytes. In vitro, TZDs also influence the lineage allocations of hematopoietic and mesenchymal cells in the bone marrow (Fig. 23.10). They decrease differentiation to osteoblasts and increase differentiation to adipocytes, resulting in fat accumulation in the bone marrow. They also decrease circulating insulin, decrease aromatase activity, and decrease circulating leptin. These three effects work to decrease osteoblast function and increase osteoclast differentiation. The result is unbalanced bone remodeling with low bone formation and high bone resorption. In clinical studies, TZDs have been found to reduce BMD and increase fractures in women with T2D. The fracture increases are substantial, with one study estimating that TZD use in high risk women causes one fracture for every 21 women who complete 1 year of therapy. The risk of fracture appears to increase with the duration of therapy. Observational studies suggest that fracture risk with TZD use also increases for males. Incretin-Based Treatments Food ingestion increases level of gut hormones, mainly of glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide 1 (GLP-1), which increases the insulin secretion from the pancreatic β cells, delays gastric emptying, and suppresses the hunger and thus contributing to glucose homeostasis. For persons with T2D, decreased secretion of and resistance to the effects of GIP and GLP-1 contributes to hyperglycemia. Both GIP and GLP-1 are rapidly inactivated by the serine protease dipeptidyl peptidase 4 (DPP-4). Both GLP-1 agonists and DPP-4 inhibitors are used therapeutically for treatment of T2D. Incretin hormones (particularly GIP) are involved in the linking of nutrient ingestion to bone formation and suppression of bone resorption. GIP receptors (GIPR) are found in osteoblast and osteocytes and modulate an anabolic bone response in vitro. GIPR −/− knockout mice have low bone formation, high bone resorption, and decreased BMD compared with GIPR +/+ controls. Additionally, in animal experiments, GLP-1 analogs are reported to improve bone formation. Recent human data suggest that shortterm infusions of GIP can work to decrease bone resorption independent of their effects on insulin. GLP-1 Receptor Agonists There are several GLP-1 receptor agonists marketed for treatment of T2D. These include exenatide, a

VI.  SKELETAL DISEASE AND TREATMENT

438

23.  SWEET BONES: DIABETES EFFECTS ON BONE

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FIGURE 23.10  The mechanisms of thiazolidinedione (TZD)-induced bone fragility. The figure illustrates the effect of type 2 diabetes on allocation of mesenchymal cells to adipocyte (green line) instead of osteoblast lineage (red line) resulting in reduced number of osteoblastic cells. In addition, reduction in amylin levels, estrogen due to reduced aromatase activity, and leptin levels increases the osteoclast cells. Thus, there is an increase in bone resorption and decrease in bone formation resulting in reduced BMD and increased fracture risk.

molecule that is highly homologous to GLP-1 but resistant to DPP-4 degradation. Additional compounds have slower absorption and longer duration of action (albiglutide, dulaglutide, liraglutide). Studies assessing bone outcomes using these compounds in persons with T2D showed modest positive to no effect on bone turnover, BMD, or fracture risk. In a recent metaanalysis of 16 randomized control trials of GLP-1 analogs, liraglutide was associated with significant reduction in fractures while exenatide was associated with increase in risk for fractures. Analogous different off-target effects of different GLP-1 analogs have been reported on cardiovascular outcomes. DPP-4 Inhibitors DPP-4 inhibitors are given orally to decrease blood sugars in persons with T2D. As outlined above, they work by slowing down the degradation of GLP-1 and GIP and decreasing glucagon release. Clinical studies have shown increased bone formation markers with DPP-4 inhibitors and reduction (or neutral effect) on bone resorption markers. Whether DPP-4 inhibitors have any effect on BMD and fracture risk in T2D has also been examined, although most of these studies have been of short duration and included fracture as a secondary

rather than primary endpoint, which sometimes leads to underreporting of total fracture numbers. Overall, the studies suggest that DPP-4 inhibitors may be protective for bone health. Sodium-Glucose Cotransporter 2 Inhibitors Sodium-glucose cotransporter 2 (SGLT-2) inhibitors (canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin) are newer agents for treatment of T2D and are starting to be used for some persons with T1D. They lower blood sugar by inhibiting the proximal tubular reabsorption of glucose. SGLT-2 inhibitors also have several effects that are potentially detrimental to bone. They increase circulating serum phosphate concentrations by promoting renal tubule phosphate reabsorption, thereby increasing PTH and FGF23 concentrations (Fig. 23.11). These effects may increase bone resorption. They have diuretic effects that can promote postural hypotension and increase fall risk. SGLT-2 inhibition also can result in hyponatremia. Decreased serum sodium may increase oxidative stress and increase bone resorption. Clinical studies of canagliflozin have demonstrated adverse effects on bone, with reports indicating an increased fracture rate as early as 12 weeks after therapy

VI.  SKELETAL DISEASE AND TREATMENT

Management and Treatment Approaches

439

FIGURE 23.11  Possible mechanisms whereby SGLT-2 inhibitors exert adverse effects on bone. (A) SGLT-2 inhibitors increase serum phosphate levels, probably by promoting renal tubular phosphate reabsorption. Because of the action of SGLT-2 inhibitors to decrease Na+ transport, this increases the electrochemical gradient for Na+, thereby driving increased cotransport of phosphate and Na+. (B) Increased serum phosphate levels increase parathyroid hormone (PTH) secretion. Either directly or indirectly (e.g., mediated by effects of PTH), the increased serum phosphate has the potential to increase FGF23 secretion by osteocytes. Both PTH and FGF23 promote phosphaturia by decreasing renal tubular phosphate reabsorption of phosphate. Note that they exert opposite effects upon 1α-hydroxylation of 25-hydroxyvitamin D with PTH increasing and FGF23 decreasing 1,25-dihydroxyvitamin D formation. FGF-23, fibroblast growth factor 23, SGLT-2, sodium-glucose cotransporter 2. Reproduced with permission from Taylor SI, Blau JE, Rother KI. SGLT2-inhibitors trigger downstream mechanisms that may exert adverse effects upon bone. Lancet Diabetes Endocrinol. January 2015;3(1):8–10.

initiation. Subsequent studies using dapagliflozin and empagliflozin generally have not shown these effects. In a multicenter, randomized control clinical trial, dapagliflozin had no effect on markers of bone formation and resorption or BMD after 50 weeks of treatment in persons with T2D. Further data are needed to determine the effects of SGLT-2 inhibitors on bone health and whether certain populations (adolescents, postmenopausal women) may be at higher risk of adverse bone effects with prolonged use. Amylin Analogs Amylin is a hormone that is co-secreted with insulin from pancreatic β cells. It has peripheral and central

effects on glucose metabolism and food intake. It is deficient in T1D and relatively deficient in insulin-requiring T2D. In vitro, physiologic amylin concentrations stimulate osteoblast proliferation. Amylin also inhibits osteoclast development and function. In vivo mouse data suggest that amylin administration increases bone formation and decreases bone resorption. Very limited clinical trial data do not suggest that amylin therapy has a clear impact on bone metabolism or bone density in persons with T1D. We are not aware of any clinical trials of amylin that have assessed bone metabolism or other bone outcomes in persons with T2D. Information on the effect of certain medications used in treatment of T2D such as alpha-glucosidase inhibitors

VI.  SKELETAL DISEASE AND TREATMENT

440

23.  SWEET BONES: DIABETES EFFECTS ON BONE

(acarbose, miglitol, and voglibose), bromocriptine (Cycloset), and meglitinides (repaglinide) on bone health is currently unknown. Bariatric Surgery Bariatric surgery is recommended in patients with T2D who are morbidly obese (BMI  >  40 kg/m2) to improve glycemic control and reduce comorbidities associated with obesity. Studies have reported declines in BMD as early as within the first year of metabolic surgery among patients with T2D. There is a strong association between bone loss and weight loss after bariatric surgery. Potential mechanisms of bone loss after bariatric surgeries are mechanical unloading due to rapid weight loss, reduced calcium and vitamin D absorption, and increase in bone resorption.

Bone Health Management in Diabetes The cornerstones of optimization and management of bone health in persons with diabetes begin with basic nutritional and lifestyle interventions that have been proven to benefit bone health. These include adequate dietary calcium, optimizing vitamin D stores through nutrition and potentially sensible sun exposure. Because high glucose concentrations increase urinary calcium excretion, it is particularly important that persons with diabetes get adequate dietary calcium. Persons with diabetes should also limit alcohol and avoid smoking. It is also important that persons with diabetes engage in adequate regular weight-bearing physical activity and decrease sedentary time. This is important not only for bone health but also for improving insulin sensitivity, muscle strength, and overall cardiovascular fitness. Physical activity generally is accompanied by the need for additional dietary carbohydrate intake and/or decreases in administered insulin. Frequent monitoring of blood sugar is often necessary to ensure that exercise is done safely. Because persons with diabetes are at increased risk of fracture with falls, therapeutic strategies that minimize the risk of severe hypoglycemia, balance training, avoidance/cautious use of psychotropic medication, and regular visual assessments are important parts of care that may reduce risk of fracture. Persons with T1D are at increased risk of celiac disease compared with the general population. As untreated celiac disease has adverse effects on bone health, regular serologic screening should be done in this population, with institution of a gluten-free diet for those who are confirmed by intestinal histology to have active disease.

Standards of care for persons with diabetes suggest that fracture histories should be taken in middle-aged and older adults with diabetes and bone density measured as appropriate. Despite studies suggesting low bone turnover in persons with T1D and T2D, both registry data and data from clinical trials of osteoporosis medications support the effectiveness of antiresorptive bisphosphonate therapy in increasing BMD in persons with diabetes. Alendronate increased bone density in 297 women with diabetes enrolled in the Fracture Intervention Trial (FIT) equivalently to normoglycemic individuals. Raloxifene reduced vertebral fracture risk by 35% in the Raloxifene Use for the Heart (RUTH) trial, with consistent effects among subgroups, including the approximately 4500 women with diabetes. However, examination of osteoporosis medication use and fractures in the Danish Registry demonstrated no difference in fracture rates during treatment with bisphosphonates or raloxifene between individuals with T1D or T2D and normoglycemic control subjects. The risk for atypical femoral fracture is higher among postmenopausal women with diabetes compared with women without diabetes. Therefore, longer duration bisphosphonate therapy should be prescribed with caution for persons with diabetes. There are only case reports available for other therapies such as strontium or teriparatide. There are also no randomized control trials comparing the efficacy and safety of antiresorptive or anabolic agents on BMD or fracture outcomes in populations of persons with T1D or T2D. Studies have also examined whether osteoporosis medications might have effects on glucose metabolism. Analyses of persons treated with bisphosphonates and denosumab in randomized fracture trials have not shown a significant effect of osteoporosis medications on glucose levels or diabetes incidence. Observational data among a small number of individuals treated with teriparatide show similar findings.

CONCLUSIONS Both T1D and T2D have effects on bone health. The pathophysiology of these effects is complex, but the end result is increased rates of fracture, which are not entirely reflected in changes in BMD and amplified by concurrent vascular complications. The best practices for fracture prevention are to optimize glycemic control, to reduce the risk of hypoglycemia, and to employ strategies similar to those used for others at high risk of fracture, including insuring adequate calcium and vitamin D intake and working to prevent falls.

VI.  SKELETAL DISEASE AND TREATMENT

Suggested Readings

STUDY QUESTIONS   

1. D  escribe the difference in pathophysiology of T1D and T2D. 2. Describe the effects of hyperglycemia on bone accrual, BMD, and fragility fracture. 3. Explain how various diabetes medications affect bone. 4. Describe how diabetes-related factors, such as duration and vascular disturbances, and factors not related to diabetes, such as age and sex, affect fracture risk in diabetes. 5. What is Charcot foot? Describe the pathophysiology.   

441

Suggested Readings 1. DiMeglio LA, Evans-Molina C, Oram RA. Seminar: type 1 diabetes. Lancet. June 16, 2018;391(10138):2449–2462. 2. Chatterjee S, Khunti K, Davies MJ. Seminar: type 2 diabetes. Lancet. June 3, 2017;389(10085):2239–2251. 3. Shah VN, Carpenter RD, Ferguson VL, Schwartz AV. Bone health in type 1 diabetes. Curr. Opin. Endocrinol. Diabetes Obes. August 2018;25(4):231–236. 4. Compston J. Type 2 diabetes mellitus and bone. J. Intern. Med. February 2018;283(2):140–153. 5. Ferrari SL, Abrahamsen B, Napoli N, et al. Diagnosis and management of bone fragility in diabetes: an emerging challenge. Osteoporos. Int. July 31, 2018. https://doi.org/10.1007/s00198-018-4650-2. 6. Gilbert MP, Pratley RE. The impact of diabetes and diabetes medications on bone health. Endocr. Rev. 2015;36:194–213. 7. Marin C, Luyten FP, Van Der Schueren B, Kerckhofs G, Vandamme K. The impact of type 2 diabetes on bone fracture healing. Front. Endocrinol. 2018;9.

VI.  SKELETAL DISEASE AND TREATMENT

Index Note: ‘Page numbers followed by “f” indicate figures, “t” indicate tables and “b” indicate boxes.’

A Abaloparatide, 406 Abnormal hormones, 382–383 Absorptiometry DXA, 105–108, 106f photon, 200–201, 431 X-ray, 431 Absorption, 122 AC. See Adenylate cyclase (AC) Ac.f. See Activation frequency (Ac.f) Acetylcholine (ACh), 356, 363 N-Acetylgalactosaminyltransferase 3 (GalNac-T3), 279–280 Acid-labile subunit (ALS), 66 Acid–base balance, 283–285, 292–293 Acidification process, 39–40 ACPAs. See Anti-citrullinated peptide antibodies (ACPAs) Acquired immunodeficiency syndrome (AIDS), 285–286 ACTH. See Adrenocorticotropic hormone (ACTH) Actin ring. See Filamentous actin (F-actin) Activation frequency (Ac.f), 146 Activator protein 1 (AP-1), 59 Active osteomalacia, 154 Active transcellular transport, 264–266 Activin receptor 2B (ACVR2B), 328–329 ACVR2B-Fc, 328–329 Activin receptor–like kinase-2 (ALK-2), 245 Activin type II receptor (ACTR-II), 245 Activin type IIB receptors (ACTR-IIB), 245 Acute hyperphosphatemia, 275b–276b ACVR2B. See Activin receptor 2B (ACVR2B) ADAMTS1, 414 Adaptive immune system, 333 Adaptive immunity, 334–337, 334t antibody effector functions, 336f Adaptive skeletal response, 204 Adaptor proteins, 38 Adenomatous polyposis coli (APC), 63 Adenosine triphosphate (ATP), 320, 366 ATP-ubiquitin-proteasome system, 329 Adenylate cyclase (AC), 356–358 Adequate Intakes (AIs), 296 Adherent junctions, 45–46 Adhesion focal tyrosine kinase. See Proline-rich tyrosine kinase 2 (Pyk2) Adhesion receptors, 81 ADHR. See Autosomal dominant hypophosphatemic rickets (ADHR) Adipocytes, 31, 312 Adipocytokines, 435 Adipokines, 294

Adiponectin, 4, 435 Adjusted appositional rate (Aj.AR), 154 Adolescence childhood and, 226–227 puberty and, 192–193 Adrenergic C1 group, 354 Adrenergic receptors (ARs), 356–358 Adrenocorticotropic hormone (ACTH), 364 Advanced glycation end products (AGEs), 7–8, 137, 428f, 430, 435–436 accumulation in collagen, 436f Adynamic bone disease, 378 Aerobic endurance exercises, 317 Aerobic exercise, 327 Age-related bone loss, 201, 391 AGEs. See Advanced glycation end products (AGEs) Aging, 34, 34f, 248–249 aging-associated osteoporosis, 330 effects on musculoskeletal system, 330–332 sarcopenia, 330–332, 331f skeletal changes with skeletal expansion and age, 199–200 skeletal mass and age, 200–201 AGM. See Aortagonad-mesonephros (AGM) AIDS. See Acquired immunodeficiency syndrome (AIDS) AIs. See Adequate Intakes (AIs) Aj.AR. See Adjusted appositional rate (Aj. AR) Akt, 44 phosphorylation, 66 ALK-2. See Activin receptor–like kinase-2 (ALK-2) Alkaline phosphatase (ALP), 10, 12, 45, 237–239 Alkaline salts, 292–293 ALP. See Alkaline phosphatase (ALP) ALS. See Acid-labile subunit (ALS) Aluminum, 278 aluminum-based antacids, 275b–276b bone disease, 378 Alveolar bone, 338 American Association of Clinical Endocrinologists, 407–408 American College of Endocrinology, 407–408 American College of Physicians, 407–408 Amino acids, 285, 285f β-Aminopropionitrile, 136–137 AMP-activated protein kinase (AMPK), 437 Amphiregulin (AREG), 414 Ampulla, 166 Amputation, 430

443

Amylin analogs, 439–440 Anabolic agents, 394 antiremodeling agent followed by, 409 followed by antiremodeling agent, 409 therapy, 157 for osteoporosis, 404–407 window, 48, 157 Androgens, 44, 302, 305 enhance periosteal bone formation, 227 Androstenedione, 302 Angiogenesis, 21, 245–247 Angiopoietin-1 (ANG-1), 247 Animal models, 371 of skeletal mechanotransduction, 206–209, 208f extrinsic loading models, 207–209 intrinsic loading models, 206–207 invasive (surgical) models, 207–208 noninvasive models, 208–209 Anlage. See Cartilage model Antagonistic processes, modeling and remodeling, 212–213 Anti-citrullinated peptide antibodies (ACPAs), 344–346 Antibiotics, 372–373 Antibodies, 334–335 Anticatabolic agents. See Antiresorptive agents Anticatabolic treatment. See Antiremodeling Antigens, 334–335 Antiinflammatory Treg CD4+ T cells, 339 Antiremodeling, 394 agent anabolic followed by, 409 followed by anabolic, 409 followed by antiremodeling agent, 409 therapies, 396–404 Antiresorptive agents, 155 Antiresorptive therapy, 155–157 Antisclerostin antibody, 406–407 Aortagonad-mesonephros (AGM), 28–29 AP-1. See Activator protein 1 (AP-1) APC. See Adenomatous polyposis coli (APC) Apolipoprotein E gene (ApoE), 179–180 Apoptotic osteocytes, 52 Apparent bone mineral density (aBMD), 133–134 ARC. See Arcuate nucleus (ARC) Arcuate nucleus (ARC), 358–359 Area under the curve (AUC), 380, 382b Areal bone mineral density (aBMD), 105, 107, 196

444 AREG. See Amphiregulin (AREG) Arg-Gly-Asp-containing matrix proteins (RGD-containing matrix proteins), 37–38 ARs. See Adrenergic receptors (ARs) Arterial calcification, 384, 385f ASBMR Task Force (2016), 403 Assessment of fracture healing biomechanical stages of fracture healing, 241 methods of evaluation, 240–241 Astrocytes, 349–350 ATP. See Adenosine triphosphate (ATP) Atrogenes, 321 Atrophic nonunion, 239–240 AUC. See Area under the curve (AUC) Autoimmune diseases, 344–346 RA, 344–346 Autophagy, 331 Autosomal dominant hypophosphatemic rickets (ADHR), 183–184, 279–280 ARHR1, 183 Average tissue mineralization, 97–98 Axial loading stress and strain in, 126–128 in tension and compression, 129–138 Axon, 349

B B cells, 335–336 and bone remodeling, 337–338, 338f B lymphocytes, 337 Back and musculoskeletal pain, 403 Bacterial bone infection, 341 Bacterial MAMPs, 374 BAG family molecular chaperone regulator 4 (BAG-4), 72 BAIBA. See Beta-aminoisobutyric acid (BAIBA) Balance bone remodeling, 96–97 hormonal or metabolic balance, 213 net negative bone, 97 of strength, stiffness, and toughness, 136 Bariatric surgery, 440 Basic multicellular unit (BMU), 37, 342 BC cells. See Breast cancer cells (BC cells) BDNF. See Brain-derived neurotrophic factor (BDNF) Beckwith–Wiedemann syndrome, 67 Bending test, 131–133 Beta-aminoisobutyric acid (BAIBA), 322–323, 325–326 Beta-tricalcium phosphate scaffold, 246 BFR. See Bone formation rate (BFR) Bglap gene, 64 BIA. See Bioelectrical impedance analysis (BIA) Biglycan, 12 Bioelectrical impedance analysis (BIA), 330–331 Biofilm formation, 341 Biological processes, 260 Biomarkers, 99 bone, 380 of cancer in bone, 421–422 serum, 409

Index

Biomechanical stages of fracture healing, 241, 241t Bipolar disorder, 367 Bisphosphonates, 251, 328–329, 394, 398–403, 399f–400f, 405–406, 422 Bivariate GWAS, 328 Blastocyst, 171–173 Blood supply to bone, 20–22 BMC. See Bone mineral content (BMC) BMD. See Bone mineral density (BMD) BMDD. See Bone mineral density distribution (BMDD) BMI. See Body mass index (BMI) BMP. See Bone morphogenetic protein (BMP) BMP type II receptor (BMPR-II), 245 BMSCs. See Bone marrow stromal cells (BMSCs) BMSi. See Bone material strength index (BMSi) Body mass index (BMI), 290–291, 291f, 431 Bone, 189 age, 191 architecture and geometry, 146–147 biomarkers, 380 blastema, 85 blood supply to, 20–22 bone-targeted therapies for bone metastasis, 422 calcium and, 3–4 and cancer, 411–424 cell contributions, 416–417 composition, 5, 5f disease, 426b endocrine effects on muscle, 326–328 FGF23, 327 osteocalcin, 327 prostaglandin E2, 328 RANKL, 326–327 sclerostin, Dkk1, and Wnts, 326 unknown factors, 328 fluid compartments, 22–23 force–displacement curve, 126, 127f functions, 3–4, 125 glue, 12–13 hierarchical organization, 4f hierarchical structure, 125–126 histology, 240, 379f histomorphometric analysis, 145–152 histomorphometry, 98, 141 imaging, 378–380 innervation, 22 lacuna-canalicular system, 218 macroscopic organization, 17–20 marrow biopsy, 141 mass and quality, 23–26 matrix proteins, 417 microdamage accumulation, 25–26, 25f microenvironment, 414–415 microstructural organization, 13–17 mineral, 9–10, 137 mineralization, 97–98 morphology, 427b as multiscale material, 4–5

nanoscale organization, 5–13 noncollagenous extracellular matrix proteins, 10, 11t–12t nutrient interventions studying effects on bone outcomes, 296–298 premetastatic niche generation in, 415–416 primary mineralization, 10f rate of turnover, 23 response, 204–205 scintigraphy, 120 solid mechanics, 126–129 strength measurement, 139–140 stress and strain in axial loading, 126–128 structure, 203 tissue material (intrinsic) properties, 23–24 TMV system, 378 trabecular architecture, 23 transgenic animals and bone biology, 179–184 turnover, 3–4 types of tumor metastases in, 413f weight loss, 294–295 Bone adaptation, rules for, 209–211 dynamic loads, responds to, 210 error driven, 212 loading period, 210 rate-related phenomena, 210–211 strain rate, 211, 211f threshold of strain or strain rate, 209, 210f Bone biopsy, 435 histomorphometry on, 141 human, 142f Bone cells, 39f, 203, 306 osteoblasts, 45–49 osteoclasts, 37–45 osteocytes, 49–55 Bone collagen abnormalities, 383 glycation, 436 Bone collar, 86 Bone drift, 90–91 Bone extracellular matrix, 136–137 inorganic hydroxyapatite, 137–138 type I collagen, 136–137 Bone formation, 210, 210f, 213, 215f, 241–242, 412 markers, 421–422 PTH effects on, 300 Bone formation rate (BFR), 149–150, 150f, 209, 378 Bone fracture, 235 Bone geometry and architecture in T1D, 431–432 in T2D, 433 Bone health, 292–293 diabetes management and, 436–440 macro-and micronutrient roles in, 283–288 management in diabetes, 440 Bone lining cells, 48–49 Bone marrow, 152 microenvironment, 34, 34f niche cells, 30–32 hematopoietic bone marrow niche cells, 32 nonhematopoietic, 30–31

Index

Bone marrow stromal cells (BMSCs), 42, 343, 361–362, 414f, 415–416 Bone mass, 57, 133–134, 288–289 Bone material strength index (BMSi), 436 Bone mechanics bending, 131–133 bone extracellular matrix Inorganic hydroxyapatite, 137–138 type I collagen, 136–137 bone tissue material properties, 135–136 cancellous architecture and anisotropy, 134 cortical bone size and architecture, 134–135 factors contributing to whole bone mechanical properties, 133–138 torsion, 130–131 Bone metabolic unit (BMU), 393 Bone metastasis, 411, 411f. See also Cancer in bone bone-targeted therapies, 422 imaging, 421 immune suppression in, 416 intrinsic properties of tumor cells enhancing, 414 mechanisms responsible for, 413–414 osteoblasts role in development and growth, 418–421 osteoclasts role in development and progression, 417–418 physical characteristics of bone contribution to, 416 Bone mineral content (BMC), 23, 105, 192–193, 193f, 195f, 210, 260, 322 Bone mineral density (BMD), 97–98, 104–105, 107, 159, 193, 249, 260, 285, 305–306, 322, 344, 359–360, 389–390, 390f, 393, 427b, 428f in T1D, 431, 432f in T2D, 431, 432f Bone mineral density distribution (BMDD), 97–98 Bone modeling, 89–91 cellular processes, 90 characteristics, 90t events that signal, 90 laboratory assessment, 98–100 during life cycle, 90 longitudinal growth, 90 radial growth, 90, 91f tooth movement, 92f Bone morphogenetic protein (BMP), 46–48, 73–74, 87–89, 242–243, 245, 293–294, 318, 320f, 419 BMP-2, 364–365 BMP-3, 73–74 Bmp2 and Bmp4, 73–74 deletion of Bmpr1a, 74 Bone multicellular unit (BMU), 91–92 Bone remodeling, 91–97, 95f, 99f, 239–240 abnormalities induced by cancer in bone, 412–413 activation stage, 93 B cells and, 337–338 balance, 96–97 characteristics, 90t cycle, 93–95

cycle duration, 95 effects on bone structure, 97 events that signal, 92–93 formation stage, 95 laboratory assessment, 98–100 material property of bone after, 97–98 myeloid cells and, 339 quiescence (resting) stage, 95 rate, 96, 96f resorption stage, 93–94 reversal phase, 94–95 T cells and, 338–339 tooth movement, 92f uncoupled in myeloma, 412f Bone remodeling compartment (BRC), 48–49, 49f Bone repair, 97 Bone resorption, 412 markers, 98–99 PTH effects on, 301 Bone sialoprotein (BSP), 12–13, 38–39 Bone tissue, 203 material properties, 435–436 Bone turnover, 433–434, 427b markers, 421–422 suppression, 389 Bone volume per unit tissue volume (BV/TV), 133–134 Bone-specific alkaline phosphatase (BSAP), 380 Boron, 286 Bound water, 23 Brain, bone and bone feedback to, 366–367 bone impact of neurological disorders, 367 functional role of central and peripheral efferent pathways, 356–364 functional role of sensory neurotransmitters in bone, 364–366 innervation of bone, 350–353, 350f nervous system, 349–350 neural connections between bone and, 353–356 Brain-derived neurotrophic factor (BDNF), 322–323, 325 BRC. See Bone remodeling compartment (BRC) Breast cancer cells (BC cells), 414 Brittle bone disease, 6b, 328–329 Brittle IV mice (Brtl mice), 6b Broadband attenuation (BUA), 122 Brown fat, 3 Brtl mice. See Brittle IV mice (Brtl mice) BSAP. See Bone-specific alkaline phosphatase (BSAP) BSP. See Bone sialoprotein (BSP) BUA. See Broadband attenuation (BUA) BV/TV. See Bone volume per unit tissue volume (BV/TV)

C C cells. See Parafollicular cells c-Fms receptor, 42–43, 61 c-Fos, 43, 46–47 c-jun (transcription factors of AP-1 family), 46–47

445 c-Jun N-terminal kinase (JNK), 63–64 c-Met, dual kinase inhibitor of, 418–419 c-terminal telopeptide (CTX), 434 C011A1 promoter, 77 Cachexia, 329–330 Cadherins, 83 domains, 83 Caffeine consumption, 289 CAFs. See Cancer-associated fibroblasts (CAFs) Calcification, 190 Calcified cartilage zone, 89 Calcified tissues, 20–21 Calciotropic hormones, 257 Calcitonin, 257, 273, 394, 398 Calcitonin gene-related peptide (CGRP), 22, 352, 364–365 Calcitriol. See 1,25-dihydroxyvitamin D (1,25-(OH)2D) Calcium, 191f, 192–193, 278, 283, 288–289, 292f, 394–396 absorption, 195 for bone health, 394f calcium–PTH–FGF23 loop, 376 1,25-dihydroxyvitamin D regulation and metabolism, 267–268 distribution in body, 257–258, 258t estrogen, 272–273 hormones controlling calcium metabolism, 266–274 25-hydroxyvitamin D metabolism, 266–267 intestinal absorption, 261–264 in nephron, 265f organ system interplay regulates metabolism, 257–260 perturbing calcium metabolism, 289–291 PTH and PTHrP, 269–272 regulation of whole body calcium metabolism, 260–274 renal reabsorption, 264–266 vitamin D and, 266–269, 267f Calcium-dependent tyrosine kinase. See Proline-rich tyrosine kinase 2 (Pyk2) Calcium-sensing receptor (CaSR), 190, 268f, 382 Cambium layer, 19–20 cAMP. See Cyclic AMP (cAMP) cAMP-response element-binding protein (CREB), 63–64, 300 Canagliflozin, 438 Canaliculi, 50–51, 216–217, 216f Cancellous bone, 17–19, 133–134 architecture, 18f subchondral plate, 19, 19f Cancer in bone, 411 abnormalities of bone remodeling by, 412–413 bone turnover markers and biomarkers, 421–422 changes in phenotype of cancer cells, 414–415 consequences, 411t intrinsic properties of tumor cells, 414 premetastatic niche generation in bone, 415–416 tumor cell homing to bone, 415f, 416

446 Cancer-associated fibroblasts (CAFs), 415–416 Cancer-related bone destruction, 411 Canonical notch signaling, 77 Canonical pathways, 48 Canonical Wnt signaling, 62f, 63 Capillaries in bone, 21 Capsaicin, 364–365 CAR cells. See CXCL12-abundant reticular cells (CAR cells) Carbonyl stress, 436f Carboxymethyl-lysine, 436 Cardiotrophin-1 (CT-1), 70 CART. See Cocaine-and amphetamineregulated transcript (CART) Cartilage model, 85–86 Cartilage oligomeric matrix protein (COMP), 247 Casein kinase I (CKI), 63 CaSR. See Calcium-sensing receptor (CaSR) β-Catenin, 43–44, 63, 224 Cation-independent mannose-6-phosphate receptor. See Insulin-like growth factors (IGFs)—IGF-IIR Cbfa1/Runx2 genes, 180 CD. See Cluster of differentiation (CD) CD4+ T cells, 338–339 CD44 receptor, 10–12, 50–51, 83 CD8+ FoxP3+ Treg cells, 339 CEE. See Conjugated equine estrogen (CEE) CEE/MPA study. See Conjugated equine estrogen/medroxyprogesterone acetate study (CEE/MPA study) Cell cell-to-cell interactions, 420–421 number and activity, 148–149 saturation, 214 sensitivity, 214 surface attachment molecules, 81–83, 81t markers, 29–30 survival, 300 types, 189–190 and environment, 216–217 Cell adhesion, 221–222, 223f cell adhesion/cytoskeletal molecules, 220 Cellular adhesion kinase. See Proline-rich tyrosine kinase 2 (Pyk2) Cellular deformation and stimulation, 218–220 Cellular events of fracture repair, 241–242 Cellular immunity, 334 Cellular polarization, 37 Cement lines, 16–17 Central nervous system (CNS), 349 Central neurotransmitters, 364 orexin, 364 oxytocin, 364 POMC and melanocortin system, 364 Central NPY, 361 Cesium chloride (CsCl), 166–167 CFU-GM. See Granulocyte-macrophage colony-forming unit (CFU-GM) CGRP. See Calcitonin gene-related peptide (CGRP)

Index

Charcot foot, 426b–427b, 427f Chemokines, 420–421 CXCL2, 30 Chemoresistance, 416–417 CHiA-PET. See Chromatin Interaction Analysis by Paired-End Tag Sequencing (CHiA-PET) Childhood, 226–227 Children, 248 fracture risk in, 197–198 Chloride ions (Cl−ions), 38–39 Cholecalciferol, 311 Cholesterol, 302 Chondrocytes, 246–247 Chondrogenesis, 248 molecular regulation, 242–244 Chromatin immunoprecipitation analysis, 419 Chromatin Interaction Analysis by Paired-End Tag Sequencing (CHiA-PET), 163 Chronic hypophosphatemia, 274 Chronic kidney disease (CKD), 375 causes and proposed mechanisms of decreased bone formation, 382t hip fracture incidence, 377f pathogenesis of abnormal bone in patients with, 380–383 regulation of serum phosphorus levels, 376f stages, 376t Chronic kidney disease–mineral and bone disorder (CKD-MBD), 377 biochemical abnormalities, 383–384 bone abnormalities, 377–383 vascular calcification, 384 Chronological assessment, 113, 115f Chylomicrons, 266 CIA. See Collagen-induced arthritis (CIA) CIC-7. See H+/Cl− exchange transporter 7 (CIC-7) Ciliary neurotrophic factor receptor (CNTFR), 57 Citrullination, 344–345 CKD. See Chronic kidney disease (CKD) CKD-MBD. See Chronic kidney disease– mineral and bone disorder (CKD-MBD) CKI. See Casein kinase I (CKI) Clear Lipid-exchanged Acrylamidehybridized Rigid Imaging/ Immunostaining/In situ hybridization-compatible Tissue hYdrogel (CLARITY), 144 Clinical diagnostic computed tomography, 108–112 Clinical scanners, 109 Clinical skeletal imaging, 103 Cluster of differentiation (CD), 27 Clustered regularly interspaced short palindromic repeat (CRISPR), 175 revolution, 175–178 CML. See Nε-carboxymethyllysine (CML) CNS. See Central nervous system (CNS) CNTFR. See Ciliary neurotrophic factor receptor (CNTFR)

Cocaine-and amphetamine-regulated transcript (CART), 362 COLIA1. See Type I collagen (COLIA1) Collagen, 5–9 cross-links, 98 enzymatically mediated cross-linking, 7 fibrils, 6f, 7 molecules, 7f nonenzymatically mediated cross-linking, 8 orientation of fiber bundles, 8–9, 9f twisted plywood configuration, 8, 9f Collagen-induced arthritis (CIA), 346 Combination treatments, 409–410 Comminuted fracture, 235 COMP. See Cartilage oligomeric matrix protein (COMP) Compact bone, 17 Complement activation, 334–335 Complex traits, 159 Compression, axial loading in, 129–138 Compressive loading, 130 Computed tomography (CT), 108–109, 111f, 330–331 appearance of images, 110f calcium suppressed image of bone, 112f clinical diagnostic computed tomography, 108–112 partial volume averaging, 112f scans, 107 voxel size and resolution, 111f Concurrent antiremodeling agents, 410 and anabolic, 409–410 Concurrent treatments. See Combination treatments Congenital leptin deficiency, 311 Conjugated equine estrogen (CEE), 396, 396f Conjugated equine estrogen/ medroxyprogesterone acetate study (CEE/MPA study), 396 Connexins (Cx), 79–80 Cx37, 79–80 Cx43, 45–46, 60–61, 79–80, 223–224 Contact radiography. See Microradiography Conventional radiography (X-ray), 103–105 microradiography, 105f radiograph of thoracic spine, 104f Coordinating biochemical response to mechanical stimulation, 223–226 Copper, 285 Cortical bone, 17 Cortical geometry, 146–147 Costochondral junctions, 270b Coupling, 96 COX. See Cyclooxygenase (COX) Cre-LoxP technology, conditional models using, 181–182 CREB. See cAMP-response element-binding protein (CREB) CRISPR. See Clustered regularly interspaced short palindromic repeat (CRISPR) CRISPR RNA (crRNA), 176 CRISPR/Cas9 system, 177–178 CsCl. See Cesium chloride (CsCl) CSF1 gene, 61 CT. See Computed tomography (CT)

447

Index

CT-1. See Cardiotrophin-1 (CT-1) CTLA-4. See Cytotoxic T-lymphocyte antigen 4 (CTLA-4) CTX. See c-terminal telopeptide (CTX) CXC motif chemokine 12 (CXCL12), 415–416 CXCL12 (SDF1), 416 CXCL12-abundant reticular cells (CAR cells), 30–31 CXCR4 gene, 416 Cyclic AMP (cAMP), 58–59, 244 Cyclooxygenase (COX), 32, 223–224, 244 Cyclophosphamide, 344 Cystitis, 403 Cytokines, 57, 325, 420–421 Cytoskeleton, 221–222 Cytotoxic T cells, 336–338 Cytotoxic T-lymphocyte antigen 4 (CTLA-4), 339, 346

D D-banding pattern, 7 DA. See Dopamine (DA) DA β-hydroxylase (DBH), 358–359 Damage-associated molecular patterns (DAMPs), 334 DAP12. See DNAX-activation protein 12 (DAP12) Dapagliflozin, 438 DAT. See Dopamine transporter (DAT) DBH. See DA β-hydroxylase (DBH) DC-STAMP. See Dendritic cell–specific transmembrane protein (DC-STAMP) DCR. See Distal control region (DCR) Decalcification, 143–144 Decorin, 12 Deep vein thrombosis, 397 Degree of mineralization heterogeneity, 97–98 Dehydroepiandrosterone, 302 11-beta-Dehydrogenase isozyme 2 (11βHSD2), 44–45 Dendrites, 349 Dendritic cells, 339 Dendritic cell–specific transmembrane protein (DC-STAMP), 41–42 Denosumab, 251–252, 394, 403–406, 422 Densitometry, 115–116, 117f Dentin matrix acidic phosphoprotein 1 (DMP-1), 10, 12–13, 51, 61, 180–181, 183, 244, 326–327 Deoxypyridinoline, 7 Dephosphorylating osteopontin, 38–39 Depolarization, 349 Derived variable, 146, 147t Designer DNA nucleases, 175–178 CRISPR-based genome editing, 168f genome engineering using ZFN and TALENs, 168f nuclease-induced DSBs, 169f Deterministic radiation effects, 103–104 Diabetes mellitus (DM), 249, 425, 426t bone health management, 440 fracture healing, 430 fractures in, 427–430 management and bone health, 436–440

osteoporosis medication effect on glucose metabolism, 440 skeletal changes in, 430–433 Dickkopf (Dkk), 64–65 Dkk1, 51, 326, 344, 383, 419 expression levels of, 419f Diet, 291–292 Western diet, 280 Dietary bioactives and mechanisms, 293–294, 294f Dietary intake, 266 Dietary phosphate, 274–276, 279 deficiency, 259 intake alter FGF23concentration, 280 Dietary potassium, 290 Dietary Reference Intakes (DRIs), 296 1,25-Dihydroxyvitamin D (1,25-(OH)2D), 69–70, 189, 260, 283 regulation and metabolism, 267–268 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), 154–155, 311, 311f Dimerized sex steroid receptors, 302 Diminished returns principle, 232 Dipeptidyl peptidase 4 (DPP-4), 437 inhibitors, 438 Direct mechanical linkage model, 222 Disease model, 160 Disheveled (Dsh), 63 Disordered osteoblast function or differentiation, 383 Distal control region (DCR), 301 Dkk. See Dickkopf (Dkk) DM. See Diabetes mellitus (DM) DMP-1. See Dentin matrix acidic phosphoprotein 1 (DMP-1) DNA homologous recombination process, 170 plasmid, 166–167 preparation for microinjection, 166 recombinant, 166–167 variants, 159 DNA-binding protein inhibitor ID (Id), 46–47 DNAX-activation protein 12 (DAP12), 41–42 Dog-bone section for tensile testing, 129, 130f Dopamine (DA), 354, 362–363 Dopamine transporter (DAT), 362–363 Dorsal root ganglia (DRG), 350 Double muscling, 324–325 Downregulation of sost/sclerostin by PTH, 300–301 Doxycycline (Dox), 181 DPP-4. See Dipeptidyl peptidase 4 (DPP-4) DRG. See Dorsal root ganglia (DRG) DRIs. See Dietary Reference Intakes (DRIs) Drosophila, 61, 77, 177 “Drug holiday”, 398, 403 Dsh. See Disheveled (Dsh) Dual energy scanning, 112–113 Dual-energy X-ray absorptiometry (DXA), 105–108, 153, 197, 330–331, 372–373, 378–380, 389–390, 432 assessment of femoral neck, 106f DXA-based BMD measurement, 393

graphical depiction of T-and Z-scores for bone density, 107f in laboratory, 108f vertebral fracture assessment, 108f DXA. See Dual-energy X-ray absorptiometry (DXA) Dynamic histomorphometry, 149–151 Dynamic loads, bone responds to, 210 Dynamic measurement, 146 Dysbiosis, 342–343

E E-cadherin, 414–415 E11/PDPN/GP38 proteins, 50–51 EARs. See Estimated Average Requirements (EARs) ECF. See Extracellular fluid (ECF) ECFCs. See Endothelial colony forming cells (ECFCs) ECM. See Extracellular matrix (ECM) EDTA. See Ethylenediaminetetraacetic acid (EDTA) Efnb1 gene, 78 EGF. See Epidermal/epithelial growth factor (EGF) eGFR. See Estimated glomerular filtration rate (eGFR) eIF4E. See Eukaryotic translation initiation factor 4E (eIF4E) Elastic region, 126 Electrical stimulation effect on fracture repair, 252 Electromagnetic therapy, 252 Electroporation, 172 Embryogenesis and development, 317–321 Embryonic stem cells (ES cells), 164, 171–172, 177f Embryonic vascular system, 189–190 Empagliflozin, 438 EMT. See Epithelial mesenchymal transition (EMT) Encyclopedia of DNA Elements (ENCODE), 163–164 Endocannabinoids, 362 Endochondral matrix, 239 Endochondral ossification, 85–89, 87f, 236–239, 246–247 Endocortical surface, 20, 212–213 Endocortical trabecular density, 229, 229f Endocrine activation of immune-mediated bone loss, 341–344 cross talk between muscle and bone, 322–326 BAIBA, 326 BDNF, 325 cytokines, 325 IGF-1, 325 irisin, 325 musclin, 325–326 myostatin, 324–325 regulators, 194–195 Endocytosis, 276–277 Endomysium, 319–320

448 Endosteal surface, 20 Endothelial cells, 31 Endothelial colony forming cells (ECFCs), 247 Endothelial progenitor cell (EPC), 247 Energy, strain, 206 Enhancer of zeste homolog 2 (EZH2), 419 EP1–4. See Heptahelical EP receptors (EP1–4) EPC. See Endothelial progenitor cell (EPC) Ephrin B2, 412 Ephrin–Ephrin receptor, 78–79 bidirectional signaling, 79f EphA and EphB receptors, 78 ephrin-A2–EphA2 complex, 79 ephrin-B-EphB family, 78–79 Epidermal/epithelial growth factor (EGF), 58, 83 Epimysium, 319–320 Epiphyseal growth plate, 86–87, 88f hypertrophic zone, 89 longitudinal growth, 90 proliferative zone, 87–89 reserve zone, 87 resting zone, 87 zone of ossification, 89 Epiphyseal line, 89 Epithelial mesenchymal transition (EMT), 414–415, 415f Equol, 294 ER. See Estrogen receptor (ER) ERK. See Extracellular signal–regulated kinases (ERK) Erlenmeyer flask, 90 Ertugliflozin, 438 ES cells. See Embryonic stem cells (ES cells) Escherichia coli, 166 Estimated Average Requirements (EARs), 296 Estimated glomerular filtration rate (eGFR), 375 Estrogen receptor (ER), 51–52, 182 ESR1, 196 Estrogen(s), 44, 272–273, 305, 394, 396 deficiency, 304–305 immune activation role in, 342–343 osteoporosis, 393 loss in women at menopause, 391–392 therapy, 195 Ethylenediaminetetraacetic acid (EDTA), 143–144 Eukaryotic translation initiation factor 4E (eIF4E), 66 Exercise aerobic, 327 aerobic endurance, 317 prescription for optimizing skeletal health, 226 protocol, 207 resistance, 331–332 skeletal benefits of physical activity and, 226–228 training and exercise recommendations principles, 228–232

Index

Extracellular factors in bone cells, 223–224 Extracellular fluid (ECF), 349–350 Extracellular glycoproteins, 217 Extracellular matrix (ECM), 37, 89 Extracellular nucleotides, 366 Extracellular signal–regulated kinases (ERK), 42–44, 66 Extrinsic loading models, 207–209 EZH2. See Enhancer of zeste homolog 2 (EZH2)

F F-actin. See Filamentous actin (F-actin) Facilitated diffusion model, 262 FAK. See Focal adhesion kinase (FAK) Falls (risk factor for fracture), 392, 431 Fanconi syndrome, 279 Farnesyl diphosphate synthase (FPPS), 398–399, 401f Fas ligand, 44 FDA. See US Food and Drug Administration (FDA) FDG-PET. See Fluorodeoxyglucose positron emission tomography (FDG-PET) FEA. See Finite element analysis (FEA) FEMs. See Finite element models (FEMs) Fetal calcium:phosphate accrual ratio, 259 Fetal mineral transport and mineral metabolism, 273 Fibroblast growth factor (FGF), 65, 68–70, 182–183 FGF5, 414 FGF18, 69 FGF21, 69, 435 FGF23, 4, 51, 55, 68–70, 183, 189, 279–281, 281f, 326–327, 375, 382, 384 iron on metabolism, 280–281 Fibroblast growth factor receptor (FGFR), 281f FGFR-1, 55 FGFR-2, 69, 242–243, 322–323 FGFR3, 182–183 Fibrocartilage, 237 Fibrolamellar bone. See Plexiform bone Fibronectin, 12 Filamentous actin (F-actin), 37 Finite element analysis (FEA), 116–118 Finite element modeling, 203–204 Finite element models (FEMs), 116–118, 117f FIT. See Fracture Intervention Trial (FIT) Flavonoids, 293 FLEX. See Fracture Intervention Trial Long-Term Extension (FLEX) “Floxed” gene, 181–182 Fluid compartments in bone, 22–23 Fluid flow in bone tissue, 217–218 mechanical stimulation, 218–220 Fluoride, 285–286 Fluorine-18–labeled sodium fluoride (18F–NaF), 120 Fluorochrome labels, 142–143, 154–155, 240, 240f Fluorodeoxyglucose positron emission tomography (FDG-PET), 421

FNDC5 protein, 325 Focal adhesion kinase (FAK), 51–52, 222, 306–307 Follistatin-like protein 1, 322–323 Follow-on treatment. See Sequential treatments Force–displacement curve, 126, 127f Formation modeling, 89 Formic acid, 143–144 Fortified foods, 291–292 FPPS. See Farnesyl diphosphate synthase (FPPS) Fracture in diabetes, 427–430 healing, 82, 235 assessment, 240–241 bone fracture types, 235 cellular events of fracture repair, 241–242 in diabetes, 430 local regulation of fracture repair, 244–248 mechanical and electrical stimulation effects, 252 molecular regulation of chondrogenesis and osteogenesis, 242–244 osteoporosis drug effects, 250–252 primary and secondary repair mechanisms, 236 incidence, 297 repair cellular events, 241–242 molecular regulation of chondrogenesis and osteogenesis during, 242–244 stages, 236–240, 238f systemic factors in, 248–249 risk, 294–295 in children, 197–198 Fracture Intervention Trial (FIT), 403 Fracture Intervention Trial Long-Term Extension (FLEX), 403 Fracture Reduction Evaluation of Denosumab in Osteoporosis (FREEDOM trial), 403, 409 Fracture Risk Assessment Tool (FRAX), 393, 432 Fracture Study in Postmenopausal women with Osteoporosis trial (FRAME trial), 406–407, 409 Fragility fractures, 390, 427 FRAME trial. See Fracture Study in Postmenopausal women with Osteoporosis trial (FRAME trial) FRAX. See Fracture Risk Assessment Tool (FRAX) FREEDOM trial. See Fracture Reduction Evaluation of Denosumab in Osteoporosis (FREEDOM trial) Frequency, strain, 205–206 Frizzled family of receptors (FZD family of receptors), 61, 63, 224 Frost, Harold, 205 Frost’s mechanostat theory, 322 Fruit and vegetable intakes, 292–293 Functional analysis, 163

Index

Furosine, 8 FZD family of receptors. See Frizzled family of receptors (FZD family of receptors)

G G protein signaling, 222–223 G protein–coupled receptors (GPCRs), 58, 222–223, 244, 363 G-CSF. See Granulocyte cell stimulating factor (G-CSF) Gadolinium (Gd3+), 221 β-Galactosidase, 164 GalNac-T3. See N-Acetylgalactosaminyltransferase 3 (GalNac-T3) GALNT3 mutations, 280 Gap junction alpha-1 protein, 45–46 Gap junction plaques, 79 Gastrointestinal tract (GI tract), 395 GCs. See Lucocorticoids (GCs) GDF-8. See Growth and differentiation factor-8 (GDF-8) GEFOS. See Genetic Factors for Osteoporosis Consortium (GEFOS) Genant method, 107 Gene targeting, 176f, 181–182 in mammals, 175 Genetic analysis of bone phenotypes, 159–164 functional analysis, 163 GWAS, 160–162 linkage analysis, 159–160 omics analysis of bone phenotypes, 163–164, 163t WES and WGS, 162–163 Genetic disorders, 159 Genetic Factors for Osteoporosis Consortium (GEFOS), 161 Genetic regulation, 195–196 Genetically modified embryonic stem cells, microinjection of, 170–175 Genome-wide association studies (GWAS), 159–162, 328 Genotype-Tissue Expression Project (GTEx), 163–164 Germ-free mice, 370b, 371, 373 Gfi-1. See Growth factor-independent 1 (Gfi-1) GFP. See Green fluorescent protein (GFP) GFP+ cells. See Green fluorescent protein+ cells (GFP+ cells) GFR. See Glomerular filtration rate (GFR) GH. See Growth hormone (GH) GI tract. See Gastrointestinal tract (GI tract) GIP. See Glucose-dependent insulinotropic peptide (GIP) GJA1 gene, 60–61, 79–80 Glandular tissues, 103–104 Glial cells, 349–350 Glomerular filtration rate (GFR), 375 Glomerulus, 375 Glucagon-like peptide 1 (GLP-1), 437 receptor agonists, 437–438 Glucocorticoid receptor, 306 Glucocorticoids, 44–45, 48, 249, 250f, 305–307, 306f

Glucose metabolism, 440 Glucose-dependent insulinotropic peptide (GIP), 437 GIPR, 437 Glutamate, 366 Glycocalyx, 217 Glycogen synthase kinase-3β (GSK-3β), 63 Glycoprotein 130 (gp130), 57, 70 Glycoproteins, 12 Glycosaminoglycans, 10–13 GM-CSF. See Granulocyte-macrophage colony-stimulating factor (GM-CSF) Gnotobiotic condition, 370b Gold thioglucose (GTG), 358–359 gp130. See Glycoprotein 130 (gp130) GPCRs. See G protein–coupled receptors (GPCRs) GPR48. See Leucine-rich repeat-containing G protein–coupled receptor (LGR4) Grading clinical data, 288b Granulocyte cell stimulating factor (G-CSF), 32 Granulocyte-macrophage colony-forming unit (CFU-GM), 41, 61 Granulocyte-macrophage colony-stimulating factor (GM-CSF), 41–42 GRB2. See Growth factor receptor–bound protein 2 (GRB2) Green fluorescent protein (GFP), 164, 242 Green fluorescent protein+ cells (GFP+ cells), 30–31 Growth and differentiation factor-8 (GDF-8), 324–325 Growth factor receptor–bound protein 2 (GRB2), 42–43, 66 Growth factor-independent 1 (Gfi-1), 419 Growth factors, 57 Growth hormone (GH), 309f, 57, 87–89, 190–191, 260, 303–304, 308–310, 309f receptor, 308 Growth plate, 86–87 GSK-3β. See Glycogen synthase kinase-3β (GSK-3β) GTEx. See Genotype-Tissue Expression Project (GTEx) GTG. See Gold thioglucose (GTG) GTP-hydrolyzing enzymes (GTPases), 38, 398–399 Guanine nucleotide exchange factor, 38 Gut microbiome, 370 GWAS. See Genome-wide association studies (GWAS)

H H-type capillaries, 21 H+/Cl− exchange transporter 7 (CIC-7), 38–39 Hairy/enhancer-of-split related with YRPW motif-like protein 1, 77 Half osteons, 17 Hard callus formation (endochondral ossification), 237–239 Haversian canal, 17, 20, 115f, 152 Haversian system, 16 HbA1c. See Hemoglobin A1c (HbA1c)

449 hCG. See Human chorionic gonadotropin (hCG) HDAC1. See Histone deacetylase 1 (HDAC1) HDR. See Homology-directed repair (HDR) Health Outcomes and Reduced Incidence with Zoledronic Acid Once YearlyPivotal Fracture Trial (HORIZONPFT), 403 Healthy musculoskeletal system, 317 Helix-loop-helix family, 46–47 Hematopoiesis, 27 Hematopoietic bone marrow niche cells, 32 Hematopoietic stem cell (HSC), 27–29, 337, 416–417 hormonal regulation, 33 HSC-supportive cells, 30–31 neuronal regulation, 33 Hematopoietin/interferon receptors, 58 Hemichannels, 79 Hemiosteon, 17, 91–92 Hemoglobin A1c (HbA1c), 425, 426b Heparan sulfate, 10–12 Hepatocyte growth factor (HGF), 418–419 Heptahelical EP receptors (EP1–4), 223–224 HGF. See Hepatocyte growth factor (HGF) HIF. See Hypoxia-inducible transcription factor (HIF) High molecular weight protein (HMW protein), 69 High-resolution MRI (hrMRI), 118 High-resolution peripheral quantitative CT machines (HR-pQCT), 109, 113–118, 378–380, 381f, 431, 433f Micro-CT, 113–115, 114f modeling analysis, 116–118 quantitative computed tomography, 115–116 Hilton’s law, 350–351 Hip fractures, 235, 389, 392, 428–430 Histologic method, 240 Histomorphometry, 98, 141 assumptions and technical aspects, 152–153 bone architecture and geometry, 146–147 cell number and activity, 148–149 dynamic, 149–151 histologic features of disease and treatment, 153–157 osteomalacia, 154 osteoporosis, 153–154 histomorphometric analysis, 145–152 hyperparathyroidism, 154–155 marrow, 152 microdamage, 152 osteocytes, 151–152 Paget disease of bone, 155–157 primary vs. derived variables, 146, 147t referents, 146 specimen collection, 141–142 specimen processing, 142–143 static vs. dynamic measurements, 146 tissue types, 147–148 in vivo labeling, 142–143 Histone deacetylase 1 (HDAC1), 419 Historical classification scheme, 377–378

450 HLAs. See Human leukocyte antigens (HLAs) HMW protein. See High molecular weight protein (HMW protein) Hole zones, 9–10 Homologous recombination, 172 Homology-directed repair (HDR), 176–177 Hooke’s Law, 129 for shear, 131 “Hoop” strain, 218–220 HORIZON-PFT. See Health Outcomes and Reduced Incidence with Zoledronic Acid Once Yearly-Pivotal Fracture Trial (HORIZON-PFT) Hormonal effects on bone cells 1,25-dihydroxyvitamin D3, 311 direct vs. indirect effects, 299 GH, 308–310 glucocorticoids, 305–307 insulin, 310–311 leptin, 311–312 PTH, 299–301 sex steroids, 301–305 thyroid hormone, 307–308 Hormone replacement therapy (HRT), 396 Hormones, 280, 412 controlling calcium metabolism, 266–274 1,25-dihydroxyvitamin D regulation and metabolism, 267–268 25(OH)D metabolism, 266–267 vitamin D actions, 268–269 vitamin D and metabolites, 266–269 hormonal mechanisms, 367 of pregnancy and lactation, 273–274 Hounsfield unit (HU), 109 HR-pQCT. See High-resolution peripheral quantitative CT machines (HR-pQCT) hrMRI. See High-resolution MRI (hrMRI) HRT. See Hormone replacement therapy (HRT) HSC. See Hematopoietic stem cell (HSC) 5-HTT. See Serotonin transporter (5-HTT) HU. See Hounsfield unit (HU) Human chorionic gonadotropin (hCG), 165–166 Human leukocyte antigens (HLAs), 336–337 Human RANKL (huRANKL), 180 Humoral immunity, 334 Hyaline cartilage, 85–86 Hyaluronan, 10–12 Hydroxylysyl (Hyl), 7 Hydroxyproline, 5–7 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1), 53 25-Hydroxyvitamin D (25(OH)D), 266–267, 283 Hypercalcemia, 261b Hypercalciuria, 433–434 Hypercholesterolemia, 403 Hyperglycemia, 428f, 433–434, 436f Hyperparathyroidism, 261b primary, 154 secondary, 154–155 Hyperphosphatemia, 274, 275b–276b Hypertrophic/hypertrophy, 89 chondrocytes, 236

Index

nonunion, 239–240 zone, 89 Hypocalcemia, 262b, 376 Hypoglycemia, 430 Hypogonadism, 367 Hypophosphatasia, 12 Hypophosphatemia, 274, 277b Hypothyroidism, 307 Hypoxia-inducible transcription factor (HIF), 80 HIF1α, 415–416 HIFα, 80–81

I IFMRS. See International Federation of Musculoskeletal Research Societies (IFMRS) IFNγ. See Interferon gamma (IFNγ) Ig. See Immunoglobulin (Ig) IGF1. See Insulin-like growth factor (IGF1) Igf1r-null mice, 67 IGFs. See Insulin-like growth factors (IGFs) IHH. See Indian hedgehog protein (IHH) IKK2. See Inhibitor of NF-κB kinase subunit beta (IKK2) Imaging techniques, 103, 146–147, 330–331 Imidazolone, 8 Immature dendritic cells, 339 Immobilization, 322–323 Immune suppression in bone metastasis, 416 Immune system, 333 Immune-mediated bone loss, endocrine activation of, 341–344 immune activation role in estrogen deficiency, 342–343 T cell role in parathyroid hormone responses, 343–344 Immunoglobulin (Ig), 81 family proteins, 83 functions, 334–335 IgG1, 59 Immunohistochemistry techniques, 144, 351 In vivo labeling, 142–143, 143f In vivo mechanical forces, 52 In vivo micro-CT measures, 113, 115f Incretin-based treatments, 437–438 Indian hedgehog protein (IHH), 87–89, 242–243 Inducible form of NOS (iNOS−/−), 224 Infancy, 226 and childhood skeletal growth, 190–191 calcium, 191f X-ray image, 191f Infections and bone cells, 339–341 acute osteomyelitis, 340f factors contributing to osteomyelitis infection/persistence, 341t Inflammatory response, 236 Inhibitor of NF-κB kinase subunit beta (IKK2), 44 Initial values principle, 232 Innate immunity, 333–334, 334t Innervation of bone, 22, 350–353, 350f Inorganic hydroxyapatite, 137–138, 137f Inorganic phosphate, 258

iNOS−/−. See Inducible form of NOS (iNOS−/−) Inositol-1, 5-trisphosphate (IP3), 4, 270–271 Inside-out signaling, 81 Insulin, 310–311, 310f, 436–437. See also Diabetes secretion, 426f Insulin receptor (IR), 310 IRS, 66 Insulin-dependent DM. See Type 1 DM (T1DM) Insulin-like growth factor (IGF1), 33, 53, 65–67, 66f, 192–193, 260, 285, 300, 322–323, 325, 343, 372, 383, 415–416, 428f, 430 IGFR-1, 66, 190–191, 273, 310 IGFR-2, 190–191 Insulin-like growth factors (IGFs), 65–67, 87–89, 179, 189 IGF-II, 65–67, 66f IGF-IIR, 66 IGFBP, 66, 190–191 IGFBP-4, 66 Int-1 gene, 61 Integrins, 81–83 integrin-cytoskeletal network, 221–222 Interferon gamma (IFNγ), 339 Interleukin (IL), 70–71 IL-1, 41–42, 272, 331, 363 IL1β, 417–418 IL-6, 70–71, 71f, 196, 246, 322–323, 331, 416, 435 Rα, 57 type I IL-1 receptors, 70 type II IL-1 receptors, 70 IL-7, 325, 342, 419 IL-8, 322–323 IL-11, 414 IL-17, 338–339 Intermittent PTH, 75, 343–344 International Federation of Musculoskeletal Research Societies (IFMRS), 163–164 Interstitial bone, 17 Intestinal calcium absorption, 261–264, 263f Intestinal phosphate absorption, 276–279 Intestinal tract, 262 Intracerebroventrical (icv) administration, 358–359 infusion, 312 Intracortical envelope, 20 Intracortical radial resorption spaces, 93–94 Intracortical remodeling, 92–93 Intracrine mechanism, 68 Intramembranous ossification, 85, 86f, 236 Intrinsic loading models, 206–207 Intrinsic protein kinase activity, receptors with, 58 Invasive (surgical) models, 207–208 Ion channels, 220–221, 222f Ionized calcium (Ca2+), 264, 283 Ionotropic channels, 220–221 IP3. See Inositol-1,4,5-trisphosphate (IP3) IR. See Insulin receptor (IR) Irisin, 323–325 Iron, 285 on FGF23 metabolism, 280–281

Index

J Jagged 1 (JAG1), 414–415 Janus kinases (JAKS), 70 JNK. See c-Jun N-terminal kinase (JNK) jun-D, 46–47

K KDIGO. See Kidney Disease Improving Global Outcomes (KDIGO) Keratocan, 51 Kidney Disease Improving Global Outcomes (KDIGO), 377–378, 377t Kidneys, 375 Kinase Src, 38, 59 Klotho, 55, 70 Knock-in technology, 170–171 Knockout technology, 170–171 Kremen proteins, 64–65

L L-type capillaries, 21 Label escape, 143 Lactate dehydrogenase (LDH), 151–152 Lactation, 199 hormones of, 273–274 Lactobacillus acidophilus, 342–343 Lacunar–canalicular systems, 22–23, 50–51 Lamellae, 8, 13 in cancellous bone, 17 in interstitial bone, 17 in plexiform bone, 14–15 primary osteons in, 15–16 Lanyon, Lance, 204–205, 209, 214 LAPs. See Latency-associated peptides (LAPs) Large latent complexes (LLCs), 75 Latency-associated peptides (LAPs), 75 Latent transforming growth factor-beta binding protein (LTBPs), 74–75 Lathyrism, 136–137 LC. See Locus coeruleus (LC) LD. See Linkage disequilibrium (LD) LDH. See Lactate dehydrogenase (LDH) Lead, 286 LEF. See Lymphoid enhancer-binding factor (LEF) Leptin, 294, 311–312, 312f, 358–360, 358f, 435 Leucine-rich repeat-containing G protein– coupled receptor (LGR4), 60, 63 Leukemia inhibitory factor (LIF), 70, 322–323, 325 Leukemia inhibitory factor, 171–172 LGR4. See Leucine-rich repeat-containing G protein–coupled receptor (LGR4) LIF. See Leukemia inhibitory factor (LIF) LIF receptor beta (LIF-Rβ), 70 Ligand-activated transcription factors, 272 Lining cells, 20, 20f Linkage analysis, 159–160 Linkage disequilibrium (LD), 160–161 Lipocalin-2, 364 Lipoprotein receptor–related protein 4 (LRP4), 50–51, 53, 63 Lipoprotein receptor–related protein 5 (LRP5), 48, 61–63, 196, 224

Lipoprotein receptor–related protein 6 (LRP6), 53, 62 LIPUS. See Low-intensity pulsed ultrasound (LIPUS) LLCs. See Large latent complexes (LLCs) LMW protein. See Low molecular weight protein (LMW protein) Local regulation of bone cells angiogenic factors, 80–81 cell surface attachment molecules, 81–83, 81t through cell–cell contact, 77–80 cytokines and growth factors and receptors, 57 differentiation and activity of osteoblasts and osteoclasts, 59 factors influencing, 59–70, 60t local factors affecting osteoclasts and osteoblasts, 70–77 membrane-bound ligands, 58 receptor classification, 58 signal transduction cascades, 58–59 soluble ligands, 57 of fracture repair bone morphogenetic proteins, 245 EPC, 247 PDGF, 245–246 prostaglandins, 244 skeletal muscle interactions, 247–248 VEGF, 246–247 Locus coeruleus (LC), 354 Log-likelihood score (LOD score), 159–160 Loosely bound water, 23 Low bone mass, 389–390, 390f Low molecular weight protein (LMW protein), 69 Low-intensity pulsed ultrasound (LIPUS), 252 Low-trauma fractures. See Fragility fractures LRP4. See Lipoprotein receptor–related protein 4 (LRP4) LT-α. See Lymphotoxin-alpha (LT-α) LTBPs. See Latent transforming growth factor-beta binding protein (LTBPs) Lucocorticoids (GCs), 344 Lymphoid enhancer-binding factor (LEF), 63 LEF-1, 43 Lymphotoxin-alpha (LT-α), 72 Lysine-specific histone demethylase 1, 419 Lysyl oxidase, 415–416

M Macro-nutrient roles in bone health, 283–288 Macrophage colony-stimulating factor 1 (M-CSF), 37–38, 42, 48–49, 58, 61 Macrophage inflammatory protein 1 alpha (MIP1α), 417–418 Macrophages, 32 MAF. See Minor allele frequency (MAF) Magnesium, 278, 283, 285, 289 deficiency, 434 Magnetic resonance imaging (MRI), 118–119, 119f, 330–331, 421 ultrashort TE sequences, 119

451 Major histocompatibility complex (MHC), 336–337 Malignancies, 33–34 MAML. See Mastermind-like (MAML) Mammalian Notch proteins, 77 Mammalian target of rapamycin (mTOR), 44, 66 MAMPs. See Microbe-associated molecular patterns (MAMPs) Manhattan plot, 161 MAPK. See Mitogen-activated protein kinase (MAPK) MAR. See Mineral apposition rate (MAR) Marrow cavity, 3, 17 MARRS protein. See Membrane-associated, rapid response steroid-binding protein (MARRS protein) Massively parallel reporter assays (MPRAs), 163 Mastermind-like (MAML), 77 Matrix calcification, 89 Matrix extracellular phosphoglycoprotein (MEPE), 10, 12–13, 51, 279, 326 Matrix gla protein (MGP), 384 Matrix metalloproteinases (MMP1, MMP13, MMP-14), 51, 414, 418 Maturation, 189–190 MBTPS1. See Membrane-bound transcription factor peptidase site-1 (MBTPS1) MC. See Methylcellulose (MC) MC3T3-E1 osteoblastic cell models, 48 MC4R. See Melanocortin receptor 4 (MC4R) McNeal tetrachrome, 142f, 144–145 MDMB. See Mean degree of mineralization of bone (MDMB) MDSCs. See Monocytic myeloid suppressor cells (MDSCs); Muscle-derived stem cells (MDSCs) Mean degree of mineralization of bone (MDMB), 97–98 Mechanical loading, 322 Mechanical stimulation effect on fracture repair, 252 Mechanobiology, 220 Mechanoreceptor, 220–223, 221f Mechanosensitive channels, 220–221 Mechanostat, 204–205, 211–213, 212f bone adaptation, 212 bone formation, 213 Frost’s, 213t modeling and remodeling antagonistic processes, 212–213 set points and usage windows, 212 Mechanotransduction, 216–217, 223 MEF2. See Myocyte enhancer factor (MEF2) MEF2C. See Myocyte enhancer factor 2C (MEF2C) Megaanalysis, 161 Megakaryocytes, 32, 421 Melanocortin receptor 4 (MC4R), 364 Melanocortin system, 364 Membrane domain, 37 Membrane-associated, rapid response steroid-binding protein (MARRS protein), 263

452 Membrane-bound ligands, 58 Membrane-bound transcription factor peptidase site-1 (MBTPS1), 328 Mendelian bone disorder, 159 Menopause, 342 MEPE. See Matrix extracellular phosphoglycoprotein (MEPE) MERA. See Multiplexed editing regulatory assay (MERA) Mesenchymal cells, 236 Mesenchymal stem cells (MSCs), 27–28, 64, 236, 383 populations, 30–31 Mesodermal germ layer, 317 Metaanalysis, 161, 395f Metabolic bone disease, 398 Metabolites, 266–269 Metabolomics, 370 Metagenome, 370 Metagenomics, 370b Metallothionein promoter (Mt promoter), 67 Metformin, 437 Methyl methacrylate, 144 Methylcellulose (MC), 218 METTL21C gene, 328 MGP. See Matrix gla protein (MGP) MHC. See Major histocompatibility complex (MHC) Micro-computed tomography (Micro-CT), 113–115, 134, 146–147 chronological assessment, 115f imaging bone vasculature, 116f scanners, 109 Micro-MRI (μMRI), 118 Microbe-associated molecular patterns (MAMPs), 373–374 Microbial metabolites, 374 Microbiome, 369–371, 370b effects on bone, 371–373 on bone mass, remodeling, and strength, 372–373 engineering, 371 mechanisms linking microbiome to bone, 373–374 preclinical manipulations, 371–372 Microbiota, 369, 370f, 370b, 374 Microdamage, 92–93, 93f, 98, 152 accumulation of bone, 25–26, 25f Microfibrils, 7 Microinjection of genetically modified embryonic stem cells, 170–175 Chimeric mice produced by, 179f mouse blastocyst, 175f, 177f simple schematic of gene targeting, 176f uterine implant surgery, 178f Micro–magnetic resonance imaging (Micro-MRI), 431 Micronutrient roles in bone health, 283–288 Microphthalmia-associated transcription factor (Mitf), 44 Microradiography, 104–105, 105f MicroRNAs, 195–196 miR-218, 414–415 Mineral accrual and loss over life span, 259–260

Index

changes in bone dimensions, 260 peak bone mass, 260 placental mineral transport, 259f premature infants, 260 recommended dietary allowances, 260t Mineral apposition rate (MAR), 149 Mineral crystallinity, 137 Mineral deficiencies, 288–289 Mineral homeostasis, 190, 257 in diabetes, 433–434 Mineralization lag time (Mlt), 148, 154 Mineralized matrix, 299 Mineralizing cartilage, 241 Minimum effective strains, 212 Minor allele frequency (MAF), 160–161 MIP1α. See Macrophage inflammatory protein 1 alpha (MIP1α) “Missing heritability” problem, 161 Mitf. See Microphthalmia-associated transcription factor (Mitf) Mitochondrial 25-hydroxyvitamin D-1 alpha hydroxylase/cytochrome p450 27B1, 267 Mitogen-activated protein kinase (MAPK), 61 Mixed uremic osteodystrophy, 378 Mlt. See Mineralization lag time (Mlt) MM. See Multiple myeloma (MM) Modeling analysis, 116–118 Modeling-based formation, 299 Modulus of elasticity, 129 Molecular regulation of chondrogenesis and osteogenesis, 242–244, 243t Monocytic myeloid suppressor cells (MDSCs), 339 Mononucleated preosteoclasts, 47 MORE trial. See Multiple outcomes of raloxifene evaluation trial (MORE trial) Mouse DXA, 372–373 embryonic fibroblasts, 171–172 models, 180 MPRAs. See Massively parallel reporter assays (MPRAs) MRI. See Magnetic resonance imaging (MRI) MSCs. See Mesenchymal stem cells (MSCs) Mt promoter. See Metallothionein promoter (Mt promoter) mTOR. See Mammalian target of rapamycin (mTOR) Multiaxial stacked rosette gauges, 204–205 Multicompartmental calcium kinetic model, 289f Multimodal exercise programs, 228 Multinucleation, 37, 41 Multiple endocrine neoplasias, 261b Multiple myeloma (MM), 411 bone lesions in, 412–413 cells, 417–418 Multiple outcomes of raloxifene evaluation trial (MORE trial), 397 Multiplexed editing regulatory assay (MERA), 163 Multiplication stimulating activity, 65 Muscle, 247

atrophy, 321 bone and, 318 aging effects on musculoskeletal system, 330–332 development and structure, 319–320 endocrine cross talk between muscle and bone, 322–326 endocrine effects, 326–328 linked diseases between bone and muscle, 328–330 mechanical interaction, 321–322, 322f bone’s endocrine effects, 326–328 FGF23, 327 osteocalcin, 327 prostaglandin E2, 328 RANKL, 326–327 sclerostin, Dkk1, and Wnts, 326 unknown factors, 328 homeostasis, 321 satellite cells, 317–318 Muscle-derived stem cells (MDSCs), 247–248 Musclin, 325–326 Muscular dystrophy, 322 Musculoskeletal disease, 328 Musculoskeletal system, 317 aging effects, 330–332 Myeloid cells and bone remodeling, 339 Myeloma bone involvement in, 413f bone remodeling uncoupled in, 412f osteocyte interactions, 420f Myf-5. See Myogenic factor 5 (Myf-5) Myocyte enhancer factor (MEF2), 301 Myocyte enhancer factor 2C (MEF2C), 328 Myogenic factor 5 (Myf-5), 63–64 Myokines, 322–323 Myostatin, 248, 322–325 normal and myostatin-null animals, 324f

N N-methyl-D aspartate (NMDA), 366 n-terminal telopeptide (NTX), 434 nAChR. See Nicotinic ACh receptors (nAChR) NaF. See Sodium fluoride (NaF) NaK-ATPase. See Potassium-transporting ATPase (NaK-ATPase) Nano-CT machines, 109, 113 National Osteoporosis Foundation (NOF), 288–289, 407–408 NCPs. See Noncollagenous proteins (NCPs) NCX1. See Sodium-calcium exchanger (NCX1) NE. See Norepinephrine (NE) Necrotic bone, 340 Needle biopsies of the iliac crest, 141 Neoangiogenesis, 242 Neomycin resistance gene (neor), 172 Nephrocalcinosis, 277b Nephron, 264, 265f Nerve fibers in bone, 22, 352–353 Nerve growth factor (NGF), 22, 351 Nervous system, 349–350 Nestin-GFP+ putative niche cells, 30–31 NET. See Norepinephrine transporter (NET) Net formation, 211–212

Index

Net resorption, 211–212 Neural cell adhesion molecule, 243 Neuroglial cells, 349–350 Neurokinin 1 receptor (NK1R), 365 Neurological disorders, bone impact of, 367 Neuromedin U (NMU), 363 Neuromodulin/GAP-43 protein, 22 Neuronal regulation of hematopoietic stem cell niche, 33 Neurons, 349, 350f Neuropathy, 352–353 Neuropeptide Y (NPY), 22, 352, 361–362 Neutral axis, 132 Neutralization of pathogens, 334–335 Neutrophils, 32 Newborn mineral homeostasis, 190 Next-generation sequencing (NextGen sequencing), 162–164 NF-κB. See Nuclear factor-kappa B (NF-κB) NFATc1. See Nuclear factor of activated T cells (NFATc1) NGF. See Nerve growth factor (NGF) NGF/TrkA signaling, 351–352, 354f NHEJ. See Nonhomologous end joining (NHEJ) Niche cells, 27–28 in development, 28–29, 29f imaging, 29–30 and malignancies, 33–34 Nicotinic ACh receptors (nAChR), 363 Nitric oxide pathway (NO pathway), 224 NK1R. See Neurokinin 1 receptor (NK1R) NKCC2. See Sodium-(potassium)-chloride cotransporter 2 (NKCC2) NMDA. See N-methyl-D aspartate (NMDA) Nmp4. See nuclear matrix protein 4 (Nmp4) NMU. See Neuromedin U (NMU) NO pathway. See Nitric oxide pathway (NO pathway) NOF. See National Osteoporosis Foundation (NOF) Noggin, 21 Nominal resolution, 110 Noncanonical BMP signaling, 73 pathways, 48 Wnt signaling, 62f, 63–64 Noncollagenous extracellular matrix proteins, 10 Noncollagenous proteins (NCPs), 5, 435 Nonenzymatic glycation, 7 Nonhematopoietic bone marrow niche cells, 30–31 Nonhomologous end joining (NHEJ), 176–177 Noninsulin-dependent DM. See Type 2 DM (T2DM) Noninvasive intrinsic loading models, 206–207 Noninvasive models, 208–209 Nonlamellar bone, 14–15 Nonmodifiable risk factors, 430 Nonparametric linkage analysis, 160 Nonsteroidal antiinflammatory drugs (NSAIDs), 244

Nonstochastic radiation effects. See Deterministic radiation effects Nonsurgical models, 208–209 Nonunion fractures, 239–240 Non–weight-bearing sports, 231 Norepinephrine (NE), 356 Norepinephrine transporter (NET), 356–358 Normal bone, 378 Normal plasma concentrations of calcium, 257–258, 258t Normal strain, 127, 128f Normal stresses, 126–127, 128f Notch, 21, 77–78 ICD, 77 Notch2 gain-of-function mutation, 78 signaling, 77, 420 NPT2b. See Sodium-phosphate transporter 2b (NPT2b) NPY. See Neuropeptide Y (NPY) NSAIDs. See Nonsteroidal antiinflammatory drugs (NSAIDs) NTX. See n-terminal telopeptide (NTX) Nuclear factor of activated T cells (NFATc1), 43, 59 Nuclear factor-kappa B (NF-κB), 43, 325 nuclear matrix protein 4 (Nmp4), 222 Nuclear medicine scanning, 121 Nucleotides, 366 Number of trabeculae (Tb.N), 18–19 Nutrient(s) artery, 21 influence bone mass, 288–289 interventions studying effects on bone outcomes, 296–298 Nutrition boron, 286 correcting nutrient deficiency vs. optimizing intake, 296 effects of obesity and weight loss on bone, 294–295 essential and nonessential bone-related nutrients, 284t flavonoids, 293 fluoride, 285–286 lead, 286 macro-and micronutrient roles in bone health, 283–288 nutrients influence bone mass, 288–289 perturbing calcium metabolism, 289–291 RDAs of bone-related nutrients, 287t strontium, 286 studying effects of nutrient interventions on bone outcomes, 296–298 vitamins C, K, A, and D, 286 whole diet matters, 291–293 zinc, 285 Nε-carboxymethyllysine (CML), 8

O OB. See Osteoblasts (OB) OB-6 osteoblastic cell models, 48 ob/ob T2DM model, 352–353 Obesity effects, 294–295, 295f osteoporosis, and fracture risk, 294–295 OC. See Osteocalcin (OC)

453 OcGFPtpz, 242 OCL. See Osteoclasts (OCL) OCT. See Optical cutting temperature compound (OCT) OI. See Osteogenesis imperfecta (OI) Omics analysis of bone phenotypes, 163–164, 163t Oncostatin-M (OSM), 70 ONJ. See Osteonecrosis of the jaw (ONJ) OP-1. See Osteogenic protein-1 (OP-1) Open biopsies, 141 OPG. See Osteoprotegerin (OPG) Opioid use, 367 Opsonization, 334–335 Optical cutting temperature compound (OCT), 144 Optimal bone health, 228–232 Optimization exercise prescription for optimizing skeletal health, 226 ptimizing intake, 296 Oral antibiotics, 371–372 Orexin, 364 Organ system interplay regulates calcium and phosphorus metabolism, 257–260, 258f Organogenesis, 245–246 Orthodontic tooth movement, 91 OSM. See Oncostatin-M (OSM) OSM-specific receptor-deficient mice (OSMR-deficient mice), 70–71 Osteitis deformans, 147–148, 155–157 Osteitis fibrosa cystica, 378 Osteoblastogenesis, 44, 180, 248, 309–310 Osteoblasts (OB), 21, 45–49, 148, 189–190, 236, 240, 341, 364 activity, 412–413 apoptosis, 47–48 inhibition by PTH, 300 bone lining cells, 48–49 cells, 30 formation and differentiation, 46–47 generation and fate, 45f morphology and function, 45–46 and osteocyte morphology by optical microscopy, 46f by transmission electron microscopy, 47f progenitor, 45 cells, 236 PTH effects, 300 regulation of osteoblast generation and survival, 48 role in development and growth of, 418–421 Osteocalcin (OC), 3–4, 13, 182, 242, 286, 327, 366–367, 434–435 Osteoclastogenesis, PTH effects on, 301 Osteoclastogenic cytokines, 334 Osteoclasts (OCL), 32, 37–45, 93, 189–190, 239–240, 334, 339 activity, 412–413 apoptosis, 44 assessment, 148–149 differentiation and fusion, 41–44

454 Osteoclasts (Continued) formation, 414f generation and fate, 42f morphology and function, 37–40, 40f–41f regulation of osteoclast generation and survival, 44–45 role in development and progression of bone metastasis, 417–418 signaling pathways, 43f surface-bound bisphosphonate, 401f Osteoconduction, 245 Osteocytes, 49–55, 151–152, 189–190, 216–217, 326, 420 aging and osteocyte apoptosis, 52 apoptosis, 51 cell processes reside, 220f hormonal regulation of osteocyte life span, 53 indirect tethering of osteocyte-associated proteins, 222f maturation, 51 morphology and functions, 50–51, 50f osteocytic gene expression, 52t osteocytogenesis, 51 preservation viability by mechanical stimuli, 51–52 regulation of bone formation, 53 mineralization, 53–55 resorption, 53 Osteogenesis, molecular regulation of, 242–244 Osteogenesis imperfecta (OI), 6b, 184, 322, 328–329 Osteogenic, 204–206 Osteogenic protein-1 (OP-1), 245 Osteoimmunology, 333 adaptive immunity, 334–337 autoimmune diseases, 344–346 B cells and bone remodeling, 337–338 distinctions between innate and adaptive immunity, 334t endocrine activation of immune-mediated bone loss, 341–344 infections and bone cells, 339–341 innate immunity, 333–334 myeloid cells and bone remodeling, 339 T cells and bone remodeling, 338–339 Osteoinduction, 245 Osteolytic metastases, 412 Osteomacs, 339 Osteomalacia, 154 Osteomimicry, 414–415 Osteomyelitis, 339–340 Osteonal lamellae, 15–16 Osteonecrosis, 402–403 Osteonecrosis of the jaw (ONJ), 422 Osteonectin, 10, 13 Osteons, 17 Osteopenia, 317 Osteopontin, 12–13 Osteoporosis, 126, 153–154, 226, 228, 285–286, 293–295 anabolic therapies, 404–407 bisphosphonate therapy, 398–403, 399f–400f

Index

calcitonin therapy, 398 calcium, 394–396 clinical diagnosis of, 201 definition, 389–390 denosumab therapy, 403–404 diagnosis, 393 drugs effects on fracture healing bisphosphonates, 251 denosumab, 251–252 PTH, 250–251 estrogen therapy, 396 medication effect on glucose metabolism, 440 osteoporosis-pseudoglioma syndrome, 61–62 osteoporosis-related fractures, 389 pathogenesis, 393–394 in pregnancy, 198–199 risk factors, 391–393, 391t selective estrogen receptor modulators, 397–398 sequential treatments, 408–409 therapies, 394–407 treatment guidelines and decisions, 407–410, 408t vitamin D, 394–396 in women, 390f Osteoporotic fractures, 390, 391f OsteoProbe, 436 Osteoprogenitor cells, 236 Osteoprotegerin (OPG), 42, 53, 59–61, 179–180, 196, 326, 337, 361, 393, 414, 417–419, 434 Osteotomy, 207, 235 Osterix (OSX). See Transcription factor Sp7 (SP7) Outside-in signaling, 81 Ovariectomy (OVX), 352–353 Oxidative stress, 435, 249, 436f Oxytocin, 364

P P1NP. See Procollagen type I propeptides (P1NP) P2Rs. See Purinoreceptors (P2Rs) P2X7R complexes, 366 p55. See TNFR-1 receptor p75. See TNFR-2 receptor PACAP. See Pituitary Adenylate cyclase– activating polypeptide (PACAP) Paget disease of bone, 155–157 anabolic therapy, 157 antiresorptive therapy, 155–157 Paired of box protein 3 (Pax3), 63–64 PAM. See Protospacer adjacent motif (PAM) PAMPs. See Pathogen-associated molecular patterns (PAMPs) Pancreatic polypeptide (PP), 361 Paraffin embedding, 143–144 Parafollicular cells, 273 Parametric linkage analysis, 160 Parasympathetic nervous system (PSNS), 363–364. See also Sympathetic nervous system (SNS) acetylcholine, 363

PACAP, 363–364 VIP, 363–364 Parathyroid hormone (PTH), 33, 43, 48, 154, 189, 212, 242–243, 250–251, 250f, 257, 269–272, 271f, 283, 299–301, 300f–301f, 339, 375, 404 control of PTH production and release, 270–272 effects on phosphate reabsorption, 279 T cell role in PTH responses, 343–344 Parathyroid hormone–related peptide receptor (PTH1-R), 68 Parathyroid hormone–related peptide/ protein (PTHrP), 68, 179, 190, 242–243, 257, 269–272, 343, 406, 414–419 Paraventricular nucleus (PVH), 354 Passive transport, 261–262 Pathogen-associated molecular patterns (PAMPs), 333 Pattern recognition receptors (PRRs), 335f pathogen-and danger-associated molecules recognized by, 336t types, 335t Pax3. See Paired of box protein 3 (Pax3) PC2. See Protein convertase 2 (PC2) PCP pathway. See Planar cell polarity pathway (PCP pathway) PCR. See Polymerize chain reaction (PCR) PDGF. See Platelet-derived growth factor (PDGF) PDK1. See 3-Phosphoinositide-dependent kinase 1 (PDK1) Peak bone mass, 260. See also Skeletal development development, 197 Pentosidine, 8, 436 Peptide YY (PYY), 361 Perimysium, 319–320 Periosteal modeling, 97 Periosteal sheath, 19–20, 20f Periosteal surface, 19–20 Periosteum, 19–20, 22, 236 Periosteum-derived SSCs, 45 Peripheral efferent pathways, functional role of, 356–364 Peripheral ganglia, 350 Peripheral nervous system (PNS), 349 Peripheral quantitative computed tomography (pQCT), 431 Peripheral scanners, 109 Perlecan (PLN), 221 Perturbing calcium metabolism, 289–291, 289f–290f PET. See Positron emission tomography (PET) PET–CT. See Positron emission tomographycomputed tomography (PET–CT) PEX. See Phosphate-regulating neutral endopeptidase (PEX) PGC1α. See Proliferator-activated receptor gamma coactivator-1α (PGC1α) Pheochromocytomas, 360 Phosphate, 190, 283–285 wasting, 277b

455

Index

Phosphate-regulating gene with homologies to endopeptidases on X chromosome (PHEX), 279–280, 327 Phosphate-regulating neutral endopeptidase (PEX), 196 Phosphate–PTH–FGF23–klotho loop, 375 Phosphatidylinositol 3,4,5-triphosphate (PIP3), 66 Phosphatidylinositol-4,5-bisphosphate (PIP2), 270–271 Phosphatonins, 279 Phosphoinositide 3-kinase (PI3-K), 42–44, 61 3-Phosphoinositide-dependent kinase 1 (PDK1), 66 Phospholipase C (PLC), 68 Phospholipase C-gamma (PLC-γ), 42–43 Phosphorus/phosphate, 283–285 dietary phosphate intake alter FGF23concentration, 280 distribution in body, 257–258, 258t FGF23, 279–281 intestinal phosphate absorption, 276–279 organ system interplay regulates metabolism, 257–260 parathyroid hormone effects on phosphate reabsorption, 279 regulation of whole body phosphate metabolism, 274–281 renal phosphate reabsorption, 279–281 in Western diet, 280 Phylogenomics, 370b Physical activity, 226–227 skeletal benefits and exercise during growth, 226–227 skeletal responses and exercise in adulthood, 227–228 Physiological processes, 283 PI3-K. See Phosphoinositide 3-kinase (PI3-K) PIP2. See Phosphatidylinositol-4,5bisphosphate (PIP2) PIP3. See Phosphatidylinositol 3,4,5triphosphate (PIP3) Pituitary Adenylate cyclase–activating polypeptide (PACAP), 363–364, 366 PKA. See Protein kinase A (PKA) PKC. See Protein kinase C (PKC) Placental mineral transport, 259f Plain radiography, 240 Planar cell polarity pathway (PCP pathway), 63 Plasma cells, 337 Plasma membrane calcium-transporting ATPase (PMCA), 259 Plasma membrane disruptions (PMDs), 223–224 Plasma phosphate concentrations, 274 Plasmid DNA, 166–167 Plastic-embedded cancellous bone sites, 144 Platelet-derived growth factor (PDGF), 13, 58, 241–242, 245–246, 246f PDGFRα, 30–31 Platelets, 421 PLC. See Phospholipase C (PLC) PLC-γ. See Phospholipase C-gamma (PLC-γ) Pleiotropic genes for bone and muscle, 328

Plexiform bone, 14–15, 15f PLN. See Perlecan (PLN) PLS3, 50–51 Plums, 293 Pluripotent ES cells, 170 PMCA. See Plasma membrane calciumtransporting ATPase (PMCA) PMDs. See Plasma membrane disruptions (PMDs) PMO. See Postmenopausal osteoporosis (PMO) PNS. See Peripheral nervous system (PNS) Podosome belt. See Filamentous actin (F-actin) Podosomes, 37 Poisson’s ratio, 129 Polar moment of inertia, 131 Polarity, 206 Polymerize chain reaction (PCR), 166–167 POMC. See Pro-opiomelanocortin (POMC) Pore water, 22–23 Positive staining, 354 Positron emission decay, 122 Positron emission tomography (PET), 121–122, 421 Positron emission tomography-computed tomography (PET–CT), 121–122, 421 Postmenopausal bone loss, 393 Postmenopausal osteoporosis (PMO), 389, 394–395 Postpartum dual energy X-ray absorptiometry, 273–274 Potassium-transporting ATPase (NaK-ATPase), 264 PP. See Pancreatic polypeptide (PP) pQCT. See Peripheral quantitative computed tomography (pQCT) Prebiotics, 370b Preganglionic fibers, 356 Pregnancy, 198–199 hormones of, 273–274 Pregnant mare serum gonadotropin, 165–166 Prelymphatic vessels, 21 Premature infants, 260 Premetastatic niche generation in bone, 415–416 Presenilin-1 (Psn1), 77–78 Presenilin-2 (Psn2), 77–78 Presynaptic terminals, 349 Primary bone, 14–16 Primary hyperparathyroidism, 154 Primary lamellar bone, 14 Primary osteons, 15–16 Primary repair mechanism, 236 Primary variable, 146, 147t Principal directions, 131 Pro-inflammatory cytokines, 344 Pro-opiomelanocortin (POMC), 364 Proapoptotic effect of glucocorticoids, 306 Probiotics, 370b Procollagen molecule, 5–7 Procollagen type I propeptides (P1NP), 406–407, 421–422, 434

Professional antigen presenting cells. See Dendritic cells Progressive overload principle, 230 Progressive resistance training (PRT), 228 Proliferative zone, 87–89 Proliferator-activated receptor gamma coactivator-1α (PGC1α), 331 Proline-rich tyrosine kinase 2 (Pyk2), 306–307 Pronuclear microinjection, 165 of embryos microinjection, 167 Propranolol, 356–358 Prostacyclin, 76 Prostaglandins, 76–77, 244, 417–418 PGE2, 32, 76–77, 223–224, 244, 328 Prosthetic joint infection, 341 Protein convertase 2 (PC2), 279–280 Protein kinase A (PKA), 63 Protein kinase C (PKC), 63–64, 266 Protein tyrosine kinase 2-beta, 38–40 Protein-calorie malnutrition, 288–289 Proteoglycans, 10–13, 11t–12t, 16–17 Proteomics, 370 Proton secretion, 38–39 Protospacer adjacent motif (PAM), 176 PRRs. See Pattern recognition receptors (PRRs) PRT. See Progressive resistance training (PRT) PRV. See Pseudorabies virus (PRV) PsA. See Psoriatic arthritis (PsA) Pseudoarthrosis, 252 Pseudorabies virus (PRV), 353–354 Psn1. See Presenilin-1 (Psn1) PSNS. See Parasympathetic nervous system (PSNS) Psoriatic arthritis (PsA), 338–339 PTH. See Parathyroid hormone (PTH) PTH receptor 1 (PTHR1), 299–300 PTH-1R. See PTHrP receptor (PTH-1R) PTH1-R. See Parathyroid hormone–related peptide receptor (PTH1-R) PTH–FGF23–1, 25(OH)2D loop, 376–377 PTHR1. See PTH receptor 1 (PTHR1) PTHrP. See Parathyroid hormone–related peptide/protein (PTHrP) PTHrP receptor (PTH-1R), 343 Puberty and adolescence, 192–193 Pulmonary embolism, 397 Pulsed electromagnetic field therapy, 252 Purinoreceptors (P2Rs), 366 PVH. See Paraventricular nucleus (PVH) Pyk2. See Proline-rich tyrosine kinase 2 (Pyk2) Pyridinoline, 7 Pyrophosphates, 12 PYY. See Peptide YY (PYY)

Q Quality of bone, 23–26 Quantitative computed tomography (QCT), 431, 115–116, 431–432 Quantitative ultrasound (QUS), 122 Quantum concept, 91–92 Quiescent osteoblasts, 20, 20f

456 R RA. See Rheumatoid arthritis (RA) Rac. See Ras-related C3 botulinum toxin substrate (Rac) Race and ethnic differences in bone strength, 193–194 Rachitic rosary, 270b Radiation effects, 103–104 Radiography, 104, 240 conventional, 103–105 DXA, 105–108, 106f vertebral fracture assessment, 108f Radionuclide imaging, 119–122 positron emission tomography and PET–CT, 121–122 RAGE. See Receptors for AGEs (RAGE) Raloxifene, 440, 394, 397–398 Raloxifene Use for the Heart trial (RUTH trial), 440 Randomized controlled trials (RCTs), 293 RANK. See Receptor activator of nuclear factor κB (RANK) RANK-Fc fusion protein, 59 RANKL. See Receptor activator of the nuclear factor NF-κB ligand (RANKL) RANKL:OPG ratio, 59, 417–418 RANKL–RANK–OPG system, 59–61 “Rapid losers”, 393–394 Ras-related C3 botulinum toxin substrate (Rac), 38 Rac-Rho, 61 Rate of bone turnover, 23 RBP-JK. See Recombining binding protein suppressor of hairless (RBP-JK) RCTs. See Randomized controlled trials (RCTs) RDAs. See Recommended Dietary Allowances (RDAs) Reactivation, 416–417 of lining cells by PTH, 301 Reactive oxygen species (ROS), 435, 249, 428f Receptor activator of nuclear factor κB (RANK), 42, 58, 179–180, 417 Receptor activator of the nuclear factor NF-κB ligand (RANKL), 37–38, 42–44, 53, 58, 179–180, 196, 224, 274, 293–294, 326–327, 356–358, 394f, 403, 414, 414f, 417–419 Receptor classification, 58 Receptor-interacting protein-1 (RIP-1), 72 Receptors for AGEs (RAGE), 436, 436f Recombinant DNA, 166–167 Recombinant human (rh), 245 Recombinant human PDGF-BB (rhPDGFBB), 249 Recombination process, 335–336 Recombining binding protein suppressor of hairless (RBP-JK), 77 Recommended Dietary Allowances (RDAs), 296 of bone-related nutrients, 287t Red muscles, 320 Reference probe indentation (RPI), 227–228 Referents, 146 Refractory periods, 214, 214

Index

Remodeling canopy, 94 remodeling-based formation, 299 transient, 97 Renal calcium excretion, 257 reabsorption, 264–266 Renal function, 120 Renal insufficiency, 422 Renal osteodystrophy, 377–378 Renal outer medullary potassium channel (ROMK), 264 Renal phosphate reabsorption, 279–281 Repair mechanism of fracture bone remodeling, 239–240 electromagnetic therapy, 252 hard callus formation, 237–239 inflammatory response, 236 LIPUS, 252 local regulation, 244–248 soft callus formation, 237 Reproduction, skeletal changes with lactation, 199 pregnancy, 198–199 Research model creation, 164–178 Reserve zone, 87 Resistance exercise, 331–332 Resorptive modeling, 89 Resting zone, 87 Reversal line. See Cement lines “Reverse” tetracycline-controlled transactivator (rtTA), 181 Reversibility principle, 232 RGD-containing matrix proteins. See Arg-Gly-Asp-containing matrix proteins (RGD-containing matrix proteins) Rheumatoid arthritis (RA), 338, 344–346 leads to bone loss and destruction of articular joints, 345f with synovial membrane pannus formation, 345f rhPDGF-BB. See Recombinant human PDGF-BB (rhPDGF-BB) Ribonucleoprotein (RNP), 177–178 Ribs, 3 Rickets/osteomalacia, 270b RIP-1. See Receptor-interacting protein-1 (RIP-1) RNP. See Ribonucleoprotein (RNP) Rodent ulna model, 209 ROMK. See Renal outer medullary potassium channel (ROMK) Romosozumab, 406–407 ROS. See Reactive oxygen species (ROS) RPI. See Reference probe indentation (RPI) 16S rRNA sequencing, 370 rtTA. See “Reverse” tetracycline-controlled transactivator (rtTA) Ruffled border, 37–39 Runt-related transcription factor 2 (RUNX2), 46–47, 85, 180, 195–196, 242, 384, 419 RUNX2 promoter 1 (RUNX2-P1), 419 RUTH trial. See Raloxifene Use for the Heart trial (RUTH trial)

S Safranin O, 144–145, 145f Salmonella enterica, 340 Sarcopenia, 317, 329–332 Sarcopenic obesity, 331 Scattering, 122 SCF. See Stem cell factor (SCF) Schizophrenia, 367 Schwann cells, 349–350 Sclerosteosis, 406 Sclerostin, 48, 53, 65, 224, 326, 383, 406 SD. See Standard deviations (SD) Sealing zone, 37–38 Secondary (spontaneous) healing of fracture, 236, 237f Secondary bone, 16–17, 16f formation, 239 Secondary hyperparathyroidism, 154–155 Secondary ossification, 86 Secondary osteon, 16 Secondary repair mechanism, 236, 237f Secreted frizzled-related protein (sFRP), 64–65 sFRP-1–5, 48, 64–65, 279 Secreted protein acidic and rich in cysteine (SPARC). See Secreted protein acidic and rich in cysteine (SPARC) Section modulus, 134 “Seed and soil” hypothesis, 413–414 Selectins, 83 Selective estrogen receptor modulators (SERMs), 394, 394f, 397–398 estrogen agonist or antagonist actions, 397t Selective serotonin reuptake inhibitors (SSRIs), 360–361, 367 Self-transcribing active regulatory region sequencing (STRR-seq), 163 Sense organs, 125 Sensitivity, 382b “Sensor” cell type, 216–217 Sensory fibers, 22 Sensory neuroinflammation, 366 Sensory neuropathy, 430 Sensory neurotransmitters in bone, 364–366. See also Central neurotransmitters CGRP, 364–365 glutamate, 366 nucleotides, 366 SP, 365–366 VIP and PACAP, 366 Sepsis, 341 Sequence kernel association test (SKAT), 162–163 Sequential treatments, 408–409 SERMs. See Selective estrogen receptor modulators (SERMs) Serotonin, 360–361, 361f Serotonin transporter (5-HTT), 361 Sex steroid hormones, 303f, 302f, 190–191, 194–195, 301–305, 302f action on membrane-bound receptors, 302–303 estrogen loss and bone loss, 304 during growth, 303 receptor signaling, 302 sexual dimorphism of skeleton, 308–309

Index

Sexual dimorphism, 303 sFRP. See Secreted frizzled-related protein (sFRP) SGLT-2 inhibitors. See Sodium-glucose cotransporter 2 inhibitors (SGLT-2 inhibitors) sgRNA. See Single-chain guide RNA (sgRNA) Shear strain, 127, 128f, 131 Shear stress, 126–127, 128f, 218–220 Short chain fatty acids, 374 SIBLING. See Small integrin-binding ligand N-linked glycoprotein (SIBLING) Signal transducers and activators of transcription (STATs), 70 STAT1, 42–43 Signal transduction cascades, 58–59 Signal-to-noise ratio, 118 Signaling proteins, 38 Silencer of death domains (SODD). See BAG family molecular chaperone regulator 4 (BAG-4) Simple fracture, 235 Single-chain guide RNA (sgRNA), 176 Single-gene disorders, 159 Single-nucleotide polymorphism markers (SNPs), 159–160 SKAT. See Sequence kernel association test (SKAT) Skeletal benefits of physical activity and exercise during growth, 226–227 Skeletal changes across life span with aging, 199–201 in diabetes, 430–433 postnatal growth development of peak bone mass, 197 endocrine regulators, 194–195 fracture risk in children, 197–198 genetic regulation, 195–196 infancy and childhood skeletal growth, 190–191 newborn mineral homeostasis, 190 puberty and adolescence, 192–193 race and ethnic differences, 193–194 timing of growth rates, 196–197 prenatal growth calcification and mineral homeostasis, 190 development and maturation, 189–190 with reproduction, 198–199 Skeletal development, 85–89 endochondral ossification, 85–89, 87f intramembranous ossification, 85, 86f Skeletal envelopes, 19–20 Skeletal genetics genetic analysis of bone phenotypes, 159–164 transgenic animals and bone biology, 179–184 transgenic technology and creation of novel research models, 164–178 Skeletal hard tissue biomechanics bone structure and function, 125–126 mechanical testing, 129–138 methods of testing mechanical properties of bone tissue, 138–140 modulus of elasticity and Poisson’s ratio, 129

Skeletal imaging computed tomography, 103–108 HR-pQCT, 113–118 MRI, 118–119 radionuclide imaging, 119–122 ultrasound, 122 X-ray, 103–108 Skeletal mechanotransduction, animal models of, 206–209 Skeletal muscle, 319, 321–322 interactions, 247–248 regulation, 321 Skeletal perfusion, 21–22 Skeletal radiographs, 421 Skeletal related events (SREs), 417 Skeletal responses to physical activity and exercise, 227–228 principles of training and exercise recommendations, 228–232 Skeletal stem progenitor cells (SSCs), 45 Skeletogenesis, 73–74, 242–243 SLC. See Small latent complex (SLC) SLE. See Systemic lupus erythematosus (SLE) SLRPs. See Small leucine-rich proteoglycans (SLRPs) Smad4 protein, 245 Small integrin-binding ligand N-linked glycoprotein (SIBLING), 10, 12–13, 53–55 Small latent complex (SLC), 75 Small leucine-rich proteoglycans (SLRPs), 10, 12 SMI. See Structural model index (SMI) SNPs. See Single-nucleotide polymorphism markers (SNPs) SNS. See Sympathetic nervous system (SNS) Sodium fluoride (NaF), 10 Sodium-(potassium)-chloride cotransporter 2 (NKCC2), 264 Sodium-calcium exchanger (NCX1), 264 Sodium-glucose cotransporter 2 inhibitors (SGLT-2 inhibitors), 438–439, 439f Sodium-phosphate transporter 2b (NPT2b), 259, 278f Soft callus formation (cartilage formation), 237 Solid mechanics, 126–129 Soluble ligands, 57 Soluble receptors, 57 Solvent drag, 264 Somatotropin/growth hormone, 87–89, 190–191, 273 Somitogenesis, 317 SOS. See Speed of sound (SOS) SOST gene, 51, 406 Sostdc1. See Wise Southern blot analysis, 167, 172 Sox genes, 195–196 SOX-9, 85–86, 242, 250–251 Soy isoflavones, 293 SP. See Substance P (SP) SP7. See Transcription factor Sp7 (SP7) Spacer sequence, 176

457 Spatially averaged, temporally averaged intensity (ISATA), 252 spCas9. See Type II CRISPR-Cas system derived from Streptococcus pyogenes (spCas9) Specificity, 382b principle, 231 Spectral imaging scanning, 112–113 Speed of sound (SOS), 122 Spinal nerves, 350 Spongy bone, 3. See also Cancellous bone Src, proto-oncogene tyrosine-protein kinase, 38, 415–416 SREs. See Skeletal related events (SREs) SSCs. See Skeletal stem progenitor cells (SSCs) SSRIs. See Selective serotonin reuptake inhibitors (SSRIs) Standard deviations (SD), 389–390 Staphylococcus aureus, 340 Staphylococcus epidermidis, 340 Static loads, 210 Static measurement, 146 STATs. See Signal transducers and activators of transcription (STATs) Stem cell niches, 27–28, 28f transplantation, 27 Stem cell factor (SCF), 30 Stereological techniques, 134 Steroid(s), 53, 375 hormones, 44 Stochastic radiation effects, 103–104 Stochastic remodeling, 92 Strain components, 204–206 Strain environment, 204 distribution, 205 duration, 205 energy, 206 frequency, 205–206 loading frequency, 211 magnitude, 204–205, 211 polarity, 206 rate, 205, 206f, 211, 211f Strain in axial loading, 126–128 Streptozotocin-induced T1DM model, 352–353 Stress, 206 in axial loading, 126–128 fracture, 235 stress-concentrating effects, 217–218 Stroma-derived factor 1 (SDF1). See Chemokines—CXCL2 Stromal cells, 415–416 Strontium, 286 STRR-seq. See Self-transcribing active regulatory region sequencing (STRR-seq) Structural model index (SMI), 113 Structural water, 23 Structure model index, 18–19 Subchondral plate, 19 Substance P (SP), 22, 352, 365–366 Sulfonylureas, 437 Sulfur-containing amino acids, 292–293

458 Sunshine vitamin, 266 Superovulation, 165–166 Supplements, 291–292 Suppression of bone turnover, 389 Surgical pin models, 207–208 Sympathetic nerve fibers, 22 Sympathetic nervous system (SNS), 33, 354, 356–363. See also Parasympathetic nervous system (PSNS) CART, 362 dopamine, 362–363 endocannabinoids, 362 leptin, 358–360 NMU, 363 NPY, 361–362 serotonin, 360–361 Sympathetic outflow, 354 Syndecans, 83 Systemic factors in fracture repair aging, 248–249 DM, 249 glucocorticoids, 249 Systemic lupus erythematosus (SLE), 344

T T cells, 335–337, 337f, 346 and bone remodeling, 338–339 role in parathyroid hormone responses, 343–344 T helpers (Th), 338 Th2 CD4+ T cells, 339 T-cell receptors (TCRs), 336–337 T-score, 105 T1D. See Type 1 diabetes (T1D) T2D. See Type 2 diabetes (T2D) Tachykinin 1-deficient mice (Tac1−/− mice), 366 Tag SNPs, 160–161 TAK1. See TGF-β-activated kinase 1 (TAK1) TALENs. See Transcription activator-like effector nucleases (TALENs) Tamoxifen, 182 Targeted remodeling, 92 Tartrate-resistant acid phosphatase (TRAP), 38–39, 144–145, 180–181, 239, 239f, 434 Taxonomic sequencing, 370 Tb.N. See Number of trabeculae (Tb.N) Tb.Sp. See Trabecular separation (Tb.Sp) TBS. See Trabecular bone score (TBS) TCIRG1 gene, mutations in, 39–40 TCRs. See T-cell receptors (TCRs) Technetium-99m methylene diphosphonate (99mTc-MDP), 120 99m Technetium-labeled bisphosphonate bone scans, 421 Telopeptide, 7 Tension, axial loading in, 129–138 Teriparatide, 157, 405–406 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), 151–152 Testosterone, 302 Tetracycline (Tet), 181 double-labeled transiliac crest bone biopsy, 377–378 labeling, 143

Index

Tetracycline-responsive element (TRE), 181 TF. See Tissue factor (TF) TGF-β-activated kinase 1 (TAK1), 73 TGF-β. See Transforming growth factor beta (TGF-β) Th. See T helpers (Th) TH. See Tyrosine hydroxylase (TH) Thiazolidinedione (TZD), 437, 438f Three-dimensional imaging approaches (3D imaging approaches), 134 Thrombospondin (TSP), 10 TSP1 and 2, 12 Thromboxane A2 (TXA2), 76 Thymidine kinase (tk), 172 Thyroid hormones and bone cells, 179, 307–308 Thyroid receptors (TRs), 307 Thyroid stimulating hormone (TSH), 308t, 307 Thyrotoxicosis, 307 Thyrotropin releasing hormone (TRH), 307 Thyroxine, 89, 190–191 Tibial diaphysis, 207–208 Tightly bound water, 23 Tissue, 147–148 quality, 136 radiosensitivity, 103–104, 109–110 strain, 218 tissue-resident macrophages, 339 Tissue factor (TF), 242 TLR. See Toll-like receptor (TLR) TmP/GFR. See Transport maximum for phosphate adjusted for glomerular filtration rate (TmP/GFR) TMV. See Turnover, mineralization, and volume (TMV) TNF. See Tumor necrosis factor (TNF) TNF receptor–associated factor 6 (TRAF6), 43 TNFR. See Tumor necrosis factor receptor (TNFR) TNFR-associated factor 2 (TRAF2), 72 TNFR1-associated DEATH domain protein (TRADD), 72 Toll-like receptor (TLR), 333 Toluidine blue, 144–145, 145f Torsion, 130–131 Tph2. See Tryptophan hydroxylase 2 (Tph2) Trabecula (trabecular tunneling), 17 Trabecular bone, 203, 204f. See also Cancellous bone adaptation, 209 architecture, 23 Trabecular bone score (TBS), 432 Trabecular separation (Tb.Sp), 18–19 Trabecular surfaces, 20, 212–213 tracrRNA. See Trans-acting RNA (tracrRNA) TRADD. See TNFR1-associated DEATH domain protein (TRADD) Traditional knockout models, 182–183 TRAF2. See TNFR-associated factor 2 (TRAF2) TRAF6. See TNF receptor–associated factor 6 (TRAF6) Trampoline, 218–220 Trans-acting RNA (tracrRNA), 176

Transcaltachia, 263 Transcription activator-like effector nucleases (TALENs), 168f, 175 Transcription factor Sp7 (SP7), 46–47, 180, 182, 419 Transcriptomics, 370 Transforming growth factor beta (TGF-β), 10–12, 32, 58, 74–75, 241–242, 318, 416–417 activation, 75 bioavailability, 75 signaling, 414–415 TGF-β-activated signaling pathway in osteoblasts, 74f TGF-β1, TGF-β2, and TGF-β3, 32, 74 Transgenic animals, 164 and bone biology, 179–184 bone cell–specific promoters, 180–181 bridging human disorders and mouse models of bone diseases, 182–183 conditional models using Cre-LoxP technology, 181–182 secondary challenges and genetic background impacts, 183–184 Transgenic rodent production, 164 Transgenic technology, 164–178 blastocysts, collection of, 171–172 chimeric animals, 174 collection of single-cell embryos, 177–178 designer DNA nucleases, 175–178 differential interference contrast (DIC) optics, 167 DNA preparation for microinjection, 167 homologous recombination, 172 homologous recombination in embryonic stem cells, 164 microinjection of genetically modified embryonic stem cells, 170–175 production of transgenic rodents, 164 pronuclear microinjection, 165 Transient receptor potential cation channel subfamily V member 1 (TRPV1), 351 Transient receptor potential cation channel subfamily V member 5 (TRPV5), 263 Transient receptor potential cation channel subfamily V member 6 (TRPV6), 196, 259 Transiliac biopsies, 141 Translocation, 374 Transport maximum for phosphate adjusted for glomerular filtration rate (TmP/ GFR), 279 TRAP. See Tartrate-resistant acid phosphatase (TRAP) Trauma, 236 TRE. See Tetracycline-responsive element (TRE) Treatment guidelines for osteoporosis, 407–410, 408t TRH. See Thyrotropin releasing hormone (TRH) Triglycerides, 3 3,5,3′-l-Triiodothyronine (T3), 308t, 307 Trimeric guanine nucleotide-binding proteins, 222–223

459

Index

Trk-A. See Tyrosine kinase receptor A (Trk-A) TRPV1. See Transient receptor potential cation channel subfamily V member 1 (TRPV1) TRs. See Thyroid receptors (TRs) Tryptophan hydroxylase 2 (Tph2), 360–361 TSH. See Thyroid stimulating hormone (TSH) TSH receptors (TSHR), 307 TSP. See Thrombospondin (TSP) Tumor cells, 415–416, 419 dormancy, 416–417 homing to bone, 415f, 416 intrinsic properties enhancing bone metastatic potential, 414 progression, 416–417 tumor-derived osteoblastic factors, 416t Tumor necrosis factor (TNF), 37–38, 72–73, 417 ligand superfamily member 11, 37–38 TNF-α, 272, 342, 417–418, 435 TNF/FasL receptors, 58 Tumor necrosis factor receptor (TNFR), 57 TNFR-1 receptor, 72 TNFR-2 receptor, 72 TUNEL. See Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Turnover, mineralization, and volume (TMV), 377–378, 380f, 380t Twist-related protein 1 (Twist), 46–47 Twist-related protein 2 (Dermo-1), 46–47 TXA2. See Thromboxane A2 (TXA2) Type 1 diabetes (T1D), 249, 310, 425, 426t BMD in, 431, 432f bone geometry and architecture, 431–432 fracture incidence by age, 429f morbidity and mortality, 430 risk in, 427–430 HbA1c and, 426b hyperglycemia in, 428f pathophysiology, 426f Type 2 diabetes (T2D), 249, 295, 425, 426t BMD in, 431, 432f bone effect and fracture risk, 437t geometry and architecture, 433 fracture morbidity and mortality, 430 risk in, 430 HbA1c and, 426b hyperglycemia in, 428f pathophysiology, 426f Type I collagen (COLIA1), 38–39, 136–137, 196 fibers, 13–14 gene, 180–181, 184 Type II collagen, 89 Type II CRISPR-Cas system derived from Streptococcus pyogenes (spCas9), 176 Type X collagen, 89 TYRO protein tyrosine kinase–binding protein, 41–42

Tyrosine hydroxylase (TH), 356 Tyrosine kinase receptor A (Trk-A), 22, 351 TZD. See Thiazolidinedione (TZD)

U ULs. See Upper Intake Levels (ULs) Ultrashort TE sequences, 119 Ultrasound, 122, 252 Ultraviolet (UV) microscopy, 152 UVB light, 266 UVB radiation, 292 Undercarboxylated osteocalcin (uOC), 434–435 uNTX. See Urinary N-telopeptide of type I collagen (uNTX) uOC. See Undercarboxylated osteocalcin (uOC) Upper Intake Levels (ULs), 296 Urban–Rifkin–Davis syndrome, 75 Uridine triphosphate (UTP), 366 Urinary N-telopeptide of type I collagen (uNTX), 422 Urinary tracer excretion, 297 US Food and Drug Administration (FDA), 246 UTP. See Uridine triphosphate (UTP)

V Vacuolar proton pump (H+/ATPase), 38–39 Van Buchem disease, 406 Vangl2 gene, 63–64 Vascular calcification, 384 Vascular endothelial growth factor (VEGF), 13, 80, 241–242, 246–247, 415–416 alpha, 418–419 binding to tyrosine kinase receptors, 80 cellular targets, 80 role in fracture repair, 236–240 and vascular development, 80–81 VEGF:BMP-4, 246–247 VEGF–Notch–Noggin signaling pathway, 21 Vascular endothelial growth factor receptor 2 (VEGFR2), 31, 418–419 Vascular smooth muscle cells (VSMC), 384 Vascularization, 246–247 Vasculogenesis, 247 Vasoactive intestinal peptide (VIP), 22, 352, 363–364, 366 vBMD. See Volumetric bone mineral density (vBMD) VDR. See Vitamin D receptor (VDR) VDREs. See Vitamin D response elements (VDREs) Vegetable intake, 292–293 VEGF. See Vascular endothelial growth factor (VEGF) VEGFR2. See Vascular endothelial growth factor receptor 2 (VEGFR2) Ventromedial hypothalamus (VMH), 358–359 Versican, 10–12 Vesicular transport model, 263 Vesperlysine, 8

Vessels in bone, 21 in haversian canals, 21 “Vicious cycle”, 417 of osteolytic bone metastasis, 418f VIP. See Vasoactive intestinal peptide (VIP) Visceral afferents, 356 Visual impairments, 430 Vitamin C, K, A and D, 286 Vitamin D, 190, 266–269, 267f, 292, 292f, 394–396 actions, 268–269 for bone health, 394f deficiency, 396 metabolites, 257 Vitamin D receptor (VDR), 196, 263, 268–269, 269f, 311 Vitamin D response elements (VDREs), 269 Vitronectin, 12 VMH. See Ventromedial hypothalamus (VMH) Volkmann’s canals, 16 Voltage-dependent L-type calcium channel subunit alpha-1D, 263–264 Voltage-gated calcium channel subunit alpha Cav1.3. See Voltage-dependent L-type calcium channel subunit alpha-1D Voltage-sensitive channels, 220–221 Volumetric bone mineral density (vBMD), 107, 195 Volumetric DXA, 107–108 Von Kossa tetrachrome, 144–145 VSMC. See Vascular smooth muscle cells (VSMC)

W Waisted section for tensile testing, 129, 130f Wall width (W.Wi), 148–149, 153–154 Water and fat suppressed proton projection MRI (WASPI), 119 WBCT. See Whole body computerized tomography (WBCT) WBSS. See Whole body skeletal surveys (WBSS) WBV. See Whole body vibration training (WBV) Weight loss effects on bone, 294–295 weight-bearing exercise, 227–228 Weinbaum model, 218–220 WES. See Whole exome sequencing (WES) Western diet, 280 WGS. See Whole genome sequencing (WGS) WHI study. See Women’s Health Initiative study (WHI study) White fat, 3 White muscle, 320 WHO. See World Health Organization (WHO) Whole body computerized tomography (WBCT), 421 Whole body skeletal surveys (WBSS), 421 Whole body vibration training (WBV), 228 Whole diet matters, 291–293

460 Whole exome sequencing (WES), 159, 162–163 Whole genome sequencing (WGS), 159, 162–163 Wingless gene (Wg gene), 61 Wise, 65 Wnt signaling, 48, 61–64, 318, 320f canonical, 62f, 63 inhibitors, 64 noncanonical, 62f, 63–64 pathway, 224, 225f, 326 WNT1, 338 Wnt–β-catenin signaling pathway, 87–89, 243–244, 326, 327f Wolff’s law, 203–204, 322

Index

algorithm demonstrating, 204f questions related to, 203 test, 214–215 Women’s Health Initiative study (WHI study), 396, 396f World Health Organization (WHO), 105–107, 389–390 Woven bone, 13–14, 14f, 85, 236, 241

X X-linked hypophosphatemia (XLH), 277b, 279–280 X-ray absorptiometry, 431 Xenotropic and polytropic retrovirus receptor 1 (XPR1), 279

Y Y1 receptors, 361–362 Yellow marrow fat, 3 Yield force, 126 Yield point, 127–128

Z Z-score, 105 Zinc, 285, 288–289 Zinc finger nuclease (ZFN), 168f, 175 Zoledronate, 422 Zoledronic acid, 402f Zone of ossification, 89 Zyxin, 222