When nutrition meets bone: modulation of inflammatory signals for skeletal homeostasis and repair Restricted; Files Only
Roberts, Joseph (Spring 2020)
Abstract
Metabolic bone diseases characterized by low bone mass are highly prevalent. Many factors contribute to the development of these diseases, including poor nutrition, gut microbiota, and inflammation. Inflammatory effectors can tip the balance between bone formation and resorption to favor osteoclastic bone resorption. As bone mass declines, the risk of experiencing a fracture increases. While most fractures heal naturally, excessive inflammation can disrupt the well- orchestrated series of cellular and molecular events that would normally heal the broken bone. However, the contributors to this excessive inflammatory response to bone fractures remains poorly understood. The overarching goal of this dissertation is to investigate the inflammatory and nutritional contributors to bone homeostasis and repair.
Using highly informative pre-clinical murine models we sought to address the following three aims: 1) establish the role of the inflammatory modulator Wnt5a in osteoblast and osteoclast coupling; 2) examine the influence of aging on the inflammatory response to fracture; and 3) determine whether probiotics regulate inflammation during fracture healing. In Aim 1 and 2, murine genetic models to conditionally delete Wnt5a in osteoclasts and IL-17ra in periosteal mesenchymal cells were employed. In Aims 2 and 3, the murine mid-diaphyseal femoral fracture model was used to assess fracture healing.
The findings from this dissertation identify inflammatory effectors as important components of bone homeostasis and repair. Aim 1 redefined the role of Wnt5a in controlling bone homeostasis by identifying a unique phosphorylated Wnt5a that is secreted by mature osteoclasts to control bone formation. Results from Aim 2 revealed that increased aged results in a unique systemic cytokine profile after fracture that includes higher levels of the anti-osteogenic proinflammatory cytokine IL-17a. Lastly, Aim 3 demonstrates that influencing the gut microbiome through supplementation with the probiotic species B. adolescentis can dampen systemic inflammation to accelerate healing and protect the intact skeleton against bone loss. The collective results of this dissertation bring novel insight to the growing body of literature which indicate that inflammation is a key negative contributor to bone maintenance and repair. Additionally, we identify probiotic supplements as a therapeutic approach to dampen the negative consequence of systemic inflammation after traumatic bone injuries.
Table of Contents
TABLE OF CONTENTS
INTRODUCTION 1
Societal and Economic Burden of Musculoskeletal Diseases 1
Bone biology 2
The Basic Multicellular Unit 3
Non-canonical Wnt5a control of bone remodeling 4
Mechanisms of fracture healing 5
Patient-specific factors that influence fracture healing 6
Role of inflammation in bone homeostasis and repair 7
Role of IL-17a in bone remodeling and repair 9
Role of the gut microbiota in bone health 10
PURPOSE OF RESEARCH 11
REFERENCES 14
CHAPTER 2: ADVANCES AND PROMISES OF NUTRITIONAL INFLUENCES ON NATURAL BONE REPAIR 19
ABSTRACT 20
INTRODUCTION 21
MECHANISMS OF BONE FRACTURE HEALING 22
METABOLIC RESPONSE TO FRACTURE 23
IMPACT OF NUTRITIONAL DEFICIENCIES ON BONE HEALING 25
INFLUENCE OF NUTRITIONAL SUPPLEMENTS ON NATURAL BONE HEALING 28
NUTRITIONAL INFLUENCES ON PATIENT OUTCOMES 32
EMERGING AREAS AND FUTURE PERSPECTIVES 33
Nontraditional Dietary Supplements 33
Precision Nutrition through Integrated Omics 34
CONCLUSION 35
REFERENCES 37
CHAPTER 3: GENERATION AND EXPERIMENTAL OUTCOMES OF CLOSED FEMORAL FRACTURE IN MICE 55
ABSTRACT 56
1. INTRODUCTION 57
2. MATERIALS 58
3. METHODS 62
4. NOTES 74
REFERENCES 78
CHAPTER 4: DELETION OF WNT5A IN OSTEOCLASTS RESULTS IN BONE LOSS THROUGH DECREASED BONE FORMATION 84
ABSTRACT 85
INTRODUCTION 86
METHODS 87
Animals 87
Lentivirus-mediated deletion of Wnt5a 88
Bone marrow macrophage (BMM) osteoclast formation assay 89
Isolation of periosteal cells 89
Periosteal cell osteogenesis assay 90
Gene expression 90
Micro-computed tomography 91
Histology and static histomorphometry 92
Dynamic Histomorphometry 92
Measurement of bone turnover markers 93
Post-translational modification (PTM) and phosphorylation detection assays for WNT5A 93
Statistics 95
RESULTS 95
Deletion of Wnt5a decreases osteoclast formation and function in vitro 95
Generation of mice with conditional deletion of Wnt5a in mature osteoclasts 95
Conditional deletion of Wnt5a in mature osteoclasts results in an unexpected decrease in bone mass 96
Decreased bone formation contributes to the low-bone-mass phenotype in Wnt5a cKO mice 98
Non-specific deletion of Wnt5a in periosteal mesenchymal cells does not affect osteogenic differentiation potential 98
Osteoclast-derived WNT5A is distinct from osteoblast-derived WNT5A 99
DISCUSSION 100
REFERENCES 106
CHAPTER 5: AGED MICE HAVE A UNIQUE SYSTEMIC CYTOKINE PROFILE DURING FRACTURE HEALING CHARACTERIZED BY HIGH LEVELS OF ANTI-OSTEOGENIC IL-17A 121
ABSTRACT 122
INTRODUCTION 123
METHODS 124
Animal husbandry 124
Fracture model 124
Micro-computed tomography 125
Histology and static histomorphometry 126
Immunohistochemistry 126
Measurement of serum cytokines 127
Periosteal cell isolation 128
Periosteal cell osteogenic differentiation 128
Gene expression 129
Statistics 129
RESULTS 129
Aged mice are more impacted by femoral fracture 129
Fracture healing is delayed in aged mice 130
The inflammatory response to fracture is exacerbated in aged mice 130
Deletion of IL-17ra in the periosteum increases callus bone volume 131
IL-17a inhibits osteogenic differentiation of fracture activated periosteal cells 131
DISCUSSION 132
REFERENCES 137
CHAPTER 6: CONTROL OF FRACTURE-INDUCED GUT LEAKINESS BY BIFIDOBACTERIUM ADOLESCENTIS 149
ABSTRACT 150
INTRODUCTION 151
METHODS 152
Animal husbandry 152
Assessment of gut permeability after fracture 153
Probiotic manipulation of gut microbiota 153
Fracture model 154
Bacterial culture 154
Micro-computed tomography 155
Histology and static histomorphometry 156
Gene expression 157
Measurement of serum inflammatory and bone turnover markers 157
Measurement of serum endotoxin/lipopolysaccharide 158
16s rRNA Sequencing 158
Statistical & Bioinformatic analysis 159
RESULTS 159
Fracture induces gut permeability 159
Probiotic supplementation accelerates callus cartilage remodeling 160
B. adolescentis dampens the systemic inflammatory response to fracture 161
Probiotic supplementation increases expression of intestinal tight junction proteins after fracture 162
B. adolescentis supplementation influences the diversity of the gut microbiome during fracture healing 163
Probiotic supplementation protects against post-traumatic bone loss 164
DISCUSSION 165
REFERENCES 172
CHAPTER 7: DISCUSSION AND CONCLUSIONS 192
KEY FINDINGS 193
STRENGTHS, INNOVATIONS, AND LIMITATIONS 194
FUTURE DIRECTIONS 195
CONCLUSIONS 198
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