Tendon function is essential for quality of life, yet the pathogenesis and healing of tendinopathy remains poorly understood compared to other musculoskeletal disorders. The aim of regenerative medicine is to replace traditional tissue and organ transplantation by harnessing the developmental potential of stem cells to restore structure and function to damaged tissues. The recently discovered interdependency of cell phenotype and biophysical environment has created a paradigm shift in cell biology. This dissertation introduces a dynamic in vitro model for tendon function, dysfunction and development, engineered to characterize the mechanobiological relationships dictating stem cell fate decisions so that they may be therapeutically exploited for tendon healing. Cells respond to mechanical deformation via a complex set of behaviors involving force-sensitive membrane receptor activity, changes in cytoskeletal contractility and transcriptional regulation. Effective ex vivo model systems are needed to emulate the native environment of a tissue and to translate cell-matrix forces with high fidelity. A naturally-derived decellularized tendon scaffold (DTS) was invented to serve as a biomimetic tissue culture platform, preserving the structure and function of native extracellular matrix. DTS in concert with a newly designed dynamic mechanical strain system comprises a tendon bioreactor that is able to emulate the three-dimensional topography, extracellular matrix proteins, and mechanical strain that cells would experience in vivo. Mesenchymal stem cells seeded on decellularized tendon scaffolds subject to cyclic mechanical deformation developed strain-dependent alterations in phenotype and measurably improved tissue mechanical properties. The relative tenogenic efficacies of adult stem cells derived from bone marrow, adipose and tendon were then compared in this system, revealing characteristics suggesting tendon-derived mesenchymal stem cells are predisposed to differentiate toward tendon better than other cell sources in this model. The results of the described experiments have demonstrated that adult mesenchymal stem cells are responsive to mechanical stimulation and, while exhibiting heterogeneity based on donor tissue, are broadly capable of tenocytic differentiation and tissue neogenesis in response to specific ultrastructural and biomechanical cues. This knowledge of cellular mechanotransduction has direct clinical implications for how we treat, rehabilitate and engineer tendon after injury. / Ph. D.

Falls among the elderly are a major source of injury, often leading to serious fractures, hospitalization, and death. Osteoporosis (OP) is a global problem intimately related with these fractures, characterized by reduced bone mass, increased bone fragility. There exists a high failure rate in the translation of treatments to osteoporotic populations. Mesenchymal stromal cell (MSC) transplantation as a therapeutic strategy for OP has not yet been examined in clinical trials. This may be attributed to the mixed findings of pre-clinical studies aimed at determining the efficacy of MSC therapy towards bone regeneration in OP. The most common animal model of OP is ovariectomy (OVX) that simulates post-menopausal estrogen loss. A plethora of bone regeneration studies have used OVX models with 12-16 weeks post-OVX periods and have generally reported positive results from a variety of treatment modalities, including MSC therapy. However, the use of the minimum post-OVX period may not be appropriate to reflect the global changes in regenerative potential of OP patients. In our research group's previous study, MSC were isolated from a minimum 60 week post-OVX rat model, representing a severe case of OP. The MSC isolated from these animals are a unique cell population that we expect may better represent the outcomes of autologous cell therapies for the older patient population in the clinic. In the present study, adipose and bone marrow derived MSC from OVX and age-matched animals were evaluated for their osteogenic and adipogenic differentiation potentials in culture through passage 10. Results from this study suggest that bone marrow derived-MSC maintain their phenotype and functionality more effectively than adipose derived-MSC in OP. Further investigations used regenerative medicine approaches for cell expansion on keratin protein coated microcarriers in static culture. Hair-derived keratin biomaterials have demonstrated their utility as carriers of biologics and drugs for tissue engineering. An optimal microcarrier was selected that demonstrated superior retention of the protein coating through electrostatic interactions and high cell viability. Finally, the integration of cell-microcarriers into a perfusion bioreactor system was explored. Preliminary results demonstrated the feasibility of MSC growth and differentiation on microcarrier based packed beds. Moreover, AD-MSC from OP rats were unresponsive to both inductive media and shear stress related osteogenic cues. These results highlight the complexity and challenges associated with the MSC regenerative strategy. / Doctor of Philosophy / Osteoporosis is a skeletal disease that results in reduced bone mass, increased bone fragility and fracture risk. Osteoporotic patients who experience falls suffer serious fractures, hospitalization, and poor bone healing. Several different therapies have been developed for the treatment of osteoporosis, though many are unable to translate from the bench to the clinical population. A popular treatment being investigated is the application of mesenchymal stromal cells (MSC) for fracture repair and the reversal of osteoporotic bone losses. However, cells isolated from aged and osteoporotic patients have been shown to have deficient bone forming properties. Nevertheless, animal models of osteoporosis applying this treatment report amelioration of bone loss. This work seeks to examine a more clinically relevant rat model of osteoporosis. Typical osteoporosis models use an ovariectomy procedure to simulate post-menopausal bone loss on relatively young animals and conduct short-term studies. These studies may not accurately reflect the global regenerative changes in osteoporosis patients or the impaired MSC properties. Adipose and bone marrow derived MSC from a long term ovariectomy model were investigated for their regenerative potentials. MSC growth and bone forming potential was evaluated on keratin protein coated microcarriers in both static and perfusion cultures. Results from this study suggest that bone marrow derived MSC maintain their phenotype and functionality more effectively than adipose derived MSC in osteoporosis. Further preliminary results demonstrated the feasibility of MSC growth and differentiation on microcarrier based packed beds. These results highlight the complexity and challenges associated with the MSC regenerative strategy.

Tendon injuries significantly impact quality of life and are often career ending. Mesenchymal stem cell (MSC) therapy is known to augment intrinsic tendon healing, however, little is known of the stem cells endogenous to tendon, the microenvironmental cues that induce tendon differentiation, and whether individual cells in an inflammatory milieu respond differently to these cues. To address these questions, a three-dimensional tenogenesis assay was developed as an efficient and reproducible metric of cellular capacity to differentiate toward tendon. In contrast to more complex assays of tenogenesis, this design incorporates a simple apparatus using commercially available plasticware for the application of uniaxial static strain in in a type I collagen cell-seeded hydrogel construct. Tendon-related gene expression, glycosaminoglycan levels, elongated cell morphologies and parallel cell alignments are enhanced with BMP-12 induction over ten days of culture. This dissertation provides novel insight to the roles of growth factors in MSC tenogenesis. Tendon healing in vivo is dependent on endogenous tendon stem cells (TSC) that mediate the inflammatory response to injury and promote synthesis of collagen and matrix remodeling, among other extracellular processes. Recent evidence suggests that these cells exist on a spectrum of differentiation potencies, and may be differently committed to the tendon fate. Individual stem cells were isolated from the tendon, and their capacities for proliferation, tri-lineage differentiation and tenogenesis were evaluated. Three distinct TSC phenotypes were revealed, and significant, positive correlations were found in quadra-differentiation potency (toward four lineages) and the expression of a strong, composite tendon phenotype. These studies have important implications in the current standard-of-care in regenerative therapies for tendon. Our benchtop tenogenesis assay can be used to determine the therapeutic potential of allogeneic MSC lines and MSCs from novel sources for 'off-the-shelf' treatments. Our study of TSCs lends valuable insight to the diversity of cell phenotypes found in tendon, and the potential contributions of each phenotype to tendon healing and homeostasis. These results further strengthen the status of tendon as a superior source of stem cells for tendon repair. / Ph. D. / Tendons are fibrous, elastic bands of collagen that connect muscles to bones and are essential to movement and proper functioning of the skeletal system. Weight-bearing tendons like the Achilles in humans and superficial digital flexor tendons in horses are particularly prone to damage and degeneration with overuse and/or aging. Bone marrow-derived stem cell treatments have shown promise in the reduction of pain and inflammation, and restoration of native tendon structure and function in cases of severe tendon injuries. However, the roles of stem cells in tendon healing, particularly their ability to transition to cell types native to tendon and integrate with an environment distinct from their own is unknown. Culturing of stem cells in three dimensional (3D) environments has enabled us to identify and understand the biochemical and mechanical signals that trigger stem cell transitions to tendon cells in tendons, but currently available 3D culture systems are complex and inefficient. In this dissertation we have developed a cost-effective and high throughput 3D culture system to assay the potential of stem cells to form tendon cells and composite tendon-like tissues. Toward this, we have also optimized the effects of known tendon proteins on the tendon fate in 3D culture of stem cells. Like most adult tissues, the tendon encompasses an in-house repository of stem cells. Tendon stem cells (TSCs) are primarily responsible for the inflammatory and reparative responses to tendon injury. Recent evidence suggests that TSCs are diverse in character, and differ from each other in their ability to form cells and tissues of fat, bone and cartilage. In this work, we provide evidence that TSCs are also differently committed to forming tendon tissue, and moreover that significant inter-relationships among gene expression patterns in these cells directly contribute to cultural diversity. In sum, our results provide novel insight to the roles of stem cells in tendon healing, particularly their response to subtle changes in their biochemical environment, and the contributions of individual cells in a milieu to a holistic reparative response.

Osteoarthritis (OA) is a degenerative disease of diarthrodial joints causing pain and loss of joint function. Etiology is heterogeneous, but commonly involves inflammation arising from impairment of normal tissue homeostasis and/or function. A cycle of low-grade inflammation and global tissue degradation causes alteration of tissue morphology and function via primary mechanisms or inability to withstand physiological forces. Current therapies variably ameliorate symptoms but do not modify progression. Mesenchymal stem cells (MSCs) have multi-modal properties but are ineffective in ameliorating equine OA. However, anti-inflammatory activities of bone marrow derived MSCs (BMSCs) are enhanced by three-dimensional spheroid culture so equine BMSC (eBMSC) spheroids could inhibit intra-articular inflammation. The overarching hypothesis is that eBMSCs can be enhanced to produce an allogeneic eBMSC therapy that inhibits intra-articular inflammation. In vitro experiments compared differences in anti-inflammatory phenotype between spheroid and traditionally cultured monolayer eBMSCs, the viability and health of eBMSC spheroids administered through needles, and the effects of allogeneic donor on the anti-inflammatory potential of eBMSC spheroids. A model of equine LPS induced synovitis was used to investigate anti-inflammatory efficacy of spheroid eBMSCs compared to placebo or monolayer eBMSCs in vivo. eBMSCs aggregate into spheroids that have stable stem cell marker expression with increased secretion and gene expression of IL-6 and PGE2, and gene expression of SDF-1 and TSG-6. IFN𝛾 and TNFα were not produced by eBMSC spheroids and IL-10 production varied between individuals. Spheroids maintain higher viability and lower senescence than monolayer eBMSCs after injection through a needle and form in high-throughput culture without detrimental effects on expression of TSG-6, IL-6 and PGE synthases that denote an anti-inflammatory phenotype. Additionally, there is significant variation in this phenotype depending on the eBMSC donor. eBMSC spheroids reduced total nucleated cell counts and objective lameness measurements at peak levels of intra-articular inflammation compared to monolayer cultured eBMSCs in vivo. In summary, spheroids increase anti-inflammatory potential of eBMSCs and are practical for clinical use. Increased anti-inflammatory efficacy was demonstrated in a model of in vivo inflammation. This dissertation provides an understanding of the anti-inflammatory activities of eBMSC spheroids that can be used to develop an OA therapy. / Ph. D. / Osteoarthritis (OA) is a progressive disease of joints causing pain and loss of function. Multiple factors cause OA including inflammation, tissue destruction from enzymes, and breakdown due to reduced strength with continued use. This cycle of inflammation and joint tissue degradation causes joint tissue damage despite treatment with symptom relieving therapies. Mesenchymal stem cells (MSCs) are a multi-modal therapy, but have been ineffective to relieve equine OA. However, MSCs derived from bone marrow (BMSCs) have enhanced anti-inflammatory activity when produced by three-dimensional culture so BMSCs from horses could reduce joint inflammation better as three-dimensional spheroids. The overarching goal of these studies was to produce an “off the shelf” horse BMSC therapy that reduces joint inflammation both for horse treatment, and as a model for human OA. These studies compared differences between spheroid and traditionally grown (monolayer) BMSCs to reduce inflammation, survival of spheroids administered through needles, and the variability between different horse donors on the ability of spheroids to reduce inflammation. The ability of spheroids to reduce joint inflammation was determined in live horses compared to control or monolayer BMSCs. Horse BMSCs form spheroids that retain the properties that define stem cells, plus spheroid BMSCs produce factors that stem cells use to reduce the inflammatory response. Spheroids have enhanced survival compared with monolayer BMSCs after injection through a needle and spheroids can be produced in large quantities without affecting their potential to reduce inflammation. Additionally, BMSCs from different horse donors have varied potential to reduce inflammation. In live horses, donor horse BMSC spheroids reduced signs of joint inflammation and pain when inflammatory levels were highest compared to monolayer BMSCs. This dissertation demonstrates enhanced ability of spheroid BMSCs to reduce inflammation and provides key information that will be used to develop OA therapies.

Gap junctions, comprised of connexin proteins, are essential for direct intercellular electrical, metabolic, and immunological coupling. Connexin43 (Cx43, gene name GJA1) is the most ubiquitously expressed gap junction protein, and Cx43 gap junctions are altered in pathological states including cardiac disease and cancer. The GJA1 mRNA undergoes alternative translation initiation to yield a truncated Cx43 isoform, GJA1-20k, that can regulate gap junction formation. Using epithelial-mesenchymal transition (EMT) as a cellular model of gap junction remodeling, we have demonstrated altered translation initiation of Gja1 as a mechanism by which cellular Cx43 gap junctions can be dynamically regulated. Suppression of Gja1 alternative translation is necessary for Cx43 gap junction loss, and stable expression of GJA1-20k rescues gap junction formation during EMT. To identify regulatory factors acting on the Gja1 mRNA, an MS2 RNA aptamer tagging system was adapted to isolate Gja1 with associated RNA binding proteins. We find the RNA binding protein IMP1 is sensitive to hypoxic stress and complexes with Gja1 mRNA, where it is necessary for alternative translation to generate GJA1-20k. We have demonstrated alterations in translation initiation of the Gja1 mRNA as a critical mechanism by which cells modulate Cx43 gap junctional coupling in changing conditions and identified a novel regulator of this process in mammalian cells. / Doctor of Philosophy / Communication between cells is necessary for healthy function of organs throughout the body. Gap junctions form conduits through which signals can pass directly between neighboring cells. Many diseases, including cancer and heart disease, involve disturbances in gap junction communication. Connexin proteins are the building blocks of gap junctions, and it was recently demonstrated that smaller fragments of connexins are synthesized by cells by a poorly understood process called alternative translation. Importantly, levels of these connexins fragments can alter gap junction formation. We have used mammalian cells to delineate the mechanism by which this alternative protein translation regulates gap junction formation and generated insight into how such protein synthesis is dynamically regulated. Harnessing this knowledge will inform development of new therapeutics inducing alternative translation to rescue gap junctions, and restore normal communication in pathological conditions.

No / There is growing interest in the use of mesenchymal stem cells (MSC) for immune therapy. Clinical trials that use MSC for treatment of therapy resistant graft versus host disease, Crohn's disease and organ transplantation have initiated. Nevertheless, the immunomodulatory effects of MSC are only partly understood. Clinical trials that are supported by basic research will lead to better understanding of the potential of MSC for immunomodulatory applications and to optimization of such therapies. In this manuscript we review some recent literature on the mechanisms of immunomodulation by MSC in vitro and animal models, present new data on the secretion of pro-inflammatory and anti-inflammatory cytokines, chemokines and prostaglandins by MSC under resting and inflammatory conditions and discuss the hopes and expectations of MSC-based immune therapy.

Hui, Fan Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 153-181). / Abstract also in Chinese. / Acknowledgements --- p.ii / Abstract --- p.iii / Abbreviations --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xv / List of Tables --- p.xv / List of Figures --- p.xvi / Major Conference Presentations --- p.xix / Publications in Preparation --- p.xxii / Chapter Chapter 1 --- Study Background --- p.1 / Chapter 1. --- Introduction --- p.2 / Chapter 1.1. --- Spinal Deformities --- p.2 / Chapter 1.1.1. --- Treatment --- p.2 / Chapter 1.2. --- Spinal fusion --- p.4 / Chapter 1.2.1. --- Gold Standard of Spinal Fusion --- p.4 / Chapter 1.2.2. --- Decortication in Spinal Fusion --- p.4 / Chapter 1.2.3. --- Autograft in Spinal Fusion --- p.4 / Chapter 1.2.4. --- Local Factors Influencing Spinal Fusion --- p.5 / Chapter 1.2.5. --- Ultimate Goals of Spinal Fusion --- p.7 / Chapter 1.2.6. --- Limitations of Spinal fusion --- p.7 / Chapter 1.3. --- Alternatives of Different Components for Enhancing Spinal Fusion / Chapter 1.3.1. --- Bone Graft Substitute --- p.9 / Chapter 1.3.2. --- Bioactive Factors --- p.15 / Chapter 1.4. --- Limitations of the Alternative Methods in Spinal Fusion Enhancement --- p.19 / Chapter 1.4.1. --- BMPs --- p.19 / Chapter 1.4.2. --- Gene Therapy --- p.20 / Chapter 1.4.3. --- Biophysical Stimulation --- p.20 / Chapter 1.5. --- Recent Methods in Enhancing Spinal Fusion --- p.21 / Chapter 1.5.1. --- Low Intensity Pulsed Ultrasound --- p.21 / Chapter 1.5.2. --- Mesenchymal Stem Cells in Spinal Fusion --- p.24 / Chapter 1.6. --- Conclusion --- p.26 / Chapter Chapter 2 --- "Hypothesis, Objectives and Plan of Study" --- p.29 / Chapter 2. --- "Hypothesis, Objectives and Plan of Study" --- p.30 / Chapter 2.1 --- Study Hypothesis --- p.31 / Chapter 2.2 --- Study Objectives --- p.31 / Chapter 2.3 --- Plan of Study --- p.32 / Chapter 2.3.1 --- For First Objective --- p.32 / Chapter 2.3.2 --- For Second Objective --- p.32 / Chapter 2.3.3 --- For Third Objective --- p.33 / Chapter Chapter 3 --- In vitro Study of Effect of Low Intensity Pulsed Ultrasound on Mesenchymal Stem Cells --- p.34 / Chapter 3.1. --- Introduction --- p.35 / Chapter 3.2. --- Materials and Methods --- p.36 / Chapter 3.2.1. --- Experimental Animal --- p.36 / Chapter 3.2.2. --- Materials and Reagents --- p.36 / Chapter 3.2.2.1. --- Dulbecco，s Modified Eagle Medium (DMEM) --- p.36 / Chapter 3.2.2.2. --- Phosphate Buffered Saline (PBS) --- p.37 / Chapter 3.2.2.3. --- Osteogenic Medium (OS) --- p.37 / Chapter 3.2.2.4. --- Alkaline Phosphatase (ALP) Buffer --- p.37 / Chapter 3.2.2.5. --- ALP Substrate Buffer --- p.38 / Chapter 3.2.2.6. --- MTT Stock Solution --- p.38 / Chapter 3.2.2.7. --- MTT Working Solution --- p.38 / Chapter 3.2.2.8. --- Lysis buffer --- p.38 / Chapter 3.2.2.9. --- Alkaline Phosphatase (ALP) Working Reagents --- p.39 / Chapter 3.2.3. --- Isolation of Bone Marrow Derived Mesenchymal Stem Cells (BM derived MSCs) --- p.39 / Chapter 3.2.4. --- In vitro Low Intensity Pulsed Ultrasound Treatment --- p.40 / Chapter 3.2.4.1. --- In vitro LIPUS Devices --- p.40 / Chapter 3.2.4.2. --- Treatment Procedure and Experimantal Groupings --- p.40 / Chapter 3.2.5. --- Effect of LIPUS on Cell Viability and Osteogenesis in bone marrow derived-MSCs --- p.41 / Chapter 3.2.5.1. --- Cell Viability Assay --- p.41 / Chapter 3.2.5.2. --- Alkaline Phosphatase (ALP) Enzyme Activity --- p.42 / Chapter 3.2.5.3. --- Cell Morphology and Alkaline Phosphatase Cytochemistry --- p.42 / Chapter 3.2.6. --- Statistical Analysis --- p.43 / Chapter 3.3. --- Results --- p.43 / Chapter 3.3.1. --- Morphology --- p.43 / Chapter 3.3.2. --- Total Number of Viable Cells --- p.44 / Chapter 3.3.3. --- ALP Activity Absorbance --- p.44 / Chapter 3.3.4. --- ALP staining --- p.45 / Chapter 3.3.5. --- Qualitative Analysis --- p.45 / Chapter 3.3.6. --- Quantitative Analysis --- p.46 / Chapter 3.4. --- Discussion --- p.46 / Chapter 3.4.1. --- LIPUS have No Enhancing Effect on Proliferation of MSCs in Basal Medium Nor Osteogenic Medium --- p.47 / Chapter 3.4.2. --- LIPUS Stimulate Proliferation of MSCs in Early Period --- p.49 / Chapter 3.4.3. --- LIPUS Further Enhanced Osteogenesis of MSCs in Osteogenic Medium --- p.49 / Chapter 3.4.4. --- 10 mins LIPUS treatment for 7 days can positively enhance osteogenic differentiation --- p.50 / Chapter 3.4.5. --- Optimum Conditions of LIPUS was Cell Type Dependent --- p.51 / Chapter 3.4.6. --- LIPUS Promoted Osteogenesis in MSCs through Accelerated Mineralization --- p.52 / Chapter Chapter 4 --- Enhancement of Posterior Spinal Fusion The Effect of Tissue-Engineered MSC and Calcium Phosphate Ceramic composite treated with LIPUS in Vivo --- p.68 / Chapter 4.1. --- Introduction --- p.69 / Chapter 4.1.1. --- TCP Biomaterials --- p.70 / Chapter 4.2. --- Materials and Methods --- p.71 / Chapter 4.2.1. --- Materials and Reagents --- p.71 / Chapter 4.2.2. --- Preparation of MSC Derived Osteogenic Cells-tricalcium Phosphate Ceramics Composite --- p.73 / Chapter 4.2.3. --- Posterior Spinal Fusion Surgery --- p.74 / Chapter 4.2.4. --- In vivo LIPUS treatment --- p.75 / Chapter 4.2.5. --- Assessment of Fusion Mass --- p.76 / Chapter 4.2.6. --- Histology --- p.77 / Chapter 4.2.7. --- Statistical Analysis --- p.79 / Chapter 4.3. --- Results --- p.79 / Chapter 4.3.1. --- Fusion by Manual Palpation --- p.79 / Chapter 4.3.2. --- pQCT Analysis --- p.80 / Chapter 4.3.3. --- Histological Analysis --- p.81 / Chapter 4.4. --- Discussion --- p.85 / Chapter 4.4.1. --- Summary of the Findings from Different Assessment Methods --- p.85 / Chapter 4.4.2. --- Addition of MSCs to TCP ceramic in Spinal Fusion --- p.87 / Chapter 4.4.3. --- The Needs of Differentiated MSC in Spinal Fusion --- p.89 / Chapter 4.4.4. --- bFGF Masked the Effect of OS in MSC --- p.91 / Chapter 4.4.5. --- LIPUS Enhanced Bone Formation --- p.95 / Chapter 4.4.6. --- LIPUS Enhanced Bone Formation through Mineralization --- p.96 / Chapter 4.4.7. --- LIPUS Enhanced Spinal Fusion through Bone Remodeling-induced Fusion Mass --- p.97 / Chapter 4.4.8. --- LIPUS Enhanced Bone Formation through Endochondral Ossification --- p.99 / Chapter Chapter 5 --- In Vivo Monitoring of Spinal Fusion in Animal Model with High-resolution Peripheral Quantitative Computed Tomography-A New Pilot Study --- p.122 / Chapter 5.1. --- Introduction --- p.123 / Chapter 5.2. --- Materials and Methods --- p.124 / Chapter 5.2.1. --- Animal Groupings --- p.124 / Chapter 5.2.2. --- Preparation of MSC Derived Osteogenic Cells-tricalcium Phosphate Ceramics Composite --- p.124 / Chapter 5.2.3. --- Posterior Spinal Fusion Operation Procedures --- p.125 / Chapter 5.2.4. --- LIPUS treatment --- p.125 / Chapter 5.2.5. --- High-resolution Peripheral Quantitative Computed Tomography …- --- p.125 / Chapter 5.2.6. --- Analysis with HR-pQCT --- p.126 / Chapter 5.3. --- Result --- p.128 / Chapter 5.3.1. --- Qualitative Observations from HR-pQCT Images --- p.128 / Chapter 5.3.2. --- Quantitative Analysis --- p.129 / Chapter 5.4. --- Discussion --- p.130 / Chapter Chapter 6 --- "Overall Summary, Discussion and Conclusion" --- p.140 / Chapter 6.1. --- Overall Summary and Discussion --- p.141 / Chapter 6.2. --- Limitations and Further Studies --- p.145 / Chapter 6.3. --- Conclusions --- p.147 / Chapter 6.4. --- Summary Flowchart of the whole thesis --- p.148 / References --- p.153

Smad7作為轉化生長因數-β信號通路中的負性調節因子為人所知，異常的Smad7表達通常會引發癌症及組織纖維化等疾病。而目前對於其在骨重建及其相關疾病中的作用尚未有研究。本研究利用Smad7部分敲除小鼠來探索Smad7在骨重建，骨質疏鬆以及間充質幹細胞分化等方面的作用。 / 本研究所用的Smad7部分敲除小鼠模型來源於已有報導過的Smad7ΔE1(KO)小鼠。該小鼠體內Smad7基因組外顯子I的翻譯區被替換，導致部分蛋白失及其功能破壞。研究結果表明，KO小鼠在6、12、24周齡時股骨遠端幹骺端均有不同程度下降的骨小梁數目、厚度，骨礦化率，骨密度，骨體積分數，及其上升的骨小梁間隙和破骨細胞表面。骨髓來源間充質幹細胞的多向分化實驗表明，KO組呈現出抑制性的成骨能力，表現為鈣結節形成減少，鹼性磷酸酶活性下降，早晚期成骨標記基因表達下降。該組亦表現出促進性的成脂能力，有較多及較早的脂滴形成，成脂標記基因表達上升。而對於骨髓來源巨噬細胞的體外破骨誘導實驗表明，KO組有更多且更大的破骨細胞形成，較大的骨吸收面積，以及上升的破骨標記基因表達。卵巢切除小鼠模型的研究表明，術後4、8、16周，KO组的股骨遠端幹骺端对比野生组有更大程度下降的骨形态学参数，以及明顯升高的破骨細胞融合標記蛋白的表達。體外實驗表明KO组有更多且更大的破骨細胞形成，以及更大面積的骨吸收。積雪草酸曾被證實在肝纖維化模型中誘導Smad7 基因的表達，也在本實驗中用以研究對骨質疏鬆疾病的作用。卵巢切除動物模型連續給藥8周後，骨質疏鬆的現象有明顯逆轉，表現為升高的骨形态学参数，及下降的股骨內破骨細胞融合標記蛋白的表達。 / 總結，本研究證實了Smad7在骨骼發育重建及骨疾病的病理機理等方面的研究提供了突破性的見解。部分敲除Smad7可以導致抑制性的成骨能力，促進性的破骨能力，以及損傷性的骨重建，亦會加速骨質疏鬆的進程，并可作為全新的藥物治療靶點，提示Smad7 本身對於骨重建及骨代謝的保護性作用，為代謝性骨疾病的研究及其臨床藥物開發提供了更廣泛的前景。 / Smad7 has been well documented as a negative regulator of TGF-β signaling, and its altered expression often leads to human diseases such as cancer and fibrosis. However, the role of Smad7 in regulating bone remodeling and related diseases remains unclear. We performed both in vivo and in vitro experiments as well as disease model and drug therapy studies using both wild-type (WT) and Smad7ΔE1 (KO) mice to investigate the functional role of Smad7 in bone remodeling, osteoporosis, and MSCs differentiation. / The Smad7ΔE1 mice were generated by replacing part of the exon1 of Smad7 gene as reported, which resulted in truncated protein and partial loss of Smad7 function. Mice were genotyped by PCR. The μ-CT, histological assays and bone histomorphometric assays in metaphysic region of the femurs showed lower trabecular number (TbN), trabecular thickness (TbTh), mineral apposition rate (MAR), higher trabecular separation (TbSp) and Osteoclast Surface (Oc.S/BS & Oc.N/BS) in the KO mice at 6, 12, to 24 weeks old; as well as lower bone mineral density (BMD) and bone volume fraction (BV/TV) at 24 weeks old in the KO mice. The in vitro BM-MSCs multi-lineage differentiation studies showed the suppressed osteogenic potential in the KO group with fewer mineralized nodules, lower ALP activity and expression of Col1A1, Runx2 and OCN; while the adipogenic potential was elevated with more lipid droplets formation and higher expression of Adipsin and C/EBPα. The osteoclastogenic potential of KO mice BMMs was also elevated, showing higher osteoclasts activity and larger resorptive areas, as well as elevated expression of TRAP and CTR. Both in vivo and in vitro studies of the osteoporotic models showed that the KO mice had lower BMD, TbTh, and higher TbSp compared to the WT mice at 4, 8, 16 weeks after OVX, similar results of lower BV/TV and TbN were observed at 4 weeks after OVX in the KO mice. The RANKL-induced osteoclastogenesis potential was elevated compared to WT mice, with more and bigger osteoclasts, larger resorptive areas, as well as elevated expression of TRAP and CTR. The osteoclastic cell fusion was also enhanced. Treatment of Asiatic acid (one traditional Chinese medicine that has been proved to induce the expression of Smad7 as reported) in the OVX mice reversed the osteoporotic process with increase BMD, BV/TV, TbN, TbTh, and decreased TbSp compared to the untreated group. The osteoclastic cell fusion was suppressed after AA treatment. / Partial loss of Smad7 function leads to impaired bone remodeling in vivo, reduced osteogenesis and enhanced osteoclastogenesis in vitro, and also accelerates the osteoporotic development and osteoclastic cell fusion. Asiatic acid may be a novel potential drug for prevention of osteoporosis. Our findings provide new evidences for a better understanding of the biological functions of Smad7 in bone remodeling and its therapeutic potential for metabolic bone diseases. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Nan. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 131-153). / Abstracts also in Chinese.

No description available.

Master of Science / Department of Anatomy and Physiology / Mark L. Weiss / Wharton's jelly is a non-controversial source of mesenchymal stromal cells. Isolation of the cells is non-invasive and painless. The cells have been shown to have a wide array of therapeutic applications. They have improved symptoms when transplanted in a variety of animal disease models, can be used in tissue engineering applications to grow living tissue ex vivo for transplantation, and can be used as drug delivery vehicles in cancer therapy. The cells have also been shown to be non-immunogenic and immune suppressive. This thesis focuses on optimizing isolation protocols, culture protocols, cryopreservation, and characterization of cells in different growth conditions. Results from the experiments indicate that isolation of cells by enzyme digestion yields cells consistently, a freezing mixture containing 90% FBS and 10% DMSO confers maximum viability, and the expression of mesenchymal stromal cell consensus markers does not change with passage and cryopreservation. The results of the experiments also show that cells grow at a higher rate in 5% oxygen culture conditions compared to 21% oxygen culture conditions, serum does not have an effect on growth of the cells, serum and oxygen do not have effects on the expression of mesenchymal stromal cell consensus markers and the cells are stable without nuclear abnormalities when grown in 5% oxygen and serum free conditions for six passages after first establishing in serum conditions.