[Truncated abstract] Intracellular communication between osteoclasts and osteoblasts is imperative to maintain bone integrity. A myriad of molecules are responsible for regulating osteoblast and osteoclast activity. In particular, it is well documented that osteoblast-derived factors are crucial in directly controlling osteoclast formation and function. Since bone formation is coupled to bone resorption, it would be expected that osteoclasts also have some role in regulating the growth and function of osteoblast cells. However, despite extensive research upon osteoclast and osteoblast biology, the mechanisms by which osteoclasts regulate osteoblast growth and function is not well understood. In an attempt to further elucidate the mechanisms by which osteoclasts and osteoblasts communicate, the technique of subtractive hybridisation was used to identify a novel osteoclastderived factor identical to that of mouse Seminal Vesicle Secretion VII (SVS VII). Previous characterisation of the gene in bone demonstrated that SVS VII was abundantly and specifically expressed by mature osteoclasts (Phan, 2004). Additional research hinted that SVS VII acted as a novel osteoclast-derived factor, that by paracrine mechanisms, targeted osteoblast function (Phan, 2004). However, it remained open as to whether the SVS VII molecule did uniquely target the osteoblast, and whether this interaction influenced bone formation in vivo. Therefore, this thesis endeavoured to functionally characterise the role of the SVS VII molecule in the bone environment. ... Further work is needed to identigy a clear consensus binding sequence, to determine the specificity of the interaction between SVS VII protein and each phage clone, and to isolate a specific binding partner for SVS VII. In conclusion, the studies of this thesis sought to characterise the significance of SVS VII expression by mature osteoclasts, relative to its effects on osteoblast behaviour, but failed to conclusively determine a role for SVS VII in bone. Given that the effects of SVS VII on in vitro osteoblast activity and function are minimal, it is doubtful that SVS VII primarily acts as a paracrine factor integral to osteoblast function. Therefore, these findings conflict with those presented previously (Phan, 2004). However, it was demonstrated that SVS VII treatment was associated with in vivo effect on the skeleton, suggesting that SVS VII may target other elements of the bone microenvironment. Via mechanisms not yet understood, which possibly involves additional factors of the bone 11 extracellular matrix, SVS VII may target a subset of osteoprogenitor cells within the bone environment and act to regulate their proliferation. Therefore, SVS VII may enhance osteogenic precursor cell number at sites of bone formation which would increase the pool of cells that can differentiate down the osteoblast linage and contribute to bone formation. In this regard, SVS VII might function in a manner homologous to the Ly-6 molecule Sca-1 and act as an important factor that maintains a balance between the bone formation and resorption process. Clearly, more work focusing on alternative facets of bone biology is needed to identify whether there is a significant role for SVS VII in skeletal tissue.

Osteoporosis is a skeletal disease affecting 44 million Americans. A primary strategy to prevent osteoporosis is to develop a high peak bone mass in youth. Oral Contraceptives (OCs) alter hormones in women and could affect bone mass development. The interaction between OCs and skeletal mineralization is poorly understood. PURPOSE: Our aims were to 1) compare bone mineral density (BMD) of young women who had a history of OC use with regularly menstruating controls, 2) compare changes in BMD in controls, women who initiate OC use, and those who have a history of use, and 3) to evaluate predictive capabilities of physical activity and years of oral contraceptives use on changes in BMD. METHODS: We recruited women, 18 to 25 years of age, with a history of OC use and controls. BMD at the hip, whole-body, and spine (AP, g/cm�� and width-adjusted lateral, g/cm��) was measured by dual-energy x-ray absorptiometry. Physical activity (METs) was measured via questionnaire and grip strength was evaluated using an isometric dynamometer. RESULTS: Groups were similar in body mass index (BMI), fat mass, grip strength, calcium intake and physical activity but controls were slightly older than OC users. In analysis of covariance (ANCOVA), controlling for age and BMI, controls had significantly greater BMD than OC users at baseline at the AP and lateral spine, hip, and whole-body (p<0.05). By ANCOVA (covariates = age at baseline, change in weight), oral contraceptive users had greater bone loss at L��� in the lateral view than controls whereas, controls had greater increases in L��� volumetric BMD, BMD of the total hip, and whole body than OC users (p<0.05). Stepwise regression results did not reveal years of oral contraceptive use, grip strength, or METs to be a significant predictor of changes in BMD at any site. CONCLUSIONS: We conclude that, in the cross-sectional analysis, oral contraceptive use by young women may compromise bone health during a time when mineral is still accruing. In the prospective analysis, regularly menstruating controls had greater BMD accrual or less bone loss over a 12-month time period than women with a history of oral contraceptive use. / Graduation date: 2005

University of Macau / Institute of Chinese Medical Sciences

A new model of a critically-sized segmental femoral bone defect in rats was developed to enable in vivo imaging and facilitate post-mortem mechanical testing of samples. The critically-sized nature of the model was assessed and confirmed. The efficacy of sustained co-delivery of osteogenic (BMP-2 and TGF- Ò3) and angiogenic (VEGF) growth factors in promoting functional bone repair was assessed. Effects of scaffold modification in terms of geometry and composition were evaluated. The results indicated that co-delivery of BMP-2 and TGF- Ò3 resulted in a dose-dependent improvement in functional bone repair. Modification of the polylactide scaffold to include an absorbable ceramic component and a cored out geometry enhanced rate of union. Addition of VEGF to the scaffold treatment did not significantly impact revascularization of the defect site or functional repair of the bone defect. These data demonstrate that the complex environment of an acute bone defect requires different treatment strategies than simple ectopic models would suggest. A positive predictive correlation between bone repair parameters measured in vivo and mechanical functionality was established. The novel defect model demonstrated robustness and reproducibility. Implications for further research are discussed.

A comprehensive understanding of the interactions between orthopaedic and dental implant surfaces with the surrounding host tissue is essential in the design of advanced biomaterials that better promote bone growth and osseointegration of implants. Dental implants with roughened surfaces and high surface energy are well known to promote osteoblast differentiation in vitro and promote increased bone-to-implant contact in vivo. In addition, increased surface roughness increases osteoblasts response to the vitamin D metabolite 1α,25(OH)2D3. However, the exact mechanisms mediating cell response to surface properties and 1α,25(OH)2D3 are still being elucidated. The central aim of the thesis is to investigate whether integrin signaling in response to rough surface microtopography enhances osteoblast differentiation and responsiveness to 1α,25(OH)2D3. The hypothesis is that the integrin α5β1 plays a role in osteoblast response to surface microtopography and that 1α,25(OH)2D3 acts through VDR-independent pathways involving caveolae to synergistically enhance osteoblast response to surface roughness and 1α,25(OH)2D3. To test this hypothesis the objectives of the studies performed in this thesis were: 1) to determine if α5β1 signaling is required for osteoblast response to surface microstructure; 2) to determine if increased responsiveness to 1α,25(OH)2D3 requires the vitamin D receptor, 3) to determine if rough titanium surfaces functionalized with the peptides targeting integrins (RGD) and transmembrane proteoglycans (KRSR) will enhance both osteoblast proliferation and differentiation, and 4) to determine whether caveolae, which are associated with integrin and 1α,25(OH)2D3 signaling, are required for enhance osteogenic response to surface microstructure and 1α,25(OH)2D3. The results demonstrate that integrins, VDR, and caveolae play important roles in mediating osteoblast response to surface properties and 1α,25(OH)2D3. Silencing of the β1 integrin in osteoblast-like MG63 cells significantly reduced osteogenic response to surface topography and 1α,25(OH)2D3. Silencing of the α5 subunit did not alter the response of MG63 cells to changing surface roughness or chemistry, although future work must confirm these results given similar cell surface α5 integrin expression observed in control and α5-silenced cells. Multifunctional RGD, KRSR, and KSSR coated surfaces show that RGD increased osteoblast proliferation and reduced differentiation, KRSR had no affect on osteoblast phenotype, and KSSR increased osteoblast differentiation. These results suggest that titanium surfaces can be modified to manipulate proliferation and differentiation and that RGD/KSSR functionalized surfaces could be further investigated for use as osteointegrative surfaces. The results using VDR deficient osteoblasts demonstrate that 1α,25(OH)2D3 acts via VDR-dependent mechanisms in cells cultured on titanium surfaces that support terminal differentiation. In caveolae deficient osteoblasts, 1α,25(OH)2D3 affected cell number, alkaline phosphatase activity, and TGF-β1 levels, although levels of osteocalcin and PGE2 were not affected. These results are consistent with the hypothesis that VDR is required for the actions of 1α,25(OH)2D3, but that caveolae-dependent membrane 1α,25(OH)2D3 signaling modulates traditional VDR signaling. The exact mechanisms for this interaction remain to be shown. Overall, these results are important in better understanding the role of β1 integrin partners in mediating osteoblast response to implant surfaces and in understanding how integrin signaling can alter osteoblast differentiation and responsiveness to 1α,25(OH)2D3 via genomic and non-genomic pathways.

The growth plate resting zone consists of hyaline-like chondrocytes disbursed in a proteoglycan rich extracellular matrix. These cells give rise to the columns of the growth zone, consisting of progressively hypertrophic cells. Proliferation of resting zone chondrocytes induced by systemic and local stimuli is the driving force of longitudinal growth of long bones. Therefore, homeostasis of this cell population has great importance. Although the regulation of proliferation and differentiation of these cells has been well studied, little is known about the regulation of their apoptosis. We have previously shown that chelerythrine and tamoxifen induce apoptosis in resting zone chondrocytes in a nitric oxide (NO)-dependent pathway. In this study we explored two physiological apoptogens: inorganic phosphate (Pi) and 17β-estradiol (E₂). We found NO production is necessary in Pi-induced apoptosis. We also found that NO donors induced chondrocyte apoptosis by up-regulating p53 expression, Bax/Bcl-2 expression ratio and cytochrome C release from mitochondria, as well as caspase-3 activity, indicating that NO induces chondrocyte apoptosis in a mitochondrial pathway. Mitogen activated protein kinase (MAPK) activity was involved. A c-Jun N-terminal kinase (JNK) inhibitor, but not inhibitors of p38 or extracellular signal-regulated kinase (ERK1/2), was able to block NO-induced apoptosis, indicating that JNK is necessary in this pathway. Taken together, Pi elevates NO production, which leads to a mitochondrial apoptotic pathway dependent on JNK. On the other hand, although E₂caused apoptosis in resting zone chondrocytes in a dose-dependent manner, up-regulated p53 and Bax, and induced release of cytochrome C from the mitochondria, which indicated a mitochondrial apoptotic pathway, the apoptosis did not involve elevated nitric oxide production or MAPK as was found in Pi-induced apoptosis. This study elucidates the signaling pathway underlying Pi and E₂-induced chondrocyte apoptosis. It has important implications on understanding the development of mammalian growth plate. It also provides further information about the physiological functions of estrogen on longitudinal bone growth.

Despite numerous studies have demonstrated an association of type 2 diabetes mellitus (T2DM) and osteoporosis, the underlying mechanism connecting these two conditions remains elusive. Clinically, combined calcium and vitamin D supplement is the commonest osteoporosis therapy; however, recent studies have suggested an increase in cardiovascular risks associated with calcium plus vitamin D supplementation. Therefore, an alternative strategy in treating osteoporosis patients with T2DM is urgently needed. In this study, we hypothesized that combined administration of menaquinone-4 (vitamin K₂, biologically active form of vitamin K) and 1α,25-dihydroxyvitamin D₃ (vitamin D₃, biologically active form of vitamin D) as a novel therapy in treating osteoporosis of T2DM patients. Anabolic effect of vitamin K₂ and vitamin D₃, alone or in combination, was assessed on primary osteoblasts harvested from the iliac crests of C57BL/KsJ lean (db⁺/m⁺) and obese/diabetic (db⁺/db⁺, leptin receptor-deficient) mice. Furthermore, the underlying cellular mechanism was also investigated. Serum undercarboxylated osteocalcin (an indication of vitamin K₂ level) level was higher whereas vitamin D₃ level was lower in db⁺/db⁺ mice, and sections of the iliac crests of db⁺/db⁺ mice illustrated extensive porous structures filled with enlarged adipocytes compared with db⁺/m⁺ mice. Lower levels of bone anabolic markers and bone formation transcription factors (osteocalcin, Runx2, Dlx5, ATF4, type I collagen, OSX, alkaline phosphatase (ALP) activity, p-Smad1/5/8 and p-ERK1/2) were observed in the osteoblasts of db⁺/db⁺ mice. Acute vitamin D₃ (10 nM) application elicited a more sustained and greater magnitude of increase of [Ca²⁺]ᵢ in osteoblasts of db⁺/m⁺ mice when compared with db⁺/db⁺ mice. A significantly higher level of calcium deposits in osteoblasts was observed in db⁺/m⁺ mice when compared to db⁺/db⁺ mice. Co-administration of vitamin K₂ (10 nM) and vitamin D₃ (10 nM) caused an enhancement of calcium deposits in osteoblasts in both strains of mice. Vitamins K₂ and D₃ co-administration time-dependently (7, 14 and 21 days) increased the levels of bone anabolic markers and transcription factors for bone formation, with a greater magnitude of increase observed in osteoblasts of db⁺/db⁺ mice. Suppressed expression of calcium-sensing receptor (CaSR), F-actin, V-ATPase, vitamin D receptor (VDR) and pregnane X receptor (PXR) observed in osteoblasts of db⁺/db⁺ mice were partially reversed by combined vitamins treatment. Moreover, combined vitamins K₂ plus D₃ treatment significantly enhanced migration and the appearance of surface microvilli and ruffles of osteoblasts of db⁺/db⁺ mice. Effects of combined vitamins K₂ plus D₃ treatment observed in osteoblasts of db⁺/db⁺ and db⁺/m⁺ mice were eradicated by warfarin (20 µM, a vitamin K epoxide reductase inhibitor). Thus, our results illustrate that vitamins K₂ plus D₃ supplementation is a novel therapeutic strategy in treating osteoporosis of T2DM patients. / 儘管大量研究已證明第二類型糖尿病和骨質疏鬆症的關聯，連接這兩個病症的基本機制仍然是難以捉摸的。在臨床上，鈣和維生素D的綜合補充劑是最常見的骨質疏鬆症治療，然而最近的研究卻表明服用鈣和維生素D的綜合補充劑會增加患者的心血管風險，因此急切需要尋找可以給予同時患有骨質疏鬆症和第二類型糖尿病患者的替代治療。在本研究中，我們假設甲萘醌-4（維生素K₂，維生素K生物活性形式）和1α，25 - 二羥基維生素D₃（維生素D₃，維生素D的生物活性形式）可以嘗試在同時患有骨質疏鬆症和第二類型糖尿病患者身上作為一種革新的療法。本研究從C57BL/KsJ瘦削/非糖尿病 (db⁺/m⁺) 的小鼠和肥胖/帶有第二類型糖尿病基因 (db⁺/db⁺) 兼有瘦素受體缺陷的小鼠的髂嵴原始成骨細胞上對維生素K₂和維生素D₃單獨或組合使用的合成代謝作用進行了評估。此外，我們也對該成骨細胞的底層機制進行了一系列的研究。 / 在肥胖/帶有第二類型糖尿病基因的小鼠血清內低羧骨鈣素水平（維生素K₂水平的指標）較高而維生素D水平較低，另外，它們的髂嵴的部分與瘦削/非糖尿病的小鼠相比，呈現出比較廣泛的多孔結構並填滿了擴大的脂肪細胞。從肥胖/帶有第二類型糖尿病基因的小鼠的成骨細胞中，可以觀察到它們的骨合成代謝的標誌物和骨骼形成的轉錄因子 (骨鈣蛋白，Runx2，Dlx5，ATF4，第一類型骨膠原，OSX，鹼性磷酸酶 (ALP) 活性，p-Smad1/5/8和p-ERK1/2) 的水平比較低。急性維生素D₃ (10 nM) 的應用在瘦削/非糖尿病小鼠的成骨細胞比起在肥胖/帶有第二類型糖尿病基因的小鼠的成骨細胞引起更持續和更大幅度的細胞內鈣變化增加。在瘦削/非糖尿病小鼠的成骨細胞中比起在肥胖/帶有第二類型糖尿病基因的小鼠的成骨細胞有顯著較高的鈣沉積形成。維生素K₂ (10 nM) 和維生素D₃ (10 nM) 的綜合藥在兩種小鼠的成骨細胞中可以有效地增強鈣沉積的形成。維生素K₂和維生素D₃的綜合藥對增加骨合成代謝的標誌物和骨形成轉錄因子的水平有時間依賴性 (7，14和21日)，療程越長至21日，在肥胖/帶有第二類型糖尿病基因小鼠的成骨細胞中有更大的幅度的增加。合併維生素治療能部分有效地逆轉在肥胖/帶有第二類型糖尿病基因小鼠的成骨細胞中被抑制表達的鈣敏感受體 (CASR)，F-肌動蛋白，V-ATP酶，維生素D受體 (VDR) 和孕烷X受體 (PXR)。此外，結合維生素K₂加維生素D₃治療顯著增強了肥胖/帶有第二類型糖尿病基因小鼠的成骨細胞的細胞遷移和增加了成骨細胞表面外觀的微絨毛和褶皺。在瘦削/非糖尿病小鼠的成骨細胞及肥胖/帶有第二類型糖尿病基因的小鼠的成骨細胞上結合維生素K₂加維生素D₃的治療效果被華法林 (20 μM，維生素K環氧化物還原酶抑製劑) 根除。因此，我們的結果証明了維生素K₂加維生素D₃補充劑的結合使用可有效地作為治療第二類型糖尿病患者並患有骨質疏鬆症的一種新的治療策略。 / Poon, Chui Wa Christina. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014.n5203 / Includes bibliographical references (leaves 135-151). / Abstracts also in Chinese. / Title from PDF title page (viewed on 26, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

[Truncated abstract] Bone remodeling is an important process to maintain mechanical integrity. It is accomplished by two important steps, bone resorption followed by new bone formation. Osteoclasts and osteoblasts are the principal cells in bone resorption and bone formation, respectively. A multitude of local and systemic factors regulates this process by controlling the cellular activities in bone remodeling compartments (BRC). An imbalance of osteoblastic bone formation and osteoclastic bone destruction will result in the development of skeletal diseases. Recent studies suggested that angiogenesis is closely associated with bone remodeling. The vasculature in bone is important for skeletal development, growth and repair. During endochondral ossification, cartilage is invaded by blood vessels which bring in osteoblast and osteoclast precursor cells, nutrients, growth factors and differentiation factors. During fracture repair, it has been demonstrated that mature osteoclasts produce heparanase which can degrade heparin sulfate proteoglycans, a major component in extracellular matrix (ECM). The process leads to the release of heparin-binding growth factors including vascular endothelial growth factor (VEGF), a potent angiogenic factor which contributes largely to local angiogenesis. In recent studies, endothelial cells have been found to produce bone morphogenetic protein (BMP)-2 and BMP-4 when they are subjected to mechanical stimuli, or a hypoxia environment. Conversely, inhibition of angiogenesis has been shown to prevent fracture healing. In a distraction osteogenesis model, either inhibition of angiogenesis or disruption of the mechanical environment prevents normal osteogenesis and results in fibrous nonunion. .... A total of 42 mice from F1 and F2 generations were genotyped as transgene positive. Preliminary analysis using radiography did not reveal any difference between the gross structures of transgenic and wild type mice. Interestingly, the preliminary histology revealed a decrease in trabecular bone and an increase of lipid space in metaphysis of transgenic mice overexpressing EGFL6. However, further studies will need to be carried out to investigate the role of EGFL6 in angiogenesis and adipogenesis using a transgenic mice model. This will be a prime focus of future work. Collectively, the results presented in this thesis have identified EGFL6, a member of the EGF-like family, as a potential angiogenic factor which may play an important role in bone remodeling. EGFL6 has been found to be expressed highly in calvarial osteoblasts and upregulated during primary murine osteoblast differentiation. EGFL6 has been 8 characterized to be a secreted homomeric complex. More importantly, EGFL6 has been shown to induce angiogenic activity in endothelial cell migration, tube formation and in vivo chick embryo chorioallantoic membrane assay. Furthermore, conditioned medium containing the EGFL6 recombinant protein was shown to induce phosphorylation of ERK in endothelial cells. Inhibition of ERK impaired EGFL6-induced ERK activation and endothelial cell migration. Taken together these studies raise the possibility that EGFL6 has a potential role in angiogenesis, and mediates a paracrine mechanism of cross-talk between vascular endothelial cells and osteoblasts during osteogenesis. An understanding of this process offers the potential to facilitate the development of therapeutic treatments for bone disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Bone is a dynamic tissue that is constantly remodeling to repair damage and strengthen regions exposed to loads during everyday activities. However, certain conditions, including long-term unloading of the skeleton, hormonal imbalances, and aging can disrupt the normal bone remodeling cycle and lead to low bone mass and osteoporosis, increasing risk of fracture. While numerous treatments for low bone mass have been devised, dynamic mechanical loading modalities, such as axial loading of long bones and lateral loading of joints, have recently been examined as potential methods of stimulating bone formation. The effectiveness of mechanical loading in strengthening bone is dependent both on the structural and geometric characteristics of the bone and the properties of the applied load. For instance, curvature in the structure of a bone causes bending and increased strain in response to an axial load, which may contribute to increased bone formation. In addition, frequency of the applied load has been determined to impact the degree of new bone formation; however, the mechanism behind this relationship remains unknown. In this thesis, the application of mechanical loading to treat osteoporotic conditions is examined and two questions are addressed: What role does the structural geometry of bone play in the mechanical damping of forces applied during loading? Does mechanical resonance enhance geometric effects, leading to localized areas of elevated bone formation dependent on loading frequency? Curvature in the structure of bone was hypothesized to enhance its damping ability and lead to increased bone formation through bending. In addition, loading at frequencies near the resonant frequencies of bone was predicted to cause increased bone formation, specifically in areas that experienced high principal strains due to localized displacements during resonant vibration. To test the hypothesis, mechanical loading experiments and simulations using finite element (FE) analysis were conducted to characterize the dynamic properties of bone. Results demonstrate that while surrounding joints contribute to the greatest portion of the damping capacity of the lower limb, bone absorbs a significant amount of energy through curvature-driven bending. In addition, results show that enhanced mechanical responses at loading frequencies near the resonant frequencies of bone may lead to increased bone formation in areas that experience the greatest principal strain during vibration. These findings demonstrate the potential therapeutic effects of mechanical loading in preventing costly osteoporotic fractures, and explore characteristics of bone that may lead to optimization of mechanical loading techniques. Further investigation of biomechanical properties of bone may lead to the prescribing of personalized mechanical loading treatments to treat osteoporotic diseases.

Indiana University-Purdue University Indianapolis (IUPUI) / Mechanical loading associated with exercise is known to benefit bone health; however, most studies explore exercise benefits on bone mass independent of bone structure and strength. The purpose of this dissertation is to explore the response of the skeleton to exercise across the translational divide between animal- and human-based studies, with a particular emphasis on exercise-induced changes in bone structure and estimated strength. To explore the skeletal benefits of exercise, models were used wherein loading is introduced unilaterally to one extremity. Unilateral exercise enables the contralateral, non-exercised extremity to be used as an internal control site for the influences of systemic factors, such as genetics and circulating hormones. In study 1, a dose response between load magnitude and tibial midshaft cortical bone adaptation was observed in mice that had their right tibia loaded in axial compression at one of three load magnitudes for 3 d/wk over 4 weeks. In study 2, the ability of peripheral quantitative computed tomography to provide very good prediction of midshaft humerus mechanical properties with good short-term precision in human subjects was demonstrated. In study 3, collegiate-level jumping (long and/or high jump) athletes were shown to have larger side-to-side differences in tibial midshaft structure and estimated strength between their jump and lead legs than observed in non-jumping athletes. In study 4, prepubertal baseball players followed for 12 months were shown to gain more bone mass, structure and estimated strength in their throwing arm relative to their nonthrowing arm over the course of 12 months. These cumulative data using a combination of experimental models ranging from animal to cross-sectional and longitudinal human models demonstrate the ability of the skeleton to adapt its structure and estimated strength to the mechanical loading associated with exercise. Study of these models in future work may aid in optimizing skeletal responses to exercise.