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Bone cell differentiation and remodelling are controlled by hormones, growth factors and specific transcriptional regulators. This thesis aims to investigate the role of two transcription factors, c-Fos/AP-1 and Msx-1/2 in osteoclast and osteoblast differentiation using in vivo and in vitro approaches. c-Fos has been shown previously to be essential for osteoclast differentiation and is overexpressed in osteoclasts of Paget's disease. To investigate the role of c-Fos in osteoclasts, transgenic mice were generated where c-fos was overexpressed in osteoclasts using the TRAP promoter. Several TRAP-c-fosLTR transgenic founders were generated which developed severe bone remodelling lesions with eventual tumour formation. Histological and in situ expression studies showed abundant osteoclasts within these lesions which expressed c-Fos, in addition to the anti-apoptotic gene Bcl-2. These features are reminiscent of Pagetic osteoclasts and suggest that these mice are useful for studying bone remodelling disorders. The effects of c-Fos on osteoblasts was next investigated in vitro using a well defined inducible expression system. Stable MC3T3-E1 osteoblastic subclones expressing a tetracycline-regulatable c-fos gene demonstrated that exogenous c-Fos appeared to augment the proliferation induced by BMP-2, and inhibited BMP-2-induced alkaline phosphatase activity during differentiation. Moreover, ectopic c-Fos expression in these cells stimulated apoptosis induced by serum withdrawal and Etoposide. This apoptosis was not effectively blocked by the caspase inhibitors Z-VAD-fmk and DEVD-CHO, but was blocked by the cell cycle dependent kinase (CDK) inhibitor, Roscovitine, and ectopic Bcl-2 or p2l^{WAF1,CIP1,SD11} expression. These results may provide a novel link between growth control and apoptosis in osteoblasts, such that under environmental stress, c-Fos may drive cell cycle progression and render the cell susceptible to apoptosis. Finally, the regulation of Msx2 by osteotropic factors using two reporter gene constructs was analysed in osteoblastic cells. PTH showed no regulation of Msx2 expression, however, small increases were observed with BMPs. The expression of Msx1 during bone development was also analysed in tissues from Msx1-lacZ transgenic mice. LacZ expression was detected in mineralising tissues of the foetus and neonate, but no LacZ expression was observed after birth. Taken together, these data further delineate the functional roles of these transcription factors in bone development and bone disease. Importantly, these studies provide a role for c-Fos in osteoblast apoptosis and osteoclast function, and may serve as a model for c-Fos overexpression in Paget's disease.

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Bone remodelling is a complex process, which involves the coupling of bone formation to completed foci of bone resorption, the balance between these 2 processes determines if bone is lost or gained at a particular site. During bone resorption osteoclasts release growth factors sequestered in bone matrix, which are thought to initiate new bone formation. On the other hand, osteoblasts can regulate osteoclast activity through the expression of the counter-acting cytokines, RANKL and OPG. The aim of this project was to determine if factors released during bone resorption impact on the RANKL/OPG system or on osteoclasts directly to regulate bone remodelling. OPG secretion was characterized in a number of osteoblast-like cells and the osteosarcoma cell line MG-63 was chosen as a model for osteoblastic cell behaviour in vitro. EDTA bone extracts prepared from normal human cortical bone powder were used to treat MG-63 cells in vitro. The response to the extract was dependent on the purification procedure used. OPG production was inhibited by partially purified extracts prepared using hydrophobic interaction chromatography, C18 SPE. In comparison extracts prepared using size exclusion centrifugal filters stimulated OPG secretion in confluent MG-63 cells. Therefore bone matrix constituents were able to influence osteoclast activity directly and indirectly through the osteoblastic cells to produce the same response. The simplest mechanism for this co-ordinated response would be the presence of one factor in the extract that is able to influence both osteoblasts and osteoclasts. The identity of the factor responsible for the opposing effects seen in the bone matrix extracts is at the moment unknown. The work presented in this thesis clearly demonstrated that unknown growth factors present in bone matrix influence bone remodelling.

Osteoarthritis (OA) is the most common joint disease in human populations with onset and severity influenced by mechanical loading, aging effects, genetics, anatomy, and body mass. Despite major advancements in knowledge, the aetiopathogenesis of OA is complex and still poorly understood. Lack of standardization in methods to quantify skeletal OA make it difficult to study the effects of interacting explanatory variables on arthritic response, and prevents comparison of results between bioarchaeological studies. Joint changes of OA as a function of both the natural aging process and of mechanical stress can make an individual appear older than their chronological age, potentially impacting current methods to derive accurate skeletal age at death estimates, particularly in load-bearing regions. This project addressed these issues through three studies, using a large skeletal sample of modern Europeans for which sex, age, and occupation were available. The first study used principal component analysis (PCA) as a standardized procedure to compute aggregate scores for joint complexes and a systemic measure of OA in each region of the lumbar spine, pelvis, and knee. The second study analyzed the composite scores with a multiple regression model to determine the relative contribution of three predictors: age, activity, and body size, and their effect on skeletal expression of OA in each region. Body size (stature and mass) was calculated from postcranial skeletal measurements; torsional strength (J) of the femoral midshaft was calculated from three-dimensional surface models, size standardized and used as a proxy for measure of activity. The third study considered the effect of OA severity on the validity and reliability of three methods to estimate age at death from load-bearing joints of the os coxa: the pubic symphysis, auricular surface, and acetabulum. The study was designed to determine whether OA in adults acts as a potential limitation or benefit in deriving accurate skeletal age at death estimates from pelvic joint morphology that will contribute to standardized methods in establishing physiological degeneration of the skeleton due to aging. Body size and activity factors did not contribute significantly to OA pathology outside of the age-related expression in either of the lumbar vertebrae or knee regions, and only demonstrated a weak association at pelvic joints. Differences in adult patterns of age are reflected in joint arthritic changes of the os coxa and OA severity has an effect on the accuracy of age estimates from the pelvis; those with OA consistently aging faster in all three joint areas. This influence is most significant for young individuals at the auricular surface and pubic symphysis, over-aging at both. Oldest persons with little arthritic patterning at the acetabulum were under-aged, but accuracy of the age estimate improved as OA severity increased. Systemic measures of OA determined through PCA as an indicator of age, appear useful to identify the very old, but may also help to distinguish between systemic age-related stresses and localized biomechanical effects. Interpreting OA as evidence for old age, measures of habitual activity, and larger body mass should be exercised with caution in skeletal populations. / Graduate / 2018-04-18 / 0327 / 0339 / 0571 / calce.stephanie@gmail.com

Nano-FET biosensors are an emerging nanoelectronic technology capable of real-time and label-free quantification of soluble biological molecules. This technology promises to enable novel in vitro experimental approaches for investigating complex biological systems. In this study, we first explored osteocyte mechanosensitivity under different mechanical stimuli and found that osteocytes are exquisitely sensitive to different oscillatory fluid flow conditions. We therefore aimed to characterize protein-mediated intercellular communication between mechanically-stimulated osteocytes and other bone cell populations in vitro to elucidate the underlying mechanisms of load-induced bone remodeling. To this end, we devised a novel nano-manipulation based fabrication method for manufacturing nano-FET biosensors with precisely controlled device parameters, and further investigated the effect of these parameters on sensor performance.

Nano-FET biosensors are an emerging nanoelectronic technology capable of real-time and label-free quantification of soluble biological molecules. This technology promises to enable novel in vitro experimental approaches for investigating complex biological systems. In this study, we first explored osteocyte mechanosensitivity under different mechanical stimuli and found that osteocytes are exquisitely sensitive to different oscillatory fluid flow conditions. We therefore aimed to characterize protein-mediated intercellular communication between mechanically-stimulated osteocytes and other bone cell populations in vitro to elucidate the underlying mechanisms of load-induced bone remodeling. To this end, we devised a novel nano-manipulation based fabrication method for manufacturing nano-FET biosensors with precisely controlled device parameters, and further investigated the effect of these parameters on sensor performance.

No description available.