Effects of Hormones and Load on Murine Trabecular and Cortical Bone Turnover

Obesity presents a number of challenges to healthcare systems worldwide, increasing the risk of several disorders, including bone disorders such as osteoporosis. The effects of impaired hormone signalling in obesity are difficult to separate from those caused by concomitant increases in mechanical load from body weight. The work presented in this thesis explored the roles of testosterone, leptin, and a high-fat diet in bone health, combining animal models and cell culture models to investigate the influence of these factors on bone morphology and metabolism, particularly in the context of hormonal deficiencies and metabolic disorders, which can occur during obesity. Testosterone's anabolic role in bone formation is well established, but this study highlights anti-resorptive effects through the RANKL pathway, which maintains bone mass by suppressing osteoclast activity. In orchiectomised mice, testosterone deficiency led to significant reductions in trabecular bone volume, which were restored by testosterone replacement therapy (TRT). Despite these improvements, TRT did not fully restore tibial mechanical strength, aligning with clinical findings that TRT does not reduce fracture risk in men. This suggests testosterone's effects on bone may involve factors beyond mineral content, such as the extracellular matrix. In vitro investigation revealed no clear relationship between testosterone and gene expression in pre-osteoblast cells, though non-significant trends toward increased RUNX2 expression were observed, suggesting a potential role in osteoblast differentiation. Testosterone also increased collagen and calcium deposition in 3D culture models, reinforcing its anabolic effects on bone. Leptin, traditionally known for regulating metabolism, also plays a crucial role in bone turnover. Leptin deficiency in Ob/Ob mice led to impaired cortical and trabecular bone structure, independent of body weight. These effects were site-specific, with differing impacts on vertebrae and tibiae. Leptin deficiency and HFD both increased marrow adiposity, suggesting a shift toward adipogenesis at the expense of osteogenesis, further complicating the relationship between obesity and bone health. Despite increased body weight, HFD did not enhance bone volume in C57 mice, indicating that obesity can negatively affect bone strength through mechanisms beyond weight-bearing. In vitro, leptin treatment had minimal effects on gene expression in pre-osteoblast cells, but enhanced observed staining intensity for collagen and calcium matrix deposition, highlighting leptin's potential role in promoting bone formation. The study also investigated how leptin and testosterone interact with mechanical loading in bone cells. In 2D cultures, fluid shear stress combined with leptin increased osteogenic marker expression, though no significant changes were observed in gene expression. In 3D cultures, hormone treatments, particularly leptin, enhanced collagen and calcium deposition under mechanical load, suggesting a synergistic effect in promoting bone formation. This research enhances our understanding of how testosterone, leptin, and HFD influence bone health, particularly their interplay with mechanical loading. It challenges existing views on obesity and bone strength, highlighting the complex relationship between metabolic health and bone morphology. The findings suggest that therapies targeting the bone-protective effects of testosterone and leptin could be beneficial for treating osteoporosis and other metabolic bone diseases. Further research is needed to explore the mechanisms underlying hormone signalling and mechanotransduction to better inform future treatments for bone-related complications in individuals with hormonal imbalances and obesity.