Advanced humanised bone model recapitulating molecular and functional signatures of osteoporotic bone for therapeutic research

Postmenopausal osteoporosis is a debilitating bone disease that is associated with bone loss and increased risk of fractures of hip, wrist and vertebrae, which impacts 200 million women worldwide with significant medical and economic burden and is expected to grow with the aging population. Although several anabolic and anti-catabolic therapeutic interventions have been developed to treat osteoporosis, 50-70% of treated individuals still experience an osteoporotic fracture depending on the therapeutic intervention. Decline in circulating estrogen levels after the menopause in women alters the cellular function of osteoblasts and osteoclasts, damages bone vasculature, reduces osteocyte mechanosensitivity, increases sclerostin production (WNT inhibitor) and impacts survival of bone cells. Overall, this leads to an increase in bone turnover, with bone resorption outpacing formation, and bone loss, but also changes in bone tissue mineral composition and distribution. The mechanisms driving these changes have been explored; estrogen deficiency alters the mechanobiological responses of osteocytes, enhances osteocyte-mediated osteoclast activity and causes heterogeneity in the mineral distribution but the underlying mechanisms are not fully understood. Romosozumab, a humanized monoclonal antibody against sclerostin, offers therapeutic potential for postmenopausal osteoporosis. It binds to sclerostin and prevents the inhibition of the WNT/β-catenin pathway and thereby promotes osteogenesis. However, the mechanisms of the therapeutic efficacy remain poorly understood. Most of our understanding about postmenopausal osteoporosis and therapeutics comes from ovariectomized animal models, which do not accurately reproduce human bone dynamics or predict clinical therapeutic responses due to interspecies differences. Although recent in vitro bone models have been developed, these simplified models cannot reproduce human multicellular responses, limiting their translational potential. Thus, there is a distinct need for advanced 3D models of human bone, which can account for paracrine regulation by multiple cells (osteocytes, osteoblasts, osteoclasts, vascular cells) and biophysical conditions. Furthermore, the mechanistic effects of estrogen deficiency on bone vasculature are not fully understood. In the first study of this thesis, (1) an advanced 3D vascularized, mineralized and humanized bone model was developed by following an endochondral ossification priming approach, and (2) this model was applied to mimic postmenopausal osteoporosis and provide a mechanistic understanding of changes in vascularization and bone mineralization in estrogen deficiency. The study confirmed the successful development of a humanised multicellular bone model, which induced formation of vasculature, associated with hypertrophy (collagen X), and promoted mineralization. When the model was applied to study estrogen deficiency, the development of distinct vessel-like structures (CD31+) in the postmenopausal 3D constructs was observed. Moreover, during estrogen withdrawal vascularized bone demonstrated a significant increase in mineral deposition and apoptosis, which did not occur in non vascularized bone. These findings reveal a potential mechanism for bone mineral heterogeneity in osteoporotic bone; whereby vascularized bone becomes highly mineralized whereas in non vascularized regions this effect is not observed. In the second study of this thesis, in vitro vascularized bone models were advanced by incorporating cyclic mechanical stimulation using a commercial bioreactor, to account for the influence of biophysical stimuli that exist in vivo. The results emphasize the need to incorporate biophysical stimulation in studies of skeletal biology and pathogenesis by demonstrating that mechanical loading with estrogen (healthy condition) enhanced mineral production, hypertrophy, apoptosis and vascularization. In contrast, mechanical loading with estrogen withdrawal (disease condition) increased collagen 1 and accelerated the transition of osteoblasts to osteocytes, which was associated with pathological hypertrophy and apoptosis. The findings revealed an interplay between estrogen signalling and mechanical loading in a 3D multicellular, vascularized and humanized bone microenvironment. In the third study, human osteoclasts were included in the bone mimetic models to study estrogen deficiency, sclerostin inhibition and osteocyte driven osteoclastogenesis. Using this advanced 3D bone mimetic model, it was demonstrated that estrogen withdrawal disrupted WNT/β-catenin signalling by enhancing sclerostin production leading to increased osteoclastogenic signalling. Importantly, treatment with an anti-sclerostin monoclonal antibody (romosozumab) partially restored WNT signalling and attenuated osteocyte induced osteoclastogenesis. Furthermore, estrogen withdrawal enhanced fibronectin production in the models, which was consistent with the transcriptomic analysis of human osteoporotic bone. This increase in fibronectin may represent a compensatory ECM response to associated with heterogeneity in the mineralization observed postmenopausal osteoporosis. Interestingly, sclerostin inhibition increased fibronectin network formation indicating a previously unexplored link between sclerostin and ECM organization. Together, this research thesis realized the generation of an advanced humanized multicellular bone mimetic platform and established its translational relevance to study postmenopausal osteoporosis therapeutics. The model elucidated the effect of estrogen withdrawal on vascularization and mineralization dynamics under physiological mechanical loading. Furthermore, role of estrogen deficiency in inhibiting WNT/β-catenin pathway and driving osteocyte-mediated osteoclastogenic signalling was explored. The therapeutic anti-sclerostin antibody (romosozumab) was tested on the models to explore anti-resorptive and anabolic potential. The translational relevance of the model was investigated by comparing its transcriptional profiles of osteoporotic human bone specimens. Together, this research provides a valuable platform for studying osteocyte-ECM-osteoclast interactions and for evaluating next generation therapies in a physiologically relevant niche.

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University of Galway

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University of Galway Theses (PhD Theses)