Estrogen Deficiency alters Vascularization and Mineralization dynamics: insight from a novel 3D Humanized and Vascularized Bone Organoid Model

Abstract Osteoporosis is not merely a disease of bone loss but also involves changes in the mineral composition of the bone that remains. In vitro studies have investigated these changes and revealed that estrogen deficiency alters osteoblast mineral deposition, osteocyte mechanosensitivity and osteocyte regulation of osteoclastogenesis. During healthy bone development, vascular cells stimulate bone mineralization via endochondral ossification, but estrogen deficiency impairs vascularization. Yet, existing in vitro bone models overlook the role of vascular cells in osteoporosis pathology. Thus, here we (1) develop an advanced 3D vascularized, mineralized and humanized bone model following the endochondral ossification process, and (2) apply this model to mimic postmenopausal estrogen withdrawal and provide a mechanistic understanding of changes in vascularization and bone mineralization in estrogen deficiency. We confirmed the successful development of a vascularized and mineralized human bone model via endochondral ossification, which induced self-organization of vasculature, associated with hypertrophy (collagen X), and promoted mineralization. When the model was applied to study estrogen deficiency, we reported the development of distinct vessel-like structures (CD31+) in the postmenopausal 3D constructs. 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-vascularised regions this effect is not observed. New and Noteworthy Here we develop an in vitro 3D vascularized and humanized bone model following an endochondral ossification approach. We applied the model to recapitulate estrogen deficiency as representative of osteoporotic phenotype. The results of this study reveal that estrogen deficiency exacerbates formation of 3D vessel like structures in vascularized models and thereby drives mineral deposition.