Mechanical loading under estrogen deficiency enhances mineralization and osteoblast maturation in an advanced 3D vascularized bone organoid driven by hypertrophy and apoptosis

This study advanced a previously developed humanized vascularized bone organoid model by adding controlled cyclic mechanical loading to investigate how estrogen supplementation versus estrogen withdrawal alters bone mechanobiology. Human bone marrow mesenchymal stem cells were first differentiated into a chondrogenic template, then embedded with HUVECs and additional HBMSCs to generate vascularized, mineralized gelatin-based 3D bone models. After mineralized vascular models were established, constructs were cultured for 21 additional days under static or mechanically stimulated conditions with either continued estrogen supplementation or estrogen withdrawal to mimic postmenopausal estrogen deficiency. Under estrogen supplementation, mechanical loading enhanced mineral production, ALP activity, collagen I synthesis, and vascularization, consistent with coordinated anabolic bone adaptation. Under estrogen withdrawal, mechanical loading produced a distinct disease-associated response, including increased collagenous matrix production, increased calcium deposition, downregulation of early osteoblast markers Runx2 and Opn, and upregulation of DMP1, suggesting accelerated osteoblast maturation. Estrogen withdrawal also increased collagen X and cleaved caspase-3, indicating pathological hypertrophy and apoptosis, while loading still enhanced vascularization. Overall, the study provides a translational 3D vascularized bone model for studying how mechanical stimulation and estrogen deficiency interact in postmenopausal osteoporosis-related osteogenic dysregulation.