Generation of 3D humanized vascularized and mineralized bone mimetic model via endochondral ossification

This protocol describes the development of a 3D vascularized and humanized bone model that recapitulates healthy bone formation through endochondral ossification. Human bone marrow stem cells (HBMSCs) are first differentiated into a chondrogenic template within gelatin-mtgase hydrogels, then embedded into a second hydrogel layer, which is co-seeded with HBMSCs and HUVECs to promote both vascularization and osteogenesis under estrogen supplementation. Human pre-osteoclasts are seeded on the mineralized vascular construct. The resulting construct shows organized vasculature, osteoclast differentiation, expression of osteogenic markers, and mineral deposition. This model provides a physiologically relevant, multicellular platform for studying human bone development and can be effectively modulated to conduct hormone withdrawal studies to model disease phenotype.