Development-Informed Microsphere Assemblies Enable Spatiotemporally Programmed Bone-Vascular Organoid-Like Constructs for Large Bone Defect Repair

Large segmental bone defects are difficult to repair because endogenous stem-cell activation, vascular ingrowth, and osteogenic maturation are insufficient to rebuild organized bone tissue. Bone organoids offer a promising regenerative strategy, but current systems are limited by Matrigel dependence, poor vascularization, low post-implantation survival, and inadequate reconstruction of hierarchical bone microarchitecture. Here, we report a development-informed, Matrigel-free bone-vascular organoid-like platform that integrates single-cell-guided gene programming with an osteoinductive and vascular-permissive microsphere matrix. Single-cell analysis identified BMP4 and VEGFA as key genes linking osteogenic commitment with vascular morphogenesis. Engineered BMSCs were used to sequentially generate osteoinductive bone organoid-like constructs(OLCs) and bone-vascular OLCs with coordinated osteogenic and angiogenic activity. Microfluidic GelMA/PEO/bioactive glass(BG) microsphere clusters provided adhesive, mineralization-promoting, and space-making cues for cell migration, matrix deposition, and endothelial invasion. Within seven days, the OLCs developed mineralized matrix, vascular-like sprouting, and early vascular-like microchannels. After implantation into rabbit femoral defects, they enhanced vascularized bone formation, defect bridging, and trabecular remodeling. This work demonstrates the power of development-informed, Matrigel-free bone-vascular OLCs in accelerating vascularized bone regeneration for large segmental bone defect repair.