Intra-spheroidal BMP-2 delivery in co-cultured spheroids enables concurrent vasculogenesis and osteogenesis in 3D engineered bone constructs.

Critical-sized bone defects demand rapid vascularization for graft success, yet conventional pre-vascularization strategies have failed to coordinate vessel formation with osteoinduction. Here, we developed a GelMA-based 3D construct encapsulating co-cultured spheroids composed of human adipose-derived stem cells and human umbilical vein endothelial cells, incorporating BMP-2-immobilized nanofibers that directly deliver BMP-2 within the spheroid microenvironment. This spatially confined delivery system allowed BMP-2 to concurrently enhance hADSC osteogenesis, HUVEC functionality, and pericyte-associated phenotype, achieving concurrent vasculogenesis and osteogenesis. The spheroids maintained high viability and supported extensive endothelial sprouting, pericyte-like outgrowth, and mineral deposition. Compared with conventional BMP-2 delivery, intra-spheroidal presentation significantly enhanced angiogenic sprouting, cellular proliferation, matrix remodeling, and osteogenic differentiation from co-culture spheroids. Upon subcutaneous implantation, the hydrogels exhibited enhanced host-graft vascular integration, functional intragraft perfusion, microvascular density, and ectopic mineralized tissue formation with functionally matured vessels averaging 16.1 ± 3.8 μm in diameter. In a calvarial defect model, the constructs further promoted defect bridging and bone regeneration, accompanied by increased vascular formation, greater retention of implanted human cells at 8 weeks, and pronounced osteogenic contributions evidenced by HNA+OPN+ cells comprising 73.5 ± 5.9% of osteogenic regions. Collectively, this nanofiber-mediated intra-spheroidal BMP-2 delivery strategy promoted concurrent vasculogenic and osteogenic responses across in vitro, ectopic, and orthotopic models, highlighting its potential for vascularized bone engineering.