Sequential angiogenic-osteogenic coupling via a spatiotemporally graded hydrogel enables vascularized bone organoids for critical-sized calvarial defect reconstruction

While bone organoids show potential in mimicking native bone microenvironments, their clinical translation is hindered by insufficient vascularization in large cellular aggregates. We present a spatiotemporally graded hydrogel system enabling sequential angiogenic-osteogenic coupling to achieve functional vascularization of bone organoids. The system integrates a gelatin methacryloyl (GelMA) matrix encapsulating mesenchymal/endothelial cells and dimethyloxalylglycine (DMOG) with silk fibroin microspheres loaded with nano-hydroxyapatite (nHAp). By leveraging differential degradation kinetics, rapid GelMA dissolution coordinates with DMOG release to initiate prevascular networks for metabolic support, while sustained silk fibroin degradation enables nHAp-mediated osteogenic maturation. Dynamic culture over 21 days demonstrated spatiotemporal synchronization of vascular perfusion and bone matrix deposition, overcoming vascularization limitations in organoid scaling. In critical-sized calvarial defects, this sequential coupling strategy achieved significantly greater bone regeneration compared to static scaffolds, with functional microvascular integration. This spatiotemporally graded hydrogel platform establishes a paradigm for engineering metabolically active bone organoids, advancing hierarchical tissue reconstruction strategies.