Hierarchically released biomimetic scaffold incorporating VEGF, bFGF, and BMP2 synergistically promotes angiogenesis and bone regeneration

• We engineered a hierarchically releasing biomimetic scaffold (GelMA microspheres/silk fibroin composite) that mimics natural bone healing: early burst release of VEGF/bFGF promotes angiogenesis, followed by sustained release of BMP2 driving osteogenesis. This spatiotemporal control overcomes limitations of single/simultaneous factor delivery. • Pretreatment of MSCs with low-dose bFGF (10–20 ng/mL) for 1 day, before BMP2 application, significantly potentiates osteogenic differentiation. This synergy operates via the PI3K/Akt-GSK3β-β-catenin pathway, enhancing β-catenin expression and nuclear translocation. • The study provides a rational design blueprint for bioactive bone grafts: optimal factor concentrations (BMP2:500 ng/mL, VEGF:20 ng/mL, bFGF:10–20 ng/mL), critical release sequence (VEGF/bFGF early, BMP2 sustained), and underlying mechanism (PI3K/Akt-β-catenin). This significantly advances vascularized bone regeneration strategies. Existing bone biomaterials inadequately simulate the intricate natural bone healing signal cascade. This study integrated vascularization and bone formation with a multi-growth factor delivery system to emulate the angiogenesis and osteogenesis signal cascade during bone repair. It was observed that VEGF (vascular endothelial growth factor) primarily stimulated the proliferation, migration, and tube formation of HUVECs (human umbilical vein endothelial cells), while not influencing BMP2 (bone morphogenetic protein 2) induced osteogenic differentiation of MSCs (mesenchymal stem cells). On the other hand, pretreatment of MSCs with low concentration bFGF (basic fibroblast growth factor) for 1 day and subsequent addition of BMP2 enhanced BMP2-induced osteogenic differentiation via regulation of the PI3K/Akt-GSK3β-β-catenin pathway and β-catenin expression and nuclear translocation. Based on these findings, a hierarchically released biomimetic scaffold composed of GelMA (Gelatin methacrylate) microspheres and silk fibroin composite material was fabricated, featuring early release of VEGF and bFGF and sustained release of BMP2. In vivo, osteogenesis and bone repair experiments demonstrated enhanced angiogenesis, osteogenesis, and bone repair capacity of this hierarchically released biomimetic scaffold, indicating promising clinical applications for bone defect repair.