A temporally functional composite hydrogel scaffold for cranial defect repair via sequential modulation of angiogenesis and osteogenesis

The repair of large cranial defects remains a major clinical challenge, as conventional materials primarily act as inert fillers and fail to meet the complex biological requirements of cranial bone regeneration. In particular, they lack the ability to temporally coordinate angiogenesis and osteogenesis. This study aimed to develop a temporally functional composite scaffold to dynamically modulate the regenerative microenvironment and promote sequential vascularized bone regeneration. <b>Methods:</b> A silk fibroin-based hydrogel system was designed, incorporating salvianolic acid B (SalB)-loaded sustained-release hydrogel and mineralized silk fibroin hydrogel microspheres (MSFM). Material characterization was performed to evaluate the structural and mechanical properties of the scaffold, as well as the drug release behavior. <i>In vitro</i> assays were conducted to assess endothelial cell migration, tube formation, and the expression of angiogenesis-related genes, along with the osteogenic differentiation potential of bone marrow-derived mesenchymal stem cells (BMSCs). <i>In vivo</i> reparative efficacy was further validated using a rat cranial defect model through morphological and histological analyses. <b>Results:</b> Characterization confirmed that OSFM microgels were uniformly spherical with a porous internal structure and exhibited sustained release of OGP. <i>In vitro</i>, OSFM showed excellent cytocompatibility with BMSCs, significantly enhancing cell proliferation, ALP activity, and mineralized nodule formation compared with SFM (p < 0.05). Tube formation and scratch assays demonstrated that OSFM-conditioned medium promoted HUVEC migration and angiogenesis. <i>In vivo</i>, implantation of OSFM+PCL scaffolds into rat calvarial defects resulted in markedly superior bone regeneration compared with control, PCL, and SFM+PCL groups. The bone volume fraction in the OSFM+PCL group reached 52.31 ± 4.27% at the 8th weeks, significantly higher than 23.65 ± 3.81%, 30.42 ± 3.96%, and 37.86 ± 4.12% in the other groups (p < 0.05). Histological staining confirmed more mature bone formation, abundant collagen deposition, and tight integration between new bone and scaffold. Immunohistochemistry revealed upregulated expression of RUNX2, OCN, and CD31, indicating enhanced osteogenesis and angiogenesis. <b>Conclusions:</b> This temporally functional composite scaffold achieved a sequential "angiogenesis first, osteogenesis later" strategy by leveraging the differential degradation kinetics of its components. The findings demonstrate a biomimetic and temporally regulated approach with strong bioactivity and translational potential for cranial bone regeneration.