A bilayered dual-peptide hydrogel for spatiotemporally coordinated repair of bone defects

• A bilayered photocurable hydrogel is innovatively designed to integrate “recruitment-induction” dual functions, addressing the performance bottleneck of single-functional bone repair hydrogels. • The material couples YLL3-mediated targeted cell recruitment and OGP-driven osteogenic induction via its layered structure, realizing synergistic regulation of bone regeneration processes. • Systematic in vitro/ in vivo characterizations confirm the hydrogel’s favorable biocompatibility, controlled functional release, and efficient bone defect repair efficacy. • This material design expands the functionalization pathway of hydrogel-based tissue engineering scaffolds and provides a practical candidate for clinical bone repair applications. The core challenge in bone defect repair lies in achieving efficient recruitment of stem cells to the defect area and their directed osteogenic differentiation. However, existing strategies fail to meet clinical needs due to limitations such as single functionality, poor stability of bioactive molecules. To address the key challenges, this study engineered a physiological bone repair processes matched dual-layer photocurable hydrogel: the outer layer, chemically grafted with YLL3 peptides-known to bind α4β1 integrin on osteoprogenitor cells- recruits endogenous stem cells; the inner layer, osteogenic growth peptide (OGP) to activate Mitogen-Activated Protein Kinase (MAPK) signaling, inducing osteogenic differentiation of recruited cells. Photocuring technology was used to precisely regulate the hydrogel’s mechanical properties (compressive modulus: 1–5 MPa) and form interconnected 3D pores (favorable for cell infiltration), while its controlled degradation profile (4–8 weeks) was synchronized with native bone regeneration kinetics. In vitro experiments confirmed the hydrogel supported high viability (>90%) and proliferation of dental pulp stem cells (DPSCs), with significantly upregulated osteogenic markers (alkaline phosphatase activity increased by 47.2%; osteocalcin expression upregulated by 53.6%) compared to non-functionalized hydrogels. In vivo, the hydrogel effectively recruited endogenous cells to the defect area and promoted osteogenic differentiation. In a rabbit calvarial bone defect model, micro-CT analysis at 8 weeks post-implantation showed the hydrogel group achieved a 60% defect repair rate, with a 38.5% increase in new bone volume compared to the blank control group. This dual-layer hydrogel, via spatiotemporal coordination of “recruitment-induction” functions, provides a promising biomaterial strategy for clinical bone defect repair.