An injectable biomimetic blood clot system for spatiotemporally programmed vascularized osteogenesis

Critical-sized bone defects pose a significant clinical challenge, with inadequate blood supply being a major factor impairing bone healing. In the initial stage of bone healing, the blood clot acts as a "natural glue," serving not only as a scaffold for stem cell growth but also providing a reservoir of cytokines and growth factors that regulate and reconstruct the osteogenic microenvironment. In this study, we hierarchically integrated platelet concentrates with distinct functions into an injectable thermosensitive hydrogel, constructing a "biomimetic blood clot" system. First, leukocyte-platelet-rich fibrin (L-PRF) lyophilized powder, which promotes the osteogenic differentiation of periosteum-derived mesenchymal stem cells (PMSCs), was incorporated into methacrylated gelatin hydrogel microspheres (GM) to form GM^L-PRF. Subsequently, advanced platelet-rich fibrin (A-PRF) lyophilized powder, which enhances the angiogenic differentiation of lamina propria-derived mesenchymal stem cells (LMSCs), was blended into a hydrogel precursor composed of chitosan (CS), polygalacturonic acid (PgA), β-glycerophosphate (β-GP), and GM^L-PRF. This yielded a thermosensitive hydrogel, designated CSPgA^A-PRFGM^L-PRF (CP^AG^L), which exhibits favorable mechanical properties at 37 °C and enables the programmed release of growth factors from both A-PRF and L-PRF. The CP^AG^L hydrogel could induce the differential differentiation of distinct types of MSCs. It facilitates vascularized bone regeneration by activating the JAK2-STAT3/HIF-1α and the TGF-β/Wnt signaling pathway, thereby effectively initiating the coupled angio-osteogenic process. In summary, the biomimetic blood clot system CSPgA^A-PRFGM^L-PRF represents a potential injectable biomaterial strategy for bone defect repair.