Gelatin-based cryogels seeded with exosomes enhance osteogenic activity and bone regeneration in a rabbit femoral defect model

The development of biomaterial scaffolds that combine structural support with biological activation remains critical for bone defect repair. In this study, gelatin-based cryogels (GBCs) that incorporate β-tricalcium phosphate (β-TCP) and hyaluronic acid (HA) were fabricated and optimized to balance porosity and mechanical stability. Increasing the HA concentration increased the water content, swelling ratio, and porosity while reducing the compressive stiffness. The cryogels that contained 0.3% HA exhibited interconnected macropores (200–300 μm after swelling), ∼30% porosity, and a compressive modulus of 3.67 MPa and were selected for biological evaluation. Human umbilical cord-derived mesenchymal stem cell (hUC-MSC)-derived exosomes were incorporated to increase osteogenic bioactivity. Compared with the control treatment, exosome treatment significantly increased MG-63 proliferation, alkaline phosphatase activity, and mineral deposition (p < 0.05). Notably, compared with exosomes alone, exosome-seeded cryogels synergistically increased osteogenic differentiation (p < 0.001). In a rabbit femoral defect model, compared with cryogels without exosomes, exosome-functionalized cryogels promoted denser bone matrix formation and neovascularization. These findings demonstrate that GBCs seeded with exosomes provide a structurally permissive and biologically active microenvironment that improves bone regeneration. This cell-free strategy represents a promising platform for translational bone repair applications.