Bone regeneration and self-repair of bone are highly challenging without surgical interventions. Currently, chitosan-based injectable hydrogels have gotten great attention in bone tissue engineering due to their biodegradability, cytocompatibility, cell adhesion, and proliferation. To improve the strength and mechanical properties of scaffolds, metal oxide nanoparticles were incorporated into a hydrogel matrix to develop a new scaffold for bone regeneration. In this work, TiO₂ nanorods were prepared using the hydrothermal method and then loaded with exosomes, which were then incorporated into chitosan hydrogel. Injectable hydrogel scaffolds were obtained by free radical polymerization of biocompatible monomers such as chitosan, acrylamide, and itaconic acid, and TiO₂ NRs were loaded, which improves the mechanical properties of CS-hydrogel. The thermal stability of nanofiller-loaded hydrogels is improved, and the differences were statistically significant (p < 0.05), confirming thermal resistance. In addition, cell adhesion, proliferation, ALP activity, and the gene expression of osteogenic genes such as OPN, BSP, and OCN and angiogenesis genes, namely VEGF, ANG-1, and CD31, by activating the Wnt/β-catenin signaling pathway in vitro, were performed for the prepared CS-hydrogel@TiO₂NRs-exo hydrogel. Cell adhesion and spreading area were significantly enhanced (p < 0.05), and in vitro studies demonstrated that the incorporation of TiO₂NRs-exo in CS hydrogel enhances the OCN, OPN, VEGF, and ANG gene expressions and promotes new bone formation. Western blotting and RT-PCR results revealed that CS-hydrogel@TiO₂NRs-exo could induce osteogenic differentiation of MC3T3-E1 cells by activating the Wnt/β-catenin pathway, while the inhibitor DKK1 significantly reduced osteogenic differentiation following stimulation with CS-hydrogel@TiO₂NRs-exo. Overall, the studies revealed that the formulated CS-hydrogel@TiO₂NRs-Exo hydrogel has superior properties and can be used as a scaffold for bone regeneration.
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