Injectable thermosensitive hydrogel incorporating exosome-loaded chitosan microspheres for immunomodulation and cartilage regeneration

ABSTRACT Background Chronic inflammation and impaired immune microenvironment are key barriers to effective cartilage regeneration. Herein, we developed an integrated, injectable thermosensitive hydrogel system termed CS@Gel-Mg. This system modularly combines exosome-loaded chitosan microspheres (CM@Exo)​ embedded within a Mg 2+ -coordinated poly(N-isopropylacrylamide)-based ionic liquid hydrogel (Gel-Mg), aiming to modulate local immune response and promote cartilage repair. Methods Hydroxypropyl-modified chitosan microspheres were synthesized via emulsion crosslinking and effectively loaded with bioactive exosomes. The Gel matrix was fabricated using an ionic liquid monomer (IL-C4) copolymerized with NIPAM and N-vinylimidazole, followed by Mg 2+ complexation. Physicochemical properties, including morphology (SEM/TEM), surface chemistry (XPS/FTIR), and thermoresponsive behavior (DSC/TGA), were systematically characterized. The effects of CS@Gel-Mg on immune and cartilage regeneration were evaluated in vivo and in vitro using Western blot, qPCR, histological staining, flow cytometry, gait analysis, and asymmetric weight-bearing analysis. Results In vitro, the system demonstrated excellent cytocompatibility, promoted ATDC5 cell proliferation and migration, and inhibited apoptosis. The hydrogel also reprogrammed macrophage polarization from M1 to M2 phenotype, as confirmed by qPCR and flow cytometry. In vivo, using a rat full-thickness cartilage defect model, CS@Gel-Mg exhibited improved weight-bearing function, reduced inflammatory infiltration, and superior cartilage histological scores compared to control groups. Mechanistically, immunofluorescence and Western blot analyses suggested enhanced expression of SOX9, COL2A1 and COL1A1, along with suppression of inflammatory cytokines. Furthermore, SOX9 knockdown in vivo diminished the therapeutic effect, confirming its regulatory role in the immuno-chondrogenic axis. Conclusion This exosome-integrated thermoresponsive hydrogel provides a multifunctional platform for immune modulation and cartilage regeneration. Our findings highlight the potential of CS@Gel-Mg as a promising therapeutic strategy for osteochondral repair.