Evaluation of the combination of chitosan hydrogel crosslinker and dental pulp stem cell-derived exosomes on osteoblast viability and differentiation

Dental pulp stem cell (DPSC) has gained attention as a cell-free therapeutic approach to enhance bone regeneration. However, the degradation of exosomal biomaterials limits their application, which may be overcome by using an injectable chitosan hydrogel crosslinker (ICHC) as a natural polymeric carrier. ICHC was prepared and loaded with exosomes derived from DPSCs. Cytotoxicity, calcium deposition, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM) were conducted to assess biocompatibility, mineralization capacity, and structural characteristics. The cell viability of ICHCs, exosome 5 ng/mL, and exosome-loaded ICHC 0.6% ranged from 95% to 132%. NTA analysis revealed particle sizes of 147.5 nm for exosomes and 377.5 nm for exosome-loaded ICHC, with concentrations of 4.6 × 10 6 and 1.4 × 10 7 particles/mL, respectively. TEM further validated effective encapsulation, revealing electron-dense particles dispersed throughout the ICHC hydrogel matrix. The combination of exosome-loaded ICHC achieved 100% viability in osteoblast cells and promoted more elongated cell morphology compared to the control, suggesting a safe and effective cell-free material for bone regeneration therapy.