Synergistic effects of modified zinc oxide nanoparticle in a hybrid chitosan-gelatin hydrogel for bone regeneration

The development of composite hydrogels with enhanced biocompatibility and osteoconductivity remains a critical focus in bone tissue engineering. In this study, we designed a double-network hydrogel composed of chitosan, and gelatin (CG), crosslinked to improve its mechanical properties for bone regeneration applications. To further enhance its bioactivity, zinc oxide nanoparticles (ZnO) were incorporated into the hydrogel matrix. Prior to incorporation, ZnO was functionalized with a mussel-inspired polydopamine (PDA) coating, forming ZnO/PDA. Subsequently, in situ mineralization facilitated the deposition of calcium and phosphate (CaP) ions, yielding ZnO/PDA/CaP (m-ZnO). To evaluate the effects of these modifications, three hydrogel formulations were prepared: pure CG hydrogel, CG hydrogel containing ZnO/PDA (CG@Z/P), and CG hydrogel incorporated with m-ZnO (CG@m-ZnO). The addition of m-ZnO significantly enhanced the compressive strength of CG@m-ZnO, increasing it from 335.05 ± 8.35 kPa to 973.31 ± 102.19 kPa, while maintaining microstructural integrity. The incorporation of ZnO also imparted antibacterial properties, whereas the PDA and CaP layers promoted cell adhesion and proliferation. Notably, CG@m-ZnO with 50 μg/mL of m-ZnO exhibited excellent biocompatibility and significantly enhanced osteogenic differentiation of MC3T3-E1 cells, as evidenced by increased alkaline phosphate (ALP) activity and Alizarin Red S (ARS) staining. In conclusion, the CG@m-ZnO hydrogel demonstrates a synergistic combination of biocompatibility, osteoconductivity, antibacterial activity, and enhanced mechanical properties, making it a promising candidate for bone tissue engineering and regenerative medicine applications.