Synergistic Oxygen and Nitric Oxide Delivery Via an Immuno Instructive Silk Fibroin Hydrogel for Chronic Diabetic Wound Repair

Abstract

Objective: Diabetic chronic wounds exhibit impaired healing due to persistent hypoxia, oxidative stress, infection, and dysregulated immune responses. Conventional wound dressings fail to address these complex pathological factors simultaneously. This study aimed to develop a multifunctional silk fibroin (SF) hydrogel capable of co-delivering oxygen (O₂) and nitric oxide (NO) to regulate immune responses and promote diabetic wound healing. Methods: An injectable silk fibroin hydrogel was enzymatically crosslinked and incorporated with calcium peroxide as an oxygen-generating agent and S-nitrosoglutathione as a nitric oxide donor. Catalase was included to regulate peroxide levels and ensure safe oxygen release. The hydrogel was evaluated for physicochemical properties, gas release kinetics, mechanical strength, and wet-tissue adhesion. In vitro studies assessed cytocompatibility, antibacterial activity, macrophage polarization, and cytokine secretion. Therapeutic efficacy was further examined in a diabetic wound mouse model over 14 d using histological and biochemical analyses. Results: The dual gas-releasing silk fibroin hydrogel exhibited rapid in situ gelation, sustained oxygen and nitric oxide release, enhanced mechanical stability and strong wet-tissue adhesion. Cytocompatibility assays demonstrated high viability of NIH-3T3 fibroblasts and HaCaT keratinocytes (>90%). The hydrogel significantly inhibited the growth of Staphylococcus aureus and Pseudomonas aeruginosa (p<0.01) and promoted macrophage polarization toward the pro-healing M2 phenotype, as evidenced by increased CD206 and IL-10 expression with concomitant reduction of iNOS and TNF-α (p<0.05). Conclusion: The multifunctional silk fibroin hydrogel integrates sustained oxygenation, nitric oxide-mediated immunomodulation, antimicrobial activity, and tissue regeneration into a single platform. This dual gas-releasing system effectively restores the diabetic wound microenvironment and represents a promising translational strategy for advanced chronic diabetic wound management.