Double Cross-linked Biomimetic Hyaluronic Acid-based Hydrogels With Thermo-stimulated Self-contraction And Tissue Adhesiveness For Accelerating Post-wound Closure And Wound Healing

Objective Based on N-isopropylacrylamide（NIPAM）and glutaraldehyde（GTA）cross-linked hyaluronic acid（HA）,a thermally responsive and tissue-adhesive hydrogel with interpenetrating polymerization network（IPN）was prepared as a novel wound dressing to accelerate wound closure and promote wound healing.Methods I.Preparation of IPN（PNI-HA）hydrogels:The cross-linking agent N,N’-methylenebisacrylamide（MBA）was used to cross-link NIPAM by free radical polymerization to form PNIPAM（PNI,poly（N-isopropylacrylamide））as the first network system,and HA formed the second network system by nucleophilic addition reaction with GTA,and the two networks were combined to form PNI-HA was formed by the nucleophilic addition reaction of HA with GTA,and the two networks were combined to form PNI-HA;the analysis was performed by Fourier Transform Infrared Spectroscopy（FT-IR）,Solid State ¹³C Nuclear Magnetic Resonance（NMR）and Scanning Electron Microscope（SEM）to analyze the composition and microstructure of PNI-HA.II.Characterization of PNI-HA hydrogels:the general properties of PNI-HA hydrogels were analyzed by swelling experiments and rheological properties;the temperature-sensitive properties of PNI-HA hydrogels were further investigated by thermal response self-shrinkage experiments and thermal response tissue adhesion experiments.III.Biocompatibility of PNI-HA hydrogels:The biocompatibility of PNI-HA hydrogels was assessed by hemolysis assay,CCK-8,and live-dead cell staining.IV.The ability of PNI-HA hydrogel to promote wound closure and wound healing:The ability of PNI-HA hydrogel to promote wound closure and wound healing was evaluated by 4 h wound contraction test,21 d wound healing test and histopathological analysis.Results I.Preparation of PNI-HA hydrogels:All characteristic peaks of PNI-HA hydrogels were shown by FT-IR and solid-state ¹³C NMR,which confirmed that PNI-HA hydrogels had an IPN network structure composed of both PNIPAM and GTA-HA networks.It was observed by SEM that the PNIHA hydrogel has a stable,uniform and interconnected three-dimensional pore structure.II.Characterization of PNI-HA:The swelling experiments confirmed the porous structure and molecular entanglement of PNI-HA hydrogel,which is conducive to maintaining a moist environment in the wound area,removing excess exudate from the wound,cooling the wound,and ensuring adequate oxygenation.The rheological properties again confirm the stability of PNI-HA hydrogel.the unique thermo-responsive self-shrinkage and thermo-responsive tissue adhesion of PNI-HA hydrogel confirm that the hydrogel gradually decreases in volume with increasing temperature while the adhesion gradually increases,showing excellent ability in thermo-response and tissue adhesion.III.Biocompatibility of PNI-HA hydrogel:The results of hemolysis experiments showed that the hemolysis rate of PNI-HA was less than 2%.CCK-8 experiments yielded that L929fibroblasts proliferated significantly at 1-5 d.The results of live-dead staining showed that L929 fibroblasts proliferated significantly at 1-5 d.Live-dead staining results showed that L929 fibroblasts survived more than 90%.The good biocompatibility of PNI-HA hydrogel was confirmed.IV.The ability of PNI-HA hydrogel to promote wound closure and wound healing:Wound closure experiments using a mouse model with total dorsal skin defect confirmed that PNI-HA hydrogel could effectively help wound closure by mechanical stretching.The results of PNI-HA hydrogel-mediated wound healing experiments showed that PNI-HA hydrogel dressing can effectively promote wound healing.Moreover,the inflammatory infiltration and collagen deposition at the wound site were analyzed by H&E staining and Masson staining.The production of neovascularization at the wound site was monitored by immunohistochemical staining for angiogenic factor（CD31）.The role of wound closure in promoting wound re-epithelialization was explored by immunohistochemical staining for integrinβ1.The results confirmed that PNI-HA hydrogel could significantly promote wound healing by reducing inflammatory cell infiltration,decreasing collagen deposition,promoting angiogenesis,and upregulating the expression level of integrinβ1.Conclusion The above results indicated that PNI-HA hydrogel could effectively promote wound closure and wound healing through its unique thermo-responsive self-contraction and thermo-responsive tissue adhesion properties.The hydrogel with thermoresponsive selfcontraction and tissue adhesion properties showed promising applications as a multifunctional dressing for wound healing.