| Wound healing is a complex biological process,and research on exosomes-based cell-free therapy for inducing wound healing has been extensively conducted.However,limitations such as rapid clearance and lack of specificity exist in exosomes therapy.To maintain the biological activity and effective concentration of exosomes while enhancing their targeting ability,it is necessary to modify exosomes and construct optimized delivery systems.Objective: In this study,we propose to construct HA-BMSC-Exos and encapsulate them in PF127 hydrogel to investigate their effects on wound healing and explore their mechanisms of action in wound healing,aiming to provide a novel approach for targeted therapy with improved efficacy.Methods: Rat bone marrow mesenchymal stem cells were extracted using a whole bone marrow extraction method.BMSC-Exos were purified from the culture medium using gradient centrifugation,and HA-BMSC-Exos were constructed by chemically self-assembling DSPE-PEG2000-HA into the surface of BMSC-Exos.The cytotoxicity of HA-BMSC-Exos was detected.HA-BMSC-Exos were co-cultured with macrophages.The effect of HA-BMSC-Exos on macrophage polarization was detected by flow cytometry and ELISA,and the expression levels of relevant molecular pathway proteins were detected by Western blot.PF127/HA-BMSC-Exos hydrogel was prepared and its physicochemical properties were examined.50 male mice aged 6-8 weeks were randomly divided into 5 groups,with 10 mice in each group:(1)control group;(2)BMSC-Exos group;(3)HA-BMSC-Exos group;(4)PF127 group;(5)PF127/HA-BMSC-Exos group.A full-thickness skin with a diameter of 1cm was surgically excised on the back of the mice.According to the random grouping situation,100 μL of PBS,BMSC-Exos(2 mg/m L),and HA-BMSC-Exos(2 mg/m L)were subcutaneously injected at four points around the wound site,including the upper,lower,left,and right sides in each group,respectively;while the PF127 group and PF127/HA-BMSC-Exos group were topically applied with 100 μL PF127 gel and PF127/HA-BMSC-Exos gel(containing 200 μg HA-BMSC-Exos),respectively.The wound healing rates were detected,and skin samples were collected during the wound healing process for histological staining analysis to evaluate inflammation,angiogenesis,collagen deposition,and M2 polarization.Results: The constructed HA-BMSC-Exos had an increased hydrated particle size and decreased Zeta potential compared to BMSC-Exos.Macrophages treated with BMSC-Exos and HA-BMSC-Exos maintained similar cell viability to untreated macrophages.HA-BMSC-Exos promoted the internalization of macrophages to exosomes and enhanced the ability of BMSC-Exos to induce M2 polarization.Western blot results showed that HA-BMSC-Exos induced M2 polarization through the AMPK/PPARγsignaling pathway,and the AMPK inhibitor Compound C could reverse the M2 polarization induced by HA-BMSC-Exos.PF127/HA-BMSC-Exos hydrogel had good injectability and was thermosensitive,and no cytotoxicity,and could achieve exosome sustained release within 72 hours.HA-BMSC-Exos effectively accelerated wound healing,and the wound healing capability of PF127/HA-BMSC-Exos hydrogel surpassed that of HA-BMSC-Exos alone.On the 10 th day after surgery,the wound in the PF127/HA-BMSC-Exos group was completely healed,and wrinkles appeared on the skin with new hair visible around the wound.Histological evaluation showed that PF127/HA-BMSC-Exos inhibited inflammation,promoted angiogenesis and collagen deposition,and promoted M2 polarization of wound macrophages to a greater extent than HA-BMSC-Exos alone.Conclusion: HA-BMSC-Exos were successfully constructed and no cellular toxicity was detected within 48 h.HA-BMSC-Exos enhanced the delivering ability of BMSC-Exos to macrophages,thereby strengthening the ability of BMSC-Exos to induce macrophage M2 polarization.HA-BMSC-Exos regulated macrophage M2 polarization through the AMPK/PPARγ signaling pathway.The acceleration of wound healing by HA-BMSC-Exos is likely mediated by inducing M2 polarization in macrophages,thereby expediting the wound healing process.PF127/HA-BMSC-Exos hydrogel accelerated wound healing,possibly through controlled release,maintaining local exosome concentration at the wound site,and enhancing the effect of HA-BMSC-Exos in accelerating wound healing. |
