A Multifunctional Antibacterial And Self-healing Hydrogel Laden With Bone Marrow Mesenchymal Stem Cell-derived Exosomes For Accelerating Diabetic Wound Healing

Objective Recently,mesenchymal stem cells have played an important role in tissue regeneration and wound repair by enhancing reepithelialization,promoting angiogenesis,regulating inflammation,and regulating extracellular matrix remodeling.Compared with cell transplantation,cell-free therapy has advantages such as higher stability and better storage,and there is no possibility of immune rejection.Recent studies have shown that adipose derived mesenchymal stem cells have the potential to promote wound repair,and exosomes derived from human umbilical cord mesenchymal stem cells play a role in promoting angiogenesis and accelerating skin wound healing.Here,we focus on the powerful regenerative role of mesenchymal stem cell exosomes in promoting various stages of wound healing.Including promoting cell proliferation and migration,angiogenesis,and regulating polarization.The good biocompatibility of CEC-DCMC HG hydrogel provides a suitable storage environment for exosomes of bone marrow mesenchymal stem cells.This study intends to use bone marrow mesenchymal derived exosomes wrapped in CEC-DCMC HG hydrogel for wound healing in chronic diabetes mellitus.The effect of MSC-Exos on the proliferation,migration and angiogenesis of HUVECs cells are discussed through in vitro experiments,and the effect of MSC-Exos on the polarization of macrophages and the regulation of inflammatory microenvironment was further discussed,which laid the foundation for the following in vivo experiments.MSC-Exos is mounted on CEC-DCMC HG hydrogel to promote angiogenesis,collagen deposition,regulate the whole stage of wound healing during the epithelialization process,maintain wound homeostasis,and greatly shorten the healing time.MSC-Exos,which is attached to CEC-DCMC HG hydrogel,is involved in all phases of healing,and may also be involved in different phases of healing,regulating the skin microenvironment and maintaining homeostasis.Methods1.Firstly,exosomes were isolated from rat bone marrow mesenchymal stem cells by serum-free and enzyme-free differential centrifuge method.The extracted exosomes were identified by Western blot,TEM and DLS,cell viability was measured by CCK-8 cell proliferation detection kit,and cell migration ability was detected by Transwell assay.The ability of cell neovasculature was verified by tubularization experiment,and the expression of macrophage inflammatory factors inhibited by bone marrow mesenchymal stem cell exosomes was observed by immunofluorescence.Secondly,SEM was used to observe the CEC-DCMC HG hydrogel loaded with bone marrow mesenchymal stem cells and the simple CEC-DCMC HG hydrogel.Temperature scanning and amplitude oscillation scanning was used to detect the CEC-DCMC HG hydrogel hydration ratio and MSC-Exos@CEC-DCMC HG hydrogel rheological curve.The release curve of exosomes in CEC-DCMC HG hydrogel was also observed.2.The diabetic wound model was constructed in SD rats.After anesthesia,shaving,and cutting a 2cm round wound,the drug was administered continuously for two weeks to observe the bacteria and healing of the wound.We verified the inflammatory expressions of IL-1β,INOS,CD206 and TNF-α in the 3-day wound by Western blot assay.The wound size was observed by H&E staining sections on day 7 and day 14,angiogenesis was observed by immunohistochemistry on day 7,and collagen deposition was observed by Masson staining on day 14.Results We successfully obtained exosomes by gradient centrifugation,and the characteristic marker proteins Alix,CD81 and CD63 of exosomes was specifically expressed by Western blot.DLS and TEM further proved that we successfully extracted exosomes.Exosomes themselves,as biological non-immunoreactive drugs,can significantly promote the proliferation and migration of human umbilical vein endothelial cells,induce neovasculangiogenesis,inhibit the inflammatory response of macrophages,increase M2 phenotype and reduce M1 phenotype.The Schiff base reaction between carboxyethyl chitosan(CEC)and dialdehyde carboxymethyl cellulose(DCMC)formed a self-healing antibacterial hydrogel.It was prove in vivo that Cec-DCMC HG hydrogel promoted the wound healing of type 1 diabete mellitus induced by streptase and mycin.Exosomes was coated with a novel Ce C-DCMC HG hydrogel.As a functional carrier of exosomes,the synergistic treatment of MSC-Exos@CEC-DCMC HG hydrogel greatly improves the rate of diabetic wounds.This new cost-effective hydrogel loading exosomes is significant for the treatment of diabetic wounds.In vivo,MSC-Exos@CEC-DCMC HG hydrogel significantly accelerated the healing of diabetic full-layer wounds,resulting in increased wound healing rate,rapid reepithelialization,good collagen deposition,and abundant wound angiogenesis.The multifunctional MSC-Exos@CEC-DCMC HG hydrogel can secrete more VEGF,enhance cell proliferation,collagen deposition,angiogenesis,and promote migration and differentiation of bone marrow mesenchymal stem cells,thus accelerating wound healing.Compared with CEC-DCMC HG hydrogel and exosome alone,it is better for skin healing.Second,hydrogels have the ability to regulate chronic inflammation and provide an optimal environment for bone marrow mesenchymal stem cell exosomes to promote wound healing.The multifunctional MSC-Exos@CEC-DCMC HG hydrogel induces exosome release,promotes the polarity transformation of macrophages at the wound site,reduces inflammation,and facilitates excessive skin injury.Conclusion We are the first toreport self-healing multifunctional CEC-DCMC HG hydrogel loaded with bone marrow mesenchymal stem cell exosomes to synergically promote diabetic wound healing and skin regeneration.The multifunctional MSC-Exos@CEC-DCMC HG hydrogel can regulate wound site inflammation.Promote cell proliferation,collagen deposition,angiogenesis,and maintain the exosome activity of bone marrow mesenchymal stem cells,thus accelerating wound healing.Compared with CEC-DCMC HG hydrogel,it is better for skin healing.In addition,the inclusion of hydrogels and exosomes collaboratively induce the local regulation of M2 macrophage polarization,which provides a new strategy for the treatment of diabetic skin wounds.