Human Umbilical Cord Mesenchymal Stem Cell Exosomes Promote Wound Healing In Diabetic Skin

Background:Diabetic skin wound（DSW）is one of the serious complications of diabetic patients,which is due to the disorder of glucose metabolism in the body of diabetic patients.Current research shows that high glucose environment can inhibit the cell function of fibroblasts and the function of vascular endothelial cells.Inhibition of fibroblasts can slow down or even stagnate collagen deposition and slow wound reconstruction.Inhibition of vascular endothelial cells leads to decreased angiogenesis and reduced blood supply and oxygen supply to the wound,making it difficult for DSW to heal.However,the current clinical treatment mainly focuses on debridement and infection control,which cannot effectively restore the function of fibroblasts and vascular endothelial cells,and the therapeutic effect is poor.In recent years,with the continuous development of regenerative medicine,exosome therapy for DSW has achieved good therapeutic effects in animal models.Exosomes（Exo）have smaller size and lower immunogenicity than mesenchymal stem cells（MSC）,and can avoid ethical issues,it is one of the most promising methods for clinical treatment in the future.However,due to the existence of a variety of bioactive substances in Exo,the mechanism of promoting wound healing is complex,involving multiple pathways and mechanisms,which limits the wider application of exosome therapy.Therefore,in order to explore whether Exo can restore the function of fibroblasts and vascular endothelial cells in the high-glucose environment of DSW.Therefore,in this study selected human umbilical cord mesenchymal stem cells（HucMSC）with a wide range of sources,simple culture and rapid expansion,were used as the cell source for the extraction of Exo.Human fibroblasts（HFB）and human umbilical vein endothelial cells（HUVEC）were used as cell lines for in vitro experiments.The effect of Exo on HFB and HUVEC was determined by simulating the environment in DSW in 30 m M and 60 m M high-glucose medium to explore the role of Exo in DSW.Objective:HFB and HUVEC were used as experimental cell lines to explore the effect of HucMSC-Exo on HFB and HUVEC under 30 m M and 60 m M high glucose conditions,and then to understand the effect of HucMSC-Exo on the cellular functions of HFB and HUVEC at different concentrations.And by establishing a DSW model,the effect of Exo on collagen deposition and angiogenesis at DSW was explored.Methods:Characterization experiments: First,the multi-lineage differentiation ability and surface markers of HucMSCs were detected by flow cytometry and trilineage differentiation experiments.Then HucMSC-Exo was extracted,the surface markers of exosomes were detected by Western Blot,and their size was detected by nanoparticle tracking analyzer,and transmission electron microscopy（TEM）was used to detect their size and morphology.In vitro experiments: HFB and HUVEC were selected as cell lines in the in vitro experiments,and glucose was added to the culture medium to establish 30 m M group and 60 m M group to simulate a hyperglycemic environment.Transwell migration assay and scratch assay were used to detect the migration ability of HFB and HUVEC.Tubule formation assay to detect the tubule formation ability of HUVEC.Subsequently,HucMSC-Exo was added to intervene,and the effects of HucMSC-Exo on the proliferation and migration of HFB and HUVEC under 30 m M and 60 m M hyperglycemia,and the effect on the tubule formation ability of HUVEC were determined by the above experiments.In vivo experiment: A rat model of type 2 diabetes mellitus（T2DM）was constructed by intraperitoneal injection of streptozotocin（STZ）.Then,full-thickness skin with a diameter of 1.5 cm was excised on the back of the constructed diabetic rat to construct a DSW model.The rat models that successfully constructed DSW were randomly divided into three groups: PBS group,HucMSC treatment group,HucMSC-Exo treatment group.Photographs of wound healing were collected at 0,1and 2 weeks.Rats were sacrificed after two weeks of treatment,and skin wounds were removed for analysis.Through HE staining,Masson staining and CD31 immunohistochemistry experiments,the degree of wound healing,the amount of collagen deposition and the amount of angiogenesis in the rat wounds were detected,and the healing effect was evaluated.Results:Firstly,HucMSCs were extracted and then subjected to three-lineage differentiation experiments and surface markers.According to the international stem cell identification method,it was confirmed that HucMSC were successfully extracted.Then,HucMSCs-Exo were extracted by ultracentrifugation.Uniform and available for use in this study.The in vitro experiments proved that the proliferation and migration ability of HFB and HUVEC and the tubule formation ability of HUVEC were inhibited to varying degrees under high glucose state,and the inhibitory effect continued to increase with the increase of glucose concentration.The proliferation and migration ability of HUVEC and HFB cells and the tubule formation ability of HUVEC were restored to varying degrees after the addition of HucMSC-Exo under the high glucose state.These results indicated that HucMSC-Exo could effectively restore the cell functions of HFB and HUVEC that were inhibited under high glucose conditions at different blood glucose concentrations.In vivo verification experiments show that HucMSC-Exo has the best therapeutic effect on DSW.The wound healing degree,collagen deposition and angiogenesis are all improved,which proves that HucMSC-Exo can promote the proliferation,migration ability of fibroblasts and improve the Collagen production,promoting the proliferation,migration and tube-forming ability of vascular endothelial cells to promote angiogenesis and ultimately promote DSW healing.Conclusion:HucMSCs were successfully extracted and had various properties of stem cells.The vigorously growing cells were selected to extract HucMSC-Exo.The size,morphology and surface markers of HucMSC-Exo were determined to meet the basic requirements of Exo,so the extraction of HucMSC-Exo was successful.HucMSC-Exo promotes DSW healing by enhancing the proliferation,migration,and tube-forming abilities of HUVEC and HFB cells under hyperglycemia.At the same time,statistical analysis shows that compared with the concentration of 30 m M,the proliferation and migration ability of HucMSC-Exo on HFB is weakened at the concentration of 60 m M,while the effect of HucMSC-Exo on HUVEC can play a better role in promoting proliferation and migration function,while the ability to form a tube is weakened.This phenomenon indicated that HucMSC-Exo exerted different effects at different blood glucose concentrations,suggesting the use of different therapeutic methods at different blood glucose concentrations.In the validation of the in vivo experiments,the results were similar to those in the in vivo experiments,and the collagen deposition and angiogenesis of DSW after HucMSC-Exo treatment were increased compared with the control group.