The Therapeutic Effect Of Canine Adipose Mesenchymal Stem Cell-derived Exosomes On Canine Skin Injury

The skin is a key structure that protects the internal tissues of the body from injury,infection,and other effects.The ultimate goal of skin wound healing is to restore the anatomical structure and physiological function of the broken skin,and skin wound treatment aims to achieve rapid wound closure and scar-free healing.Current methods of treating skin wounds suffer from problems such as easy infection,long wound healing time,and scar tissue.Therefore,it is important to develop a novel treatment modality for skin injuries in animals.To date,various approaches have been explored,among which Mesenchymal stem cells(MSCs)therapy has great therapeutic potential.Adipose-derived mesenchymal stem cells(ADSCs)are adult stem cells derived from adipose tissue and are derived from the mesoderm.Although ADSCs have many advantages,their clinical application is limited by the high preservation and transportation requirements and the potential risks of tumorigenicity and low survival rate of implanted cells.Therefore,the development of cell-free therapies with similar effects to MSCs is essential.It has been shown that exosomes,the paracrine product of stem cells,have similar functions as stem cells and have many advantages,such as sufficient source,easy storage,low or no immunogenicity,which are promising in the field of regenerative medicine.Based on this,we selected experimental dogs to establish a full-layer skin injury model and administered canine adipose mesenchymal stem cell-derived exosomes(c ADSC-EXOs)subcutaneously to investigate the efficacy and feasibility of its treatment for skin injury.In this experiment,a male Chinese field dog was used to isolate canine adipose-derived mesenchymal stem cells(c ADSCs),and the isolated primary c ADSCs were cultured in vitro and passed to the fourth generation.cell treatment.Nine dogs were randomly divided into three groups: model group,control group,and exosome treatment group.The canine skin injury model was treated with exosomes on days 1,3,5 and 7.The dogs in the control group all received equal saline injections.After treatment,the wounds were observed and new skin was collected for testing.Two cell lines,human immortalized keratinocyte(Ha Ca T)and human umbilical vein endothelial cells(HUVEC),were divided into control and exosome-treated groups for the scratch test,and the cells were photographed and measured at different time points of treatment.The cells were photographed and the scratch width was measured at different time points of treatment,and the following results were obtained.(1)After the completion of the modeling,the model dogs showed different degrees of mental depression and decreased appetite due to the pain caused by the wound.The speed of repair was significantly faster,as reflected by the speed of scabbing and wound healing compared to the model group.(2)The results of HE staining showed that the exosome-treated group was better than the model group in terms of morphology and number of skin attachments,which tended to be similar to the normal control group.(3)Immunohistochemical staining showed that the expression of PCNA and α-SMA in the exosome-treated group was higher than that in the model group,indicating that exosome treatment promoted cell proliferation and angiogenesis in the dermis.(4)The m RNA expression levels of FGF2 and VEGFA were detected by q RT-PCR,and the results showed that the expression levels of VEGFA and FGF2 were significantly upregulated in the exosome treatment group compared with the model group.(5)The cell scratch assay showed that exosomes accelerated HUVEC scratch closure at 12 h and 24 h after exosome treatment compared with the control group and the difference was highly significant;exosomes significantly accelerated Ha Ca T scratch closure at 6 h and 14 h after exosome treatment,and exosomes could improve the migration rate of Ha Ca T and HUVEC.This experiment clarified that c ADSCs exosomes can play a certain therapeutic effect on mechanical total skin injury in dogs,which may be due to the fact that c ADSCs exosomes promote cell proliferation in the dermis,accelerate angiogenesis,promote migration of keratinocytes and endothelial cells thereby accelerating skin wound healing by promoting regeneration and reconstruction of skin adnexa.This study provides a basis and reference for the clinical application of MSCs exosomes for skin injury treatment in small animals.