The Effect And Mechansim Of Antler Stem Cell And It's Secretome On Cutaneous Wound Healing

BackgroundSkin is the largest organ in our body,and is the first barrier against pathogen invasion.Cutaneous wound healing is a body's stop gap measure that quickly restores the skin barrier in order to reduce the risk of infection and further injury.As the trade-off for speed,even under optimal conditions,wound healing normally leads to fibrosis or a scar formation.A number of approaches have thus far been attempted to try to achieve optimal outcome of healing,i.e.regeneration of the normal architecture and function of the skin.Amongst these approaches,mesenchymal stem cells(MSCs)with their unique properties of self-renewal and differentiation into multiple somatic cell lineages,are emerging as a promising candidate for cell-based therapy for the treatment of cutaneous wounds.Although the bone marrow,umbilical cord and adipose tissue have proved to be sources for the most types of MSCs,alternative sources for effective MSCs have never been stopped for exploring.In further questing the new sources of MSCs for the treatment of cutaneous wounds,one cell type comes into light: the antler stem cells(ASCs),the permanent cranial bony protuberance from which an antler casts and regenerates annually.Studies show that ASCs are unique in that they can not only promote perfect cutaneous wound healing(healing tissue consists of hair follicles,sebaceous glands and blood vessels)in a very rapid manner(healing of a wound with the diameter exceeds 10 cm within a week of time),but also initiate full regeneration of antlers(a type of complex mammalian organ)immediately following completion of wound healing.ASC plays an essential role in the regenerated healing of antlers after falling off,which may provide a new sight on regeneration of damaged skin.ObjectiveThe aim of the present study was to use radiation-induced cutaneous injury model to investigate whether the regenerative property of the antler stem cells is species-specific,i.e.if the antler stem cells could promote regenerative healing on other mammalian cutaneous wounds;furthermore,we investigate the repair function and mechanism of antler stem cell conditioned medium(ASC-CM)on mechanically skin damaged;finally,we identified and analyzed the components of ASC-CM.MethodsIn vitro studies,we compared the proliferation rates of ASCs and the other types of MSCs;we prepared the ASC-CM and investiagated the effects of the ASC-CM on HUVEC and NIH-3T3 proliferation via flow cytometry,CCK-8 assay and IF;we application of Akata to analysis the ASC-CM components.In vivo studies,we established two animal models of radiation-induced(Sr90)and mechanically skin injury in rats;at the same time,ASCs through tail vein injection and ASC-CM through topical application was used to treat two types of skin wounds respectively;then,calculated the wound area and healing rate,and the quality of skin healing was examined by histological and qRT-PCR.ResultsIn vitro results showed that the doubling times of ASCs was shorter than the other two types of MSCs;ASC-CM significantly promoted the HUVEC and NIH-3T3 cell proliferation;finally,we found that ASC-CM had more components than MSC-CM.Radiation-induced cutaneous injury results showed that,the ASCs group had complete healing on day 51(±3 days);the rB-MSCs and hU-MSCs groups had complete healing on day 61(±3 days)and 73(±3 days)respectively;the control group had complete healing on day 84(±3 days);ASCs treated rats exhibited faster healing rate compared than rB-MSCs and hU-MSCs;histological results showed that ASCs treated rats exhibited higher healing quality than the other two MSCs groups,accompanied by more skin appendages regeneration(Hair follicles and sweat glands);in addition,compared with the control group,the expression of Col1A2 and Col3A1 was significantly decreased in the three stem cell treatment groups,while the expression level in the ASCs group was the lowest;the expression level of FGF2 was increased and ILrap was decreased;HGF was increased while TGF-? was only in reduced in the ASCs group.More importantly,ASCs promote wound healing ability is superior to the other types of MSCs,and no or less scar formation.Mechanically skin damaged results showed that the wound of ASC-CM treated rat healed completely on day 16(±3 days),and was 9 days(±2 days)earlier than NC group;the wound area of ASC-CM treated rats was smaller than MSC-CM and PC groups;in addition,ASC-CM treated rats exhibited higher healing quality with more cutaneous appendages(Hair follicle and sebaceous gland)and vessels regenerated than the other 3 groups;moreover,the ratio of Col3A1/Col1A2,TGF-?3/TGF-?1,MMP1/TIMP1,and MMP3/TIMP1 was significantly increased in ASC-CM group.ConclusionsThe above results indicate that ASCs significantly promoted cell proliferation;ASCs treatment of rat skin injury showed that shorter recovery time,faster healing rate and higher healing quality than the other groups;Our results demonstrate tha ASCs might be the better stem cell source than the currently available ones for skin tissue regeneration;ASC-CM promotes gene expression associated with fetal-like skin regeneration and serves as a new cell-free treatment for skin wound healing,and without scarring.These results are extremely promising for the translation of the full regenerative healing property of the ASCs to clinics,providing new strategies for the treatment of refractory skin injury and ultimately benefit human health.ASC-CM for treating wounds in clinic could allow precise dosing with purified paracrine components;thus leading to the development of safe and effective cell-free regenerative reagents with predictable and controllable outcomes.