Study On The Role Of Conditioned Medium Generated From Umbilical Cord Mesenchymal Stem Cells Incubated Via A Serum-free Culture System In The Repair/Regeneration Of Photoaging Skin

Objective:Mesenchymal stem cells（MSCs）can repair photoaging skin by paracrine,but the cells cultured in vitro prone to aging in the process of replication,and quality of conditioned medium（CM）secreted by MSCs will be reduced,lowing the effect of the aging skin treatment.Umbilical cord mesenchymal stem cells（UCMSCs）cultured in a new serum-free（SF）culture system developed in our laboratory exhibited a better cell morphology,viability compared with UCMSCs cultured in fetal bovine serum（FBS）culture system according to our research.Therefore,we hypothesize that the CM collected from UCMSCs cultured in SF culture system（SF-CM）has superior anti-photoaging effect compared with the CM collected from UCMSCs cultured in FBS culture system（FBS-CM）.FBS-CM or SF-CM were used treating Ultraviolet B-induced premature senescence of HDFs and skin photoaging model of ICR mice established by our laboratory,respectively,to verify the effectiveness of SF-CM in skin anti-photoaging treatment compared with FBS-CM.Our cultivation system provides a reference for the future clinical application of SF-CM.Methods:CM collected from the two culture methods was prepared,and the experiment was carried out in two parts.In terms of in vitro experiment,ultraviolet B radiation（UVB）was used to irradiate human dermal fibroblasts（HDFs）into premature senescence,and the premature senescence HDFs was treated with FBS-CM or SF-CM.Detection of cell proliferation,collagen typeⅠlevel,MMP-1 level,NAD+synthesis,cell senescence rate,senescence-associated proteins of p16^INK4a,p21,and p53,senescence-associate gene p16,p21,and p53 were performed after CM treatment.In terms of in vivo experiment,the mice dorsal skin were exposed to UVB radiation until photoaging.SF-CM,or FBS-CM was subcutaneously injected under the dorsal skin of UVB-irradiated mice.Skin samples were taken from the injection site of mice after treating for ten weeks.HE staining,Masson’s staining,immunohistochemical staining were performed to observe dermal thickness,dermal morphology and skin angiogenesis.Western blot detection was performed for the expression of senescence-associated proteins P16^INK4a,P21,and P53.Results:The in vitro results showed that SF-CM treatment promoted cell proliferation,increased the collagen type I level and NAD+level,decreased the MMP-1 level,lowered cell senescence rate,decreased the expression of senescence-associated proteins and gene of UVB-irradiated HDFs significantly.The in vivo results showed that SF-CM significantly improved skin appearance,increased dermal thickness,induced skin angiogenesis,decreased the expression of senescence-associated proteins in photoaging mice.Conclusion:These findings in vitro and in vivo both showed that SF-CM significantly recovered photoaging skin and had a better anti-photoaging effect compared with FBS-CM.SF-CM may be used as an alternative medicine for future aging-skin treatment.