Rejuvenated mesenchymal stem cell conditioned medium in promoting wound healing in mice
Background Mesenchymal stem cell(MSC)conditioned medium without cell therapy is a novel treatment approach in the field of trauma repair.However,its therapeutic efficacy is influenced by the state of MSCs.Objective To explore the effect of rejuvenated mesenchymal stem cell conditioned medium(rejuvenated-MSC-CM)on wound healing in mice.Methods A replicative senescence model of mesenchymal stem cells(MSCs)up to 15-20 passages was established and the indicators of cellular senescence were assessed.Then small molecule compounds,Valproic acid(VPA)and Repsox,were used to reverse the senescence of MSCs and the anti-aging effects were validated.The conditioned medium from senescent MSCs(aging-MSC-CM)and rejuvenated MSCs(rejuvenated-MSC-CM)were collected.Human fibroblasts were cultured with these two conditioned media,and their proliferation,migration,and invasion abilities were evaluated using CCK-8 assay,Ki-67 immunofluorescence,scratch assay,and Transwell assay.In a full-thickness skin defect model on C57BL/6 mice,the wounds were treated with aging-MSC-CM,rejuvenated-MSC-CM,and normal saline,and the effects of these treatments on wound healing were analyzed.Results The expression of SA-β-gal positive cells in senescent MSCs was reduced after treating with small molecules(P＜0.01),and immunofluorescence assay showed that the expression levels of senescence genes P21 and P16 were reduced(P21:39.34％±3.72％;P16:17.30％±2.01％;P＜0.01).The proliferative,migratory,and invasive capacities of fibroblasts treated with rejuvenated-MSC-CM were enhanced(P＜0.01).The C57BL/6 mouse wound model showed increased wound healing in rejuvenated-MSC-CM-treated mice(P＜0.01).HE staining of the wounds of mice treated with rejuvenated-MSC-CM showed accelerated re-epithelialization of epidermal cells(P＜0.01),Masson staining showed an increase in the amount of collagen deposition on the wounds(P＜0.01),and immunofluorescent staining for CD31 showed an increase in the amount of vascularization on the wounds(P＜0.01).Conclusion Rejuvenated-MSC-CM may have a promoting effect on wound repair in mice.
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