Promotion Of Hair Growth By Conditioned Medium From Extracellular Matrix/stromal Vascular Fraction Gel In C57BL/6 Mice

Objective:Comparing the effect of ECM/SVF-CM and SVF-CM on hair growth in mice and exploring its mechanism,in order to provide an effective strategy for clinical treatment of alopecia..Methods:1.In vitro experiments1.1 Preparing conditioned medium and detecting the growth factor concentration of b FGF,VEGF,PDGF and KGF:ECM/SVF-gel and SVF cells were prepared according to the methods reported in the literature.The ECM/SVF-gel and SVF cells with the same number of cells were inoculated and cultured,and then their supernatant were collected,after that,the conditioned medium was isolated and filtered from the supernatant.Finally,the growth factor concentration of b FGF,VEGF,PDGF and KGF were detected by ELIAS.1.2 Dermal Papilla Cell was isolated,cultured and identified in vitro,then detect the effect of conditioned medium on the proliferation of DPC in vitro.2.In vivo experiments2.1 Experimental intervention:7-week-old C57BL/6 mice were anesthetized by intraperitoneal injection,the back hair was shaved with clippers and completely removed using hair remover cream.Then,0.6 ml of ECM/SVF-CM,SVF-CM and PBS(0.075/cm~2),were subcutaneously injected into the back of the mice once a week for 3 weeks.2.2 Evaluation indicators:The change of skin color and hair growth on the back of mice were monitored,besides,the hair growth score was scored.Two weeks after injection,the subcutaneous angiogenesis was observed and the samples were taken for CD31 immunohistochemical staining to evaluate angiogenesis.What,s more,the back skin samples were taken for Ki-67 immunohistochemical staining to detect the proliferation activity of cells in the hair follicle protuberance area.There weeks after injection,the back skin samples were taken for Western blotting to detect the expression of Wnt 5a and Wnt10b.3.Statistical analysisSPSS21.0 statistical software was used for statistical processing,and the data were expressed in the form of mean±standard deviation(±s).Table 2 Experimental data using T test,if it conforms to the normal test and the variance is homogeneous,the single factor analysis of variance is used.LSD method was used to test the variance homogeneity of multiple comparisons between groups,and Dunnett’s T3method was used to test the variance when the variance was uneven.The difference was statistically significant(P<0.05).Results:1.Enzyme-linked immunosorbent assay showed that the levels of VEGF,b FGF,PDGF and KGF in ECM/SVF-CM were significantly higher than those in SVF-CM.2.The dermal papillae isolated from normal human hair follicles were oval in shape.Two days after inoculation,most of the DPCs migrated around DP.DPC proliferated in an adherent way,and the cells were spindle-shaped and arranged regularly when they were passaged to P3.Immunofluorescence staining showed that DPC specific markers alkaline phosphatase andβ-catenin were highly express in DPC.3.CCK8 assay indicated that the proliferation activity of DPC in CMs group was significantly higher than that in control group,while the cell proliferation activity in ECM/SVF-CM group was significantly higher than that in SVF-CM group.4.One week after the injection,dark black pigmentation appeared on the back skin of mice in ECM/SVF-CM group and light black pigmentation in SVF-CM group,while pink in control group.Two weeks after injection,the hair growth rate of the ECM/SVF-CM group was significantly faster than each other.Three weeks after injection,95%of the hair was regenerated in the shaved area of the back of the ECM/SVF-CM group,while 70%of the hair regeneration was observed in the SVF-CM group.The hair growth score showed that the hair growth rate of mice injected with CM was significantly faster than that of the control group,and the hair growth rate of ECM/SVF-CM group was faster than that of SVF-CM group.5.Compared with the control group,the subcutaneous vessels in the back of the CM injection group were significantly increased,and the ECM/SVF-CM treatment group showed clearer vascular branches and more mature angiogenesis.CD31 immunohistochemical staining further confirmed that there was more angiogenesis in ECM/SVF-CM group than in SVF-CM group and control group.6.Ki67 immunohistochemical results showed that the proliferation rate of hair follicle niche in ECM/SVF-CM group and SVF-CM group was higher than control group,and the cell proliferation activity in ECM/SVF-CM group was the strongest.7.Western blotting showed that Wnt5a and Wnt10b were highly express in CMs group,and the expression level of Wnt5a and Wnt10b in ECM/SVF-CM group was higher than SVF-CM group.Conclusion:1.ECM/SVF-CM secretes higher concentration of hair growth promoting factor than SVF-CM and promotes the proliferation of dermal papilla cells in vitro.2.ECM/SVF-CM can effectively promote hair growth,the mechanism lies in promoting the proliferation of DPCs and cells in hair follicle niche,neovascularization as well as the transformation of hair follicle to the growth phase,and the effect is significantly stronger than that of SVF-CM.