Experimental Study Of Adipose-Derived Stem Cell Conditioned Medium Combined With Microneedle To Promote Hair Regeneration In C57BL/6 Mice

Background : Hair loss is a kind of disease caused by various hair growth disorders,which has a great impact on the psychology of patients.More than 50% of people suffer from hair loss.Androgenic alopecia(AGA)and alopecia areata(AA)are the two most common types of hair loss.Currently,the FDA-approved AGA treatments include oral finasteride and topical minoxidil,regarding the medical treatment of hair loss.However,oral medication only delays the progression of losing hair and has the potential of adverse effects.Commonly-used treatment for AA is systemic or topical application of hormones or immunosuppressants.Patients often experience immunosuppression-related adverse reactions after treatment.The research of using stem cells to regenerate hair follicles by bioengineering provides a new way for the treatment of hair loss.For example,adipose-derived stem cell conditioned medium(ADSC-CM)has no cellular components and is not immunogenic,while requires no donor-acceptor matching,making it a superior choice.As a minimally invasive treatment for skin diseases,microneedling therapy is increasingly used in hair loss treatment.Studies have confirmed that microneedling therapy is associated with other hair growth-promoting therapies such as minoxidil,platelet-rich plasma,and topical steroid therapy.Combined,it can effectively stimulate hair follicle growth.At present,the regeneration effect of single treatment on hair loss is limited,which limits the application of the technology.The application of many combined treatment provides safer and more novel treatment for hair loss patients.PURPOSE: We designed this experiment to clarify the combined application of ADSC-CM and microneedling therapy in C57BL/6 mice,to observe the efficacy and safety of the combined treatment on hair regrowth in mice,and to explore the mechanism of how the treatment affects hair growth.In the end,it provides a theoretical basis for clinical effectiveness,with the hope of providing a more efficient treatment modality for patients with alopecia.METHODS: 1.In vitro experiment: Prepare conditioned medium and detect the growth factor concentration of VEGF,FGF and IGF-1: Prepare ADSC-CM according to the method reported in the literature,and add adipose-derived stem cells to the basal medium containing 10% fetal bovine serum.After culturing to the fourth passage,replace the basal medium with serum-free medium,and collect the supernatant after 72 hours of culturing,which is the ADSC-CM.The growth factor concentrations of VEGF,FGF and IGF-1 were detected by ELISA method.Dermal papilla cells(DPCs)were cultured and identified in vitro,and the effect of conditioned medium on the proliferation and migration of DPCs in vitro was detected.2.In vivo experiment: 16 7-week-old C57BL/6 mice were divided into four groups.After intraperitoneal injection of pentobarbital sodium,the hair was shaved with a razor and the back hair of the mice was completely removed with depilatory cream.Four groups of mice were deal with PBS,microneedling therapy,ADSC-CM,ADSC-CM combined with microneedling therapy respectively.The hair growth of mice in each experimental group was dynamically observed(detection indicators including mice back skin color,hair growth changes,and hair growth score).Three weeks(21 days)after the experiment,the back skin samples were collected by histological detection to count the number of hair follicles in each group.CD31 and VEGF immunohistochemical staining was used to detect the angiogenesis of the subcutaneous of the mice,and Ki-67 immunohistochemical staining was used to detect the proliferation of cells in the hair follicle bulge area.Western blotting was used to detect the expression of Wnt 5a and β-catenin,which are hair growth-related signaling molecules.RESULTS: 1.According to the instructions of the enzyme-linked immunosorbent assay kit to measure the concentrations of FGF,VEGF and IGF-1 in ADSC-CM,it can be concluded that there are more FGF,VEGF and IGF-1 in ADSC-CM,and these growth factors matters in promoting hair growth.The activity and mobility of dermal papilla cells treated with ADSC-CM can be enhanced,compared with the control group,the difference was statistically significant,P<0.05.2.Four groups of 7-week-old female C57BL/6 mice were depilated with PBS,microneedling therapy,ADSC-CM,ADSC-CM combined with microneedling therapy respectively.The results showed that after the intervention,the hair of mice in the combined treatment group grew first.The number of hairs and hair follicles was the most,and the expressions of Ki67,CD31 and VEGF were the highest,and the difference was statistically significant,P<0.05;the hair growth rate,the number and expression of hair follicles in the microneedling therapy group and the CM group were similar,and the rate of the control group was the smallest,the number was the least,and the expression was the lowest,and the difference was statistically significant,P<0.05.The result of Western blot showed that the expression of Wnt5 a and β-catenin in the combined treatment group was the highest,followed by the microneedling therapy group and the CM group,and the expression in the control group was smallest,the difference was statistically significant,P<0.05.Conclusion: This study proposes a new method for the treatment of hair loss.On the basis of microneedle treatment,the ADSC-CM is used in combination.This combined application can promote hair growth,and the effect is better than the single application of microneedle or ADSC-CM.This study provides new evidence for the clinical application of stem cells to patients with alopecia,and new ideas for the treatment of patients with alopecia.