| Objectives:Hair has many physiological functions,including resisting ultraviolet radiation,regulating body temperature,draining sweat,tactile sensation and so on.For human body,hair plays an important role in personal image.Hair loss caused by different reasons may not have a significant impact on physical function,but it will seriously affect people's social behavior and the quality of life.The treatment of alopecia has been a cutting-edge research area in recent years.Hair loss can be divided into cicatricial hair loss and non-cicatricial hair loss.In cicatricial type of hair loss,the structure of the hair follicle is usually permanently damaged and replaced by fibrous tissue.However,in the other type,the hair follicle structure is usually complete.Androgenetic alopecia is the most common type of non-cicatricial hair loss.In the early stage of androgenetic alopecia,hair cycle changes are the main factor,characterized by a shortened anagen period and an extended telogen period.Drugs or treatments that induce hair from telogen to anagen phase or extend anagen phase can be used to treat androgenetic alopecia.In recent years,energy-based devices have grown in popularity in dermatologic treatment and cosmetic application,with interest increasing in the potential role of energy-based treatments for hair loss.However,the outcomes often seem to be limited.As a result,adjuvant therapies and procedures are in urgent need of proposal and detailed investigation.Ito M et al.found that after a massive whole-layer skin injury,intact hair follicles and sebaceous glands could regenerate de novo in the process of wound healing.However,the massive wound usually leads to scar formation in human,therefore the induction of hair follicle regeneration through this method is not suitable in clinical use.Fractional radiofrequency microneedle,with minimum and appropriate wound,is an ideal choice to achieve the purpose of wound-induced hair regeneration.Fractional radiofrequency microneedle is a newly proposed,micro-invasive device,which can release bipolar RF current through microneedle and produce a thermal damage zone with a controllable depth and width.The periodical growth of hair follicle is regulated by various signaling pathways,in which Wnt signaling pathway plays a vital role.The Wnt signaling pathway is a very conservative pathway,including the Wnt/?-catenin pathway,the Wnt/cell polarity pathway and the Wnt/Ca2+pathway,where the Wnt/?-catenin pathway is the classical pathway.If the?-catenin or wntless in the hair follicle stem cells of the adult mouse was knocked out,the hair follicle would be arrested at the telogen stage and fail to enter anagen stage,which suggested that the Wnt/?-catenin activation had an important effect on the periodical regeneration of the hair follicle.In recent years,more and more studies have found that hair follicle growth is closely related to inflammatory response and immune cells.Studies have shown that inflammatory cells,especially macrophages,play a key role in inducing hair to enter the anagen phase.Through the release of energy to target tissue,fractional radiofrequency microneedle can produce a small skin thermal damage zone,and the necrotic or coagulation tissue can trigger local inflammatory response,start skin repair mechanism,and finally induce local inflammatory reaction to promote hair into anagen phase.The purpose of this research is to study the effect of fractional radiofrequency microneedle on hair growth and the related mechanisms,and to observe the clinical efficacy and safety of the therapy with a combination with topical minoxidil by a clinical trial.Methods:After being shaved,healthy female 7-week-old C57BL/6J mice were treated with fractional radiofrequency microneedle with a combination of different power(6W,8W,10W,12W,14W)and output time(100ms,200ms,300ms)to determine the best parameters on the dorsal skin.The best parameters of laser therapy were then applied to treat the dorsal skin of 30 mice,and the hair growth condition was photographed and documented.The mice were executed at the time point as follows:before treatment,1day,3 days,5 days,7 days,9 days,11 days and 13 days after laser treatment by cervical vertebra dislocation method.The growth of hair follicle was observed by HE staining,and the activation of hair follicle stem cells was observed by immunofluorescence staining.The changes of Wnt signal pathway molecules were analyzed by PCR Array technique screening,real-time fluorescence quantitative PCR and immunohistochemical staining.Real-time fluorescence quantitative PCR was used to detect the mRNA expression of inflammatory factors,such as Ccl2,iNOS and Arg1,at different time point.F4/80positive macrophages were observed by immunohistochemical staining method.The inflammatory inhibition effect of fractional laser therapy on normal mouse model and de-macrophage mouse model was finally observed.19 male subjects aged 22-55 who were diagnosed with androgenetic alopecia were recruited in the clinical trial.The baldness area was divided into two regions,and randomly assigned to be combination therapy side and monotherapy of minoxidil side.Subjects received 5 treatments with fractional radiofrequency microneedle at 4-week intervals on one side of the scalp combined with 5%topical minoxidil on both sides.Objective index including hair density and hair diameter,global assessment by the investigators,subject satisfaction degree and adverse effects evaluation were assessed.Results:10W and 200ms turned out to be the ideal parameter for hair growth,under which condition,the hair follicles of the treatment area showed to enter anagen phase first.So these treatment parameters were applied to on mice.At the 9th day after treatment,the back skin of mice began to turn gray.After 15 days of treatment hair started to grow out,and at the 18th day,the hair on the back skin grew out nearly completely.HE staining results showed that on the 5th day,the volume of dermal papilla increased;on the 7th day dermal papillae was close to the junction of dermis and subcutaneous;the 9th day dermal papillae entered into the subcutaneous tissue with an increasing number of hair matrix cells that wrapped around the dermal papillae;on the11th day most part of the dermal papillae was surrounded by matrix cells with the observation of melanin granules above the dermal papillae;on the 13th day,dermal papillae was observed to enter into the subcutaneous tissue with a numerous melanin and hair shaft occurrence.Quantitative analysis of hair length in mice showed that hair length increased gradually on the 9th day after treatment(P<0.05).The results of immunofluorescence showed that the nucleus of stem cells at hair germ side started to express BrdU on the 7th day after treatment,and the BrdU+cells appeared at follicle bulge site on the 9th day after treatment.11th and 13th days after treatment,a large number of BrdU+cells were found,mainly distributed in the outer hair root sheath and the hair matrix cells of hair follicle.Before and 5 days after treatment,a significant increase of five genes and a decrease of one was screened by PCR Array method and the results were verified by real-time PCR.The expression of Porcn and Bcl9 gene showed a significant increase(P<0.05)after 5 days treatment of fractional radiofrequency microneedle.Immunohistochemical results showed that on the 7th day and 14th day after treatment,?-catenin and Lef1 protein expression were higher than that before treatment.Intraperitoneal injection of dexamethasone can inhibit the hair growth after the treatment of fractional radiofrequency microneedle treatment.Immunohistochemical results showed an increase in the number of F4/80 positive macrophages in the treated area three days after treatment.Real-time PCR results showed that M1 macrophage marker(iNOS)mRNA levels increased significantly,and inflammatory factors Ccl2,Tnf-?,Il-1?mRNA level was significantly increased after treatment(P<0.05).The removal of macrophage in mice by the clodronate liposome revealed that the hair growth phenomenon disappeared after fractional radiofrequency microneedle treatment.Conclusion:Fractional radiofrequency microneedle can activate hair follicle stem cells to promote hair entering anagen phase.Wnt signaling pathway participates and plays an important role in this process.Inhibition of inflammatory response after treatment can make the hair growth disappear.There is an increase in the amount of macrophage 3 days after the treatment.Besides the related inflammatory cytokines increased after the treatment.The hair failed to enter anagen phase after the treatment under the condition of removing macrophage,which indicating that fractional radiofrequency microneedle may promote hair growth via inflammation and macrophages play an important role.The clinical trials showed that combined treatment of fractional radiofrequency microneedle and 5%topical minoxidil could be an effective and safe treatment option for men with male pattern hair loss. |
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