A Series Of Basic And Clinical Studies Related To Photomedicine In The Field Of Dermatology

Background and objective:Photoaging is the mainly reason of exogenous aging,which participate in the aging of the skin aging process with endogenous aging.It has been also reported that dedifferentiated adipocytes(DA)could by got by "ceiling culture" method from purified mature adipocytes.DA has many similar characteristics as stem cells,and also has also been confirmed to participate in a variety of tissue repair process.Meanwhile the usage of DA in the research filed of anti photo-aging has never been reported.In the first part of our study,the anti-ageing effect of DA and the conditioned medium of DA were studied in vitro and in vivo,and the underlying mechanism was investigated and discussed.The main manifestations of skin ageing include pigmented skin lesions,vascular lesions,skin laxity and wrinkles.Fractional carbon dioxide resurfacing(FxCR)focuses on the symptoms of photoaging as pigmented skin lesions,skin laxity and wrinkles,while long pulse Nd:YAG 1064nm laser is mainly targeted on vascular lesions together with skin laxity and wrinkles.Carbon dioxide laser is based on the theory of fractional photothermolysis(FP),which is reported to have some side effects,such as post-inflammatory hyperpigmentation,erythema and scars.Recent studies show that the conditioned medium of ASC(ASC-CM)can stimulate the synthesis of dermal collagen and the migration of fibroblasts in dermis.The second part of our study aims to assess the effect of topical ASC-CM on the wound healing process,erythema,pigmentation and other adverse effects after FxCR.In addition to photo-aging treatment,recent studies have shown that long-pulse Nd:YAG 1064nm laser can also be used for onychomycosis.In the previous literature most studies focused on the effect of long pulse Nd:YAG 1064nm laser alone,without comparing with other therapy.In the third part of this study we aimed to assess the effect and side effects of combined therapy of long-pulse Nd:YAG 1064nm laser and oral terbinafine,compared with either therappy alone.Methods:In the first part of our study,we collected DAs after mature adipocytes being cultured by ceiling culture method for 2 weeks,and confirmed DAs by multi-potential differentiation and stem-cell like cell membrane markers.The secreted cytokines in supernatant of DAs were measured by ELISA.DAs fluorescently labeled by PKH26 were subcutaneously injected into nude mice skin,and after 4 weeks and 8 weeks,skin samples were taken to observe the colonization and proliferation of DA.The unlabeled DAs were also injected subcutaneously into nude mice skin,and after 4 and 8 weeks skin samples were taken to observe the histopathological changes of dermis caused by DA.Repeated chronic UVB irradiation method was used to induce photo-ageing mice model.The 10-fold concentrated DA-CM was collected and subcutaneously injected into the photo-aged mice skin.After 4 and 8 weeks,the histopathological and immunohistochemical changes in type I and III collagen and matrix metalloproteinase(MMP)-1,-3 were measured.Stress-induced premature senescence(SIPS)in human dermal fibroblasts(HDF)was induced by five-day irradiation of UVB,and DA-CM was added during the process.Then CCK-8,EdU staining and ?-galactosidase staining were used for each group to detect cell proliferation and cell senescence.The concentrations of type ? and ? collagen,MMP-1 and MMP-3 in supernatant of HDFs were measured by ELISA,after irradiated by a single dosage of UVB as 30mJ/cm2,and the neutralizing antibody of TGF-?1 was added to detect the role of TGF-?1 in the photo-protection effect of DA-CM.In the second part,nine subjects were treated with carbon dioxide fractional laser on bilateral forearms.ADSC-CM was applied on FxCR site of one randomly selected arm.Transepidermal water loss(TEWL),skin color,and gross-elasticity of FxCR site of both arms were measured.Skin samples were taken by biopsy from three subjects 3 weeks after treatment for histopathological manifestations.In the third part of this research,we randomly divided 53 patients with a total of 90 infected nails into three treatment groups.T group received oral terbinafine,L group received long-pulsed Nd:YAG laser treatment,and T+L group received both treatments.We evaluated the mycological clearance rate(MCR)and the clinical clearance rate(CCR)of the three groups at weeks 4,8,12,16,and 24.Results:1.DAs exhibit multipotent differentiation potential and stem cell-like characteristics,and the secretary factors in DA-CM can increase the thickness of dermis and density of collagen in nude mice skin.The DAs isolated and cultured from adult human fat tissue expanded easily in vitro and exhibited fibroblast-like morphology.The cells also exhibited multipotent differentiation potential when cultured in specialized medium for 2 weeks.A variety of cytokines can be measured in the supernatant of DA,including PDGF-BB,bFGF,VEGF,KGF,TGF-?1 and HGF.Under a fluorescence microscope,intensive red fluorescence was only observed in the fat layer where the injection were performed after 4 and 8 weeks,and no fluorescence were found in epidermis or dermis,indicating that the subcutaneously injected DAs colonized and proliferated in the fat layer at least up to 8 weeks.Four and eight weeks after subcutaneously injection of DAs,the pathological finding showed DAs could significantly improve the content of collagen in the dermis.2.DA-CM mitigates the reduction of collagen type ? and ? by UVB irradiation in mice.After weekly subcutaneous injection of 10-fold concentrated DA-CM into photoaging mice skin,we determined the DA-CM effects on the photo-aged mouse model,and a significant increasing of collagen amount and improved organization of collagen fibrils in dermis were observed after 4 and 8 weeks post DA-CM injection.The section from DA-CM-injected side exhibited deeper brown staining for collagen type I and type III proteins,compared with the control side.The observations were further confirmed by quantitative assessment of the percentage of positive collagen staining.However,no obvious changes in the expression of MMP-1 and MMP-3 were observed compared to the control.3.DA-CM mitigates UVB-induced changes in cell proliferation,premature senescence and collagen type ? and ?,MMP-1 and MMP-3 proteins secreted by HDFs,in which TGF-?1 is a major regulator.After UVB irradiation,less cell proliferation was observed compared to control cells,while with DA-CM treatment,the decreased proliferation by UVB irradiation was dramatically increased.HDFs,experienced UVB irradiation for 5 consecutive days,exhibited significant changes in appearance from spindle-like to large,flat,and irregular in shape.Premature senescence staining revealed that more cells presented SA-?-gal staining after UVB irradiation,and addition of DA-CM to HDFs,and cells with SA-?-gal staining were reduced.We observed that,from 24 to 48 hours after UV irradiation,both collagen type ?and ? were dramatically reduced,compared to non UV irradiated HDFs.However,addition of DA-CM to cultured HDFs largely prevented this reduction by UV irradiation.Administration of DA-CM also effectively prevented induction of MMP-1 and MMP-3 by UV irradiation.These results indicate that the reduction of collagens and induction of MMPs by UVB irradiation are largely preventable by DA-CM.And the DA-CM effects,described above,were terminated,at least partially,by addition of TGF-?1 neutralizing antibody,suggesting that TGF-?1 in the DA-CM plays an important role in the protecting collagen from reduction caused by UVB in HDFs.4.Application ASC-CM is an effective method for enhancing wound healing after FxCR,by reducing transient adverse effects.The index of erythema,melanin,and TEWL of the ASC-CM-treated skin were significantly lower than those of the control side.The histological manifestations were not changed significantly.5.Combined oral terbinafine and long-pulsed 1064 nm Nd:YAG laser treatment is more effective for onychomycosis than either treatment alone.We did not observe complete mycological or clinical clearance for any of the patients within the first two weeks of treatment.The MCR and CCR increased in all three groups in a time-dependent manner.The MCR and CCR of the T+L group were significantly higher than those of T group and L group at weeks 8,12,16,and 24.There were no significant differences in MCR or CCR after 24 weeks of treatment between the T group and the L group.Conclusion:1.DAs can be useful for aging skin,and their effects are mainly due to secreted factors,especially TGF-?1,which stimulate collagen synthesis and alleviate collagen degradation in HDFs.2.Application of allograft ASC-CM is an effective method for enhancing wound healing after FxCR,by reducing transient adverse effects such as erythema,hyperpigmentation,and increased transepidermal water loss.3.Twelve-week combined treatment with long-pulsed Nd:YAG laser and oral terbinafine produces more rapid and effective mycological and clinical clearance in patients with onychomycosis than either alone,without any obvious side effects.