Experimental Research Of Melanocyte In Chitosan Composite Membrane And Graft Site

Vitiligo is a depigmenting skin disorder affecting approximately0.5-4%of thepopulation[1], characterized by depigmented white patches in a localized or generalizeddistribution. Although vitiligo is neither life-threatening nor symptomatic, the effects ofvitiligo can be cosmetically and psychologically devastating. The pathogenesis iscomplex and it is difficult to treat. Despite the variety of treatment of vitiligo, butnone of them can achieve the purpose of healing. For the treatment of vitiligo, severalmodalities including drug therapy, phototherapy and surgical techniques.Transplantation therapy is a fast and efficient method for the stability of the vitiligo. But“difficult” areas, such as joints, perioral, which the melanocytes attachment weredifficult, so tissue engineering was used in the field of the treatment of vitiligo.ObjectiveTo assess the best time of transplant when the melanocytes were inoculated ontochitosan/gelatin composite membranes(CCGM)， and observed the dynamics ofmelanocyte cells which growed in the dynamics of the basal layer of the animal woundsite. MethodsMelanocytes were inoculated onto CCGM and cultured for different times, then,MTT assay and NaOH assay were carried out to detect the growth of melanocytes, andmelanocyte growth dynamics were detected in different time. Skin wound surface wasprepared in20nude mice, which were equally divited into3groups, test groupinoculated with melanocytes on the CCGM; control group only inoculated withmelanocytes; negative control group treated with chitosan membrane withoutmelanocytes. Both confocal laser microscopy and immunohistochemistry wereperformed to observe the migration of melanocytes into the skin wound surface indifferent times. To estimated the best time of transplant when the melanocytes wereinoculated onto CCGM by this methods and observe the dynamic changes of melancytein animal skin basal.ResultsWhen melanocytes were inoculated on the CCGM and ordinary dish for4hours、1day、2days、3days、4days、5days, we found that the melancytes were in state ofproliferation, and the trend of two line proliferation were consistent. Melanocytesmanufacturing showed a gradual increase, especially48hours which melanocytes wereinoculated into CCGM, The proliferation of melanocytes and melanocytesmanufacturing entered into the platform. Thereforce, the activity of the melanocyteswas best when melanocytes were inoculated to the CCGM.Melanocytes inoculated on CCGM for48h and ordinary petri dish, then transplantedinto nude mice transplanted parts of7days,14days,21days,28days aftertransplantation, skin confocal laser scanning microscopy and immunohistochemicaldetection of graft site had melanocyte colonization,28days after transplantation, the melanocyte most significant.ConclusionThe best time of melanocyte transplant was48hours which the melanocytes winoculated on the CCGM and ordinary dish, Chitosan gelatin film Containimelanocytes applied to nude mice skin, after7days melanocytes will transfer to in the animal wound basal layer parts. With the extension of time, melanocytes incregradually.