A Clinical Approach For Treating Vitiligo With Allogenic Melanocyte Transplantation Using Tissue-engineering Technique

Vitiligo is a common pigment disorder, which is difficult to be cured. The melanocyte is significantly deficient or absent in the pathological examination of vitiligo skin. Autologous melanocytes transplantation is currently effective method for stable vitiligo patients. In this therapy, however, the patients have to suffer from surgical operation on their own normal skin. In addition, the melanocytes obtained are insufficiency to meet the needs of large area transplantation.Recent studies suggested that allograft melanocyte transplantation may solve the problem of melanocyte shortage and yield a better therapeutic effects, thus it is of great value to test its feasibility in clinical trial. Six years ago we had carried out a preparatory research on treeting of vitiligo by transplantating of allograft melanocytes , in which donors and receptors had no genetic connection, their MHC were not matched before transplantation, and no immunosuppressive agents were administered. Three of 11 volunteers got some repigment effect 90 days aftertransplantation and 1 of them got steady repigment. Later, some researchers of Huashan Hospital in Shanghai and Third Hospital in Hangzhou, also reported that visible repigment were found in a dozen of patients after allogenic melanocytes transplantation at several meetings.But it is regretted that except our paper we cannot find any published clinical research paper about allogenic melanocytes transplantation all over the world until now. The reason may be unideal prognosis and immature method. These results attribute to immunological rejection, the number or melanin underproduction of the allogenic melanocytes, and vitiligo local environment.In order to improve the results of this treatment method and explore which is the main affecting factor, we launched another better-designed trial by Tissue-engineering technique. Firstly we tried to culture seed cell, and then selected suitable material to compound melanocyte and prepare for cell transplantation, at last, we began the further clinical trail.Methods Section One Preparation of seed cell in three ways1 Cultured suitable melanocytes by a two-step method. First, pure human melanocytes were obtained in the medium supplemented with TPA at first. Then they were sub-passaged and cultured in the medium without TPA. The melanocytes' morphology, growth curve and cell cycles were investigated.2 Culture melanoblast from human outer root sheath. After digest dermal hair follicle and were tried to purify melanocyte (melanoblasts) in outer rooting sheath cell.3 Human ectomesenchymal cells were separated from abortus facial process.LNGFR(+)cell were sorted by immunomagenetic and identified byHNK-1 (CD57) monoclonal antibody ant Nestin monoclonal antibody. At the same time we try to induce the ectomesenchymal cells differentiate to melanocytes (or melanoblast).Section Two Selected suitable materials to compound melanocytes1 Determined the growth and the melanin synthesis function of melanocytes adding different concentration hyaluronic acid. To observe hyaluronic acid could repression melanocytes cohere and growth.2 The human melanocytes were bred on BioFOLIE membrane and its affinity was observed by microscope and electron microscope. The melanocytes graft's livingness was observed after been frozen.3 To construct three dimensions tissue engineering epidermis containing melanocytes we co-cultured keratinocytes and melanocytes and injected their suspension onto the collagens gel support. The colors of epidermis substitutes were observed before being exposed to UVA and after. It was pathological examined.Section Three Clinical trail to treating vitiligo with allogenic melanocyte transplantationThe allogenic purified melanocytes were cultured from the foreskin of healthy children. Several days before transplant melanocytes deposition were re-suspended with gel and laid in 37C .Thirty minutes later the gel containing melanocytes coagulated, then inject melanocytes onto t...