Production Film By Chitosan And Gelatin As Transporter For Melanocytes: In Vivo And In Vitro Studies

Objective Guidance in tissue engineering, we use chitosan, gelatin and other tissue engineering materials construct film and we discover the feasibility of these types of tissue engineering of film carrying, transiting melanocytes.This provide the basis for further used in clinical treatment for the depigmentation diseases.Method Chitosan and gelatin in different proportions were melted to gel with the acid and put those gel into cell culture plate baking to films; We detected the histocompatibility of the membrane material by using films leaching solution to cultured melanocytes; we observed cross-section structure of the films body by transmission electron microscopy (TEM) , make sure three-dimensional shape of tissue engineering films. Cultured melanocytes were inoculation on the films, the time that melanocytes attached on the membrane were counted. The cases of melanocytes adhesion on the films were observed by the inverted microscope and scanning electron microscopy (SEM) . MTT method, NaOH lysis methods to determination the melanocyte growth on the films and observed whether cells could synthesis melanin. The films that carrying melanocytes were transplanted to the nude mice, and then we detected the transplanted parts by confocal laser scanning microscopy and immunohistochemical. Residual melanocytes on the films were observed by fluorescent in order to judge the metastasis of melanocytes on the films.Result The films that were made by different proportions of chitosan and gelatin, did not affect the normal growth of melanocytes, indicating the films of chitosan and gelatin the present study had a good histocompatibility with melanocytes; the different proportion of mixed films had obvious three-dimensional body structures, while three-dimensional body structure of pure chitosan membrane was not obvious, the obvious three-dimensional structure was conducive to keeping the culture medium and provided better micro-environment for the melanocyte colonization in the graft site . The time that melanocytes adhered to a variety of membrane is within 3 hours and that melanocytes adhered to the 2:1 ratio of chitosan - gelatin membrane required the shortest time,and 95% melanocytes adhered to this film after 2 huors. Gelatin may have a good affinity for cell. Melanocytes attached to the pure chitosan membrane needed a longer time, 3 hours and after 1 hours only 50% cell attached. We can see melanocyte distributed uniformly and adhered to the films very well by inverted microscope and scanning electron microscope(SEM). MTT proliferation curve showed melanocytes on the different percentage of films could grow well, in which a 2:1 ratio of chitosan - gelatin flim supported melanocyte grow better than the other films, melanocytes reproducted by 1 times about 4 days, while other types of films needed 5-6 days. the melanin content curve made by NoOH lysis method was the same with the growth curve by MTT and that further confirmed the different tissue engineering support growth and proliferation of melanocytes. Confocal microscope we can see the transplanted position of each group in nude mice contained melanocytes, but not in the control; graft site tissue sections were stained with Melan-A monoclonal antibody by immunohistochemistry and the result is positive and post-transplant membrane by fluorescence staining was observed under inverted microscope shows a little residual melanocytes.Conclusion Chitosan film and the radio of 2:1,1:1,1:2 chitosan-gelatin films have good affinity with melanocytes. They can support melanocyte adhesion, growth and proliferation on them and they can support the carrying melanocytes divert to the transplanted sites of animals.So these films can be as a carrier material for melanocytes grafting,in which the film of 2:1 ratio has the best performance. It provids a good prospect of clinical application.