Primary Study On Application Of Melanocytes And Bone Marrow Mesenchymal Stem Cells In Tissue-engineered Skin Construction

Wound repairing for skin defect is one of the most common problems for plastic surgeons. Although auto-skin or allo-skin tissue grafting is commonly used as a therapy method, there still exists many disadvantages, such as insufficient harvesting tissue and following harm to patients. The research and application on tissue-engineered skin provides an alternative therapy strategy. The production of tissue-engineered dermal or full-thickness skin can be found in the market. However, these "artificial skin substitutes" are only competent to cover the wound and to accelerate the heeling. It is still not a kind of real "skin organ" with pigmentation, secretion of sweat and hair growing. Therefore, it is important to study on tissue-engineered skin with normal biological functions. The melanocyte in the skin plays a key role in adjusting the skin color and preventing the cell from ultraviolet radiation injury. Based on the previous study that the full thickness tissue-engineered skin were constructed with the bone marrow mesenchymal stem cells(BMSCs) as seed cells, we co-cultured the melanocytes and BMSCs in optimal proportion and grafted them into type I collagen membrane to construct the tissue-engineered skin with the pigmentation. Thus this study could provide a theory evidence to the further research.ObjectiveTo establish a method to acquire numerous melanocytes and BMSCs, then to identify the characteristics of these cells.To establish a way to co-culture the melanocytes and BMSCs in optimal proportion, and to construct the tissue-engineered skin in vitro.To evaluate the effect of the tissue-engineered skin constructed with melanocytes and BMSCs in vivo.MethodsCells were isolated from human foreskin and prepared as cell suspension by the digesting method, which then were cultured in the complete melanocytes medium and subcultured by trypsinizing with low concentration. After that, the DOPA staining, S-100 protein immunohistochemistry staining and the transmission electron microscope observation were performed, and growth curve was drawn as well. Meanwhile, isolation and culturing of BMSCs, as well as surface antigen recognization and multi-potent differentiation ability were researched.The type I collagen membrane was remolded. The opitimization on the medium for the mixture of the melanocytes and BMSCs according to the growth curve was carried out to validate the the proposal of the constructing tissue-engineered skin with melanocytes and BMSCs in collagen membrane, which then was tested by the S-100 protein immunohistochemistry staining and observed by the transmission electron microscope.The wound model was achieved via skin tissue ablating on the back of the nude mice. The skin substitute constructed in vitro was transplanted to the wound region. The specimens on different time were acquired and the function of skin substitutes was observed via the systemic specimen observation, the melanocyte tracking on the DAPI fluorescent staining, HE staining, S-100 protein immunohistochemistry staining and transmission electron microscope observation.ResultsThe primary passage melanocytes showed polydendritic appearance but fusiform when passaged to the second generation. They showed positive in S-100 protein immunohistochemistry staining and DOPA staining. Numerous melanosome were detected in the plasma through transmission electron microscope observation. The primary passage BMSCs from the bone marrow showed fibroblast-like colony forms. After passaging, the cell morphology of the BMSCs was uniform, with flat fusiform shape, without CD34 and CD11a expression in cell surface, and with strong CD29 staining, but light CD71 staining. More than 80% of cells was in G₀/G₁ stage, 11% in G₂/M stage and about 7% in S stage. The chromosome karyotype analysis appeared the normal female type. Lipid droplet appeared in the BMSCs plasma on the fifth to sixth day after the induction. On the fourteenth day, the differentiation reached peak level, the size of the cell were biggest and the number of lipid droplet reached to the maximum. On the third week, the cells could be stained red by the oil red O. The morphology of the cells began to change in the osteoblast condition medium for induction seven days later, which looked like osteoblast on the fourteenth day. Darkish area could be found in pericell body area when stained by alkaline phosphatase. BMSCs were negative in S-100 protein immunohistochemistry.Uncross-linked type I collegen membrane was like a white sponge and poor to tear, but the cross-linked collegen membrane was like a semitransparent achromatic film, soft and flexible, competent for tearing. On the first day, 2×10⁶ BMSCs were implanted into the collegen membrane, the size of which was the same as a foramen of 24 shadow mask, and cultured in BMSCs complete medium; then 2×10⁵ melanocytes were implanted into the collegen membrane and cultured in melanocyte complete medium on the second day. By this way, the tissue-engineered skin with mixed cells could be constructed. Melanocytes and BMSCs were seen growing well in the collegen membrane through the scanning electron microscope, melanocyte in tissue-engineered skin were positive in S-100 immunohistochemistry staining.The wound began to heal after two weeks of the operation. The two groups with BMSCs were better than the group with the pure melanocytes, the wound became smaller, the crust was also as well. Under HE staining, epithelia of the former two groups were thicker and the arrangement of epithelial cells was ordered than the pure melanocyte group. S-100 immunohistochemistry reaction was positive in the two groups with melanocytes, but negative in the group with pure BMSCs. The melanocytes were observed in the tissue-engineered skin in vivo through the transmission electron microscope, which showed the connection with the base membrane through the semi-desmosome, the epithelial cells were ordered, tonofilament were seen in the cytoplasma, the cells were connected with each other through the desmosome. Melanocytes can be tracked through DAPI fluorescent staining on the first and second week after the operation.ConclusionNumerous pure melanocytes in good state can be isolated from the human foreskin and cultured through the digestion by low desity trypsin. The BMSCs isolated from the bone marrow are multipotent and stable in the inherent state. Both melanocytes and BMSCs can be chosen as the seed cells for the tissue engineering. S-100 protein immunohistochemistry staining can be applied to identify the melanocytes in the mixture of the both cellsTwo complete medium of the melanocytes and BMCSs can replaced with each other in the first three days of culturing. The melanocytes (with 10⁵ / cm² level) and BMSCs( with 10⁶ / cm² level) can be mixed to construct the tissue-engineered skin in vitro. Both cells grow well in the collagen membrane.The constructed tissue-engineered skin which survived with not only the good bioactivity but also possessed structure and function to help producing the melanin can repair the skin wound of the experimental animals in vivo.