Reconstruction Of A Pigmented Tissue Engineering Skin Containing Capillary-like Network

The ultimate objective for tissue engineering skin is restoration of the anatomy and physiology of uninjured skin after treatment and healing of the wound. At present, only an autologous full-thickness skin grafts, free flaps or pedicle flaps restore all of the structures and functions of uninjured skin but donor sites and treatment sites must be equal in size. Angiogenesis (the formation of new blood vessels) is essential for the growth of new tissue, tissue repair and wound healing. Tissue engineering, the construction of new tissue and organs for reparative purposes, relies on angiogenesis for the vascularisation of these new grafts. In tissue engineering, the emphasis to date has been on vascularisation of newly constructed tissue grafts by an extrinsic blood supply, and relatively littleattention has been given to the possibility of building these grafts around an intrinsic blood supply. In this study, two kinds of skin substitutes were reconstructed by employing tissue engineering method:1. To develop a rapid and reproducible method for the isolation of pure melanocytes in good conditions from foreskin: The melanocytes were isolated and purified by a method employed in our lab. Two different media MCI and MC2 were used to culture the cells. The biological conditions of the cells were examined by MTT detection and flowcytometry (FCM) analysis. The melanocytes obtained by this method were free of keratinocytes and fibroblasts contamination. The cells maintained in MC2 showed better proliferation ability than those cultured in MCI. The results showed that pure melanocytes culture in good conditions can be obtained rapidly by our method.2. To construct a tissue engineering skin containing melanocytes: The keratinocytes, dermal fibroblasts and melanocytes were isolated and purified from a single foreskin biopsy. Then the cells were used to construct a tissue engineering skin containing melanocytes. The location of melanocytes in the tissue engineering skin were detected byDopa staining, transmission electron microscopy (TEM) and S-100immunostaining. The reconstructed skin was used to repair the nude mice skin defects. Results demonstrated that the artificial skin containing melanocytes was successfully constructed in vitro and can be used to repair the full-thickness skin defects successfully.3. To develop a rapid and reproducible method for the isolation of pure human umbilical vein endothelial cells (HUVECs) in good conditions: The HUVECs were isolated and purified by a method employed in our lab. The HUVECs were identified by cell morphology, immunostaining and transmission electron microscopy. The HUVECs obtained by this method were free of fibroblasts contamination. And the cells showed very good proliferation ability. The pure HUVECs in good conditions can be attained rapidly and easily by our method.4. To construct a tissue engineering skin containing capillary-like network: The HUVECs were isolated and purified from a new-born umbilical cord. The keratinocytes and dermal fibroblasts were cultivated from a new-born foreskin biopsy. The skin equivalent containing capillary-like network was constructed by employing tissue engineering method. The reconstructed skin was used to repair the nude mice skin defects.Results showed that the artificial skin containing capillary-like networkwas successfully reconstructed in vitro and repaired the full-thicknessskin defects successfully.