Impact of ultraviolet radiation on dermal and epidermal DNA damage in a human pigmented bilayered skin substitute

Our laboratory has developed a scaffold-free cell-based method of tissue engineering to produce bilayered tissue-engineered skin substitutes (TESs) from epidermal and dermal cells. However, TES pigmentation is absent or heterogeneous after grafting, due to a suboptimal number of melanocytes in culture. Our objectives were to produce TESs with a sufficient quantity of melanocytes from different pigmentation phototypes (light and dark) to achieve a homogeneous color and to evaluate whether the resulting pigmentation was photoprotective against ultraviolet radiation (UVR)-induced DNA damage in the dermis and the epidermis. TESs were cultured using different concentrations of melanocytes (100, 200, and 1,500 melanocytes/mm2 ), and pigmentation was evaluated in vitro and after grafting onto an athymic mouse excisional model. Dermal and epidermal DNA damage was next studied, exposing pigmented TESs to 13 and 32.5 J/cm2 UVR in vitro. We observed that melanocyte cell density increased with culture time until reaching a plateau corresponding to the cell distribution of native skin. Pigmentation of melanocyte-containing TESs was similar to donor skin, with visible melanin transfer from melanocytes to keratinocytes. The amount of melanin in TESs was inversely correlated to the UVR-induced formation of cyclobutane pyrimidine dimer in dermal fibroblasts and keratinocytes. Our results indicate that the pigmentation conferred by the addition of melanocytes in TESs protects against UVR-induced DNA damage. Therefore, autologous pigmented TESs could ensure photoprotection after grafting.