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.

中文翻译：

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紫外线对人类有色双层皮肤替代品中真皮和表皮DNA损伤的影响。

我们的实验室已经开发出一种基于无支架细胞的组织工程方法，可以从表皮和真皮细胞生产双层组织工程皮肤替代品（TES）。但是，由于培养中黑素细胞数量不足，移植后TES色素沉着不存在或不均一。我们的目标是从不同色素沉着的光型（浅色和深色）中生产出具有足够数量黑素细胞的TES，以达到均一的颜色，并评估所得色素沉着是否对紫外线对真皮和真皮中DNA的DNA损伤具有保护作用。表皮。使用不同浓度的黑素细胞（100、200和1,500黑素细胞/ mm2）培养TES，并在体外和移植到无胸腺小鼠切除模型上评估色素沉着。接下来研究皮肤和表皮DNA的损伤，将有色的TESs在体外暴露于13和32.5 J / cm2的UVR中。我们观察到黑素细胞的密度随着培养时间的增加而增加，直到达到与天然皮肤的细胞分布相对应的平台。含黑色素细胞的TES的色素沉着类似于供体皮肤，可见黑色素从黑色素细胞转移至角质形成细胞。TES中黑色素的含量与UVR诱导的皮肤成纤维细胞和角质形成细胞中环丁烷嘧啶二聚体的形成呈负相关。我们的结果表明，通过在TES中添加黑素细胞而赋予的色素沉着可以防止UVR诱导的DNA损伤。因此，自体着色的TES可以确保接枝后的光保护。我们观察到黑素细胞的密度随着培养时间的增加而增加，直到达到与天然皮肤的细胞分布相对应的平稳期。含黑色素细胞的TES的色素沉着类似于供体皮肤，可见黑色素从黑色素细胞转移至角质形成细胞。TESs中黑色素的含量与UVR诱导的皮肤成纤维细胞和角质形成细胞中环丁烷嘧啶二聚体的形成呈负相关。我们的结果表明，通过在TES中添加黑素细胞而赋予的色素沉着可以防止UVR诱导的DNA损伤。因此，自体着色的TES可以确保接枝后的光保护。我们观察到黑素细胞的密度随着培养时间的增加而增加，直到达到与天然皮肤的细胞分布相对应的平台。含黑色素细胞的TES的色素沉着类似于供体皮肤，可见黑色素从黑色素细胞转移至角质形成细胞。TESs中黑色素的含量与UVR诱导的皮肤成纤维细胞和角质形成细胞中环丁烷嘧啶二聚体的形成呈负相关。我们的结果表明，通过在TES中添加黑素细胞而赋予的色素沉着可以防止UVR诱导的DNA损伤。因此，自体着色的TES可以确保接枝后的光保护。可见的黑色素从黑色素细胞转移到角质形成细胞。TESs中黑色素的含量与UVR诱导的皮肤成纤维细胞和角质形成细胞中环丁烷嘧啶二聚体的形成呈负相关。我们的结果表明，通过在TES中添加黑素细胞而赋予的色素沉着可以防止UVR诱导的DNA损伤。因此，自体着色的TES可以确保接枝后的光保护。可见的黑色素从黑色素细胞转移到角质形成细胞。TES中黑色素的含量与UVR诱导的皮肤成纤维细胞和角质形成细胞中环丁烷嘧啶二聚体的形成呈负相关。我们的结果表明，通过在TES中添加黑素细胞而赋予的色素沉着可以防止UVR诱导的DNA损伤。因此，自体着色的TES可以确保接枝后的光保护。