117 Light or Dark Pigmentation of Engineered Skin Substitutes Containing Melanocytes Protects Against UV-Induced DNA Damage in Vivo

Introduction Engineered skin substitutes (ESS) were developed to meet the need for prompt wound closure in patients with large full thickness burns. ESS containing autologous fibroblasts and keratinocytes were shown to provide stable wound closure in burn patients, but are limited by hypopigmentation. DNA damage caused by ultraviolet (UV) radiation is a known risk factor for development of skin cancer. In normal human skin, epidermal melanocytes provide pigmentation, helping to shield skin from UV-induced DNA damage. The current study investigated inclusion of human melanocytes (hM) and their role in the response of ESS to UV light in vivo. Methods Primary cells were isolated from skin of healthy de-identified human donors with IRB approval. Three groups of ESS were prepared with fibroblasts and keratinocytes, +/- hM, and were grafted orthotopically to immunodeficient mice: ESS without hM; ESS with light skin-derived (Caucasian) hM (ESS+hML); and ESS with dark skin-derived (African American) hM (ESS+hMD). After 8 weeks in vivo, grafts were irradiated with 135 mJ/cm2 UV, and mice were euthanized after 2 or 24 hours; non-UV treated mice served as controls. Pigmentation and erythema were measured with a Mexameter. Melanocytes and cyclobutane pyrimidine dimers (CPDs) were quantified by immunostaining with anti-TYRP1 and anti-CPD antibodies, respectively, followed by image analysis (Nikon Elements). Statistical analyses (SigmaPlot) utilized t-test or one-way ANOVA; P< 0.05 was considered significant. Results At 8 weeks post-grafting, mean hM density in ESS+hML and ESS+hMD was not significantly different from normal human skin samples. Pigmentation (in Mexameter units) before UV irradiation was significantly different among groups (ESS+hMD > ESS+hML > ESS no hM). UV irradiation did not increase erythema in any group, but resulted in significantly increased pigmentation in ESS+hML and ESS+hMD at 2 hours, but not 24 hours, post-UV. CPDs, the most prevalent form of UV-induced DNA damage, were significantly elevated 24 hours post-UV in ESS without hM. DNA damage was significantly lower 24 hours post-UV in ESS+hML and ESS+hMD compared with ESS without hM. No differences in DNA damage were observed between ESS+hML and ESS+hMD. Conclusions Pigmentation of ESS+hML and ESS+hMD in vivo varied according to the skin phototype of the hM donor, with no difference in melanocyte density, which was similar to normal human skin. Inclusion of either light or dark hM decreased UV-induced DNA damage, suggesting that hM in ESS play a photoprotective role, as in normal human skin. Applicability of Research to Practice Protection against UV-induced DNA damage may reduce the risk of skin cancer in patients grafted with ESS containing melanocytes.