191 Full Skin Equivalent Model for Burn Wound Healing human skin (left) and full skin equivalents generated from de-epidermalized dermis, MatriDerm and Mucomaix (right). Level of (A) IL-6; (B) IL-8; (C) MCP-1; (D) IL-1β; (E) IL-10; (F) IP-10 in the culture medium at T0, T + 1-4 days, T + 5-7 days and T + 8-11 days (after burn injury). Samples from biological duplicates were pooled per donor (n = 3 donors) and re-calculated into pg/ml per day of culture to compensate for differences in intervals of medium changes. Striped line indicates the highest or lowest level of quantification. Because ex vivo human skin models were started on the first day, no levels are shown for day 0. DISCUSSION Due to issues in the translation of animal data to the human situation, as well as ethical concerns, there is a growing demand for more appropriate, animal-free approaches in preclinical research [11]. Organotypic skin models, such as FSEs, are promising alternatives to animal models because they are more standardized, controllable, and easy to customize with relevant components such as specific cell types [20,21,33]. FSE models are more realistic than models that only use an epidermal layer, as the interplay between keratinocytes and fibroblasts affects skin development and healing [34–36]. Here, we validated FSEs generated from commercially available dermal substitutes MatriDerm and Mucomaix for the use as in vitro skin models to study skin development and burn wound healing. Additionally, we investigated the effect of longer culture times (up to a total of 5 weeks). Studies usually culture organotypic skin models up to 3 weeks [37–40], but in light of preclinical studies, culture times longer than 3 weeks could be required. In the development of 3D models, predominantly the histology, composition of the extracellular matrix, or cell survival is studied [41,42]. Our study not only showed that the FSEs were capable of forming a functional epidermis, but also showed that these models were able to regenerate after thermal injury. Epidermal morphology of the FSE models after 3 weeks of culture was similar to that of ex vivo human skin. Normal epidermal differentiation was present during this period of culture, as shown by consistent expression of early and later differentiation markers cytokeratin 10 and involucrin. Extending the culture time by 1 or 2 weeks improved the organization of the epidermal structure and led to flattening of the stratum spinosum, as displayed by cytokeratin 10 expression, while the stratum corneum thickened. In FSE models, the expression of several markers of epidermal development was similar to the expression in ex vivo human skin and skin equivalent models from other researchers [20,21,43]. Involucrin was also present in the suprabasal layers of the FSEs, an observation similar to that of Coolen et al. and Thakoersing et al. [20,43]. Premature expression of involucrin is indicative of overactivated cell differentiation and is likely caused by an excess of growth factors in the culture medium or by an imbalance in the ratio of fibroblasts to keratinocytes [35,43,44]. With the extension of culture time, 6
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