ABSTRACT
Introduction: In skin traumas, such as burns, epidermal homeostasis is affected, often requiring clinical approaches. Different therapeutic strategies can be used including transplantation, besides the use of synthetic or natural materials with allogeneic cells. In this context, tissue engineering is an essential tool for skin regeneration, and using mesenchymal stem cells (MSC) from the umbilical cord appears to be a promising strategy in regenerative medicine due to its renewal and differentiation potential and hypo immunogenicity. We evaluated the transdifferentiation of MSC from umbilical cord into keratinocytes in three-dimensional (3D) in vitro skin models, using dermal equivalents composed by type I collagen with dermal fibroblasts and a commercial porcine skin decellularized matrix, both cultured at air-liquid interface (ALI). Methods: The expression of epidermal proteins cytokeratins (CK) 5, 14 and 10, involucrin and filaggrin was investigated by real-time PCR and immunofluorescence, in addition to the activity of epidermal kallikreins (KLK) on the hydrolysis of fluorogenic substrates. Results and discussion: The cultivation of MSCs with differentiation medium on these dermal supports resulted in organotypic cultures characterized by the expression of the epidermal markers CK5, CK14, CK10 and involucrin, mainly on the 7th day of culture, and filaggrin at 10th day in ALI. Also, there was a 3-fold increase in the KLK activity in the epidermal equivalents composed by MSC induced to differentiate into keratinocytes compared to the control (MSC cultivated in the proliferation medium). Specifically, the use of collagen and fibroblasts resulted in a more organized MSC-based organotypic culture in comparison to the decellularized matrix. Despite the non-typical epithelium structure formed by MSC onto dermal equivalents, the expression of important epidermal markers in addition to the paracrine effects of these cells in skin may indicate its potential use to produce skin-based substitutes.
ABSTRACT
Currently, there is a strong global trend towards the development of in vitro models to replace the use of animals in safety evaluation tests. Reconstructed Human Epidermis (RHE) models have been employed as an alternative method to animal testing of skin corrosion and irritation potential of chemical compounds. However, the consequences of an absence of the dermal compartment in these models should be considered since the cross-talk between fibroblasts and keratinocytes is fundamental for promoting proper epidermal stratification, homeostasis, inflammatory response and wound healing. In this study, we compare in-house developed models of Reconstructed Human Epidermis (i.e. USP-RHE) and full thickness skin (i.e. USP-FTS) regarding their response when submitted to skin corrosion assays, based on Guideline 431 (OECD). The results show that both models correctly classified the four substances tested (2-phenylethyl bromide, benzylacetone, lactic acid, octanoic acid) as corrosive or non-corrosive. Furthermore, we have demonstrated higher cell viability of the USP-FTS model compared to the USP-RHE model, a sign of its improved barrier function, following the exposure to the substances test on the corrosion assay. This emphasizes the importance of employing in vitro models that are more physiologically relevant and that better mimic the in vivo situation for the toxicological screening of substances.
