| Περίληψη: | Three-dimensional (3D, 3 Dimensional) reconstructed human skin equivalents (HSEs, Human Skin Equivalents), (i.e. epidermal models, 3D reconstructed full-thickness skin) are extremely valuable tools for in vitrο research, as they are scientifically valid alternatives to animal experimentation. Consequently, in 2013, testing cosmetic products and ingredients in animals was banned by the European Commission (Niehues et al., 2018). HSEs mimic the native skin, structurally and biochemically, with comparable cellular heterogeneity and structural complexity (Niehues et al., 2018). Thus, they play a key role in preclinical research for drug development (Mathes et al., 2014), while they are also absolutely essential to the cosmetics industries (Gabbanini et al., 2009). Importantly, 3D reconstructed human skin equivalents (RHS, Reconstructed Human Skin) are very useful in studying rare skin diseases, such as the recessive dystrophic epidermolysis bullosa (RDEB, Recessive Dystrophic Epidermolysis Bullosa) (Mittapalli et al., 2016) but also common skin diseases like psoriasis (Tjabringa and Bergers, 2008; Chiricozzi et al., 2014).
The present study aims to develop and characterize a 3D RHS model by use of immortalized normal human dermal fibroblasts (iNHFs, immortalized Normal Human Fibroblasts) and immortalized normal human epidermal keratinocytes (iNHKs, immortalized Normal Human Keratinocytes) and, subsequently, characterize the developed RHS, structurally and biochemically, by parallel comparison with native skin tissue. Furthermore, the generated RHS was exploited for testing cosmetic micro- and nano-emulsion formulations, as well as their constituent essential oils (EOs) and surfactants. These were initially tested here in two dimensional (2D, 2 Dimensional) cultures for potential cytotoxicity and will be subsequently evaluated in our “in-house” 3D RHS.
For generation of the 3D RHS, iNHFs were mixed with collagen and placed into a 6-well plate that allowed the intake of growth factors, hormones and nutrients suitable for the development of the 3D RHS, then, iNHKs were seeded on the reconstructed dermis. After four weeks in culture, the 3D RHS was ready to be characterized using established microscopic, histological and molecular assays.
Staining of the prepared 3D RHS with hematoxylin and eosin (H&E) showed that its microstructure mimics that of the native skin, and it had a physiological epidermal architecture containing the characteristic distinct epidermal layers, i.e. the stratum corneum (SC), stratum granulosum (SG), stratum spinosum (SS) and stratum basale (SB). Consistently, normal expression of key skin proteins was confirmed by immunohistochemistry (IHC) using specific antibodies. In particular, structural proteins of desmosomes, such as desmoglein1 (DSG1), desmocolin1 (DSC1), and corneodesmosin (CDSN), as well as the characteristic cell adhesion molecule E-cadherin displayed similar expression profiles in the 3D RHS and native skin. Furthermore, proteins related to the regulation of skin’s exfoliation/desquamation, such as the epidermal proteases KLK5, KLK6, and KLK7, as well as proteins related to the skin differentiation process, such as the involucrin, loricrin and keratin 5, were similarly expressed in both 3D RHS and native skin. Elevated expression of Ki67 in 3D RHS indicated increased proliferation compared to native skin as also observed by others. Cumulatively, the developed 3D RHS mimics native skin, thus, it can be used for the evaluation of cytotoxicity and moisturizing action of cosmetic oil in water (o/w, oil in water) micro- and nano-emulsion formulations (ΕΥΔΕ ΕΤΑΚ-ΕΥΔ ΕΠΑνΕΚ ΕΣΠΑ 2014-2020/ QFytoTera-Τ1ΕΔΚ-00996).
The neutral red uptake (ΝRU, Neutral Red Uptake) assay and colorimetric MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay were applied for testing the cytotoxicity of EOs, surfactants and micro- and nano-emulsions on 2D cultures of iNHKs. The majority of EOs and cosmetic micro-emulsion formulations tested were found non-cytotoxic, with values of iNHKs viability higher than 95% at the lower EOs concentration range. Therefore, their cosmetic properties, such as their moisturizing action, will be evaluated by application on the 3D RHS but also to test whether they may cause undesired skin irritation and corrosion, as part of their integrated assessment in vitro.
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