RESUMEN
Reconstructed skin models are suitable test systems for toxicity testing and for basic investigations on (patho-)physiological aspects of human skin. Reconstructed human skin, however, has clear limitations such as the lack of immune cells and a significantly weaker skin barrier function compared to native human skin. Potential reasons for the latter might be the lack of mechanical forces during skin model cultivation which is performed classically in static well-plate setups. Mechanical forces and shear stress have a major impact on tissue formation and, hence, tissue engineering. In the present work, a perfusion platform was developed allowing dynamic cultivation of in vitro skin models. The platform was designed to cultivate reconstructed skin at the air-liquid interface with a laminar and continuous medium flow below the dermis equivalent. Histological investigations confirmed the formation of a significantly thicker stratum corneum compared to the control cultivated under static conditions. Moreover, the skin differentiation markers involucrin and filaggrin as well as the tight junction proteins claudin 1 and occludin showed increased expression in the dynamically cultured skin models. Unexpectedly, despite improved differentiation, the skin barrier function of the dynamically cultivated skin models was not enhanced compared with the skin models cultivated under static conditions.
Asunto(s)
Modelos Biológicos , Absorción Cutánea , Piel/metabolismo , Animales , Bovinos , Células Cultivadas , Claudina-1/genética , Claudina-1/metabolismo , Fibroblastos/metabolismo , Proteínas Filagrina , Humanos , Técnicas In Vitro , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Queratinocitos/metabolismo , Ocludina/genética , Ocludina/metabolismo , Perfusión , Permeabilidad , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismoRESUMEN
Mutations in the filaggrin (FLG) gene are strongly associated with common dermatological disorders such as atopic dermatitis. However, the exact underlying pathomechanism is still ambiguous. Here, we investigated the impact of FLG on skin lipid composition, organization, and skin acidification using a FLG knockdown (FLG-) skin construct. Initially, sodium/hydrogen antiporter (NHE-1) activity was sufficient to maintain the acidic pH (5.5) of the reconstructed skin. At day 7, the FLG degradation products urocanic (UCA) and pyrrolidone-5-carboxylic acid (PCA) were significantly decreased in FLG- constructs, but the skin surface pH was still physiological owing to an upregulation of NHE-1. At day 14, secretory phospholipase A2 (sPLA2) IIA, which converts phospholipids to fatty acids, was significantly more activated in FLG- than in FLG+. Although NHE-1 and sPLA2 were able to compensate the FLG deficiency, maintain the skin surface pH, and ensured ceramide processing (no differences detected), an accumulation of free fatty acids (2-fold increase) led to less ordered intercellular lipid lamellae and higher permeability of the FLG- constructs. The interplay of the UCA/PCA and the sPLA2/NHE-1 acidification pathways of the skin and the impact of FLG insufficiency on skin lipid composition and organization in reconstructed skin are described.
Asunto(s)
Ácidos/metabolismo , Dermatitis Atópica/metabolismo , Proteínas de Filamentos Intermediarios/deficiencia , Proteínas de Filamentos Intermediarios/genética , Metabolismo de los Lípidos/fisiología , Piel/metabolismo , Dermatitis Atópica/patología , Ácidos Grasos no Esterificados/metabolismo , Proteínas Filagrina , Técnicas de Silenciamiento del Gen , Fosfolipasas A2 Grupo II/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Permeabilidad , Ácido Pirrolidona Carboxílico/metabolismo , Piel/citología , Intercambiadores de Sodio-Hidrógeno/metabolismo , Ácido Urocánico/metabolismoRESUMEN
INTRODUCTION: As humans can come into contact with xenobiotics intentionally or accidentally, knowledge about the skin absorption of these substances is crucial and requires reliable models and test procedures. Animal experiments should be avoided whenever possible, instead of making the use of in vitro systems. Furthermore, due to limited availability of normal and especially diseased human skin, alternative test systems such as reconstructed skin models are urgently required. AREAS COVERED: This article discusses the advantages and limitations of excised human skin, animal skin and reconstructed skin models for absorption testing in vitro. Furthermore, the authors also describe the standard procedure for skin absorption testing and give an excursion to the applicability of artificial membranes. Finally, the article highlights the progress in the development of reconstructed disease models and provides an extensive overview about past and ongoing research in this field. EXPERT OPINION: The development and validation of in vitro systems for skin absorption testing is inevitable. More research efforts are required for the development of reconstructed disease models. Reconstructed skin models need to be improved, especially in terms of complexity to mimic the in vivo situation better. It should not, however, be the main goal to imitate the in vivo situation exactly, but to establish reliable systems that ensure predictive and reliable data.