Vascularised human skin equivalents as a novel in vitro model of skin fibrosis and platform for testing of antifibrotic drugs.

OBJECTIVES: Fibrosis is a complex pathophysiological process involving interplay between multiple cell types. Experimental modelling of fibrosis is essential for the understanding of its pathogenesis and for testing of putative antifibrotic drugs. However, most current models employ either phylogenetically distant species or rely on human cells cultured in an artificial environment. Here we evaluated the potential of vascularised in vitro human skin equivalents as a novel model of skin fibrosis and a platform for the evaluation of antifibrotic drugs. METHODS: Skin equivalents were assembled on a three-dimensional extracellular matrix by sequential seeding of endothelial cells, fibroblasts and keratinocytes. Fibrotic transformation on exposure to transforming growth factor-ß (TGFß) and response to treatment with nintedanib as an established antifibrotic agent were evaluated by quantitative polymerase chain reaction (qPCR), capillary Western immunoassay, immunostaining and histology. RESULTS: Skin equivalents perfused at a physiological pressure formed a mature, polarised epidermis, a stratified dermis and a functional vessel system. Exposure of these models to TGFß recapitulated key features of SSc skin with activation of TGFß pathways, fibroblast to myofibroblast transition, increased release of collagen and excessive deposition of extracellular matrix. Treatment with the antifibrotic agent nintedanib ameliorated this fibrotic transformation. CONCLUSION: Our data provide evidence that vascularised skin equivalents can replicate key features of fibrotic skin and may serve as a platform for evaluation of antifibrotic drugs in a pathophysiologically relevant human setting.

Reepithelialization in focus: Non-invasive monitoring of epidermal wound healing in vitro.

Up to today, in vivo studies are the gold standard for testing of new therapeutics for cutaneous wound healing. Alternative in vitro studies are mostly limited to two-dimensional cell cultures and thus only poorly reflect the complex physiological wound situation. Here we present a new three-dimensional wound model based on a reconstructed human epidermis (RHE). We introduce impedance spectroscopy as a time-resolved test method to determine the efficacy of wound healing non-destructively by focusing on the barrier function of the RHE as a main feature of intact skin. We assessed the skin barrier quantitatively and qualitatively by calculating the transepithelial electrical resistance (TEER), by fitting an equivalent circuit and by analyzing the single characteristic frequency. Upon wounding using a 2 mm biopsy punch, the impedance dropped significantly to 3.5% of the initial value. Impedance spectroscopy thereby proved to be a sensitive tool to distinguish between wounds of different sizes. The glucose and lactate concentration in the medium revealed an acute stress reaction of the wounded RHE (wRHE) in the first days after wounding. During monitoring of reepithelialization over fourteen days, the barrier fully recovered. Microscopy and histology images correlate well with these findings, revealing an active wound closure mostly completed by day seven after wounding. These wounded epidermal models can now be applied in therapeutic screenings and with the help of rapid screening by impedance spectroscopy, expensive and time-consuming imaging and histological methods as well as the use of animal models can be reduced.