This is the first reconstructed Chinese human epidermis model reported to meet the high quality standard with industrialized production criteria. This China EpiSkin model can be used for both skin research and safety assessment in vitro.
The European Regulation on Cosmetics (no. 1223/2009) has prohibited the use of animals in safety testing since March 2009 for ingredients used in cosmetics. Irreversible events at the chromosome level (clastogenesis and aneugenesis) are commonly evaluated by scoring either micronuclei or chromosome aberrations using cell-based genotoxicity assays. Like most in vitro genotoxicity assays, the 2D in vitro micronucleus assay exhibits a poor specificity and does not mimic the dermal route. To address these limitations, the current project aims to develop and validate a 3D micronucleus assay using the EpiSkin™ model. This project is scientifically supported by the Cosmetics Europe Genotoxicity Task Force. In a first step, two key criteria for the development of micronucleus assay, namely, the sufficient yield of cells from the EpiSkin™ model and an acceptable proliferation rate of the basal layer, were assessed and demonstrated. Subsequently, six chemicals (vinblastine, n-ethylnitrosourea, β-butyrolactone, 2-acetylaminofluorene, 2,4-dichlorophenoland d-limonene) were evaluated in the EpiSkin™ Micronucleus Assay. At least two independent experiments using 48- and 72-h incubations were performed for each chemical. Results showed good inter-experimental reproducibility, as well as the correct identification of all six tested chemicals. The metabolism of 2-acetylaminofluorene on the EpiSkin™ model was also investigated and confirmed by the formation of an intermediate metabolite (2-aminofluorene). These preliminary results from the EpiSkin™ Micronucleus Assay indicate that it is a promising in vitro assay for assessing genotoxicity. The availability and suitability of this test method contribute significantly to the development of non-animal testing methods in China and its impact on the worldwide field.
Objective
This study describes the development and characterization of a novel in vitro wound‐healing model based on a full‐thickness reconstructed skin by exposing the tissue to fractional ablative laser treatment.
Method
A 3D full‐thickness skin model was fabricated and treated with fractional ablative CO2 laser. Wound‐healing process was characterized by HE staining, noninvasive OCT imaging, immunostaining, as well as transepidermal water loss measurement. Cytokines and proteins involved in the inflammatory and dermal remodeling process were studied by ELISA and protein array assays.
Results
Fractional ablative CO2 treatment induced a wound zone of 9 mm in diameter, containing 56 micro‐wounds with 200 μm diameter and 500–700 μm in depth on reconstructed full‐thickness skin model. HE staining revealed a typical wound morphology and healing process with migration of keratinocytes, formation and extrusion of necrotic tissue, and cell inclusion in dermis, which correlates with clinical observations. Based on OCT and TEWL measurements, the re‐epithelialization took place over 2 days. Laser‐triggered keratinocytes proliferation and differentiation were demonstrated by activated Ki67 and Filaggrin expression respectively. Injury‐invoked cytokine ICAM‐1 showed instant upregulation on Day 1. Decreased epidermis thickness and depression of IGFBP‐2 protein level synergistically indicated the unavoidable thermal side effects from laser treatment. Downregulated DKK‐1 protein level and upregulation of α‐SMA together implicated the risk of potential fibrosis post‐laser treatment.
Conclusion
This in vitro laser wounded reconstructed skin model captured the key events of wound‐healing process, could be used to investigate the mechanisms of wound‐healing triggered by a commonly used beauty procedure, and also provides a valuable tool for evaluating the efficacy of novel actives for the post‐procedure application.
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