A Novel Assay for Evaluating Wound Healing in a Full-Thickness in vitro Human Skin Model

Wound healing is a fundamental process to re-establish tissue integrity and skin barrier function. A model of wound healing was created by introducing epidermal wounds in a full thickness in vitro human skin model (EpiDermFT) using a 3-mm punch biopsy and subsequently evaluated at multiple recovery time points. EpiDermFT exhibits stratified epidermal components and a fully developed basement membrane resembling in vivo skin in regard to both morphology and barrier function. Historically, EpiDermFT has been used to evaluate re-epithelization of the wound by a) manually bisecting the tissues through the center of the wound, b) staining with hematoxylin and eosin, and c) quantifying migration from the wound origin. Accurate bisection of the wound is difficult and often leads to variability in assay results. Here we describe a novel method of visualizing wound re-epithelization in situ simplifying analysis and reducing the introduction of variables inherent in tissue processing that could potentially confound data. Following wounding, tissues were fixed and immunostained with markers of epidermal differentiation as well as a marker of fibroblasts allowing simultaneous visualization of migrating keratinocytes (keratin 14), differentiated suprabasal cells (involucrin), and dermal fibroblasts (vimentin) within the wound. Histological and immunohistochemical analysis showed keratinocyte migration at 2 days following wounding. In both methods, wounded tissues cultured without growth factors (2% human serum) had a reduced healing rate in which keratinocytes did not cover the entire wound within a 6-day timeframe. In contrast, wounded tissues cultured with growth factors demonstrated a dramatic increase in healing rate as keratinocyte migration completely covered the wounded area by day 6. In conclusion, this novel method of evaluating re-epithelization by utilizing immunohistochemical markers of differentiation is a quicker and more reproducible method of analyzing wound healing.

wound healing, EFT-400, punch biopsy, cytokeratin K14, involucrin, vimentin, re-epithelization, wound closure, confocal microscopy, dermal-epidermal interactions

human serum, punch biopsy wound