Three-dimensional explants of normal human skin/oral mucosa as a research tool for predicting the effects of exogenous stimuli

Explants of normal human skin cultured at the air-liquid interface and of normal human oral mucosa submerged by the culture medium represent a valid choice for improving knowledge about the immediate response of human stratified epithelia versus a large spectrum of exogenous stimuli. The main advantages consist in the preservation of the three-dimensional arrangement and the epithelial/mesenchymal cross-talk. We performed a time course study focused on a quantitative analysis of proliferation after 5-bromo-2’-deoxyuridine (BrdU) incorporation and a qualitative analysis of the expression of biomarkers of epidermal terminal differentiation and adhesion (1). Biopsies were obtained from healthy non smoking women after breast cosmetic surgery for the skin (n=20) and during wisdom teeth extraction for keratinized oral mucosa (n=15), after written informed consent. Samples were harvested immediately after the excision (B) or 6 (T6), 24 (T24), 48 (T48) hours after overnight incubation. Each subject was represented at all time points. Biopsies were cultured in a Transwell system and processed for paraffin embedding. Results were expressed as number of BrdU-positive-cells/mm2 of the living epithelium. BrdU nuclear staining was always detected in the basal layer. Keratinocyte proliferation progressively decreased both in epidermis (53.7±28.8, 47.2±21.6, 16.6±5.5 for B, T6, and T24) and in oral epithelium (265.6±101.1, 135±48.4, 36.2±25.8 for B, T6, and T24). The viability and the structural integrity of the epithelial compartment were maintained up to 72 (skin) and 48 (oral mucosa) hours. Differentiation/adhesion biomarkers were unchanged during the time-course experiment. In the last decade this experimental setting, as close as possible to the physiological condition, allowed us to study the early response of: - ionizing radiations in both models (2-3); - cigarette smoke in oral mucosa (4); - inflammatory cytokines involved in psoriasis in skin. The response was specific for each epithelium and stimulus, strengthening the hypothesis that the epidermis and the oral epithelium share morphological similarities, but posses an intrinsic regulation for keratinocyte differentiation.