Human skin equivalents (HSEs) show great similarities to human native skin. However, one of the key processes impaired under in vitro conditions is desquamation. Desquamation involves the degradation of the corneodesmosomes, in which various enzymes participate. Activation of these enzymes is affected by several microenvironmental factors such as pH and water level. The water level is assumed to depend on the presence of natural moisturizing factors (NMF). In this study, the levels of water and one of the prominent NMF components--pyrrolidone carboxylic acid (PCA)--were examined. In HSE generated under normal culture conditions (93% relative humidity (RH)), the water level and PCA content appeared to be much lower than in the native counterpart. To increase the water and PCA levels in HSE, a culture method was established in which HSE was reconstructed under reduced RH. Although at 40% RH the PCA levels in reconstructed and native tissue are similar, the hydration levels in reconstructed tissue remain still lower. Only topical application of water induced marked swelling of corneocytes. This clearly shows that the stratum corneum water level in HSE is regulated by other, still unknown, factors, in addition to NMF.
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