3D-resolved investigation of the pH gradient in artificial skin constructs by means of fluorescence lifetime imaging.

PURPOSE: The development of substitutes for the human skin, e.g., artificial skin constructs (ASCs), is of particular importance for pharmaceutical and dermatologic research because they represent economical test samples for the validation of new drugs. In this regard, it is essential for the skin substitutes to be reliable models of the genuine skin, i.e., to have similar morphology and functionality. Particularly important is the barrier function, i.e., the selective permeability of the skin, which is strongly related to the epidermal pH gradient. Because the pH significantly influences the permeation profile of ionizable drugs such as nonsteroidal anti-inflammatory drugs, it is of major importance to quantitatively measure the epidermal pH gradient of the ASC and compare it to that of genuine skin.
METHODS: Using three-dimensional fluorescence lifetime imaging combined with two-photon scanning microscopy, we measured with submicron resolution the three-dimensional pH gradient in the epidermis of ASCs stained with 2',7'-bis-(2-carboxyethyl)-5/6-carboxyfluorescein.
RESULTS: Similar to genuine skin, the surface of the artificial epidermis has an acidic character (pH 5.9), whereas in the deeper layers the pH increases up to 7.0. Moreover, the pH gradient differs in the cell interior (maximally 7.2) and in the intercellular matrix (maximally 6.6). Apart from the similitude of the pH distribution, the genuine and the artificial skin prove to have similar morphologies and to be characterized by similar distributions of the refractive index.
CONCLUSIONS: Artificial skin is a reliable model of genuine human skin, e.g., in permeability studies, because it is characterized by a similar pH gradient, a similar morphology, and a similar distribution of the refractive index to that of genuine skin.