Influence of Endothelial Cells on Structure, Biochemistry and Functionality of Epidermis Reconstructed on Synthetic Porous Membrane

Abstract

The model of keratinocytes cultured on a synthetic porous membrane at the air-liquid interface leads to the formation of a pluristratified and cornified epidermis with histological and biochemical characteristics near those observed in vivo. In the present study, we evaluated the effect of proliferative endothelial cells on epidermalization. Keratinocytes were grown in three culture conditions: in defined medium (DM; control), in medium previously conditioned by proliferative endothelial cells (CM) and in medium with proliferative endothelial cells (pEC). The structures of reconstructed epidermis were analyzed by electron microscopy, their biochemistry by DNA, protein and cytokine analyses and finally their functionality was evaluated by estradiol and water absorption testing. Ultrastructural analysis showed a well-developed and cornified epidermis for each culture condition. In addition, living epidermis was thinner in the presence of endothelial cells, revealing faster epidermal differentiation. DNA and protein analyses were in accordance with these results. Secreted soluble factors varied according to culture conditions. At 37°C, the permeability of reconstructed epidermis in DM, in CM or with pEC was 5- to 10-fold higher than that of native human epidermis with both tracers. Laminin coating of the inserts led to similar absorption results except for the DM where the barrier function to estradiol was decreased 2-fold. At 32°C, reconstructed and native epidermis were, respectively, 1.5- and 2-fold less permeable to estradiol compared to 37°C. In conclusion, this model is adequate for fundamental and pharmacological studies since it allows the study of interactions between two cell types without their direct contact as well as percutaneous absorption tests directly performed in the modified culture chamber.