Personne : Pouliot, Roxane
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Pouliot
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Roxane
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Université Laval. Faculté de pharmacie
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ncf11849049
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Publication Restreint Characterization of a new tissue-engineered human skin equivalent with hair(springerLink, 1999-06-01) Michel, Martine; Germain, Lucie; Xu, Wen; L'Heureux, Nicolas; Auger, François A.; Pouliot, RoxaneWe designed a new tissue-engineered skin equivalent in which complete pilosebaceous units were integrated. This model was produced exclusively from human fibroblasts and keratinocytes and did not contain any synthetic material. Fibroblasts were cultured for 35 d with ascorbic acid and formed a thick fibrous sheet in the culture dish. The dermal equivalent was composed of stacked fibroblast sheets and exhibited some ultrastructural organization found in normal connective tissues. Keratinocytes seeded on this tissue formed a stratified and cornified epidermis and expressed typical markers of differentiation (keratin 10, filaggrin, and transglutaminase). After 4 wk of culture, a continuous and ultrastructurally organized basement membrane was observed and associated with the expression of laminin and collagen IV and VII. Complete pilosebaceous units were obtained by thermolysin digestion and inserted in this skin equivalent in order to assess the role of the transfollicular route in percutaneous absorption. The presence of hair follicles abolished the lag-time observed during hydrocortisone diffusion and increased significantly its rate of penetration in comparison to the control (skin equivalent with sham hair insertion). Therefore, this new hairy human skin equivalent model allowed an experimental design in which the only variable was the presence of pilosebaceous units and provided new data confirming the importance of hair follicles in percutaneous absorption.Publication Restreint Physical characterization of the stratum corneum of an in vitro human skin equivalent produced by tissue engineering and its comparison with normal human skin by ATR-FTIR spectroscopy and thermal analysis (DSC)(ScienceDirect, 1999-08-18) Germain, Lucie; Juhász, Julianna; Auger, François A.; Tremblay, Nathalie; Pouliot, RoxaneAn in vitro human skin equivalent may be obtained by culturing human keratinocytes on a collagen gel containing fibroblasts. The anchored skin equivalent cultured at the air-liquid interface closely resembles human skin and is acceptable for in vitro percutaneous absorption. However, it is still more permeable than human skin. Since intercellular lipids have been recognized to play an important role in skin permeability, infrared spectroscopy and differential scanning calorimetry were performed on the stratum corneum of bovine or human skin equivalents grown at different days of air-liquid culture. The symmetric and asymmetric CH(2) stretching vibrations suggested that for all days observed, the intercellular lipids were less organized than those in human skin, irrespective of whether bovine or human collagen was used. Different culture conditions were also tested and the medium without serum and no epidermal growth factor at the air-liquid culture showed results significantly more comparable to human skin. Actually, the thermal behavior of in vitro stratum corneum showed transitions at lower temperatures than human skin. The transition around 80 degrees C, in the form of a lipid-protein complex, was absent. These results showed that the structural arrangement of intercellular lipids and their thermodynamic properties hold a crucial role in the barrier function of the stratum corneum.Publication Restreint Influence of endothelial cells on structure, biochemistry and functionality of epidermis reconstructed on synthetic porous membrane(1998-10-01) Cutler, Robert M.; Germain, Lucie; Juhász, Julianna; Noël-Hudson, Marie-Sophie; Auger, François A.; Lindenbaum, Albert; Pouliot, Roxane; Wepierre, JacquesThe 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.