Epidermal barrier function evaluation in an artificial autologous skin model based on hyaluronic acid biomaterial

Abstract

Background & Aim The aim of this study was evaluated the epidermal barrier function in an artificial bioengineered skin substitutes (ABSS) based on hyaluronic acid biomaterial. The study was performed evaluating specific skin ointments at different test period. Methods, Results & Conclusion Dermal fibroblasts (DF) and Keratinocytes (KT) were enzymatically isolated from 9 cm2 skin biopsies. After 4-5 weeks in culture, DF were part of dermal fibrin-hyaluronic acid stroma. KT (obtained using feeder-free method) were seeded on the surface to establish ABSS. Flow cytometry assay was carried out to phenotypically evaluate of keratinocytes: CD49f (integrin α6) and CD90 (fibroblast cell surface marker). Cell viability (LIVE/DEAD® assay), structural integrity (histological evaluation) and epidermal barrier function (transepidermal water loss, TEWL: Tewameter®) were measured after 5, 15 and 30 minutes to apply beclomethasone, mupirocin and vaseline. Acellular ABSS were used as controls. Specific cell culture parameters were evaluated after final recovery of keratinocytes. The cell expansion rate was significantly higher (p≤0.001%) with the CNT-57S media were supplemented with human platelet lysate. CD49f percentage was higher in keratinocytes with CNT-57S (95.2%). The CD90 percentage was lower in keratinocytes expanded with CNT-57S human lysate supplemented (1.60%). A viability assay was carried out in ABSS at first (85.1%) and second week (87.8%). Finally, barrier function evaluation of ABSS was carried out by measuring TEWL in the first and in the second week of maintenance. TEWL values in acellular ABSS were higher than in cellular ABSS, although the difference was not significant. Cells improved ABSS barrier function, thus favouring the reduction of water loss. Regarding the evaluation period, significant differences of TEWL were observed, decreasing the values in the second week. This may be due to the growth of the epithelial layer in cellular ABSS, which contributes to improvement of the barrier function. TEWL measures also decreased over the measurement time, although the differences were not significant. No difference was observed with respect to the type of ointment. In conclusion, keratinocytes culture was optimized using animal component-free and feeder-free method. A novel way to evaluate keratinocytes contribution to epidermal barrier function has been development in vitro.