Influence of dermal equivalent maturation on the development of a cultured skin equivalent

Histologic and immunofluorescence methods were used to analyse the presence of fibronectin, chondroitin-4-sulphate and chondroitin-6-sulphate, type III and IV collagens, laminin, and keratins to assess the maturation level of cultured dermal and skin equivalents. In a first phase, fibroblasts in monolayer culture were compared with dermal equivalents in which fibroblasts are embedded in a type I collagen gel. Different fluorescent patterns were observed depending on the culture system used. A sequential appearance of macromolecules was noticed in dermal equivalents. Fibronectin was first detected after 4 days of culture, whereas chondroitin-4-sulphate and chondroitin-6-sulphate and type III collagen were present after 7 days. In contrast, all three macromolecules were detected at 24 h of culture in fibroblastic monolayer cultures. In a second phase, the quality of our skin equivalents was evaluated according to the seeding time of epidermal cells upon dermal equivalents (1, 4, or 7 days). A satisfactory stratification was obtained when keratinocytes were seeded after 4 and 7 days of dermal equivalent culture. Laminin and fibronectin were detected at the dermo-epidermal junction, but type IV collagen was absent. Various keratins, as detected by the AE1, AE2, and AE3 antibodies, were present in the epidermal layer. Following keratinocyte confluence, a change in the organization pattern of type III collagen in the dermal fraction of the skin equivalent was also noticed. Our comparative results show that seeding of epidermal cells on a more mature dermal equivalent leads to improved differentiation status of the epidermal layer.