The effects of epidermal keratinocytes and dermal fibroblasts on the formation of cutaneous basement membrane in three-dimensional culture systems

Abstract

The cutaneous basement membrane (BM) plays an important role in normal and pathological conditions. However, few studies have addressed the formation of the cutaneous BM using three-dimensional culture systems. In this study, to elucidate the effects of human epidermal keratinocytes and dermal fibroblasts on the formation of the cutaneous BM, keratinocytes were cultured on several dermal substrates in the presence or absence of fibroblasts at the air-liquid interface. After 2 weeks of culture, immunohistochemical stainings for the components of the BM and electron microscopic studies of the BM zone (BMZ) were performed. In cultures of keratinocytes alone on dead reticular dermis or collagen gel without fibroblasts, beta4 integrin chain, laminin, type IV and VII collagens were all expressed. However, ultrastructurally, BMZ was not formed. In cultures of keratinocytes on fibroblast-populated collagen matrix, laminin, and type IV and VII collagens were expressed more strongly than in the absence of fibroblasts. In addition, elements of the BMZ such as hemidesmosomes, lamina lucida, lamina densa and anchoring fibrils were formed, although it was still incomplete. In the culture of keratinocytes alone on de-epidermized dermis (DED) (surface up), beta4 integrin chain, laminin, and type IV and VII collagens were strongly expressed. Also, the BMZ appeared similar to that in normal skin. In cocultures of keratinocytes and fibroblasts on DED or cultures of keratinocytes on DED combined with fibroblast-populated collagen matrix, type IV collagen was expressed more strongly than in cultures of keratinocytes alone. Ultrastructurally, similar findings to those of cultures of keratinocytes alone on DED were seen. Interestingly, when keratinocytes and fibroblasts were cocultured on DED, some fibroblasts were seen in the upper dermis as a result of migration into the dermis through partial loss of the lamina densa. These results show that keratinocytes produce most of the components of the BM such as laminin, and type IV and VII collagens. In addition, fibroblasts stimulate the expression of the components of the BM and the formation of a BMZ, suggesting that fibroblasts may produce laminin, and type IV and VII collagens or influence the effects of keratinocytes on the formation of the BM through a keratinocyte-fibroblast interaction.