Fibronectin, as well as other extracellular matrix proteins, mediate human keratinocyte adherence

Abstract

During the reepithelialization of cutaneous wounds, the migratory epidermis transits over a provisional matrix of fibronectin and fibrin in the absence of laminin and type IV collagen as well as ultrastructurally identifiable basement membrane. Since significant quantities of fibronectin occur at these sites of reepithelialization, we surmised that fibronectin is a suitable substrate for keratinocyte adherence and therefore undertook the in vitro investigations reported here. Purified human plasma fibronectin precoated on bacteriologic microtiter wells was demonstrated to mediate human keratinocyte adherence when concentrations greater than 10 μg/ml fibronectin were used. Maximal keratinocyte adherence was obtained in wells precoated with 100 μg/ml fibronectin and when cells were incubated with substrate for 60 min or longer at 37° C. Both primary and second-passaged human keratinocytes adhered as well or better to fibronectin than to types I and III collagen, laminin, or type IV collagen under both high- and low-Ca++ culture conditions. However maximal adherence to all substrates occurred when second-passaged keratinocytes were assayed in low-Ca++ medium. Under these latter culture conditions, keratinocyte phenotype resembles the phenotype of cells in the migrating epidermis. To determine specificity of these adherence reactions, antifibronectin antibodies were shown to block keratinocyte adherence to fibronectin but not to laminin substrates. Conversely, antilaminin antibodies blocked adherence to laminin but not fibronectin substrates. Thus, human keratinocytes demonstrate specific adherence to fibronectin in a time- and dose-dependent fashion and this adherence relies on the novo protein synthesis. We take these observations to support our hypothesis that the provisional fibronectin matrix observed beneath the migrating epithelium during tissue repair plays a functional role in the reepithelialization process.