RESULTS

To test whether fibronectin beneath the reepithelializating epidermis was plasma-derived or produced in situ, we wounded rat ear skin transplanted onto the flanks of immunosuppressed mice and stained the healing wounds with reciprocal species-specific anti-fibronectin antibodies. Within this time period, mouse anti-rat fibronectin antisera would identify fibronectin produced in situ by donor (rat) cells, while rat anti-mouse fibronectin antisera would identify plasma-derived fibronectin deposited under the migrating epithelial tongue from the recipient (mouse) circulation [21].

Since the graft sites were healed within 2 weeks after surgery (Fig. 1), it was possible to extirpate a 3-mm punch biopsy from the center of each graft site at that time. Two days after excisional wounding, the migrating epidermis could be seen descending vertically at the sides of the wound along a track of fibrillar material that separated the necrotic desiccated dermis at the wound surface from the inner viable dermis (Fig. 2, A). The same fibrillar material seemed to provide a provisional matrix over which the migrating epithelial cells coursed (Fig. 2, B). On immunofluorescence studies, this matrix stained for fibrinogen (data not shown), as previously demonstrated in wound healing experiments using guinea pigs [18]. Staining with reciprocal species-specific anti-fibronectin antibodies gave quite different results depending on the age of the wound. In early wounds (2 to 4 days after extirpation), the matrix beneath the wound always stained with anti-mouse fibronectin (Fig. 3, A), often without positive fluorescence for rat fibronectin (Fig. 4, B), but sometimes also positive for rat fibronectin (not shown). When the matrix did stain for rat fibronectin during these early time points, the fluorescence was usually less intense than that for the mouse fibronectin. In contrast, wounds that were; studied 7 and 10 days after excision were positive for both mouse and rat fibronectin (Fig. 4, A, B) or positive for rat fibronectin alone (data not shown). When the wound was completely reepithelialized, neither fibronectin was present.