Fate of human keratinocytes during reepithelialization in an organotypic culture model.

Reepithelialization of an incisional wound in a stratified squamous epithelium is accomplished by mobilizing keratinocytes from the wound margins. In vitro models to study this phenomenon have been limited by incomplete differentiation of the cultured epithelium. In addition, it has been difficult to follow fate of recruited keratinocytes, since techniques for marking cells have not been available. We have adapted an organotypic culture model in which keratinocytes are fully differentiated and have utilized a genetic marking protocol with retroviral vectors to study reepithelialization after an incisional wound. The fully differentiated epithelium of an organotypic culture model was incised, supported on a collagen matrix, and allowed to reepithelialize. At various times after wounding, healing cultures were monitored for migration, differentiation, and proliferation by immunohistochemical staining. Histochemical staining specific for the genetically marked cells assisted in the determination of how these cells behaved during reepithelialization. The first event observed (at 8 hours) was migration of suprabasal keratinocytes into the wound followed by a transient proliferative burst at the wound margin. Reepithelialization was complete by 24 hours. Proliferation in the wound epithelium persisted during stratification and terminal differentiation. Genetically marked cells in the wound epithelium were present in clusters demonstrating that proliferation and displacement of cells occurred near the edge of the epithelial tongue. Individual genetically marked cells were also found in the wound epithelium, indicating that individual cells had migrated a considerable distance from the wound edge without having undergone replication. This is the first report of an organotypic model for reepithelialization, and we demonstrate that migration, proliferation, and differentiation occur during this process. The proliferative response which follows initial cell migration at the wound margins suggests that these events are temporally coordinated as phenotypically different populations of cells are sequentially activated. By following the distribution of genetically marked cells in the wound, it is evident that at least two types of cells repopulate a wound-proliferative and migratory cells.