FATE OF HUMAN KERATINOCYTES DURING REEPITHELIALIZATION IN AN ORGANOTYPIC CULTURE MODEL

BACKGROUND: 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 a ddition, it has been difficult to follow fate of recruited keratinocyt es, since techniques for marking cells have not been available. We hav e adapted an organotypic culture model in which keratinocytes are full y differentiated and have utilized a genetic marking protocol with ret roviral vectors to study reepithelialization after an incisional wound . EXPERIMENTAL DESIGN: The fully differentiated epithelium of an organ otypic culture model was incised, supported on a collagen matrix, and allowed to reepithelialize. At various times after wounding, healing c ultures were monitored for migration, differentiation, and proliferati on by immunohistochemical staining. Histochemical staining specific fo r the genetically marked cells assisted in the determination of how th ese cells behaved during reepithelialization. RESULTS: The first event observed (at 8 hours) was migration of suprabasal keratinocytes into the wound followed by a transient proliferative burst at the wound mar gin. Reepithelialization was complete by 24 hours. Proliferation in th e wound epithelium persisted during stratification and terminal differ entiation. Genetically marked cells in the wound epithelium were prese nt in clusters demonstrating that proliferation and displacement of ce lls occurred near the edge of the epithelial tongue. Individual geneti cally marked cells were also found in the wound epithelium, indicating that individual cells had migrated a considerable distance from the w ound edge without having undergone replication. CONCLUSIONS: This is t he first report of an organotypic model for reepithelialization, and w e 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 te mporally coordinated as phenotypically different populations of cells are sequentially activated. By following the distribution of genetical ly marked cells in the wound, it is evident that at least two types of cells repopulate a wound-proliferative and migratory cells.