Development of a multilayered in vitro model for studying events associated with wound healing

Simplified in vitro models such as cellular monolayer cultures have only limited usefulness in the study of cutaneous wound repair processes. This has stimulated the investigation of three-dimensional tissue equivalent systems such as the dermal and skin equivalent models. With the use of a wound system constructed of rat tail type I collagen and human dermal fibroblasts, experimental wounding was accompanied by problems with mechanical scoring of the plastic substratum which prevented cell migration. These problems were overcome with the use of a multilayered model in which a punch biopsy-wounded dermal equivalent (bilayered model) or skin equivalent (tri-layered model) was placed onto an acellular collagen lattice and fixed in place with polymerizing collagen. This model permitted observation of the process of cellular repopulation of the "wound space," into which both fibroblast and keratinocyte migration commenced within 1 day. The number of fibroblasts in this space increased dramatically over a period of 9 days, the cells appearing to migrate both over and through the acellular lower collagen layer. Keratinocyte reepithelialization of the "wound space" was completed after 5 days. With the model it was shown that platelet-derived growth factor--AB and epidermal growth factor had positive effects in increasing fibroblast number within the wound space. In conclusion, the model described here should facilitate the study of fibroblast and keratinocyte responses to a wound stimulus in vitro and be a plausible in vitro system for evaluating agents which may have a potential stimulatory or inhibitory effect on numerous cellular responses associated with wound healing.