A reproducible laser-wounded skin equivalent model to study the effects of aging in vitro

Skin aging involves both chronological and photoaging processes. The effects of these processes are often overlapping and include changes in both the stratified epithelium and the fibroblast-rich dermis. Wound healing is frequently delayed with aging and can result in scarring. A skin equivalent model can be used to study the role of cells and the extracellular matrix in the process of wound healing. Current studies using this model employ a full-thickness wound placed atop a nonwounded dermis to mimic a partial-thickness wound. However, a true reproducible partial-thickness wound model has yet to be described. In this study, we investigated whether a laser-wounded skin equivalent would be a useful partial-thickness wound healing model. Three lasers were compared for the ability to generate a reproducible wound: an erbium-YAG, a high-powered excimer, and a low-powered excimer laser. Reepithelialization ability was tested using newborn and adult skin keratinocytes, adult esophageal keratinocytes, and cdk4-overexpressing newborn keratinocytes. Keratinocyte compartmentalization and basement membrane formation were assessed by immunofluorescence. The erbium-YAG and high-powered excimer laser cut reproducible wounds but left the remaining surface either discolored due to thermal damage and/or ragged; keratinocytes were unable to migrate into the wound area. The low-powered excimer laser cut reproducible wounds, leaving the cut surface intact and visibly unaltered; keratinocytes reepithelialized the wound in a collagenase-dependent manner within 3 days; and return of compartmentalization and basement membrane occurred within 14 days. The laser-wounded skin equivalent is an adjustable, reproducible partial-thickness wound model where keratinocyte biology akin to in vivo can be studied, and will be useful to study the effects of aging on wound healing.