549 In vitro superficial and full thickness wound models to study re-epithelialization processes

In vitro models are an excellent tool to study wound healing processes since they are cost effective and serve as an ethical alternative to animal experimentation. By using human primary cells, these in vitro models mimic the human wound better than animal models. To gain more insight in the wound healing process, more advanced models are needed in the dermatology field. For this reason we developed a representative human full thickness model (FTM) that recapitulates characteristics of superficial and full thickness wounds. To this end, standardized superficial wounds were generated by mechanically removing the epidermis, while full thickness wounds were made using liquid nitrogen. Three days after wounding, FTMs were harvested and wound closure was measured using ImageJ software. Cell proliferation in the re-epithelialization tongue was estimated by Ki67 immunohistochemical analysis. Combined PDGF and EGF supplementation resulted in almost complete wound closure and abundant cell proliferation in both types of wounds. In superficially wounded untreated FTMs, wound closure was less pronounced but cell proliferation was still observed. The basement membrane (BM) was evaluated by the expression of collagen type IV and laminin β3, while epidermal cell activation was evaluated by expression of keratin 17. Both BM proteins were clearly expressed at the epidermal-dermal junction underneath the newly formed epidermis, irrespective of the wound type. PDGF and EGF treated FTMs showed a strong expression of keratin 17 in unwounded areas and newly formed epidermis when compared to the untreated FTMs, which showed a weak expression in unwounded areas with an increase along the re-epithelialization tongue. These data demonstrate that the developed FTMs mimicking superficial and full thickness wounds are robust and functional in vitro models that can be used to study the effect of compounds on the re-epithelialization process in wound healing studies.