Preserved extracellular matrix in non-enzymatically detached cultured keratinocyte sheets bolsters the healing effects on grafted burn wounds

After escharotomy, full thickness large burn wounds are temporarily covered with cadaver skin because of the limited availability of autologous skin. However, cadaver skin is eventually rejected still requiring autologous skin grafting. To overcome this obstacle, cultured autologous keratinocyte sheets (KS) have been proposed to substitute the autologous skin; however, this technology has not been translated to clinical practice because of the poor quality of KS. Although the exact reason remains unknown, it is suggested that the use of enzyme for the KS detachment may have negatively impacted the overall quality of KSs. The current work is designed to address this pitfall, offering novel technology for detachment of cultured KS. Our studies demonstrate that non-enzymatic detachment of cultured KS, using temperature responsive dishes preserves extracellular matrix (ECM) (such as collagen IV and laminin 5) in KS by activating the MAPK pathway which plays an important role in cells survival and proliferation. We have shown that the cytoskeleton of keratinocytes in cultured sheets was disrupted with Dispase treatment resulting in sheet shrinkage. Importantly, we did not observe the same shrinkage if the cells were cultured in temperature-responsive dishes and detached by culture temperature reduction (T-KS). It also appears that integrity of T-KS is preserved after shear stress. Additionally, we have determined proliferation rates of KS in novel in vitro wound healing models that mimic burn wound harsh environments; the T-KS proliferation capacity was significantly higher in T-KS than the KS detached using Dispase treatment (D-KS) with or without coincubation with burn wound exudates collected from ovine third degree burn wounds. Our major finding is that T-KS overlaid onto third degree burns grafted with cadaver skin (which is rejected) resulted in better burn wound healing than D-KS. The advanced wound healing was evidenced with a greater epithelialization rate and well defined dermal-epidermal junction with continuous and better-defined lamina densa and significantly higher numbers of hemi-desmosomes. Additionally, the wounds were more mature with T-KS treatment which was evidenced by reversed keratinocyte growth factors to normal ranges after its transient increase. Furthermore, vascularization percentage was higher in wounds treated with T-KS compared to wounds covered with D-KS. Taken together, our findings point out the critical importance of non-enzymatic detachment of cultured KS and demonstrate that the effects of non-enzymatically detached KSs are superior to those of enzymatically detached sheets on ovine grafted burn wounds healing. We believe that the results of our present study will potentially enable the successful translation of KS to clinical practice for treating burn wounds.