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Improving full thickness burn wound healing by using non-enzymatic detachment of keratinocyte sheets

Suzan Alharbi

University of Texas Medical Branch

Introduction: Large burn wounds are temporarily covered with cadaver skin due to lack 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 poor quality of KS, which is suggested to be related to the use of enzyme their detachment. We hypothesized that non-enzymatic detachment using temperature responsive dishes will preserve extracellular matrix of KS and result in better burn wound healing than those cultured in conventional dishes. Methods: To compare the efficacy of KS cultured on temperature-responsive dish (T-sheet) and KS cultured on conventional dish and detached using enzyme Dispase (D-sheet), six full thickness skin burns were induced in sheep dorsum under anesthesia and analgesia. After 24 hrs, burned skin was excised and wounds were grafted with ovine cad aver skin. Autologous keratinocytes were isolated the same day of cadaver skin grafting mimicking clinical situations. KS-T were detached by temperature reduction to 20°C without using enzyme. After 3 weeks, when cadaver skin was rejected, wounds were randomly allocated groups: covered with T or D sheets or wounds without any cover (served as a control). After KS treatment, wounds were monitored for additional 14 days and wound samples were collected postmortem. Results: Wound re-epithelialization rate was significantly higher at days 7 and 14 in wounds grafted with T sheets compared to D sheets. Keratinocyte sheet thickness was significantly higher in T sheets compared to D sheets before grafting. On the other hand, epidermis thickness was comparable in wounds treated with both types of sheets at 14th day. However, the dermal-epidermal junction was well defined in wounds covered with T sheets at 14th day as evidenced by continuous and better-defined lamina densa and significantly higher numbers of hemi-desmosomes. Wounds treated with T sheets had significantly less ulceration and hemorrhage compared to control, and less neutrophil infiltration. Collagen density and vascularization percentage were significantly higher in the wounds treated with T sheets compared to wounds covered with D-sheets and control. Conclusion: Novel non-enzymatic method for KS detachment provides better sheet quality and accelerates healing of grafted burn woun

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