Non-enzymatic Detachment For Keratinocyte Sheets Using Temperature Responsive Dishes For Treating Burn Wound
Suzan Alharbi1, Yosuke Niimi1, Dominique Wiener2, Hal Hawkins1, Robert Cox1, Vsevolod Popov1, Atsuyoshi Osada3, Koji Ihara3, Hiroyuki Sakurai3, David Herndon1, Donald Prough1, Perenlei Enkhbaatar1.
1University of Texas Medical Branch, Galveston, TX, USA, 2Texas A&M University, College Station, TX, USA, 3Tokyo Women’s Medical University, Tokyo, Japan.
The use of enzymatic detachment for keratinocyte sheets (KS) negatively affects their quality which prevents their translation to clinical practice for treating burn wounds. We hypothesized that non-enzymatic detachment will preserve extracellular matrix of KS and result in better burn wound healing. To test the efficacy of KC sheets, 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 cadaver skin. Autologous keratinocytes were isolated the same day of cadaver skin grafting mimicking clinical situations. The efficacy of KS, cultured on conventional and temperature-responsive dishes, on burn wound healing was compared. Cells cultured in temperature-responsive dishes were detached by temperature reduction to 20°C (T sheets) without using enzyme. While the keratinocytes cultured in conventional dishes were detached by enzyme (Dispase) (D sheets). After 3 weeks, when cadaver skin was rejected, wounds were covered with either T or D sheets. Additionally, wounds without any cover/treatment were served as a control. After KS treatment, wounds were monitored for additional 14 days and samples were collected postmortem for various assays. Keratinocyte sheet thickness was significantly higher in T sheets compared to D sheets before grafting. Wound re-epithelialization rate was significantly higher at days 7 and 14 in wounds grafted with T sheets compared to D sheets. Wounds treated with T sheets had less hemorrhage, ulceration and neutrophil infiltration. Epidermis thickness was comparable in wounds treated with both types of sheets, while 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 higher number of hemi-desmosomes. Novel non-enzymatic method for KS detachment provides better sheet quality and accelerates healing of grafted burn wounds.
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