Abstract
Cell sheets with pre-vascularization have recently been developed but remain relatively untested in oral wound healing. Therefore, we examined the potential utility of our newly developed pre-vascularized mucosal cell sheets in oral wound healing. Mucosal keratinocytes, fibroblasts, and endothelial progenitor cells were primarily cultured for in vitro cell expansion from mucosa and blood of Sprague-Dawley rats. Mucosal cell sheets were generated using cultured keratinocytes and plasma fibrin (K sheet) or keratinocytes and a mixture of fibrin, fibroblasts, and endothelial cells (PV sheet). Autologous sheets were transplanted on deep wounds in the buccal region of rats. The gross and histological characteristics of wound healing were compared among control wound, K sheet, and PV sheet groups. We successfully cultured and expanded keratinocytes, fibroblasts, and endothelial progenitor cells in vitro for generating mucosal cell sheets with or without pre-vascularization. In the in vivo oral wound model, compared with the control wound, the PV sheet group exhibited rapid wound closure more prominently than the K sheet group. The histological healing in the PV sheet group was similar to that in rat normal buccal mucosa without fibrosis. The pre-vascularized mucosal cell sheet exhibited in vivo efficacy in oral wound healing by promoting accelerated healing.
Highlights
T progenitor cells were primarily cultured for in vitro cell expansion from mucosa and blood of Sprague
The in vitro culture and expansion of oral keratinocytes, fibroblasts, and endothelial progenitor cells were successful after harvesting the oral mucosa and peripheral blood samples
R cessful using the mixture of plasma fibrin, oral fibroblasts, and endothelial cells underneath the keratinocyte layer
Summary
In vitro mucosal and endothelial progenitor cell culture. Keratinocytes and fibroblasts from the oral mucosa of all experimental rats were successfully cultured in vitro. A A mucosal cell sheet with pre-vascularization (PV sheet) including the mixture of plasma fibrin, oral fibroblasts, and endothelial progenitor cells beneath the keratinocyte layer. During the later stage of wound healing, the epithelial and subepithelial compositions in the cell sheet groups were more similar to those of the unwounded normal buccal mucosa, whereas the epithelium and subepithelium. The relative intensity of α-smooth muscle actin was increased in the wounds (with or without cell sheets) compared with that in the normal oral mucosa; this intensity was higher in the wound control than in the K and the PV sheet groups (Fig. 6E,F). T The subepithelium exhibited thickening and increased collagen deposition in the wound control on days 14 and 28 compared with those of the normal buccal mucosa (P < 0.05) (Fig. 6G). Microscopic muscular disruption was identified in the buccal mucosa of the Rwounded controls but not in the PV sheet group
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