Abstract: BACKGROUND: In previous studies, the equivalent structure of three-dimensional cell reconstruction of tissue engineering oral mucosa is similar to normal oral mucosa, including epithelial-like structure, lamina propria-like structure, and vascular lumen-like structure, and has initially achieved the establishment of vascular equivalent, but its vascularization characteristics are not very clear. 
OBJECTIVE: Vascular-like structures of vascularized oral mucosa equivalent were obtained by targeting vascular endothelial cells specific marker expression profiles correlated with laser capture microdissection system, and their vascularization ability was evaluated to reveal their vascularization characteristics.
METHODS: Human gingival epithelial cells were cultured from human gingival epithelium and human gingival fibroblasts, human gingival mesenchymal stem cells were cultured from human gingival lamina propria. Human gingival mesenchymal stem cells were induced to differentiate into vascular endothelial-like cells after monoclonal expansion culture. Human gingival epithelial cells, human gingival fibroblasts, and vascular endothelial-like cells were loaded with acellular vascular matrix-0.25% human-like collagen type I scaffold to construct the vascularized oral mucosa equivalent. The layered structure of oral mucosa equivalent (experimental group) and the acellular vascular matrix-0.25% human-like collagen type I scaffold (control group) were implanted subcutaneously into the back of nude mice, respectively. 14 days later, the incision surface of the two groups was coated with biogel. The biogel surface of the experimental group was inoculated with human gingival epithelial cells, while the control group was not inoculated with cells. The samples were collected after 14 days of feeding. The layered structure of oral mucosa equivalent was observed by morphology. The neovascular-like structures in oral mucosa equivalents were labeled by immunohistochemistry and immunofluorescence with a more comprehensive expression profile of vascular endothelial cells, and the vascularization characteristics were analyzed. A laser capture microdissection system was used to capture the neovascularization structures in the oral mucosa equivalents specifically labeled by immunohistochemistry and analyze their vascularization characteristics.
RESULTS AND CONCLUSION: (1) The morphology showed that the cell level of oral mucosa equivalent was clear, and the structure was similar to that of normal oral mucosa, that is, there were epithelioid structures, lamina-like structures, and vascular cavelike structures, and there were scattered erythrocytes in the vascular cavelike structures. (2) The results of EdU Apollo tracer seed cells in the oral mucosa equivalent group showed that human gingival epithelial cells labeled with EdU Apollo 488 showed green fluorescence expression. DAPI labeled human gingival fibroblasts showed blue fluorescence expression and formed lamina-like structures in vivo. EdU Apollo 567 labeled vascular endothelial-like cells showed red fluorescence expression and formed a vascular-like structure in vivo. (3) Vascular endothelial cell specific marker expression profile immunofluorescence labeling of vascular structure showed that compared with normal oral mucosa, the expressions of CD31, CD51, CD54, CD105, Tie-2, VWF, vascular endothelial growth factor receptor 1, and vascular endothelial growth factor receptor 2 in oral mucosa equivalents were increased (P < 0.000 1). There were no significant changes in CD34 expression (P > 0.05). (4) Compared with the specifically labeled oral mucosal vascular structures, the expression levels of CD51, CD54, CD105, Tie-2, VWF, vascular endothelial growth factor receptor 1, and vascular endothelial growth factor receptor 2 of the oral mucosa equivalents targeted by the laser capture microdissection system were increased (P < 0.000 1). There were no significant changes in expression of CD31 and CD34 (P > 0.05). (5) The results showed that the oral mucosa equivalent reconstructed by three-dimensional cell stratification could achieve good vascularization, and its vascularization characteristics were consistent with the immunological function and characteristics of neovascularization. Vascularization helps three-dimensional cell layer reconstruction of oral mucosa equivalent regeneration.

Key words: acellular vascular matrix, vascular endothelial-like cell, three-dimensional cell reconstruction, vascularization, oral mucosa equivalent, laser capture microdissection system