Prefabrication Of The Tissue-engineered Oral Mucosa

Recent years, the tissue-engineered oral mucosa has become a new hotspot for the researchers of stomatology. In this study, we try to explore the method of fabricating tissue-engineered oral mucosa by establishing an in-vitro three-dimension culture model of epithelium and fibroblasts. The three parts of content were summaried as follows:1. Cells culture Objective:To cultivate human oral musosal epithelial cells and fibroblasts by a new method, which combined with tissue explants method and enzymatic digestion method. Materials and methods:The tissues size of 1×0.5 cm2 were harvested from retromolar trigone while the impacted tooth were extracted in 10 patients. The tissues were explanted with epithelial layer and lamina propria together. The epithelial cells and fibroblasts were dissociated by trypsin digestion method. The two kinds of cells were purified in different culture medium. The cells were identified with immunofluorescence technique and hematoxylin-eosin staining. Results:After 2-3 days the tissue explanted, new cultivated cells were seen around the tissues. About 2 weeks later, subculture could be taken when the radius of cellular proliferate to 0.5 cm～ lcm. The epithelial cells and fibroblasts were well dissociated by trypsin digestion method. Contusion:The tissue explant method combined with enzymatic digestion method is an adoptable way to culture the oral mucosal cell, which could cultivate epithelium and fibroblasts efficiently.2. Scaffold screening Objective:To screen out the scaffold which is more suitable for tissue-engineered oral mucosa. Materials and methods:5 samples epithelium and fibroblasts were seeded onto three scaffolds respectively:heterogeneous acellular dermal matrix、 PEG and PVA. After 2 days’culture, CCK-8 were used to test the cell activity, and the data were analyzed by two-way ANOVA of randomized block design. Then the epithelium and fibroblasts were seeded onto the three scaffolds in order, which was fibroblasts seeded first and epithelium a week later. After another week’s culture, Hematoxylin-eosin staining and immunohistochemical were used to display the cells cultured in the scaffolds. Results:There were no difference of the activity of epithelium and fibroblasts among the three materials (P>0.05). The results of Hematoxylin-eosin and immunohistochemical staining showed:compared with other two scaffolds, there were more cells in heterogeneous acellular dermal matrix with normal histology and protein staining. Conlusion:In the three scaffolds, heterogeneous acellular dermal matrix is the most suitable material for epithelium and fibroblasts culture in vitro.3. Prefabricate the tissue-engineered oral mucosa Objective:To explore the method of fabricating tissue-engineered oral mucosa by establishing an in-vitro three-dimension culture model of epithelium and fibroblasts. Materials and methods: epithelium and fibroblasts were seeded onto heterogeneous acellular dermal matrix in order, which was fibroblasts seeded first and epithelium a week later. After a period of 2 weeks’submerged culture, the scaffold were cultuered at the air-liquid interface for another 5 days. Immunofluorescence technique and hematoxylin-eosin staining were used to observe the result. Results:Cells were found in every layer of the scaffold heterogeneous acellular dermal matrix, with a location of epithelium on the surface and the fibroblasts in the deep layer. Conlusion:The disposition of the epithelium and fibroblasts is similar to the epithelial layer and lamina propria of oral mucosa. But the cells were in low viability, and the number was not much enough. It’s necessary to take futher study on the method of fabricating tissue-engineered oral mucosa.