壳多糖-胶原凝胶体外构建口腔黏膜固有层的可行性研究

Study on the feasibility of constructing the lamina propria of oral mucosa with chitosan and collagen gel in vitro

目的探讨以壳多糖-胶原凝胶为支架材料,利用口腔黏膜成纤维细胞(oral mucous fibroblast,OMF)在体外构建口腔黏膜人工固有层的可行性。方法将2×DMEM培养液、胎牛血清、胶原溶液、壳多糖等按一定比例配置成凝胶溶液,大鼠OMF与上述凝胶溶液迅速混合制成口腔黏膜人工固有层,动态观察细胞生长状况和凝胶收缩情况。结果成功建立大鼠OMF种子细胞库,原代细胞与传代细胞在形态和生长速度差异无统计学意义。壳多糖-胶原-OMF复合物(FPCCL)体外培养10d,OMF在支架材料中生长良好,细胞呈长梭形,排列无一定方向。随时间推移,OMF逐渐增多,凝胶颜色变浅,厚度变薄,3d后发生收缩,7d后则凝胶人工固有层稳定,无明显收缩。结论 OMF可在壳多糖-胶原凝胶形成的三维空间结构生长并分泌基质,形成类似黏膜固有层的致密结缔组织。壳多糖-胶原凝胶是构建组织工程化口腔黏膜固有层的合适材料。

Objective To investigate the feasibility of constructing the lamina propria of oral mucosa with the artificial scaffold of chitosan-collagen gel and oral mucous fibroblasts （OMF） in vitro. Methods Under sterile condition, 2 x DMEM solution, serum of embryo cattle, collagen solution and chitosan were mixed together into gel solution pro rata. Then, the rat OMFs were added into the above gel solution to form the artificial lamina propria of oral mucosa. The cell growth and the gel constriction were observed continuously. Results The rat germ storage was successfully constructed, and primary and progeny cells were not significantly different in morphology and growth rate. After ten days of in vitro culture of the chitosa-collagen-OMF complex （FPCCL）, the OMFs could adhere to artificial scaffold and grow well in the way of the spindle-like and disorder. The OMFs proliferated in the following days, whereas the gel became pale and thin, then constricted after 3 days. Conclusions The OMFs may grow and secrete substrate in three-dimensional space formed by chitosan-collagen gel, and then form compact connective tissue similar to oral mucous propria. Chitosan-collagen gel is a suitable material for constructing oral mucous propria of tissue engineering.