人工生物可降解支架构建组织工程食管的实验研究

EXPERIMENTAL STUDIES ON TISSUE ENGINEERED ESOPHAGUS RECONSTRUCTED WITH ARTIFICIAL BIODEGRADABLE SCAFFOLD

目的研究食管上皮细胞在聚乳酸-聚乙醇酸[po ly(lactic-co-g lyco lic ac id),PLGA]三维支架材料上的黏附和生长情况,探索利用组织工程技术构建组织工程食管的可行性。方法应用粒子浸出-常温模压法制作PLGA三维多孔支架材料;分离培养6只犬食管上皮细胞,体外扩增后,种植到预涂Ⅳ型胶原的PLGA支架材料上。在体外和犬腹腔内分别培养食管上皮细胞-支架复合物,分期终止培养,体外培养3 d,1、2、3和4周,其中将体外培养3 d后细胞-支架复合物植入同只犬腹腔内,于体内培养1、2、3和4周后行组织学、扫描电镜观察,以及细胞角蛋白抗体免疫组织化学检测。结果分离培养的犬原代食管上皮细胞呈铺路石样,体外可大量扩增,细胞角蛋白抗体免疫组织化学染色呈阳性;体外及体内培养可见食管上皮细胞在支架材料上黏附、生长良好,持续培养仍保持食管上皮细胞特性;犬腹腔内培养4周后,可形成食管黏膜样组织。结论预涂Ⅳ型胶原的PLGA支架材料适合食管上皮细胞黏附生长,可作为组织工程食管的支架材料。利用组织工程的方法在体外和犬腹腔内培养有可能获得适于移植的组织工程食管。

Objective To verify adhesion and growth ability of canine esophageal epithelial cells （EECs） on the poly （lactic-co-glycolic acid） （PLGA）, a three-dimensional biodegradable polymer scaffold, and to reconstruct the canine esophagus by the tissue engineering. Methods Free canine EECs isolated from adult dogs by esophagoscopy were seeded onto the PLGA scaffolds prccoated with collagen type Ⅳ after the first passage by the in vitro culture. Then, the composites of the cell-scaffold were respectively cultured in vitro and in the abdominal cavity of the dog in vivo. After different periods, the cell seeded scaffolds were assessed by histological HE staining, scanning electron microscopy, and immunohistochemical analysis. Results The cells displyed a cobblestone-shaped morphology that was characteristic of the epithelial cells and were stained to he positive for cytokeratin, which indicated that the cells were EECs. The canine EECs were well distributed and adhered to the PLGA scaffolds, and maintained their characteristics throughout the culture period. After the culture in vivo for 4 weeks, the cell-seeded scaffolds looked like tissues. Conclusion PLGA scaffolds precoated with collagen type Ⅳ can he suitable for adhesion and proliferation of EECs, and can he used as a suitable tissue engineering carrier of an artificial esophagus.