一体化层状梯度修复体用于骨软骨组织工程实验探究.docVIP

一体化层状梯度修复体用于骨软骨组织工程实验探究 　 作者：白雪东，胡蕴玉，严乐平，任力，吴刚，李丹，孙效棠，白建萍 【摘要】 ［目的］探索胶原/壳聚糖/纳米β磷酸三钙一体化层状梯度修复体在组织工程中用作骨软骨缺损修复的可行性。［方法］以Ⅰ型胶原、壳聚糖、纳米β磷酸三钙（βTCP）为基本原料，采用无毒交联并通过冷冻干燥法成型；扫描电镜观察，测定支架材料的孔径、孔隙率以及交通孔情况；分离扩增兔骨髓间充质干细胞（BMSC），将BMSC接种于材料上，扫描电镜观察细胞在材料上的黏附状态，MTT法测定细胞在材料上的生长曲线；以软骨诱导液体外诱导细胞支架复合物向软骨分化，2周后植入自体股部肌袋，6周取材，HE、甲苯胺蓝染色、Ⅱ型胶原免疫组化鉴定诱导分化效果。［结果］材料疏松多孔，孔径大于100μm，孔隙率大于95％。细胞在材料上黏附状态良好，并且增殖迅速。BMSC材料复合体经体外三维诱导后可在自体异位向软骨分化。 ［结论］Ⅰ型胶原/壳聚糖/纳米TCP一体化层状梯度修复体具有良好的孔隙结构和生物相容性，有望成为一种新型的组织工程支架材料用于骨软骨缺损修复。 【关键词】 组织工程； 骨软骨； 骨髓间充质干细胞； 胶原； 纳米β磷酸三钙 Abstract：［Objective］To explore the potential of a novel collagen I / chitosan /Nanoβtricalcium phosphate(βTCP)bilayered scaffold for being used in tissue engineering(TE)of osteochondral repair.［Method］Bilayered scaffolds were produced with collagen Ⅰ,chitosan andβTCP,using a special crosslinking and freeze drying method.The pore size,porosity and interpores of the scaffold were observed by scanning electron microscopy(SEM).Rabbit bone mesenchymal stem cells （BMSC）were isolated and amplified,then inoculated onto the scaffold.By SEM scanning,the condition of the cells adhering onto the scaffold was observed.The proliferation of the cells on the scaffolds was examined using MTT method,and the growth curve was drawn.The cellscaffold composite were then induced to differentiate towards cartilage by 3D culturing,and then implanted into muscle pouches 2 weeks later.The result was observed 6 weeks later by HE staining,toluidine blue staining and type Ⅱ collagen immunohistochemistry.［Result］The scaffold possessed high porosity and proper pore size,the porosity was above 95%.BMSC could adhere onto the scaffold well,and the proliferation rate of the cells on the scaffolds was perfectly good.After in vitro induction,BMSCscaffold composite can differentiate toward cartilage ectopicly.［Conclusion］The novel collagen I / chitosan /βTCP bilayered scaffold possesses good pore structure and biocompatibility,and will possibly become a new biomaterial of TE used f