三相蚕丝组织工程韧带构建及其生物相容性的研究  

Construction of three-phase silk tissue-engineered ligament and preliminary analysis of its biocompatibility

作　　者：孙立国[1,2] 范宏斌[2] 李宏国[2] 屈玲[3] 仓宝成 薛英森 刘鑫成 李丹[2] 

机构地区：[1]北京军区天津疗养院,天津300081 [2]第四军医大学西京医院全军骨科研究所,西安710032 [3]第四军医大学西京医院检验科,西安710032 [4]解放军一五三中心医院检验科,郑州450042

出　　处：《中国骨与关节杂志》2015年第11期820-825,共6页Chinese Journal of Bone and Joint

基　　金：国家自然科学基金(31170936;31470316)

摘　　要：目的评价三相蚕丝组织工程韧带的理化性质及生物相容性。方法编织蚕丝网状支架,提取丝素蛋白,利用硫酸软骨素、透明质酸钠、羟基磷灰石（HA）及丝素蛋白修饰三相支架,测定支架材料形貌及表面元素。支架材料接种骨髓间充质干细胞（bone marrow mesenchymal stem cell,BMSCs）后利用扫描电镜（SEM）、激光共聚焦显微镜,观察细胞的黏附及增殖情况。结果蚕丝去除丝胶后,表面变光滑,脱丝胶前蚕丝纤维的直径为（9.89±1.98）μm,脱丝胶后大小为（9.14±1.75）μm,表面经脱丝胶处理后蚕丝纤维直径没有显著改变（P=0.221）。表面元素分析表明B区中的S和Na元素及C区中的Ca元素比A区有显著提高,并且材料的孔径在60-80μm。A、B、C区的细胞在培养后4 h与培养后2 h相比,细胞黏附率分别为：A区（83.2±3.4）%,B区（81.8±2.8）%,C区（71.1±5.4）%,差异有统计学意义（P=0.0001）,表明细胞培养后4 h大部分细胞在材料上有较好的黏附。扫描电镜观察,接种后1天,BMSCs在材料上黏附良好,接种后7天细胞爬满材料表面并可见细胞伪足深入材料内部生长。活/死细胞染色利用激光共聚焦显微镜观察培养的7天比1天的细胞增殖明显,且未见明显死亡,细胞在材料的多个层次可见。结论构建的三相蚕丝组织工程韧带具有良好的生物相容性,利于BMSCs的黏附增殖。Objective To evaluate physicochemical properties and biocompatibility of the three-phase silk tissue-engineered ligament. Methods The silk mesh was woven, and silk fibroin was extracted. The three-phase scaffold was modified with chondroitin sulfate, hyaluronic acid sodium, hydroxyapatite（ HA） and silk fibroin. The surface morphology and element of the scaffold were determined. After bone marrow mesenchymal stem cells（ BMSCs） seeding, the cell＇s proliferation and adhesion were observed by scanning electron microscope（ SEM） and laser confocal microscopy. Results After the removal of sericin, silk surface became smooth. The silk fiber diameter was（ 9.89±1.98） μm. After the removal of sericin, the diameter was（ 9.14±1.75） μm. No significant differences existed in the diameter of silk fiber（ P=0.221）. A surface element analysis showed the Ca element in area C, S and Na elements in area B were all higher than those in area A. The pore size of the material was 60-80 μm. The cell adhesion rates of area A, B and C 4hrs later were（ 83.2±3.4） %,（ 81.8±2.8） % and（ 71.1±5.4） %, which were significantly different from those 2hrs later（ P=0.0001）. It indicated that most of the cells were adherent to the material after 4hrs＇ cells culture. One day after the seeding, the BMSCs in the material adhered well by SEM. Seven days later, the cells covered the material surface and the pseudopodia grown into internal were visible. Using life / dead cell staining, the cells cultured 7d were of higher proliferation than 1d by laser confocal microscopy. The cells were visible in multiple levels of the material. Conclusions The constructed three-phase silk tissue-engineered ligament has good biocompatibility, which is conducive to the proliferation of BMSCs. It lays the foundation for the vivo experiment in the next.

关 键 词：组织工程 材料试验 生物相容性材料 蚕丝 

分 类 号：R318.08[医药卫生—生物医学工程]