纳米羟基磷灰石/壳聚糖支架与诱导骨髓间充质干细胞体外构建软骨支架复合体  

Construction of composite graft by using bone marrow stem cells and nano-hydroxyapatie/chitosan scaffold

作　　者：李劼若[1] 查振刚[1] 姚平[2] 束志勇[1] 吴昊[1] 屠美[3] 熊高鑫[1] 梁耀中[1] 

机构地区：[1]暨南大学附属第一医院骨科,广东省广州市510630 [2]暨南大学医学院生理教研室,广东省广州市510630 [3]暨南大学理工学院材料系,广东省广州市510630

出　　处：《中国组织工程研究与临床康复》2009年第3期411-416,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家863高技术研究发展计划资助项目(2007AA09Z440);广州市科技计划项目(2006Z3-E5211)~~

摘　　要：背景:国内外许多研究通过不同的构建方法构建组织工程化软骨支架复合体修复骨软骨联合缺损,且取得了一定的进展,但目前各种方法存在的问题突出表现为组织工程化的软骨和骨组织之间的界面、移植体和宿主骨和软骨之间的界面耦合不够理想。目的:将体外提纯、扩增的骨髓间充质干细胞诱导成软骨细胞,将其接种于穿"靴"的纳米羟基磷灰石/壳聚糖支架的底部上联合培养,探索其用于组织工程软骨复合体的可行性。设计、时间及地点:细胞和材料复合的体外观察实验,于2008-03/07在暨南大学附属第一医院中心实验室和暨南大学理工学院材料系实验室完成。材料:通过原位复合和冷冻干燥结合的方法制备纳米羟基磷灰石/壳聚糖支架。健康新西兰兔10只由广东省医学实验动物中心提供。方法:密度梯度离心法提取分离骨髓间充质干细胞,软骨诱导液诱导骨髓间充质干细胞2周后甲苯胺蓝染色检测。把诱导得到的软骨细胞接种于穿"靴"的纳米羟基磷灰石/壳聚糖支架的底部,将细胞-支架复合物置入成软骨条件培养液中培养2周。主要观察指标:倒置相差显微镜下观察细胞形态特征,鉴定CD29,CD44,CD34和CD45抗原的表达,观察细胞生长特性,测定细胞活力和生长周期,扫描电镜观察纳米羟基磷灰石/壳聚糖支架结构和细胞与支架的复合情况。结果:骨髓间充质干细胞可在体外分离扩增,表达CD29和CD44,不表达CD34和CD45,细胞活力为95.27%,G0～G1期细胞占94.68%。经软骨诱导液诱导后骨髓间充质干细胞转化成软骨细胞;制备的纳米羟基磷灰石/壳聚糖多孔支架孔隙率为90%,平均孔径为150μm,与软骨细胞有较好的黏附性。结论:初步证实纳米羟基磷灰石/壳聚糖支架与骨髓间充质干细胞诱导成的软骨细胞复合可以在体外构建组织工程软骨复合体。BACKGROUND： Many studies have achieved certain progress in repair of the osteochondral defect by tissue-engineered chondral composite graft which was constructed with different methods. But the existing problems of these methods are presented that the coupling of interfaces of bone and cartilage in graft and host is not ideal. OBJECTIVE： After purification and amplification in vitro, the bone marrow mesenchymal stem cells （BMSCs） were induced into chondrocytes, and then implanted them at the bottom of nano-hydroxyapatie/chitosan （nano-HA/CS） scaffold for combined culture. The aim was to investigate the feasibility of them used in tissue-engineered chondral composite graft. DESIGN,TIME AND SETTING： An in vitro observational experiment of cytology and the compositing with biomaterial was performed at the Central Laboratory in the First Affiliated Hospital of Ji＇nan University and the Laboratory of Department of Material Science in College of Science and Engineering of Ji＇nan University from March to July 2008. MATERIALS： Nano-HA/CS scaffolds were prepared by using the in-situ composite technique and freeze-drying method. A total of ten healthy New Zealand rabbits were provided by Experimental Animal Center of Guangdong Province. METHODS： BMSCs were isolated from rabbits with density gradient centrifugation. BMSCs were cultivated with chondrogenic differentiation medium and tested by stained with toluidine blue after 2 weeks. The chondrocytes were implanted at the bottom of the Nano-HA/CS scaffold with a boot-shaped structure, and then cultivated the composite in the chondrogenic differentiation medium for 2 weeks. MAIN OUTCOME MEASURES： Cellular configuration was observed under inverted phase-contrast microscope. The CD29, CD44, CD34, and CD45 antigen expressions were indentified. The growth characteristics of cells, cell viability and growth period were analyzed. The nano-HA/CS scaffolds and the composite graft were observed by scanning electron microscope. ~i ：~ . ：L,： BMSCs can be

关 键 词：纳米羟基磷灰石/壳聚糖 骨髓间充质干细胞 组织工程 软骨复合体 

分 类 号：R318[医药卫生—生物医学工程]