体外构建可注射性组织工程软骨的初步研究  

Construction of injectable tissue engineered cartilage in vitro

作　　者：王东武[1] 杨柳[1] 段小军[1] 李忠[1] 

机构地区：[1]第三军医大学西南医院关节外科中心,重庆400038

出　　处：《第三军医大学学报》2006年第11期1145-1147,共3页Journal of Third Military Medical University

基　　金：国家自然科学基金资助项目(30270375);全军医学科学技术研究"十五"计划重点课题(01Z072);重庆市重点科技攻关项目(2003-13-6)~~

摘　　要：目的利用关节软骨细胞复合壳聚糖-磷酸甘油(ch itosan/glycerophosphate,C/GP)凝胶,体外构建可注射性组织工程软骨,为微创方式治疗关节软骨缺损提供依据。方法以体外培养扩增的兔关节软骨细胞为种子细胞,以自制温固化可注射C/GP凝胶为支架,按5×107/m l细胞浓度复合体外培养。于倒置显微镜下观察细胞形态与分布,四甲基偶氮唑盐(MTT)法计算培养1周时种子细胞存活率,培养4周时样本进行组织学及扫描电镜观察。结果软骨细胞在7∶1(体积分数)C/GP凝胶中保持球形,细胞存活率95%以上;HE染色可见软骨较为典型的陷窝样结构、软骨囊及同源细胞群等现象;甲苯胺蓝异染及免疫组化Ⅱ型胶原染色呈强阳性。结论软骨细胞在C/GP凝胶中可存活并保持分泌软骨基质的功能,形成类软骨组织。温固化可注射性C/GP凝胶能作为软骨组织工程的载体材料。Objective To observe the biological behavior of chondrocytes in chitosan based hydrogel in vitro and to evaluate the possibility of chitosan and glycerophosphate mixed gel using as injectable scaffolds in tissue engineering. Methods A novel injectable thermosetting gel was made of chitosan and glycerophosphate mixture. Chondrocytes isolated by enzymic digestion from the articular cartilage of the young rabbits aged 3 -4 weeks were cultured in monolayer system. After primary culture and subculturing, the ceils were seeded in chitosan based gel in concentration of 5 ×10^7/ml in vitro. Characteristics of chondrocytes during 4-week culture were observed with invert microscope. MTr was used to detect the seed growth after one-week culture. In 4th week, after haematoxylin eosine and toluidine blue staining, the artifical cartilage was performed type 11 collagen immunohistochemistry and observed by scanning electron microscope. Results Chondrocytes retained their round morphology, multiplied, and produced matrix in chitosan and glycerophosphate mixture gels. The results of immunohistochemistry were strongly positive. Conclusion That most chondrocytes appeared to occupy large areas of rough chitosan based gels, guarantees a suitable environment where chondrocytes secrete matrix macromolecules and preserve their round morphology in vitro. Chitosan and glycerophosphate mixture gel may be a vehicle for injectable tissue -engineered cartilage.

关 键 词：关节 软骨细胞 壳聚糖 甘油磷酸钠 支架/组织工程 

分 类 号：R-33[医药卫生] R318.08