壳聚糖和软骨细胞外基质复合组织工程骺软骨支架的性能观察  

Performance of epiphyseal cartilage tissue engineering scaffold constructed by chitosan and extracellular matrix of cartilage

作　　者：卢强[1] 彭江[1] 黄靖香[1] 张莉[1] 郭全义[1] 卢世璧[1] 

机构地区：[1]解放军总医院骨科,北京市100853

出　　处：《中国组织工程研究与临床康复》2009年第16期3022-3026,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：全军十一五专项项目(06Z057);国家自然科学基金重点项目(30330570);解放军总医院科技创新基金(06ZS03)~~

摘　　要：背景:在组织工程骺软骨构建过程中,采用何种载体承载已经扩增了的软骨细胞越来越受到人们的关注。目的:探讨软骨细胞外基质与壳聚糖复合构建组织工程骺软骨支架的可行性并对其性能进行初步观察。设计、时间及地点:体外观察实验,于2007-12/2009-03在解放军总医院骨科研究所完成。材料:壳聚糖(脱乙酰度90%,Mr10×105)由青岛海汇生物工程公司提供。市售猪关节软骨。方法:将新鲜的猪关节软骨在液体中粉碎,梯度离心后制备成3%软骨微丝悬液,将其按1︰1的比例与2%的壳聚糖溶液混合,采用冷冻冻干法制备脱细胞软骨基质三维多孔支架。主要观察指标:对梯度乙醇处理再次干燥后支架材料进行组织学及扫描电镜观察,测定支架孔径和孔隙率、吸水率,并采用MTT法分析支架浸提液毒性。分离培养兔骨髓间充质干细胞,用转化生长因子β1成软骨诱导后种植至支架,倒置显微镜、电镜观察细胞在支架上的生长、分化情况。结果:支架内孔洞相互连通,孔大小(161±31)μm,孔隙率为(90.1±1.6)%,吸水率为(2361±132)%。组织学观察显示,三维多孔支架甲苯胺蓝染色、番红O染色、Ⅱ型胶原免疫组织化学染色均呈阳性。MTT法结果显示支架无细胞毒性。细胞在支架表面黏附良好,分布均匀,并有基质分泌。结论:制备的软骨细胞外基质和壳聚糖复合三维多孔支架结合良好,含有软骨细胞外基质主要成分,无毒,具备合适的孔径和孔隙率,生物相容性良好。BACKGROUND： Choose an ideal vector for amplified chondrocytes arouses more and more attention during the construction of epiphyseal cartilage tissue engineering. OBJECTIVE： To explore the feasibility and performance of epiphyseal cartilage tissue engineering scaffold constructed by chitosan and extracellular matrix of cartilage. DESIGN, TIME AND SETTING： An in v#ro study was performed at Department of Orthopedics, General Hospital of Chinese PLA from December 2007 to March 2003. MATERIALS： Chitosan （deacetylation： 90%; Mrl0Xl05） was provided by Haihui Bioengineering Company, Qingdao; articular cartilage of swine was collected from market. METHODS： Fresh porcine articular cartilages were obtained and shattered in the iso-osmia liquid. After pulverization and gradient centrifugation, 3% artilage microfilament suspension was equally mixed with 2% chitosan. Three-dimensional porous scaffolds were fabricated using a simple freeze-drying method. MAIN OUTCOME MEASURES： After the second gradient ethanol treatment, the scaffolds were investigated by histological staining and scanning electron microscopy to measure aperture, porosity, and water absorption rate. MTT test was also done to assess cytotoxicity of the scaffolds. After induced by transforming growth factor-β1 （TGF- β1）, bone marrow mesenchymal stem cells （BMSCs） of rabbits were incubated onto the scaffolds. Cell proliferation and differentiation were analyzed using inverted microscopy and scanning electron microscopy. RESULTS： The three-dimensional porous scaffold had good pore interconnectivity with pore diameter （161±31） μm, （90.1±1.6）% porosity and （2 361±132）% water absorption rate. The histological staining showed that toluidine blue, safranin O and anti-collagen Ⅱ immunohistochemistry staining were positive. The intrinsic cytotoxicity assessment of the scaffolds using MIT test showed that the scaffolds had no cytotoxic effect on BMSCs. Most of the BMSCs attached and covered the surface of the scaffolds with m

关 键 词：支架 软骨 骺损伤 组织工程 

分 类 号：R318[医药卫生—生物医学工程]