体外模拟压应力三维动态培养构建组织工程软骨的实验研究

发布时间：2018-04-02 10:00

本文选题：组织工程软骨　切入点：生物反应器　出处：《中国修复重建外科杂志》2014年09期

【摘要】：目的通过体外模拟压应力刺激兔软骨细胞复合脱细胞软骨细胞外基质(extracellular matrix,ECM)源性三维多孔支架培养组织工程软骨,探讨生物反应器中三维动态培养对软骨生长的促进效应。方法取健康成年新西兰大白兔四肢关节软骨分离培养软骨细胞,将第2代细胞复合于脱细胞软骨ECM源性三维多孔支架,静态预培养5 d后随机分为2组,A组继续静态培养,B组于生物反应器中进行动态压应力加载(频率1 Hz,15%压缩应变)培养。3周后采用活-死细胞染色试剂盒检测细胞活性,扫描电镜观察细胞在支架上的生长及分布,定量检测支架中细胞的糖胺聚糖(glycosaminoglycan,GAG)、胶原蛋白及DNA含量,进行力学分析检测弹性模量。结果激光共聚焦显微镜观察示,B组支架内细胞生长良好且分布均匀;A组细胞生长较差,中心区域失染。扫描电镜观察示,B组软骨细胞在支架上黏附、增殖、生长良好;A组软骨细胞明显减少,且分布散乱。生化成分分析示,B组胶原蛋白、GAG及DNA含量分别为(675.85±27.93)μg/mg、(621.72±26.75)μg/mg、(16.98±3.23)μg/样本,A组分别为(438.72±6.35)μg/mg、(301.63±30.51)μg/mg、(10.18±4.39)μg/样本,B组各成分含量均明显高于A组(t=18.512,P=0.000;t=17.640,P=0.000;t=2.790,P=0.024)。力学性能测试示,B组弹性模量(0.67±0.09)MPa显著高于A组(0.49±0.16)MPa和空白支架(0.43±0.12)MPa差异有统计学意义(P0.05)。结论生物反应器模拟压应力三维动态培养优于普通静态三维培养,可提供有利于软骨细胞在ECM支架中生长的最佳环境,是体外构建组织工程软骨的良好方法。
[Abstract]:Objective to culture tissue engineered cartilage by stimulation of rabbit chondrocytes with extracellular matrix extracellular matrix (ECM) under simulated compressive stress in vitro. To investigate the effect of three-dimensional dynamic culture on the growth of cartilage in bioreactor. Methods chondrocytes were isolated and cultured from articular cartilage of healthy adult New Zealand rabbits. The second passage cells were combined with acellular cartilage ECM derived three-dimensional porous scaffold. After 5 days of static preculture, two groups were randomly divided into two groups: group A and group B were cultured in a bioreactor under dynamic compressive stress (frequency of 1 Hzn 15% compression strain) for 3 weeks, and then the cell activity was detected by living and dead cell staining kit. The growth and distribution of cells on the scaffold were observed by scanning electron microscope. The contents of glycosaminoglycan, collagen and DNA in the scaffold were measured quantitatively. Results the results of laser confocal microscopy showed that the cells in group B grew well and the cells in group A grew poorly. SEM observation showed that chondrocytes of group B adhered to scaffold, proliferated, and chondrocytes in group A decreased obviously. The biochemical composition analysis showed that the contents of gag and DNA in group B were 621.72 卤26.75 渭 g / 渭 g / 渭 g / group respectively: 438.72 卤6.35 渭 g / g, respectively. The mechanical properties of group B were significantly higher than those of group B (10.18 卤4.39) 渭 g / group, respectively. The elastic modulus of group B was significantly higher than that of group A (18.512 渭 g / L) 0.000t (17.640g / t) 0.000t 2.790P240.The mechanical properties of group B were significantly higher than that of group B (P < 0.05). The results of mechanical tests showed that the elastic modulus of group B was significantly higher than that of group B (P < 0.01). The content of gag and DNA in group B was significantly higher than that in group B (P < 0.05). The content of gag and DNA in group B was significantly higher than that in group A (621.72 卤26.75) 渭 g / g / group (16.98 卤3.23) 渭 g / group, respectively. The mechanical properties of group B were 0.67 卤0.67 卤4.39 渭 g / group respectively. There was significant difference between group A (0.49 卤0.16)MPa) and blank scaffold (0.43 卤0.12)MPa). Conclusion Three-dimensional dynamic culture of simulated compressive stress in bioreactor is superior to static three-dimensional culture. It can provide the best environment for the growth of chondrocytes in ECM scaffold and is a good method to construct tissue engineered cartilage in vitro.
【作者单位】：解放军总医院全军骨科研究所;
【基金】：国家自然科学基金资助项目(81071458) 国家高技术研究发展计划(863)资助项目(2012AA020502)~~
【分类号】：R318.08

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