生物反应器中的力学刺激促进组织工程软骨再生

Bioreactor cultivation and mechanical stimulation for regeneration of tissue-engineered cartilage

背景:软骨组织工程已被广泛用来实现体外软骨组织再生,并用来修复软骨缺损。软骨组织工程主要由软骨细胞、软骨支架和体外培养环境3部分组成。目的:实验通过体外模拟体内关节软骨生长环境,为进一步提高组织工程软骨的仿生性及更好地修复损伤软骨提供理论依据。方法:2月龄新西兰大白兔,用于分离培养膝关节软骨细胞,取第2代细胞以1×106 L-1细胞浓度接种于去细胞软骨基质支架上制备细胞-支架复合物。实验分为实验组和对照组,对照组单纯细胞支架复合静态培养1 d,实验组于Instron生物反应器中培养,加以力学刺激(力学加载参数为3 h/d,1 Hz,压缩量为10%)7,14,21,28 d。结果与结论:(1)随着培养时间的延长,实验组细胞-支架复合物厚度、弹性模量及最大负荷均显著高于对照组(P<0.05)。(2)实验组组织切片苏木精-伊红染色均可见软骨细胞增殖及软骨陷窝形成,番红"O"染色见细胞-支架复合物的细胞外基质中有大量蛋白聚糖形成,随力学刺激时间延长而逐渐增加,并与蛋白聚糖试剂盒检测结果相符。(3)实时荧光定量PCR检测示,实验组Ⅰ,Ⅱ型胶原表达均高于对照组(P<0.05),实验组中力学刺激21 d时Ⅰ型胶原表达最高(P<0.05);Ⅱ型胶原在力学刺激28 d表达最高(P<0.05)。(4)结果证实细胞-支架复合物通过生物反应器的力学加载,可以产生更多的胶原和蛋白聚糖等细胞外基质成分,并增强其力学特性,使其更加复合人体软骨生长环境,并提高其与正常软骨的仿生性,从而使细胞支架复合物能更好的修复损伤软骨。

BACKGROUND： Cartilage tissue engineering has been widely used to achieve cartilage regeneration in vitro and repair cartilage defects. Tissue-engineered cartilage mainly consists of chondrocytes, cartilage scaffold and in vitro environment. OBJECTIVE： To mimic the environment of articular cartilage development in vivo, in order to increase the bionic features of tissue-engineered cartilage scaffold and effectiveness of cartilage repair. METHODS： Knee joint chondrocytes were isolated from New Zealand white rabbits, 2 months old, and expanded in vitro. The chondrocytes at passage 2 were seeded onto a scaffold of articular cartilage extracellular matrix in the concentration of 1×106/L to prepare cell-scaffold composites. Cell-scaffold composites were cultivated in an Instron bioreactor with mechanical compression（1 Hz, 3 hours per day, 10% compression） as experimental group for 7, 14, 24, 28 days or cultured statically for 1 day as control group. RESULTS AND CONCLUSION： Morphological observations demonstrated that the thickness, elastic modulus and maximum load of the composite in the experimental group were significantly higher than those in the control group, which were positively related to time（P〈0.05）. Histological staining showed the proliferation of chondrocytes, formation of cartilage lacuna and synthesis of proteoglycan in the experimental group through hematoxylin-eosin staining and safranin-O staining, which were increased gradually with mechanical stimulation time. These results were consistent with the findings of proteoglycan kit. Real-time quantitative PCR revealed that m RNA expressions of collagen type I and collagen type Ⅱ were significantly higher in the experimental group than the control group（P〈0.05）. The experimental group showed the highest m RNA expression of collagen type I and collagen type Ⅱ at 21 and 28 days of mechanical stimulation, respectively（P〈0.05）. With the mechanical stimulation of bioreactor, the cell-scaffold composite can produce more extracellular matrix, such as collagen and proteoglycan, strengthen the mechanical properties to be more coincident with the in vivo environment of cartilage development, and increase the bionic features. With the progress of tissue engineering, the clinical bioregeneration of damaged cartilage will be achieved.