旋转生物反应器培养对组织工程气管软骨力学强度的影响

Effects of rotating bioreactor on mechanical characteristics of tissue-engineered tracheal cartilage

目的研究旋转生物反应器培养对组织工程气管软骨力学强度的影响,探索适宜的组织工程气管软骨培养方法。方法分离2周龄Lewis大鼠剑突软骨细胞传代培养,收集第3代软骨细胞种植到DegraPol管状支架上,静态培养7d,然后将软骨细胞-支架复合物分别置于旋转生物反应器内培养(生物反应器组)或继续静态培养培养3周(静态培养组)。取出软骨细胞-支架复合物,以噻唑蓝(MTT)法测定软骨细胞增殖活性,结果以吸光度(A)值表示(每组n=6);以Zwick1445型材料试验机测定软骨细胞-支架复合物的最大应变值和应力值(每组n=4);并制备扫描电镜标本,观察软骨细胞在DegraPol支架中培养后的超微结构变化。结果不同条件下培养3周,生物反应器组和静态培养组A值分别0.17±0.05、0.12±0.01,最大应力值分别为(0.33±0.04)和(0.26±0.01)MPa,最大应变值分别为(3.53±0.91)和(1.71±0.13)mm/mm,2组间3项指标的差异均有统计学意义(均P<0.05)。扫描电镜观察显示生物反应器组获得更好的软骨样结构和更多的细胞外基质。结论旋转生物反应器能够提供更好的体外培养条件,有利于组织工程气管软骨的形成。

Objective To study the effects of rotating bioreactor on the mechanical intensity of tissue-engineered tracheal cartilage, and to investigate an optimum method for constructing tissue-engineered tracheal cartilage. Methods Xiphoid chondrocytes were harvested from 2-week-old Lewis rat and serially passaged. The third passage cells were collected, seeded onto DegraPol scaffolds and cultivated in static conditions for 7 days. Afterwards, the chondrocytes-DegraPol constructs were cultured in a rotating bioreactor （bioreactor group） or static conditions （static group）. After 3-week cultivation, the constructs were examined by 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenylter-trazolium bromide （MTT） assay （n = 6 for each group）, and the results were expressed as light absorption （A）. The tension tests of maximum stress and maximum strain were carried out with a Zwick 1445 material-testing machine （n = 4 for each group）. Ultrastructure of the constructs was observed under a scanning electron microscope. Rusults After being cultured for 3 weeks, the A values of bioreactor and static groups were significantly different （0.17±0.05 vs. 0.12±0.01, P 〈 0.05）. The maximum stress and maximum strain in bioreactor group were （0.33±0.04） MPa and （3.53±0.91） mm/mm respectively, which were significantly higher than those in static group [（0.26±0.01） MPa and （1.71±0.13） mm/mm, P 〈 0.05]. Scanning electron microscopy showed that the constructs of bioreactor group achieved better chondrocyte-like ultrastructure and more extracellular matrix than static group. Conclusion Rotating bioreactor can provide better in vitro cultural conditions, which are good for the formation of tissue-engineered tracheal cartilage.