不同持续时间的周期性压力对构建组织工程软骨的影响

Tissue engineered cartilage in response to different durations of the cyclic pressure

目的探讨在周期性压力条件下构建组织工程软骨每天加压的最佳持续时间。方法构建自行设计的生物反应器和往复式加压泵组成的“周期性压力场培养系统”，将体外培养的第二代乳兔关节软骨细胞接种到聚乳酸一聚羟基乙酸共聚物（PLGA）支架上，随机分成四组。第一、二、三组分别在每天持续时间为4、8、12h的周期性压力（强度0～200kPa，频率为0．1Hz）下培养，第四组（对照组）为不加压的静态培养，各组培养时间均为2周。2周后肉眼大体观察，HE染色组织学观察工程软骨细胞增殖及分布；甲苯胺蓝染色法观察硫酸糖氨多糖（GAG）的分泌及分布，并用1，9-二甲基亚甲蓝法定量检测GAG的含量；采用Ⅱ型胶原免疫组织化学法观察Ⅱ型胶原的分泌及分布，并用image—proplus图像分析系统对Ⅱ型胶原染色面积行半定量分析。结果在强度0～200kPa，频率为0．1Hz周期性压力作用下，8h组支架．细胞复合体体积最大，表面光滑、有光泽、有弹性，支架内软骨细胞数量最多，排列最为规则，Ⅱ胶原和GAG含量也最高（P〈0．01）。结论软骨细胞的新陈代谢受周期性压力持续时间的影响，在0～200kPa、0．1Hz频率作用下，每天持续8h的周期性压力能更好地促进软骨细胞增殖，合成Ⅱ型胶原、GAG等细胞外基质。

Objective To determine the best duration for exerting the cyclic pressure under which the tissue-engineered cartilage is constructed. Methods Free ehondroeytes isolated from rabbit articular cartilage were seeded into polylaetic aeid-eo-g]ycolie aeid（PLGA） scaffolds after expansion in vitro, and randomized into 4 groups. In Groups 1 to 3, chondrocytes were cultured under daily cyclic pressure （0 ~ 200 kPa, 0. 1Hz） for 4 hours, 8 hours, 12 hours respectively; Group 4 was a control in which no pressure was exerted. In each group, after 2 weeks of culture, the tissue engineered cartilages were observed in vitro and assessed by his- tological staining of HE. Next, the content of DNA and the secretion of typeⅡ collagen and GAG in cartilages were detected quantitatively. Results Under the daily cyclic pressure （0-200 kPa, 0. 1 Hz）, the scaffold-chondrocytes complex in the group of 8 hours got the largest volume, smooth, lucidus, and elastic surface, the most queuing chondrocytes, and the highest content of type 11 collagen and GAG （ P 〈 0.01 ）. Conclusions Since chondrocytes are baro-senstive, the metabolism of chondrocytes can be affected by the time of cyclic pressure. Under the effect of 0-200 kPa, 0. 1Hz, the daily cyclic pressure of 8 hours may be optimal for ehondroeytes to multiply and synthesize extracellular matrixes such as type Ⅱ collagen and GAG.