灌流-液压式反应器体外构建组织工程化软骨  

Constructing Tissue Engineered Cartilage in Vitro Using a Perfusion-Hydrodynamic Pressure Bioreactor

作　　者：吕晓杰[1] 周广东[1] 李宏[1] 曹珲[1] 刘霞[1] 刘凯[1] 崔磊[1] 刘伟[1] 曹谊林[1] 

机构地区：[1]上海交通大学医学院第九人民医院整复外科上海市组织工程研究与开发中心,上海200235

出　　处：《上海交通大学学报（医学版）》2006年第9期1006-1010,共5页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家重点基础研究发展规划("973"项目)(G1999054308-2);国家高技术研究发展计划("863"计划)(2002AA205021);上海高等学校青年科学基金(03BQ32)资助项目

摘　　要：目的初步探讨应用灌流-液压式反应器体外构建组织工程化软骨的可行性及其效果。方法消化、分离猪耳廓软骨细胞,接种于聚羟基乙酸支架上培养1周,形成细胞-支架复合物。实验分2组(每组8个样本):实验组放入灌流-液压式反应器连续培养3周;反应器主要参数设置:灌流量100μL/m in,顺、逆时针方向各30 m in,灌流时间开/关各8 h/16 h,液压强度100 kPa,频率0.5 Hz,作用时间4 h/d。对照组则继续在培养皿内培养。第4周时取材,分别从大体、组织学、Ⅱ型胶原免疫组化和生物化学等方面对实验组和对照组形成的软骨组织进行检测和比较。结果大体观察见实验组和对照组均形成了软骨样组织,组织湿重检测实验组为(191.03±18.55)mg,对照组为(130.78±10.33)mg(P<0.01)。组织学观察显示,两组软骨组织内软骨细胞分布均匀,包埋于软骨陷窝内,软骨基质分泌旺盛,局部还有未降解的支架材料。免疫组化染色证实两组形成的软骨组织均有Ⅱ型胶原分布,软骨组织厚度实验组为(3.04±0.13)mm,对照组为(2.14±0.16)mm,两组间比较有显著性差异(P<0.01)。生物化学检测证实,实验组蛋白聚糖含量为(12.63±0.62)mg/g,明显高于对照组的(8.23±0.81)mg/g(P<0.01)。结论利用灌流-液压式反应器能够在体外构建组织工程化软骨,并且可以在一定程度上弥补传统构建方法的不足,提高体外构建软骨的质量。Objective To explore the feasibility and efficacy of constructing tissue engineered cartilage in vitro using a perfusion-hydrodynamic pressure bioreactor. Methods Chondrocytes isolated from swine＇s auricular cartilage were seeded onto polyglycolic acid （PGA） to be cultured in a three dimensional environment for 1 week. Then the chondrocyte-polymer constructs were divided into two groups： the experimental group and control group （8 constructs in each group）. The experimental group was put into the perfusion-hydrodynamic pressure bioreactor to be cultured for another 3 weeks. The parameters of bioreactor were set as follows： flow rate of 100 mL/min, clockwise and anticlockwise 30 rain respectively, on/off 8 h/16h, hydrodynamic pressure of 100 kpa with 0.5 Hz for 4 h/d. The control group was cultured with the routine method. Specimens were harvested and analyzed by gross observation, histology, type Ⅱ collagen immunohistochemistry and biochemistry after 4 weeks. Results After 4 weeks, gross observation showed cartilage-like tissue was formed in both groups, and tissue wet weight of experimental group and control group were （ 191.03 ± 18.55） mg and （ 130.78 ± 10.33 ） mg, respectively （ P 〈 0.01 ）. Histological observation showed cartilage was formed in both groups, and chondrocytes were enclosed in lacuna and surrounded by rich cartilage ECM. Immunohistochemical staining proved the presence of type I1 collagen. Cartilage of experimental group （ [ 3.04 ± 0.13 ] mm） was significantly thicker than that of control group （ [ 2.14 ± 0.16 ] mm） （ P 〈 0.01 ）. In addition, the biochemical examination showed that the proteoglycans content in cartilage of the experimental groups （ [12.63 ±0.62] mg/g）was higher than that of the control group（ [8.23 ±0.81] mg/g） （P 〈0.01）. Conclusion The tissue engineered cartilage can be constructed in a perfusion-hydrodynamic pressure bioreactor,which can make up the shortage of traditional method to some extent and improve the qua

关 键 词：灌流-液压式反应器 软骨细胞 组织工程 体外构建 

分 类 号：R318[医药卫生—生物医学工程]