利用温敏胶原动态培养构建组织工程复合物的初步研究  

CONSTRUCTION OF TISSUE ENGINEERED COMPOSITE WITH THERMOSENSITIVE COLLAGEN HYDROGEL IN DYNAMIC CULTURE SYSTEM

作　　者：许飞翔[1] 赵劲松[2] 郭斌[1] 马健超[1] 黄岚峰[1] 

机构地区：[1]吉林大学第二医院骨科,长春130041 [2]吉林大学第二医院眼科,长春130041

出　　处：《中国修复重建外科杂志》2016年第3期309-313,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(31071222);吉林大学白求恩前沿交叉学科创新项目(2013106023)~~

摘　　要：目的研究动态培养条件下以BMSCs为种子细胞、温敏胶原(thermosensitive collagen hydrogel,TCH)为细胞黏附基质和聚乳酸(poly-L-lactic acid,PLLA)为支架材料体外构建的细胞-支架复合物形态及功能特征。方法分离Fischer344大鼠长骨来源的BMSCs,并体外扩增至第3代作为种子细胞,利用PLLA纤维编织材料作为支架,结合TCH为细胞黏附基质构建细胞-支架复合物。实验组和对照组细胞-支架复合物分别行动态培养和静态培养。体外培养7 d对两组复合物行大体观察和扫描电镜观察,检测培养0、1、3、7 d后复合物中总DNA含量。结果两种方式培养后胶原均包绕PLLA支架材料并形成致密的膜状结构。扫描电镜观察示,实验组复合物表面BMSCs呈梭形沿长轴排列整齐,细胞活性良好,表面可见绒毛凸起,纵断面见PLLA纤维之间充满胶原纤维和细胞;对照组复合物表面BMSCs呈延展状态,PLLA之间的细胞相对较少。体外培养1、3、7 d实验组细胞-支架复合物中总DNA含量均明显高于对照组(P<0.05)。与培养0 d比较,两组培养1、3、7 d的总DNA含量均逐渐升高,但仅7 d时总DNA含量与培养0 d比较差异有统计学意义(P<0.05)。结论利用TCH和PLLA纤维联合构建的复合细胞外基质具有良好的生物相容性,动态培养可促进BMSCs在材料表面和内部的分布,促进细胞增殖,可用于组织工程复合物的体外构建。Objective To explore the morphological and functional features of tissue engineered composite constructed with bone mesenchymal stem cells（BMSCs） as seeding cells, thermosensitive collagen hydrogel（TCH） and poly-L-lactic acid（PLLA） as the extracellular matrix（ECM） scaffolds in the dynamic culture system. Methods BMSCs were separated from long bones of Fischer344 rat, and cultured; and BMSCs at the 3rd generation were seeded on the ECM scaffold constructed with braided PLLA fiber and TCH. The BMSCs-ECM scaffold composite was cultured in the dynamic culture system which was designed by using an oscillating device at a frequency of 0.5 Hz and at swing angle of 70°（experimental group）, and in the static culture system（control group） for 7 days. The general observation and scanning electron microscopy（SEM） observation were performed; total DNA content was measured at 0, 1, 3, and 7 days. Results PLLA was surrounded by collagen to form translucent gelatiniform in 2 groups; and compact membrane developed on the surface of PLLA. SEM observation showed that BMSCs had high viability and were fusiform in shape with microvilli on the surface of cells, and arranged in line; collagen and cells filled in the pores of PLLA fiber in the experimental group. The cells displayed a flat shape on the surface; there were less cells filling in the pores of PLLA fiber in the control group. At 1, 3, and 7 days, total DNA content in the experimental group was significantly higher than that in control group（P〈0.05）. The total DNA content were increased gradually with time in 2 groups, showing significant difference between at 0 day and at 7 days（P〈0.05）. Conclusion The ECM constructed with TCH and PLLA has good biocompatibility. The dynamic cultivation system can promote the cell proliferation, distribution, and alignment on the surface of the composite, so it can be used for tissue engineered composite in vitro.

关 键 词：动态培养 组织工程 细胞外基质 种子细胞 支架材料 

分 类 号：R318.08[医药卫生—生物医学工程]