摘要
目的探讨纤维蛋白凝胶种植技术在组织工程骨体外构建中的应用,从而改善目前常规构建技术的缺陷。方法首先采用逆转录病毒pLEGFP-N1对人骨髓间充质干细胞进行荧光蛋白标记,经成骨诱导培养作为种子细胞,然后分别应用纤维蛋白凝胶种植技术(A组)和沉淀种植技术(B组),体外构建细胞-材料复合体,通过倒置荧光显微镜和扫描电镜等观测种子细胞在支架材料内的分布与数量。结果逆转录病毒载体pLEGFP-N1成功标记骨髓间充质干细胞,并应用于工程化组织体外构建的示踪;A组细胞在材料内分布更均匀、数量更多,构建第2天细胞数量为(6.83±0.15)×105/个复合体,而B组细胞为(3.28±0.18)×105/个复合体,两组数据有非常显著统计学意义(P<0.01)。结论纤维蛋白凝胶种植技术简单易行,不仅改善种子细胞在骨支架材料内的分布,而且提高细胞种植效率,为体外高效构建组织工程骨创造了条件。
Objective To establish a new technique of construction the cell-scaffold complex in vitro with fibrin gel in bone tissue engineering. Methods The human marrow mesenchymal stem cells （MMSCs） were isolated from healthy volunteers and expanded culture. Then the MMSCs transfected with the recombinant retrovirus pLEGFP-N1 containing enhanced green fluorescent protein （EGFP） and G418 resistant clone were expanded and the labeled cells were differentiated into osteoblasts as seeding cells. The cell-scaffold complexes were constructed with fluorescent labeled cells and the scaffold of hydroxyapatite in vitro by different seeding methods. The complexes were constructed with fibrin gel in group A and the control samples were prepared by MMSCs dropped into the scaffold directly in group B. The tissue development was observed by fluorescence microscope and scanning electron microscope. The number of seeding cells in the complex was analysed after digestion with trypsin and EDTA. Results EGFP was successfully expressed after MMSCs being transfected with the retroviral vector pLEGFP-N1 and applied for labeling cells in bone tissue engineering. The distribution of cells throughout the porous scaffold was more homogeneous in group A than in group B. After the 2nd day, the number of cells in each complex was significantly higher （P 〈 0.01） in group A [（6.83±0.15）×10^5] than that in group B [（3.28±0.18）×10^5]. Conclusion The seeding method of construction with fibrin gel is simple and easy to operate. The distribution of seeding cells in the fibrin gel is homogenous and the concentration of the cells is high. This technique is useful for regeneration tissue engineering bone by application of this technique.
出处
《生物医学工程与临床》
CAS
2006年第5期271-274,F0003,共5页
Biomedical Engineering and Clinical Medicine
基金
国家自然科学基金资助项目(30300079
30270375)
全军十五医药卫生科研重点基金资助项目(01Z072)
关键词
骨髓间充质干细胞
组织工程
纤维蛋白
骨
marrow mesenchymal stem cells
tissue engineering
fibrin
bone
