兔骨髓间充质干细胞在藻酸锶凝胶中体外三维培养的实验研究

Three- dimensional culture of rabbit bone marrow mesenchymal stem cells in Sr- alginate gels

[目的]观察兔骨髓间充质干细胞(BMSCs)于藻酸锶凝胶中体外三维培养的形态与活性变化,为藻酸锶凝胶复合干细胞应用于骨组织工程提供理论依据。[方法]分离培养原代兔BMSCs并扩增,取第3代BMSCs以细胞密度为5×105个/ml复合于藻酸锶凝胶中三维培养,每日于倒置相差显微镜下观察细胞形态,在不同时间点对其进行Live/Dead染色测细胞存活,CCK-8检测细胞增殖,并用扫描电镜观察凝胶的超微结构及细胞于凝胶上的黏附情况。[结果]藻酸锶凝胶质软、透明、渗透性较好;BMSCs接种于藻酸锶凝胶中初始形态为圆形、呈悬浮生长,后细胞逐渐贴壁生长并呈星形或多角形;接种第1 d和第14 d凝胶中活细胞率分别为(88.61±4.08)%和(92.16±2.10)%,两者间无显著统计学差异(P>0.05);第4、7、10、13 d时二维培养组中OD值显著高于三维培养组(P<0.05);扫描电镜示藻酸锶凝胶呈典型的"鸡蛋箱"样结构,孔隙直径大小平均为150μm;接种于凝胶中7 d后可见细胞均匀黏附于凝胶壁生长。[结论]藻酸锶凝胶是一种优良的细胞支架,将其复合种子细胞应用于骨组织工程具有广阔的前景。

[Objective] To examine the cell morphology and viability of bone marrow stem cells （BMSCs） cultured on strontium （Sr） - crosslinked alginate hydrogel and to provide experimental foundation for using Sr - alginate gel - based 3 - D BMSC cultures in bone tissue engineering. [ Methods ] Primary rabbit BMSCs were isolated and expanded in culture. The third passage BMSCs were seeded into Sr - alginate gels at a density of 5 x 105/ml. In - gel BMSC morphology and growth were ob- served under inverted phase contrast microscope; cell viability and proliferation were analyzed by LIVE/DEAD staining and cell counting kit - 8 （ CCK - 8） colorimetric assay, respectively; and BMSC substrate adhesion and Sr - alginate gel microstructure were observed under a scanning electron microscope. [ Results ] Sr - alginate gel represented a soft and transparent material with good permeability and typical ＂egg box＂ - like structure, with a mean pore diameter of 150 μm. Initially, BMSCs exhibited a spherical morphology because of lack of interactions between the ceils and Sr - alginate gel, but on adherence to the gel, they gradually assumed a polygonal shape. BMSCs fully adhered to the inner wall of Sr - alginate gels 7 days after seeding. Cell viabili- ty at day 1 and 14 was 88. 61% ＋4.08% and 92. 16% ＋2. 10%, respectively （P 〉0. 05） . Cell proliferation at day 4, 7, 10, and 13 was significantly higher in the 2 - D monolayers than in the 3 - D cultures （ P 〈 0. 05 ） . [ Conclusion] Sr - alginate gels represent a promising cell scaffolding matrix that holds potential for use in bone tissue engineering. Key words： mesenchymal stem cells, Sr- alginate gel, tissue engineering