骨髓基质干细胞与聚丙交酯/乙交酯/天冬氨酸/聚乙二醇体外复合培养的实验研究

目的探讨骨髓基质于细胞（marrow stromal stem cells，MSCs）与聚丙交酯／乙交酯／天冬氨酸／聚乙二醇[poly（lactic acid／glycolic acid／asparagic acid—co—polyethylene glycol），PLGA—ASP—PEG]的生物相容性，为构建组织工程骨进行骨缺损修复提供理论基础，以及为组织工程支架材料的优化提供实验依据。方法本体开环共聚法合成PLGA—ASP—PEG三嵌段共聚物。取4周龄新西兰大白兔骨髓，体外分离培养MSCs。将第3代MSCs以1．0×10^6／ml接种到PLGA—ASP—PEG材料上，测定细胞黏附力和黏附率；扫描电镜观察细胞的形态学特征；MTT法检测细胞增殖；流式细胞仪检测细胞周期、增殖指数、DNA指数及凋亡；通过考马斯亮蓝测定法和^3H-脯氨酸掺入实验观察细胞的蛋白合成和胶原合成情况。以PLGA支架材料作为对照。结果MSCs在PLGA—ASP—PEG支架材料上贴附、生长良好，其黏附、增殖和蛋白、胶原合成能力均显著高于PLGA组（P〈0.05），细胞凋亡率显著低于PLGA组（P〈0.05）。DNA指数显示两组细胞均为正常的二倍体细胞。结论PLGA—ASP—PEG的生物学性能与PLGA相比得到显著改善和提高，可作为MSCs的理想载体构建组织工程骨进行骨缺损修复研究。

EXPERIMENTAL STUDIES ON A NEW BONE TISSUE ENGINEERED SCAFFOLD BIOMATERIALS COMBINED WITH CULTURED MARROW STROMAL STEM CELLS IN VITRO

Objective To explore the biocompatibility of poly(lactic acid/glycolic acid/asparagic acid-co-polyethylene glycol) biomaterials (PLGA-ASP-PEG) and biological behaviors of cultured marrow stroml stem cells (MSCs) combined with this new type of scaffold in tissue engineering. Methods The PLGA-ASP-PEG tri-block copolymers were obtained through bulk ring-opening copolymerization method. MSCs were isolated from the bone marrow of 4-week-old New Zealand rabbits. The 3rd-generation MSCs were cultured combining with PLGA-ASP-PEG in vitro, while cells cultured in PLGA as control group. The cell adhesion rate and the adhesive power were examined by conventional precipitation method and micropipette aspiration technique respectively. The morphological features were studied by scanning electron microscope. The proliferation behavior of the cells was analyzed by MTT assay. The cell cycle, proliferation index, DNA index and apoptosis of the cells were detected by flow cytometry. The synthesis of protein and collagen were examined by Coomassie Brilliant Blue dyes and 3H-Proline incorporation test. Results The MSCs adhered and grew well on the surface of the biomaterial PLGA-ASP-PEG. The powers of cell adhesion, proliferation and protein and collagen synthesis of the cells were all significantly higher than those of PLGA group (P<0.05), but the apoptosis rate was significantly lower than that of PLGA group (P<0.05). The DNA indexes showed the cells of both PLGA-ASP-PEG group and PLGA group were normal diploid cells. Conclusion PLGA-ASP-PEG showed good biocompatibility and the biological properties improved greatly compared with the PLGA scaffold materials. These results demonstrated that the promise of PLGA-ASP-PEG can be used as an ideal scaffold material for construction of tissue engineered bone to restore bone defects in bone tissue engineering.