PLGA-[ASP-PEG]三嵌段基质材料对骨髓间充质干细胞黏附增殖及诱导成骨分化的研究

目的：探讨聚乳酸聚乙醇酸共聚物（poly lactic acid-co-glycolic acid,PLGA）-天冬氨酸（asparagic acid,ASP）-聚乙二醇（poly ethylene glycol,PEG）三嵌段多元共聚物上骨髓间充质干细胞（mesenchymal stem cells,MSCs）的黏附、增殖及成骨分化情况。方法：在PLGA支架材料中引入PEG和含有多个功能位点的ASP,制成PLGA-ASP-PEG]三嵌段高分子支架材料。将材料与MSCs复合培养,以未改性的PLGA支架材料作对照,通过沉淀法、MTT法和考马斯亮蓝法分别检测MSCs的黏附和增殖变化。用成骨诱导培养基培养14d和28d,碱性磷酸酶（alkaline phosphatase,ALP）染色和钙结节染色了解MSCs成骨分化情况。结果：MSCs在PLGA-ASP-PEG]材料表面贴壁生长,细胞数目明显多于对照组。细胞黏附率检测显示,PLGA-ASP-PEG]表面MSCs的黏附性能和增殖能力明显高于对照组,差异有统计学意义（P〈0.05）。MTT比色实验显示MSCs在PLGA-ASP-PEG]三嵌段材料上培养20d后,吸光度（A）值为1.336,约为对照组0.780的2倍。培养12d时,PLGA-ASP-PEG]材料组的细胞蛋白含量为66.44μg/孔,对照组为41.23μg/孔。成骨诱导培养基培养后,ALP染色和钙结节染色均为阳性,PLGA-ASP-PEG]三嵌段材料及其降解产物不影响MSCs的成骨分化。结论：PLGA-ASP-PEG]能促进组织工程种子细胞在骨基质材料表面的黏附、增殖,并能较好地保持细胞的形态,对成骨分化无明显影响。

Adhesion,proliferation and osteodifferentiation of bone mesenchymal stem cells on PLGA-[ASP-PEG] tri-bolck polymer scaffolds

Objective：To explore the adhesion,proliferation and osteodifferentiation of bone mesenchymal stem cells （BMSCs）on the prepared lactic acid/glycolic acid/asparagic acid-co-polyethylene glycol（PLGA-ASP-PEG]）tri-block polymer scaffolds. Methods：Modified PLGA with polyethylene glycol （PEG） and asparagic acid（ASP）that has many ligands,and then the synthesis PLGA-ASP-PEG] tri-block polymer material was prepared. BMSCs were cultured in PLGA-ASP-PEG] polymer material and poly lactic acid-co-glycolic acid（PLGA）were used as control group. Precipitation method,MTT assay and total cellular protein detection were used to test the adhersion and proliferation of BMSCs. After the third generation of BMSCs was cultured on PLGA-ASP-PEG]tri-block polymer scaffolds for 14 day and 28 day with osteogenic supplements,the osteodifferentiation of MSCs were observed through alkaline phosphatase（ALP） staining and calcium tubercle staining. Results：BMSCs grew adherent to the surface of PLGA-ASP-PEG] polymer scaffolds and the number of BMSCs was much higher than that of PLGA. The precipitation method suggested that adhesion and proliferation of BMSCs on the surface of PLGA-ASP-PEG] was much higher than the control group（P 〈0.05）. MTT assay showed that after BMSCs were cultured for 20 days,the absorbance A of PLGA-ASP-PEG] polymer scaffolds and PLGA were 1.336 and 0.780 respectively. Total cellular protein could image the adhersion and proliferation of BMSCs indirectly. After BMSCs were cultured for 12 days,the total cellular protein of PLGA-ASP-PEG] and PLGA were 66.44 μg/pore and 41.23 μg/pore respectively. PLGA-ASP-PEG] polymer scaffolds had well biocompatibility and cell adhersion. The positive results with ALP staining and calcium tubercle staining in both groups indicated tri-block polymer scaffold and its degradations had no effect on osteodifferentiation. Conclusion：PLGA-ASP-PEG]could improve the adhesion and proliferation of seed cells on bone-matrixmaterial,maintain the morphous of s