壳聚糖及其复合物对小鼠成骨细胞增殖及分化的影响

Study of the effect of chitosan and its composites on proliferation and differentiation of mouse osteoblasts

目的:用含壳聚糖(chitosan,CS)、Ⅰ型胶原蛋白和重组人骨形态发生蛋白2(recombinant human bone morphogenetic protein-2,rhBMP-2)的培养液体外培养成骨细胞MC3T3-E1,评价3种因素对成骨细胞增殖及分化的影响。方法:实验分为4组,实验组A:CSα-MEM培养基;实验组B:CS+Ⅰ型胶原蛋白溶液的α-MEM培养基;实验组C:CS+Ⅰ型胶原蛋白溶液+rhBMP-2溶液的α-MEM培养基。对照组为含1%FBS的α-MEM培养基。采用MTT法检测加入处理因素后1、3、5、7 d的吸光度(OD)值,并绘制细胞生长曲线,观察成骨细胞的增殖情况。采用碱性磷酸酶活性测定、碱性磷酸酶染色和茜素红钙结节染色观察成骨细胞的分化作用:检测加入处理因素后1、3、5、7 d的碱性磷酸酶活性,并在细胞培养的第7天进行碱性磷酸酶染色,第14天进行茜素红钙结节染色。采用SPSS13.0软件包对所得数据进行单因素方差分析,2组之间比较采用Post Hoc检验。结果:MTT检测结果显示,实验组C的OD值高于其他组,实验组C与其他组间的两两比较具有显著差异(P<0.05)。碱性磷酸酶活性测定结果显示,实验组C的活性高于其他组,实验组C与实验组A、对照组间两两比较具有显著差异(P<0.05),与实验组B两两比较无显著差异(P>0.05)。茜素红染色和碱性磷酸酶染色结果显示,实验组C可见更多的钙盐沉积,且蓝染颗粒多于其他组。结论:壳聚糖、Ⅰ型胶原蛋白和rhBMP-2共同作用,更能促进成骨细胞的增殖与分化。

PURPOSE： A mouse osteoblast cell line, MC3T3-E1, was cultivated in the medium that contained chitosan, type I collagen and recombinant human bone morphogenetic protein-2 in vitro to evaluate the effect of chitosan and its composites on proliferation and differentiation of mouse osteoblasts. METHODS： This study was categorized into 4 groups based on the medium used. Group A： ^-MEM medium; group B： CS, type I collagen and a-MEM medium; group C： CS, type I collagen, rhBMP-2 and a-MEM medium, a-MEM medium containing I%FBS was used in the control group. Cells of each group were cultivated for 1,3,5 and 7 days. The optical density （OD） value at each time point was evaluated with MTI＇ assay and growth curve was drawn to observe the proliferation of osteoblasts. Differentiation of osteoblasts was determined with alkaline phosphatase （ALP） activity assay, alkaline phosphatase staining and alizarin red staining. Alkaline phosphatase activity of each group was measured at day 1, 3, 5 and 7 days. After 7 days of culture, the cells were stained with alkaline phosphatase, and at day 14, the mineralized nodules were stained with alizarin red. Statistical analysis was performed using SPSS13.0 software package. RESULTS： The MTT assay results showed that the OD value was maximal when osteoblasts were cultured in group C. The difference were statistically significant between group C and others （P〈0.05）. The ALP activity showed that the result of group C was significantly higher than other groups. The increase of ALP activity was significant between group C and control group （P〈0.05）. However, no significant difference was found between group C and group B （P〉0.05）. Compared with the control group, group C had more calcium nodules and blue particles than others. CONCLUSIONS： The incorporation of type I collagen and bone morphogenetic protein- 2 into chitosan can promote MC3T3-El cell proliferation and differentiation better. Supported by Major Science and Technology Project of Liaoning Province（2010225001）.