不同孔径CPC材料对大鼠骨髓间充质干细胞增殖的影响

目的：评价不同大小孔径的磷酸钙骨水泥（Calcium phosphate cement,CPC）材料对大鼠骨髓间充质干细胞（Bone mesenchymal stem cells,BMSCs）增殖能力的影响。方法：用盐析法制备三种不同孔径的（200-300μm、300-450μm、450-600μm）CPC材料,利用Micro-CT测量三种材料的平均孔径、孔隙率。无菌条件下取新生大鼠BMSCs原代培养并传代;将三组材料分别放置于24孔板内,每个材料接种5×104个细胞后,37℃、饱和湿度环境下静置培养。于接种后第1、4、7、14、21天用picogreen试剂盒测定细胞增值率;并在第14天、21天戊二醛固定材料并干燥喷金,扫描电镜观察材料表面细胞生长情况。结果：micro-CT测量结果显示：三种CPC材料孔径间相互连通,孔隙率均大于68%,平均孔径分别为235μm、422μm、505μm。细胞在三组材料上均呈对数增长趋势,在第14天到达平台期,在第1天三组细胞数量无明显差异,第4天450-600μm组细胞数量明显高于其余两组（P〈0.05）,在第7天细胞数量随孔径的增加而增加,3组间均有统计学差异（P〈0.05）,第14天和第21天200-300μm组细胞数量明显少于其余两组（P〈0.05）,300-450μm组和450-600μm组间无统计学差异（P〉0.05）。结论：孔径大小可影响大鼠BMSCs在多孔CPC材料上的增殖能力,随着孔径增大,细胞增殖力增高。本研究为进一步研究孔径结构对细胞的影响提供了实验依据。

Effect of different sizes of aperture of calcium phosphate cement scaffolds on cell proliferation of bone marrow mesenchymal stem cells in rats

Objective：To study the effect of CPC material with different pore sizes on cell proliferation.Methods：Primary cul-tured rat bone mesenchymal stem cells were used in the present study.Three kinds of CPC scaffolds with different pore sizes（200-300μm,300-450μm,450-600μm） were divided into three groups.Micro-CT was used to analyze the average pore size and porosity.The three kinds of CPC material were placed in 24-well plates and 5×104 cells were incubated per-well.The cells were har-vested 1,4,7,14 and 21 days after the incubation,and then use the PicoGreen dsDNA Quantitation Reagent kit to test the cell proliferation on the three group of scaffolds.The cell morphology and cell condition were observed by scanning electron microscope.Results：The pore sizes of CPC material used in this study were interconnected.Porosity of the three groups was greater than 68% with the average pore size of 235μm,422μm and 505μm,respectively.The amount of cell in the 3 groups has no significant in day 1.In the 4th day the amount of cell in the 450-600μm group more than that of B and C groups.The number of cell necrosis in A group was significantly more than that of B and C groups.In the 7th day,increase of the pore size,the amount of cell increased gradually.In the 14th and 21th day,the amount of cell in 200-300μm group was significantly less than that the other two groups.Conclusion：The pore size may affect cell proliferation.Along with the increase of the spore size,cell adhesion rate increased gradually.The present study provides the founda-tion for further study on the effect of aperture structure on cell viability.