凹凸棒石/Ⅰ型胶原/聚乙烯醇复合支架材料制备与表征及体外成骨诱导性能研究

Preparation,Characterization and Osteogenic lnduction Properties of Attapulgite/Ⅰ Collagen/Polyvinyl Alcohol Composite Scaffold

为比较不同比例凹凸棒石(ATP)复合支架材料的理化特征及其体外成骨诱导性能,并筛选出复合材料中最佳的ATP含量,采用冷冻干燥法制备0%～70%八个比例的凹凸棒石/Ⅰ型胶原/聚乙烯醇(ATP/ColⅠ/PVA)复合支架材料。通过宏观和SEM (Scanning electron microscope)观察不同材料的形貌特征;采用干湿质量法检测材料的吸水膨胀率、孔隙率、透气性;使用生物微力试验机检测复合支架材料弹性模量;利用CCK-8法检测细胞在支架材料中的增殖;运用Real-time PCR检测间充质干细胞(MSCs)与支架材料复合培养7d、14d和21d时与成骨细胞分化相关基因Runx-2、Osterix、OPN、OC和ALP mRNA表达的影响。扫描电镜结果表明,随着ATP含量的增加,复合支架孔径增加;在测定物理性质时,透气性20%～60%ATP相比于0%ATP下降极其显著(P<0.01);吸水膨胀率30%、40%、60%、70%ATP相比于0%ATP上升极显著(P<0.01);20%、30%、40%ATP相比于0%ATP弹性模量增加极显著(P<0.01);孔隙率40%ATP与0%ATP相比下降显著(P<0.05)。在CCK-8检测结果中ATP含量在30%、40%的支架材料上增殖率显著高于其他六组材料。Real-time PCR检测结果显示,30%、40%ATP含量与其他组相比,OPN、OC表达量明显升高,10%～50%ATP支架材料Runx-2、ALP、Osterix随时间延长和ATP含量增加而上调,60%、70%ATP含量的支架材料各基因含量缓慢增长。30%、40%ATP支架材料具有合适的孔隙率、良好的吸水膨胀性能和弹性模量,细胞在支架上增殖率最佳,且成骨诱导性能良好。

In order to compare the physical and chemical characteristics of different attapulgite composite scaffold materials and their in vitro osteogenic induction properties,and selected the best ATP content in the composites.Eight different proportions of attapulgite/typeⅠcollagen/polyvinyl alcohol(ATP/ColⅠ/PVA)composite scaffolds were prepared by freeze-drying method and the morphology characteristics of different materials were tested through macroscopic and scanning electron microscope(SEM).The absorbing water expansion rate,porosity,and gas permeability of the material were tested by dry-wet mass method.Utilizing the bio-micro force testing machine to test the elastic modulus of the composite scaffold.Using CCK-8 method to detect the proliferation of cells in the scaffold materials.Using Real-time PCR,the expression of relative bone-related gene including Runx-2,OPN,OC,Osterix and ALP extracted from mesenchymal stem cells(MSCs)cultured in different proportion composite scaffolds at individual 7 d,14 d,21 d was measured.The results of scanning electron microscopy showed that the pore size of the composite scaffold increased with the increase of ATP content.When the physical properties were measured,the permeability of 20%-60%ATP was significantly lower than that of 0%ATP(P<0.01).The absorbing water expansion rate of 30%,40%,60%,70%ATP went up significantly compared to 0%ATP(P<0.01).The modulus of elasticity of 20%,30%,40%went up very significantly compared with 0%ATP(P<0.01).The porosity of 40%ATP decreased significantly compared with 0%ATP(P<0.05).The result of CCK-8 suggested 30%,40%were the best proportion of attapulgite,which showed the significant proliferation compared with others.The statistics of Real-time PCR results showed that the expression of OC,OPN of 30%,40%were higher than that of the other six groups,the expression of Runx-2,ALP,Osterix of 10%-50%ATP grew with time and the increasing dose of attapultige.Increa-singly,the gene content of the scaffold materials with 60%and 70%ATP content increased slowly.with the suitable porosity,favorable water absorption property and evaluate modulus of elasticity,the optimum cell proliferation and osteoinductivity,the 30%,40%were the potential proportion scaffolds.