3D电喷印法制备凹凸棒石/聚己内酯支架及体外成骨诱导性能

Three-dimensional electrohydrodynamic printing of attapulgite/polycaprolactone scaffolds and osteogenic differentiation ability in vitro

背景:静电纺丝制备的聚己内酯纳米纤维支架具有仿细胞外基质的结构,但其亲水性较差,不利于细胞的黏附、迁移、增殖和分化。目的:利用3D电喷印法制备凹凸棒石/聚己内酯支架,评价其对小鼠骨髓间充质干细胞的生物学影响及成骨诱导分化作用。方法:采用3D电喷印法制备含不同质量比例凹凸棒石(0%,0.5%,1%,2%)的凹凸棒石/聚己内酯支架,扫描电镜观察其形貌结构。将4组支架分别与骨髓间充质干细胞共培养,第7天时扫描电镜下观察细胞黏附,第2天时Live/Dead染色观察材料表面细胞生长状态,第1,3,5天时利用CCK-8法检测细胞增殖,第3,7,14天时采用Real-time PCR检测成骨标志基因的表达水平,第3,7,10天时检测细胞碱性磷酸酶活性。结果与结论:①扫描电镜显示,4组支架均呈多孔隙的网状结构,平均孔隙约450μm,其中0%,0.5%凹凸棒石组支架表面光滑,1%,2%凹凸棒石组支架表面粗糙;②扫描电镜显示,细胞能在4组材料表面黏附和生长,其中1%凹凸棒石组细胞数量多于其他3组;③Live/Dead染色显示,细胞在4组材料表面黏附和生长,其中2%凹凸棒石组细胞数量最少;④CCK-8检测结果显示,0.5%凹凸棒石组的细胞增殖速率快于其他3组;⑤Real-time PCR检测显示,0.5%凹凸棒石组共培养14 d的碱性磷酸酶、骨钙素、Osterix和Runx-2基因表达量均高于其他3组;⑥0.5%,1%,2%凹凸棒石组的碱性磷酸酶活性均不同程度地高于0%凹凸棒石组,共培养3,7 d时0.5%凹凸棒石组最高,共培养10 d时1%,2%凹凸棒石组最高;⑦结果表明,加入质量比例0.5%凹凸棒石的聚己内酯支架具有良好的生物相容性和一定的成骨诱导效应。

BACKGROUND:The polycaprolactone nanofiber scaffolds prepared by electrostatic spinning have a structure resembling extracellular matrix,but it is not conducive to cell adhesion,migration,proliferation and differentiation due to the poor hydrophilicity.OBJECTIVE:To fabricate attapulgite/polycaprolactone scaffolds via three-dimensional(3D)electrohydrodynamic printing and evaluate the biological effects and osteogenic induction ability on mouse bone marrow mesenchymal stem cells.METHODS:Attapulgite/polycaprolactone scaffolds with different concentration of attapulgite(0%,0.5%,1%,2%)were prepared by 3D electrohydrodynamic,and the morphology and structure were observed by scanning electron microscope.The mouse bone marrow mesenchymal stem cells were co-cultured with four groups of scaffolds.The growth and morphology of bone marrow mesenchymal stem cells were observed with scanning electron microscope and Live/Dead staining at 7 and 2 days,respectively.The proliferation of bone marrow mesenchymal stem cells was detected by CCK-8 assay at 1,3 and 5 days.The mRNA expression levels of osteogenic marker genes were detected by real-time PCR at 3,7,and 14 days.Alkaline phosphatase activity was detected at 3,7 and 10 days of co-culture.RESULTS AND CONCLUSION:(1)Scanning electron microscopy results showed that four groups of scaffolds presented macropore morphology with the pore size of about 450μm,and the surfaces of 0% and 0.5% attapulgite were smooth,while the scaffold surfaces in 1% and 2% attapulgite groups were rough.(2)Scanning electron microscopy showed that cells adhered and grew well on the surface of these scaffolds,and there were more cells grow in 1% attapulgite group than in other groups.(3)Live/Dead staining showed that cells adhered and grew well on the surface of four groups of scaffolds,among which 2%attapulgite group had the least cells.(4)The results of CCK-8 assay showed that the cell proliferation rate of 0.5%attapulgite group was higher than that of the other groups.(5)Real-time PCR showed that the expressions of alkaline phosphatase,osteocalcin,osteoblast-specific transcription factor and Runx-2 genes in 0.5%attapulgite group for 14 days were higher than those in the other groups.(6)The alkaline phosphatase activities of 0.5%,1% and 2% attapulgite groups were all higher than 0% attapulgite group,in which the 0.5% attapulgite group was the highest at 3 and 7 days but 1% and 2% attapulgite groups were higher than other groups at 10 days of co-culture.(7)The 0.5% attapulgite scaffold has better biocompatibility and osteogenic induction potential.