TiO_(2)纳米管表面聚合物辅助沉积Ta_(2)O_(5)涂层对MC3T3-E1细胞生物学活性的影响

Effects of polymer assisted deposition of Ta_(2)O_(5) coating on the surface of TiO_(2) nanotubes on improving the biological activity of MC3T3-E1 cells

目的探讨TiO_(2)纳米管表面聚合物辅助沉积五氧化二钽(Ta_(2)O_(5))涂层对小鼠成骨前体细胞MC3T3-E1生物学活性的影响。方法制备出Ta/TiO_(2)纳米管(Ta/NT组)、Ta_(2)O_(5)/纯钛(Ta_(2)O_(5)/PT组)及Ta_(2)O_(5)/TiO_(2)纳米管(Ta_(2)O_(5)/NT组)3组样本,其中后2组的Ta_(2)O_(5)涂层通过聚合物辅助沉积法制备。对3组样品进行表征检测:扫描电镜(SEM)观察表面形貌,X射线光电子能谱(XPS)分析表面元素组成,X射线衍射(XRD)检测表面化合物。选用MC3T3-E1细胞探讨3组样品对细胞生物学活性影响;荧光显微镜观察细胞骨架和细胞核的黏附;CCK-8法测定细胞活性;检测细胞碱性磷酸酶(ALP)活性;茜素红染色后半定量分析钙沉积;Real-time PCR检测成骨标志基因重组人骨形态发生蛋白-2(BMP-2)、ALP、骨钙蛋白(OCN)和骨桥蛋白(OPN)的基因表达。结果SEM图像显示Ta_(2)O_(5)/NT组表面可见TiO_(2)纳米管基底表面覆盖片状、带状交联的微-纳米级Ta_(2)O_(5)涂层结构;XRD显示Ta_(2)O_(5)/NT组的TiO_(2)纳米管呈现微量的脱矿钛及金红石晶体结构;与Ta/NT组比,Ta_(2)O_(5)/NT组和Ta_(2)O_(5)/PT组黏附细胞核密度较高,细胞骨架块状、丝状结构相对较大,交联、团簇状较多,细胞骨架张力较大,具有较高的细胞活力;茜素红染色样本半定量分析显示Ta_(2)O_(5)/NT组、Ta/NT组、Ta_(2)O_(5)/PT组钙沉积量依次降低(P<0.01);Ta_(2)O_(5)/NT组、Ta/NT组和Ta_(2)O_(5)/PT组7d和14d的ALP活性、钙沉积量和ALP、OCN、OPN mRNA含量依次降低,14 d Ta_(2)O_(5)/NT组的BMP-2 mRNA含量在3组中最高(P<0.05)。结论Ta_(2)O_(5)/TiO_(2)纳米管复合涂层可促进MC3T3-E1细胞黏附、细胞活性、ALP活性、矿化及成骨相关基因的表达,有利于其生物学活性的发挥。

Objective To explore the effect of polymer assisted deposition of tantalum pentoxide(Ta_(2)O_(5))coating on the surface of TiO_(2) nanotubes on the biological activity of mouse bone forming precursor cells,MC3T3-E1 cells.Methods Three sets of samples were prepared,including Ta/TiO_(2)nanotubes(the Ta/NT group),Ta_(2)O_(5)/pure titanium(the Ta_(2)O_(5)/PT group)and Ta_(2)O_(5)/TiO_(2) nanotubes(the Ta_(2)O_(5)/NT group).The Ta_(2)O_(5)/PT group and the Ta_(2)O_(5)/NT group were prepared by polymer-assisted deposition.The three sets of samples were characterized and tested by scanning electron microscopy(SEM)to observe the surface morphology.X-ray photoelectron spectroscopy(XPS)was used to analyze surface element composition,and X-ray diffraction(XRD)was used to detect surface compounds.MC3T3-E1 cells were selected to explore the effect of three groups of samples on cell biological activity.Fluorescence microscope was used to observe the adhesion of cytoskeleton and nucleus.Cell activity was determined by CCK-8 method.Cell alkaline phosphatase(ALP)activity was detected.Calcium deposition was detected by alizarin red staining and semi-quantitative analysis.The expression levels of recombinant human bone morphogenetic protein-2(BMP-2),ALP,osteocalcin(OCN)and osteopontin(OPN)were detected by real-time PCR.Results SEM images showed that on the surface of the Ta_(2)O_(5)/NT group,the TiO_(2) nanotube substrate was covered with a sheet-like,banded cross-linked micro-nano Ta_(2)O_(5) coating structure.XRD showed that the TiO_(2) nanotubes in the Ta_(2)O_(5)/NT group presented a trace of demineralized titanium and rutile crystal structure.Compared with the Ta/NT group,the Ta_(2)O_(5)/NT group and the Ta_(2)O_(5)/PT group showed higher cell density,larger cytoskeletal mass and filamentous structure,more crosslinking and clusters,higher cytoskeletal tension and higher cell activity.The semi-quantitative analysis of alizarin red staining samples showed that calcium deposition decreased successively in the Ta_(2)O_(5)/NT group,the Ta/NT group and the Ta_(2)O_(5)/PT group(P<0.01).The ALP activity,calcium deposition and ALP,OCN and OPN mRNA contents on day 7 and 14 were decreased successively in the Ta_(2)O_(5)/NT group,the Ta/NT group and the Ta_(2)O_(5)/PT group,and the BMP-2 mRNA content on day 14 in the Ta_(2)O_(5)/NT group was the highest in the three groups(P<0.05).Conclusion Ta_(2)O_(5)/TiO_(2) nanotube composite coating can promote the adhesion of MC3T3-E1 cells,the expression of mineralization and osteogenic genes,and enhance the activities of cell and ALP,which is conducive to the play of biological activities.