钛合金表面纳米化对大鼠成骨细胞的影响

The effect of nanoporous surface on titanium alloy on rat osteoblast

目的探讨钛合金表面纳米化对成骨细胞黏附、增殖、分化和成骨矿化能力的影响。方法采用塑性变形和化学处理方法在Ti6Al4V合金上制得一种新型多孔纳米晶体的表面结构。将原代培养的大鼠成骨细胞与纳米表面、光滑表面及粗糙表面钛合金共培养。分别通过细胞计数、扫描电镜、MTT比色法、逆转录聚合酶链反应（reverse transcriptase—polymerase chain reacdon，RT—PCR）分析和钙结节荧光染色等方法观察成骨细胞在3组钛合金表面的黏附、增殖、分化及成骨能力，所得数据进行统计学分析。结果黏附能力实验结果显示早期纳米表面黏附的成骨细胞数多于其它两组（P〈0．05）。扫描电镜观察显示纳米表面上成骨细胞可以较其它两组更早的伸展黏附。增殖能力实验显示，3组细胞的生长曲线大体形态基本相同，但纳米表面成骨细胞的指数增殖期比光滑表面和粗糙表面长3-10d，且最高峰更高。RT—PCR研究表明，共培养14d后，纳米表面黏附的成骨细胞中碱性磷酸酶、Ⅰ型胶原和骨钙素mRNA的转录量均升高（P〈0．05）。另外，与光滑和粗糙表面相比，纳米表面形成钙结节面积显著增大（P〈0．05）。结论纳米多孔表面技术能促进成骨细胞的体外早期黏附、增殖、分化和成骨能力，为纳米技术进一步应用到人工关节提供新的方向。

Objective To evaluate the effect of nanoporous surface of titanium alloy on adhesion, proliferation, differentiation and mineralization of osteoblasts. Methods The present study was to prepare a novel nanostructured surface of Ti6Al4V alloy by the severe plastic deformation （SPD）. Osteoblasts were cultured on nanoporous, smooth and rough titanium alloy in vitro. The adhesion, proliferation, differentiation and mineralization of osteoblasts were observed by cells counting, scanning electron micrography （SEM）, standard MTT assay, reverse transcriptase-polymerase chain reaction （RT-PCR） analysis and fluorescent labeling of calcium deposition. Results The number of cells on the nanoporous titanium alloy was higher than any that of other substrates. The early cellular extension on the nanophase surfaces was observed under SEM. The growth curve of osteoblasts cultured on different surfaces had similar appearance, however, longer exponential phase of growth and higher cell count was observed in osteoblasts cultured on nanoporous surface. Of the examined genes, collagen Ⅰ （COL- Ⅰ ）, alkaline phosphatase（ALP） and ostcocalcin （OC） showed a change in expression with different surfaces. All of them were upregulated on the nanophase surface. In addition, a larger area of calcified nodules on nanophase surface was also observed, compared with the other groups. Conclusion Nanoporous surface technology can improve the early adhesion, proliferation, differential and mineralization of osteoblasts in vitro, which suggests nanometer technology should be further considered for orthopaedic implant applications.