酸蚀阳极氧化处理钛表面对骨髓间充质干细胞内Wnt信号通路的影响

Titanium surface modification by acidic oxidation treatment activates the Wnt signaling pathway in bone marrow mesenchymal stem cells

背景:课题组前期研究发现采用阳极氧化方法在纯钛表面制备出的二氧化钛纳米管阵列,能显著影响成骨细胞的分化功能。目的:观察酸蚀阳极氧化处理钛表面对骨髓间充质干细胞Wnt信号通路的影响。方法:采用酸蚀和阳极氧化方法在钛表面制备微米级粗糙度表面复合纳米管形貌,设置氧化电压分别为5 V和20 V,分别记为5 V阳极氧化组和20 V阳极氧化组,以抛光的纯钛作为对照组。将原代培养的SD大鼠骨髓间充质干细胞分别接种到对照组、5 V阳极氧化组和20 V阳极氧化组钛表面,接种3 d后,采用实时定量PCR方法检测细胞内经典和非经典Wnt信号通路蛋白的基因表达水平;接种7 d后,采用Western blot方法检测β-catenin蛋白细胞核内表达量。结果与结论:(1)经典Wnt信号蛋白表达:与对照组比较,5 V阳极氧化组、20 V阳极氧化组细胞内Wnt1和Wnt3a表达水平升高(P<0.05);(2)非经典Wnt信号通路蛋白表达:与对照组比较,5 V阳极氧化组、20 V阳极氧化组细胞内Wnt4表达下降(P<0.05);3组细胞内Wnt5a表达无差异;与对照组比较,5 V阳极氧化组细胞内Wnt7a表达下降(P<0.05),20 V阳极氧化组细胞内Wnt7a表达升高(P<0.05);与对照组比较,5 V阳极氧化组、20 V阳极氧化组细胞内Wnt11表达升高(P<0.05);(3)细胞核内β-catenin表达:与对照组比较,5 V阳极氧化组、20 V阳极氧化组细胞核内β-catenin表达明显升高(P<0.05);(4)结果表明:钛表面微米级粗糙度复合纳米管形貌可激活骨髓间充质干细胞内经典Wnt信号通路。

BACKGROUND:Preliminary findings have shown that titanium dioxide nanotube array prepared on the titanium surface by an anodizing method can significantly affect the osteoblast differentiation.OBJECTIVE:To observe the effect of titanium surface modification by acidic oxidation treatment on the Wnt signaling pathway in bone marrow mesenchymal stem cells.METHODS:The micro/nano-structure composed of nanotubes was prepared by acidic oxidation method.The oxidizing voltage was set to 5 V and 20 V.The polished pure titanium was used as control.Primary cultured Sprague-Dawley rat bone marrow mesenchymal stem cells were seeded onto the titanium surface of the three groups.After 3 and 7 days of incubation,real-time quantitative PCR was used to detect the genes levels of canonical Wnt and non-canonical Wnt signaling pathway and western blot method was used to detect(3-catenin expression level in the nuclei,respectively.RESULTS AND CONCLUSION:(1)Canonical Wnt signaling expression:Wnt1 and Wnt3a expression on the micro/nano-structure surfaces significantly increased compared to the control surface(P<0.05).(2)Non-canonical Wnt signaling expression:compared with the control surface,Wnt4 expression decreased on the two kinds of micro/nano structure surfaces(P<0.05);Wnt7a decreased on the small-diameter nanotube(R5)surface(P<0.05),increased on the big-diameter nanotube surface(R20)(P<0.05);Wnt11 elevated on both two micro/nano structure surfaces(P<0.05).Wnt5a expression level had no obvious change in the three groups.(3)(3-catenin expression level in the nuclei significantly increased after titanium surface modification by different acidic oxidation treatments compared to the control surface.These results show that the micro/nano-structure titanium surface activates the canonical Wnt signaling pathway in the bone marrow mesenchymal stem cells.