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

doi:10.3969/j.issn.2095-4344.0404

Abstract

Abstract:

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 β-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) β-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.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Dental Implants, Titanium, Nanotubes, Stem Cells, Tissue Engineering

CLC Number:

R394.2

Wang Wei, Du Yuan-hong. Titanium surface modification by acidic oxidation treatment activates the Wnt signaling pathway in bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(1): 13-19.

Figures/Tables 2

2.1 钛表面形貌观察在低倍镜下，对照组表面为平行的抛光划痕，5 V阳极氧化组和20 V阳极氧化组表面为凹凸不平的微米坑形貌。在高倍数显微镜下，5 V阳极氧化组和20 V阳极氧化组表面均匀分布着纳米管阵列，5 V阳极氧化组表面纳米管直径约为30 nm，20 V阳极氧化组表面纳米管直径约为100 nm，对照组表面没有明显的纳米级形貌(图1)。 2.2 骨髓间充质干细胞的形态及生长情况观察在骨髓间充质干细胞培养过程中，采用倒置显微镜观察细胞生长情况及形态，并拍照记录。细胞培养14 d后，骨髓间充质干细胞紧贴培养瓶生长，呈较为均匀的成纤维细胞样，旋涡状或辐射状整齐有序排列(图2A)。使用成脂诱导液诱导成脂分化10 d后，油红O染色可见细胞内及细胞间分布着大小不等的红色脂滴(图2B)。使用成骨诱导液成骨诱导 21 d后，经茜素红染色后可见细胞内形成大小不规则的红色矿化结节(图2C)。 2.3 微米纳米形貌表面骨髓间充质干细胞内经典Wnt信号通路蛋白表达水平骨髓间充质干细胞在3组材料表面培养3 d后，5 V阳极氧化组和20 V阳极氧化组细胞内Wnt1和Wnt3a表达量较对照组明显增高(P < 0.05)，且20 V阳极氧化组表面表达量最高(图3)。5 V阳极氧化组和20 V阳极氧化组Wnt1表达量分别为对照组的2.1和2.5倍，Wnt3a表达量分别为对照组的4.2和5.1倍。 2.4 微米纳米形貌表面骨髓间充质干细胞内非经典Wnt信号通路蛋白表达水平 3组材料表面骨髓间充质干细胞的非经典Wnt信号蛋白表达没有明显规律。与对照组相比，5 V阳极氧化组和20 V阳极氧化组细胞内Wnt4表达量下降(P < 0.05)，分别为对照组的77%和69%；3组细胞内Wnt5a表达无明显差别。与对照组比较，5 V阳极氧化组细胞内Wnt7a表达量下降44%(P < 0.05)；20 V阳极氧化组细胞内Wnt7a表达量升高，为对照组的1.56倍(P < 0.05)。5 V阳极氧化组和20 V阳极氧化组细胞内Wnt11表达水平升高(P < 0.05)，分别是对照组的1.25倍和1.625倍(图4)。 2.5 微米纳米形貌表面骨髓间充质干细胞细胞核内β-catenin蛋白表达水平骨髓间充质干细胞在3组材料表面培养7 d后，5 V阳极氧化组和20 V阳极氧化组细胞核内β-catenin的表达量高于对照组(P < 0.05)。条带灰度半定量分析显示与对照组相比，5 V阳极氧化组表达量升高2.3倍，20 V阳极氧化组升高3.6倍(图5)。"

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