JZUS - Journal of Zhejiang University SCIENCE

Journal of Zhejiang University SCIENCE B 2017 Vol.18 No.9 P.778-788

Adhesion of bone marrow mesenchymal stem cells on porous titanium surfaces with strontium-doped hydroxyapatite coating

Author(s): Dan-li Fu, Qiao-hong Jiang, Fu-ming He, Bai-ping Fu
Affiliation(s): Department of General Dentistry, West Branch of Stomatology Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China; more
Corresponding email(s): fbp@zju.edu.cn
Key Words: Cell adhesion, Bone marrow mesenchymal stem cell, Strontium-doped hydroxyapatite, Titanium

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Abstract: Objective: To determine the adhesion behavior of bone marrow mesenchymal stem cells (MSCs) on a titanium surface with a nanostructured strontium-doped hydroxyapatite (Sr-HA) coating. Methods: Sr-HA coatings were applied on roughened titanium surfaces using an electrochemical deposition method. Primary cultured rat MSCs were seeded onto Sr-HA-, HA-coated titanium, and roughened titanium surfaces, and they were then cultured for 1, 6, and 24 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the metabolic condition of the cells. Scanning electron microscopy (SEM) was used to observe the cell morphology. The cytoskeletal structure was analyzed using fluorescence actin staining to characterize cell adherence. Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) was used to analyze the gene expression levels of FAK (focal adhesion kinase), vinculin, integrin β1, and integrin β3 after culturing for 24, 48, and 72 h. Results: MSCs cultured on the Sr-HA surface showed better cell proliferation and viability. Improvement of cell adhesion and structural rearrangement of the cytoskeleton were observed on the Sr-HA surface. The gene expression of FAK, vinculin, integrin β1, and integrin β3 was also elevated on the Sr-HA surface. Conclusions: Cell viability, adhesion, cell morphology, and the cytoskeletal structure were all upregulated considerably by the titanium surface modified with a Sr-HA coating.

多孔纯钛表面掺锶羟基磷灰石涂层对骨髓间充质干细胞粘附的影响

目的：观察电化学沉积的纳米结构掺锶羟基磷灰石涂层表面对大鼠骨髓间充质干细胞早期粘附行为的影响。
创新点：首次观察电化学沉积的掺锶羟基磷灰石涂层表面骨髓间充质干细胞的早期粘附行为，并对相关基因进行了检测。
方法：纯钛表面经过喷砂和双重酸处理，形成多孔粗糙结构。用电化学方法在其粗糙表面沉积羟基磷灰石涂层（HA）和掺锶羟基磷灰石涂层（Sr-HA）。用贴壁法将4周大鼠股骨骨髓间充质干细胞分离进行原代培养后将细胞接种到多孔纯钛，HA和Sr-HA表面培养1、6和24小时。用场发射扫描电镜（SEM）观察细胞的形貌特点。用异硫氰酸荧光素（FITC）标记的鬼笔环肽进行免疫荧光染色标记细胞骨架，Hoechst 33258进行细胞核染色，激光共聚焦荧光显微镜进行拍照后使用ImageJ进行细胞的计数和图形分析。用实时荧光定量逆转录聚合酶链式反应（RT-qPCR）测定24、48和72小时不同实验组中骨髓间充质干细胞中FAK、vinculin、integrin β1和integrin β3的基因表达，并进行统计学分析。
结论：SEM观察结果显示，骨髓间充质干细胞在三种表面都能正常黏附、生长和增殖。在Sr-HA组表面，细胞粘附、细胞活力和细胞骨架的铺展都较粗糙表面组和HA组有较显著的提高。RT-qPCR结果显示，在各个时间点Sr-HA组表面骨髓间充质干细胞的FAK、vinculin、integrin β1和integrin β3的基因表达与粗糙组有显著性差异，且在24小时后与HA组亦有显著性差异。综上所述，掺锶羟基磷灰石纳米涂层具有较好的生物相容性，可以促进大鼠骨髓间充质干细胞在纯钛表面的早期粘附。

关键词：细胞粘附；骨髓间充质干细胞；掺锶羟基磷灰石；钛

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多孔纯钛表面掺锶羟基磷灰石涂层对骨髓间充质干细胞粘附的影响

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