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

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Journal of Zhejiang University-SCIENCE B Aims and scope Submit manuscript

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.

中文概要

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。

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Authors and Affiliations

  1. Department of General Dentistry, West Branch of Stomatology Hospital, School of Medicine, Zhejiang University, Hangzhou, 310006, China

    Dan-Li Fu & Qiao-hong Jiang

  2. Department of Oral Implantology, Stomatology Hospital, School of Medicine, Zhejiang University, Hangzhou, 310006, China

    Fu-ming He

  3. Department of Prosthodontics, Stomatology Hospital, School of Medicine, Zhejiang University, Hangzhou, 310006, China

    Bai-ping Fu

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Project supported by the Zhejiang Provincial Natural Science Foundation of China (No. LQ13H140001)

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Fu, DL., Jiang, Qh., He, Fm. et al. Adhesion of bone marrow mesenchymal stem cells on porous titanium surfaces with strontium-doped hydroxyapatite coating. J. Zhejiang Univ. Sci. B 18, 778–788 (2017). https://doi.org/10.1631/jzus.B1600517

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