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中华口腔医学研究杂志(电子版) ›› 2017, Vol. 11 ›› Issue (02) : 93 -99. doi: 10.3877/cma.j.issn.1674-1366.2017.02.005

所属专题： 文献；

基础研究

钛表面聚合物静电作用固定骨形态发生蛋白2的实验研究
代国荣1, 张新春1, 万文清2, 王焱1,†()   
  1. 1. 510055　广州，中山大学光华口腔医学院·附属口腔医院，广东省口腔医学重点实验室
    2. 510275　广州，中山大学材料科学与工程学院，聚合物复合材料及功能材料教育部重点实验室
  • 收稿日期:2017-02-02 出版日期:2017-04-01
  • 通信作者: 王焱
  • 基金资助:
    国家自然科学基金(81550013、81628005)

The study of bone morphogenetic protein 2 immobilization on titanium surface with polymer electrostatic interaction

Guorong Dai1, Xinchun Zhang1, Wenqing Wan2, Yan Wang1,†()   

  1. 1. Guanghua School of Stomatology, Hospital of Stomatolagy, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
    2. Key Laboratory for Polymeric Composite and Functional Materials of Ministry of Education, School of Material Science and Engineering, Sun Yat-sen University, Guangzhou 510275, China
  • Received:2017-02-02 Published:2017-04-01
  • Corresponding author: Yan Wang
  • About author:
    Corresponding author: Wang Yan, Email:
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引用本文：

代国荣, 张新春, 万文清, 王焱. 钛表面聚合物静电作用固定骨形态发生蛋白2的实验研究[J]. 中华口腔医学研究杂志(电子版), 2017, 11(02): 93-99.

Guorong Dai, Xinchun Zhang, Wenqing Wan, Yan Wang. The study of bone morphogenetic protein 2 immobilization on titanium surface with polymer electrostatic interaction[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2017, 11(02): 93-99.

目的

探索以纳米金颗粒结合静电吸附方法及化学反应将骨形态发生蛋白2（BMP-2）固定于钛（Ti）种植体表面的技术，为钛表面改性提供新的生物处理技术。

方法

利用化学方法合成负电聚合物BPP[BA-Pasp（MEA-DDP）-Pasp（DAP-CA）]。直径10 mm、厚度0.3 mm的钛箔随机分为3组，分别进行处理：（1）纯Ti抛光组；（2）Ti/BPP组，磁控溅射法在纯钛表面喷涂纳米金颗粒，并连接BPP聚合物；（3）Ti/BPP/BMP-2组，在Ti/BPP组基础上通过静电吸附连接BMP-2蛋白。扫描电镜（SEM）观察各组表面形貌，检测表面亲水性、粗糙度和表面电位。荧光标记观察BMP-2连接及分布情况，并比较异硫氰酸荧光素（FITC）-BMP-2与Ti/BPP连接前后荧光强度的变化。Elisa法测试Ti及Ti/BPP表面BMP-2的整合率。单因素方差分析及独立样本t检验对数据进行统计分析，Bonferroni法进行两两比较。

结果

荧光标记和SEM结果均显示BMP-2可有效连接在纯钛表面，FITC-BMP-2与Ti/BPP连接前荧光强度为124.000 ± 0.968，连接后荧光强度为84.060 ± 0.495，差异具有统计学意义（F= 37.727，P<0.001）。以Ti组电势为0时，Ti/BPP组表面电势为-31.2 mV，连接BMP-2蛋白后表面电势明显上升至+9.3 mV。Ti/BPP表面连接BMP-2整合率[（14.883 ± 0.916）%]高于纯Ti抛光表面[（5.990 ± 0.306）%；F= 6.000，P<0.001]。Ti/BPP组与Ti/BPP/BMP-2组表面亲水性明显高于Ti组，而三组表面粗糙度无明显差异。

结论

联合运用纳米金颗粒及负电聚合物可有效将BMP-2固定于纯钛表面。

关键词: 钛, 骨形态发生蛋白2, 纳米粒，金, 静电
Objective

To explore the methods and surface characteristics of bone morphogenetic protein 2 (BMP-2) immobilizcd on titanium (Ti) surface by electrostatic interaction and to offer a new biochemical surface modification technology of Ti surface.

Methods

Pure Ti foils (10 mm in diameter and 0.3 mm in thickness) were randomly divided into 3 groups for the following processing: polished Ti surface (blank control) ; negative polymer on Ti surface after coating gold nanoparticles by magnetron sputtering and connecting polymer BPP (negative control, Ti/BPP) ; the immobilization of BMP-2 protein by electrostatic adsorption and chemical reaction on Ti/BPP (experimental group, Ti/BPP/BMP-2) . Surface morphology, static water contact angle, surface roughness and Zeta potential were observed, respectively. The connection and distribution of BMP-2 was confirmed by fluorescence labeling, and the differences of the fluorescence intensity before and after the linking of fluorescein isothiocyanate (FITC) -BMP-2 on Ti/BPP were tested by fluorescence spectrophotometer. The integration rate of BMP-2 on Ti and Ti/BPP was measured with enzyme linked immunosorbent assay (Elisa) . The data analysis was conducted using One-Way ANOVA and independent sample T-test, while Bonferroni test was used for a multiple comparison procedure.

Results

The results of SEM and fluorescence labeling showed that BMP-2 was successfully grafted on Ti surfaces. The fluorescence intensity of FITC-BMP-2 decreased from 124.000 ± 0.968 to 84.060 ± 0.495 after the reaction with Ti/BPP (F= 37.727, P<0.001) . The surface zeta potential of Ti/BPP was -31.2 mV, while Ti/BPP/BMP-2 was +9.3 mV, when that of Ti was 0 mV. The integration rate of BMP-2 on Ti/BPP (14.883 ± 0.916) %, was significantly higher than that on Ti [ (5.990 ± 0.306)%; F= 6.000, P<0.001]. The hydrophilic property of Ti/BPP and Ti/BPP/BMP-2 group was much higher than that of Ti group, while there was no significant difference in the surface roughness of the three groups.

Conclusion

BMP-2 can be successfully grafted on Titanium surface by electrostatic adsorption and chemical reaction using negative polymer BPP.

Key words: Titanium, Bone morphogenetic protein 2, Nanoparticles, gold, Static electricity
图1 负电巯基聚合物（BPP）的合成过程
图2 钛试件分组及处理示意图
图3 各组钛试件表面形貌SEM图
表1 各组钛试件表面静态水接触角值（°，±s）
组别 试件数 水接触角
Ti组 5 67.2 ± 0.4a
Ti/BPP组 5 32.2 ± 1.1b
Ti/BPP/BMP-2组 5 28.8 ± 1.1b
F值 - 307.753
P值 - <0.001
图4 各组钛试件表面静态水接触角值
表2 各组钛试件表面粗糙度值（μm，±s）
组别 试件数 Ra Rp
Ti组 5 0.377 ± 0.023 3.3 ± 0.5
Ti/BPP组 5 0.358 ± 0.024 3.0 ± 0.4
Ti/BPP/BMP-2组 5 0.384 ± 0.031 3.7 ± 0.4
F值 - 0.829 0.269
P值 - 0.481 0.773
图5 激光共聚焦显微镜下观察各组钛试件三维重建图像
图6 激光共聚焦测量各组钛试件表面粗糙度值
图7 固体表面电位仪测定各组钛试件表面电势
图8 FITC-BMP-2在Ti/BPP试件上的荧光分布（× 200）
图9 FITC-BMP-2与Ti/BPP试件连接前后的荧光强度的变化
图10 试件Ti与Ti/BPP表面BMP-2整合率
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