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中华临床医师杂志(电子版)

中华临床医师杂志(电子版) ›› 2018, Vol. 12 ›› Issue (02) : 98 -102. doi: 10.3877/cma.j.issn.1674-0785.2018.02.008

所属专题： 文献；

基础研究

亲水性钛表面微纳米形貌对大鼠骨髓间充质干细胞增殖与成骨分化的影响
周敏1, 武东辉1, 吴亚霖2, 庞静雯1, 庄秀妹3,†()   
  1. 1. 510220　广州市海珠区口腔医院
    2. 510040　广州，至壹口腔门诊部
    3. 510120　广州，中山大学附属孙逸仙纪念医院口腔科
  • 收稿日期:2017-09-21 出版日期:2018-01-15
  • 通信作者: 庄秀妹
  • 基金资助:
    国家自然科学基金青年基金项目(81600899)

Hydrophilicity of titanium with micro/nanotopographical surface promotes proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells

Min Zhou1, Donghui Wu1, Yalin Wu2, Jingwen Pang1, Xiumei Zhuang3,†()   

  1. 1. Guangzhou Haizhu District Hospital of Stomatology, Guangzhou 510220, China
    2. Top One Dental Clinic, Guangzhou 510040, China
    3. Department of Stomatology, Sun Yat-sen Memorial Hospital Affiliated to Sun Yat-sen University, Guangzhou 510120, China
  • Received:2017-09-21 Published:2018-01-15
  • Corresponding author: Xiumei Zhuang
  • About author:
    Corresponding author: ZHUANG Xiu-mei, E-mail:
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引用本文：

周敏, 武东辉, 吴亚霖, 庞静雯, 庄秀妹. 亲水性钛表面微纳米形貌对大鼠骨髓间充质干细胞增殖与成骨分化的影响[J]. 中华临床医师杂志(电子版), 2018, 12(02): 98-102.

Min Zhou, Donghui Wu, Yalin Wu, Jingwen Pang, Xiumei Zhuang. Hydrophilicity of titanium with micro/nanotopographical surface promotes proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells[J]. Chinese Journal of Clinicians(Electronic Edition), 2018, 12(02): 98-102.

目的

探讨亲水性钛表面微纳米形貌对大鼠骨髓间充质干细胞（rBMSCs）增殖与成骨分化的影响。

方法

采用阳极氧化法和喷砂碱热法分别构建微纳米形貌钛表面，检测其亲水性。钛片表面接种rBMSCs，采用细胞计数试剂盒-8（CCK8）法在培养1、3、5、7 d时检测rBMSCs细胞活性；第7天和14天时检测总蛋白浓度与碱性磷酸酶（ALP）活性，并在第7天时检测成骨标志物ALP、I型胶原（COL1）与成骨特异性转录因子2（RUNX2）的mRNA表达水平。采用t检验比较2组水静态接触角、CCK-8、总蛋白浓度以及成骨分化PCR检测指标。

结果

阳极氧化组钛表面呈规则有序纳米管阵列，喷砂碱热组呈三维网状纳米多孔结构，二者具有相似的微纳米形貌，阳极氧化组水静态接触角大于喷砂碱热阻[（83.3±2.3）° vs （47.7±2.0）°]，差异具有统计学意义（t=11.54，P<0.001）。相比阳极氧化组，喷砂碱热组rBMSCs细胞增殖能力均有所增强，接种3、5、7 d后喷砂碱热组rBMSCs细胞活性均明显高于阳极氧化组（0.66 ±0.03 vs 0.52 ±0.03；1.15 ±0.06 vs 0.85 ±0.05；1.58 ±0.07 vs 1.26 ±0.07），且差异具有统计学意义（t=2.962、3.845、3.183，P=0.042、0.018、0.033）；培养7、14 d后，喷砂碱热组rBMSCs的总蛋白浓度高于阳极氧化组[（389±45）μg/ml vs（226±32）μg/ml；（1070±59）μg/ml vs （760±65）μg/ml]，差异具有统计学意义（t=3.319、3.518，P=0.029、0.025）；第7、14天时喷砂碱热组钛表面rBMSCs的ALP活性高于阳极氧化组[（2.11±0.32）U/gprot vs （1.00±0.21）U/gprot；（6.13±0.57）U/gprot vs （3.92±0.51）U/gprot]，差异具有统计学意义（t=2.912、2.976，P=0.043、0.041）；第7天时，喷砂碱热组rBMSCs中ALP、COL1与RUNX2的mRNA表达水平均高于阳极氧化组（1.86 ±0.24 vs 1.00 ±0.15；2.05 ±0.16 vs 1.00 ±0.14；2.28 ±0.18 vs 1.00 ±0.12），差异具有统计学差异（t=3.383、5.012、5.710，P=0.028、0.007、0.005）。

结论

相比阳极氧化组，喷砂碱热组钛表面微纳米形貌具有更强的促rBMSCs增殖与成骨分化能力，该过程可能与其良好的亲水性有关。

关键词: 大鼠骨髓间充质干细胞, 成骨分化, 微纳米形貌, 亲水性
Objective

To investigate the effect of hydrophilicity of titanium with micro/nanotopographical surface on the cell viability and osteogenic differentiation in rat bone marrow mesenchymal stem cells (rBMSCs).

Methods

Methods of anodic oxidation and sandblast-alkali heat were utilized to establish scale structures. rBMSCs were seeded on these two titanium discs, cell viability was detected at 1, 3, 5 and 7 days by CCK8 assay. Total protein values and alkaline phosphatase (ALP) activity were examined at 7 and 14 days. ALP, collagen-I(COL1) and runt related transcription factor 2 (RUNX2) mRNA expression at 7 days were detected by qRT-PCR. The t test was used to compare water static contact angle, CCK8, total protein concentration and PCR detection index of osteogenic differentiation.

Results

The titanium surface treated by anodic oxidation was regular ordered nanotube arrays, while that treated by sandblast-alkali heat exhibited a three dimensional mesh nano porous structure, which both groups presented a similar micro-nanotopographical feature. However, the static contact angle of water in anodic oxidation group was significantly enhanced than that of sandblast-alkali heat group (83.3±2.3 vs 47.7±2.0, t=11.54, P<0.001). Compared with anodic oxidation group, rBMSCs on the discs modified by sandblast-alkali heat showed enhanced cell viability at 3, 5 and 7 days (0.66 ±0.03 vs 0.52 ±0.03, 1.15 ±0.06 vs 0.85 ±0.05, 1.58 ±0.07 vs 1.26 ±0.07), and these differences were statistically significant (t=2.962、3.845、3.183, P=0.042、0.018、0.033). After 7 and 14 days, the total protein concentration of rBMSCs in sandblasting alkali heat group was higher than that of anodic oxidation group [(389±45) μg/ml vs (226±32) μg/ml, (1070±59) μg/ml vs (760±65) μg/ml], whose differences were statistically significant (t=3.319、3.518, P=0.029、0.025). Moreover, ALP activity of rBMSCs of sandblasting alkali heat group at 7 days and 14 days was higher than that of anodic oxidation group [(2.11±0.32) U/gprot vs (1.00±0.21) U/gprot, (6.13±0.57) U/gprot vs (3.92±0.51) U/gprot], which showed statistically significant difference (t=2.912、2.976, P=0.043、0.041). Finally, mRNA levels of ALP, COL1 and RUNX2 in rBMSCs of sandblasting alkali heat group is higher than that of anodic oxidation group at 7 days (1.86 ±0.24 vs 1.00 ±0.15; 2.05 ±0.16 vs 1.00 ±0.14; 2.28 ±0.18 vs 1.00 ±0.12), and these difference was statistically significant (t=3.383、5.012、5.710, P=0.028、0.007、0.005).

Conclusion

Micro-nanotopographical surface of titanium implant in sandblast-alkali heat group promotes cell viability and osteogenic differentiation of rBMSCs, which may be affected by its hydrophilicity.

Key words: Rat bone marrow mesenchymal stem cells, Osteogenic differentiation, Micro-nanotopographical surface, Hydrophilicity
图1~2 钛表面形貌特点（电镜，×30 000）　图1为阳极氧化组，图2为喷砂碱热组
图3~4 2组钛表面的亲水性比较　图3为阳极氧化组，图4为喷砂碱热组
表1 阳极氧化组与喷砂碱热组不同时间钛表面rBMSCs的吸光度值（±s）
组别 第1天 第3天 第5天 第7天
阳极氧化组 0.36±0.04 0.52±0.03 0.85±0.05 1.26±0.07
喷砂碱热组 0.39±0.04 0.66±0.03 1.15±0.06 1.58±0.07
t值 0.567 2.962 3.845 3.183
P值 0.601 0.042 0.018 0.033
表2 阳极氧化组与喷砂碱热组培养7、14 d后钛表面rBMSCs总蛋白浓度（μg/ml，±s）
组别 第7天 第14天
阳极氧化组 226±32 760±65
喷砂碱热组 389±45 1070±59
t值 3.319 3.518
P值 0.029 0.025
表3 阳极氧化组与喷砂碱热组钛表面rBMSCs的ALP、COL1与RUNX2表达（±s）
组别 ALP COL1 RUNX2
阳极氧化组 1.00±0.15 1.00±0.14 1.00±0.12
喷砂碱热组 1.86±0.24 2.05±0.16 2.28±0.18
t值 3.383 5.012 5.710
P值 0.028 0.007 0.005
1
Liang J, Xu S, Shen M, et al. Osteogenic activity of titanium surfaces with hierarchical micro-/nano-structures obtained by hydrofluoric acid treatment [J]. Int J Nanomedicine, 2017, 12: 1317-1328.
2
Zhang W, Cao H, Zhang X, et al. A strontium-incorporated nanoporous titanium implant surface for rapid osseointegration [J]. Nanoscale, 2016, 8(9): 5291-5301.
3
Zhuang XM, Zhou B, Ouyang JL, et al. Enhanced MC3T3-E1 preosteoblast response and bone formation on the addition of nano-needle and nano-porous features to microtopographical titanium surfaces [J]. Biomed Mater, 2014, 9(4): 45001.
4
Lotz EM, Olivares-Navarrete R, Berner S, et al. Osteogenic response of human MSCs and osteoblasts to hydrophilic and hydrophobic nanostructured titanium implant surfaces [J]. J Biomed Mater Res A, 2016, 104(12): 3137-3148.
5
Alfarsi MA, Hamlet SM, Ivanovski S. Titanium surface hydrophilicity enhances platelet activation [J]. Dent Mater J, 2014, 33(6): 749-756.
6
Kopf BS, Ruch S, Berner S, et al. The role of nanostructures and hydrophilicity in osseointegration: In-vitro protein-adsorption and blood-interaction studies [J]. J Biomed Mater Res A, 2015, 103(8): 2661-2672.
7
Dohan ED, Vazquez L, Park YJ, et al. Identification card and codification of the chemical and morphological characteristics of 14 dental implant surfaces [J]. J Oral Implantol, 2011, 37(5): 525-542.
8
庄秀妹, 邓飞龙. 钛表面及其涂层纳米化对骨结合的影响和机制 [J]. 国际口腔医学杂志, 2014, 41(4): 427-430.
9
Wennerberg A, Jimbo R, Stubinger S, et al. Nanostructures and hydrophilicity influence osseointegration: a biomechanical study in the rabbit tibia [J]. Clin Oral Implants Res, 2014, 25(9): 1041-1050.
10
Lotz EM, Olivares-Navarrete R, Hyzy SL, et al. Comparable responses of osteoblast lineage cells to microstructured hydrophilic titanium-zirconium and microstructured hydrophilic titanium [J]. Clin Oral Implants Res, 2016, 28(7): e51-e59.
11
Hong YS, Kim MJ, Han JS, et al. Effects of hydrophilicity and fluoride surface modifications to titanium dental implants on early osseointegration: an in vivo study [J]. Implant Dent, 2014, 23(5): 529-533.
12
杜娟, 姜焕焕, 莫嘉骥, 等. 钛表面形貌和亲水性表面对成骨细胞增殖、分化的影响 [J]. 中国口腔颌面外科杂志, 2012,10(3): 182-187.
13
姜焕焕, 刘鑫, 乔士冲, 等. 亲水性纯钛表面对成骨细胞黏附行为的影响 [J]. 中国口腔颌面外科杂志, 2011, 9(1): 18-22.
14
Hotchkiss KM, Reddy GB, Hyzy SL, et al. Titanium surface characteristics, including topography and wettability, alter macrophage activation [J]. Acta Biomater, 2016, 31: 425-434.
15
Alfarsi MA, Hamlet SM, Ivanovski S. Titanium surface hydrophilicity modulates the human macrophage inflammatory cytokine response [J]. J Biomed Mater Res A, 2014, 102(1): 60-67.
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