口腔医学研究

口腔医学研究 ›› 2025, Vol. 41 ›› Issue (7): 607-614.DOI: 10.13701/j.cnki.kqyxyj.2025.07.011

• 口腔种植学研究 • 上一篇    下一篇

3D打印钛合金表面氢化TiO2纳米管对人牙龈成纤维细胞生物学行为影响的实验研究

郭雅彤1,2, 张正1,2*, 陈溯3*, 张研1,2   

  1. 1.天津市口腔医院,南开大学医学院 天津 300041;
    2.天津市口腔功能重建重点实验室 天津 300041;
    3.首都医科大学附属北京口腔医院 北京 100050
  • 收稿日期:2025-01-23 出版日期:2025-07-28 发布日期:2025-07-24
  • 通讯作者: *张正,E-mail:zhangzheng@nankai.edu.cn; 陈溯,E-mail:13910164776@163.com
  • 作者简介:郭雅彤(1996~ ),女,河北沧州人,硕士,医师,研究方向:种植体表面改性。
  • 基金资助:
    天津市自然科学基金(编号:23JCYBJC01130); 天津市卫生健康科技项目(编号:TJWJ2023MS034)

Influence of Hydrogenated TiO2 Nanotube of 3D-printed Titanium Alloy on Biological Behavior of Human Gingival Fibroblasts

GUO Yatong1,2, ZHANG Zheng1,2*, CHEN Su3*, ZHANG Yan1,2   

  1. 1. Tianjin Stomatological Hospital, Tianjin 300041, China;
    2. Beijing Stomatological Hospital, Beijing 100050, China;
    3. Capital Medical University School of Stomatology, Beijing Stomatological Hospital, Beijing 100050, China
  • Received:2025-01-23 Online:2025-07-28 Published:2025-07-24

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摘要： 目的: 研究在3D打印钛合金(Ti6Al4V)表面制备氢化TiO2纳米管,材料的表面性能及其对人牙龈成纤维细胞(human gingival fibroblasts,HGFs)生物学行为的影响。方法: 应用选择性激光熔融技术(selective laser melting technology,SLM)制备3D打印钛合金(3D-Ti),并进行电化学阳极氧化在其表面制备TiO2纳米管得到3D-TNTs,对3D-TNTs进一步高温氢化处理,从而获取3D-H2-TNTs。选取光滑的钛合金(MP-Ti)作为对照组,应用场发射扫描电镜(scanning electron microscopy,SEM)、接触角测量仪、表面粗糙度测量仪及原子力显微镜对MP-Ti、3D-Ti、3D-TNTs、3D-H2-TNTs的表面形貌、亲水性、粗糙度进行测量及分析。在4组材料表面培养HGFs,应用SEM观察细胞黏附情况;应用荧光染色、细胞计数试剂盒检测细胞的早期黏附及增殖情况;应用荧光定量聚合酶链反应(fluorescent quantitative polymerase chain reaction,RT-qPCR)和酶联免疫吸附反应(enzyme-linked immunosorbent assay,ELISA)分别检测细胞黏附相关基因及纤维连接蛋白(fibronectin,FN)的表达情况。结果: 3D-Ti的微米钛球表面经过阳极氧化处理,得到了呈放射状、排列规则有序的纳米管结构,经过进一步氢化处理后,材料表面获得了接触角小于5°的超亲水特性。SEM可观察到在培养早期4 h内,HGFs在3D-H2-TNTs表面的形态最为铺展,有更多更长的伪足,且细胞的黏附数量显著高于其他3组(P＜0.05);当培养时间延长至1、3、5 d,HGFs在3D-Ti、MP-Ti、3D-H2-TNTs和3D-TNTs上的增殖呈依次递减趋势,各组间比较均有统计学差异(P＜0.05),3D-H2-TNTs在细胞培养初期促进细胞生长的优势作用在后期逐渐消失;在3D-H2-TNTs表面培养4 h和1 d时,HGFs的黏附相关基因整联蛋白-β1(integrin-beta 1,ITG-β1)、黏着斑激酶(focal adhesion kinase,FAK)、黏着斑蛋白(vinculin,VCL)的表达量及FN分泌量较对照组均明显增多(P＜0.05)。结论: 应用电化学阳极氧化与高温氢化处理相结合的方法,可以在3D打印钛合金表面构建 超亲水性的微纳复合生物层,可促进HGFs的早期黏附和增殖,增强细胞黏附相关基因及纤维连接蛋白的表达。

关键词: 3D打印钛合金, TiO2纳米管, 氢化处理, 人牙龈成纤维细胞

Abstract: Objective: To study the surface properties of 3D-printed titanium alloy (Ti6Al4V) with hydrogenated TiO2 nanotubes (TNTs) prepared by selective laser melting (SLM) technology, and its influence on the biological behavior of human gingival fibroblasts (HGFs). Methods: 3D-printed titanium alloy specimens (3D-Ti) were prepared by SLM technology and subjected to electrochemical anodic oxidation. 3D-TNTs were formed, which were further subjected to high-temperature hydrogenation treatment to obtain 3D-H2-TNTs. Mimic polished titanium alloy (MP-Ti) were used as the control group. The surface morphology, hydrophilicity, and roughness of MP-Ti, 3D-Ti, 3D-TNTs, and 3D-H2-TNTs were measured and analyzed by field emission scanning electron microscopy (SEM), contact angle measurement instrument, surface roughness measurement instrument, and atomic force microscope. HGFs were cultured on the surfaces of the four groups materials, and the cell morphology was observed by SEM. The early adhesion and proliferation of cells were detected by fluorescence staining (DAPI) and cell counting kit-8 (CCK-8). Real-time quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of adhesion-related genes and fibronectin (FN). Results: The surface of the nano-titanium spheres of 3D-Ti was anodized to obtain a radial, regular, and ordered nanotube structure. After further hydrogenation, a superhydrophilic surface with a contact angle less than 5° was obtained. Cell experiments showed that HGFs on the surface of 3D-H2-TNTs were the most spreading, with more and longer pseudopodia, and the number of early cell adhesion was significantly higher than the other three groups (P<0.05) in the early 4 hours of culture. The proliferation of HGFs on 3D-Ti, MP-Ti, 3D-H2-TNTs, and 3D-TNTs decreased gradually when the culture time was extended to 1, 3, 5 days. Compared to the early stage of cell culture, the dominance of 3D-H2-TNTs in promoting cell growth disappeared. The expression of integrin-beta 1 (ITG-β1), focal adhesion kinase (FAK), vinculin (VCL), and FN secretion of HGFs on the 3D-H2-TNTs were significantly increased compared with the control group (P<0.05) at 4 hours and 1 day. Conclusion: The superhydrophilic micro/nano composite morphology can be formed on the surface of 3D printed titanium alloy by electrochemical anodization and high temperature hydrogenation, which can promote the early adhesion and proliferation of HGFs and enhance the expression of adhesion-related genes and fibronectin.

Key words: 3D-printed titanium alloy, TiO2 nanotubes, hydrogenation treatment, human gingival fibroblasts