口腔疾病防治 ›› 2021, Vol. 29 ›› Issue (4): 226-233.DOI: 10.12016/j.issn.2096-1456.2021.04.002

• 基础研究 • 上一篇    下一篇

新型钛表面微纳米共存梯度仿生结构对骨髓间充质细胞黏附、增殖及成骨分化的影响

王旻(),姜楠,祝颂松()   

  1. 口腔疾病研究国家重点实验室 国家口腔疾病临床医学研究中心 四川大学华西口腔医院口腔医院颌面外科,四川 成都(610041)
  • 收稿日期:2020-08-24 修回日期:2020-10-19 出版日期:2021-04-20 发布日期:2021-02-26
  • 通讯作者: 祝颂松
  • 作者简介:王旻,医师,硕士研究生在读,Email:15882167039@163.com
  • 基金资助:
    国家自然科学基金(81901026)

A novel biomimetic micro/nano hierarchical interface of titanium enhances adhesion, proliferation and osteogenic differentiation of bone marrow mesenchymal cells

WANG Min(),JIANG Nan,ZHU Songsong()   

  1. State Key Laboratory Oral Diseases, National Clinical Reasearch Center for oral Diseases, Department of oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
  • Received:2020-08-24 Revised:2020-10-19 Online:2021-04-20 Published:2021-02-26
  • Contact: Songsong ZHU
  • Supported by:
    National Natural Science Foundation of China(81901026)

摘要:

目的 制备一种新型微纳米共存梯度仿生表面结构,并探究其对骨髓间充质细胞生物学活性的影响。方法 通过放电等离子烧结法和阳极氧化处理分别在纯钛表面(纯钛组)制备微米骨小梁样结构(微米骨小梁组)和TiO2纳米管形貌(TiO2纳米管组);再通过阳极氧化法在微米骨小梁样结构上制备TiO2纳米管结构,形成新型微纳米共存梯度仿生表面结构,命名为微纳米复合钛组。应用扫描电子显微镜(scanning electronmicroscopy,SEM)和原子力显微镜(atomic force microscopy,AFM)、接触角(contact angle,CA)测量对纯钛组、微米骨小梁组、TiO2纳米管组和微纳米复合钛组进行表征观察。将大鼠骨髓间充质细胞(bone marrow mesenchymal cells,BMMCs)接种于4组材料上,SEM观察细胞黏附情况;MTT法检测细胞在材料表面增殖能力;碱性磷酸酶(alkaline phosphatase,ALP)活性检测细胞在材料表面分化能力;共聚焦显微镜(confocal laser scanning microscope,CLSM)下观察细胞黏附、免疫荧光检测相关蛋白肌动蛋白(F-actin)、黏着斑蛋白(vinculin)、骨钙素(osteocalcin,OCN)、骨桥蛋白(osteopontin,OPN)表达;qRT-PCR观察细胞成骨转录因子RUNX2、OCN、OPN、I型胶原(collagen I,COLI)表达情况。结果 微纳米复合钛组表面亲水性最好(CA为9° ± 2.1°);MTT结果示5~9 d,微纳米复合钛组和TiO2纳米管组的细胞增殖活性显著高于纯钛组和微米骨小梁组(P<0.001);ALP结果示14 d时,微纳米复合钛组表面ALP活性最高;CLSM结果示培养24 h后,微纳米复合钛组肌动蛋白(F-actin)染色最深;培养72 h后,微纳米复合钛组OCN、OPN表达强于微米骨小梁组和TiO2纳米管组。qRT-PCR结果示TiO2纳米管组和微纳米复合钛组表面细胞所有成骨转录因子RUNX2、OCN、OPN、COLI的基因表达水平强于纯钛组和微米骨小梁组,其中I型胶原COLI基因表达水平差异有统计学意义(P<0.001)。结论 微纳米复合钛这种新型微纳米共存梯度仿生表面结构能有效促进骨髓间充质细胞黏附、增殖及成骨向分化。

关键词: 微纳米结构, TiO2纳米管, 仿生, 表面处理, 种植体, 骨髓间充质细胞, 细胞黏附, 细胞增殖, 成骨分化, 放电等离子烧结, 阳极氧化处理

Abstract:

Objective To design a novel biomimetic micro/nano hierarchical interface on endosseous titanium implants and investigate its effect on the biological activity of bone marrow mesenchymal cells. Methods Electrochemical anodization and spark plasma sintering were used to modify smooth titanium (untreated Ti group) with a microporous trabecular bone-like architecture (micro-Ti group) and TiO2 nanotube architecture (nano-TiO2 group). Additionally, electrochemical anodization was employed to prepare TiO2 nanotubes on microporous trabecular bone-like architectures, which formed a novel biomimetic hierarchical interface (micro/nano-TiO2 group). Four groups of titanium samples were characterized by field emission scanning electron microscopy (SEM), atomic force microscopy (AFM) and contact angle (CA). Bone marrow mesenchymal cells (BMMCs) were seeded on four groups of titanium samples. Scanning electron microscopy (SEM) was employed to observe cell morphology. Cell proliferation was determined by MTT assay. The expression of focal adhesion proteins (F-actin; vinculin; osteocalcin, OCN; osteopontin, OPN) were observed under a confocal laser scanning microscope (CLSM). The mRNA expression levels of osteogenic factors (runt-related transcription factor 2, RUNX2; osteocalcin, OCN; osteopontin, OPN; collagen I, COL I) were assessed by qRT-PCR. Results The micro/nano- TiO2 group featured a hydrophilic surface (CA=9° ± 2.1°). The results of the MTT assay indicated that the relative cell proliferation rates for the nano- TiO2 and micro/nano-TiO2 samples were significantly increased compared with those for the untreated-Ti and micro-Ti samples (P<0.001) after 5-9 days. The ALP results indicated that the micro/nano-TiO2 sample gained the highest value at 14 days. After 72 h of incubation, the expression of osteocalcin (OCN) and osteopontin (OPN) on micro/nano-TiO2 was the strongest. After 24 h incubation, the expression of F-actin on micro/nano-TiO2 was the strongest. In comparison with untreated-Ti and micro-Ti samples,the mRNA expression levels of all the osteogenic factors (runt-related transcription factor 2, RUNX2; osteocalcin, OCN; osteopontin, OPN; Collagen I, COL I) were markedly increased on the nano-TiO2 and micro/nano-TiO2 samples, the mRNA expression levels of collagen I (COL I) were significantly different between the nano-TiO2 and micro/nano-TiO2 samples versus the untreated-Ti and micro-Ti samples (P<0.001). Conclusion The novel biomimetic micro/nano hierarchical interface has a positive effect on cell attachment, viability and osteogenic differentiation of bone marrow mesenchymal cells.

Key words: micro/nano structure, TiO2 nano-tubes, bionic, surface treatment, implants, bone marrow mesenchymal cells, cell adhesion, cell proliferation, osteogenic differentiation, spark plasma sintering, electrochemical anodization

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