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

doi:10.12016/j.issn.2096-1456.2021.04.002

Abstract

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

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

CLC Number:

WANG Min,JIANG Nan,ZHU Songsong. A novel biomimetic micro/nano hierarchical interface of titanium enhances adhesion, proliferation and osteogenic differentiation of bone marrow mesenchymal cells[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2021, 29(4): 226-233.

王旻,姜楠,祝颂松. 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[J]. 口腔疾病防治, 2021, 29(4): 226-233.

Figures/Tables 10

Table 1 Specific prime sequences of PCR

Gene	Forward primer 5′-3′	Reverse primer 5′-3′
COL I	GCTGGCAAGAATGGCGAC	AGCCACGATGACCCTTTATG
OCN	GGAGGGCAGTAAGGTGGTGA	ACGGTGGTGCCATAGATGC
OPN	AACAGTATCCCGATGCCACA	TGGCTGGTCTTCCCGTTG
RUNX2	CAGGCGTATTTCAGATGATGACA	TAAGTGAAGGTGGCTGGATAGTG
Actin	CCCATCTATGAGGGTTACGC	TTTAATGTCACGCACGATTTC

Figure 1 SEM images of the surface of titanium samples a: untreated-Ti (× 1 000); b: microporous trabecular bone-like architecture (×1 000). It resembled a trabecular bone; c: TiO2 nano-pores arranged uniformly (× 80 000); d: TiO2 nano-pores arranged uniformly (× 160 000); SEM: scanning electronmicroscopy

Figure 2 AFM images of samples in the untreated-Ti group and nano-TiO2 group a: untreated-Ti group, which showed a relatively rough surface; b: nano-TiO2 group; nano-pores (diameter: 50-120 nm) were observed on its surface; AFM: atomic force microscopy

Table 2 Surface parameters of samples in the untreated-Ti group and nano-TiO2 group

Group	Surface roughness （nm）	Vertical range （nm）	Surface difference （%）
Untreated-Ti Nano-TiO₂	15.89 ± 2.5 46.30 ± 2.18¹⁾	156.53 ± 30.31 340 ± 33.56¹⁾	7.35 ± 2.18 13.78 ± 2.58¹⁾

Figure 3 The static contact angles on the samples in the four groups a: untreated-Ti group, 35° ± 2.3°; b: micro-Ti group, 18° ± 1.6°; c: nano-TiO2 group, 14° ± 1.6°; d: micro/nano-TiO2 group, 9° ± 2.1°

Figure 4 SEM images of BMMCs 4 h after culture on the surfaces of samples in the four groups a: untreated-Ti group, most of the cells were fusiform, some of which can be seen with pseudopodia and antennas; b: micro-Ti group, the cells were flat and polygonal, with pseudopodia and antennas clearly visible; c: nano-TiO2 group, the cells were flat and polygonal, closely attached to the surface of the titanium samples; d: micro/nano-TiO2 group, cells were connected with each other and grew together flakily, with large numbers of pseudopodia and antennas; SEM: scanning electronmicroscopy

Figure 5 The cell proliferation rate of BMMCs cultured on the samples in the four groups *: vs. untreated-Ti group, P＜0.05; $: vs. micro-Ti group, P＜0.05. The relative cell proliferation rates of samples in the four groups increased between days 1-9, and the relative cell proliferation rates of samples in the nano-TiO2 group and the micro/nano-TiO2 group significantly increased compared with those in the untreated-Ti group and the micro-Ti group(P＜0.001) between days 5-9

Figure 6 ALP activity of BMMCs cultured on the samples in the four groups *: vs. untreated-Ti group, P＜0.05; $: vs. micro-Ti group, P＜0.05; #: vs. nano-TiO2 group, P＜0.05. The relative cell proliferation rates of samples in the four groups increased between days 7-14, and the samples in the micro/nano-TiO2 group gained the highest value on the 14th day

Figure 7 Immunofluorescence staining images of BMMCs cultured on the samples in the four groups （× 400） a: the expression of F-actin and vinculin after 24 h incubation. The expression of F-actin in the micro/nano-TiO2 group was the strongest. b: the expression of OCN and OPN after 72 h incubation. The expression of OCN and OPN in the micro/nano-TiO2 group was the strongest. Blue: cell nucleus; green: either F-actin or OPN; red: either vinculinor or OCN; OCN: osteocalcin; OPN: osteopontin

Figure 8 mRNA expression levels of various osteogenic transcription factors in the four groups a: RUNX2; b: OCN; c: OPN; d: COL Ⅰ (collagen I). *P＜0.05 vs. untreated-Ti group, #: P＜0.05 vs. micro-Ti group, $: P＜0.05 vs. Nano-TiO2 group. Compared with the untreated-Ti group and the micro-Ti group, the mRNA expression levels of all the osteogenic factors in the nano-TiO2 and micro/nano-TiO2 groups markedly increased; compared with the untreated-Ti group and micro-Ti group, the mRNA expression levels of COL I in the nano-TiO2 and micro/nano-TiO2 groups significantly increased (P < 0.001)

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