Journal of Prevention and Treatment for Stomatological Diseases ›› 2021, Vol. 29 ›› Issue (4): 226-233.DOI: 10.12016/j.issn.2096-1456.2021.04.002
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WANG Min(),JIANG Nan,ZHU Songsong(
)
Received:
2020-08-24
Revised:
2020-10-19
Online:
2021-04-20
Published:
2021-02-26
Contact:
Songsong ZHU
Supported by:
通讯作者:
祝颂松
作者简介:
王旻,医师,硕士研究生在读,Email:基金资助:
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.
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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 |
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
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) |
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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