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. 2023 Apr 3;24(7):6675.
doi: 10.3390/ijms24076675.

Endothelial Cell Behavior and Nitric Oxide Production on a-C:H:SiOx-Coated Ti-6Al-4V Substrate

Igor A Khlusov  1 , Alexander S Grenadyorov  2 , Andrey A Solovyev  2 , Vyacheslav A Semenov  2 , Maksim O Zhulkov  3 , Dmitry A Sirota  3 , Aleksander M Chernyavskiy  3 , Olga V Poveshchenko  3   4 , Maria A Surovtseva  3   4 , Irina I Kim  3   4 , Natalya A Bondarenko  3   4 , Viktor O Semin  5
Affiliations

Endothelial Cell Behavior and Nitric Oxide Production on a-C:H:SiOx-Coated Ti-6Al-4V Substrate

Igor A Khlusov et al. Int J Mol Sci. .

Abstract

This paper focuses on the surface modification of the Ti-6Al-4V alloy substrate via a-C:H:SiOx coating deposition. Research results concern the a-C:H:SiOx coating structure, investigated using transmission electron microscopy and in vitro endothelization to study the coating. Based on the analysis of the atomic radial distribution function, a model is proposed for the atomic short-range order structure of the a-C:H:SiOx coating, and chemical bonds (C-O, C-C, Si-C, Si-O, and Si-Si) are identified. It is shown that the a-C:H:SiOx coating does not possess prolonged cytotoxicity in relation to EA.hy926 endothelial cells. In vitro investigations showed that the adhesion, cell number, and nitric oxide production by EA.hy926 endothelial cells on the a-C:H:SiOx-coated Ti-6Al-4V substrate are significantly lower than those on the uncoated surface. The findings suggest that the a-C:H:SiOx coating can reduce the risk of endothelial cell hyperproliferation on implants and medical devices, including mechanical prosthetic heart valves, endovascular stents, and mechanical circulatory support devices.

Keywords: a-C:H:SiOx coating; adhesion; cell number; endothelial cells EA.hy926; in vitro cytotoxicity; local atomic order; nitric oxide production.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
SEM image of the a-C:H:SiOx coating surface on Ti-6Al-4V substrate.
Figure 2
Figure 2
Cross-sectional bright-field TEM image of a-C:H:SiOx coating (a), NBD at the coating center (b).
Figure 3
Figure 3
RDF of atoms in a-C:H:SiOx coating and peak resolution into pair atomic spacings using the Gaussian function. Five areas were examined for statistics. X axis indicates the radial distance (r) (given in angstrom). Y axis indicates radial distribution function g(r) (given in arbitrary units).
Figure 4
Figure 4
The model (pattern) of the short-range order atomic structure of a-C:H:SiOx coating.
Figure 5
Figure 5
Viability of EA.hy926 cells after 24 and 72 h incubation with extracts: (a)—T1 substrates, (b)—T2 substrates. Data are presented as mean ± SD. 1—p < 0.05 compared to 24 h, 2—p < 0.02 compared to 100% extract after 72 h, 3—p < 0.05 compared to 50% extract after 72 h. Each group consists of six substrates for testing.
Figure 6
Figure 6
Density distribution of EA.hy926 cells depending on days of substrate cultivation: 1—T1 vs. T2 after 3, 7, and 14 days (p < 0.02), 2—T1 vs. T2 after 1 day (p = 0.01), 3—T2 vs. T1 after 1 day (p = 0.01), 4—T2 vs. T1 substrate after 3 days (p < 0.02), 5—T1 vs. T2 substrate after 3 days (p = 0.01), 6—T2 vs. T1 substrate after 7 days (p < 0.05), 7—T1 vs. T2 substrate after 7 days (p = 0.01). Data are presented as mean ± SD. Each group consists of six substrates for testing.
Figure 7
Figure 7
EA.hy926 cell distribution maps on substrates: (a)—T1 surface, (b)—T2 surface after culturing of 1 to 14 days. Scale bar: 50 µm. Adherent cells, actin filaments, and nuclei are stained with Vybrant™ CFDA SE Cell Tracer Kit (green), phalloidin (green), and DAPI (blue), respectively.
Figure 8
Figure 8
Optical density distribution of EA.hy926 cells depending on days of substrate cultivation: 1—T2 vs. T1 after 1, 3, 5, and 7 days (p = 0.0004), 2—T1 vs. T2 after 1 day (p = 0.04), 3—T1 vs. T2 after 3 days (p < 0.04), 4—T1 vs. T2 after 5 days (p = 0.008). MTT staining is presented as mean ± SD. Six measurements were performed in each group. Y axis indicates dissolved formazan crystals.
Figure 9
Figure 9
Block diagram (a) of EA.hy926 cell death on Ti substrates: 1—necrotic cells vs. those after 7 days (p = 0.01), 2—live cells vs. those on T1–T2 substrates after 3 days (p < 0.05), 3—live cells vs. those on T2 substrate after 7 days (p = 0.04). Data are presented as mean ± SD. Six measurements were performed in each group. Cell distribution maps (b) on T1 and T2 substrates. Staining: acridine orange and ethidium bromide. Scale bar: 50 µm. Magnification: 200×.
Figure 10
Figure 10
NO production by EA.hy926 cells cultured on substrates: 1—24 h vs. 72 h (p < 0.01), 2—T2 vs. EA.hy926 cells on culture plastic (p = 0.01), 3—T2 vs. T1 (p = 0.02). Data are presented as mean ± SD. Six measurements were performed in each group. Control-EA.hy926 cells cultured on plastic.
Figure 11
Figure 11
Stages of sample preparation and a-C:H:SiOx coating deposition.
See this image and copyright information in PMC

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