Restor Dent Endod.  2020 Nov;45(4):e54. 10.5395/rde.2020.45.e54.

Effects of zinc oxide and calcium–doped zinc oxide nanocrystals on cytotoxicity and reactive oxygen species production in different cell culture models

• Souza GLd
• Gomes Moura CC
• Almeida Silva AC
• Marinho JZ
• Silva TR
• Dantas NO
• Bonvicini JFS
• Turrioni AP

• KMID: 2512045
• DOI: http://doi.org/10.5395/rde.2020.45.e54

Abstract

Objectives
This study aimed to synthesize nanocrystals (NCs) of zinc oxide (ZnO) and calcium ion (Ca²⁺ )-doped ZnO with different percentages of calcium oxide (CaO), to evaluate cytotoxicity and to assess the effects of the most promising NCs on cytotoxicity depending on lipopolysaccharide (LPS) stimulation.
Materials and Methods
Nanomaterials were synthesized (ZnO and ZnO:xCa, x = 0.7; 1.0; 5.0; 9.0) and characterized using X-ray diffractometry, scanning electron microscopy, and methylene blue degradation. SAOS-2 and RAW 264.7 were treated with NCs, and evaluated for viability using the MTT assay. NCs with lower cytotoxicity were maintained in contact with LPS-stimulated (+LPS) and nonstimulated (−LPS) human dental pulp cells (hDPCs). Cell viability, nitric oxide (NO), and reactive oxygen species (ROS) production were evaluated.Cells kept in culture medium or LPS served as negative and positive controls, respectively. One-way analysis of variance and the Dunnett test (α = 0.05) were used for statistical testing.
Results
ZnO:0.7Ca and ZnO:1.0Ca at 10 µg/mL were not cytotoxic to SAOS-2 and RAW 264.7. +LPS and −LPS hDPCs treated with ZnO, ZnO:0.7Ca, and ZnO:1.0Ca presented similar NO production to negative control (p > 0.05) and lower production compared to positive control (p < 0.05). All NCs showed reduced ROS production compared with the positive control group both in +LPS and −LPS cells (p < 0.05).
Conclusions
NCs were successfully synthesized. ZnO, ZnO:0.7Ca and ZnO:1.0Ca presented the highest percentages of cell viability, decreased ROS and NO production in +LPS cells, and maintenance of NO production at basal levels.

Keyword

Cell survival; Nanoparticles; Zinc oxide

Figure

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  Figure 1 X-ray diffractograms of the zinc oxide (ZnO):calcium (xCa) nanocrystals. X-ray diffraction patterns of pure and Ca-doped ZnO nanocrystals (A), variation in angular distance confirming the incorporation of Ca2+ ions into ZnO nanocrystals (B), calcium oxide (CaO) nanocrystal patterns observed in 0.7 wt.% Ca (C), increase in the concentration of CaO with Ca doping, from 3.8% to 6.7% for the 0.7 to 9.0 wt.% samples (C).

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  Figure 2 Scanning electron microscopy and energy dispersive X-ray spectrometry (EDS) results of the zinc oxide (ZnO):calcium (xCa) nanocrystals. ZnO nanocrystals (A) with spherical morphology predominated. In the rod-shaped nanocrystals, the rod length grew with the incorporation of Ca (B-E). In EDS, only Zn and O atoms were observed (F).

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  Figure 3 Methylene blue degradation in aqueous solution and ultraviolet-visible (UV-Vis) light irradiation of the zinc oxide (ZnO):calcium (xCa) nanocrystals.A, the absorbance of the solution at time > 0; Ao, the initial absorbance.

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  Figure 4 Cell viability determination of SAOS-2 and RAW 264.7 after treatment with zinc oxide (ZnO), ZnO:0.7 calcium (Ca), ZnO:1.0Ca, ZnO:5.0Ca and ZnO:9.0Ca by the MTT formazan assay. (A-C) The effects of nanoparticles at the concentrations of 10 µg/mL, 50 µg/mL, and 100 µg/mL, respectively, on SAOS-2 viability. (D-F) The effects of nanoparticles at the concentrations of 10 µg/mL, 50 µg/mL, and 100 µg/mL, respectively, on RAW 264.7 viability. Capital letters allow comparisons among different nanocrystals and with the control group (Dulbecco's Modified Eagle's Medium).

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  Figure 5 Cell viability determination of human dental pulp cells (hDPCs) after treatment with zinc oxide (ZnO), ZnO:0.7 calcium (Ca) and ZnO:1.0Ca by the MTT formazan assay. Effects of nanocrystals at the concentrations of 10 µg/mL on lipopolysaccharide-nonstimulated (−LPS) and -stimulated (+LPS) hDPCs. Capital letters allow comparisons among different nanocrystals for the same condition (−LPS or +LPS) and with control groups (positive control group: +LPS; and negative control group: Dulbecco's Modified Eagle's Medium, −LPS).

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  Figure 6 Nitric oxide (NO) production by lipopolysaccharide-nonstimulated (−LPS) and -stimulated (+LPS) human dental pulp cells (hDPCs) after treatment with zinc oxide (ZnO), ZnO:0.7 calcium (Ca), and ZnO:1.0Ca. The effects of nanocrystals at the concentration of 10 µg/mL on NO production in −LPS and +LPS hDPCs. Capital letters allow comparisons among different nanocrystals for the same condition (−LPS or +LPS) and with control groups (positive control group: +LPS; and negative control group: Dulbecco's Modified Eagle's Medium [DMEM], −LPS).

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  Figure 7 Reactive oxygen species (ROS) production by human dental pulp cells (hDPCs) after the treatment with zinc oxide (ZnO), ZnO:0.7 calcium (Ca), and ZnO:1.0Ca. The effects of nanocrystals at the concentration of 10 µg/mL on ROS production in lipopolysaccharide-nonstimulated (−LPS) and -stimulated (+LPS) hDPCs. Capital letters allow comparisons among different nanocrystals for the same condition (−LPS or +LPS) and with control groups (positive control group: +LPS; and negative control group: Dulbecco's Modified Eagle's Medium [DMEM], −LPS).DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate.

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  Figure 8 Images representative of reactive oxygen species (ROS) production by human dental pulp cells (hDPCs) treated with zinc oxide (ZnO), ZnO:0.7 calcium (Ca) and ZnO:1.0Ca. Images in the column on the left represent the groups in which the cells were notstimulated by lipopolysaccharide (−LPS), while in the images in the column on the right, the cells were kept in contact with LPS (+LPS). Dulbecco's Modified Eagle's Medium (DMEM) (A), LPS (B), ZnO (C, D), ZnO:0.7Ca (E, F), ZnO:1.0Ca (G, H). The intensity of fluorescence emitted by the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe quantified ROS production, with higher fluorescence indicating higher levels of production of these oxidant agents.

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