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This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
Graphene oxide (GO), a product of graphite, is a candidate for nano-reinforcing cement-based materials due to its good water dispersibility and excellent mechanical properties. On the other hand, zinc oxide (ZnO) is well-known for its antibacterial characteristics as well. Therefore, we aimed to evaluate the impacts of adding ZnO and GO nanoparticles on the antibacterial properties of flowable composites.
In this, in vitro experimental study was designated into five groups containing: (1) no nanoparticles as control group, (2) 1 wt.% ZnO nanoparticle, (3) 1 wt.% GO, (4) 1 wt.% physical compound of ZnO and GO, and (5) 1 wt.% chemical compound of ZnO and GO. The antibacterial properties of composite resin discs were evaluated by direct contact test. Data were analyzed using a one-way analysis of variance, followed by Tukey's post hoc tests (P = 0.05).
Streptococcus mutans colony counting in the first 24 h showed the least growth rate in the chemical compound group (2.2 × 10 [5]). However, in 7 days, the least colony number was observed in the GO group (2 × 10 [3]). Moreover, the physical compound showed the least bacterial adhesion.
Adding GO alone to composites, compared to adding ZnO or physical and chemical compounds of GO-ZnO, was more helpful to increase the antimicrobial characteristics.
Composite resins are now used extensively in restorative dentistry. Appropriate mechanical properties, low polymerization shrinkage, and high wear resistance, as well as antibacterial activity, are the essential properties for composite resins.
The antibacterial function of composite resins can be effective in controlling secondary caries adjacent to the filling.
There are different approaches for the antibacterial activity of composite resins and adhesives.
The other approach was incorporating quaternary ammonium into resin monomers. The permanent positive charge of these composites helps in electrostatic-based bacterial eradication.
The third approach was mixing metal particles (oxides) or ions into the restorative materials.
Zinc has an antibacterial effect against many bacteria, including Streptococcus mutants.
Raw materials
In this in vitro experimental study, spherical ZnO nanoparticles with an average particle size of 20 nm
Scanning electron microscopy images of nanoparticle powders (×35K). (a) Zinc oxide powder, (b) Graphene oxide powder, (c) Physical compound of graphene oxide and zinc oxide, (d) Chemical compound of graphene oxide and zinc oxide.
For the synthesis of the chemical compound, 0.5 wt.% of ZnO nanoparticles and aminopropyl triethoxysilane were mixed in dimethyl sulfoxide solvent and activated through the ultrasonic method. The obtained sediment was rinsed with ethanol and centrifuged. The defined value of the sediment was added to 0.5 wt. % of GO in dimethylformamide solvent. With the combination of ultrasonic, alcohol purification method, and drying in oven, the chemical compound was obtained
Preparation of the specimen (adding nanoparticles to the composite)
Grandio Flow Composite Shade A2 (VOCO GmbH, Germany) was selected for this study. One percent by weight (1 wt.%) of each nanoparticle was weighed by a digital scale (with the accuracy of 0.0001) (A and D Company, Japan) and mixed manually with a spatula for 15 min under the red light on a vibrator so that homogeneity obtained. Hence, the groups (1) as the control (without adding nanoparticle), (2) containing 1 wt.% of ZnO nanoparticles, (3) containing 1 wt.% of GO, (4) containing 1 wt.% of the physical compound of ZnO-GO, and (5) containing 1 wt.% of the chemical compound of ZnO and GO were formed. SEM images of mixed composites were taken.
Characterization
SEM (JSM 6701F, JEOL) was used to observe the morphology of nanoparticles-contained cured composites discs and GO, ZnO, physical, and chemical compounds of nanoparticle powder. X-ray photoelectron spectroscopy (XPS, PHI-5702, Physical Electronics) was used to analyze homogeneity, purity percentage, and level percent of materials on the surface of specimens by Al-Ka radiation as the excitation source and the bonding energy of Au (Au 4f7/2: 84.00 eV) as reference.
Antibacterial test
The antibacterial activities of composite resins containing different nanoparticles were evaluated using the direct contact test. Initially, three 500 μL sterile microtubes were selected for each group. Then, 200 μL of prepared resin composite was added to each microtube. A predesigned Teflon jig was pressed into microtubes, and composites were cured by light cure unit (Bluephase 8, 800 mW/cm
Bacterial adhesion to composite
One piece of each composite group (2 mm × 2 mm × 1 mm) was light-cured for 20 s and placed in the phosphate-buffered saline (137 mM NaCl, 207 mM Na
Statistical analysis
One-way analysis of variance followed by Tukey's post hoc comparison test was used to test the differences between the control and experimental groups at the level of significance of P < 0.05 with SPSS (SPSS, IBM Corp, IBM, USA).
Scanning electron microscopy evaluation of the powder of nanoparticle
The SEM was used to observe the morphology of nanoparticles and composite discs. In
Scanning electron microscopy images of composites discs (×15K). (a) Without powder, (b) With zinc oxide powder, (c) With graphene oxide powder, (d) With physical compound of graphene oxide and zinc oxide, (e) With chemical compound of graphene oxide and zinc oxide.
Energy dispersive X experiment and X-ray diffraction sample chart
X-ray diffraction analysis. Zinc oxide nanoparticles powder (a), Graphene oxide nanoparticles powder (b), Physical compound of zinc oxide and graphene oxide powder (c), Chemical compound of zinc oxide and graphene oxide powder (d). X-ray diffraction charts of composites discs. (a) Without powder, (b) With zinc oxide powder, (c) With graphene oxide, (d) With physical compound of graphene oxide and zinc oxide, (e) With chemical compound of graphene oxide and zinc oxide.
Elemental analysis in energy dispersive spectroscopy X
According to
The number of colonies in all groups decreased in a week in comparison to 24 h. This decrease was particularly significant in Group 3 (GO), followed by Group 2 (ZnO).
Scanning electron microscopy images of bacterial adhesion
Scanning electron microscopy images of microbial adherence to composite discs (×35K). (a) Without powder, (b) With Zinc oxide powder, (c) With graphene oxide, (d) With physical compound of graphene oxide and Zinc oxide, (e) With chemical compound of graphene oxide and zinc oxide.
According to Dias et al., S. mutans is the main cause of dental caries around the world and was also recognized as the most cariogenic streptococcal species in that study.
Although carbon-based nanomaterials such as GO potentially fight against multidrug-resistant bacteria. The antibacterial activity and mechanism are far from explicit molecular views.
In the present study, a concentration of 1% of GO was used to avoid the toxicity of zinc.
Another study by Brandão et al. showed that the incorporation of 2–5 wt.% of ZnO-NP could endow antibacterial activity to composite resins without jeopardizing their physicochemical properties.
Zhang et al. reported the concentration and time dependency of antibacterial activity of GO. They evaluated the effect of GO on Escherichia coli and reported tension in the membrane of E. coli as soon as GO nanorods contacted with the cells. By interacting with the phospholipid membrane of E. coli, the membrane was damaged due to the increase in the amount of reactive oxygen species followed by the glutathione reduction. This can lead to bacterial death.
In the present study, there was no significant difference in the antibacterial activity between the nanoZnO and control groups, which could be due to the spherical form of nanoZnO.
ZnO has a broad-spectrum antibacterial activity and has a wide range of nanoscale forms, such as nanowires, nanoparticles, nanobelts, nanosprings, nanopencils, nanocomposites, nanoboxes, and nanorings. The morphology of ZnO is determined by the condition and method of synthesis. Some parameters such as pH, temperature, solvents, various precursors, and physicochemical settings can be controlled to obtain the best antibacterial properties. It has been shown that the shape of ZnO can affect the internalization mechanism which indicates rods and wires can penetrate the bacterial cells more easily than spherical-shaped particles. According to this concept, the properties of the surface of particle are likely to play a crucial role in the production of reactive oxygen species, but the antimicrobial activity of the substances may depend on the shape. Shape-dependent activity is explained by the percentage of active facets on the nanoparticles which can be synthesized as a function of growth parameters. It has been shown that rod structures have more active aspects that increase antibacterial activity compared to spherical nanostructures.
Two pathways have been described as the possible mechanisms for the antibacterial activity of ZnO. The first advocates that ZnO reacts with the water of environment. Releasing Zn
2+into the growth media may interfere with the bacterial metabolism by displacing Mg
2+, which is extremely necessary to the enzymatic activity of the biofilm. The second advocates that ZnO can also generate reactive peroxides that penetrate the membrane cell, causing damage, and inhibiting bacterial growth. Taking into account that both mechanisms involve the release of active species from ZnO surfaces, it is clear that the high surface area to volume ratio of the 10–50 nm ZnO-NP particles in this study affected the behavior of the experimental composites.
Dias et al. reported the antibacterial effect of adding ZnO to resin composites on S. mutants.
Tavassoli Hojati et al. showed that the added ZnO nanoparticles could effectively prevent the growth of S. mutans. By increasing the amount of ZnO nanoparticles, the growth of bacteria significantly decreased, and the composition of these nanoparticles did not show adverse effects on the mechanical properties of composites. The flexural strength and compression modulus with the nanoparticles remained unchanged, while these composites exhibited lessened depth of light penetration and increased bond strength, with no significant change in the degree of conversion between the groups. The results of this study indicated that adding a low concentration of nanoparticles led to homogeneous distribution, while the higher mass fraction of nanoparticles resulted in heterogeneous distribution which reduced the mechanical properties of the composite. The reduced mechanical properties in this study were probably related to the effect of nanoparticles on composite polymerization, rather than the formation of structural defects due to the heterogeneous distribution of particles.
Kasraei et al. found that the composites containing nanoZnO particles exhibited higher antibacterial activity against S. mutans and Lactobacillus compared to the control group.
Based on our results, adding GO alone to composites, rather than ZnO, or the physical and chemical compounds of GO-ZnO was more helpful to increase the antimicrobial characteristics.
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Conflicts of interest
The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or nonfinancial in this article.