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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 19  |  Issue : 1  |  Page : 81

Antimicrobial effect of different physical and chemical compounds of zinc oxide and graphene oxide added to composite resins


1 Department Operative of Dentistry, Faculty of Dentistry, Kermanshah University of Medical Sciences, Kermanshah, Iran
2 Dental Materials Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran
3 Department of Microbiology, Faculty of Medicine, Babol University of Medical Sciences, Babol, Iran

Date of Submission19-Dec-2021
Date of Acceptance03-Mar-2022
Date of Web Publication26-Sep-2022

Correspondence Address:
Dr. Homayoon Alaghehmand
Department Esthetic and Restorative Dentistry, Faculty of Dentistry, Babol University of Medical Sciences, Babol
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1735-3327.356820

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  Abstract 


Background: 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.
Materials and Methods: 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).
Results: 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.
Conclusion: 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.

Keywords: Composite resins, graphite, nanoparticles, zinc oxide


How to cite this article:
Farhangian Z, Alaghehmand H, Tashakkorian H, Mokhtarpour F, Davoodabadi A. Antimicrobial effect of different physical and chemical compounds of zinc oxide and graphene oxide added to composite resins. Dent Res J 2022;19:81

How to cite this URL:
Farhangian Z, Alaghehmand H, Tashakkorian H, Mokhtarpour F, Davoodabadi A. Antimicrobial effect of different physical and chemical compounds of zinc oxide and graphene oxide added to composite resins. Dent Res J [serial online] 2022 [cited 2022 Dec 6];19:81. Available from: https://www.drjjournal.net/text.asp?2022/19/1/81/356820




  Introduction Top


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.[1] Therefore, researchers aimed to produce composite resins with antibacterial along with the desired mechanical properties.

There are different approaches for the antibacterial activity of composite resins and adhesives.[2] The first approach was adding different antibacterial agents to the matrix to inhibit bacterial growth over time. Fluoride and chlorhexidine were the most common antimicrobial agents.[3]

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.[4],[5] Gold, silver, and zinc are among the most prominent employed metals.[6],[7] Antimicrobial properties of metals are directly related to their contact surface. Furthermore, nanoparticles interact widely with microorganisms.[2]

Zinc has an antibacterial effect against many bacteria, including Streptococcus mutants.[8],[9] Zinc oxide (ZnO), besides the mentioned benefits, is insoluble and white.[10] Furthermore, the ability of nanoparticles to be absorbed in cell membranes results in several intracellular processes that increase reactivity and antibacterial activity.[11] Graphite nanoparticles, which include carbon nanotubes, fluorine, and graphene, due to their innovative features with antimicrobial properties, are considered new and upcoming agents.[12],[13],[14] Graphene is a solid two-dimensional and single-atomic substance combining with carbon hybridization SP2, has attracted much attention over the past decade. Its unique properties and prominent features are high electrical conductivity, optimal mechanical properties, large surface area, low thermal expansion coefficient, and very high aspect ratio.[15],[16],[17] Graphene is also compatible with the environment and provides a suitable substrate for biological-chemical functions.[18],[19] The antimicrobial effect of graphene oxide (GO) particles on both Gram-negatives (e.g., Pseudomonas aeruginosa) and Gram-positives (e.g., S. mutants) has been investigated in previous studies[20],[21] and its low cellular cytotoxicity has been proved in the in vitro studies.[20] Furthermore, the ZnO in graphene is more penetrative in the cell wall and consequently more destructive to the bacteria.[20] However, the main limitation of the graphene compounds is its grey color and tendency to aggregate when added to a colloidal suspension.[22] Thus, the composition of GO with composite resins might have antibacterial activity,[10],[22] but the opacity of the filler particles prevents visible light from passing through and also affects the mechanical properties of the composites.[23] By combining the antimicrobial properties of GO with the bright color of ZnO, the problem will be solved.[22] The aim of this study was to evaluate the effect of adding ZnO nanoparticles and GO on the antibacterial properties of flowable composites.


  Materials and Methods Top


Raw materials

In this in vitro experimental study, spherical ZnO nanoparticles with an average particle size of 20 nm [Figure 1]a and GO nanoparticles with a size range of 3.4–7 nm and layering structure [Figure 1]b were purchased from Merck Company (Germany). To synthesize the physical compound, 0.5 wt.% of ZnO and 0.5 wt.% of GO were used, the ZnO solution was, initially, dissolved in methanol and chloroform and then mixed with an alcoholic solution of GO for 24 h. The specimen was, then, separated by the solvent propagation method in a centrifuge. After solvent evaporation, the obtained sediments were dried in an oven [Figure 1]c.
Figure 1: 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.

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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 [Figure 1]d. Scanning electron microscopy (SEM) images of nanoparticle powders were taken [Figure 1].

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[2], Ivoclar Vivadent, Lichtenstein) for 20 s. 10 μL of standard S. mutans suspension ATCC 35668 (purchased from Pasteur Institute, Iran) (1.5 × 10[8]) was added to the microtubes and shaken. Microtubes were incubated for an hour for solution evaporation. Then, 300 μL of brain–heart infusion broth was added to them. Ten μL from each solution was collected after incubation in 37°C for 24 h and 1 week, 10 μL from each solution was selected, and bacterial colonies in the blood agar culture were counted. The number and average of colonies in three microtubes were reported.

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 Na2HPO4, 2Mm KH2PO4) containing the standard S. mutans suspension ATCC 35668 (1.5 × 10[8] bacteria) and was shaken in 37°C for 24 h. At that moment, the specimens were dried and fixed with methanol. FE-SEM was used to observe the bacterial adhesion to the composite specimens.

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).


  Results Top


Scanning electron microscopy evaluation of the powder of nanoparticle

The SEM was used to observe the morphology of nanoparticles and composite discs. In [Figure 1]a, it can be seen the thickness and length of ZnO nanoparticles for almost 20 nm and 40–200 nm, respectively. Most of the nanoparticles are seen in irregular plate forms. In [Figure 1]b, GO nanoparticles are seen in very thin (almost 4–7 nm) but very wide (10–50 μ) sheets. In [Figure 1]c, physical compound of ZnO + GO is seen in agglomerated great amounts of ZnO on GO sheets. In [Figure 1]d, chemical compound of ZnO + GO can be seen in irregular plates of ZnO connected to GO sheets.

[Figure 2] shows SEM images of composites discs ([Figure 2]a shows powder-free composite). Black arrows show ZnO nanoparticles. None of [Figure 2]c, [Figure 2]d, [Figure 2]e is not showing graphene oxide nanoparticles. A little of porosities are observed in [Figure 2]b, [Figure 2]c, [Figure 2]d, [Figure 2]e.
Figure 2: 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.

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Energy dispersive X experiment and X-ray diffraction sample chart

[Figure 3] shows X-ray diffraction (XRD) analysis of four types of powders that were used in this study. Vertical axis is intensity (a.u.), and horizontal axis is energy (eV). Zn is being observed in [Figure 3]a, [Figure 3]c, and [Figure 3]d. Amount of Zn in [Figure 3]a is very more. In [Figure 3]b, carbon is being seen very more. [Figure 4] shows XRD analysis of composite discs ([Figure 4]a shows powder-free composite). Zn is being observed in [Figure 4]b, [Figure 4]d, and [Figure 4]e, and is not being observed in [Figure 4]c.
Figure 3: 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).

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Figure 4: 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.

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Elemental analysis in energy dispersive spectroscopy X [Table 1] and [Table 2] is being seen. According to [Table 2], in Groups 2,4 and 5, Zn is considerably more than the others.
Table 1: Energy dispersive spectroscopy elemental analysis to weight percentage of four types of powders that used in study

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Table 2: Energy dispersive spectroscopy elemental analysis to weight percentage of composite discs: Group 1 (control), Group 2 (with zinc oxide), Group 3 (with graphene oxide), Group 4 (physical compound of zinc oxide and graphene oxide), Group 5 (chemical compound of zinc oxide and graphene oxide)

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  Antimicrobial test results Top


According to [Table 3], the number of colonies in each group had approximately the same range in 24 h. The number of colonies in Groups 3 and 5 was lower than that of the other groups, but this difference was not significant.
Table 3: Colony count of microbial culture after 24 h and 7 days

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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

[Figure 5] shows SEM images of microbial adherence to composite discs after 24 h. Black arrows are showing S. mutants coccies. The least amounts of adhesion were observed in Groups 4 and 3, respectively.
Figure 5: 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.

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  Discussion Top


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.[24] Furthermore, S. mutans is the first agent involved in bacterial colonization and biofilm growth.[25] This was the rationale behind using a single S. mutans in the present 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.[26] GO is synthesized by oxidation of graphite through the Hummers method. It was believed earlier that graphene nanoparticles were not small enough to be released and perfused into surroundings and in turn, reducing the effects of fluoride and other ions on the bacteria. However, our study indicated that GO could reduce bacterial adhesion and growth on the composite surface. This can be important because the bacterial colonization on composite surfaces is significantly more than on other surfaces such as dental amalgam and glass ionomers resulting in early recurrent caries.

In the present study, a concentration of 1% of GO was used to avoid the toxicity of zinc.[27] Furthermore, previous studies showed that incorporation of ZnO nanoparticles for up to 1% into the composite resins could significantly inhibit the colonization of cariogenic bacteria without sacrificing the mechanical properties such as flexural strength, flexural modulus, compressive strength, and micro-shear bond strength of the composite resins.[2]

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.[25]

Zhang et al. reported the concentration and time dependency of antibacterial activity of GO. They evaluated the effect of GO on  Escherichia More Details 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.[26]

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.[24]

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 Zn2+ into the growth media may interfere with the bacterial metabolism by displacing Mg2+, 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.[25]

Dias et al. reported the antibacterial effect of adding ZnO to resin composites on S. mutants.[24]

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.[2]

Kasraei et al. found that the composites containing nanoZnO particles exhibited higher antibacterial activity against S. mutans and Lactobacillus compared to the control group.[27]


  Conclusion Top


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.

Financial support and sponsorship

Nil.

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.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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