|Year : 2019 | Volume
| Issue : 2 | Page : 81-87
Antibacterial effect of fluorinated graphene and zinc oxide nanoparticles incorporated in zinc oxide-based sealers on Enterococcus faecalis (in vitro study)
Aaripaka Sridevi, J Sindhu, DN Naveen, DN Nirupama, Mohan Thomas Nainan
Department of Conservative Dentistry and Endodontics, Vydehi Institute of Dental Sciences and Research Centre, Bengaluru, Karnataka, India
|Date of Web Publication||19-Aug-2019|
Dr. J Sindhu
Vydehi Institute of Dental Sciences and Research Centre, # 82, EPIP Area, Nallurahalli, Whitefield - 560 066, Bengaluru, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Incorporation of nanoparticles with antibacterial properties plays an essential role in the success of endodontic therapy.
Aim: The aim of this study is to evaluate the antimicrobial efficacy of zinc oxide (ZnO)-based sealer incorporated with fluorinated graphene and ZnO nanoparticles against Enterococcus faecalis.
Subjects and Methods: Sixty human extracted mandibular premolars with single canal were selected. After biomechanical preparation of all teeth and inoculation with E. faecalis, teeth were divided into three groups according to the type of the tested sealer, Group 1: Teeth were obturated with ZnO-based sealer only, Group 2: Teeth were obturated using sealer incorporated with fluorinated graphene, and Group 3: Teeth were obturated using sealer incorporated with ZnO nanoparticles. Each group was subdivided into two subgroups according to the timing of filling removal and culture sample. Subgroup A: Obturation removal and culture sample were taken after 1 week. Subgroup B: Obturation removal and culture sample were taken after 3 weeks. The number of colonies forming units was counted to assess the effect against E. faecalis. Data were recorded, tabulated, and statistically analyzed using the Kruskal–Wallis test was applied to find significance among the groups (1, 2, and 3). The Mann–Whitney U-test was applied to find significance between the groups. The Wilcoxon signed-rank test was applied to find significance between two-time intervals (24 h to 1 week, 24 h to 2 weeks) in each group. P < 0.05 was considered as statistically significant.
Results: After 3 weeks from obturation, fluorinated graphene had a better antibacterial effect and there was a significant difference between the three tested groups.
Conclusion: After 1- and 3-weeks samples after obturation fluorinated graphene had a better antibacterial effect and there was a significant difference between the three tested groups.
Keywords: Enterococcus faecalis, fluorinated graphene, zinc oxide eugenol sealer, zinc-oxide nanoparticles
|How to cite this article:|
Sridevi A, Sindhu J, Naveen D N, Nirupama D N, Nainan MT. Antibacterial effect of fluorinated graphene and zinc oxide nanoparticles incorporated in zinc oxide-based sealers on Enterococcus faecalis (in vitro study). Saudi J Oral Sci 2019;6:81-7
|How to cite this URL:|
Sridevi A, Sindhu J, Naveen D N, Nirupama D N, Nainan MT. Antibacterial effect of fluorinated graphene and zinc oxide nanoparticles incorporated in zinc oxide-based sealers on Enterococcus faecalis (in vitro study). Saudi J Oral Sci [serial online] 2019 [cited 2020 Feb 19];6:81-7. Available from: http://www.saudijos.org/text.asp?2019/6/2/81/264760
| Introduction|| |
Endodontic success depends on the elimination of microorganisms from the root canal system and prevent recolonization. To accomplish this desired result, a union of chemical and mechanical approaches is used. Due to complex canal anatomy microbe can linger even after rigorous disinfections regime, thus complete sterilization of the canals is not achieved.
Enterococcus faecalis, a Gram-positive facultative anaerobe is found abundantly in untreated canals and failed root canals. Its capability in tolerating unfavorable environmental conditions results in high survival rates and made these bacteria to be more resistant to intracanal medications.
At present, most sealers fail to completely seal dentinal walls of the root canal, thus micro spaces always retain within the canal walls. Microbes gain access to this space headlining the need to have antibacterial properties in the sealers used in obturations.
Nanotechnology is defined as a science that deals with the development of new materials with new properties and functions through controlling and restructuring of the materials on a nanometer scale of “<100 nm.”
Nanoparticles with the antibacterial properties show assuring effect against rebellious microorganism. These particles dismantle the bacterial cells through various mechanisms. The notion of using nanoparticles in endodontics as modern treatment technique was developed afresh.,
Among metal oxide nanoparticles, zinc oxide (ZnO) nanoparticles have received much attention in the recent past. They have showed high antibacterial activity destructing microbial cells in a higher pH environment. ZnO nanoparticles has shown to be efficient against both Gram-negative and Gram-positive pathogens.
ZnO nanoparticles also has other various significant features such as high catalysis activity, chemical, and physical stability, good antibacterial property as well as intensive ultraviolet and infrared adsorption with a broad range of applications as semiconductors, sensors, transparent electrodes, solar cells, etc.
Graphene consists of monolayer of carbon atoms arranged in the two-dimensional body. It has many unique properties such as excellent antimicrobial property, good mechanical properties, biocompatibility, and chemical stability. Over the past few years, appreciable research has been concentrated on this blooming synthetic antibacterial polymer to avert the bacterial infection and protect human health.,,
Since fluorinated graphene is a member of graphene-family, we could wager that FG has the same effect as graphene. Research on fluorinated graphene is still at the beginning and less reports of its application in the biomedical field.
The objective of this study was to investigate the antibacterial effect of fluorinated graphene on E. faecalis when incorporated into ZnO-based sealer. The null hypothesis was that fluorinated graphene has no antibacterial efficacy against E. faecalis.
| Subjects and Methods|| |
Preparation of fluorinated graphene
They are several methods to prepare fluorinated graphene. In this study, FG was prepared using Graphene oxide (GO) (HENGQIU TECH.INC.CHINA) by a hydrothermal method.
A total of 100 mg of GO was mixed in 80 ml of ultrapure water under magnetic stirring and the dispersion was ultrasonically treated for 60 min. The dispersion was then centrifuged to remove the insoluble substance. After this, 10 mL of hydrofluoric acid (40 wt%) and 10 ml of nitric acid (65 wt%) were added in the GO dispersion under magnetic stirring. The resulted clear solution was directly heated to evaporate water in an oil bath and white fluorinated graphene powder was achieved.
Sixty extracted human mandibular premolars with single canals were used (extracted for periodontal or orthodontic reasons). Tissue remnants and debris were removed from all teeth surfaces and sterilized in an autoclave at 120°C for 20 min. The teeth were stored in normal saline solution to avoid any dehydration till used.
Access cavity preparation was done in each tooth using high speed round and endo-z burs under water coolant, the pulp was extirpated and the working length was measured by penetration of # 10 K-file into the root canal until appeared from the apical foramen and then 1 mm was subtracted from the measurement to confirm the working length.
All the canals were enlarged till size# 35 K-file with irrigation by normal sterile saline for 2 min. Canals were then dried using sterile paper points # 35.
Epoxy resin was coated on the outer surface at apical 3 mm and left to dry to block the root canal foramina thus eliminating any chance of bacterial leakage during inoculation, bio-mechanical and obturation procedures.
For easy handling and identification, each tooth was set vertically inside a sterile Eppendorf by friction. The interface between the tooth and Eppendorf was filled with epoxy resin to prevent leakage and then sterilized to avoid any contamination with other microorganisms.
Preparation of the Enterococcus faecalis suspension 
The bacterial suspension was prepared by sub-culturing the standard strain of E. faecalis bacteria on blood agar plates and was incubated in aerobic condition for 24 h at 37°C.
The suspension was vortexed to achieve a homogeneous suspension of bacterial solution and was calibrated compared with 0.5 McFarland turbidity standards (1.5 × 108 CFU/ml).
Inoculation of the root canals with the Enterococcus faecalis suspension 
Outer surface of the Eppendorf's was covered with parafilm to avoid contamination of the bacterial suspension.
Using 1 ml tuberculin syringe the root canals were completely filled with 250 μL of bacterial suspension, a sterile K-file #15 was used to carry suspension to the full working length of the canals. Then, the samples were placed vertically inside a closed sterile plastic container.
Samples were incubated at 37°C for 24 h. Initial culture sample was taken using sterile paper points after 24 h. Each sample was then taken and placed on blood agar plate surface, and again incubated aerobically at 37°C for a day.
Each root canals were then obturated using the lateral condensation technique.
A ZnO sealer mixed with different nanoparticles and standardized gutta-percha was used.
Paper points were placed inside capped tubes containing 0.5 ml sterile saline and vortexed to form homogeneous mix. Teeth were placed in incubator at 37°C till the testing period.
The root canals were divided into three groups according to the type of tested sealer.
- Group 1: ZnO-based sealer (control group) (Eugentin, Techno Dent Co., Ltd., Russia)
- Group 2: ZnO-based sealer mixed with fluorinated graphene
- Group 3: ZnO-based sealer mixed with ZnO nanoparticles. (Sigma Aldrich Pvt. Ltd.).
According to the timing of filling removal and culture sample each group was subdivided in to two subgroups each of ten teeth:
- Subgroup A: Obturation was removed and culture sample were taken after 1 week
- Subgroup B: Obturation was removed and the culture sample was taken after 3 weeks
- Standard H-file # 35 was used to remove gutta-percha and sealer, and then final irrigation was performed using sterile saline solution.
Sampling technique and microbiological processing
Three bacterial samples were taken for each tooth:
- S1: After incubation period of 24 h and before biomechanical preparation
- S2: After 1 week of removal of tested sealer
- S3: After 3 weeks of removal of tested sealer.
Each paper point was placed in the canal to the full working length and left for 10s to gather the root canal substances and then shifted into the screw-capped tube. The homogeneous suspension was obtained by vertexing the screw-capped tube with 0.5 ml saline and paper point taken from each canal. 10 μ of bacterial suspension was obtained from each screw-capped tube by streaking on the corresponding blood agar plates. Moreover, then samples were incubated aerobically at 37°C for 24 h. After the incubation period, the grown colony-forming units (CFUs) were counted manually.
Data were statistically analyzed using the Kruskal–Wallis test to compare the significance among the groups (1, 2, and 3), Mann–Whitney U-test was applied to find significance between the groups and Wilcoxon signed-rank test was applied to find significance between the time intervals in each group. P < 0.05 was considered as statistically significant.
| Results|| |
In comparison to initial samples, the CFU of 1 week and 3 weeks samples taken after obturation showed a significant reduction in the number of bacterial counts [Table 1] and [Figure 1] but when compared with Group 2 and 3, CFU was significantly high (P < 0.01) [Table 2]. The CFU values were seen to increase in the 3rd week (P < 0.01).
|Table 1: Comparison between the percentage of colony forming units of Enterococcus faecalis 24 h after inoculation versus 1 week and 3 weeks after obturation with zinc oxide sealer only|
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|Figure 1: Comparison between the percentage of colony forming units of Enterococcus faecalis 24 h after inoculation versus one week and three weeks after obturation with zinc oxide sealer only|
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In comparison with the initial samples, CFU of 1 week and 3 weeks after obturation showed a significant reduction in the number of bacterial counts [Table 3] and [Figure 2]. Statistically less CFU were seen when compared with Group 1 and 2 (P < 0.01) [Table 2], and the counts remained constant during the 1st and 3rd week (P > 0.01).
|Table 3: Comparison between the percentage of colony forming units of Enterococcus faecalis 24 h after inoculation versus 1 week and 3 weeks after obturation with zinc oxide sealer mixed with fluorinated graphene|
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|Figure 2: Comparison between the percentage of colony forming units of Enterococcus faecalis 24 h after inoculation versus 1 week and 3 weeks after obturation with zinc oxide sealer with fluorinated graphene|
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In comparison to initial samples, CFU of 1 week and 3 weeks after obturation showed a significant reduction in the number of bacterial counts [Table 4] and [Figure 3]. CFU values was low when compared with Group 1 (P < 0.01) but slightly high when compared to Group 2 (P < 0.01) [Table 2]. There was a slight increase in the CFU counts in the 3rd week which was insignificant (P > 0.01).
|Table 4: Comparison between the percentage of colony forming units of Enterococcus faecalis 24 h after inoculation versus 1 week and 3 weeks after obturation with zinc oxide sealer mixed with zinc oxide nanoparticles|
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|Figure 3: Comparison between the percentage of colony forming units of Enterococcus faecalis 24 h after inoculation versus 1 week and 3 weeks after obturation with zinc oxide sealer mixed to zinc oxide nanoparticles|
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| Discussion|| |
Sealers with antimicrobial properties are used nowadays to eliminate microbes like E. faecalis after the chemo mechanical preparation of the root canal system. The antimicrobial activity of endodontic sealers, could arrest the re-penetration of bacteria and prevent their regrowth in the root canal, thus preventing recontamination.,
Of late, incorporation of nanoparticles has shown brilliant results like the enhanced spectrum of activity when conjugated with other agents.
Fluorinated graphene, a member of graphene family has recently found to have an effective antibacterial activity against dental pathogens. Emami-Karvani et al., 2011 showed that ZnO nanoparticles have effective action against both Gram-positive and Gram-negative microorganisms. Hence, the future application of nanoparticles appears bright.
In the present study, the efficacy of fluorinated graphene and ZnO nanoparticles as antibacterial agents was evaluated against E. faecalis. These materials were compared with the traditional endodontic sealer, i.e., ZnO-eugenol sealer.
From the present study, it can be inferred that sealer mixed with fluorinated graphene (Group 2) showed the highest antibacterial activity against E. faecalis followed by sealer mixed with ZnO nanoparticles (Group 3) and ZnO-based sealer (Group 1) showed the lowest antibacterial activity [Table 5] and [Figure 4], [Figure 5].
|Table 5: Comparison between the amount of bacterial count reduction 1 week and 3 weeks after obturation in comparison to bacterial count 24 h after inoculation of bacteria between the three tested groups|
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|Figure 4: Comparison between the amount of bacterial count reduction one week and three weeks after obturation in comparison to bacterial count 24 h after inoculation of bacteria between the three tested groups|
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|Figure 5: Colony forming units of the all the groups. (a) After 24 h, (b) after one week and (c) after three weeks of obturation|
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There was a significant reduction of CFUs after 1 week from obturation with conventional ZnO-eugenol sealer. This was in accordance with studies done by Kaiwar et al., Anumula et al., Gomes et al., and Ibraheem and Salman. The antibacterial activity of ZnO eugenol-based sealer against E. faecalis could be accredited to the presence of eugenol as the main element. Eugenol, a phenolic compound denatures proteins present in the microorganisms leading to its death.
Fluorinated graphene incorporated to ZnO-based sealer (Group 2), showed a significant reduction in the CFUs after 1 week of obturation compared to Group 2 and Group 3. Sun et al., 2017 found that the antibacterial activity of GIC can be enhanced by adding the fluorinated graphene. Akhavan and Ghaderi, 2010 and Pham et al., 2015 believed that the antibacterial property of fluorinated graphene is caused by both physical and chemical effects. The physical action is by the destruction of the bacteria cell membrane by the sharp edges of graphene nanosheets in suspensions., The chemical effect is primarily due to the oxidative stress created by reactive oxygen species and extraction of phospholipids from the cell membrane., Wang et al., 2016 showed a high antibacterial activity of fluorinated graphene with 99.9% viability loss in Escherichia More Details coli.
ZnO nanoparticles incorporated in ZnO eugenol sealer (Group 3), showed a significant reduction in the CFUs after 1 week of obturation compared to Group 1. This could be attributed to the destructive action of ZnO nanoparticles with the bacterial cells. ZnO nanoparticles penetrates the bacteria and causes cell death by interacting with their enzymes leading to the increased production of reactive oxygen such as hydrogen peroxide., These results were in accordance with Kishen et al. and Shrestha et al.
Among all experimental groups, 3 weeks after obturation the CFUs were significantly increased in Group 1 and very slightly increased in Group 3 (P > 0.05). In Group 2, they were nearly constant (P > 0.05). The results in Group 2 and Group 3 could also be attributed to their particle sizes. Size of the nanoparticles is also responsible for its antibacterial activity. This leads to significant increase in the surface contact and the interaction between the negatively charged bacterial cell and the positively charged nanoparticles.
The ZnO-based sealer incorporated with fluorinated graphene showed better antibacterial properties when compared with the plain sealer and the sealer which was incorporated with ZnO Nanoparticles at both 1 week and 3 weeks intervals this may be due to the reason that graphene derivatives locate at the site where the bacterial cells were enriched. This shows that there is a strong interaction between the graphene and the bacterial cells. The hydrophobic sp2 carbon atoms in fluorinated graphene have a strong interaction with the lipid bilayer of cell membranes of bacteria. There are many studies that reports the strong affinity of bacteria toward hydrophobic surfaces than hydrophilic surfaces. Highly fluorinated graphene sheets have many hydrophobic areas which attract the bacteria onto the sheets. Furthermore, Akhavan et al. and Liu et al. reported that graphene sheets have the capability of wrapping the bacteria, isolating them from any growth medium, thus leading the bacterial cells to neither consume the nutrient nor proliferate.
| Conclusion|| |
Within the limitations of this study all the sealers used proved to be having antibacterial properties.
Sealer mixed with different nanoparticles has a superior antibacterial effect than pure ZnO-based sealer.
Among the three groups, Group 2, i.e., sealer mixed with fluorinated graphene showed the best antibacterial effect during all time periods. Further studies on fluorinated graphene are necessary to determine its long-term clinical performance.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]