Original Article |
Corresponding author: Abbas Bahador ( abahador@sina.tums.ac.ir ) © 2022 Maryam Pourhajibagher, Roghayeh Ghorbanzadeh, Abbas Salehi-Vaziri, Abbas Bahador.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Pourhajibagher M, Ghorbanzadeh R, Salehi-Vaziri A, Bahador A (2022) In vitro assessments of antimicrobial potential and cytotoxicity activity of an orthodontic adhesive doped with nano-graphene oxide. Folia Medica 64(1): 110-116. https://doi.org/10.3897/folmed.64.e58600
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Introduction: Formation of white spots and initial carious lesions are the most important complications of fixed orthodontic treatment. Preparation of orthodontic adhesives containing antimicrobial agents might be a practical solution for the prevention of the mentioned defects.
Aim: The current study aims to assess the antimicrobial and cytotoxicity effects of a conventional orthodontic adhesive containing different concentrations of nano-graphene oxide (N-GO).
Materials and methods: 50 Transbond XT orthodontic adhesive discs containing 0, 1, 2, 5, and 10% N-GO were prepared and sterilized by 25 kGy Gamma irradiation. After determination of cytotoxicity potential of modified orthodontic adhesive on human gingival fibroblast (HGF) cells, antimicrobial effects of the modified orthodontic adhesive against Streptococcus mutans in the preformed cariogenic biofilms was investigated using eluted components from composite discs by comparing the viable counts of bacteria after 3, 7, 15, 30, and 60 days of the aging process in artificial saliva.
Results: Based on the results, there was no cytotoxic effects of modified orthodontic adhesive on HGF cells (p>0.05). Transbond XT orthodontic adhesive containing 5 and 10 wt% N-GO reduced considerably the mean total viable counts of S. mutans up to 30 days (p<0.05). However, at 60 days, only 10 wt% N-GO could statistically decrease the colony-forming unit (CFU)/mL of test microorganisms. Antimicrobial activity of eluted components from modified orthodontic adhesive discs against S. mutans was in line with the concentration of N-GO.
Conclusions: At 5% and 10% concentrations, a modified orthodontic adhesive containing N-GO has a significant antimicrobial activity against S. mutans in cariogenic biofilms.
Abbreviations: N-GO: nano-graphene oxide; CFU: colony-forming unit; WSL: white spot lesion; NPs: nanoparticles; S. mutans: Streptococcus mutans; BHI: brain heart infusion; SD: standard deviation; ZnO: zinc oxide
antimicrobial agents, composite resins, eluted component test, nano-graphene oxide, orthodontics, Streptococcus mutans
Enamel decalcification or a white spot lesion (WSL) adjacent to fixed orthodontic appliances are the most common complications (up to 96%) of orthodontic treatment which may increase the cost and increase the time of patient visits.[
Deliberate embedding of nanoparticles (NPs) in dental materials including resin-based composites and application of nanomaterials for improvement in dentistry has been much lauded and rapidly gaining significant importance. Inorganic NPs, including nano-graphene oxide (N-GO), are the most commonly used as antimicrobial agents, which are more encouraging.[
Ever since the observation of the antimicrobial and anti-biofilm activities of N-GO in 2010, the interest in antimicrobial N-GO materials is rapidly growing. It has been shown that the sharp edges of N-GO can vigorously rupture and puncture the microbial membrane via physicochemical processes (a.k.a. “nanoknife” mechanism) resulting in microbial cells death.[
Consequently, the current experimental study aimed to assess the antimicrobial activity of a conventional orthodontic composite resin blended with N-GO against S. mutans in the preformed cariogenic biofilms.
N-GO was prepared using Hummers method.[
Field emission scanning electron microscopy (FESEM; Zeiss, Sigma VP, Germany, 15 kV accelerating voltage) with ImageJ program was used to determine the morphology of N-GO.
Orthodontic adhesive Transbond XT (3 M Unitek, Monrovia, CA) and Transbond XT supplemented with different concentration of N-GO (0, 1, 2, 5, and 10%) were used as the original and the test materials, respectively. The orthodontic adhesive was prepared using a mixing spatula on a glass slab in a moderately dark room until a uniform consistency was achieved. FESEM was used to determine the uniform consistency of modified adhesive. Metal molds (5 mm in diameter and 1 mm thick) were used to make disc-shaped orthodontic adhesive samples containing different concentrations of N-GO (Fig.
Human gingival fibroblast cells (HGF, CELL No. IBRC C10459) were obtained from the National Center for Biological Genetic Resources of Iran. The cell line was cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium (Gibco, Germany) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/mL penicillin, 100 μg/mL streptomycin, and 100 μg/mL amphotericin B (all purchased from Sigma-Aldrich, Steinheim, Germany). Cells were grown at 37°C with 5% CO2/95% air in a humidified incubator. The culture medium was changed every three days. In the two subculture, HGF cells with 1×105 cells/mL were seeded in a flat-bottomed 96-well cell culture microplate (JET BIOFIL®, Jet Bio-Filtration Co., Ltd, and Guangzhou, China) and allowed to attach for 24 h in a humidified incubator at 37°C in the presence of 5% CO2. After 24 h of incubation, HGF cells were treated with different concentrations of N-GO (0, 1, 2, 5, and 10%). Finally, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to determine the cytotoxicity effects of N-GO on HGF cells and cell viability was measured at 570 nm using a spectrophotometric microplate reader (BioTek Elx 808, USA).[
Streptococcus mutans
(ATCC 35668) was grown in brain heart infusion (BHI) broth (Merck, Darmstadt, Germany) under capnophilic atmosphere (aerobic plus 5% CO2) at 37°C for 48 h. To assay the antimicrobial efficacy of the modified orthodontic adhesive doped with N-GO by an eluted component test against S. mutans, the cariogenic biofilms were prepared as described previously.[
This assay was used to evaluate the antimicrobial effect of N-GO released from modified orthodontic adhesive samples according to a previous study.[
Statistical analysis was performed using SPSS version 22.0 (SPSS Inc., Chicago, IL, USA). Tukey’s post hoc analysis was used for the pairwise comparisons of the experimental groups. The significance level was set at p<0.05.
The morphology of N-GO was confirmed by FESEM analysis. As demonstrated in Fig.
The cytotoxicity of HGF cells non-targeted and HGF cells-targeting N-GO was evaluated. The results of MTT assay showed that there was no significant cytotoxic effects against HGF cells (p>0.05), which suggested that N-GO had no effect on eukaryotic cells (Fig.
Table
Number of colony-forming units (CFU) per milliliter of S. mutans (mean ± SD) in the different treatment groups compared with the control group in eluted component test
Days | Concentration (%) | S. mutans | |
Mean ±SD* of Log10 CFU/mL | P value | ||
Control | 6.23±0.07 | - | |
3 | 1 | 5.88±0.38 | 0.122 |
2 | 5.53±0.40 | 0.071 | |
5 | 2.97±0.09 | 0.001* | |
10 | 2.65±0.09 | 0.001* | |
7 | 1 | 5.50±0.41 | 0.068 |
2 | 5.26±0.50 | 0.064 | |
5 | 3.05±0.15 | 0.001* | |
10 | 2.83±0.12 | 0.000* | |
15 | 1 | 5.18±0.77 | 0.141 |
2 | 4.83±0.48 | 0.055 | |
5 | 3.01±0.12 | 0.000* | |
10 | 2.56±0.14 | 0.000* | |
30 | 1 | 5.36±0.50 | 0.072 |
2 | 4.65±0.80 | 0.070 | |
5 | 3.19±0.13 | 0.002* | |
10 | 2.97±0.13 | 0.001* | |
60 | 1 | 5.77±0.36 | 0.125 |
2 | 5.31±0.38 | 0.064 | |
5 | 4.57±0.59 | 0.051 | |
10 | 2.84±0.09 | 0.000* |
To avoid enamel decalcification or WSL adjacent to fixed orthodontic appliances and maintain treatment success during fixed orthodontic treatment, modified orthodontic adhesive containing antimicrobial agents are used to resist the accumulation and formation of biofilm of cariogenic bacteria including S. mutans, followed by white spot lesions and the risk of tooth decay around the brackets. Thus, adequate control of oral cariogenic bacteria has a critical role in successful orthodontic treatments.[
In the current study, we assessed the effect of incorporation of N-GO on antimicrobial effects of the orthodontic adhesive. N-GO has an intrinsic antimicrobial effect, minimum cytotoxicity, high biocompatibility, and gray colour which are less likely to alter its esthetic.[
The results of the current study revealed that adding up to 10% wt. N-GO, the antimicrobial effect of Transbond XT composite against S. mutans changes significantly in comparison with the control group. This clinically important property of N-GO incorporated modified orthodontic adhesive is in line with the study conducted by Sodagar et al.[
Our findings are consistent with a recent Sodagar et al. study[
Passariello et al.[
In the current study, despite applying the ageing process of modified test material and the use of artificial saliva, in interpreting the results, it should be highlighted that the study design did not cover all variability of the physico-chemical conditions of the oral environment including complex oral microbiome that may affect the antimicrobial effectiveness of the test materials in vivo. Colour stability and polymerization shrinkage of adding N-GO on orthodontics adhesive could be considered in the future.[
We present evidence that 5% and 10% wt. N-GO can serve as an orthodontic adhesive additive with antimicrobial effect for controlling S. mutans growth in cariogenic biofilms. However, low-solubility of N-GO remains a major drawback.
This study was supported by a grant from the National Institute for Medical Research Development (NIMAD, Grant No. 971197).
This study was approved by the Ethics Commission of IR.NIMAD.REC.1397,101, 25.
Not applicable.
The authors declare that they have no competing interests.