Physico-Mechanical Properties, Antimicrobial Activities, and Anti-Biofilm Potencies of Orthodontic Adhesive Containing Cerium Oxide Nanoparticles against Streptococcus mutans

Introduction: White spot lesions around orthodontic brackets may lead to the formation of dental caries during and following fixed orthodontic treatment. Aim: This study aimed to evaluate the physico-mechanical properties and antimicrobial potencies of orthodontic adhesive doped with cerium oxide nanoparticles (CeO 2 -NPs) against Streptococcus mutans . Materials and methods: After synthesis and conformation of CeO 2 -NPs by transmission electron microscope (TEM), shear bond strength (SBS) and adhesive remnant index (ARI) of modified orthodontic adhesive containing different concentrations of CeO 2 -NPs (0, 1, 2, 5, and 10 wt%) were measured. The antimicrobial effects of modified orthodontic adhesive were evaluated by disk agar diffusion method and biofilm formation inhibition assay. Results: The pseudo-spherical shapes of CeO 2 -NPs were observed in TEM micrographs. The physico-mechanical finding showed that 5 wt% CeO 2 -NPs showed the highest concentration of CeO 2 -NPs and SBS value (18.21±9.06 MPa, p <0.05) simultaneously with no significant differences in ARI compared with the control group ( p >0.05). There was a significant reduction in cell viability of S. mutans with increasing CeO 2 -NPs concentration. The 3.1 Log 10 and 4.6 Log 10 reductions were observed in the count of treated S. mutans with 5 and 10 wt% CeO 2 -NPs, respectively ( p <0.05). Conclusions: Overall, an orthodontic adhesive containing 5 wt% CeO 2 -NPs had antimicrobial properties against S. mutans without adverse effects on SBS and ARI.


INTRODUCTION
The composite resin bonding method in orthodontics is mainly used to attach the bracket to the tooth surface. [1] In direct orthodontic bonding, the brackets are placed directly and individually on the enamel of each tooth. This method requires more time than the indirect bonding method. The most important advantage of direct bonding is to make sure that the brackets are properly attached and placed on the tooth. [2,3] Unfortunately, despite the advantages of bonding such as high beauty and easy technique, this method has some disadvantages such as plaque accumulation, white lesions, and broken band. [4] These disadvantages prolong treatment, increase the duration of clinical work and the cost of treatment. Several methods have been introduced to prevent biofilm formation and tooth decay. One method is to add antimicrobials to the composite resin. [5] Nanoparticles have high antimicrobial properties due to their small size. In addition to knowing the antimicrobial effects of nanoparticles, their effects on the bond strength between the bracket and the composite or bond strength of orthodontic cement are also important. [6] Oral streptococci, especially Streptococcus mutans as the most important member of Viridans streptococci, are known to cause tooth decay and subsequent diseases by synthesizing extracellular polymers and forming biofilms on dental surfaces. [7] Recently, the antimicrobial properties of cerium oxide nanoparticles (CeO 2 -NPs) and their applications in the field of medicine and other sciences have been considered, and the decision to combine these nanoparticles with resin composites used in orthodontics was taken. [8] To the best of our knowledge, no studies are available regarding the antimicrobial efficacy of orthodontic adhesive doped with CeO 2 -NPs against any cariogenic bacteria.

AIM
Therefore, this study aimed to investigate the antimicrobial and anti-biofilm properties of orthodontic adhesive doped with CeO 2 -NPs, as well as maintenance of sufficient shear bond strength (SBS) of orthodontic light-curing composite toward the eradication of S. mutans. Therefore, we tested the hypothesis that CeO 2 -NPs can act as an antimicrobial and anti-biofilm agent against S. mutans biofilm culture.

Synthesis of CeO 2 -NPs
CeO 2 -NPs were synthesized using a modified hydrothermal method. [9] Briefly, 0.22 g of cerium(III) nitrate hexahydrate (Sigma-Aldrich, Steinheim, Germany) was dissolved in 75 μL of trisodium phosphate dodecahydrate (0.02 g/ mL) (Sigma-Aldrich, Steinheim, Germany) and 20 mL of deionized water and stirred vigorously for 4 h at room temperature. The mixture was then placed into a stainless steel autoclave at 170°C for 12 hours. The mixed solution was cooled and a white precipitate was isolated by centrifugation at 10000 rpm for 10 min. The supernatant was decanted away and the white CeO 2 was then washed repeatedly with deionized water and ethanol. Finally, the product was freeze-dried overnight. Synthesized CeO 2 -NPs morphology was observed and photographed using transmission electron microscopy (TEM; Zeiss EM10C, Germany) with an accelerating voltage of 100 kV.

Shear bond strength (SBS) testing
Twenty freshly extracted bovine incisors with intact buccal enamel and with no cracks or any lesions were collected, immersed in 0.5% of chloramine T trihydrate (Sigma Aldrich Chemie GmbH, Taufkirchen, Germany) at 4°C for seven days, and embedded in cold-cure acrylic resin according to ISO/TS 11405:2003. The surfaces of all teeth were cleaned, polished, and rinsed with air-water spray for 10 s and air-dried for 10 s. Thirty-five percent of phosphoric acid gel (Ultra etch; Ultradent Products Inc., South Jordan, UT, USA) was used to etch the buccal surfaces of all teeth. After 20 s, the teeth were rinsed with water for 10 s, and dried with air for 10 s. Then, the etched area of the buccal surface of all teeth was covered with a thin layer of CeO 2 -NPs at the different concentrations (1, 2, 5, and 10 wt%) and cured with a LED light-curing unit (Demetron, Kerr, Orange, CA, USA). According to the Felemban and Ebrahim study [11] , the orthodontic metal brackets were used to bond all teeth. Based on the ISO/TS 11405:2015 guideline, all teeth were stored in distilled water at 37°C for 24 hours, thermocycled 3000 times in a water bath between +5°C and +55°C, and remained in each reservoir for 30 s after the bonding procedures. Zwick/Roell, Germany with a speed of 1.0±0.1 mm/min in occlusal-gingival direction at the bracket-tooth interface was used as a mechanical testing machine for SBS testing (Fig. 1). Finally, the values of SBS were calculated in MPa as described previously. [11] Folia Medica I 2022 I Vol. 64 I No. 2

Adhesive remnant index (ARI)
ARI score in debonding of stainless-steel brackets from enamel surface was assessed by a stereomicroscope (SMZ800, Nikon, Tokyo, Japan) at ×10 magnification based on the Oliver and Griffiths study. [12] Microorganism and growth conditions S. mutans (ATCC 35668) was cultured in brain heart infusion (BHI) broth (Merck, Darmstadt, Germany) supplemented with 0.1% sucrose and incubated in an aerobic atmosphere with 5% CO 2 at 37°C. A 0.5 McFarland standard bacterial suspension (1.5×10 8 colony forming units (CFUs)/mL) was prepared to examine the antimicrobial efficacy of orthodontic adhesive doped with CeO 2 -NPs.

Preparation of modified orthodontic adhesives samples
Disk-shaped modified orthodontic adhesive patterns were prepared using metal molds 5 mm in diameter and 1 mm thick. Modified orthodontic adhesive disks containing different concentrations of CeO 2 -NPs were made by the fabrication of CeO 2 -NPs adhesives section. According to the ISO 11135:1994, prepared disks were then exposed to light cure for 40 s, de-molded, polished, and sterilized. [10] Antimicrobial testing

Disk agar diffusion test
Disk agar diffusion test by the Kirby-Bauer method was done to determine the antimicrobial features of orthodontic adhesive by zones of growth inhibition around each of the samples. [13] 1.5×10 8 CFU/mL of S. mutans was swabbed onto the surface of Mueller-Hinton agar (Merck, Darmstadt, Germany) plates (i.e., 100-mm plate diameter). The size of the growth inhibition zone around the disks was measured after overnight incubation at 37°C in an aerobic atmosphere with 5% CO 2 .

Biofilm inhibition test
Twenty-five orthodontic adhesive disks with different concentrations of CeO 2 -NPs were placed in flat-bottomed 48well microtiter plates containing S. mutans suspension with a concentration of 1.5×10 8 CFU/mL. To form biofilms on the disks, the microtiter plate was incubated under the aerobic atmosphere with 5% CO 2 at 37°C for 72 hours. Afterwards, disks were rinsed in 1 mL of sterile deionized water for 1 min to remove planktonic microbial cells. Orthodontic adhesive disks were then vortexed severely in 1 mL of BHI broth for 30 s. The obtained bacterial suspensions were serially diluted, cultured in mitis salivarius agar (Merck, Darmstadt, Germany), and the microbial colony counts were determined as mentioned in the previous study. [14,15]

Statistical analysis
The microbial experiments were done in triplicate and the data were analysed using one-way analysis of variance (ANOVA). Statistical analysis was performed using SPSS for windows version 23.0 (SPSS Inc., Chicago, IL, USA). P values <0.05 were considered statistically significant.

The morphologies of synthesized CeO 2 -NPs
TEM analysis has confirmed the general structure of the synthesized CeO 2 -NPs (Fig. 2). The presence of aggregates with pseudo-spherical shapes was observed in TEM micrographs.

SBS test
The data of SBS of orthodontic adhesive doped with different concentrations of CeO 2 -NPs are presented in Table  1. As shown, 1 wt% CeO 2 -NPs revealed the highest value of SBS (30.42±11.15 MPa, p>0.05). Also, the lowest SBS value (7.75±2.43 MPa, p<0.05) was reported in 10 wt% CeO 2 -NPs. According to the results, the SBS values decreased following an increase in the concentration of CeO 2 -NPs.

ARI score
The frequencies of ARI scores in the test groups are shown in Table 2. There was no significant difference in the ARI scores between different concentrations of CeO 2 -NPs in orthodontic adhesive disks and the control group (p<0.05).

Disk agar diffusion assay
The antimicrobial property of orthodontic adhesive disks containing different concentrations of CeO 2 -NPs was assessed using the release of nanoparticles from the disks. No growth inhibition zone was observed around any of the disks. This indicates that the CeO 2 -NPs were not able to be released at the plate surface.

Effects of CeO 2 -NPs on biofilm formation inhibition
As shown in Fig. 3A, there was a considerable decrease in cell viability of S. mutans with increasing CeO 2 -NPs concentration. The results exhibited that 5 and 10 wt% CeO 2 -NPs significantly reduced S. mutans to 3.1 Log 10 and 4.6 Log 10 , respectively (p<0.05). The effect of different concentrations of CeO 2 -NPs on cell viability of S. mutans is shown in Fig.  3B.

DISCUSSION
Bonding brackets to teeth is one of the common methods in orthodontic treatment. [16] One of the important complications of fixed orthodontic appliances, such as brackets, in which dental composites are used to attach them, is the breaking of the composite bond due to the forces applied to the bracket, as well as increasing the risk of plaque accumulation and expansion and subsequent caries around the brackets. [17] Therefore, it is necessary to have a composite which, while having suitable mechanical properties, can reduce the amount of dental plaque microorganisms and reduce dental demineralization. Another complex problem in fixed orthodontic treatments is the control of enamel demineralization around the brackets used during treatment. Brackets and various tools used in orthodontic treatments make oral hygiene more difficult and, according to some studies, increase the number of oral bacteria. [18] Previous studies have also shown that the rate of demineralization of enamel, white spots, and tooth decay are much higher in people who have received orthodontic treatment. [19][20][21][22] It should be noted that the composites used in orthodontics have a polymer matrix that is involved in the accumulation of aerobic and anaerobic microorganisms. The formation of supragingival biofilm is mainly seen around orthodontic attachments in clinical studies. The main cause of dental caries is S. mutans. S. mutans is a Gram-positive, optional anaerobic coccus that is the oral cavity flora in humans. [23,24] Any imbalance of the oral microflora will lead to oral diseases. S. mutans causes damage to tooth enamel by fermenting sucrose and producing lactic acid. The bacterium also uses sucrose to make dental plaque. Dental plaque is made from dextran, a type of polysaccharide. [25] Chin et al. [26] found that S. mutans and Lactobacillus acidophilus have the ability to bind to orthodontic bonding agents and colonization during orthodontic treatment, and with increasing the load of these bacteria, the prevalence of caries increases.
Nanotechnology can be used effectively to maintain oral health. In particular, nanoparticles are useful antimicrobial agents for bonding and orthodontic appliances. They can also be used in dental restorations such as cavities, sealants, and root canals. Its antimicrobial ability reduces plaque around the brackets, which can prevent decay during treatment. [27,28] Due to the problem of decalcification and decay around orthodontic brackets, various research studies have been done on the effect of using antimicrobial and anti-decay materials. [29][30][31][32] Due to the variety of studies performed so far, no study has been performed on the antimicrobial effect of CeO 2 -NPs on reducing the rate of S. mutans in patients using fixed orthodontic appliances.
The relevant literature shows that CeO 2 -NPs are widely used as a catalyst in industry and as an antioxidant in applied nanomedicine. The antimicrobial mechanism of CeO 2 -NP action probably occurs via oxidative stress of components in the microbial cell membrane and accumulation of oxygen reactive species in microbial cells. [33][34][35] In the present study, the effect of CeO 2 -NPs on physico-mechanical properties including SBS testing and ARI were de-termined. Based on the results, by addition of CeO 2 -NPs up to 5%, the SBS of Transbond XT composite to enamel was within the clinically acceptable score without remarkable changes. In contrast, the SBS in 10% CeO 2 -NPs was significantly lower than that in the control group. As previously reported, the SBS in clinical conditions can be 40% less in vitro conditions. [36] Therefore, this makes our SBS results in the acceptable score that will be in vivo, adding 5 wt% CeO 2 -NPs can maintain the SBS for optimum clinical applications. Also, there is no considerable difference between the different concentrations of CeO 2 -NPs in terms of ARI scores, which was in agreement with the other studies. [36][37][38] Moreover, in this study, the antimicrobial potential of orthodontic adhesive doped with different concentrations of CeO 2 -NPs was evaluated. Similar to previous studies [36][37][38][39][40][41] , a progressive increase in the inhibition of microbial biofilm growth was revealed with increasing the concentration of CeO 2 -NPs. Although concentrations higher than 5 wt% CeO 2 -NPs could considerably decrease the growth and biofilm formation of S. mutans, the mean of SBS decreased.
Pelletier et al. [39] evaluated the growth and viability of Escherichia coli and Bacillus subtilis as the Gram-negative and Gram-positive species, respectively, relative to CeO 2 -NP. Bactericidal effects of CeO 2 -NP were determined using the minimum inhibitory concentrations (MIC) and CFU/mL measurements, disk agar diffusion test, and live/dead assays. Their results showed that the bacterial growth rates depended on CeO 2 -NP concentrations in the range of 50 to 150 mg/L. Also, the growth inhibition of E. coli and B. subtilis was observed. There was no significant difference in inhibiting the microbial growth of bacteria by CeO 2 -NPs at 50 µg/mL. In another study, the efficient and antibacterial application of CeO 2 -NP against Gram-positive and Gram-negative pathogens was assessed by Pop et al. [40] According to their findings, the growth inhibition toward all five pathogens tested including E. coli, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, and B. cereus was observed with notable results. The MICs of CeO 2 -NPs against pathogenic bacteria were 2.15, 1.07, 1.07, 10, and 4.3 mg/mL for E. coli, S. typhimurium, L. monocytogenes, S. aureus, and B. cereus, respectively. Several other studies have reported the antimicrobial effect of CeO 2 -NPs against E. coli and S. aureus. [8,34,35,41,42] The results of these studies showed that CeO 2 -NPs with the lowest concentration are able to inhibit significantly the growth of these microorganisms.
One of the important aspects of using nanoparticles is their toxicity. Although the evidence is insufficient, nanoparticles do not appear to be more toxic than conventional materials. Due to the wide scope of nanotechnology and the lack of studies on the effects of nanoparticles on SBS of orthodontic adhesives, as well as the physical properties of orthodontic acrylics, further studies are proposed to clarify these aspects. Also, clinical trial studies should be performed to confirm the anti-caries properties of orthodontic adhesive doped with CeO 2 -NPs.

CONCLUSIONS
In overview, this study demonstrated that 5 wt% CeO 2 -NPs as an orthodontic adhesive with a clinically acceptable score of SBS and ARI had antimicrobial and anti-biofilm activities against S. mutans. However, we acknowledged that further evaluation of these activities of cerium oxide nanoparticles against cariogenic bacteria in multi-species biofilm structure is warranted.