The Antibacterial Effects of Resin-Based Dental Sealants: A Systematic Review of In Vitro Studies

This review aimed to assess the antimicrobial effects of different antibacterial agents/compounds incorporated in resin-based dental sealants. Four databases (PubMed, MEDLINE, Web of Science and Scopus) were searched. From the 8052 records retrieved, 275 records were considered eligible for full-text screening. Nineteen studies met the inclusion criteria. Data extraction and quality assessment was performed by two independent reviewers. Six of the nineteen included studies were judged to have low risk of bias, and the rest had medium risk of bias. Compounds and particles such as zinc, tin, Selenium, chitosan, chlorhexidine, fluoride and methyl methacrylate were found to be effective in reducing the colony-forming unit counts, producing inhibition zones, reducing the optical density, reducing the metabolic activities, reducing the lactic acid and polysaccharide production and neutralizing the pH when they are added to the resin-based dental sealants. In addition, some studies showed that the antibacterial effect was not significantly different after 2 weeks, 2 months and 6 months aging in distilled water or phosphate-buffered saline. In conclusion, studies have confirmed the effectiveness of adding antibacterial agents/compounds to dental sealants. However, we should consider that these results are based on laboratory studies with a high degree of heterogeneity.


Introduction
Dental caries is a highly prevalent chronic disease, affecting more than 60% of school children [1,2]. Caries is a biofilm-sugar-dependent disease. The bacteria in the biofilm over the teeth metabolize fermentable carbohydrates and produce acids [3]. The acids demineralize the hydroxyapatite (HAp) of dental tissues (enamel and dentin), leading to an irreversible process over time [3].
The loss of minerals is considered as an unbalance in the natural demineralizationremineralization process of teeth, and it causes caries lesions to have several stages. In the early stages, carious lesions have subclinical characteristics [4]. However, if the biofilm persists over the teeth due to poor oral hygiene and frequent consumption of fermentable carbohydrates such as sucrose, there will be a periodic reduction in the pH of the oral environment [4]. In such scenarios, dental caries becomes clinically visible in the form of white-spot lesions [4]. Initial non-cavitated white lesions may become cavitated if the process of demineralization continues without the process of remineralization. Although these carious lesions usually get restored, recurrent caries could appear around the restorations [5]. Each time the restoration is replaced, dental tissue is eventually removed. This

Search Strategies
The search strategies of the four electronic databases are described in Table 1. They were developed and applied by three authors (M.S.A, M.I.A. and M.S.I.). The last search was run on 1 June 2020. No date or language restriction was applied at this stage. The resulting citations from all databases were imported to Covidence online platform for screening. (sealant* OR sealing* OR (pit AND fissure)) #3 #1 and #2 Database: SCOPUS #1 (TITLE-ABS-KEY (sealant* OR sealing* OR (pit AND fissure))) #2 (TITLE-ABS-KEY (antibacterial OR antimicrobial OR remineral* OR hydroxyapatite OR calcium* OR fluorid* OR bioactiv* OR biofilm*)) #3 #1 and #2

Inclusion and Exclusion Criteria
Studies included in this review were in vitro, laboratory studies that assessed the antimicrobial activities of resin-based dental sealants. Studies that are not laboratory studies, studies that did not have resin-based sealants, studies that only tested resinmodified glass ionomer and studies that did not assess any antibacterial activity were excluded. The compositions on commercial resin-based sealants were searched through the safety data sheet of the material itself or the literature to confirm that the sealant is a resin-based material.

Studies Screening and Selection
Selection of the studies was performed using a pair of reviewers (M.S.A., M.I.A. and M.A.A.). Reviewers were not blinded to the identity of the authors or journal. The selection process consisted of title and abstract screening then full text screening. If any of the exclusion criteria were found, the record was excluded. Disagreements among reviewers were solved by a senior reviewer (M.S.I.).

Data Extraction
Two reviewers (S.S.A and M.S.I.) extracted data regarding various variables from the included studies using customized data collection forms. The extracted data included qualitative and quantitative data. The following data were extracted: the details of the sample including sample size, type, measurements and curing time, assessment methods used to assess the antibacterial effect, other properties assessed beside antibacterial properties, the antibacterial agents, bacteria or type of inoculum used and the control and tested (interventional) groups details. In addition, data about the outcome measures, in regard to units and effects, were extracted.

Quality Assessment
The risk of bias of the included studies was assessed by three independent reviewers (S.S.A, M.S.A and M.S.I.). The assessment tool was adapted from previously published scoping and systematic reviews [25,26]. Studies with one to three "Yes" only were considered to have a low risk of bias. Studies scoring four to six "Yes" or seven to nine "Yes" were considered to have a medium risk of bias or a high risk of bias, respectively.

Assessment of Heterogeneity
Two reviewers (S.S.A. and M.S.I.) extracted data about the interventional, methodological and statistical heterogeneities of the included studies. Interventional heterogeneity was assessed by checking the differences in compositions of the tested groups and control groups among the included studies, such as the percentage and type of the fillers and incorporation of other agents such remineralizing agents. Methodological heterogeneity was assessed by extracting data in regard to the sample types, curing time, bacteria type or inoculum source, the methods of outcome assessment and the study's overall risk of bias.

Data Synthesis
A qualitative summary of the assessment methods, interventions, outcomes and any additional relevant information was planned to be reported. We also planned to perform a quantitative meta-analysis using a fixed-effect model or a random-effect model if an I 2 statistics at or below 50% was found with no significant methodological heterogenicity or an I 2 statistics was found to be above 50% with no significant methodological heterogenicities, respectively. On the other hand, if a significant statistical heterogeneity or a methodological heterogenicity was found, a meta-analysis was not planned to be conducted.

Studies Selection
A total number of 10,103 potentially relevant records were retrieved from the four databases. Duplicates were initially removed by Covidence.org and 8052 records included for abstract and title screening. Two hundred and seventy-five records were eligible for full-text screening. Nineteen records met the inclusion criteria and were included in the review. The screening process is reported in Figure 1.

Risk of Bias Appraisal
Out of the 19 included studies, six were judged to have low risk of bias, and the rest were of medium risk of bias (Table 2). Sample size calculation and blinding were not reported in all of the included studies, which led to a positive risk of bias in these two parameters ( Figure 2). Studies always reported the sample preparation or measurements and there was always at least one method of quantitative assessment ( Figure 2).

Risk of Bias Appraisal
Out of the 19 included studies, six were judged to have low risk of bias, and the rest were of medium risk of bias (Table 2). Sample size calculation and blinding were not reported in all of the included studies, which led to a positive risk of bias in these two parameters ( Figure 2). Studies always reported the sample preparation or measurements and there was always at least one method of quantitative assessment ( Figure 2).

Studies Characteristics
A summary about the characteristics of the 19 included studies is reported in Table 3.

Samples
Out of the 19 included studies, only one study [29] used human teeth samples and one study used bovine teeth samples [37], while most of the studies used discs samples of the same material. There was a large variation between the studies in regard to the number of samples used to assess the antibacterial effect. This variation was also found between different tests in the same study. The light-curing/polymerization time was reported in most of the studies and showed variation. However, five studies [10,13,32,39,40] did not report any specific time.

Bacteria and Inoculum
Most studies (17 studies) tested the antibacterial activities of the sealants against S. mutans. One study [21] tested the effect against multispecies biofilms cultured from pooled saliva of high-caries risk and low-caries risk pediatric dental patients, and another study tested the effect against Enterococcus faecalis [29]. Other bacteria such as L. acidophilus [28,32,37,38], S. oralis [10], C. albicans [10], S. salivarius [13] and S. sobrinus [40] were also used to test the antibacterial effects.

Other Properties and Tests
Beside assessing the antibacterial effects, some studies investigated other properties such as fluoride release, degree of conversion, shear-bond strength, microhardness, compressive strength, tensile strength, flexural strength, depth of cure, softening, cytotoxicity and microleakage (Table 3).

Control and Tested Groups
There was a variation in the materials used as control groups between the included studies (Table 3). Tested groups were either commercially available sealant materials or newly developed sealant materials.
The results of other studies that used optical density, bacterial leakage, scanning electron microscopy, confocal laser scanning microscopy and bacterial genomic profiling to report the antibacterial effects of the tested materials are reported in Table 4. Other bacterial activities such lactic acid production, metabolic activities, polysaccharide production and pH after exposure to various tested antibacterial resin-based sealants were reported in a few studies (Table 5).

Biofilm (log CFU/mL) Biofilm (log CFU/mL)
The sealant containing 2 wt % α-TCP + 2 wt % TAT showed a significant reduction in CFU counts in comparison to the control group (p < 0.05).

Clinpro™ Sealant
The sealant containing 4% MAE-DB showed a significant reduction in CFU count in comparison to the controls (p < 0.05).

(CFU/mL) 0 wt % chitosan(-)
The sealants containing 2, 3, 4 and 5% of CH showed a significant reduction in CFU count in 1 month in comparison to the control and 1% CH groups (p < 0.001). In general, there were significant differences between the groups (p < 0.001).  The 1% DMAE-CB sealant showed a significant reduction in CFU count in comparison to the controls with or without aging (p < 0.05). There was no significant difference between the aged and non-aged samples in each group (p > 0.05).

Inhibition zone (mm) Inhibition zone (mm)
The sealants containing 1% CHX showed significant increase in the inhibition zones against S. mutans and L. acidophilus at 0 day in comparison to the controls. These differences were still observed after 7 and 30 days but less pronounced (p < 0.05).

Width of inhibition zone (mm) Width of inhibition zone (mm)
The 2%, 2.5% chitosan, Teethmate™ F-1 and Seal&Protect™ sealants showed a higher inhibition zone in comparison to the other groups. Seal&Protect showed the highest bacterial reeducation rate.

Inhibition zone in mm Inhibition zone in mm
The Teethmate-F1 showed a significant difference in the inhibition zone between S. mutans and L. acidophilus.

Width of inhibition zone (mm) Width of inhibition zone (mm)
The Teethmate-F sealant was the only material that showed an inhibition zone against all strains of S. mutans.
L. acidophilus suspensions exposed to pit and fissure sealants No statistical analysis was mentioned in this study.

Fresh material Fresh material
The Dyract Seal showed the highest antibacterial affect in comparison to other groups; this difference was significant at 0 timepoint and for 2-week aged samples but not the 1-month aged samples (p < 0.0001). Leakage frequency after 90 days (n) Leakage frequency after 90 days (n) For the leakage frequency there was no significant difference between EWBMCP and CLPR sealants (p > 0.05). However, both showed a significant difference in leakage frequency in comparison to EWB sealant (p< 0.05).
The EWBMCP sealant showed a higher median survival time in comparison to the other sealants.   Table 5. Antibacterial effects of dental sealants measuring the metabolic activity, lactic acid production, pH, polysaccharide production and acid stress and oxygen stress tolerance.

Assessment Study
Intervention (Mean ± SD) Control (Mean ± SD) Summary of Results

Metabolic Activity
Ibrahim et al., 2020 [21] -- The sealants containing 5% DMAHDM + 0% NACP showed significant reductions in metabolic activity in saliva-derived biofilm from both high and low caries-risk pediatric patients in comparison to the control (p < 0.05). However, the sealant containing DMAHDM + NACP showed less reduction in comparison to the sealant containing only DMAHDM (p < 0.05). There was no significant difference in the same group regarding the type of the saliva inoculum (p > 0.05).

S. mutans S. mutans
There was no significant difference between the pH of the biofilm cultured on the sealants containing 2.5% and 5% ZnM in comparison to the control group.
There was a slight significant difference between 2.5% SnM and the control group The 5% SnM containing sealant kept the pH level close to the neutral.

Poly-saccharide Production
Ibrahim et al., 2019 [27] --The sealants containing 5% DMAHDM with or without NACP showed significant reduction in polysaccharide production in comparison to the other sealants (p < 0.05).

Discussion
The incorporation of antibacterial agents into dental sealants has sought to develop resins with improved therapeutic properties to prevent dental caries. The present review mapped the studies that evaluated dental sealants containing antibacterial agents. This review's findings evidenced how researchers have been assessing dental sealants and summarized the outcomes of the previous studies. Furthermore, this review summarized the main compounds that have been tested as antibacterial agents in dental sealants and evaluated the quality of the current evidence.
From the nineteen studies included in this systematic review, most of them (thirteen) showed a medium risk of bias. This finding was mainly based on the fact that the sample calculation and blinding were not included in all of them. It is common to observe that researchers consider previous studies to define the sample size, without critical thinking or indication of the sample calculation. This is a problem, especially when no statistically significant differences are detected in the test. The authors must be aware that a small sample size can decrease the statistical power and result in type II error. As the statistical power is not always reported, it is suggested that the sample size calculation and the power of the study are written in the manuscripts about this subject to improve the quality of research reports. Blinding is another parameter that is commonly not followed up [26]. The addition of antibacterial agents in dental resins frequently alters their physical properties, such as the color and viscosity of dental sealants. These characteristics can hinder the blinding process. As well as the reporting of sample size calculation, this parameter could assist in increasing the quality of studies.
The methods for analyzing the antibacterial activity of restorative materials have been criticized in the literature [26,41,42]. This stems from the lack of standardization of the tests, the misinterpretation of purely qualitative analyses or the misuse of quantitative tests. The methods addressed to analyze the dental sealants involved different outcomes. A meta-analysis was not performed due to the methodological variations, which should be more standardized to produce comparable data. Fortunately, all included studies used at least one quantitative test. In addition, the CFU counting test, which is the gold standard for evaluating microorganisms' viability, was the most used. The inhibition zone test was the second most used. For some years now, some journals have been asking that authors not use this method.
A clear example of this was a publication by the Editorial Board of the Journal of Endodontics in 2007 [43]. The editors argued that this journal would no longer accept inhibition zone tests because antimicrobial agents can interact with the agar medium and remove ions from the environment. Furthermore, the agar medium's buffering activity and the chemical interaction between the agar and the antibacterial agents can change the inhibition zone diameter and, consequently, the conclusions about the antibacterial effect. In addition, the zone of inhibition depends on the agent's ability to permeate through the agar, which is essentially hydrophilic. Therefore, agents with greater hydrophobicity may induce a smaller inhibition zone, despite having a positive effect against the formation of biofilms. In conclusion, this is a test that provides uncertain information about the materials' antimicrobial capacity, not necessarily reflecting the antimicrobial activity neither in vitro nor in vivo [43]. Therefore, we suggest that readers must have caution when reading and interpreting studies of resin-based sealants with antimicrobials that used only the zone of inhibition test to assess the materials' antimicrobial activity.
Other methods such as scanning electron microscopy, confocal laser scanning microscopy, bacterial genomic profiling and tests to analyze the metabolic activity or the capacity to produce essential compounds for biofilm structure are interesting to improve the investigations' quality findings. Few studies used a set of methods to analyze the dental sealants. However, as previously stated, most of them used CFU counting as the primary outcome. This method provides the most predictable effect because it does not depend solely on bacterial metabolic activity, such as the MTT test, or bacterial membrane integrity, such as microscopy with a live/dead kit [44]. CFU essentially relies on bacterial viability. Therefore, it is indicated that the CFU results are the main ones considered when analyzing the antimicrobial activity of dental materials with antimicrobial agents [44].
The other concern about the antimicrobial activity tests related to the characteristics of the microbial inoculum used [26,41,42]. From the nineteen studies selected, seventeen tested the dental sealants against S. mutans. This bacterium is directly related to the development of caries [45,46]. However, dental biofilm is much more complex, involving several microorganisms and microbial interactions, which increases the challenge for antimicrobial dental materials. Although some studies used other microorganisms, such as L. acidophilus, S. oralis, C. albicans, S. salivarius and S. sobrinus, only one study considered the complexity of dental biofilm [21]. This study used multispecies biofilms cultured from pooled saliva of high-caries risk and low-caries risk pediatric dental patients. Currently, we need better in vitro studies that use biofilms with a longer maturation time and microorganisms directly associated with dental caries. The use of biofilms cultured from pooled human saliva seems to be the best alternative for in vitro evaluations to better predict in vivo outcomes. Pooled saliva is recommended to be used to reduce the variation in thickness and densities of biofilms between individuals [47,48].
Among the antibacterial agents/compounds evaluated, those based on quaternary ammonium compounds stand out. This finding is similar to the previous one in the scoping review about antimicrobial agents in restorative resin-based materials, when many studies have tested adhesive systems containing methacryloyloxidodecylpyridinium bromide (MDPB) [26]. Another material frequently found was that composed of fluoride. Fluoride can reduce the metabolic activity of microorganisms or induce the formation of fluorapatite after dental demineralization occurs [49]. Fluorapatite is more resistant to demineralization than hydroxyapatite, making the tooth less susceptible to demineralization when fluoride is present in the environment [49]. However, the study with the highest level of evidence on resinous materials with fluoride shows that other antimicrobial agents, such as MDPB, induce a better antimicrobial effect than materials containing only fluoride [50]. This finding must occur due to the low fluoride concentration capable of being incorporated into resinous materials. With regard to dental sealant types, there is insufficient evidence about the effectiveness of sealants with fluorine compared to other types of sealants [7].
In addition to antimicrobial analyses, many studies have evaluated the physicochemical properties by incorporating antimicrobial agents. Since dental sealants must remain in function, with adhesion to the tooth, and without suffering fractures, the authors must analyze the materials' physicochemical properties. A material that rapidly suffers degradation or exhibits mechanical properties that lead to a loss of function will likely need to be replaced or repaired. The degree of conversion, which analyzes the carbon-carbon double bonds' capacity to convert into carbon-carbon single bonds [51], was the most commonly used test among the studies.
This chemical property is frequently altered by incorporating antimicrobial agents and bioactive charges due to the difference in the refractive index between the resin matrix and the added compounds [52,53]. The viscosity of resins after incorporating agents can also be modified, changing the degree of conversion [54] and the adhesion to dental tissues, which is the primary outcome to be assessed. The studies that tested the degree of conversion showed promising results with the incorporation of antimicrobial agents such as ZnM, SnM, DMAHDM, TAT, chitosan and DMAE-CB. The same effect was also found when the adhesion to the tooth structure was tested, such as the shear-bond strength. These findings may be due to the low concentration of agents incorporated in the sealants, which was up to 5 wt % for most studies. Overall, acceptable physicochemical and antimicrobial properties were found for the sealants with most of the studied antibacterial agents.
There is a trend toward formulating and evaluating dental resin-based sealants with antibacterial agents. Even though there is no standardization among the studies, and most of the studies showed issues related to the risk of bias, most of the studies used the gold-standard test to evaluate the antimicrobial activity, which is a very positive finding. The improving of inoculum source to produce a high challenge scenario for the novel dental sealants should be a fundamental goal of the future in vitro studies. The use of more complex methods to better predict antimicrobial results may be the way to accelerate the translation of knowledge from the bench to the clinic.

Conclusions
In summary, based on the included in vitro studies, the addition of antibacterial agents in resin-based dental sealants could have promising antimicrobial effects. These effects may improve the function of sealants as materials for caries prevention and improve their therapeutic activity. However, standardization of the in vitro studies' protocols and in situ studies and clinical trials to assess these effects and support the findings are recommended.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.