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Abstract

Background:

Streptococcus mutans (S. mutans) participates in the dental caries process. Titanium dioxide (TiO₂) nanoparticles produce reactive oxygen species capable of disrupting bacterial DNA synthesis by creating pores in cell walls and membranes.

Objective:

The objective of this study was to determine the effect of TiO₂ on the disruption of S. mutans biofilm.

Methods:

This study was conducted in four phases involving a TiO₂-containing toothbrush and TiO₂ nanoparticles. Each phase was completed using 24 h established S. mutans biofilm growth. Phase one data was collected through a bacterial plating study, assessing biofilm viability. Biofilm mass was evaluated in phase two of the study by measuring S. mutans biofilm grown on microtiter plates following crystal violet staining. The third phase of the study involved a generalized oxygen radical assay to determine the relative amount of oxygen radicals released intracellularly. Phase four of the study included the measurement of insoluble glucan/extracellular polysaccharide (EPS) synthesis using a phenol-sulfuric acid assay.

Results:

Both exposure time and time intervals had a significant effect on bacterial viability counts (p = 0.0323 and p = 0.0014, respectively). Bacterial counts after 6 min of exposure were significantly lower than after 2 min (p = 0.034), compared to the no treatment control (p = 0.0056). As exposure time increased, the amount of remaining biofilm mass was statistically lower than the no treatment control. Exposure time had a significant effect on oxygen radical production. Both the 30 and 100 nm TiO₂ nanoparticles had a significant effect on bacterial mass. The silver nanoparticles and the 30 and 100 nm TiO₂ nanoparticles significantly inhibited EPS production.

Conclusion:

The TiO₂-containing toothbrush kills, disrupts, and produces oxygen radicals that disrupt established S. mutans biofilm. TiO₂ and silver nanoparticles inhibit EPS production and reduce biofilm mass. The addition of TiO₂ to dental products may be effective in reducing cariogenic dental biofilm.

Keywords

Oral bacteria dental plaque toothbrush viability oxygen radicals nanoparticles

Introduction

Despite advances in oral health technology and research over the years, approximately 60%–90% of children, and many adults, are affected by dental caries.¹ Streptococcus mutans (S. mutans) is generally accepted as one of the most common bacteria associated with dental caries.² Insoluble glucan, a key component of extracellular polysaccharides (EPS), has been shown to be important for the colonization of S. mutans to enamel surfaces. EPS increases the pathogenesis of dental caries by creating a structural scaffolding that promotes bacterial adherence and enhances biofilm matrix acidogenicity.³ Studies have shown that S. mutans colonization occurs anywhere between 6 and 36 months of age, and often is associated with eruption of primary teeth.^4,5 The earlier the colonization, the higher the caries risk, and potential development of early childhood caries (ECC).⁶

To reduce caries prevalence, prevention technologies aim to modify and improve oral hygiene and plaque control. Titanium dioxide (TiO₂) use is being explored in dentistry, and has been added to glass ionomer (GI), resin composite, orthodontic adhesive, and orthodontic brackets.⁷

One innovative technology is the Soladey™ ion5 toothbrush, which uses a chemically inert TiO₂ semiconductor, instead of toothpaste, to reduce caries.⁴ The head appears like a normal toothbrush, the neck contains a TiO₂ rod, the handle is composed of stainless steel, and the base contains a solar panel.⁸ There is no on/off switch, rather, in the presence of light (either artificial or natural), the TiO₂ generates an excess of negatively charged electrons which attach to hydrogen ions in the bacterial plaque, producing oxygen radicals which have a bactericidal effect (Figure 1).^9,10 Oxygen radicals disrupt bacterial DNA synthesis and create pores in bacterial cells walls and membranes.

Figure 1.

This diagram depicts the mechanism of action of the TiO₂ semiconductor. In the presence of light, there is a generation of negatively charged electrons. These electrons attach to the hydrogen ions present in bacterial plaque to form hydroxyl (OH⁻) ions. This interaction leads to the production of oxygen radicals inside biofilm, specifically a superoxide ion, which ultimately disrupts DNA synthesis and creates pores in bacterial cell walls and membranes, thus increasing the bactericidal effect.⁹

TiO₂ occurs in three forms: rutile, anatase, and brookite.¹¹ This study utilized the anatase form of TiO₂, which is considered the most photoreactive form.¹² The mechanism of action involves the production of reactive oxygen species (ROS), depletion of cellular antioxidants, and production of superoxide ions protecting tooth surface from bacterial adhesion.^10,12

The Food and Drug Administration (FDA) has approved TiO₂ for use in toothpastes, food (E171 additive), pharmaceutical additives, glazes, plastics, paper fibers, cosmetics, and air and water purification.^12,13,14

Dental caries remains a significant public health issue and research is needed to determine cost-effective strategies for caries prevention and management.¹⁵ Research involving TiO₂ may serve as a novel approach to biofilm regulation and caries management strategies. This study aims to evaluate the effects of TiO₂ on biofilm viability and mass, the mechanistic properties of the ion5 toothbrush and its effect on EPS. Furthermore, this study may translate to new practical and evidence-based treatment recommendations using TiO₂ nanoparticles in future products such as toothpastes and restorative materials.

Materials and methods

This study was conducted in four phases involving a TiO₂-containing toothbrush and TiO₂ nanoparticles. Phase I evaluated biofilm viability using the toothbrush and TiO₂ nanoparticles. Phase II assessed biofilm mas using the toothbrush and TiO₂ nanoparticles. Phase III examined the mechanistic properties of the toothbrush through a generalized oxygen radical assay. Phase IV measured insoluble glucan/extracellular polysaccharide (EPS) synthesis following TiO₂ nanoparticle treatment using a phenol-sulfuric acid assay. Each experiment was completed three times. Negative controls consisted of 0-time interval and no light treatments.

Exposure method for experiments involving biofilm viability using the ion5 toothbrush

The ion5 toothbrush brush head was removed and the TiO₂ rod inside the toothbrush was used. The stainless-steel handle of the toothbrush was positioned in a test tube clamp on a ring stand. During exposure, the ring clamp was loosened and the TiO₂ rod lowered to contact the biofilm. A lamp (40 W soft white bulb) acted as the light source to activate the TiO₂ semiconductor within the rod and positioned level with the solar panel in the toothbrush (10 cm from light to toothbrush). It was turned on during the various times of exposure. Between each well treatment, the TiO₂ rod was cleaned utilizing an alcohol soaked Kimwipe and allowed to dry.

Biofilm viability

Spiral plate method using the ion5 toothbrush

To correlate killing of biofilm cells with oxygen radical production, a biofilm viability assay was conducted. An overnight culture of S. mutans (strain UA159; ATCC 700610) was made. To a six well tissue culture plate, S. mutans (50 µL) and tryptic soy broth (TSB) were supplemented with 1% sucrose as a supplemental carbohydrate source (TSBS; 3 mL) and incubated for 24 h at 37°C and 5% CO₂ to establish the biofilm. Wells were gently rinsed with saline to remove unbound bacteria and exposed to the toothbrush for 0 (no treatment), 2, 4, and 6 min. After treatment, wells were rinsed again and S. mutans biofilm was scraped, 1:100 and 1:10,000 dilutions plated onto blood agar plates to determine colony forming units (CFU) and incubated for 24–48 h.

Spiral plate method using TiO₂ nanoparticles

In six well plates, S. mutans (strain UA159) and TSB were added, as described above, and incubated for 24 h at 37°C and 5% CO₂ to establish the biofilm. The wells were rinsed with saline. Two concentrations of TiO₂ nanoparticles were tested. 2.5% and 1.25% nanoparticle suspensions were prepared using both 30 nm (US Research Nanomaterials TiO₂, Anatase, 99.98%, 20 wt% in water) and 100 nm (US Research Nanomaterials TiO₂, Anatase, 99.9%, 20 wt% in water)^16,17 TiO₂ nanoparticles and saline. One milliliter of the suspensions were added for 2 and 6 min increments to the corresponding wells. After each time interval, the wells were rinsed three times and scraped into test tubes. Each treatment group had duplicate sets of tubes (untreated, 1.25% suspension of 30 nm, 2.5% suspension of 30 nm, 1.25% suspension of 100 nm, and 2.50% suspension of 100 nm TiO₂ nanoparticles). 1:1000 and 1:100,000 dilutions were plated onto blood agar plates. After 24–48 h, the CFU were determined.

Biofilm mass measurements

Crystal violet staining biofilm formation assay using the ion5 toothbrush

To correlate reduction of biofilm mass with oxygen radical production, a biofilm mass crystal violet staining (CV) assay was conducted. This assay examined S. mutans in a 96 well microtiter plate model. Planktonic bacteria were removed and the wells rinsed. The biofilm was treated with the toothbrush for time intervals of 0 (no treatment), 30 s, 2, 4, and 6 min. After treatment, the wells were washed and 10% formaldehyde added to fix and kill the biofilm cells, stained with CV and the stain extracted using 2-propanol and analyzed at 490 nm as described previously.²

Crystal violet staining biofilm formation assay using TiO₂ nanoparticles

To determine the mechanistic effect of the TiO₂ nanoparticles on established S. mutans biofilm, a CV staining assay was conducted. S. mutans (strain UA159) biofilm was established in TSBS in quadruplicate for 24 h at 37°C using a 96-well microtiter plate. Dilutions of the 30 or 100 nm TiO₂ particles ranging from 0.156%−5% were added and incubated for 2 min. Planktonic bacteria were removed and the wells rinsed. The wells were stained with CV and the stain was extracted using 2-propanol and analyzed at 490 nm.

Generalized oxygen radical assay on biofilm using the ion5 toothbrush

To determine the relative amount of oxygen radicals released intracellularly in the biofilm bacterial cells following TiO₂ treatment, a fluorescent dye (CellROX green; Invitrogen; ThermoFisher Scientific) was used to measure the ROS release.^18–20 Following exposure of biofilm cells to the toothbrush, CellROX green reagent was added in quadruplicate in a similar manner as the biofilm viability and mass protocols. After biofilm growth for 24 h, the biofilm was treated with the ion5 toothbrush for several different time intervals. Immediately after treatment, the CellROX green reagent was added (200 µL; 5 µM) to each well and plates were incubated for 30 min at 37°C. After the 30-min incubation, biofilm was washed three times to remove excess reagent. The reagent that entered the biofilm cells in the wells was excited at 485 nm and the emission measured at 520 nm using a fluorescent microtiter plate reader (Molecular Devices, Sunnyvale, California). Controls included wells without CellROX reagent, wells with only TSBS and without bacterial cells but with CellROX reagent and wells with TSBS and without both bacteria and CellROX reagent.

Isolation of insoluble and soluble glucan from TiO₂ nanoparticle treated biofilm

Biofilm was established in TSBS in six well plates for 24 h at 37°C. The wells were washed with sterile saline two times. Particles (30 and 100 nm TiO₂ and silver nanoparticles (Ag 99.99%, 50–80 nm, metal basis)) were added for 2 min in duplicate.²¹ Wells were washed three times with sterile saline. Three milliliter of TSBS was added to each well, and incubated again for 24 h at 37°C. The wells were scraped into 1 mL of water. The biofilm was sonicated and centrifuged at 10,000g for 10 min at 4°C. The supernatant containing soluble glucan was collected. The pellet containing the biofilm cells was washed twice in water and all three supernatants pooled (soluble glucan component). This was repeated three times. The biofilm pellet and pooled supernatant were saved for the next step. For the extractions, three volumes of cold ethanol (9 mL) was added to the supernatant and incubated on ice for 5 min for precipitation of glucan and the solution centrifuged to collect the soluble glucan. The precipitated soluble glucan was washed three times in cold ethanol (3 mL). It was re-centrifuged and dissolved in 1 mL of deionized water for analysis. One molar NaOH was added to the biofilm pellet (1 mL) from above (1 mg pellet/0.3 mL of 1 M NaOH) and shaken for 2 h at 37°C, centrifuged and the supernatant containing the insoluble glucan collected. Three volumes of cold ethanol were added to the supernatant to precipitate the insoluble glucan and centrifuged for 10 min. The precipitate was dissolved in 1 mL of 1 M NaOH. The reducing sugars in the soluble and insoluble glucan preparations were measured using the tube-based phenol sulfuric acid assay.^22,23,24

Phenol sulfuric acid assay (EPS) for measurement of isolated EPS [tube-based] following TiO₂ treatment

Soluble and insoluble glucan preparations were treated with 30 and 100 nm anatase TiO₂ and silver nanoparticles were incubated in 96 well plates for 2 min using two different volumes of nanoparticles (50 and 100 μL). Immediately following the exposure, the nanoparticles were removed and 150 μL of concentrated sulfuric acid was added to each well, and followed by 30 μL of 5% phenol. The 96 well plate was floated in a water bath (90°C) and removed after 5 min. Following the removal from the water bath, the plates were cooled to room temperature for another 5 min. Each sample was transferred to a new 96 well plate and the absorbance was measured at 490 nm.

Biofilm phenol sulfuric acid assay for measurement of EPS in situ

To determine the in situ amount of EPS present in biofilm, S. mutans (strain UA159) biofilm was established for 24 h at 37°C. The media was removed and wells rinsed. 50 μL of 30 and 100 nm TiO₂ and silver nanoparticles were added to the wells in quadruplicate for 2 min. The wells were rinsed and media was replaced and incubated again overnight for 24 h at 37°C. The next day, the media was removed and the EPS assay protocol as described above was followed and absorbance was measured at 490 nm.

Results

Effects on S. mutans biofilm following treatment with the ion5 toothbrush

The ANOVA analysis indicated that exposure time of the toothbrush on S. mutans biofilm had a significant effect on bacterial viability counts (p = 0.0323) and treatment time intervals had a significant effect on outcomes (p = 0.0014). Bacterial viability counts after 6 min of exposure was significantly lower than after 2 min of exposure and the no light negative control (p = 0.0304 and p = 0.0056, respectively). Overall, time had a significant effect on viability outcomes (p = 0.0263) (Figure 2). The negative control group consisting of biofilm mass treated with the TiO₂ toothbrush at 0 s was significantly higher than after 30 s, 2, 4, and 6 min (p = 0.0008, p = 0.0016, p = 0.0002, and p = 0.0022, respectively) of exposure. Biofilm mass was not significantly different among the 30 s, 2, 4, and 6 min time intervals (Figure 3). The generation of oxygen radicals with no TiO₂ toothbrush treatment (negative control) was significantly lower than 2, 4, and 6 min (p = 0.0045, p = 0.0319 and p = 0.0071, respectively). There was no significant difference in generation of oxygen radicals between no treatment and 30 s (Figure 4).

Figure 2.

Effect of TiO₂ toothbrush on established S. mutans biofilm viability. The mean exposure (+SE) of established S. mutans to the TiO₂-containing toothbrush for 0, 2, 4, and 6 min is shown. Following the exposure times, the wells were rinsed, scraped, and 1:100 and 1: 10,000 dilutions spiral plated onto blood agar plates. The number of colony forming units (CFU/mL) was determined. The asterisk (*) indicates a statistically significant difference between the 6 min exposure time and the no treatment control. Each experiment was repeated three times. For spiral plate data, ANOVA was run to test for fixed effects by exposure time. Analyses were performed using the ranks of the data. A 5% significance level was used for all the tests.

Figure 3.

Effect of TiO₂ toothbrush on established S. mutans biofilm mass. The mean absorbance (+SE) at 490 nm reflects the exposure of the TiO₂ -containing toothbrush to established S. mutans biofilm for time intervals of 0, 30 s, 2, 4, and 6 min. The asterisks (*) indicate a statistically significant difference between the 30 s, 2, 4, and 6 min exposure times compared to the no treatment control. Each experiment was repeated three times. For CV staining data, ANOVA was used to test for fixed effects for each outcome. Analyses were performed using the ranks of the data. A 5% significance level was used for all the tests.

Figure 4.

Effect of TiO₂ toothbrush on generation of oxygen radicals in S. mutans biofilm. Mean intracellular release of oxygen radicals (+SE) in response to the titanium-dioxide containing toothbrush for time intervals of 0, 30 s, 2, 4, and 6 min is shown. Following exposure, CellROX reagent added and biofilm cells were excited at 485 nm and emission measured at 520 nm. The asterisks (*) indicate a statistically significant difference between the no treatment control and 2, 4, and 6 min of exposure. No statistical difference was noted between the no treatment and 30 s. Each experiment was repeated three times. Analyses were performed using the ranks of the data. A 5% significance level was used for all the tests.

Effects of S. mutans Biofilm Following Treatment with TiO₂ Nanoparticles

Both the 30 and 100 nm TiO₂ nanoparticles at concentrations ranging from 0.16% to 5% had a significant inhibiting effect on bacterial mass. However, there was no statistical difference of the 30 nm nanoparticles at a concentration of 2.50% (p = 0.06) (Figure 5). There was no statistical difference in biofilm viability between the 30 and 100 nm TiO₂ nanoparticles compared to the untreated negative control (Figure 6). The silver nanoparticles, and the 30 and 100 nm TiO₂ nanoparticles significantly inhibited EPS production in the tube-based assay. Additionally, the 100 nm TiO₂ nanoparticles exhibited statistically significant greater EPS inhibition than the silver nanoparticles (Figure 7). The 30 and 100 nm TiO₂ nanoparticles had significantly less EPS production in the in situ assay than the negative control and silver nanoparticle groups. The 100 nm nanoparticles exhibited greater biofilm EPS production than the 30 nm nanoparticles. Silver nanoparticles were statistically more inhibitory to EPS than the 30 and 100 nm TiO₂ nanoparticles (Figure 8).

Figure 5.

Effect of 30 and 100 nm TiO₂ nanoparticles on established S. mutans biofilm mass. Dilutions of 30 or 100 nm TiO₂ particles ranging from 0.156%–to 5% were exposed to established S. mutans biofilm. Wells were rinsed, stained with crystal violet (CV) and analyzed at 490 nm. The mean values (+SE) are shown. The asterisks (*) indicate a statistically significant difference between no treatment control compared to concentrations of 30 and 100 nm TiO₂ concentrations ranging from 0.16%–5%. Each experiment was repeated three times. For CV staining data, ANOVA was used to test for fixed effects of particle size and concentration for each outcome. Analyses were performed using the ranks of the data. A 5% significance level was used for all the tests.

Figure 6.

Effect of TiO₂ nanoparticles on established S. mutans biofilm viability. 2.5% and 1.25% TiO₂ nanoparticle suspensions were added for 2 and 6 min increments to established S. mutans biofilm. Each treatment group had duplicate sets of tubes and 1:1000 and 1: 100,000 dilutions were plated. Mean colony forming units (CFU/mL; +SE) were determined. There are no asterisks present as there was no statistical difference in biofilm viability between the 30 and 100 nm TiO₂ nanoparticles compared to the untreated control. Each experiment was repeated three times. For spiral plate data, ANOVA was run to test for fixed effects of particle size and concentration. Analyses were performed using the ranks of the data. A 5% significance level was used for all the tests.

Figure 7.

Effect of TiO₂ and silver nanoparticles on S. mutans tube-based phenol sulfuric acid assay. Following the isolation of soluble and insoluble glucan, 30 and 100 nm TiO₂ nanoparticles and silver nanoparticles were exposed to S. mutans. Following exposure, sulfuric acid was added, followed by phenol. Samples were transferred to a new plate and mean absorbance (+SE) measured at 490 nm. The asterisks (*) indicate that 30 and 100 nm TiO₂ nanoparticles and silver nanoparticles significantly inhibit EPS production compared to untreated control. The @ symbol indicates a statistically significant difference between the 100 nm TiO₂ nanoparticles and the silver nanoparticles. Each experiment was repeated three times. For tube EPS data, ANOVA was run to test for fixed effects of particle size, concentration, and glucan status. Analyses were performed using the ranks of the data. A 5% significance level was used for all the tests.

Figure 8.

Effect of TiO₂ and silver nanoparticles on S. mutans biofilm phenol sulfuric acid assay in situ. Established S. mutans biofilm was exposed to 30 and 100 nm TiO₂ nanoparticles and silver nanoparticles. The wells rinsed, media replaced and incubated again overnight. EPS protocol followed and mean absorbance (+SE) measured at 490 nm. The asterisk (*) indicates that the 30 and 100 nm TiO₂ had statistically significant differences between the control and silver groups. The @ symbol indicates a statistically significant difference between the silver nanoparticles and the 30–100 nm titanium dioxide nanoparticles with greater EPS enhancement. Each experiment was repeated three times. For microplate EPS, ANOVA was used to test for fixed effects of particle size and dilution. Analyses were performed using the ranks of the data. A 5% significance level was used for all the tests.

Discussion

Various studies have incorporated TiO₂ nanoparticles into glass ionomer (GI) and resin composites. However, no studies have evaluated the effect of TiO₂ on S. mutans EPS production. The previous studies in dentistry have sought to evaluate the antibacterial properties of TiO₂.

TiO₂ serves as a chemically stable, biocompatible and nontoxic filler.²⁵ Elsaka et al.²⁵ and Dias et al.²⁶ incorporated TiO₂ nanoparticles into GI and resin, respectively. The authors found that TiO₂ nanoparticle-containing GI and resin modified with TiO₂ demonstrated bacterial inhibition.^25,26

While our study did not evaluate GI or resin composite, the bacterial inhibition demonstrated in these studies may be attributed to the role of oxygen radical production and inhibition of EPS, specifically insoluble glucan production. When placing a restorative material, it is advantageous to control plaque accumulation, and the inhibition of insoluble glucan prevents S. mutans from adhering to not only the restoration, but also tooth structure. Additionally, if oxygen radicals are being released from the restorative material, we know that S. mutans can be disrupted in as little as 30 s, and significantly inhibited within 6 min.²⁷

A limitation of the study by Elsaka et al.,²⁵ is that it is difficult to discern whether the bacterial inhibition of the GI is due to the TiO₂ nanoparticles alone, the fluoride releasing mechanism of GI, or both. Our results indicate that TiO₂ itself is capable of bacterial inhibition. Further studies would be needed to determine the difference when incorporated into GI. However, this would not be as pertinent to resin composite studies. Elsaka et al.²⁵ stated that the smaller the nanoparticle, the greater the intracellular damage. This study is unable to provide any statistically significant data to support this concept. Additional studies would be needed to evaluate the size of TiO₂ nanoparticles within restorative materials and its influence on the inhibitory, physical, and mechanical properties.

TiO₂ has also been incorporated into orthodontic adhesives and coating of orthodontic brackets and archwires. Patients undergoing comprehensive orthodontic treatment often have difficulty maintaining proper oral hygiene. As a result, many develop white spot lesions, due to enamel demineralization. Andriani and Purwanegara²⁸ hypothesized that by adding TiO₂ nanoparticles to orthodontic adhesive this will prevent enamel demineralization. It has been found that TiO₂ nanoparticles “exhibit better antibacterial properties compared to chlorhexidine.²⁸” The authors found that 2% TiO₂ nanoparticles in orthodontic adhesive resin can increase the antibacterial effect of orthodontic adhesive, but this activity was not enough to prevent decreased enamel microhardness.²⁸

Salehi et al.,²⁹ evaluated the long term (90 days) benefit of N-doped (nitrogen-doped) TiO₂ coated stainless steel brackets against S. mutans biofilm. The results of this study found that the N-doped TiO₂ film prevents the growth of S. mutans biofilm for at least 3 months.²⁹ Therefore, its use decreases the chance of developing dental caries post orthodontic treatment.

Like these previous studies, our study evaluated 24 h established S. mutans. While these studies did not utilize a toothbrush, they ultimately found a decreased amount of S. mutans. TiO₂ nanoparticles are efficacious at reducing and inhibiting established S. mutans biofilm. A TiO₂-containing toothbrush may prove beneficial in providing not only mechanical removal of S. mutans, but also release oxygen radical species that reduce EPS production leading to decreased caries.

The results of this study indicate that the TiO₂-containing toothbrush kills and produces oxygen radicals that disrupt established S. mutans biofilm. The reactive oxygen species (ROS) produced from the TiO₂ nanoparticles contributed to a detrimental effect on S. mutans biofilm. To further evaluate the mechanistic properties of TiO₂ nanoparticles we assessed the effects of 30 and 100 nm anatase TiO₂ on established S. mutans biofilm. We found that the TiO₂ nanoparticles decreased the mass of S. mutans and reduced the amount of insoluble glucan/EPS activity in a microtiter plate-based study.

TiO₂ nanoparticles and silver nanoparticles demonstrate the inhibition of EPS production and reduce biofilm mass. This means that these nanoparticles have the capability to disrupt the scaffolding, and thus, reduce the bacterial adherence of S. mutans to tooth structure. Therefore, like previous studies found, there is an antibacterial effect that will reduce caries risk as the established biofilm is altered. However, the overall viability was not altered. Plaque control remains an essential part of oral health and treatment should be designed to control the plaque, rather than eliminate it.⁵

The results of this study benefit dentistry and the overall community by creating awareness for novel technologies that may benefit preventive dental care, education, and the management of decay. This technology ultimately may change the way people brush their teeth, influence future toothbrush design, and could potentially decrease the need for dental restorations. While we would not necessarily recommend the use of the ion5 toothbrush due to the need for additional research, this study suggests there is merit behind incorporating TiO₂ and other various nanoparticles such as silver into dental instruments and dental materials to manage dental caries.

The limitations of the study include the inability to fully separate the nanoparticles from the media. Despite thoroughly rinsing the wells, it often appeared that small particles of TiO₂ remained in the washed wells. When using the toothbrush, it is possible, although unlikely, there was some form of mechanical removal of the biofilm when placed in contact with the established biofilm. Additionally, the same effects may not be observed when the head of the toothbrush is placed back onto the rod. Despite these limitations, this study has proven that TiO₂ and silver nanoparticle technology should be further explored and implemented in dentistry.

Conclusion

This study concludes that the TiO₂-containing toothbrush kills, disrupts, and produces oxygen radicals that disrupts established S. mutans biofilm. Furthermore, TiO₂ nanoparticles demonstrate inhibition of EPS production and reduce biofilm mass by changing the overall bacterial physiology leading to enhanced antimicrobial effects. However, no significant reduction in biofilm viability was observed with nanoparticles. Silver nanoparticles exhibit a similar effect to the TiO₂ nanoparticles and had increased inhibition of EPS production compared to the TiO₂ nanoparticles.

Footnotes

Author contributions

MS contributed to conception, design, data acquisition, analysis and interpretation, drafted and critically revised the manuscript. SD contributed to conception, design and data acquisition, critically revised the manuscript. HA contributed to conception, design, data acquisition, and interpretation and drafted the manuscript. AS contributed to conception, design, and drafted and critically revised the manuscript. LV contributed to interpretation, drafted and critically revised the manuscript. RG contributed to conception, design, data acquisition and interpretation, and drafted and critically revised the manuscript. All authors gave their final approval and agree to be accountable for all aspects of the work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: We would like to thank the Indiana University School of Dentistry Graduate Research Fund for providing the funding for this study.

Guarantor

ORCID iDs

Molly K Sanders

Allison C Scully

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