No-Ozone Cold Atmospheric Plasma Makes Distilled Water an Effective Tooth Bleaching Gel

Abstract

Background: Tooth whitening with peroxide is linked to a number of negative consequences. This study aimed to evaluate the effect of tooth bleaching and color safety using no-ozone cold atmospheric-pressure plasma (NCP) with distilled water (DW) and to assess the microhardness and mineral composition of the treated teeth. Methods: The enamel of 90 extracted human teeth were randomly assigned to three groups (n = 30): Group 1 (no treatment), Group 2 (NCP + DW), and Group 3 (only DW). The overall color change (ΔE) was observed at 10 min, 20 min, 30 min, 1 day, 7 days, 1 month, 3 months, and 6 months after treatment and quantified. Six cadavers treated with NCP and DW for 30 min were evaluated for color change using ShadeEye NCC and the VITA Easyshade. Furthermore, the microhardness and mineral composition of the teeth were evaluated using a Vickers hardness tester and an electro micro analyzer, respectively. All statistical data were analyzed at a significance level of 5%. Results: Group 1 showed no color change. The ΔE values of Group 2 were 17.67 and 5.62 times higher than those of Group 3 after 30 min and 6 months, respectively. Methylene blue solution revealed that the concentration of hydroxyl radicals was higher in Group 2. The average ΔE value using ShadeEye NCC for cadaveric teeth was 4.0. Evaluation via VITA Easyshade showed that the brightness was increased by 5 levels. No significant differences were observed among the groups in terms of the microhardness of the enamel and dentin or the Ca, P, Na, Cl, Zn, and Mg values in the enamel and dentin. Conclusions: Tooth bleaching using NCP with DW exhibited high efficacy without inducing damage or demineralization in the dental hard tissue.

1. Introduction

Hydrogen peroxide (HP) and carbamide peroxide (CP) have been commonly employed in various tooth-bleaching systems as effective agents with which to whiten teeth. HP and CP whiten teeth via chemical reactions that lead to the production of free radicals [1]. However, the exact mechanism underlying tooth bleaching has not been fully elucidated. HP and CP, as oxidizing agents, penetrate the tooth, and free radicals, which are produced during the bleaching procedure, interact with the adsorbed colored organic molecules to oxidize and break down the long-chain dark-colored organic chromophore molecules to remove tooth staining, resulting in the brighter appearance of the teeth [2,3,4]. However, the free radicals generated can be transmitted to deeper layers of the material according to the principle of diffusion, negatively affecting the bond between the filling material and the tooth [5,6].

The use of peroxide for tooth whitening, however, is associated with several adverse effects, such as tooth hypersensitivity, gingival irritation, deterioration of esthetic restorations, demineralization of the teeth, and a decrease in enamel hardness [7,8,9,10]. A decrease in enamel hardness can weaken the tooth and lead to other problems. Consequently, previous studies have investigated the chemical toxicity of peroxide bleaching agents and their effects on dental hard tissue, smear layer, and bonding strength [11,12].

Several studies investigating the effectiveness of HP-based tooth bleaching agents have revealed the significant adverse effects of these agents on the teeth. Further studies on the use of non-peroxide-based tooth bleaching agents, with a focus on tooth safety, are underway. The use of alternative materials, such as saline and chlorine dioxide, instead of peroxide agents, has increasingly been explored in recent studies [13,14,15], which aimed to identify a bleaching agent with minimal harmful adverse effects and maximum efficiency.

Chlorine dioxide has strong oxidizing properties and has been considered an alternative technology for whitening teeth in a shorter period of time [15], but there is no published evidence to support its safety. Concentrated chlorine dioxide is not suitable for human consumption and may cause acute toxicity [16]. On the other hand, tooth whitening using saline solution has been reported to be safe and not harmful to enamel [13]. Various light sources have been used in tandem with bleaching gels to increase their efficacy. However, the use of these light sources has given rise to adverse effects which are different from those associated with the use of bleaching gels owing to the high temperatures to which the oral tissues are subjected [17].

The safety and superiority of the whitening effect achieved with the plasma-based tooth whitening method, even with low concentrations of HP gel, has been demonstrated in our previous studies [18,19]. Compared with existing tooth bleaching methods, bleaching gel containing 5.4% HP exhibits excellent whitening efficacy within a short duration [20]. This finding may be attributed to the rapid decomposition of HP into hydroxyl groups (•OH) by the powerful electronic energy generated in the plasma [21]. Thus, water alone, rather than low concentrations of HP, could act as a sufficient bleaching gel if more-powerful plasma devices can produce higher amounts of •OH.

This study aimed to investigate whether distilled water (DW) can be used as a potential bleaching agent in bleaching gel using no-ozone cold atmospheric-pressure plasma (NCP). In addition, this study also investigated its effect on tooth tissue, including the bleaching effect, color stability, and microhardness and mineral composition of the teeth.

2. Materials and Methods

2.1. Cold Atmospheric-Pressure Plasma Device

A new cold atmospheric plasma that generates almost no ozone, named NCP, was used in this study. This plasma device consists of a main body consisting of smps, a solenoid valve, a gas flow regulator, a high voltage circuit, etc., and a handpiece, which is the part that generates NCP. Inside the handpiece, there is a coaxial dielectric barrier discharge (DBD)-type plasma source, which consists of a stainless steel (Stainless Steel 306) inner electrode and an outer electrode surrounding the outer diameter of the ceramic nozzle (Al₂O₃). When the start button on the main body is pressed, argon gas flows between the internal electrode and the ceramic nozzle, and an output voltage of 3 kVpp is applied to the electrode at a frequency of 20 kHz, generating plasma. The argon gas rate in this device can be adjusted through a gas flow controller, and the gas flow rate was fixed at 1 slm (standard litter per min) in this study. An illustration of the described device has been presented in a previous paper [21].

2.2. Sample Preparation

Ninety extracted human teeth were used in this study. The protocol for the use of human teeth was approved by the Ethics Committee of the Dental Hospital of Pusan National University (PNUDH-2014-025).

All extracted teeth were examined to detect the presence of caries, cracks, fractures, and erosion, and defective teeth were excluded from the experiment. A low-speed saw (Minitom, Struers, Copenhagen, Denmark) was used to section each tooth at the cementoenamel junction. The teeth were subsequently thoroughly polished with an ultrasonic scalar to remove calculi and periodontal remnants. The polished samples were immersed in 0.4% sodium azide to inhibit the growth of microorganisms and stored at room temperature until further use.

2.3. Tooth Bleaching Procedure In Vitro

The samples were randomly divided into three groups (n = 30) after tooth preparation. The teeth included in the control group (Group 1) were not treated with any material or device; rather, they were simply washed and immersed in artificial saliva (Talivar, Hanlim, Seoul, Republic of Korea) after 30 min. The teeth included in Group 2 were treated with DW and NCP for 30 min. The tooth surface was treated with DW every 30 s while maintaining a distance of 1 cm between the tip of the device and the enamel of the tooth. The teeth were immersed in artificial saliva after the completion of the procedure. The teeth included in Group 3 were treated with DW for 30 min. The teeth were subsequently immersed in artificial saliva after the completion of the procedure.

The specimens stored in artificial saliva after completing the treatment procedures (10, 20, 30 min) were evaluated after 1 day, 7 days, 1 month, 3 months, and 6 months to determine the changes in color. The artificial saliva was replaced daily.

2.4. Assessment of Tooth Color

The changes in the color of the samples were evaluated as described in a previous study [18]. Tooth color measurements were performed by acquiring images using a digital camera (Pixel link PL-B686CU, Pixelink, Ottawa, Canada) connected to a stereomicroscope (40× magnification, SZ-CTV, Olympus, Tokyo, Japan). The color of the tooth samples in each group was compared before and after treatment. The changes in the color of the tooth samples were evaluated using Adobe^® Photoshop CS2 (Adobe System Inc., San Jose, CA, USA) according to the Commission International de L’Eclairage Lab (CIELAB) Color System. The overall change in color (ΔE) was calculated using the baseline color parameters. CIELAB represents the L*, a*, and b* parameters for lightness–darkness, redness–greenness, and yellowness–blueness, respectively. The ΔE value was calculated using the following equation:

$$\Delta E=\sqrt{{\left({\Delta L}^{*}\right)}^2+{\left({\Delta a}^{*}\right)}^2+{\left({\Delta b}^{*}\right)}^2}.$$

2.5. Previous Clinical Tests Using Cadavers

The cadavers used in this study were donated to the School of Medicine, Pusan National University, for educational purposes. The teeth of six cadavers were subjected to the proposed bleaching method to determine the efficacy of this method in clinical practice. Calculus was manually removed using a scaler, and the teeth were subsequently treated with NCP and DW for 30 min. The tooth-color-measuring instruments evaluated were the following two devicesL the ShadeEye NCC (Shofu Inc., Kyoto, Japan) and the VITA Easyshade (VITA Zahnfabrik, Bad Säckingen, Germany).

2.6. Measurement of •OH

The amount of •OH produced in the two groups was measured using methylene blue (MB; Sigma-Aldrich, St. Louis, MO, USA). The MB solution test consisted of making a simple comparison between two groups undergoing MB degradation. An MB solution of 0.1 mM concentration was used in this study, and the absorbance of the solution was measured using a spectrophotometer (UV mini-1240, Shimadzu, Kyoto, Japan) at 665 nm. DW (0.6 mL) was added to the 24-well plate. NCP was and was not applied under the same conditions for 1 min. The amount of MB solution in contact with each group was subsequently measured to determine optical density.

2.7. Measurement of Microhardness

Immediately after application for 30 min, the Vickers Hardness Number (VHN) of the enamel surfaces of the samples in each group (n = 10) was measured using a Vickers hardness tester (microhardness; Akashi MWK-III, Tokyo, Japan) placed perpendicular to the tooth surface under a 100-g load for 10 s. The average value was calculated four times for each sample at a magnification of 400×.

2.8. Electro-Micro Analysis

The levels of Ca, P, Na, Cl, Zn, and Mg were measured using an electro micro analyzer (EPMA; SX100, CAMECA, Corbevoie, France) for quantitative analysis of the changes in the surface composition of the enamel after 30 min of application in each group (n = 10). The samples with carbon coating were evaluated under the following conditions: 10-µm; accelerating voltage, 15 keV; and beam electric current, 20 nA. The enamel and dentinal surfaces were evaluated at four points per sample to determine the weight%.

2.9. Statistical Analysis

All statistical analyses were performed using SPSS version 18 software (SPSS Inc., Chicago, IL, USA). The color of the tooth samples and the amount of •OH were compared using Student’s t-test for independent samples in each group. The tooth color change, mineral composition, and microhardness in each group were analyzed using one-way analysis of variance (ANOVA) and a post hoc Tukey’s test. All statistical data were analyzed at a significance level of 5%.

3. Results

3.1. Efficacy of Bleaching

Figure 1 depicts the bleaching efficacy of the experimental and control groups. Group 1 is the blank control group, meaning there was no treatment and there was no change. The ΔE value of Group 2 was 17.67 times higher than that of Group 3 at 30 min. The ΔE value was 12.89 after 24 h, which was higher than that of the other group. The ΔE value of Group 2 subsequently reduced to 6.96, which was 5.62 times higher than the Group 1 (ΔE values = 0) at 3 and 6 months. A significant change in color was observed in the experimental group compared with that in the other groups (p < 0.05).

Each tooth was treated with DW and NCP for 30 min. The ΔE values of the samples immediately after treatment for 30 min were higher than the ΔE values of the samples following treatment with DW only. The ΔE value following treatment with DW and NCP was greater than that in the other groups after 24 h. The ΔE values were maintained at 4.16 for up to 6 months of long-term observation.

3.2. Color Change in Cadaveric Teeth

Tooth bleaching was performed using a multi-jet device with DW for 30 min. Color evaluation by VITA Easyshade increased brightness by 5 levels from A4 to D3 (Figure 2a,b). The average overall change in color using ShadeEye NCC was 4.00, indicating bleaching efficacy. The highest and lowest ΔE values of the tooth samples were 7.86 and 2.34, respectively (Figure 2c).

3.3. OH Detection

The optical density values of Groups 2 and 3 were 0.63 and 0.97, respectively (Figure 3). A significant difference was observed between the teeth that had been treated with NCP and those that had not been treated with NCP (p < 0.05). The NCP-treated group exhibited a lower absorbance than the non-NCP group. Therefore, the light color of the MB solution at the completion of the experiment was considered to indicate a higher amount of •OH.

3.4. Microhardness

Figure 4 presents the microhardness results of the enamel immediately after 30 min of tooth bleaching. Comparison of the VHN values revealed no significant differences (p > 0.05) among the tooth bleaching groups in terms of the microhardness of the enamel. Thus, the microhardness was considered to exhibit no significant changes following treatment with NCP.

3.5. EPMA

No significant differences were observed among the tooth bleaching groups after 30 min of tooth bleaching in terms of the mineral compositions of the enamel (Figure 5). Furthermore, no significant changes in the Ca, P, Na, Cl, Zn, or Mg levels in the enamel were observed in any of the groups (p > 0.05).

4. Discussion

This study was an in vitro study that used a plasma source instead of the existing light source and performed teeth bleaching with DW instead of an oxidizing bleaching agent. A significant advantage of the plasma bleaching method is that it does not require the use of a high-temperature light source. Moreover, it exhibits a substantial bleaching effect within a short duration even when a bleaching gel containing a low concentration of HP is used. Furthermore, it does not induce any significant side effects on the dental hard tissues and the soft tissues surrounding the teeth at this level [20]. These findings indicate that the plasma tooth bleaching method may be a good alternative to the existing tooth bleaching methods. •OH released from HP-containing bleaching gels plays a crucial role in the tooth whitening mechanism. The electrons in the plasma facilitate the effective breakdown of the hydrogen bonds of HP, thereby producing a large amount of •OH [21]. Thus, a better whitening effect can be expected in a shorter duration if a plasma device more powerful than existing devices is developed. Ordinary DW may be a suitable agent for tooth whitening if bleaching gels can be replaced with DW without HP, and •OH can be produced in DW at a level similar to that of a bleaching gel containing HP at low concentrations.

The present study aimed to identify alternatives to peroxide-based bleaching agents, which are associated with several drawbacks and limitations. Bleaching with NCP and DW has been used to address this problem. The efficacy and safety of NCP and DW, and whether this method can be employed efficiently and safely, with no adverse effects on dental hard tissues, was evaluated in the present study.

Performing tooth bleaching using NCP with DW, a non-peroxide method, for 30 min led to a significant improvement in the appearance of the tooth. Furthermore, this method also yielded higher brightness values than the other methods for up to 6 months. Thus, Figure 2 demonstrates that the combination of DW and NCP may be an effective and accelerative method for tooth bleaching, compared with existing procedures.

The findings of the present study are consistent with those of earlier experimental studies on tooth bleaching using non-peroxide materials. Sun et al. [22] reported that the tooth whitening effect achieved after 20 min via direct-current atmospheric-pressure cold-air plasma was superior to that of the effect achieved with the existing HP gel treatment. However, these results did not reveal the effect of the chemical composition of saline on the mechanism of tooth bleaching. Nevertheless, the use of pure DW in the present study ensured that uncontrolled chemical components had no effect on the results.

A high concentration of the HP bleaching agent yields an excellent effect; however, high concentrations of HP are known to have adverse effects on the tooth and surrounding tissue. Kawamoto et al. [23] reported that treatment with 30% HP for 10 days yielded significantly higher efficacy than treatment with a low concentration of HP. However, several treatment sessions were required to achieve the desired results despite the use of a high concentration of the HP bleaching agent. The present study proposed the use of a new method, whose efficacy surpasses that of all previous methods, without harming dental tissue. In the case of DW and NCP treatments, the number of treatments has increased, but this represents a great advantage in that it can avoid various serious effects of HP.

Several instruments and devices developed for the color matching of teeth are commercially available for use in the field of esthetic dentistry. The CIELAB color system is used most widely for matching the color of the teeth [20,24,25,26]. The changes in the color of each tooth sample were documented by acquiring an image with a digital camera in the present study. The images were subsequently analyzed using Adobe^® Photoshop CS2 to determine the changes in the color of the tooth. This analysis was unaffected by external colorimetric factors such as the illumination and the color of gingival surfaces. The ShadeEye NCC is a type of colorimeter that reads visible light using a filtered photodetector and calculates the information of chroma, value, and hue. It is expressed as L*a*b* colorimetric or XYZ colorimetric values [27]. However, since it is a device that measures a small area of the tooth, color errors may occur if the measuring tip is not always located in the same area. The VITA Easyshade is an effective method of observing color tone differences by directly comparing teeth with existing color samples, and it is the most commonly used method in clinical practice [28]. However, there are problems such as limitations in the color tone range, subjective factors when selecting a color tone, and inconsistency in selection due to ambient light sources, etc. In this study, three methods of measuring tooth color were used. The CIELAB color system was used to measure color change due to whitening in extracted teeth, and the color change in the cadavers was performed using ShadeEye NCC and VITA Easyshade, which are measurement methods mainly applied in clinical practice.

Cadavers have been used to evaluate the suitability and efficacy of drugs in clinical research. A measurable change in the brightness of the teeth was determined using ShadeEye NCC and the VITA Easyshade method in the present study. In the evaluation using VITA Easyshade, it was confirmed that the brightness of the teeth increased by 5 levels. Cadaver teeth treated with DW and NCP using ShadeEye NCC showed an ΔE value of 4.0. Considering that the change in tooth color can be seen with the naked eye when the ΔE values is 3, the highest ΔE value is 7.86, which can be considered a high value. Although the experimental NCP device could not treat the entire tooth with plasma evenly, it was found to have a high tooth bleaching effect. In future human clinical studies, NCP will be applied evenly to all teeth through improvements in equipment to ensure the same whitening effect. This result not only means that the quantity of •OH produced by NCP decomposing water could be sufficient for teeth whitening; it also means that if NCP is applied to teeth whitening, water can be used instead of dangerous HP.

Ablal et al. [15] conducted additional research on alternative materials and reported that tooth bleaching performed using chlorine dioxide is faster than tooth bleaching performed using HP. However, Cheng et al. [13] reported that the use of chlorite-based bleaching agents can lead to the erosion of minerals and the formation of cracks in the tooth structure. Several materials that can be used as alternatives to peroxides have been investigated; however, no effective alternatives with no harmful side effects have been identified. In contrast, several studies have demonstrated the efficacy and safety of the application of plasma with existing tooth-bleaching agents [13,14,24,25,26]. The present study demonstrated that treatment with NCP combined with DW can yield the desired tooth-bleaching effect without the inclusion of the adverse effects associated with HP treatment, underscoring an advancement in the field of dentistry.

The amount of •OH generated was investigated to support the bleaching mechanism, as NCP generates many active species, including OH, O, H, O₃, and NO. OH reacts with chromophores as macromolecular stains switch to small molecular stains. Several previous studies have reported results similar to those of the present study. Lee et al. [24] and Sun et al. [22] reported that •OH was an important reactive species in tooth bleaching. MB solution, which was introduced in some recent articles, was used in the present study owing to its ability to indicate the presence of •OH. The color of the MB solution was used to detect the •OH concentrations. The MB cations reacted with •OH to generate hydroxyl ions and colorless MB radical cations, resulting in a color change from dark blue to colorless [29]. In this study, it was observed that in DW treated with NCP, the color of the MB solution became lighter, and the absorbance decreased. It can be assumed that DW is decomposed by NCP, and a large amount of •OH is generated.

Several studies have been conducted to determine the effects on dental hard tissues; however, the results are highly controversial. Some previous studies have demonstrated that bleaching with HP and CP has harmful effects on dental hard tissue, especially on the mineral composition and microhardness of the tooth. Cakir et al. [30] and Li and Greenwall [31] reported mineral loss in teeth after peroxide-based bleaching; thus, the use of a peroxide-based bleaching agent can affect the mineral composition of dental hard tissue. Mondelli et al. [32] reported that the use of a peroxide bleaching agent that included an acid as an activator led to a decrease in the microhardness of the dental hard tissue. In addition, Pinto et al. [33] reported that the use of 10% CP resulted in a reduction in the microhardness and an increase in the surface roughness. In this study, changes in the hardness and mineral composition of dentin and enamel could not be observed after DW and NCP treatment. These results mean that the DW and NCP treatment does not have any detrimental effects on the hard tissues of the tooth. Because even low concentrations of HP cause side effects on teeth, alternative materials that do not contain HP are needed for teeth whitening.

Considering these results, the present study suggests that DW can act as an effective whitening gel when combined with an optimal plasma device. Therefore, the tooth whitening method using DW and NCP can be a reliable and novel tooth bleaching method that does not affect the properties of the dental hard tissue. Although human teeth were used in this experiment, extracted teeth or the teeth of a cadaver were used. Therefore, for a more accurate tooth whitening effect, the next study should be conducted directly through clinical trials.

5. Conclusions

The present study demonstrated that tooth bleaching using DW and NCP resulted in a substantial whitening effect without inducing a significant change in the mineral composition and microhardness of the dental hard tissue. These results underscore a great advancement in the field of modern dentistry as they provide a novel method for tooth bleaching without the use of a peroxide-based agent.