Plasma technology is becoming more and more part of our daily lives. Electrical, thermal, chemical and physical properties of plasma have been widely used in various fields including electronics, manufacturing, energy, aerospace and environmental industry. Recently, plasma has attracted increased attention in the biomedical field. Plasma is a partially ionized gas containing a large amount of highly reactive species including ions, electrons, free radicals and electronically excited neutrals. Plasmas can be classified according to thermodynamic equilibrium. Temperatures in the so called thermal plasma, which is in thermodynamic equilibrium, can reach as high as several 10,000 K. In non-thermal plasma, plasma which is not in thermodynamic equilibrium, temperatures can be as low as 300 K (1). Non-thermal plasmas can be divided according to pressure into the atmospheric pressure plasmas (APP) and low pressure plasmas. For generating low pressure plasmas, vacuum chamber and vacuum pumps are required which can be very expensive and complicated (2). Various types of plasma sources have been developed to generate plasmas at atmospheric pressure. One of them is the non-thermal APP jet, which can be further subdivided according to their electrode design: dielectric-free electrode (DFE) jets, dielectric barrier discharge (DBD) jets, DBD-like jets and single electrode (SE) plasma jet (3).
The APP jet contains all reactive species as mentioned above including active radicals such as: Reactive Oxygen Species (ROS) O, OH-, HO2-, O2, HO2, H2O2 and Reactive Nitrogen Species (RNS) NO, N2, NO2. Both ROS and RNS have the potential to react with biological materials while their temperature remains near room temperature. Because the APP jet can be applied to desired sites in open space without damaging the surrounding tissues, it has recently attracted much interest in biomedicine due to its potential applications in bacteria inactivation, tissue sterilization, blood coagulation, wound healing, suppressing the melanoma cancer cell and treatment of corneal infections (4-8). Also, in dentistry the plasma technology has shown great potential (9).
One of the most popular esthetic procedures in dentistry is tooth bleaching and every day more and more patients are seeking to improve their smiles with an effective and safe method. Tooth bleaching can be generally divided into two types: “in home bleaching” in which the patient uses a tray containing a low concentration of carbamide peroxide (CP) gel for two or three weeks and “in office bleaching” in which dental practitioners use a higher concentration of hydrogen peroxide (HP) gels 15-45% and higher concentrations 30-37% of CP gels, which is directly applied to the teeth for a total period of 30-60 minutes (10-12). The mechanism of bleaching is based on the HP or CP gel’s ability to penetrate tooth structure and produce free radicals that oxidize organic stains within the tooth (13). For better results in office, tooth bleaching products available on the market can be combined with a supplementary light source (i.e. laser, LED, OLED, halogen lamp or plasma arc) (14, 15). However, information on the effectiveness of these light sources in the literature is conflicting. Some authors claim that use of the light sources increases the efficiency of tooth bleaching (16) while others reported it had no clinically significant effect on tooth bleaching (17). The success of tooth bleaching depends on the concentration of the gel and total treatment duration (18). The time needed for tooth bleaching is of great importance, longer time produces better bleaching results. However, increased application time may cause enamel surface alterations, such as loss of mineral content and microhardness (19, 20).
The aim of this study was to evaluate if APP jet as activator can accelerate the degradation of hydrogen peroxide and provide more effective results in a shorter period of time without a significant temperature increase which may cause damage of the tooth and surrounding tissue. For this purpose pastilles were used because they provide more uniformly colored test sample than teeth. Research hypotheses: (H1) The optical effect of bleaching is not affected by APP jet activation (H2) There is no change in pH values of bleaching gels activated by APP jet during bleaching (H3) Additional APP jet activation can lead to surface temperature increase.
Materials and Methods
Experimental setup for treating gel-pastille samples is presented in Figure 1. Atmospheric pressure plasma (APP) jet used in this investigation was a single electrode jet also known as plasma needle. Copper electrode (wire 100 microns in diameter) inserted in a glass tube (outer diameter 1.5 mm, length 5 cm) was connected to a 25 kHz and 2.5 kV power supply (21). Normally, around 1 W of power is transferred to the sample. Helium (4.6 purity), at flow rate 2 l/min, was used as a working gas. Samples (pastille, bleaching gel) were placed on a non-conductive holder approximately 13 mm from the tip of the electrode to the gel surface.
During the treatment, surface temperature of the sample and the optical emission spectroscopy (OES) of the APP jet were measured. Fiber optic spectrometer Avantes AvaSpec 3648 (Avantes Inc., Apeldoorn, The Netherlands) with a 0.8 nm spectral resolution in the range from 200 to 1100 nm was used. Collimating lens at the end of the optical fiber was placed at the beginning of the jet, perpendicular to the jet axis as seen in Figure 1. The integration time was set to 250 ms. Miniature Infrared Sensor (MI, Cole-Parmer, Vernon Hills, Illinois, USA) pyrometer with 10:1 distance-to-target ratio was mounted approximately 10 cm from the sample at an angle of about 45°. A pyrometer was used because it provides non-contact measurement and is not affected by plasma as thermocouples. Because the emissivity of the used samples is not known, a calibration using K-type thermocouple was performed. Samples (pastille and pastille with bleaching gel applied) were heated in an oven and then the temperature was measured simultaneously with pyrometer and thermocouple while the samples were cooling down. From these measurements, pyrometer calibration curves for our samples were determined.
Samples and experimental procedure
35 pastilles were made out of 400 mg hydroxylapatite powder (Hydroxylapatite for analysis, ACROS Organics Co., Fair Lawn, NJ, USA). Pastilles 12.5 mm in diameter and 2.5 mm thick were compressed under the pressure of 20 bar (Universal GP1, Banja Luka, Bosnia and Herzegovina) out of hydroxylapatite powder weighted with scale (Mettler PM200, Greifensee, Switzerland). In order to obtain strength, the pastilles were then dried for 2 hours at the temperature of 150°C in a dry sterilizer (Instrumentaria, ST-01/02, Zagreb, Croatia) (22). Solution was made from 2 g of green tea (Fanning’s, Cedevita d.o.o., Zagreb, Croatia) boiled in 100mL of distilled water for 5 minutes and after that the tea was cooled to room temperature. The pastilles were then immersed into the tea for 8 hours in order to gain color (18).
In this study, we used two different bleaching gels: DASH (Discus Dental, LLC, Los Angeles, California, USA), which is a 30% HP gel and BOOST (Ultradent Products, Inc., South Jordan, Utah, USA), which is a 40% HP gel. For measuring the bleaching effect of each gel, specimens were divided into two groups (n=10) depending on used bleaching gel (DASH or BOOST). Each group was then divided into two subgroups: (G1) control - bleaching gel without APP jet activation and (G2) bleaching gel + APP jet activation. One separate group (n=5) was formed for treatment with only APP jet without bleaching gel. As for the pH measurement, two groups were formed (n=5); each for different gel in combination with APP jet.
Whitening procedure, using only bleaching gels, was performed according to the manufacturer’s instructions. 1.5 mm thick layer was applied on the pastille, left for 15/20 minutes (DASH/BOOST) and then removed using a plastic spatula. Immediately after the gel was removed, RGB of the pastille was measured. Three such cycles were performed on the same pastille. In the second procedure, first a 1.5 mm layer of bleaching gel was applied on the pastille and then treated with APP jet for 9 minutes. After the treatment, the gel was removed and RGB of the pastille was measured. In the third whitening procedure, the pastilles were treated for 9 minutes only with APP jet. RGB was measured immediately after that.
Bleaching evaluation, pH measurements and statistical analysis
Instruments such as spectrophotometers and colorimeters are used to measure the colors of different materials as well as tooth color (23, 24). Immediately after the treatment, RGB (red, green, blue) values of pastilles were measured with colorimeter (PCE-RGB 2, PCE Instruments, Southampton, United Kingdom). RGB values of each pastille were measured 5 consecutive times before the treatment and 5 consecutive times after the treatment in order to minimize the measurement error. Because only the differences between bleaching efficiency of different procedures were evaluated, RGB color space could be used. The RGB color model is based on mixing red, green and blue with different intensities thus forming the final color. In RGB space the color is therefore presented as an RGB triplet. Each of the three colors can vary from zero to maximum value (in our case 1023), where black is represented as point (0, 0, 0) and white is at a point (1023, 1023, 1023). Euclidian distance between measured RGB and white point can be defined as white distance. The color change ΔRGB can therefore be defined as a difference between the white distance of pastille before the treatment and the white distance of the same pastille after the treatment. Arithmetic mean value of ΔRGB and its standard deviation were evaluated over 5 consecutive measurements of 5 different pastilles treated under the same conditions.
The pH was measured using ExStik EC 500 pH meter with contact pH electrode (Flir Commercial Systems, Inc., Nashua, New Hampshire, USA). The pH meter was standardized by Hanna instruments buffer solutions of pH 4, 7 and 10 (Hanna instruments, Ann Arbor, Michigan, USA) and was re-calibrated after each measurement. Initial pH value of both bleaching gels was measured on a glass plate prior to the treatment. In order to measure pH value of bleaching gels after the APP jet treatment, 5 pastilles were used for each type in total of 10 pastilles. The bleaching gel was placed on pastille and after the APP jet treatment the pH electrode was placed in contact with the gel for a period of 10 minutes at room temperature 21˚C and humidity 56%. Each minute the pH value was recorded for total of 10 measurements for each sample. The electrode was washed between samples measurements under a stream of water to remove gel debris. The electrode was then rinsed using deionized water and dried with sterile gaze (Lola Ribar d.d., Zagreb, Croatia). After re-calibration, the procedure was repeated for the next pastille.
Data analysis and graphical presentation of the results was performed with Origin Pro 8.5 (OriginLab Corporation, North Hampton, MA, USA).
Typical optical emission spectra for APP helium jet without a sample and for jet during the treatment of BOOST gel on pastilles are presented in Figure 2. The strongest emission lines in the presented OES spectra belong to helium atom and nitrogen molecule. Apart from these two sets of lines, OH, N2+, O and H emission lines are also visible. It can be observed that intensities of the emission lines of the APP+BOOST spectrum are slightly higher than the ones of the free APP spectrum. Similar OES results can be observed for APP jet during the DASH gel treatment.
In Figure 3, the results of color change ΔRGB immediately after the treatments are shown. The ΔRGB color change for BOOST gel showed better results than for DASH. The best results were obtained when APP jet was in conjunction with bleaching gels. Slightly better results were obtained when APP jet was in conjunction with BOOST than with DASH.
The pH values of DASH and BOOST gel were measured before the treatment and after the treatment with APP (Table 1). After the APP treatment, the pH value of DASH gel was approximately 2 times lower than before the treatment, while in the case of BOOST gel, the pH value dropped to about 75% of its initial value. The pH was also measured each minute for another 10 minutes after the treatment with APP jet. These values did not change dramatically and no trend was observed.
|DASH before the treatment||4.64||0.08|
|DASH after the treatment||2.05||0.15|
|BOOST before the treatment||7.25||0.01|
|BOOST after the treatment||5.34||0.08|
The non-contact temperature measurements of the pastille or gel surface show that the treatment with APP rises the surface temperature of the samples. In the case of the pastille to about 30°C, the surface temperature of DASH and BOOST gel rises to a slightly higher temperature of about 32°C and 30.5°C, respectively (Figure 4).
OES spectra of the APP jet during the treatment of bleaching gels on a pastille shows the intensive increase of emission lines compared to APP jet without the sample (Figure 2). However, the ratio between intensities of APP jet without the sample and with the sample is not the same for all spectral features. While gels used in this study consist of hydrogen peroxide, one of the biggest relative increases can be observed for OH emission lines (308 nm). Even though the working gas is helium, because of the diffusion of the ambient air into the plasma, there is a lot of nitrogen in the OES spectra (25). When the surrounding air changes its composition, because of the chemically active role of plasma on the gel, some molecules evaporate from the sample and the OES spectra of the light emitted by the plasma also changes. This is why the change in OH emission lines is also observed about 10 mm above the treated gels, while as previously mentioned, the OES spectra were measured at the beginning of the jet and the optical fiber was positioned perpendicular to the jet. Moreover, if OES would be measured lower along the jet or if the angle between jet and the fiber was different, the ratios between spectral features would change (25). Therefore, if the collimating lens of the OES spectrometer was pointed down toward the sample, the intensity of the OH emission lines compared to e.g. nitrogen emission lines would probably increase.
In order to evaluate the bleaching effect of different procedures, RGB color change of the pastilles before and after the treatment was determined. In case of treatment with only bleaching gels, ΔRGB results are presented after the first, second and third cycle. As mentioned before, one cycle is 15 and 20 minute long treatment with DASH and BOOST, respectively. The whitening of the pastilles after the third DASH treatment is not considerably better than with only two consecutive 15 minute treatments. However, two 15 minutes DASH treatments are almost 20% better than only one. When using APP in combination with DASH gel for 9 minutes there were 32% better results in comparison with only DASH gel (3 x 15 minutes). In the case of BOOST gel, whitening is approximately 8% better with each additional cycle. When BOOST is treated for 9 minutes with APP jet the ΔRGB is about 15% higher than with three 20 minute BOOST treatment cycles. Here it should be mentioned that after prolonged plasma treatment, the bleaching gel deformed and changed viscosity. However, this did not reduce the bleaching effect. The bleaching effect can also be achieved with APP treatment only, but it is almost 3 times lower than plasma in conjunction with bleaching gels. Similar whitening effects were also reported in the study by Choi et al. (26) where they used APP with different gases. In the study by Nam et al. (27), three different sources (APP, plasma arc lamp and diode laser) for activation of carbamide peroxide were used, and APP obtained the best bleaching results.
The manufacturer suggested that the products have relatively neutral pH to minimize the potential damage which could be caused by highly acidic or highly basic solutions. However, most of the bleaching gels were found to be acidic, especially in-office bleaching products (28). The effects of acidic or basic solutions depend on the exposure time and how often the product is used. Whitening toothpastes, which are used every day, should also have a neutral pH; it is reported that some toothpastes had a highly acidic pH as low as 3.67±0.06 (29). Acidic and neutral in-office bleaching agents have the same whitening efficiency in situ and in vitro (30). In this study DASH gel had pH values of 4.64 ± 0.08 while BOOST gel was close to neutral with pH 7.25 ± 0.01. The acid pH measured for 30% HP (DASH) was below the critical level for enamel which is in between 4.5-5.5 and can cause hard tissue demineralization. According to the literature, this can be attributed to the low concentrations of calcium and phosphate ions and high concentrations of sodium and chloride ions in bleaching gels which can cause under-saturation with respect to hydroxyapatite (31). After the APP jet treatment in conjunction with bleaching gels, the pH values dropped for both types of gels (Table 1). This drop is related to chemical activity of APP jet. Active radicals such as Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) are present in plasma and can react with bleaching gel thus lowering pH value of the gel which can lead to potentially less harmful effect on the tooth surface without alterations of the enamel structure. Further research is needed to investigate the main chemical interaction which can lower pH value of bleaching gels. Subjecting the teeth and oral tissues to a low or high pH for an extended period of time may cause adverse side effects such as enamel demineralization (32) and root resorption (33). Therefore the reduction of bleaching time with APP jet treatment, as presented in this study, and consequently shortening the exposure of teeth to low pH can decrease the side effects described above.
According to the available literature, the use of the light activation in conjunction with bleaching gels generates higher intrapulpal temperatures (34-36). In the present study, during the bleaching procedure, the heat generated by APP jet was measured on the surface of the treated samples and in all cases it did not pass 33˚C. The maximum temperature increase was 8˚C and 10˚C above baseline for BOOST and DASH gel, respectively. In the case of BOOST gel, in a few minutes of treatment the temperature increased from room temperature to slightly above 30°C and remained constant throughout the treatment, while in the case of DASH gel the surface temperature increased to 30°C in about 3 minutes and continued to rise slowly to about 32°C in 9 minute treatment. The effects of various lights used to accelerate the bleaching process on surface and pulp chamber temperature increases have been investigated (18). Zach and Cohen found in a monkey model that a 5.5°C temperature rise was likely to cause irreversible pulpal damage (37). Since dental tissue has a low thermal conductivity, superficial heating of a tooth does not significantly heat the pulp (38). It was found that the increase in the pulp chamber temperature with most bleaching lights was above this critical threshold. In the study by Eldeniz et al. (39), the diode laser induced significantly higher temperature increases than any other curing unit (11.7°C) while the light emitting diode unit produced the lowest temperature change (6.0°C). In a study by Luk et al. (16) the application of infrared laser, CO2 laser, halogen lamp and argon laser in combination with 35% hydrogen peroxide gel and 10% carbamide peroxide gel caused significant temperature increase inside the pulp chamber and on the tooth surface. Klaric et al. (34) found that the ZOOM2 light source led to the largest increase in mean pulpal and tooth surface temperatures of 21.1 and 22.8 °C, followed by focused femtosecond laser which increased the pulpal and surface temperatures by up to 15.7 and 16.8 °C. Also, the effect of an intact pulpal circulation or simulated blood flow is able to dissipate some of the applied heat before pulpal cells are damaged (40, 41). Our in vitro model was not able to replicate this, so it is possible that the 10°C surface temperature rise is in reality lower and therefore below the threshold for possible pulp damage.
Another shortage in interpretation of the results in this study is that, apart from the surface experiment, this type of study could not yet be conducted in vivo. Where light is used to accelerate bleaching, the intrapulpal and surface temperatures also depend on the light application period and type of light used. Usually, a longer light irradiation produces a higher temperature rise because more light energy is converted into heat (42). The application time for APP jet was 9 minutes which is considerably shorter than the time proposed by the manufacturer of bleaching gels used in the study. Clinical outcome of this heat generation is expansion of the liquids inside the dentinal tubules and the pulp which in combination with dehydration of the bleached hard dental tissues result in hyperemia and post-operative sensitivity (43). As the temperature of the sample surface with APP jet treatment is under the body temperature, which means that a patient could not feel a sense of pain from cold or heat and the tooth pulp or tooth surrounding tissue will not be thermally damaged (27). For this reason, APP jet is applicable to therapies that require chemical activation without temperature increase such as tooth bleaching.
This study has a few limitations. The research was conducted on pastilles of hydroxylapatite, which are different from human teeth in their chemical composition, surface color or physical characteristics. The pastilles were used as an experimental model to investigate the potential bleaching effect of APP jet. They served as experimental and uniform models and were equal in their composition, size and color. That is why the pastilles serve as a good and suitable choice for comparison of the effects of different bleaching treatments. On the other hand, human or bovine teeth are all different in their chemical, physical and optical characteristics and the true effect of the APP jet cannot be reliably determined. Pastilles also have a flat surface, which is important for color measurement when using the RGB colorimeter. Also, the temperature values measured in this study cannot be directly applied to temperature changes in vivo. Further studies with different concentrations of bleaching agent in conduction with APP jet or other types of plasma using human teeth are necessary to characterize the precise surface temperature rise.
In this study it was proved that APP jet can be used as a chemical activator which can accelerate the degradation of hydrogen peroxide. This provides more effective results of tooth bleaching in a shorter period of time than the procedures proposed by the manufacturers. The measurements of surface temperature during the APP treatment suggest that this kind of treatment has a greater capability than conventional light sources. APP jet treatment combined with bleaching gels (30 and 40% HP) could therefore become a procedure that could be safely used for in office tooth bleaching without a significant temperature increase.