Hiromichi Yumoto, Department of Conservative Dentistry, The University of Tokushima Graduate School, Institute of Health Biosciences, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan. E-mail: firstname.lastname@example.org
Aims: The aim of this work was to clarify the effects of electromagnetic wave irradiation (EMWI) on oral bacterial pathogens.
Methods and Results: A Gram-negative (Porphyromonas gingivalis) or Gram-positive (Streptococcus mutans, S. intermedius, Enterococcus faecalis) bacterial suspension was irradiated by EMW apparatus (500–1000 kHz, 5–15 times, 1 s time−1). Quantification of survival bacteria by CFU counting revealed that EMWI exhibited marked bactericidal activity against all tested bacteria and bactericidal activity at 500 kHz increased in an irradiation number-dependent manner. After EMWI at 500 kHz, scanning electron microscopic observations showed that the chain of S. mutans cells was shortened after 5 irradiations and the outlines of bacterial cells (S. mutans and P. gingivalis) were unclear after 5–10 irradiations. EMWI inhibited the inductive effect of S. mutans on pro-inflammatory cytokine production in human monocytes and this inhibitory effect was comparable with that of heat-killed bacteria. Furthermore, using an enzyme activity assay, EMWI partially inactivated the activities of gingipains from P. gingivalis.
Conclusions: These findings demonstrated that EMWI has inactivation and bactericidal activities against single microbial species among four kinds of oral pathogens.
Significance and Impact of the Study: Electromagnetic wave irradiation may be applicable for medical disinfection and sterilization, such as refractory periapical periodontitis.
A large number of reports using conventional culture and molecular biologic techniques have shown that root canal infection is mainly caused by a mixture of Gram-positive and Gram-negative bacteria and various bacterial species are associated with primary endodontic infections (Baumgartner and Falkler 1991; Dougherty et al. 1998; Rolph et al. 2001; Siqueira et al. 2001; Fouad et al. 2002; Munson et al. 2002; Sunde et al. 2003; Siqueira et al. 2007). Disinfection within the root canal system is the most important point in endodontic treatment; however, it is quite difficult to completely remove bacteria and all causal agents from the root canal system because of the anatomical and morphological complexities. We previously suggested that infected root canal treatment consisting of cleaning, shaping and irrigation could not completely remove bacteria from the root canal system (Matsuo et al. 2003); therefore, it is necessary to develop new methods of bacterial control against infected root canal-associated bacteria.
Various sterilization and disinfection technologies are generally used in our daily life, especially in hospitals and other health care settings. Methods widely used for sterilization and disinfection include chemicals, heat and UV irradiation (Muraca et al. 1987); however, the widespread use of antibiotics as chemicals causes the emergence of more-resistant and more-virulent strains of microorganisms (Aiello and Larson 2003; Russell 2003; 2004) and heat application at temperatures above 120°C, such as steam, dry-heat and soft hydrothermal treatment, is restricted to use for the depyrogenation of parenterals, such as medical devices, and cannot be applied in medical practice. UV and UV-light-emitting diode (LED) irradiation are effective disinfection methods because they are easy to apply, require no additional chemical input and produce no hazardous by-products (Hamamoto et al. 2007; Mori et al. 2007). The antimicrobial effects of various lasers in the root canal have also been studied in vitro, and most reports have indicated that laser irradiation is useful for bacterial suppression (Mehl et al. 1999; Folwaczny et al. 2002; Schoop et al. 2006; Wang et al. 2007; Kivanc et al. 2008; Noiri et al. 2008); however, the antimicrobial effects of both UV and laser irradiation are inversely proportional to the square of the distance from the light source to the target area and it is also impossible to insert their light source tips near the apical root portion according to the curvature of the root; therefore, it remains necessary to improve the sharpness and material of the laser tip for application to various complex forms of root canals. Several in vitro studies have shown that high-energetic extracorporeal shock waves had a bactericidal effect on planktonic micro-organisms, such as Staphylococcus aureus, Streptococcus epidermidis, Pseudomonas aeruginosa and the MRSA 27065 strain (Kerfoot et al. 1992; von Eiff et al. 2000; Gollwitzer et al. 2004; Gerdesmeyer et al. 2005), but this is difficult to use as intracorporeal application for narrowing and complex regions like a root canal; thus, it is necessary to develop new methods of bacterial control at any infectious sites.
Methods widely used for sterilization and disinfection as described above need prolonged application (several tens of minutes or hours); therefore, it is necessary to reduce the duration for clinical application, such as in root canal treatment. It has been recently reported that microwave irradiation (≤9 min) with several advantages (for example, reduction of application time by rapid heating, pathogen destruction, ease of control and compactness) effectively disintegrated cells of both Gram-positive and Gram-negative bacteria, and this damage was more severe in Gram-negative than Gram-positive cells (Zhou et al. 2010). In addition to a reduction of application time, another interesting in vivo study from a clinical point of view has shown that high-frequency electrical pulses (high-frequency electric waves; 312·5 kHz and application time; 0·14 s) may be utilized as a supplement to traditional endodontic techniques to improve the cleansing and elimination of organic residues within the root canal system (Lendini et al. 2005); however, no reports to date have demonstrated the effects of electromagnetic wave irradiation (EMWI) on the viability of pathogenic bacteria and the healing of infected tissues resulting from inflammation triggered by bacterial infection.
Given these reports, we focused on the potential usefulness of EMWI as a disinfection apparatus against pathogens localized in the root canal. Using a thin and flexible endodontic hand-operated instrument (K-file No. 10), which is applicable for narrow and curved roots, we tested several oral bacterial pathogens, S. mutans, S. intermedius, Enterococcus faecalis and Porphyromonas gingivalis, which have the ability to colonize and form extracellular matrices on the surface of gutta-percha points (Takemura et al. 2004), are consistently isolated from endodontic infections (Sundqvist 1994; Le Goff et al. 1997) and are associated with extraradicular biofilm formation and refractory periodontitis (Noguchi et al. 2005). The aims of this study were to investigate the inactivation and bactericidal activities by EMWI on oral bacterial pathogens and to characterize the EMW-irradiated bacterial cells morphologically and biochemically.
Materials and methods
Bacterial strains and growth conditions
Streptococcus mutans MT8148 (kindly provided by Dr T. Ooshima, Osaka University), S. intermedius UNS46 (kindly provided by Dr H. Nagamune, The University of Tokushima) and E. faecalis ATCC19433 were cultured in brain-heart infusion (BHI) broth (Difco, Detroit, MI, USA) or on BHI agar at 37°C. In an anaerobic environment (>16% CO2) using AnaeroPack anaerobic atmosphere generation systems (Mitsubishi Gas Chemical Co. Inc., Japan), P. gingivalis ATCC33277 and W83 (kindly provided by Dr I. Takazoe, Tokyo Dental College) were grown in BHI broth containing 0·5% yeast extract (Difco), 10 μg ml−1 hemin and 1 μg ml−1 vitamin K or on CDC anaerobic blood agar (BD, Franklin Lakes, NJ, USA).
Electromagnetic wave (EMW) source
Electromagnetic waves (EMWs) were generated with high-frequency therapy equipment (J. Morita Mfg. Corp., Kyoto, Japan) (Fig. 1). The frequency of EMWs from this treatment device was set to 500–1000 kHz, and tone-burst waves with a large crest factor were employed to efficiently generate arch discharges. In preliminary experiments, we previously identified that minimizing the diameter of the active electrode tip and taper as much as possible and maintenance of the contact depth between the active electrode and tissue to within 5 mm converged the electric current leading to the efficient generation of Joule heat; therefore, we employed an endodontic hand-operated instrument (K-file No. 10; Mani, Inc., Utsunomiya, Japan) (Fig. 1).
Bactericidal activity by electromagnetic wave irradiation
All tested bacteria were grown in a liquid medium, washed and resuspended in phosphate-buffered saline (PBS) to a final concentration of approximately 1·0 × 109 CFU ml−1. The bacterial concentrations were determined spectrophotometrically according to standard curves. A microbial suspension of 125 μl was transferred into a 0·2-ml thin-walled tube and irradiated with EMW. It has been considered that the lower frequency (up to 100 kHz) has the possible biological effects such as the reduction of heart rate and the disturbance of nerve and muscle responses. Moreover, it has been presented that the intermediate frequency (100 kHz–300 GHz) produces heat in a frequency-dependent manner and has the harmful effects such as tissue and body heating by the absorption of produced electromagnetic energy (ICNIRP 1998). Therefore, considering a clinical application, the frequency was set to 500, 750 and 1000 kHz, and the current was intermittently passed for 1 s time−1 at intervals of about 2 s to protect against these adverse health effects. Appropriate dilutions of irradiated and unirradiated control microbial suspensions were spread onto BHI agar or CDC anaerobic blood agar as described in previous paragraph, and CFU was enumerated after incubation at 37°C for 1 day (for S. mutans, S. intermedius and E. faecalis) or 7 days (in an anaerobic environment for P. gingivalis). The % survival ratio was calculated.
The temperature of the microbial suspension during EMWI was monitored using a thermometer (CUSTUM CT-1300 Type K; CUSTUM, Tokyo, Japan). To determine whether the bactericidal activity by EMWI was owing to the increased temperature of the microbial suspension, thermal cycler (Takara PCR Thermal Cycler Dice TP600, Shiga, Japan) raised S. mutans suspension of 125 μl from room temperature to 74·5°C over 90 s and maintained for 1 s in a similar temperature change to EMWI. After this heat treatment, appropriate dilutions of heat-treated S. mutans suspensions were spread onto BHI agar. After incubation at 37°C for 2 days, CFU was enumerated and the % survival ratio was calculated.
Scanning Electron Microscopy
Scanning electron microscopy (SEM) of bacteria was performed as follows. Briefly, 50-μl bacterial suspension after EMWI as described above was placed on cell desks (Cell Desk LF1; Sumitomo, Tokyo, Japan) in 24-well plates. After 1 h, the cell desks were washed twice with d-H2O and were fixed by adding 2·5% glutaraldehyde (TAAB Laboratories Equipment Ltd, Berks, UK) in 0·1 mol l−1 phosphate buffer. The cell desks were left to stand overnight at 4°C, and then the solution was removed and the cell desks were washed with 0·1 mol l−1 phosphate buffer, pH 7·4. The attached bacterial cells on the cell desks were postfixed with 1% osmium acid solution for 1 h. The bacterial cells were dehydrated through a graded series of ethanol solutions to 100% ethanol. After dehydration, the bacterial cells were treated with 50%t-butylalcohol for 15 min and twice with 100%t-butylalcohol for 30 min. The cell desks with fixed bacterial cells were lyophilized with a freeze-drying device (JFD 300; JEOL, Tokyo, Japan), and coated with Au ion at a thickness of 100 Å for SEM analysis. SEM was carried out with a Miniscope TM-1000 (Hitachi High-Technologies Corp., Tokyo, Japan).
THP-1 cell culture
THP-1 cells, a human monocytic cell line, were cultured in RPMI 1640 medium supplemented with 10% (v/v) foetal bovine serum (FBS; JRH Biosciences, Lenexa, KS, USA), 0·05 mmol l−1 2-mercaptoethanol, 100 μg ml−1 streptomycin and 100 U ml−1 penicillin. Cells (500 μl well−1) were seeded in 48-well tissue culture plates at a concentration of 1 × 106 cells ml−1 and were cultured in 5% CO2 at 37°C.
Stimulation assay with S. mutans irradiated by EMWI
Streptococcus mutans MT8148 were collected by centrifugation, washed in PBS and suspended in medium devoid of antibiotics at a concentration of 2·5 × 108 or 2·5 × 109 cells ml−1 as live bacteria. Bacterial concentrations were determined spectrophotometrically according to a standard curve. Heat-inactivated bacteria were prepared by boiling for 10 min at 100°C and bacteria irradiated by EMW were prepared as described above. Live, heat-inactivated or irradiated bacteria were directly added to THP-1 cell culture in 48-well culture tissue plates and incubated for 6 h. After incubation, the cell culture supernatants were collected and used to quantify the levels of pro-inflammatory cytokines using an enzyme-linked immunosorbent assay (ELISA), and stored at −20°C until assayed.
ELISA for pro-inflammatory cytokines
The concentrations of interleukin (IL)-1β, IL-8 and tumour necrosis factor (TNF)-α in cell culture supernatants were determined using commercially available ELISA kits (Duo Set ELISA Development System; R&D Systems, Minneapolis, MN, USA) as described in the manufacturer’s instructions.
Bacterial enzyme activity assay
The amidolytic activity of arginine-specific (Rgp) and lysine-specific (Kgp) gingipains in the whole culture of P. gingivalis ATCC33277 was determined with either N-benzoyl-l-arginine-4-nitroanilide hydrochloride (BApNA) or l-lysine-p-nitroanilide dihydrobromide (Z-Lys-pNa) (both from Sigma, St Louis, MO, USA). Samples were preincubated in 200 mmol l−1 Tris-HCl, 100 mmol l−1 NaCl and 5 mmol l−1 CaCl2 (pH 7·6), supplemented with 10 mmol l−1 cysteine, for 10 min at room temperature and assayed for amidase activity with 2 mmol l−1 substrate. The formation of p-nitroanilide was monitored spectrophotometrically at 405 nm.
All data were analysed using one-way anova and Tukey’s multiple comparison tests with SPSS software (ver. 12·0; SPSS Japan Inc., Tokyo, Japan). Differences were considered significant when the probability value was <5% (P < 0·05).
Bactericidal activity by EMWI
We first estimated the frequency effect of EMWI on the bactericidal activities to kill oral bacteria. A 1·0 × 109 CFU ml−1 oral bacterial suspension (125 μl in 0·2-ml tube) was irradiated with EMW at 500, 750 and 1000 kHz five times (1 s time−1) and the levels of surviving bacteria were quantified. EMWI at 500–1000 kHz was significantly sufficient to inactivate all tested bacteria (Fig. 2). EMWI exhibited greater bactericidal activity to kill Gram-negative bacteria, P. gingivalis (ATCC33277; survival ratio 0·01–0·78%). Moreover, we compared the heavily encapsulated and essentially afimbriated P. gingivalis strain W83 with the nonencapsulated and fimbriated P. gingivalis strain ATCC33277, to determine if there was a protective effect afforded by the capsule (Aduse-Opoku et al. 2006). The bactericidal activity (0·9–6·1%) to kill the encapsulated strain after EMWI was less than that to kill the nonencapsulated strain. EMWI also had bactericidal activity against Gram-positive bacteria, S. mutans (0·32–0·81%) and S. intermedius (0·32–6·49%), and S. mutans was slightly sensitive to EMWI than S. intermedius. Among tested Gram-positive bacteria, it has been demonstrated that Enterococci can grow in the range of 10–45°C and survive very harsh environments, including extreme alkaline pH (9·6), high salt concentrations and a temperature of 60°C for 30 min (Tendolkar et al. 2003). In particular, E. faecalis is a microorganism commonly detected in a high percentage of asymptomatic, persistent endodontic infections and is implicated in the aetiology of failing root canal treatment. Interestingly, EMWI had bactericidal activity to kill heat-resistant E. faecalis (4·35–6·47%). Overall, there was little difference in bactericidal activity among the frequencies tested (500–1000 kHz).
Therefore, we next estimated the effect of the number of EMWI (1 s time−1) at 500 kHz on bactericidal activities to kill oral bacteria. Bactericidal activity to kill both Gram-negative and Gram-positive bacteria by EMWI at 500 kHz increased in an irradiation number-dependent manner, with the exception of heat-resistant E. faecalis (Fig. 3).
Temperature change during EMWI
To determine whether these bactericidal activities of EMWI were owing to the increased temperature of the microbial suspension, we monitored the temperature of the microbial suspension during EMWI at 500 kHz. As shown in Fig. 4(a), the temperature of the microbial suspension increased with the irradiation number at the rate of approximately 4·5°C per irradiation. After 10 consecutive irradiations, the temperature of microbial suspension was still 74·5°C. As the irradiation current was intermittently passed for only 1 s time−1, the temperature decreased immediately after halting the irradiation. We next determined whether the heat treatment and a similar temperature change to EMWI can affect the viability of S. mutans cells. Although a temperature increase to 74·5°C resulted in a significant reduction of viable S. mutans cells, EMWI had greater bactericidal activity than this heat treatment (Fig. 4b).
Aggregate morphological change by EMWI
We also observed morphological changes of S. mutans MT8148 and P. gingivalis ATCC33277 as Gram-positive and Gram-negative bacteria, respectively, after EMWI at 500 kHz by SEM (Fig. 5). The chain of S. mutans cells was shortened after five irradiations. After 10 irradiations of S. mutans and five irradiations of P. gingivalis, the outlines of bacterial cells were unclear and observed as denatured aggregates. These observations of aggregate morphological changes after EMWI correlated with the data of bactericidal activities showing that most bacterial cells were killed (Fig. 3).
Inactivation of pro-inflammatory activity by EMWI
The ability of EMW-irradiated oral bacteria to elicit a pro-inflammatory reaction may remain an important clinical problem. Next, we determined the ability of EMW-irradiated oral bacteria to induce the production of pro-inflammatory cytokines in human monocytic cells. In THP-1 cells, EMWI (500 kHz, five times) significantly reduced the pro-inflammatory activity of S. mutans to produce IL-1β, IL-8 and TNF-α to a similar degree as when killed by heat treatment (Fig. 6). These results suggest that EMWI diminishes the ability of oral bacteria to elicit inflammation.
Decreased gingipain activity by EMWI
We further determined whether EMWI can also decrease the amidolytic activity of oral bacteria. EMWI (500 kHz, five times) significantly reduced the activity of two major proteinases, RGP and KGP, in P. gingivalis (Fig. 7). In particular, both RGP and KGP activities of P. gingivalis after irradiation were less than that of heat-killed bacteria. These data indicate that EMWI reduces the virulence of oral pathogens.
This study successfully demonstrated that EMWI for 5 s at 500–1000 kHz has bactericidal activity against both Gram-positive oral bacteria (S. mutans, S. intermedius and E. faecalis) and Gram-negative oral bacterium, P. gingivalis, and EMWI for 5 s at 500 kHz reduces the pro-inflammatory activity of S. mutans and the proteinase activity of P. gingivalis (Figs 2, 3, 6 and 7).
In general, our results show that EMWI for the range of bacteria tested exhibits greater bactericidal activity to kill Gram-negative bacteria than Gram-positive bacteria. The present results are in agreement with a previous report showing that the damage was more severe in Gram-negative than Gram-positive cells by microwave irradiation for several minutes and this difference in cell disintegration efficiency is likely due to substantial differences in the bacterial cell wall structure (Zhou et al. 2010). Similarly, it has been suggested that the comparatively rigid peptide interbridge structure in the thicker peptidoglycan layer of Gram-positive bacteria may resist damage to the cell wall caused by microwave irradiation (Woo et al. 2000). On the other hand, another report has shown that Gram-positive species are more susceptible to 405-nm LED array illumination than Gram-negative bacteria and added strength to the probability that the inactivation of the bacteria through exposure to 405-nm light is the result of the photostimulation of endogenous intracellular porphyrin molecules, which leads to the production of reactive species such as singlet delta oxygen, a well-recognized trigger of cell death (Hamblin and Hasan 2004; Maclean et al. 2009). Therefore, the divergence between our results and the previous report may reflect differences in the irradiation source and the mechanism of inactivation and bactericidal activity. Recent studies have shown that extracorporeal shock-wave therapy at any energy flux densities and pulse combination has no significant effect on the viability of P. gingivalis W83, which is a heavily encapsulated and essentially afimbriated strain (Novak et al. 2008), and exposure of heat-resistant E. faecalis suspension to 405-nm LED at 10 mW cm−2 for up to 120 min resulted in negligible inactivation (Maclean et al. 2009). Interestingly, we demonstrated a significant change in the viability of P. gingivalis W83 besides the nonencapsulated and fimbriated P. gingivalis ATCC33277 strain. Moreover, EMWI had bactericidal activity to kill heat-resistant E. faecalis. These findings indicate that EMWI can reduce the bacterial protective ability afforded by the capsule and has potential for use in the development of applications for disinfection. In particular, E. faecalis is a microorganism commonly detected in a high percentage of asymptomatic, persistent endodontic infections and is implicated in the aetiology of failing root canal treatment because it can survive very harsh environments, including extremely alkaline pH (9·6) and high salt concentrations (Tendolkar et al. 2003); therefore, this EMWI may be applicable to refractory periapical periodontitis.
Methods widely used for sterilization and disinfection, such as chemicals, heat and UV radiation, need prolonged application (several minutes or hours); therefore, it is necessary to reduce the duration of clinical application. Only five irradiations (1 s time−1) with EMW at 500–1000 kHz have marked inactivation and bactericidal activity against oral bacteria. This result suggests that EMWI for several seconds could be applied in a clinical setting as an effective and rapid disinfection method against microorganisms surviving at infected sites. Considering the effect of heat generated by EMWI on bactericidal activity, we monitored the temperature of the microbial suspension during EMWI at 500 kHz (Fig. 4a). The temperature increased with the number of irradiations, but was still 74·5°C after 10 consecutive irradiations. Owing to intermittent irradiation (1 s time−1), the temperature decreased immediately after halting the irradiation. The heat treatment at 74·5°C and a similar temperature change to EMWI resulted in a significant reduction of viable S. mutans cells. Interestingly, EMWI had greater bactericidal activity than this heat treatment (Fig. 4b). This result suggests that the instantaneous increased level of heat by EMWI after several irradiations is not enough to completely kill bacteria, and other factors may affect the inactivation and bactericidal activity against oral bacteria. In accordance with this result, previous study showed that microwave-irradiated bacterial cells exhibited a greater metabolic imbalance than conventionally heat-treated cells and the destruction of microorganisms by microwave at temperatures lower than the thermal destruction point (Kozempel et al. 1998). Another report suggested that microbial cells exposed to microwave irradiation suffer from nonthermal as well as thermal (heating) effects resulting in cell disintegration (Woo et al. 2000). However, the mechanisms of bactericidal activity by EMWI are not fully understood and then further studies on the microbiological effects by EMWI are needed. Hayashi et al. (2010) reported that heating at 110°C for 10 min and heating to 140°C were the most favourable conditions to strengthen human dentin judging from the increased flexural strength and stabilization of linear shrinkage and may be useful to prevent tooth fracture (Hayashi et al. 2008); therefore, local elevation of temperature by EMWI may have the ability to strengthen human teeth.
Scanning electron microscopy photographs of nonirradiated and irradiated pure S. mutans and P. gingivalis culture cells showed clear damage (i.e. shortened chain of S. mutans cells, denatured aggregates) caused by EMWI treatment, indicating the cell disintegration effects by EMWI (Fig. 5). As shown, both S. mutans and P. gingivalis collapsed cell populations by EMWI are consistent with the results of the pure culture test by CFU determination after EMWI.
After damaging bacterial cells by EMWI, it was determined whether the remaining denatured and aggregated bacterial cells have the capability to induce pro-inflammatory responses and the activity of the pathogenic factor. We previously reported that live, not heat-killed, S. mutans can induce the production of pro-inflammatory cytokines and chemokines, such as IL-6, -8, monocyte chemoattractant protein-1 and CC chemokine ligand 20, in human dental pulp fibroblasts and THP-1 cells differentiated to macrophage-like cells (Takahashi et al. 2008; Hirao et al. 2009). Here, the capability of S. mutans irradiated with EMW to induce the production of IL-1β, IL-8 and TNF-α in THP-1 cells was compared with that of live and heat-killed S. mutans. The present findings demonstrated that EMWI (500 kHz, five times) abolishes the pro-inflammatory activity of S. mutans almost completely (Fig. 6). A recent report showed that heat-killed P. gingivalis does not induce the expression of cellular adhesion molecules on endothelial cells (Takahashi et al. 2006). Moreover, the effect of EMWI on amidolytic activities, major pathogenic factors, of P. gingivalis, was compared with that of heat treatment. Our findings showed that EMWI (500 kHz, five times) can partially reduce the enzymatic activity of P. gingivalis and this effect of EMWI is greater than that of heat treatment. These data indicate that EMWI considerably diminishes the virulence of oral pathogen to elicit inflammation at infected sites.
This present introductive report of EMWI demonstrated that EMWI has inactivation and bactericidal activity against oral pathogenic bacteria, and suggests that EMWI has several advantages, such as rapid heating, pathogen destruction and inactivation, ease of control and compactness, over previously developed treatments. In addition, using a thin and flexible instrument (K-file no. 10) which has suitable mechanical strengthen than smaller files (nos. 6 and 8) as an active electrode, EMWI may be applicable to tiny, narrowing, curved or complex morphological regions infected with bacteria as well as the root canal. Therefore, EMWI has a great advantage over laser applications because it remains necessary to improve the sharpness and material of laser tips for application in complex regions, and EMWI has potential in the development of applications for medical disinfection and sterilization.
This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (21592423 and 23390435). We thank J. Morita Mfg. Corp. (Kyoto, Japan) for providing the high-frequency therapy equipment. The authors declare that they have no conflicts of interest.