Assessment of the functional efficacy of root canal treatment with high-frequency waves in rats.

The purpose of this study was to develop a high-frequency wave therapy model in rats and to investigate the influence of high-frequency waves on root canal treatment, which may provide a novel strategy for treating apical periodontitis. Root canal treatments with and without high-frequency wave irradiation were performed on the mandibular first molars of 10-week-old male Wistar rats. The mesial roots were evaluated radiologically, bacteriologically, and immunohistochemically. At 3 weeks after root canal treatment, lesion volume had decreased significantly more in the irradiated group than in the non-irradiated group, indicating successful development of the high-frequency therapy model. The use of high-frequency waves provided no additional bactericidal effect after root canal treatment. However, high-frequency wave irradiation was found to promote healing of periapical lesions on the host side through increased expression of fibroblast growth factor 2 and transforming growth factor-β1 and could therefore be useful as an adjuvant nonsurgical treatment for apical periodontitis.

Introduction

Apical periodontitis is a disease caused by bacterial infection in the root canal system [1]. Treatment methods for apical periodontitis include mechanical removal of the infection source in the root canal; chemical cleaning of the root canal with sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic acid (EDTA); and dense, hermetically sealed obturation. Root canal treatment (RCT) can fail due to the anatomical complexity of the root canal system, which includes lateral branches and isthmuses [2], and extraradicular biofilms [3-6], which make it difficult to completely remove the source of bacterial infection [7]. The balance between bacteria and the host immune system is a key element in biofilm-related infections [8]. Therefore, alternatives to removing the source of infection have been investigated, such as gene therapy [9-11]. Recent research has explored how strengthening the host immune system can lead to healing; this approach is the basis of the current study.

This study focused on high-frequency wave (HFW) apical therapy, an adjuvant nonsurgical treatment for apical periodontitis. HFW therapy is widely used as a diathermy therapy and has curative properties in human hard tissue, such as promoting bone union after fracture [12-14]. HFW irradiation generates heat as a result of vibration energy induced by free electrons colliding with molecules in a conductor as electric current flows. In dentistry, this principle is applied in electric cauterization [15, 16]. One indirect application of HFW is its use to promote bone healing; in Japan, HFW is applied in orthopedic surgery to treat slow-healing fractures and nonunion [17-21]. In the dental field, HFW therapy has also recently been used to promote healing after implant surgery [22].

HFW irradiation exhibits bactericidal effects on various free-floating bacteria, such as Porphyromonas gingivalis, Staphylococcus intermedius, Enterococcus faecalis, and Streptococcus mutans [23]. It can also induce gene expression of growth factors, such as transforming growth factor-β1 (TGF-β1) and fibroblast growth factor 2 (FGF2), in mouse osteoblasts [24].

In humans, HFW irradiation has been shown to significantly promote healing of periapical lesions [25]. However, the mechanism of action of HFW in the root canal—whether it targets the tissue around the apex, the bacteria, or both—remains unknown. The current study investigated the adjuvant effect of HFW irradiation in the nonsurgical treatment of apical periodontitis, which consisted of mechanical removal and chemical cleaning of the source of infection.

Recently, Yoneda et al. [26] developed a model for the treatment of infected root canals in rats. In this model, apical periodontitis was induced in rat mandibular first molars and RCT was then performed under rubber dam isolation. The volume of the periapical lesion and its change over time were observed with micro-computed tomography (micro-CT). RCT significantly reduced the bacterial count in the root canal, establishing a system leading to clinical healing. With this model it is possible to evaluate the changes and reactions occurring as a result of HFW irradiation after RCT.

In the present study, we developed a new high-frequency therapy model in rats by modifying the model of Yoneda et al. [26]. We used this new model to elucidate the mechanism of action of HFW therapy in apical periodontitis. The influence of HFW as a supplement to conventional RCT in treating periapical lesions was evaluated with three-dimensional micro-CT analysis, bacteriological evaluation, and immunohistochemical analysis.

Materials and methods

Ethics statement

This study was approved by the Animal Care and Use Committees of the Osaka University Graduate Schools of Dentistry and Engineering (Permit Nos. 26-016-0 and 26-1-0). All animal experiments were carried out in accordance with the Guidelines for Animal Experiments of Osaka University. Surgical procedures were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize the animals’ suffering.

Animals

Thirty-four 10-week-old male Wistar rats (Clea Japan, Tokyo, Japan) were used in this study, as shown in Fig 1. The rats were divided into three groups: micro-CT (n = 12), bacterial quantification in the root canal (n = 10), and immunohistochemical observation (n = 12).

Fig 1

Experimental schedule of high-frequency wave root canal therapy in rat model.

The animals were maintained in the animal facility of the Osaka University Graduate School of Dentistry under a 12-h light/12-h dark cycle. Food and water were freely available. The sample size of each experimental group was set according to the method of Kadam et al. [27].

High-frequency wave treatment device

HFW irradiation was performed with a prototype electrosurgical unit system (J. Morita Manufacturing, Kyoto, Japan) [23-25]. The system consists of a main body with a root canal length-measurement function, an active electrode, and a counter electrode. HFW irradiation is initiated by stepping on a foot pedal. A stainless steel #10 K-file (MANI, Tochigi, Japan) was used as the active electrode, and a stainless-steel hook was used as the counter electrode. Irradiation conditions were set according to the method of Tominaga et al. [25]: frequency 510 kHz, voltage 35 V, energization time 1 s, and irradiation interval 4 s.

Root canal treatment in rats

Experimental periapical lesions were induced according to the methods of Kawahara et al. [28] and Kuremoto et al. [29]. Periapical lesions were induced by exposing the pulp of all right and left mandibular first molars. The rats were divided into an irradiated group and a non-irradiated group. In both groups, the root canals of the right mandibular first molars were prepared as in conventional RCT. RCT was performed according to the method of Yoneda et al. [26]. In short, the tooth was isolated with a custom-made rubber dam clamp (YDM, Tokyo, Japan) and rubber dam sheet (Heraeus Kulzer, Hanau, Germany). The pulp chamber was opened and necrotic coronal pulp was removed with a round bur and a micro-excavator (OK Micro-exca, Seto, Ibaraki, Japan). The working length was set to the level of 1.0 as indicated on an electronic apex locator (Root ZX, J. Morita Manufacturing) by using K-files (Dentsply Maillefer, Ballaigues, Switzerland); the root canal was then enlarged up to a #20 file. Root canals were irrigated with 0.5 ml each time, in total 2 ml of 2.5% NaOCl (Neo Dental Chemical Products, Tokyo, Japan) [30]. The canals were dried with sterilized paper points (VDW, Munich, Germany) and filled with #20 gutta-percha points (SybronEndo, Orange, CA, USA) and root canal sealer (RealSeal SE, SybronEndo) according to the single-cone obturation technique [31]. After processing with a bonding system (Clearfil Bond SE ONE, Kuraray Noritake Dental, Tokyo, Japan), the pulp chamber was filled with flowable composite resin (MI FLOW, GC, Tokyo, Japan). In the irradiated group, HFW irradiation was applied once to the periapical lesion and twice to the inside of the root canal immediately before root canal obturation. The root canals of the non-irradiated group were obturated as in conventional RCT without HFW irradiation. The left mandibular first molars of the irradiated group were used as a control group without RCT.

Three-dimensional measurement of periapical lesion volume

Twelve rats were divided equally into an irradiated group and a non-irradiated group (n = 6 in each group). At 0, 1, 2, 3, 4, 6, and 8 weeks after root canal filling, the periapical lesions of the rats were scanned with a micro-CT scanner (R_mCT2; Rigaku, Tokyo, Japan) under the following conditions: field of view: φ10 × 10 mm; voxel size: 20 × 20 × 20 μm; tube voltage: 90 kV; and tube current: 160 μA. After scanning, 512 consecutive tomographic slice images, each with a thickness of 20 μm, were obtained. The images were then reconstructed with the Three-dimensional Reconstruction Imaging for Bone (TRI/3D-BON) system (Ratoc System Engineering, Tokyo, Japan). The volume of the periapical lesion at the mesial root was measured as previously described [26]. In short, a calibration curve for converting CT values into bone mineral density (BMD) values was prepared by using a phantom with a known BMD. Then, BMD images of each sample were obtained. The threshold for hard tissue extraction was set with discriminant analysis, and binarization was performed. The area of the apical lesion was measured on the binarized image. The space of the periodontal ligament around sound teeth in rats of the same age was determined. The apical lesion volume was obtained by subtracting the periodontal ligament space from all lesions in the apical area, and volume differences were compared among the groups.

Quantification of bacteria in the root canal

Ten rats were divided into an irradiated group and a non-irradiated group (n = 5 in each group). All rats in both groups were euthanized immediately after RCT and the mandibular first molars were extracted. The teeth were cut at the mesial root furcation and bacteria were removed from the root surface by curetting with a sterilized spoon excavator (YDM). The mesial root was then frozen in liquid nitrogen and crushed with an SK mill (Tokken, Chiba, Japan). The samples were divided into two equal parts: one for the ATP assay to measure the number of viable bacteria in the root canal and the other for real-time polymerase chain reaction (PCR) to measure the total number of bacteria in the root canal. Following the method previously described by Yoneda et al. [26], DNA extraction was performed on a powdered sample with the InstaGene Matrix (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. Assays were performed with a 20-μl solution containing 1 μl of DNA extract (Applied Biosystems Power SYBR Green PCR Master Mix; Life Technologies, Grand Island, NY, USA) and bacterial universal primers 357F and 907R [32] (0.5 μl each), which were prepared in parallel reaction mixtures for each target sequence. The thermal cycling conditions for the Applied Biosystems 7500 Fast Real-Time PCR system (Life Technologies) were 95°C for 10 min, 40 cycles at 95°C for 15 s, and 65°C for 1 min, with recording of the fluorescence signal at the end of each cycle. Melting-curve analysis consisted of a denaturation step at 95°C for 15 s and a temperature reduction to 60°C for 1 min followed by a temperature increase to 95°C at a rate of 1%, with continuous fluorescence reading. Data were acquired and analyzed with Applied Biosystems 7500 system SDS v2.0.2 software (Life Technologies). Enterococcus faecalis SS497 was used as a standard curve. The amount of ATP in the root canal, representing the number of live bacteria, was measured. The ATP eliminator kit (AF-3X2; DKK-TOA, Tokyo, Japan) and ATP analyzer (AF-100; DKK-TOA) were used as previously described [33].

Immunohistochemical observation

All 12 rats from both the irradiated group and the non-irradiated group (n = 6 in each group) were euthanized at 2 or 3 weeks after root canal filling. Pentobarbital sodium was intraperitoneally administered, and perfusion fixation was performed with periodate lysine paraformaldehyde (PLP) fixative (Wako Pure Chemical Industries, Osaka, Japan). Mandibular samples containing the first molars were dissected, fixed in PLP for 12 h at 4°C, and decalcified in 10% EDTA containing 15% glycerol at 4°C. Serial sections of 5-μm thickness were stained with an enzyme antibody method using specific antibodies against interleukin-1β (IL-1β) (ab 9787; Abcam, Cambridge, England), TGF-β1 (MAB 240; R & D systems, Minneapolis, MN, USA), and FGF2 (ab 16828; Abcam) and then observed under a light microscope (Optiphot-2; Nikon, Tokyo, Japan).

Statistical analysis

Tukey’s test was used to detect statistically significant differences in the volume change of the periapical lesions. The Steel–Dwass test was used for statistical analysis of the quantification of root canal bacteria. Each risk factor was evaluated as 5%. IBM SPSS Statistics (version 22.0, IBM SPSS, Chicago, IL, USA) was used for statistical analyses.

Results

Representative micro-CT images of the buccolingual cross-section of the mesial root of the mandibular first molar at each timepoint are shown in Fig 2A. Compared with the control group, the volume of periapical lesions in the mesial root in the irradiated group was significantly smaller at 2 weeks after RCT (p = 0.009) and at 6 weeks after RCT (p = 0.013; Fig 2B). Furthermore, the lesion volume had decreased significantly more at 3 weeks after RCT in the irradiated group than in the non-irradiated group (p = 0.019; Fig 2B).

Fig 2

Three-dimensional measurement of periapical lesion volume.

a: Representative micro-CT images of mesial root of mandibular first molar at each timepoint. b: The volume of the periapical lesion of the mesial root was measured at each timepoint (n = 6 each). The graph shows the mean and standard deviation of the ratio of the volume at each timepoint to the volume at week 0 after root canal treatment (RCT). Significant differences are represented by different letters in the same week group (Tukey’s test, p < 0.05).

Quantification of bacteria in root canal

The number of live bacteria and the total number of bacteria in the root canal were significantly lower in both the irradiated and non-irradiated groups, compared with numbers in the control group (p = 0.043 and 0.035, respectively). No significant difference in bacterial numbers was observed between the irradiated group and the non-irradiated group ().

Immediately after RCT, (a) the number of live bacteria in the root canal after high frequency wave irradiation was quantified with an ATP assay and (b) the number of total bacteria was quantified with real-time PCR. The number of bacteria in the root canal of the left first molar of rats in the irradiated group was simultaneously determined as the control group. The graph shows the average value and standard deviation. Significant differences can be seen between different characteristics in the same graph (n = 5 each, Steel–Dwass test, p < 0.05).

Immunohistochemical analysis of periapical lesions

At 2 weeks after RCT, IL-1β-positive cells were widely expressed in the periapical lesions in both the irradiated group and the non-irradiated group (Fig 4A, 4C, 4E and 4G). However, at 3 weeks after RCT, the expression was limited in both groups (Fig 4B, 4D, 4F and 4H). Additionally, the expression of IL-1β decreased with HFW irradiation (Fig 4B, 4D, 4F and 4H). At 3 weeks after RCT, there was more expression of FGF2-positive cells within the periapical lesions in the irradiated group than in the non-irradiated group (Fig 4J, 4I, 4N and 4P). The irradiated group showed more expression of TGF-β1-positive cells at the boundary between the periapical lesion and the alveolar bone at 3 weeks after RCT, compared with the non-irradiated group (Fig 4R, 4T, 4V and 4X).

Fig 4

Immunohistochemical analysis of mesial root lesions.

Immunohistochemical analysis was performed for IL-1β (a–h), FGF2 (i–p), and TGF-β1 (q–x) in the periapical area at 2 weeks (a, c, e, g, i, k, m, o, q, s, u, w) and 3 weeks (b, d, f, h, j, l, n, p, r, t, v, x) after root canal treatment. Panels e–h, m–p, and u–x show high-magnification views of the framed areas in panels a–d, i–l, and q–t, respectively. AB: alveolar bone, C: cementum, D: dentin, P: pulp, arrowheads: positive cells.

Discussion

This work assessed the influence of HFW irradiation in vivo in a rat model and investigated the target of HFW.

Studies of HFW therapy have focused on the disinfection properties of HFW [23] and its promotion of tissue regeneration [17-22]. In human clinical studies, healing of periapical lesions is seen by 1 month after RCT [25]. In our study, the lesion volume in the irradiated group had decreased significantly at 3 weeks after RCT with HFW irradiation (Fig 2). Human studies have also shown that HFW consistently promotes healing within a certain time period following RCT [25]. Other research has suggested that bone regeneration in skull defects may be promoted by irradiation with 510 kHz HFW, and that excessive irradiation conversely inhibits bone formation [34]. In the present study, we used a frequency of 510 kHz, which is the same frequency as that used for electric cauterization. In previous reports, no differences were found in the bactericidal effect of HFW at frequencies above 500 kHz [23].

It has been reported that HFW exerts a bactericidal effect against free-floating bacteria [23]. The mechanism by which HFW stimulation achieves sterilization may be related to thermal energy and electrical energy. Because joule heat is generated by energizing, thermal energy is generated during HFW irradiation. The temperature rise resulting from a single HFW irradiation of 125 μl of phosphate-buffered saline is approximately 4.1°C; the temperature of the liquid drops to the original temperature after an interval of 4 s [35]. In the present study, no adverse events were observed throughout the observation period. Similarly, no adverse events were reported in the human clinical study by Tominaga et al. [25]. Therefore, HFW irradiation and the associated temperature increase were determined not to be harmful under the conditions tested; this finding suggests the possibility of clinical application of the procedure as adjunctive therapy for apical periodontitis.

No significant change was observed in the number of bacteria after HFW irradiation during RCT in the rat model (Fig 3). The reason for this lack of change may be that it is difficult to irradiate the biofilm directly inside the root canal and the apex. Furthermore, residual bacteria decreased significantly after RCT alone, compared with the level before treatment. Yoneda et al. reported that RCT removed 75% of bacteria in the root canal and that 75% of cases were cured with conventional RCT; however, the remaining 25% of bacteria were difficult to remove [26]. Our findings suggest that HFW irradiation had no effect on the number of remaining bacteria in this experimental setting. However, because of differences in physical size, a lower voltage (35 V) was used in rats compared with that used in humans; therefore, the treatment may have been compromised. HFW irradiation at a higher voltage may have bactericidal effects.

Fig 3

Quantification of bacteria in root canal.

a: ATP level (number of live bacteria) in root canal immediately after root canal treatment (RCT). b: Total number of bacteria in root canal after RCT.

Yumoto et al. [24] reported that HFW irradiation induced gene expression of growth factors, such as TGF-β1, VEGF, and FGF2, in mouse osteoblasts in vitro. In the present study, the expression of TGF-β1 was upregulated at the border between the alveolar bone and the lesion in the HFW-irradiated group (Fig 4R and 4V); FGF2 expression was also upregulated within the lesion (Fig 4J and 4N). These data suggest that HFW promoted the healing of apex lesions by promoting the expression of TGF-β1 and FGF2. In addition, IL-1β was downregulated in the HFW-irradiated group, suggesting that HFW irradiation suppressed inflammation in the periapical lesions. Further evaluation of the expression of osteoblastic markers and proliferating cells in the lesions is needed.

For the treatment of biofilm infections, it is critical to remove the infectious source mechanically or chemically; however, it is impossible to completely remove the source from the interior and exterior of the root canal, as previously indicated. In this study, HFW irradiation promoted the healing of apical lesions via interaction with the host side. Therefore, HFW irradiation offers the possibility of promoting healing through enhancement of the host immune system in apical lesions after some of the infectious source is successfully removed through RCT.

Much research has been conducted on biofilm infections; however, these infections arise from a complex variety of factors, many of which have not yet been fully elucidated. Our study focused on the essential conditions in situ/in vivo, and our findings indicate that HFW may be effective in stimulating immune-response cells in a rat model for treatment of infected root canals. Of particular interest is the effect of HFW on host cells. Therefore, we suggest that RCT with HFW may increase healing speed and thus merits development as a new adjuvant treatment for apical periodontitis.

27 Apr 2020

PONE-D-20-08421

Assessment of the functional efficacy of root canal treatment with high frequency waves in rats

PLOS ONE

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Reviewer #1: The purpose of this study was to investigate the potential effects of high frequency wave (HFW) irradiation in healing outcomes of experimental apical periodontitis in vivo and in vitro conditions. Several methodological concerns and lack of clarity in details jeopardize proper interpretation of study findings. The results need careful interpretation in light of the lack of translatability of methods to be used in clinical practice, and the lack of a true biofilm in the experiments. It is not clear why the authors went into great lengths with the in vivo study to then turn into in vitro studies to assess expression of growth factors. It would make more sense to use periapical lesions/normal tissues from the animals for these assays.

Extensive grammatical and technical revisions are necessary.

line 48 – What are the authors trying to say with: “It has been reported that the average success rate for initial root canal treatment (RCT) was lower than that of pulpectomy [2-5].” Pulpectomy is part of RCT and these procedures are not alternatives of each other. This does not make sense.

Line 48- Revise ‘tight’ obturation with ‘dense, hermetically-sealed’ obturation

Line 58 – a reference is needed at the end of the sentence

Line 59 – HFW is NOT an accepted method for NSRCT, therefore the authors should limit their purpose to analyse HFW as an ADJUVANT in NSRCT procedures.

Line 113 – add some brief details of the lesion induction protocol for clarity. Were lesions induced on the right and left side of all animals in both irradiated and non irradiated groups? Clarify

Lines 118-120 – Please revise this sentence. “enlarging’ a canal means the apical diameter established during instrumentation. I assume “enlarged to the level of 1.0” refers to working length distance to the apical foramen, as determined by an electronic apex locator (which the authors erroneously call ‘root canal meter’. The size of apical enlargement, which should be standardized between groups and of utmost importance to this study however is not mentioned in the text.

Justify the use of 0.5ml of 2.5% NaOCl

Justify the use of single cone obturation technique and provide details of the gutta percha cones used

Line 126, add number of rats divided in irradiated and non irradiated groups each

Line 128 – HFW was irradiated – was this through the foramen? How far out into the periapical lesion and how was this measured? Clarify

Line 203 – Biofilm formation was described to occur using 7-day polymicrobial cultures. However true biofilms have been shown to require a minimum of 21 days of culture. Hence the authors cannot state that a biofilm was used, only a polymicrobial mixture.

The results presented do not provide clear direction as if the images in Fig. 2 and 4 come from one animal or if representative sections of various animals are presented.

Line 351 – this does not reflect the study purpose stated in the introduction

The conclusions are not supported by the data presented – e.g. “it was proved that HFW irradiation promoted periapical lesion healing”. The authors fail to recognize that the success of endodontic treatment alone is >90%, therefore the HFW if any, might have helped the process by stimulating immune-response cells.

Lastly, HFW cannot be suggested as a new treatment for apical periodontitis as standalone. Root canal treatment is still necessary, as shown by their own results in which HFW had little to no activity on bacteria. Therefore the conclusions need to be toned down.

Figure 1 – add the number of animals used in each experiment

Figure 2 – include representative microCT images for each test and control site in irradiated and non irradiated groups that correlate with the results graph presented. Ideally the microCT images in which the obturation of the root canal can be visible as well as the periapical lesion size must be included.

Figure 4 – include lower magnification images showing the level of root canal obturation in irradiated and non irradiated groups.

Reviewer #2: I would like to thank the authors for submitting their article to PLOS ONE. It was my pleasure to review the manuscript entitled “Assessment of the functional efficacy of root canal treatment with high frequency waves in rats”. Undoubtedly, a lot of work was done to carry out this study and I commend the authors on their efforts in preparing the manuscript. Several analyses were performed in this study, mixing in vivo and in vitro assays, which makes it too long, difficult to read and sometimes even confusing. I suggest the authors to divide this paper in two: in vivo and in vitro assessment. Although the manuscript is very interesting, there are significant shortcomings that deter my enthusiasm.

- Overall, the entire manuscript needs to be revised for proper English/grammar/syntax/spelling

Introduction

- "RCT often 50 fails because the anatomical complexity of the root canal system, which includes lateral branches and isthmuses". This is information is not correct.. the success rate of primary root canal treatment is high, around 90-95%

- "This study focused on high frequency wave (HFW) apical therapy, which is a nonsurgical treatment for apical periodontitis" This kind of sentence should not be in this section

Methods

- This section is confusing and also the experiments need to be better described instead of using the expression "according to the method"

- Did the authors measured the periapical lesions before root canal treatment?

- Page 8, line 118. Eletronic apex locator instead of electrical root canal meter

- Which file was used to enlarge the root canals?

- Page 9, line 135. "the periapical lesions were scanned with a micro-135 CT scanner" the rats were scanned, not the periapical lesions"

- What were the parameters used during the scanning procedure?

- What about the radiation in which these rats where submitted during all these scans?

Results

- Is there a control group in the micro-ct analysis? The authors did not mention it on the MM section. What is the N of this group?

- This section also needs to be rewritten

Discussion

- This section is poorly written

- The authors did not discuss the results of immunohistochemical analysis of the periapical lesion and the quantification of growth factors expression with rat osteoblasts and fibroblasts

Figures

- A micro-CT image showing the reduction of the periapical lesion is relevant to present herein

Reviewer #3: The use of high frequency waves in endodontic treatment is a hot topic nowadays and this study was well designed. This manuscript focused on the assessment of the functional efficacy of RCT with HFW in vivo with rat model and also in vitro. I appreciate the hard work of the authors to provide important information to the endodontic field regarding HFW.

I have some recommendations and concerns:

1. In introduction line 48, why did authors compared the success rate for initial root canal treatment with pulpectomy? What was the purpose of the comparison of these two procedures?

2. Please check grammar and abbreviations, eg. line 76 micro-computed tomography should be (Micro-CT)

3. The description of materials and methods needs to be reorganized. The paragraph describing root canal treatment in rats (line 107-131) was unclear and was repeated in the paragraph of quantification of bacteria in the root canal (line 148-173)

4. The paragraph of statistical analysis (line 246-252) needs to be revised. In scientific writing, authors should avoid using "we used...".

5. In the discussion, authors discussed the mechanism of HFW's bactericidal effect and its thermal energy generation. Could authors discuss the potential damage of periodontal ligament by using HFW given to the rise of temperature?

Again, I appreciate the authors' contribution and looking forward to your responses.

Reviewer #4: The aim of this paper is interesting and the study may provide a new non-surgical treatment for apical periodontitis, however some flaws need to be addressed.

Abstract:

1. Line 38-40, the authors concluded that “high frequency wave irradiation promoted healing of the periapical lesion through increased expression of VEGF, FGF2 and TGF-β1”,however, in the part of ”Immunohistochemical observation” (methods), the samples were stained with specific antibodies against IL-1β, TGF-β1 and FGF2. Please explainhow these results lead to the conclusion described above.

Introduction:

2. Line 48-49, “It has been reported that the average success rate for initial root canal treatment (RCT) was lower than that of pulpectomy”. This sentence is quite confusing. Please explain this in detail.

Methods:

3. In the parts of “Root canal treatment in rats”, line 128-129, “HFW irradiation was applied three times each to the periapical lesion and the inside of the root canal immediately before the root canal obturation”, however, in the part of “Quantification of bacteria in the root canal”, line 150-151, “HFW irradiation was applied three timesto the periapical lesion and the root canal after the root canal preparation”. Are these two time point the same?

4. Line 153-154, “Immediately following the RCT, 10 rats were sacrificed and the mandibular first molars were extracted”, the rats were sacrificed and bacteria were collected immediately following the RCT, however, in the corresponding part of result, line 267, “At 4 weeks after RCT”. Authors described the ATP and PCR results for “Quantification of bacteria in the root canal” was from samples from 4 weeks after RCT. Please confirm the descriptions and make the unification.

Results:

5. Line 255-258, “Compared with the controlgroup, the volume of the periapical lesions in the mesial root was significantly smaller in the irradiated group at 2 weeks after the RCT (p = 0.009), and in the non-irradiated group at 6 weeks after the RCT(p = 0.013, Fig. 2)”, authors described that the volume of periapical lesions was significantly smaller in non-irradiated group at 6 weeks after RCT, but form Fig. 2, we could find that at 2 weeks after RCT, the volume of periapical lesions was also significantly smaller in non-irradiated group when compared with the control group (“a” and “b” meant significant difference). Please confirm the result.

6. Line 333-335, “In rat osteoblasts, the expression of FGF2 was significantly higher in HFW irradiated osteoblasts than in non-irradiated osteoblasts at 3 days (irradiation applied 5 and 10 times, p = 0.010) and 5 days (irradiation applied 10 times, p = 0.013)”, the authors obtained the result that the expression of FGF2 was significantly higher in HFW irradiated osteoblasts than in non-irradiated osteoblasts at 5 days with 10 times irradiation, however, from Fig. 7(b), we could observed that both for irradiation with 5 times and 10 times at 5 days, expressions of FGF2 showed no significant difference (the same letter “a” meant no significant difference). Please confirm the result.

7. Line 344-345, “By comparing the expression level of (a) FGF2, (b) VEGF, and (c) TGF-β1 in rat fibroblasts, and (d) FGF2, (e) VEGF, and (f) TGF-β1 in rat osteoblasts”, the authors described that (a), (b) and (c) meant the expression level in rat fibroblasts, and (d), (e) and (f) meant the expression level in rat osteoblasts. However, in Fig. 7, we found t (a), (b) and (c) meant the expression level in rat osteoblasts, and (d), (e) and (f) meant the expression level in rat fibroblasts, which was quite contrary to the description. Please confirm it.

Discussion:

8. The irradiation times in all parts of in vivo were consistent (three times each to the periapical lesion and the inside of the root canal), however, in the in vitro assays, the irradiation times were various (15 times for biofilm discs, 5 and 10 times for cells). The authors should discuss why they chose these different irradiation times and whether the different irradiation times affected the therapeutic effect.

Reviewer #5: The central idea of the manuscript was to access antibacterial/antibiofilm efficacy and growth factors expression to high frequency waves applied during endodontic treatment in teeth with periapical lesions. The manuscript proposal is interesting and focuses on a new possibility for root canal treatment. However, some major changes should be considered.

Abstract

1) Objective is not clear – to “investigate the influence of root canal treatment using high-frequency waves” on…? Antibiofilm effect was not mentioned. Growth factors expression was note mentioned.

2) Methodology should be better explained. It is too superficial. It is not possible to understand how the in vitro e in vivo experiments were performed. It is too confusing.

Introduction

1) Introduction should be more concise and objective.

2) Authors reported: “Recently, it was reported that the balance between bacteria and the host immune system is important for biofilm related infections [13].” Recently, from 2002? It is not recent information.

3) The reference is missing: “The initial choice for the treatment of apical periodontitis is to remove the source of infection; however, it is speculated that strengthening the host immune system can also lead to healing.”

4) Authors reported: “This study focused on high frequency wave (HFW) apical therapy, which is a nonsurgical treatment for apical periodontitis.” Is HFW a nonsurgical treatment for apical periodontitis? Reference?

5) Authors reported: “In the dental field, this principle is applied in electric cauterization.” Reference?

6) TGF-β1, VEGF and FGF2. Abbreviation should be described first.

Materials and Methods

1) Authors reported: “Root canal treatment in rats: Thirty-four 10-week-old male Wistar rats were used for this experiment, and the experimental design is shown in Figure 1.” Information about rats had already been mentioned - unnecessary.

2) Figure 1 should be mentioned before the topic “root canal treatment in rats”.

3) Why only the left mandibular first molars of the irradiated group were used as control group without RCT?

4) Authors reported: “In both groups, the root canals of the right mandibular first molars were prepared as in conventional RCT.” Which groups?

5) What was the tip of the endodontic file used?

6) Both topics “Root canal treatment in rats” and “Quantification of bacteria in the root canal” present information about root canal treatment and HFW.

7) About the topic “Biofilm formation” – “the HA discs…had been treated with saliva for more than 8 h.” Sterile saliva? Normal saliva? More than 8h means 9h, 10h, 24h?? Number of samples?

8) “S. mutans and E. faecalis were cultured for 2 days, and P. intermedia, P. gingivalis and F. nucleatum were cultured for 7 days.” What does it mean?

9) Authors used different times of HFW irradiation for different experiments (root canal and periapical lesions, biofilm on HA discs, rat cells). Why?

Results/figure

1) “The effect of HFW radiation on bacterial biofilm in vitro: Observation of the biofilm on a hydroxyapatite (HA) disc with CLSM revealed that P. gingivalis and P. intermedia showed an antibiofilm effect after direct HFW irradiation (Fig. 5a)”. It should be better described. Bacterial species showed antibiofilm effect or HFW irradiation promoted an antibiofilm effect on P. gingivalis and P. intermedia?

2) Figure

Discussion

1) “This work assessed the influence of HFW irradiation by studying the general frequency, voltage, energization time, and interval of HFW.” It was not the objective of the present study. Authors did not test different frequencies, voltage, energization time and interval…at least it was not mentioned at the methodology. According to HFW treatment device: “Irradiation conditions were set according to the method of Tominaga et al: frequency 510 kHz, voltage 35 V, energization time 1 s, irradiation interval 4 s.” Authors only varied the number of application.

2) Authors reported: “In previous human studies, as in this rat study, the promotion of healing by HFW after a certain time period following RCT has been consistent [20].” However, reference 20 is a review article related to bone fracture and the conclusion refers to the uncertainty surrounding the use of electrical stimulation and fracture healing.

3) Authors did not mention anything about possible side effects or consequences of the HFW.

References

1) Most references are out of date.

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Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

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Submitted filename: review.docx

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11 Jun 2020

Thank you very much for your e-mail of April 28th, 2020 concerning our manuscript Submission ID [PONE-D-20-08421] - [EMID:97f63f5e89367a4c] entitled " Assessment of the functional efficacy of root canal treatment with high frequency waves in rats ".

According to the suggestions, we modified the text and figures. We also appreciate the comments made by the editors and the reviewers. As reviewers pointed out, our in vitro experimental results caused some confusion so we clarified the points by focusing on in vivo experiments and modifying the body substantially. The point-by-point responses to each question or suggestion raised by the reviewers are attached as another file. In revising the manuscript, we paid much attention to the points suggested. We hope that these responses will prove satisfactory.

Submitted filename: Response to Reviewers.docx

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15 Jul 2020

PONE-D-20-08421R1

Assessment of the functional efficacy of root canal treatment with high frequency waves in rats

PLOS ONE

Dear Dr. Maezono,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

While the revised manuscript is significantly improved, there are some clarifications needed throughout the manuscript. There are some wordings and clarification in all sections of the manuscript. Please review the comments from the reviewers and address them accordingly.

==============================

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We look forward to receiving your revised manuscript.

Kind regards,

Sompop Bencharit, DDS, MS, PhD, FACP

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

While the revised manuscript is significantly improved, there are some clarifications needed throughout the manuscript. There are some wordings and clarification in all sections of the manuscript. Please review the comments from the reviewers and address them accordingly.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: N/A

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Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

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Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: It was my pleasure to revise again this paper. The authors tried to answer all the questions raised by the reviewers, which improved the quality of this paper. Therefore, I commend the authors on their efforts. However, I still have several concerns/suggestions that need to be adressed

1. Overall, English still needs to be revised

2. Abstract: this section is confusing. I suggest the authors to start this section with the aim of the manuscript. Then, explain, in a concise way, the methods used.

3. Introdution: another section that needs to be revised in order to make it

- Page 9, line 52. Root canal system instead of root canal

- Page 9, line 54. Remove hand and rotary files as the root canal can be prepared using other kinematics as reciprocation, oscillatory

- Page 9, line 56. Remove the word often

- Page 10, line 60. "Removing the source of infection is considered the choice treatment" The authors mentioned it a few line above

4. Methods:

- Please provide the total amount of irrigation used per tooth (the authors mentioned it in a reply to a reviewer

- Why did the authors use RealSeal? This is not fabricated anymore

- How was the HFW irradiation applied to the periapical lesion?

- How was the procedure to assess the same cross-section at the same point in all time-points? Did the authors register the image-stacks?

5. Discussion:

- Page 20, line 246. This is not correct, as this was not the aim of this paper. Please, rewrite it. Besides, do not use the word biofilm

- Page 22, line 271. "RCT successfully removes most bacteria leaving only that which has invaded deep inside the dentinal tubules". This is incorrect, as several studies already demonstrated that 30-60% of the root canal walls remain untouched after root canal preparation

Reviewer #3: Thank you for revising your manuscript and your hardworking is appreciated by the reviewer.

Although the authors addressed most of the previous comments from the reviewer, it is still unclear in the M&M session.

For example, in each paragraph, authors used the same expression of " Twelve rats were divided equally into an irradiated group (n = 6) and a non-irradiated group (n = 6)" twice, and "The rats were divided into an irradiated group (n = 5) and a non-irradiated group (n = 5)." once.

I understand that there were 34 rats in total included in this study, and they were divided into three groups: MicroCT, Quantification of bacteria in the root canal, and Immunohistochemical observation. For each method, there were 12, 10, and 12 subjects included, respectively.

However, the M&M was still not clear and the repetitive description of "Twelve rats were divided equally into an irradiated group (n = 6) and a non-irradiated group (n = 6)" will cause confusion and may require readers to go back and forth to check the subject allocation.

Please revise this session accordingly.

Also, the sentence in Line 55 "treatment (RCT) often fails because the anatomical complexity of the root canal system, which includes lateral branches and isthmuses [2] and extraradicular biofilms [3-6], makes it difficult to completely remove the source of the bacterial infection [7]." is still inappropriate. The meaning of this sentence is inexplicit because it can be interpreted as "RCT often fails". In fact, initial NSRCT has a success rate around 95%.

Reviewer #4: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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27 Aug 2020

Reviewer #2: It was my pleasure to revise again this paper. The authors tried to answer all the questions raised by the reviewers, which improved the quality of this paper. Therefore, I commend the authors on their efforts. However, I still have several concerns/suggestions that need to be adressed

1. Overall, English still needs to be revised

Proofreading by an English native speaker was performed and attached please find the certificate of proofreading.

2. Abstract: this section is confusing. I suggest the authors to start this section with the aim of the manuscript. Then, explain, in a concise way, the methods used.

Following your suggestion, we changed abstract to start with the aim, and then explained the methods used more concisely.

3. Introdution: another section that needs to be revised in order to make it

- Page 9, line 52. Root canal system instead of root canal

We changed the sentence following your advice.

- Page 9, line 54. Remove hand and rotary files as the root canal can be prepared using other kinematics as reciprocation, oscillatory

We removed the phrase “hand and rotary files” following your suggestion.

- Page 9, line 56. Remove the word often

We removed the word “often” following your advice.

- Page 10, line 60. "Removing the source of infection is considered the choice treatment" The authors mentioned it a few line above.

We deleted that part to avoid duplication.

4. Methods:

- Please provide the total amount of irrigation used per tooth (the authors mentioned it in a reply to a reviewer

We revised this part as below.

“Root canals were irrigated with 0.5 ml each time, in total 2ml of 2.5% NaOCl.”

- Why did the authors use RealSeal? This is not fabricated anymore

Real Seal SE (Pentron) was used in this experiment because it was used in the previous study by Yoneda et al. Real Seal SE sealer is currently available for purchase in Japan.

- How was the HFW irradiation applied to the periapical lesion?

We applied once to the periapical lesion and twice to the inside of the root canal as described in materials and methods section as below.

“HFW irradiation was applied once to the periapical lesion and twice to the inside of the root canal immediately before root canal obturation.”

- How was the procedure to assess the same cross-section at the same point in all time-points? Did the authors register the image-stacks?

Image processing software we used this time (TRI/3D-BON) didn’t have the function to register the image-stacks, therefore stacked images were reconstructed by determining a reference point so that they had the same cross section.

5. Discussion:

- Page 20, line 246. This is not correct, as this was not the aim of this paper. Please, rewrite it. Besides, do not use the word biofilm

Following your advice, we revised this part as below.

“This work assessed the influence of HFW irradiation in vivo in a rat model and investigated the target of HFW.”

- Page 22, line 271. "RCT successfully removes most bacteria leaving only that which has invaded deep inside the dentinal tubules". This is incorrect, as several studies already demonstrated that 30-60% of the root canal walls remain untouched after root canal preparation

As this reviewer suggested, it has been reported that residual bacteria in isthmus and lateral branches are difficult to remove by conventional RCT. In a rat root canal treatment model, Yoneda et al. reported that 75% of the bacteria from untreated root canals were eradicated by conventional root canal treatment and led to healing, but the remaining 25% were difficult to remove by conventional RCT only. We modified this part as below.

“Furthermore, residual bacteria decreased significantly after RCT alone, compared with the level before treatment. Yoneda et al. reported that RCT removed 75% of bacteria in the root canal and that 75% of cases were cured with conventional RCT; however, the remaining 25% of bacteria were difficult to remove [26]”

Reviewer #3: Thank you for revising your manuscript and your hardworking is appreciated by the reviewer.

Although the authors addressed most of the previous comments from the reviewer, it is still unclear in the M&M session.

For example, in each paragraph, authors used the same expression of " Twelve rats were divided equally into an irradiated group (n = 6) and a non-irradiated group (n = 6)" twice, and "The rats were divided into an irradiated group (n = 5) and a non-irradiated group (n = 5)." once.

I understand that there were 34 rats in total included in this study, and they were divided into three groups: MicroCT, Quantification of bacteria in the root canal, and Immunohistochemical observation. For each method, there were 12, 10, and 12 subjects included, respectively.

However, the M&M was still not clear and the repetitive description of "Twelve rats were divided equally into an irradiated group (n = 6) and a non-irradiated group (n = 6)" will cause confusion and may require readers to go back and forth to check the subject allocation.

Please revise this session accordingly.

Thank you very much for your comments. Following your suggestion, we revised the description about the number of rats allocated in each experiment. In materials and methods “Animals” section, allocation of all 34 rats are provided and also in each experiment, we modified the description of the number of rats used for better understanding.

Also, the sentence in Line 55 "treatment (RCT) often fails because the anatomical complexity of the root canal system, which includes lateral branches and isthmuses [2] and extraradicular biofilms [3-6], makes it difficult to completely remove the source of the bacterial infection [7]." is still inappropriate. The meaning of this sentence is inexplicit because it can be interpreted as "RCT often fails". In fact, initial NSRCT has a success rate around 95%.

Some have reported a success rate of approximately 95% for NSRCT, but this represents the success rate for pulpectomy in which no lesions have formed at the root apex, and does not represent the success rate of infected root canal treatment of teeth with pulp necrosis and apex lesion formation. The success rate of treatment of pulp-necrotic teeth with preoperative lesions is reported in the following article to be 82%.

Treatment outcome in endodontics: the Toronto study--phase 4　J endod,34(3):258-263 2008.

However, we thought it would be better to weaken the tone of the argument, so we modified that part as “Root canal treatment (RCT) can fail due to the anatomical complexity of the root canal system, which includes lateral branches and isthmuses [2], and extraradicular biofilms [3-6], which makes it difficult to completely remove the source of the bacterial infection [7].” to tone down. We appreciate your comments.

Submitted filename: Response to Reviewers.docx

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11 Sep 2020

Assessment of the functional efficacy of root canal treatment with high frequency waves in rats

PONE-D-20-08421R2

Dear Dr. Maezono,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Sompop Bencharit, DDS, MS, PhD, FACP

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have sufficiently addressed all comments.

Reviewers' comments:

18 Sep 2020

PONE-D-20-08421R2

Assessment of the functional efficacy of root canal treatment with high-frequency waves in rats

Dear Dr. Maezono:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr. Sompop Bencharit

Academic Editor

PLOS ONE