Sonic irrigation for removal of calcium hydroxide in the apical root canal: A micro-CT and light-coupled tracking analysis.

OBJECTIVE: This study aimed to evaluate the efficacy of three sonic irrigation systems for removal of calcium hydroxide dressing from the apical root canal.
MATERIALS AND METHODS: A total of 96 single-rooted teeth in three categories of root canal curvatures (straight: 0-5°, moderate: 6-20°, and severe: > 20°) were allocated to four groups: conventional needle irrigation, EndoActivator, EQ-S, and Vibringe. The root canals were instrumented using Protaper NEXT and filled with calcium hydroxide. After removal of calcium hydroxide, the remaining volume of calcium hydroxide was measured by micro-CT analysis. Data were compared among root canal curvatures and irrigation systems using the Kruskal-Wallis test and Mann-Whitney test (p < .05). The oscillating range of each irrigation system was measured using light-coupled motion tracking.
RESULTS: The volumes of calcium hydroxide remaining in the canals with severe curvature were significantly higher than in those of straight curvature (p < .05). In the canals of moderate or severe curvature, EQ-S showed the highest removal percentage, followed by EndoActivator, Vibringe, and needle irrigation (p < .05). Light-coupled tracking showed the largest oscillating range in EQ-S (p < .05).
CONCLUSIONS: Sonically activated irrigation systems with a flexible tip can be beneficial for calcium hydroxide intracanal dressing removal in the curved apical canals.

Introduction

Calcium hydroxide is the most common intracanal medicament used during endodontic treatment [1]. It has diverse physical and biological advantages such as antibacterial effects, ability to dissolve tissue, promotion of hard tissue formation, reduction of bacterial toxic products, and healing of periapical tissues [2]. However, complete removal of calcium hydroxide might not clinically achievable, particularly in root canals with anatomical complexities [3]. Remnants of calcium hydroxide can hinder penetration of sealers into dentinal tubules and negatively affect sealing of canal filling materials [4].

For effective removal of calcium hydroxide, various technologies were incorporated as adjunctive methods to conventional needle irrigation by activating fluid movement within the canal space. Effective cleaning requires the ability to bring the irrigant in contact with the elements to be removed while not damaging the root dentin structures [5]. Ultrasonic devices were first introduced in mechanical debridement in endodontics and developed into passive ultrasonic irrigation (PUI) for enhancement of intracanal debridement [6]. PUI systems were designed to introduce a smooth file or wire with a noncutting tip into the canal, and energy transmission is controlled to avoid intentional contact with the canal walls. However, in a small constraint in the canal, wall contact is unavoidable, and free oscillation is inhibited, particularly in the apical roots with geometrical complexities [7]. Consequently, risks can arise such as needle binding within the irregular dentinal walls, deforming the root canal morphology, and weakening the apical constriction [8]. An alternative option, passive sonic activation, incorporates non-cutting plastic tips oscillating at much lower frequencies (150–200 Hz) compared to PUI (25,000–30,000 Hz). In addition, low electric current-assisted sonic irrigation has been introduced to even increase the efficiency compared to conventional sonic irrigation and PUI [9]. These sonic systems attempted to avoid excessive cavitation and produce sufficient fluid agitation that removes the smear layer and dislodges intraradicular biofilm [10].

Previous evaluations of activation systems for root canal irrigation have noted the major obstacle against efficient cleaning as complicated root canal anatomy [10, 11]. This limited cleaning effect is more pronounced below the root canal curvature narrowing to the apical constriction [12]. Considering the three-dimensional complexities of the apical third of the root canal system, clinical outcomes can be evaluated based on both qualitative and quantitative measurements [13]. Several studies have focused on apical third of root canals and have introduced micro-CT analysis as an outcome measurement regarding each step of clinical procedures: canal preparation and shaping [14, 15], removal of previously filled materials [16, 17], removal of hard tissue debris [3, 18], and final intracanal obturation [19, 20]. The removal of the calcium hydroxide dressing at the interappointment session is another critical part of ensuring clinical expertise and can be clearly confirmed by 3D image construction of the root canal structures.

In this study, we applied conventional needle irrigation and three types of sonic irrigation systems (EndoActivator, EQ-S, and Vibringe) for removal of intracanal calcium hydroxide and evaluated the volumes of calcium hydroxide remaining in the apical space among different root canal curvatures using micro-CT. We also measured oscillating ranges of sonically-activated irrigation tips by a light-coupled motion tracking. The null hypothesis of this study was that removal efficacy of calcium hydroxide at the apical root canal would not be affected by sonic irrigation systems.

Materials and methods

Preparation of specimens

Ninety-six extracted single-rooted teeth with completed apices and no visible caries, cracks, or other defects were used in the study. The Institutional Review Board of Seoul National University School of Dentistry approved this study (IRB No. S-D20200031). All procedures performed in this study involving human teeth were in accordance with relevant guidelines and regulations of the institutional research committee, and informed consent was obtained from all individual participants. The teeth were decoronated and sectioned to a length of 12 mm. The roots of the teeth were ultrasonically cleaned and stored in 0.9 g/L thymol solution (Sigma-Aldrich, St. Louis, MO, USA) [21]. Micro-CT scans (Skyscan 1172, Bruker, Kontich, Belgium) were performed to obtain sagittal images of each root. The scanning parameters were 100kV and 100μA at the Al + Cu filter with an exposure time of 632ms. The pixel size was 30μm with a rotation of 0.70 and an average frame number of three. The 3D images were acquired by 3D reconstruction (NRecon, Bruker, Kontich, Belgium), modeling (CTAn, Bruker, Kontich, Belgium), and analysis (CTVol, Bruker, Kontich, Belgium). Based on the sagittal images of each root, the curvatures were calculated with the Schneider method [22]. Considering their root curvatures, the 96 teeth were assigned into one of three categories: straight (0–5°), moderate (6–20°), and severe (> 21°) (n = 32/category). The 32 teeth in each category were then randomly distributed into four groups of irrigation systems: Group 1 (control), Group 2 (EndoActivator), Group 3 (EQ-S), and Group 4 (Vibringe) (n = 8/group). The sample size was determined based on a previous study that reported means and SDs for removal efficacy of calcium hydroxide using micro-CT imaging [23].

Root canal preparation and calcium hydroxide filling

Canal patency was confirmed with a #K-10 file (K-file, Maillefer Instruments, Ballaigues, Switzerland) until the tip was just visible at the apical foramen [18]. The working lengths were determined as 1 mm less than that length. All canals were prepared with Ni-Ti files using a rotary system (X-Smart, Dentsply Maillefer, Ballaigues, Switzerland) up to the X3 file (Protaper Next, Dentsply Maillefer, Ballaigues, Switzerland), ISO size 30, and taper 0.07, resulting in standardized root canals. During instrumentation, the root canals were irrigated with 2 mL 1% NaOCl solution at each change of instrumentation. After canal preparation, the canal space was dried with paper points (Absorbent Paper Points, Meta Biomed, Cheongju, Korea). The tip of the syringe containing paste was inserted into the canal to 1 mm shorter than the binding point [23]. The 0.1 mL of calcium hydroxide paste (Calcipex II, Nippon Shika Yakuhin, Shimonoseki, Japan) was injected slowly with minimal pressure to fill the space. The calcium hydroxide paste used in this study consisted of calcium hydroxide, barium sulfate, and distilled water and is easy to handle as a root canal filling material [24]. The medicament was then condensed using a paper point and a dry cotton pellet. The orifice was closed with a cotton pellet and a temporary restorative material (MD Temp Plus, Meta Biomed, Cheongju, Korea). The canal filling state was confirmed by secondary micro-CT scanning. The proper filling state was determined by adherence of the paste to the canal walls with the absence of voids. If some specimens failed to have such filling quality, they were discarded and replaced by new specimens selected by the abovementioned methods. The specimens were stored at 37°C in 100% relative humidity for one week.

Irrigation procedures

After one week of intracanal medicament application, the temporary restorative material was removed, and calcium hydroxide was removed by one of the four protocols (Table 1) [8]. All canals were irrigated with 3% NaOCl solution at 2 mm shorter than the working length with up-and-down motions. A total volume of NaOCl solution used in irrigation by each protocol was 10 ml.

Table 1

Irrigation systems used in the study.

Irrigation systems		Tip characteristics	Tip sizes	Frequency	Irrigation methods [8]
Control	Needle irrigation	Side-vented needle (NaviTip, Ultradent Products, South Jordan, UT, USA)	30G; straight	N/A	Canals were irrigated using a total of 10 mL of NaOCl for 60 seconds
EndoActivator (Dentsply Maillefer, Santa Barbara, CA, USA)	Sonic irrigation	Flexible, non-cutting polymer tip (Dentsply Maillefer, Santa Barbara, CA, USA)	#15/.02	166 Hz (10,000 cpm)	Canals were irrigated using a total of 10 mL of NaOCl, applying the device three times for 20 seconds each
EQ-S (Meta Systems, Seongnam, Korea)	Sonic irrigation	Flexible, non-cutting polymer tip (Meta Systems, Seongnam, Korea)	#15/.02	217 Hz (13,000 cpm)	Same as in EndoActivator
Vibringe (Cavex, Haarlem, Netherlands)	Sonic irrigation	Side-vented needle (NaviTip, Ultradent Products, South Jordan, UT, USA)	30G; straight	150 Hz	Same as in Control

Micro-CT analysis

The specimens were scanned using a micro-CT scanner at three time points: root curvature determination, after filling the canal with calcium hydroxide, and after removing the calcium hydroxide from the canal. The scanning parameters were the same for all three times of scans. The 3D images were acquired in the same manner at each time. The region of interest was the apical 3 mm of the root canal structure. Within the range of analysis, the volume of calcium hydroxide after filling and after removal was calculated. Percentage removal was determined according to the following equation:

Light-coupled tracking of oscillation

Each irrigation system was fixed so that it could stand alone and reveal its lateral face toward the camera. The tip of each irrigation system was 90 degrees to the camera at a 20 cm distance. The tips and the camera were set at the same height. The irrigation system was activated or left as is depending on the type. Ambient light was blocked with a cloth, and blue light from the LED unit (Elipar DeepCure-S, 3M ESPE, St. Paul, MN, USA) of 1,470 mW/cm2 was shone onto the tip. As light was coupled along the tip to track its oscillating motion, real time images were collected. Also, the same images were obtained for tip oscillation within an artificial block with a curved (10°) canal. The entire procedure was repeated three times, each time with a new tip. Based on the motion traces of oscillating tips, maximum oscillating ranges were calculated with computer software (ImageJ, NIH, Bethesda, Maryland, MD, USA).

Statistical analysis

For comparison of remaining volumes and removal percentages of calcium hydroxide among the irrigation systems and the root canal curvatures, the data did not follow a normal distribution, as confirmed by Shapiro-Wilk test. Thus, a nonparametric Kruskal-Wallis test and Mann-Whitney test with Bonferroni correction were used. All statistical analyses were performed at a significance level of 0.05 using IBM SPSS Statistics software Version 26.0 (IBM, Armonk, NY, USA).

Results

The median percentage removal of intracanal medication from the part of apical 3 mm was shown in Table 2. In the straight canals, no significant differences existed among the groups, with all exhibiting nearly complete (100%) removal of calcium hydroxide. In the canals with severe curvature, the percentage removal was highest in Group 3 (EQ-S), followed by Group 2 (EndoActivator), Group 4 (Vibringe), and Group 1 (control). Significant differences existed between Group 1 and Groups 2 and 3, and between Group 3 and Group 4 (p < 0.05). The remaining volumes of calcium hydroxide in the canals of straight or moderate curvature were not significantly different among the groups (Table 3). Instead, significant differences existed in the canals based on severe curvature, in the opposite order as percentage removal (p < 0.05). Graphs in Fig 1 depicted the comparison among the different curvatures; all groups had significantly lower removal percentages and higher remaining volumes with severe curvature compared to non-severe curvature, except Group 3 (p < 0.05), which revealed no significant differences among the three curvatures.

Table 2

Median removal percentages of intracanal medication.

Groups	Median Removal Percentage (%)
	Root canal curvatures
	Straight	Moderate	Severe
Group 1 Control	99.95 [99.61, 100] A	99.88 [94.55, 99.92] AB	80.17 [71.05, 92.66] A
Group 2 Endoactivator	100 [99.93, 100] A	99.91 [99.51, 99.99] A	99.53 [90.94, 99.84] BC
Group 3 EQ-S	100 [97.77, 100] A	99.95 [99.59, 100] A	99.95 [99.38, 100] C
Group 4 Vibringe	100 [99.19, 100] A	96.19 [89.10, 99.32] B	92.09 [86.21, 96.17] AB

Values with the same subscripts are not significantly different compared within columns (P > 0.05)

Interquartile ranges [first quartile, third quartile] are shown in parentheses.

Table 3

Median remaining volumes of intracanal medication.

Groups	Median Remaining Volume (103mm3)
	Root canal curvatures
	Straight	Moderate	Severe
Group 1 Control	0.83 [0, 1.84] A	1.34 [0.40, 63.16] A	94.60 [47.32, 156.86] A
Group 2 Endoactivator	0 [0, 0.12] A	0.42 [0.05, 1.91] A	3.18 [1.27, 30.08] BC
Group 3 EQ-S	0 [0, 9.99] A	0.36 [0, 2.30] A	0.72 [0, 1.95] C
Group 4 Vibringe	0 [0, 2.44] A	12.96 [3.18, 40.92] A	26.38 [17.84, 81.99] AB

Values with the same subscripts are not significantly different when compared within columns (P > 0.05)

Interquartile ranges [first quartile, third quartile] are shown in parentheses.

Fig 1

Removal efficacy of intracanal medication in different curvatures and devices.

(a) Removal percentage of intracanal medication. (b) Remaining volume of intracanal medication. *: Values are significantly different within the same irrigation devices (p < 0.05).

Cross-sectional micro-CT images obtained at the apical 2-mm level (Fig 2) and 3D images (Fig 3) exhibited calcium hydroxide remaining in the canal space. In canals with severe curvature, remnants of calcium hydroxide were noticeable at the inner and outermost parts of the curvature in Groups 1 and 4.

Fig 2

Cross-sectional micro-CT images at 2 mm from the apical constriction.

The white mass inside the wall indicates remaining intracanal medication.

Fig 3

3D demonstration of intracanal medication within the severe curvatures after filling and after removal by different irrigation systems.

Intracanal medication is designated in blue, and the region of interest (3 mm from the apical constriction) is shown in dashed lines.

The maximum oscillation range of each system is demonstrated in Fig 4. The lateral extent of oscillation was measured by light-coupled tracking of movement in free space and within the artificial canal block. Group 3 exhibited the largest extent of movement, followed by Group 2, while the needle tips in Groups 1 and 4 showed minimal movement (Figs 4 and 5A). Oscillation was restricted in the artificial block, resulting in near absence of movement for all groups (Fig 5B).

Fig 4

Light-coupled tracking of the operating irrigation tips in free-range and inside the artificial block with a curved (10°) canal.

Fig 5

Maximum oscillation widths of the light-coupled tips.

(a) Maximum oscillation widths outside the canal. (b) Maximum oscillation widths inside the artificial block with a curved (10°) canal. *: Values are significantly different among the irrigation devices (p < 0.05).

Discussion

This study evaluated the efficacy of sonic irrigation systems for removing calcium hydroxide in the apical root canal. All three sonic systems exhibited removal capacity significantly higher than that of conventional needle irrigation. Also, free-oscillating ranges of the irrigation tips differed among the systems. The EQ-S showed the largest oscillating range and the highest percentage removal of calcium hydroxide. Therefore, the null hypothesis that removal of calcium hydroxide at the apical root canal would not be affected by the sonic irrigation system was rejected.

Previous studies evaluating canal irrigation methods have mainly focused on removal of debris and smear layer during chemomechanical cleaning of root canal systems. When root canal treatment is extended to multiple sessions, the canal space must be filled with interim medicament to induce the pharmaceutical effect. At the time of final canal obturation, the medicament should be removed to expose dentinal tubules for application and penetration of sealers. Quantitative evaluation of cleaning efficacy often is presented as percentage removal of intracanal medication as outcome values. Compared to the original volume of filled medication, the remaining volume was minimal, resulting in approximately 100% removal rate, as shown in the current study results. However, even traces of calcium hydroxide remnants adhering to the wall surface can negatively affect effective sealing of the intraradicular structure against microbial ingress from the oral cavity or the periapical tissues [24]. Another consideration is that the geometry of the canal is difficult to be standardized in the pooled samples of diverse configurations. Therefore, we performed an additional comparison of the remaining volumes of calcium hydroxide among the groups. The pre-packaged calcium hydroxide paste used in this study, Calcipex, is a water-based mixture with high flowability. A flexible and tapered plastic needle is attached to the syringe to allow the paste to easily flow into the space. In previous in vitro studies comparing the removal efficiency of Calcipex [25, 26], additional instrumentation or agitation was not applied after injecting of Calcipex paste. We confirmed the filling quality of the intracanal medication using micro-CT. The samples with proper adaptation of the paste to the canal walls and no detectable voids were selected for the further analysis. The serial images of micro-CT provided qualitative visualization and quantitative measurement of remaining calcium hydroxide. In cross-sectional images, apical canals with severe curvature resembled oval-shaped canals or an isthmus which is difficult to access by instruments and to flush out with irrigants. The three sonic systems revealed superior cleaning efficacy to conventional needle irrigation at the curved apex.

Many previous studies have evaluated the cleaning activity of various irrigation systems, often indiscriminately including PUI and sonic irrigation, and compared those with needle irrigation methods. In this study, 3% NaOCl solution was used as an irrigant during canal preparation and removal of intracanal medication. Calcium-chelating agent such as 17% EDTA solution may be used as a final rinse to remove the smear layer as recommended in clinical practice [27]. But it might not be a primary option as demonstrated in a systematic review evaluating ultrasonic irrigant activation and syringe irrigation [28]. In this study, we have focused on a broader scale of removal efficiency of calcium hydroxide using sonic irrigation systems, which is more practical in clinical circumstances. We compared three sonically activated systems with similar frequency (150–217 Hz), but different tip types: EndoActivator and EQ-S with flexible polymer tips and Vibringe with a stainless-steel needle tip. The aim of this study was to demonstrate the flexibility of each irrigation tip during oscillation using a light-tracking method. EQ-S had a more flexible and softer type of polymer tip with a larger extent of movement compared to EndoActivator. When an irrigation needle or wire is incorporated into a narrow canal and vibrated, simultaneous contacts with rigid walls are inevitable. The irrigation tip would not be freely displaced, and fluid movements would be subjected to frictional forces between a boundary and vibrating medium to produce acoustic microstreaming [29]. The intensity of microstreaming is related directly to the streaming velocity and displacement that is proportional to the square of the displacement amplitude of the oscillating tips [5]. EQ-S had the largest range of oscillation, relating to more enhanced streaming effect for cleaning. The stainless-steel tip used with Vibringe yielded a minimal range of transversal movement, which might be related to its lower cleaning effect at the apical portion, particularly in severely curved canals. Our speculation was that limitedly flexible metal tips, as are often used in PUI, will be more highly affected by the confined geometry of canal walls. A previous study on file-to-wall contact during PUI showed that the file hit the wall and reversed production of a low-frequency secondary oscillation (6 kHz) that seemed to have more profound impact on activation of irrigant than did its primary oscillation (30 kHz) [7]. Another mechanism of ultrasonically or sonically activated irrigation was explained as acoustic cavitation that produces pressure fluctuations and nucleates vapor bubbles [5]. The intensity of cavitation is affected by several factors such as diameters and shapes of tips, oscillating amplitude, surrounding geometry, and properties of medium [29]. The softness and flexibility of irrigation tips seemed to be a critical part contributing to enhance vibration impact separately from the frequency and power of the oscillating instruments.

Another consideration in terms of clinical aspect is how the irrigation tip can be directed into the apical canal, particularly with narrow and curved configurations. In this study, we used single-rooted teeth, in which the tip would be more easily inserted than into multi-rooted teeth. Nevertheless, at the canal below the severe curvature, calcium hydroxide remnants were less completely washed out by a rigid stainless-steel tip, although the streaming was sonically-activated as with Vibringe. In clinical practice, insertion and retraction of irrigation tips deep into and out of a narrow and curved canal spaces are demanding procedural steps. Even when the tip can be readily inserted into the apical canal, clinicians might be concerned about the risk of tip separation similar to that when using the NiTi rotary files in curved narrow canals. In this light, flexible polymer tips of sonic irrigation systems have a major advantage over rigid metal tips equipped in other systems. And this could provide additional benefits for clinicians performing endodontic therapy in everyday practice.

Root canal geometry is variable based on individual anatomy of teeth. Clinical implications in endodontic research rely on representation of anatomical complexities and how relevantly specimens are assorted under experimental settings. In a systematic and critical review on measurement of root canal curvature, 3D imaging was valued over 2D microscopic evaluation to increase precision of both qualitative and quantitative measurements [30]. In evaluation of root curvature, we reviewed a stack of images from the sagittal view rotating 360° around the tooth axis and captured the most severe angulation of images. In terms of root curvature determination, this study was based on a methodology with high accuracy. Still, to obtain highly standardized specimens, root canal models must have uniform severity of curvatures and simulate anatomical complexities such as anastomoses and ramifications. Also, using a light-transmissible medium for tooth models will incorporate more easily a sonoluminescence technique to capture the oscillating modalities. These experimental setups are obtainable by current techniques of computer-aided designing and manufacturing. Enhancement of debridement and disinfection without damage to anatomical structure is a goal of activated irrigation systems. With standardized root canal models, the integrity of radicular structures will be confirmed accurately before and after irrigation procedures. This provides another topic of interest for future studies.

Conclusions

The three sonically activated irrigation systems used in this study showed increased removal capacity of calcium hydroxide in the apical root canal compared to conventional needle irrigation. EQ-S had an extended range of oscillation with a flexible irrigation tip and higher cleaning capacity at the curved apex compared to other sonic irrigation systems. Sonically activated irrigation systems with a flexible tip can be beneficial for removal of intracanal dressing materials in the curved apical canals.

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16 Mar 2022

Sonic irrigation for removal of calcium hydroxide in the apical root canal: A micro-CT and light-coupled tracking analysis

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Reviewer #1: This manuscript constitutes an attempt to evaluate the efficacy of three different sonic irrigation agitation systems on calcium hydroxide removal from apical third of the root canals using micro-CT. The study was well performed and clinically relevant. However, English needs to be improved. Some of the specific queries have been addressed below.

*Introduction:

- Line 49- the sentence- (Remnants of calcium hydroxide…filling materials)- needs reference.

- In the aim of the study, mention the three different types of sonic irrigation systems used.

*Methodology:

- How was sample size calculated?

- Posterior teeth with anastomosis would have been more relevant than using single rooted teeth.

- How were the teeth cleaned and stored?

- How was working length determined?

- In the beginning, it is mentioned 96 teeth were used. However, later it is mentioned 36 teeth were subjected to 3 categories based on root canal curvature. Its not clear, exactly what was the sample size used. Mention in detail.

- Why canals were not irrigated during instrumentation?

- How was smear layer removed which was formed during instrumentation?

- Mention the manufacturer’s details of paper points and NaOCl.

- What was the volume of calcium hydroxide placed inside each sample?

- How was calcium hydroxide placed inside each root canal?

- In control and vibringe groups, what was the time period of irrigation agitation?

- A chelator should have been used along with NaOCl to remove calcium hydroxide efficiently.

- Mention the details of calcium hydroxide paste used.

- How was normality of data evaluated?

- Do not insert tables and figure legends in between the text.

*Results:

- Results of the volume of calcium hydroxide in all the samples in different groups before removal has to be mentioned to ensure uniform distribution of calcium hydroxide.

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Reviewer #2: General Comments: Thank you for your submission to the Plos One. The purpose of the present study was to evaluate the efficacy of three sonic irrigation systems for removal of calcium hydroxide dressing from the apical root canal. The premise of the study is sound, and the article is well written. Furthermore, the experimental procedures are properly delineated; and it has merits to be published in the Plos One. However, there are some drawbacks which prevent its publication in the current form. The article needs a major revision by authors prior acceptance for publication.

- “Sonically activated irrigation systems with a flexible tip can be beneficial for cleaning of intracanal medication in the curved apical canals.” Please, rephrase it as suggested: “Sonically activated irrigation systems with a flexible tip can be beneficial for calcium hydroxide intracanal dressing removal in curved apical root canals.”

- “Calcium hydroxide is the most commonly used as intracanal medicament during endodontic treatment because of its physical and biological advantages such as antibacterial effect, tissue dissolving, promoting hard tissue formation, reducing bacterial toxic products, and healing periapical tissues (1).” This sentence is too long and confusing. Please, rephrase it.

- “This limited cleaning effect is more pronounced below the root canal curvature narrowing to the apical constriction. Considering the three-dimensional complexities of the apical third of the root canal system, clinical outcomes can be evaluated based on both qualitative and quantitative measurements.” References are missing for these two sentences.

- The originality, the state of the art of the studied subject and the scientific contribution of the study were not clear.

- Kindly check the grammar and written style of the whole manuscript.

- The sample size calculation was not described.

- According to the authors, “The curvatures of each root curvatures were determined in sagittal views from the micro-CT images.” However, as stated at the next sentence, “The curvatures were calculated with the Schneider method (17).” The authors should explain such a procedure with more details for better understanding by the readers.

- Did the authors perform a micro-CT analysis considering the anatomical features of the teeth for specimens grouping?

- The randomization of the specimen’s distribution into the experimental groups should be clearly described in the main text.

- The manufacturer details of the irrigation systems are missing.

- What was the working length of the root canals? Please, add this information.

- “…..and filled with calcium hydroxide paste (Calcipex II, Nippon Shika Yakuhin, Shimonoseki, Japan), a dry cotton pellet, and a temporary restorative material (MD Temp Plus, Meta Biomed, Cheongju, Korea).” This sentence does not make sense.

- “The canal filling state was confirmed by the secondary micro-CT scanning.” What variables were considered to confirm the filling quality? Were specimens discarded from the final sample after such analysis?

- The correct form is 3% NaOCl “solution”!!! Please, check the whole text.

- The way in which the examiners were trained/calibrated and the result of inter- and intra-examiner agreement are not shown.

- The statistical analysis should be described with more details. How were sample normality and homogeneity tested?

- According to the authors “However, even traces of calcium hydroxide remnants adhering to the wall surface can negatively affect hermetic sealing of the intraradicular

structure against microbial ingress from the oral cavity or the periapical tissues (18).” Hermetic sealing is a falacy. Please, change this term.

- The Conclusion Section should be more concise, and for this reason, it must be rewritten for better understanding by the readers.

**********

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4 May 2022

Authors ‘responses to the reviewers

Title: Sonic irrigation for removal of calcium hydroxide in the apical root canal: A micro-CT and light-coupled tracking analysis

Reviewer #1:

This manuscript constitutes an attempt to evaluate the efficacy of three different sonic irrigation agitation systems on calcium hydroxide removal from apical third of the root canals using micro-CT. The study was well performed and clinically relevant. However, English needs to be improved. Some of the specific queries have been addressed below.

*Introduction:

#1-1. Line 49- the sentence- (Remnants of calcium hydroxide…filling materials)- needs reference.

Author response to #1-1

We newly added the following reference (#4) at the end of the above-mentioned sentence.

Ref #4 Kim SK, Kim YO. Influence of calcium hydroxide intracanal medication on apical seal. Int Endod J. 2002 Jul;35(7):623-8.

#1-2. In the aim of the study, mention the three different types of sonic irrigation systems used.

Author response to #1-2

We rewrote the aim of the sentence as follows:

“In this study, we applied conventional needle irrigation and three types of sonic irrigation systems (EndoActivator, EQ-S, and Vibringe) for removal of intracanal calcium hydroxide and evaluated the volumes of calcium hydroxide remaining in the apical space among different root canal curvatures using micro-CT.”

*Methodology:

#1-3. How was sample size calculated?

Author response to #1-3

We referred to a previous study on calcium hydroxide removal using PUI that used n = 8 per each group. The following sentence was inserted in the Materials and Methods.

“The sample size was determined based on a previous study that reported means and SDs for removal efficacy of calcium hydroxide using micro-CT imaging (Ref #23).”

Ref #23. Silva LJM, Pessoa OF, Teixeira MBG, Gouveia CH, Braga RR. Micro-CT evaluation of calcium hydroxide removal through passive ultrasonic irrigation associated with or without an additional instrument. Int Endod J. 2014;48(8):768-73.

#1-4. Posterior teeth with anastomosis would have been more relevant than using single rooted teeth.

Author response to #1-4

We agree with the reviewer’s point that the isthmus between two canals is difficult to access and would require more competency of intracanal irrigation. In this study, we sorted natural tooth specimens into three different types of root canal curvatures and performed volumetric evaluations of the canal spaces before and after cleaning and removal of intracanal medication. For comparison of removal efficacy, anatomical irregularities had to be minimized among the included specimens. Therefore, the anatomical uniqueness and complexity had to be avoided as much as possible for our quantitative analysis. Agreeing with the reviewer’s comments, we rewrote the related sentence in the Discussion.

“Still, to obtain highly standardized specimens, we will consider constructing root canal models with a uniform severity of curvature.” was rewritten as follows:

“Still, to obtain highly standardized specimens, root canal models must have uniform severity of curvatures and simulate anatomical complexities such as anastomoses and ramifications.”

#1-5. How were the teeth cleaned and stored?

Author response to #1-5

We rewrote the related sentence as follows:

“The roots of the teeth were ultrasonically cleaned and stored in 0.9 g/L thymol solution (Sigma-Aldrich, St. Louis, MO, USA) (Ref #21).”

Ref #21. Yang Q, Liu MW, Zhu LX, Peng B. Micro‐CT study on the removal of accumulated hard‐tissue debris from the root canal system of mandibular molars when using a novel laser‐activated irrigation approach. Int Endod J. 2020;53(4):529-38.

#1-6. How was working length determined?

Author response to #1-6

We added more description about working length determination and the relevant reference as follows:

“Canal patency was confirmed with a #K-10 file (K-file, Maillefer Instruments, Ballaigues, Switzerland) until the tip was just visible at the apical foramen (Ref # 18). The working lengths were determined as 1 mm less than that length.”

Ref #18. Yang Q, Liu MW, Zhu LX, Peng B. Micro‐CT study on the removal of accumulated hard‐tissue debris from the root canal system of mandibular molars when using a novel laser‐activated irrigation approach. Int Endod J. 2020;53(4):529-38.

#1-7. In the beginning, it is mentioned 96 teeth were used. However, later it is mentioned 36 teeth were subjected to 3 categories based on root canal curvature. Its not clear, exactly what was the sample size used. Mention in detail.

Author response to #1-7

Thanks for the pointing out the error. We amended the sentence as follows.

“Considering their root curvatures, the 96 teeth were assigned into one of three categories: straight (0-5°), moderate (6-20°), and severe (> 21°) (n = 32/category). The 32 teeth in each category were then randomly distributed into four groups of irrigation systems: Group 1 (control), Group 2 (EndoActivator), Group 3 (EQ-S), and Group 4 (Vibringe) (n = 8/group).”

#1-8. Why canals were not irrigated during instrumentation?

Author response to #1-8

We appreciate for the reviewer’s comment pointing out the incomplete descriptions of the experimental procedures. The following sentence was newly inserted:

“During instrumentation, the root canals were irrigated with 2 mL 1% NaOCl solution at each change of instrumentation.”

The Materials and Methods section was substantiated with additional statements describing the procedures in detail. Please see the response to #1-9.

#1-9. How was smear layer removed which was formed during instrumentation?

Author response to #1-9

A chelating agent such as 15-17% EDTA is generally recommended as a final rinse to remove the smear layer prior to canal obturation. In addition, many in vitro studies evaluating the effectiveness of irrigating methods tended to use EDTA as a conjunctive irrigant, particularly for microscopic confirmation of the microbial and physical cleanliness of the dentinal tubules. In a systematic review of 48 in vitro studies comparing ultrasonic irrigant activation and syringe irrigation (Ref #28), NaOCl (1-10%) was the most frequently used irrigatns (46 studies), while EDTA was used in fewer studies (21 studies). In this study, we have focused on the broad-range of removal efficiency of calcium hydroxide using sonic irrigation systems, which is a practical issue in clinical circumstances. Still, we understand the reviewer’s concern, and added the following sentence in the Discussion.

“In this study, 3% NaOCl solution was used as an irrigant during canal preparation and removal of intracanal medication. Calcium-chelating agent such as 17% EDTA solution may be used as a final rinse to remove the smear layer as recommended in clinical practice (Ref #27). However, it might not be a primary option, as demonstrated in a systematic review evaluating ultrasonic irrigant activation and syringe irrigation (Ref #28). In this study, we have focused on a broader scale of removal efficiency of calcium hydroxide using sonic irrigation systems, which is a practical issue in clinical circumstances.”

Ref #27. Haapasalo M, Shen Y, Wang Z, Gao Y. Irrigation in endodontics. Br Dent J. 2014 Mar;216(6):299-303.

Ref #28. Căpută PE, Retsas A, Kuijk L, Chávez de Paz LE, Boutsioukis C. Ultrasonic Irrigant Activation during Root Canal Treatment: A Systematic Review. J Endod. 2019 Jan;45(1):31-44.e13.

#1-10. Mention the manufacturer’s details of paper points and NaOCl.

Author response to #1-10

The manufacturer’s details were inserted as follows:

“After canal preparation, the specimens were dried with paper points (Absorbent Paper Points, Meta Biomed, Cheongju, Korea).”

#1-11. What was the volume of calcium hydroxide placed inside each sample?

Author response to #1-11

We substantiated the required information as follows.

“The 0.1 mL of calcium hydroxide paste (Calcipex II, Nippon Shika Yakuhin, Shimonoseki, Japan) was injected slowly with minimal pressure to fill the space.”

#1-12. How was calcium hydroxide placed inside each root canal?

Author response to #1-12

We added the following sentences and references in the Discussion.

“The pre-packaged calcium hydroxide paste used in this study, Calcipex, is a water-based mixture with high flowability. A flexible and tapered plastic needle is attached to the syringe to allow the paste to easily flow into the space. In previous in vitro studies comparing the removal efficiency of Calcipex (Ref #25, #26), additional instrumentation or agitation was not applied after injecting of the Calcipex paste.”

Ref #25. Kim T, Kim MA, Hwang YC, Rosa V, Del Fabbro M, Min KS. Effect of a calcium hydroxide-based intracanal medicament containing N-2-methyl pyrrolidone as a vehicle against Enterococcus faecalis biofilm. J Appl Oral Sci. 2020 Mar 27;28:e20190516.

Ref #26. Lim MJ, Jang HJ, Yu MK, Lee KW, Min KS. Removal efficacy and cytotoxicity of a calcium hydroxide paste using N-2-methyl-pyrrolidone as a vehicle. Restor Dent Endod. 2017 Nov;42(4):290-300.

In addition, the following sentence was inserted in the Materials and Methods and in the Discussion.

“The proper filling state was determined by the adherence of the paste to the canal walls and absence of voids.”

“The samples with proper adaptation of the paste to the canal walls and no detectable voids were selected for the further analysis.”

#1-13. In control and vibringe groups, what was the time period of irrigation agitation?

Author response to #1-13

As in the other groups, the Control and Vibringe groups were irrigated for 60 seconds. We revised Table 1 accordingly and mentioned the reference (Ref #8).

#1-14. A chelator should have been used along with NaOCl to remove calcium hydroxide efficiently.

Author response to #1-14

We understand the reviewer’s concern. Please refer to the response to #1-9

#1-15. Mention the details of calcium hydroxide paste used.

Author response to #1-15

We added the following sentence with a new reference in the Materials and Methods.

“The calcium hydroxide paste used in this study consisted of calcium hydroxide, barium sulfate, and distilled water and is easy to handle as a root canal filling material (Ref #24).”

Ref #24. Hosoya N, Kurayama H, Iino F, Arai T. Effects of calcium hydroxide on physical and sealing properties of canal sealers. Int Endod J. 2004;37(3):178-84.

#1-16. How was normality of data evaluated?

Author response to #1-16

We added a description about the normal distribution of the data as follows:

“For comparison of remaining volumes and removal percentages of calcium hydroxide among the irrigation systems and root canal curvatures, the data did not follow a normal distribution, as confirmed by the Shapiro-Wilk test.”

#1-17. Do not insert tables and figure legends in between the text.

Author response to #1-17

We removed the inserted tables and figures from the text. However, in the submission guideline shown below, we were asked to insert figure captions in the text. Based on the editorial office requirement for authors, we will be happy to revise it accordingly.

“Authors’ guideline: Figure captions must be inserted in the text of the manuscript, immediately following the paragraph in which the figure is first cited (read order).”

*Results:

#1-18. Results of the volume of calcium hydroxide in all the samples in different groups before removal has to be mentioned to ensure uniform distribution of calcium hydroxide.

Author response to #1-18

We additionally mentioned the volume of calcium hydroxide injected. Please refer to the response to #1-11.

Reviewer #2:

General Comments: Thank you for your submission to the Plos One. The purpose of the present study was to evaluate the efficacy of three sonic irrigation systems for removal of calcium hydroxide dressing from the apical root canal. The premise of the study is sound, and the article is well written. Furthermore, the experimental procedures are properly delineated; and it has merits to be published in the Plos One. However, there are some drawbacks which prevent its publication in the current form. The article needs a major revision by authors prior acceptance for publication.

#2-1. “Sonically activated irrigation systems with a flexible tip can be beneficial for cleaning of intracanal medication in the curved apical canals.” Please, rephrase it as suggested: “Sonically activated irrigation systems with a flexible tip can be beneficial for calcium hydroxide intracanal dressing removal in curved apical root canals.”

Author response to #2-1

We appreciate the reviewer’s input. The sentence was corrected as suggested.

#2-2. “Calcium hydroxide is the most commonly used as intracanal medicament during endodontic treatment because of its physical and biological advantages such as antibacterial effect, tissue dissolving, promoting hard tissue formation, reducing bacterial toxic products, and healing periapical tissues (1).” This sentence is too long and confusing. Please, rephrase it.

Author response to #2-2

We amended the sentence as follows:

“Calcium hydroxide is the most common intracanal medicament used during endodontic treatment (1). It has diverse physical and biological advantages such as antibacterial effects, ability to dissolve tissue, promotion of hard tissue formation, reduction of bacterial toxic products, and healing of periapical tissues (2).”

#2-3. “This limited cleaning effect is more pronounced below the root canal curvature narrowing to the apical constriction. Considering the three-dimensional complexities of the apical third of the root canal system, clinical outcomes can be evaluated based on both qualitative and quantitative measurements.” References are missing for these two sentences.

Author response to #2-3

We cited the following references related to the above-mentioned sentences:

Ref #12. Caron G, Nham K, Bronnec F, Machtou P. Effectiveness of different final irrigant activation protocols on smear layer removal in curved canals. J Endod. 2010 Aug;36(8):1361-6.

Ref #13. Kirmizi D, Aksoy U, Orhan K. Efficacy of Laser-Activated Irrigation and Conventional Techniques in Calcium Hydroxide Removal from Simulated Internal Resorption Cavities: Micro-CT Study. Photobiomodul Photomed Laser Surg. 2021 Oct;39(10):674-681.

#2-4. The originality, the state of the art of the studied subject and the scientific contribution of the study were not clear.

Author response to #2-4

We agree with the reviewer’s point that the research interest was unclear. There are many previous studies regarding activation systems for root canal irrigation. Ultrasonic, passive ultrasonic, and sonic irrigation systems were indiscriminately compared among the different systems. We attempted to compare the devices with a similar range of working frequency, focusing on the characteristics of the irrigation tips. This was why we measured the extent of the movement of the irrigation tips. We clarify our research interest and background using the following sentences in the Discussion.

“Many previous studies have evaluated the cleaning activity of various irrigation systems, often indiscriminately including PUI and sonic irrigation, and compared those with needle irrigation methods.”

“The aim of this study was to demonstrate the flexibility of each irrigation tip during oscillation using a light-tracking method.”

“In clinical practice, insertion and retraction of irrigation tips deep into and out of narrow and curved canal spaces are demanding procedural steps…. And this could provide additional benefits for clinicians performing endodontic therapy in everyday practice.”

#2-5. Kindly check the grammar and written style of the whole manuscript.

Author response to #2-5

We received English proof reading and editing of the revised manuscript through a language editing service and corrected accordingly. The editorial certificate was uploaded with the revised documents.

#2-6. The sample size calculation was not described.

Author response to #2-6

Please refer to response #1-3.

#2-7. According to the authors, “The curvatures of each root curvatures were determined in sagittal views from the micro-CT images.” However, as stated at the next sentence, “The curvatures were calculated with the Schneider method (17).” The authors should explain such a procedure with more details for better understanding by the readers.

Author response to #2-7

We modified the following sentences for clarification.

“Micro-CT scans (Skyscan 1172, Bruker, Kontich, Belgium) were performed to obtain sagittal images of each root. The scanning parameters were 100kV and 100μA at the Al + Cu filter with an exposure time of 632ms. The pixel size was 30 μm with a rotation of 0.70 and an average frame number of three. The 3D images were acquired by 3D reconstruction (NRecon, Bruker, Kontich, Belgium), modeling (CTAn, Bruker, Kontich, Belgium), and analysis (CTVol, Bruker, Kontich, Belgium). Based on the sagittal images of each root, the curvatures were calculated with the Schneider method (22).”

#2-8. Did the authors perform a micro-CT analysis considering the anatomical features of the teeth for specimens grouping?

Author response to #2-8

Yes. The first micro-CT analysis was performed to obtain refined sagittal images of the roots to calculate accurate root curvature. Please refer to the author response to #2-7.

#2-9. The randomization of the specimen’s distribution into the experimental groups should be clearly described in the main text.

Author response to #2-9

We clarified the random distribution process:

“Considering their root curvatures, the 96 teeth were assigned into one of three categories: straight (0-5°), moderate (6-20°), and severe (> 21°) (n = 32/category). The 32 teeth in each category were then randomly distributed into four groups of irrigation systems: Group 1 (control), Group 2 (EndoActivator), Group 3 (EQ-S), and Group 4 (Vibringe) (n = 8/group).”

#2-10. The manufacturer details of the irrigation systems are missing.

Author response to #2-10

As suggested, we provided the manufacturer’s details in Table 1.

#2-11. What was the working length of the root canals? Please, add this information.

Author response to #2-11

“Canal patency was confirmed with a #K-10 file (K-file, Maillefer Instruments, Ballaigues, Switzerland) until the tip was just visible at the apical foramen (Ref #18). The working lengths were determined as 1 mm less than that length.”

Ref #18. Yang Q, Liu MW, Zhu LX, Peng B. Micro‐CT study on the removal of accumulated hard‐tissue debris from the root canal system of mandibular molars when using a novel laser‐activated irrigation approach. Int Endod J. 2020;53(4):529-38.

#2-12. “…..and filled with calcium hydroxide paste (Calcipex II, Nippon Shika Yakuhin, Shimonoseki, Japan), a dry cotton pellet, and a temporary restorative material (MD Temp Plus, Meta Biomed, Cheongju, Korea).” This sentence does not make sense.

Author response to #2-12

We substantiated the materials and methods used in canal filling with the following sentence:

“After canal preparation, the canal space was dried with paper points (Absorbent Paper Points, Meta Biomed, Cheongju, Korea). The tip of the syringe containing paste was inserted into the canal to 1 mm shorter than the binding point (Ref #23). The 0.1 mL of calcium hydroxide paste (Calcipex II, Nippon Shika Yakuhin, Shimonoseki, Japan) was injected slowly with minimal pressure to fill the space… The medicament was then condensed using a paper point and a dry cotton pellet. The orifice was closed with a cotton pellet and a temporary restorative material (MD Temp Plus, Meta Biomed, Cheongju, Korea).”

Ref #23. Peters CI, Koka RS, Highsmith S, Peters OA. Calcium hydroxide dressings using different preparation and application modes: density and dissolution by simulated tissue pressure. Int Endod J. 2005 Dec;38(12):889-95.

#2-13. “The canal filling state was confirmed by the secondary micro-CT scanning.” What variables were considered to confirm the filling quality? Were specimens discarded from the final sample after such analysis?

Author response to #2-13

We added the following sentences to better explain the process:

“The proper filling state was determined by adherence of the paste to the canal walls with the absence of voids. If some specimens failed to have such filling quality, they were discarded and replaced by new specimens selected by the abovementioned methods.”

#2-14. The correct form is 3% NaOCl “solution”!!! Please, check the whole text.

Author response to #2-14

We corrected it throughout the entire manuscript.

#2-15. The way in which the examiners were trained/calibrated and the result of inter- and intra-examiner agreement are not shown.

Author response to #2-15

We agree that the examiner training/calibration and inter-/intra-examiner agreements are essential information for evaluations based on ratings. However, as our analysis was based on quantitative calculations performed by the computer software, we thought that we could omit such information. All the experiments and calculations were performed by a single experienced operator.

#2-16 The statistical analysis should be described with more details. How were sample normality and homogeneity tested?

Author response to #2-16

Please refer to the response #1-16.

#2-17. According to the authors “However, even traces of calcium hydroxide remnants adhering to the wall surface can negatively affect hermetic sealing of the intraradicular

structure against microbial ingress from the oral cavity or the periapical tissues (18).” Hermetic sealing is a falacy. Please, change this term.

Author response to #2-17

We agree with the reviewer’s comment. The above-mentioned term was corrected as follows:

“… can negatively influence effective sealing of….”

#2-18. The Conclusion Section should be more concise, and for this reason, it must be rewritten for better understanding by the readers.

Author response to #2-18

The paragraph in the Conclusions was re-written for more clarification as suggested.

“Sonically activated irrigation systems exhibited increased removal capacity of calcium hydroxide in the apical root canal compared to conventional needle irrigation. EQ-S had an extended range of oscillation with a flexible irrigation tip and higher cleaning capacity at the curved apex compared to other sonic irrigation systems.”

“The three sonically activated irrigation systems used in this study showed increased removal capacity of calcium hydroxide in the apical root canal compared to conventional needle irrigation. EQ-S had an extended range of oscillation with a flexible irrigation tip and higher cleaning capacity at the curved apex compared to other sonic irrigation systems. Sonically activated irrigation systems with a flexible tip can be beneficial for removal of intracanal dressing materials in the curved apical canals.”

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Author response:

Using the PACE system, we adjusted all the figures.

Submitted filename: response_reivewer_220504.docx

Click here for additional data file.

9 May 2022

Sonic irrigation for removal of calcium hydroxide in the apical root canal: A micro-CT and light-coupled tracking analysis

PONE-D-21-30735R1

Dear Dr. Juhea Chang ,

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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Thank you for revising your manuscript appropriately based on the referees comments.

Thank Youi

Reviewer #2: The authors performed all the suggested modifications. The improvement in the manuscript is remarkable. Therefore, I would like to recommend the publication of this manuscript.

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23 May 2022

PONE-D-21-30735R1

Sonic irrigation for removal of calcium hydroxide in the apical root canal: A micro-CT and light-coupled tracking analysis

Dear Dr. Chang:

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