Effectiveness of Passive Ultrasonic Irrigation Protocols in Simulated Complex Root Canal Cavities

Round 1

Reviewer 1 Report

The paper is well written throughout with a well planned experiment beforehand.

The research results represent the basis for the implementation of the proposed irrigation protocols in clinical practice.

Author Response

The authors are grateful for the reviewer's comments.

Reviewer 2 Report

Dear authors, thank you for the submission. Your study is interesting; however, several flaws were detected and are described below:

The introduction, specifically the second and third paragraphs, is structured as a literature review rather than an introduction to the main subject of this study. A proper introduction to conventional irrigation and PUI needs to be done, informing the reader about the effects and reasons for both techniques to be performed, based on the literature.

The objective of this study was to evaluate the effectiveness of different PUI protocols on debris removal and the exposure of dentinal tubules in simulated complex root canal cavities using scanning electron microscopy (SEM). However, other evaluation protocols were also analyzed, as conventional irrigation with different protocols. Considerer including all protocols.

What is the difference between group 1 and 2? Both groups presented artificial cavities and no irrigation was performed. So, how can you ensure that one group has debris and the other does not?

Experimental group design: for each protocol of irrigation times and cycles there was difference between NaOCl and EDTA; why was there no such distinction in the 60s and 180s (groups 7 and 9)?

Table 1: what is the difference between the two “cleaning” columns, according to the research design?

Statistical analysis: In table 1, groups 5 and 6 are statistically different in the second column, however, both groups presented 1 as the median result. Could you explain? Consider the inclusion of minimum and maximum values, since non-parametric analyzes were performed.

The explanation of the results in table 1 are extremely confusing. Consider reviewing. Example: “CI groups (Groups 3 and 4) were superior to the negative control group (Group 2).” Groups 3 and 4 presented 2 and 3 as medium results, respectively; and group 2 presented 4 as medium; so, it is impossible the interpretation to be “superior”.

What was the statistical analysis performed for the table 2? Kruskal-Wallis test mentioned in the statistical analysis topic seems to be insufficient to analyze two variables. Were post-hoc tests performed?

“In this study, the same tooth was used in five different groups to reduce the interference of anatomical variation in the results.” Please, insert this information in the methodology topic. If possible, explain how is possible since the evaluation method used is destructible?

Please, insert limitations of your study in discussion topic.

It is unclear the distinction between table 2 and 3, based on the methods topic explanation.

Consider updating the references used, given that only 7 of 30 references are recent.

Author Response

The authors are grateful for the reviewer's comments. Please find below the point-by-point response to the reviewer’s comments.

Comment: The introduction, specifically the second and third paragraphs, is structured as a literature review rather than an introduction to the main subject of this study. A proper introduction to conventional irrigation and PUI needs to be done, informing the reader about the effects and reasons for both techniques to be performed, based on the literature.

Response: The introduction was rewritten as follows: Removing root canal debris is a challenging step during root canal treatment, and irrigation is a fundamental technique to achieve this objective once it favors cleaning areas where the mechanical instrumentation cannot reach [1]. Conventional irrigation (CI) is the most used method; however, it is inefficient for cleaning the apical portion of the root canal and isthmus since, in the best scenario, it carries the solution just 1mm beyond the needle tip [2]. This inappropriate disinfection leaves microbes alive that thrive after treatment leading to the persistent apical lesion and root canal treatment failure [3].

Recently, Passive Ultrasonic Irrigation (PUI) has been used to improve root canal system cleaning [4]. This technique uses an ultrasonic device to promote the movement of the irrigation solution within the root canal through ultrasonic waves produced by acoustic energy, facilitating the contact of the irrigation solution with irregularities and the apical portion of the root canal [4]. The acoustic flow promoted by PUI leads to the rupture of bacterial aggregations and removal of the smear layer or biofilm, associated with minimal periapical extrusion. However, conflicting results in the literature are reported compared to CI [5-8]. One of the possibilities to explain these contradictory findings is that the PUI protocols are largely flexible, ranging from the type of irrigant used and the concentration of the solution to the ultrasonic device's application time.

PUI has been employed through several protocols. Some studies use intermittent activation of 3 cycles of 20 seconds for each cycle [9,10]. On the other hand, it can be used by continuous activation of only one cycle of 60 seconds [11,12]. Continuous activation of the 3-min ultrasound has also been proposed [5,13]. In addition, there still needs to be a standard regarding the use of EDTA in the ultrasonic activation protocol. Some studies did not include it in the PUI protocol [8,14]. Differently from others who included it in the protocol [15,16]. The efficacy of the use of EDTA in the PUI protocol has been controversial in the literature. For some authors, the debris removal efficiency with EDTA's help in the PUI protocols was increased [7,17]. In contrast, other authors did not obtain the same results, showing no difference in results with and without EDTA [17,18]. Considering all this information, it is clear that a paramount protocol still needs to be standardized.

Comment: The objective of this study was to evaluate the effectiveness of different PUI protocols on debris removal and the exposure of dentinal tubules in simulated complex root canal cavities using scanning electron microscopy (SEM). However, other evaluation protocols were also analyzed, as conventional irrigation with different protocols. Considerer including all protocols.

Response: The sentence was rewritten as follows: This study aimed to evaluate the effectiveness of different CI and PUI protocols on cleaning ability by evaluating debris removal and the exposure of dentinal tubules in simulated complex root canal cavities using scanning electron microscopy (SEM).

Comment: What is the difference between group 1 and 2? Both groups presented artificial cavities and noirrigation was performed. So, how can you ensure that one group has debris and the other does not?

Response: These two groups are internal controls, positive and negative, respectively. Group 2, negative control, was filled with debris to simulate the worst-case scenario, showing cavities completely full, as evidenced by SEM.

Comment: Experimental group design: for each protocol of irrigation times and cycles there was difference between NaOCl and EDTA; why was there no such distinction in the 60s and 180s (groups 7 and 9)?

Response: The groups were assembled based on the most used clinical protocols. Group 7 is one single cycle of NaOCl for 60s; and Group 9 is 2 cycles: the first cycle is 17% EDTA alone for 60s, and the second cycle NaOCl for another 60s. They are not used combined.

Comment: Table 1: what is the difference between the two “cleaning” columns, according to the research design?

Response: The column on the left side (debris removal) assesses the smear layer's general removal, while on the right column (dentinal tube exposure), a higher magnification SEM was employed to see if the dentinal tubes were, in fact, cleaned.

Comment: Statistical analysis: In table 1, groups 5 and 6 are statistically different in the second column, however, both groups presented 1 as the median result. Could you explain? Consider the inclusion of minimum and maximum values, since non-parametric analyzes were performed.

Response: The upper and lower confidence intervals were inserted to better elucidate the differences.

Comment: The explanation of the results in table 1 are extremely confusing. Consider reviewing. Example: “CI groups (Groups 3 and 4) were superior to the negative control group (Group 2).” Groups 3 and 4 presented 2 and 3 as medium results, respectively; and group 2 presented 4 as medium; so, it is impossible the interpretation to be “superior”.

Response: The sentence was rewritten as follows: Statistical analysis was performed evaluating the protocols' general cleaning and dentinal tubules exposure abilities including all cavities (Table 1). The CI groups (Groups 3 and 4) showed better debris removal than the negative control group (Group 2). All groups using PUI (Groups 5, 6, 7, 8, 9, and 10) were more effective in removing the debris compared to the negative control group (Group 2) and CI (Groups 3 and 4) and similar to the positive control group (Group 1).

Comment: What was the statistical analysis performed for the table 2? Kruskal-Wallis test mentioned in the statistical analysis topic seems to be insufficient to analyze two variables. Were post-hoc tests performed?

Response: The Kruskal-Wallis test was used. First, we analyzed the differences between protocols in every single cavity alone (vertically). And second, we analyzed the differences between cavities in every tested protocol alone.

Comment: “In this study, the same tooth was used in five different groups to reduce the interference of anatomical variation in the results.” Please, insert this information in the methodology topic. If possible, explain how is possible since the evaluation method used is destructible?

Response: The information was added into methodology section. In this study, the same prepared tooth with cavities was used in five different groups to reduce the interference of anatomical variation in the results. To further elucidate that the method is not destructible, we re-used the prepared cavities, filling them again with debris and employing a different test protocol.

Comment: Please, insert limitations of your study in discussion topic.

Response: The following information was added: The limitations of this study include the lack of antibacterial reduction evaluation evoked by the different tested protocols.

Comment: It is unclear the distinction between table 2 and 3, based on the methods topic explanation.

Response: The table’s titles were amended: Table 2: Analysis of the general cleaning achieved for each protocol used in every individual cavity (p <0.05).Table 3: Analysis of the dentin tubule exposure for each protocol used in every individual cavity (p <0.05).

Comment: Consider updating the references used, given that only 7 of 30 references are recent.

Response: The references were updated.

Reviewer 3 Report

This study aimed to evaluate the effectiveness of different Passive Ultrasonic Irrigation protocols on debris removal and exposure of dentinal tubules in simulated complex root canal cavities.

The major strength of the paper is systematic in-vitro study.

Weakness includes the confusion in protocols for the general reader.

To improve the manuscript, please provide table in material methods. It will lead to a flow in the material methodology section.

Also provide some photographs of prepared samples so that reader can understand the methodology without much of efforts.

Author Response

The authors are grateful for the reviewer's comments. Please find below the point-by-point response to the reviewer’s comments.

Comment: To improve the manuscript, please provide table in material methods. It will lead to a flow in the material methodology section. Also provide some photographs of prepared samples so that reader can understand the methodology without much of efforts.

Response: The tables were improved, and figures about sample preparation were inserted along Figure 1.

Reviewer 4 Report

In the abstract

Group 10- 2 cycles of PUI for the 60s (NaOCl-EDTA) 25 line

While in line 31, the sequence of medicaments is changed in the 10^th group. The change of sequence is important in such studies.

“However, only groups 6, 7, and (29 line) 10 were statistically different from the CI and negative control (p<0.05). The protocols using PUI, 30 comprising groups with 3 cycles of 20s (NaOCl-EDTA-NaOCl), 2 cycles of 60s (EDTA-NaOCl),(is it 10 group or 9? needs clarification) or 1 cycle of 60 s (NaOCl), were more effective at removing debris and increasing the exposure of 32 dentinal tubules. “

Author Response

The authors are grateful for the reviewer's comments. Please find below the point-by-point response to the reviewer’s comments.

Comment: In the abstract: Group 10- 2 cycles of PUI for the 60s (NaOCl-EDTA) 25 line. While in line 31, the sequence of medicaments is changed in the 10^th group. The change of sequence is important in such studies.

Response: In line 31, we refer to group 9, which employs the solution in the opposite order.

Comment: “However, only groups 6, 7, and (29 line) 10 were statistically different from the CI and negative control (p<0.05). The protocols using PUI, 30 comprising groups with 3 cycles of 20s (NaOCl-EDTA-NaOCl), 2 cycles of 60s (EDTA-NaOCl),(is it 10 group or 9? needs clarification) or 1 cycleof 60 s (NaOCl), were more effective at removing debris and increasing the exposure of 32 dentinal tubules. “

Response: We refer to group 9, which first uses EDTA and later NaOCl.

Round 2

Reviewer 2 Report

Dear author, I have no more questions. Thank you.