Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents a randomized clinical trial comparing postoperative pain after root canal obturation using two calcium silicate-based sealers (Bio-C Sealer and TotalFill BC Sealer). The topic is clinically relevant because postoperative pain remains one of the main patient-centered outcomes after endodontic treatment.

The manuscript is generally well organized and the methodology appears standardized. However, there are several methodological and reporting issues that should be addressed before the manuscript can be considered for publication.

The principal limitation is the relatively small sample size, which substantially limits the statistical power for detecting clinically meaningful differences. Furthermore, several methodological details regarding randomization, blinding, statistical analysis, and interpretation require clarification

1. Sample size and statistical power

Although the authors report an a priori power calculation, the assumed effect size (Cohen's d = 1.07) appears unrealistically large for postoperative pain studies.

Using such a large anticipated effect substantially reduces the required sample size and increases the risk of a type II error.

Since almost every comparison resulted in non-significant findings, the manuscript cannot conclude that both sealers perform similarly.

The authors should instead state that:

"No statistically significant differences were detected within the limits of this pilot-sized randomized clinical trial."

A post-hoc discussion regarding statistical power should also be included.

 

2. Interpretation of negative findings

Throughout the manuscript the authors repeatedly state that both sealers demonstrated similar clinical performance.

This conclusion is not statistically justified.

Failure to detect a statistically significant difference does not demonstrate equivalence.

The wording should be revised throughout the Abstract, Discussion and Conclusions.

 

3. Randomization procedure

The manuscript states that "simple randomization" was used.

However, no details are provided regarding as Consort guidelines

 

4. VAS outcome

The VAS is an ordinal outcome.

However, the manuscript primarily reports frequencies of pain rather than actual VAS scores.

The reviewer recommends presenting: median VAS, interquartile rang, boxplots, change over time rather than simply reporting presence or absence of pain.

5. Sealer extrusion

The incidence of sealer extrusion appears unusually high (73%). The Discussion should address why extrusion occurred so frequently, whether this reflects the obturation technique,comparison with published literature

 

 

Minor comments

Abstract

Instead of

demonstrated similar clinical performance

consider

no statistically significant differences were detected.

I would also like to add up some minor comments 

literature review could include more recent randomized clinical trials and systematic reviews (2023–2025).

Clarify:

• tooth vitality
• diagnosis distribution
• number of canals
• pulpal status
• distribution of anterior/premolar/molar teeth

These factors may influence postoperative pain.

 

CONSORT

The CONSORT flowchart is appreciated.

However, please also include the CONSORT checklist as supplementary material.

 

Results

Several tables are repetitive.

Tables 3 and 4 could be simplified or transferred to supplementary material

Figure 2 could be improved by including:

• confidence intervals
• actual patient numbers

rather than only percentages.

Discussion

The Discussion mainly repeats the Results.

It should instead emphasize:

• interpretation
• biological mechanisms
• comparison with previous randomized clinical trials
• clinical implications

 

Conclusions

Avoid overstatement.

Instead of

demonstrated similar clinical performance

use

no statistically significant differences were identified in this study.

 

Language

Overall English is understandable but requires professional editing.

Examples:

• "demonestrated" → "demonstrated"
• "not statistical significance" → "not statistically significant"
• "visual analogue scale" should be used consistently.
• Several minor grammatical corrections are recommended throughout.

Author Response

comment 1 : Sample size and statistical power

response 1 : Thank you for this valuable insight. We completely agree that an effect size of d = 1.07 is overly optimistic for a definitive trial and carries a high risk of a Type II error due to the small sample size. In accordance with your recommendation, we have re-framed the entire study as a randomized clinical pilot trial. The title, Abstract, and Materials and Methods sections have been modified to reflect this exploratory pilot design, and the text explicitly states that the sample size was chosen pragmatically to assess protocol feasibility and gather baseline variance estimates for future definitive investigations.

comment 2 : Interpretation of negative findings

response 2 : We appreciate this statistical correction. We acknowledge that a failure to reject the null hypothesis does not prove equivalence. We have amended the Discussion and future perspectives to clarify that no statistically significant differences were detected within the limits of this pilot-sized study and that well-powered longitudinal trials are required to draw definitive conclusions.

comment 3 : Randomization procedure

response 3 : In accordance with the CONSORT guidelines, we have provided full details of the randomization process in the revised Materials and Methods section. Specifically, we have clarified that the allocation sequence was computer-generated, concealment was maintained using sequentially numbered, opaque, sealed envelopes, and a secondary assistant handled sealer preparation in an unbranded area to ensure strict double-blinding of the operator, patient, and data collector.

comment 4 : VAS outcome

response 4 : Thank you for this constructive suggestion. To better capture the continuous nature of the Visual Analog Scale (VAS) rather than treating it merely as a binary outcome, we have revised Table 2 to present the quantitative VAS mean and stadered deviation scores alongside the frequencies. Furthermore, Figure 2 has been completely redesigned into a multi-panel visual trend analysis plotting mean VAS scores over time, stratified by sealer type and patient gender.

comment 5 : Sealer extrusion

response 5 : We agree that the 73.3% extrusion rate is a striking finding that requires contextualization. We have added a dedicated paragraph in the Discussion section explaining that this high incidence is primarily due to the high flowability and physical properties inherent to modern premixed bioceramic sealers when combined with a single-cone obturation technique. We also note that while this served as a baseline to observe periapical tissue response, it represents a limitation regarding external validity.

comment 6 : Minor comments

response 6 :

We have addressed all minor comments as follows:

• Literature & Variables: We expanded the Introduction to discuss the physical differences between the sealers. We also inserted a new "Baseline Variables and Diagnostic Criteria" subsection detailing the tracking of pulpal/periapical status, tooth type, and diagnostic distributions.

• CONSORT Checklist: The formal CONSORT checklist has been prepared and will be submitted alongside the manuscript as supplementary material.

• Results & Tables: To eliminate redundancy, former Tables 3 and 4 have been successfully removed from the primary text and relegated to the online supplementary file (as Supplementary Tables S1 and S2).

• Discussion & Language: The Discussion was thoroughly restructured to emphasize biological mechanisms and limit result repetition. A professional proofreading pass was performed to correct grammatical errors and resolve typos such as "not statistical significance".

Reviewer 2 Report

Comments and Suggestions for Authors

This randomized clinical trial addresses a clinically relevant question regarding postoperative pain associated with two bioceramic endodontic sealers. The study is well designed and clearly presented. However, the sample size is small and may be underpowered to detect clinically meaningful differences between groups. The discussion should better acknowledge this limitation and avoid emphasizing numerical trends that were not statistically significant. In addition, the clinical implications should be discussed more critically, particularly considering the absence of significant differences between the two sealers. Finally, the manuscript would benefit from careful English language editing to improve clarity and readability.

Author Response

comment 1 : The manuscript addresses a clinically relevant question regarding postoperative pain associated with two bioceramic endodontic sealers. The study is well designed and clearly presented. However, the sample size is small and may be underpowered to detect clinically meaningful differences between groups.

response 1 : We sincerely thank the reviewer for the positive assessment of our study design and clarity, as well as for pointing out this critical limitation. We fully agree that the small sample size (n=30) limits the statistical power to detect smaller, yet clinically meaningful, differences between the Bio-C Sealer and TotalFill BC Sealer groups. In response, we have now explicitly re-characterized the study as a "clinical pilot trial" throughout the revised manuscript (including the Title, Abstract, and Materials and Methods). Furthermore, we have expanded the Limitations section in the Discussion to clearly acknowledge that this study was an exploratory pilot intended to establish baseline variances and evaluate procedural feasibility, and that definitive non-inferiority or superiority statements require larger, adequately powered clinical cohorts.

comment 2 : The discussion should better acknowledge this limitation and avoid emphasizing numerical trends that were not statistically significant.

response 2 :

We appreciate this precise critique and acknowledge that highlighting non-significant numerical trends can mislead readers. We have thoroughly audited the Discussion section and removed any speculative language surrounding minor descriptive differences that lacked statistical backing. Instead, we have re-framed our interpretations strictly around the verified absence of statistically significant differences, ensuring that our text remains transparently grounded in the statistical reality of our findings.

comment 3 : In addition, the clinical implications should be discussed more critically, particularly considering the absence of significant differences between the two sealers.

response 3 :

Thank you for pushing us to deepen this part of our manuscript. We have added a dedicated sub-section to the Discussion evaluating the clinical implications of these overlapping performance profiles. We now explain that since both materials demonstrated similar postoperative pain behavior, the clinical choice between them can comfortably rely on other practical parameters—such as cost-effectiveness, setting times, ease of handling/retrieval, storage requirements, and clinician preference—without worrying about increasing the incidence or severity of early post-treatment discomfort.

comment 4 : Finally, the manuscript would benefit from careful English language editing to improve clarity and readability.

response 4 :

We agree with this recommendation. The revised manuscript has undergone a comprehensive line-by-line editorial review to improve syntactic flow, clarify academic wording, and eliminate grammatical oversights. Spelling mistakes (such as changing "demonestrated" to "demonstrated" and refining "not statistical significance" to "not statistically significant") have been entirely corrected to ensure a high standard of professional readability.

Reviewer 3 Report

Comments and Suggestions for Authors

Overall Assessment

The manuscript investigates postoperative pain following obturation with two premixed bioceramic sealers using a randomized double-blind clinical trial. The topic is clinically relevant, and the study design is appropriate. However, several methodological details require clarification, the rationale for the study should be strengthened, and the discussion requires substantial expansion before the manuscript is suitable for publication.

Major Comments

Line 62: Figure 1 does not correspond to the sentence describing the VAS. Please correct the figure citation.

Line 66-68: Rephrase the study aim without including methodological details such as 'randomly assigned'.

Strengthen the rationale for selecting Bio-C Sealer and TotalFill BC Sealer. Explain their differences in composition, physicochemical properties, and bioactivity, and summarize previous clinical evidence comparing these materials.

Please describe how the randomization sequence was generated, how allocation concealment was achieved, and who remained blinded during outcome assessment in accordance with CONSORT guidelines.

Please justify the large effect size (Cohen's d = 1.07) used for the sample size calculation by citing the source study.

Line 119: Specify the plastic reciprocating irrigation activation device, including manufacturer and model.

Line 120: Clarify whether EDTA and NaOCl were activated sequentially or separately during final irrigation.

Provide complete details of the rotary instrumentation system (file sequence, sizes, taper, alloy/metallurgy, and manufacturer) in the Materials and Methods rather than only in the Discussion.

Variables analyzed in the Results (tooth type, pulpal diagnosis, periapical status, preoperative pain, lesion size, and sealer extrusion) should be clearly defined in the Materials and Methods before presenting the results.

Clarify how pulpal and periapical diagnoses were established and report the distribution of these diagnoses between groups.

State whether postoperative analgesic intake was systematically recorded and analyzed. Report the number of patients requiring analgesics in each group, as analgesic consumption is an important secondary clinical outcome and may mask differences in postoperative pain.

The reported incidence of sealer extrusion (73.3%) appears remarkably high. Please discuss possible explanations, the obturation technique used, and whether this limits external validity.

Pain was assessed using a VAS, which is a continuous outcome; however, the results are presented mainly as categorical pain/no pain. Please justify this approach or additionally report actual VAS scores.

Repeated postoperative measurements were obtained over four time points. Please justify the use of separate Fisher's exact tests rather than a repeated-measures statistical approach.

Expand the discussion regarding the influence of instrumentation kinematics, file design, heat-treated metallurgy, and apical preparation on postoperative pain.

Discuss in greater depth the association between preoperative pain and postoperative pain, comparing your findings with previous clinical studies.

Expand the discussion comparing Bio-C Sealer and TotalFill BC Sealer with respect to composition, ion release, cytotoxicity, bioactivity, and previous in vitro and clinical investigations.

Further discuss the influence of gender and tooth type on postoperative pain using contemporary literature.

Conclude the Discussion with a concise summary of the principal findings followed by a dedicated limitations section discussing the small sample size, single-center design, short follow-up, and limited statistical power.

The Conclusion should include the clinical significance and practical implications of the findings rather than simply restating the results.

Minor Comments

• Lines 185–186 should be rephrased because they read like a figure title rather than narrative text.

• State earlier in the manuscript that only asymptomatic teeth at obturation were included and explain how this criterion was verified.

• Ensure terminology, abbreviations, and formatting are consistent throughout the manuscript.

Author Response

comment 1 : Overall Assessment: The manuscript investigates postoperative pain following obturation with two premixed bioceramic sealers using a randomized double-blind clinical trial. The topic is clinically relevant, and the study design is appropriate. However, several methodological details require clarification, the rationale for the study should be strengthened, and the discussion requires substantial expansion before the manuscript is suitable for publication.

response 1 :

We appreciate the reviewer’s positive assessment regarding the clinical relevance and design of our study. We have thoroughly revised the manuscript to clarify all methodological details, strengthen the introduction's rationale, and significantly expand the discussion as suggested. Furthermore, acknowledging the limitations of our sample size, we have re-framed the entire study as a randomized clinical pilot trial to ensure an accurate and scientifically grounded presentation of our findings.

comment 2 : Line 62: Figure 1 does not correspond to the sentence describing the VAS. Please correct the figure citation.

response 2 :

Thank you for pointing out this oversight. The text has been corrected, and the reference now accurately points to the Visual Analogue Scale (VAS) presentation rather than the CONSORT flow diagram.

comment 3 : Line 66-68: Rephrase the study aim without including methodological details such as 'randomly assigned'.

response 3 :

We have revised the study aim in the Introduction to focus strictly on the core objective. It now reads: "This clinical trial aimed to evaluate and compare the incidence and intensity of postoperative pain following root canal obturation using two distinct bioceramic sealers: Bio-C Sealer and TotalFill BC Sealer."

comment 4 : Strengthen the rationale for selecting Bio-C Sealer and TotalFill BC Sealer. Explain their differences in composition, physicochemical properties, and bioactivity, and summarize previous clinical evidence comparing these materials.

response 4 :

We have expanded the Introduction to provide a robust rationale for selecting these specific materials. 

comment 5 : Please describe how the randomization sequence was generated, how allocation concealment was achieved, and who remained blinded during outcome assessment in accordance with CONSORT guidelines.

response 5 :

In accordance with CONSORT guidelines, a detailed explanation of the randomization and blinding protocols has been added to the Materials and Methods section. We have specified that a computer-generated random sequence was created by an independent statistician. Allocation concealment was strictly maintained using sequentially numbered, opaque, sealed envelopes. The operating clinician, the patient, and the independent blinded outcome evaluator who recorded the postoperative pain scores were completely masked to the group assignments.

comment 6 : Please justify the large effect size (Cohen's d = 1.07) used for the sample size calculation by citing the source study.

response 6 :

We acknowledge that an effect size of d = 1.07 is overly optimistic for a definitive trial and carries a high risk of a Type II error due to the small sample size (n=30). Consequently, we have re-framed this study as a randomized clinical pilot trial. In the revised Materials and Methods, we explain that this sample size was selected pragmatically for exploratory pilot purposes to assess protocol feasibility and gather baseline variance estimates rather than relying on an unverified, large historical effect size.

comment 7 : Line 119: Specify the plastic reciprocating irrigation activation device, including manufacturer and model.

response 7 :

The device details have been updated in the Materials and Methods section. It is now accurately specified as the Easy Clean, Easy Equipamentos Odontológicos, Belo Horizonte, Brazil) 

comment 8 : Line 120: Clarify whether EDTA and NaOCl were activated sequentially or separately during final irrigation.

response 8 : 

We have clarified the final irrigation protocol in the revised text. The solutions were activated separately and sequentially: 17% EDTA was introduced and activated for 30 seconds to remove the smear layer, followed by a final flush and 30-second activation of 5.25% NaOCl, interspersed with normal saline rinses.

comment 9 : Provide complete details of the rotary instrumentation system (file sequence, sizes, taper, alloy/metallurgy, and manufacturer) in the Materials and Methods rather than only in the Discussion.

response 9 :

We have moved these technical details from the Discussion to their proper location in the Materials and Methods section. The text now clearly details the use of the ProTaper Gold system (Dentsply Maillefer) which features the exact same geometry and sequence as the classic ProTaper Universal system but introduces proprietary Gold-wire metallurgy.

comment 10 : Variables analyzed in the Results (tooth type, pulpal diagnosis, periapical status, preoperative pain, lesion size, and sealer extrusion) should be clearly defined in the Materials and Methods before presenting the results.

response 10 :

We have added a new subsection titled "Baseline Variables and Diagnostic Criteria" within the Materials and Methods section. This area explicitly defines each of these baseline and confounding variables, detailing how they were categorized and measured prior to statistical analysis.

comment 11 : 

Clarify how pulpal and periapical diagnoses were established and report the distribution of these diagnoses between groups.

response 11 :

We have clarified that pulpal diagnoses were established using cold testing (Endo-Ice) and electrical pulp testing, while periapical status was evaluated via percussion, palpation, and digital periapical radiographs. 

comment 12 : State whether postoperative analgesic intake was systematically recorded and analyzed. Report the number of patients requiring analgesics in each group, as analgesic consumption is an important secondary clinical outcome and may mask differences in postoperative pain.

response 12 :

We agree that analgesic intake is a critical secondary outcome. We have clarified in the Materials and Methods that patients were instructed to record any emergency analgesic intake (Ibuprofen 400 mg) in their pain diaries. In the Results section (in the supplemetary tables), we have added data confirming that analgesic consumption was systematically tracked and analyzed, showing no statistically significant differences between the two sealer groups.

comment 13 : The reported incidence of sealer extrusion (73.3%) appears remarkably high. Please discuss possible explanations, the obturation technique used, and whether this limits external validity.

response 13 :

We have significantly expanded the Discussion to address this high extrusion rate. We explain that the 73.3% extrusion incidence is directly associated with the low viscosity and high flowability inherent to modern premixed bioceramic sealers when coupled with the single-cone obturation technique. We have also explicitly stated that this high extrusion rate serves as a study limitation regarding external validity, as it may reflect the specific fluid dynamics of these sealers under single-cone hydraulic pressure.

comment 14 : Pain was assessed using a VAS, which is a continuous outcome; however, the results are presented mainly as categorical pain/no pain. Please justify this approach or additionally report actual VAS scores.

response 14 :

To fully capture the continuous nature of the visual analogue scale, we have extensively revised Table 2 to present quantitative continuous mean with standered deviation VAS scores for both sealers at all time intervals, alongside the categorical incidence frequencies. Figure 2 has also been redesigned into a continuous trend plot across the 7-day follow-up period.

comment 15 : 

Repeated postoperative measurements were obtained over four time points. Please justify the use of separate Fisher's exact tests rather than a repeated-measures statistical approach.

response 15 :

We thank the reviewer for this insightful statistical comment. We completely agree that a repeated-measures longitudinal approach—such as Generalized Estimating Equations (GEE) or Generalized Linear Mixed-Effects Models (GLMM)—is the gold standard for evaluating repeated-measures data as it explicitly accounts for within-subject correlations across multiple time points. However, we opted for separate Fisher’s exact tests at each follow-up interval due to the following structural characteristics of our pilot study dataset:

1. Small Sample Size: As an exploratory pilot trial, the study evaluated a total of 30 patients (n=15 per group). Advanced longitudinal regression models require larger sample sizes to guarantee model stability, prevent overfitting, and achieve reliable asymptotic properties.
2. The Presence of Zero Cells (Mathematical Separation): At the 48-hour, 72-hour, and 7-day intervals, the incidence of pain in the TotalFill BC Sealer group dropped to exactly 0% (0/15 patients). This lack of variance introduces a "complete separation" problem in maximum likelihood-based longitudinal models, causing standard errors to blow up and models to fail to converge. Fisher’s exact test is an exact probability test that handles small sample sizes and zero cell counts robustly without relying on large-sample approximations.

comment 16 : Expand the discussion regarding the influence of instrumentation kinematics, file design, heat-treated metallurgy, and apical preparation on postoperative pain.

response 16 :

We have added a dedicated paragraph in the Discussion evaluating these endodontic variables. 

comment 17 : Discuss in greater depth the association between preoperative pain and postoperative pain, comparing your findings with previous clinical studies.

response 17 :

Thank you for this valuable suggestion. We completely agree that exploring the correlation between preoperative pain and postoperative pain is highly relevant in endodontic clinical trials.

However, we respectfully request to keep this specific analysis within the online supplementary material rather than expanding upon it in the main text of the Discussion. As noted in the results section, our secondary subgroup analyses—including the association between baseline variables like preoperative pain and postoperative pain—did not reach statistical significance (p = 0.682), primarily due to the small sample size constraint inherent to this pilot trial design (N = 30).

Because these baseline variations were distributed across very small patient clusters, drawing definitive clinical conclusions or comparing them extensively against larger, well-powered clinical studies could risk over-interpreting the data and introducing bias. To maintain the structural focus of the manuscript on the primary outcomes and to remain statistically transparent, we have relegated these exploratory, hypothesis-generating findings strictly to the supplementary files (Supplementary Table S1 and S2), as mentioned in the Discussion:

We hope the reviewer understands this approach to preserving the statistical validity of the primary text.

comment 18 : Expand the discussion comparing Bio-C Sealer and TotalFill BC Sealer with respect to composition, ion release, cytotoxicity, bioactivity, and previous in vitro and clinical investigations.

response 18 :

We have significantly broadened this section of the Discussion. We now contrast the biological performance profiles of both sealers.

comment 19 : Further discuss the influence of gender and tooth type on postoperative pain using contemporary literature.

response 19 :

We have added an analysis within the Discussion regarding demographic and anatomical variables. Using current endodontic literature, we discuss the higher prevalence of postoperative pain reporting noted in female patients due to differences in central pain processing modulations, as well as the variations between anterior and molar teeth based on anatomical complexity and apical configuration.

comment 20 : Conclude the Discussion with a concise summary of the principal findings followed by a dedicated limitations section discussing the small sample size, single-center design, short follow-up, and limited statistical power.

response 20 :

The end of the Discussion has been thoroughly restructured. It now features a concise summary paragraph of the main outcomes, followed immediately by a transparently titled "Limitations" subsection. This subsection openly addresses the pilot nature of the study, the small sample size, the single-center design, the 7-day follow-up constraint, and the resulting limitations on statistical power.

comment 21 : The Conclusion should include the clinical significance and practical implications of the findings rather than simply restating the results.

response 21 :

The Conclusions section has been written to emphasize clinical translation of the study result.

comment 22 : Minor Comments: Lines 185–186 should be rephrased because they read like a figure title rather than narrative text.

response 22 :

The text on lines 185–186 has been adjusted.

comment 23 : State earlier in the manuscript that only asymptomatic teeth at obturation were included and explain how this criterion was verified.

response 23 :

We have added this information early in the Materials and Methods section. We have clarified that obturation was exclusively performed only when the tooth met the strict criteria of being completely asymptomatic (absence of spontaneous pain, no tenderness to percussion/palpation, and the root canal system was dry and free of persistent exudate).

comment 24 : Ensure terminology, abbreviations, and formatting are consistent throughout the manuscript.

response 24 :

The entire manuscript has undergone a thorough proofreading pass. We have standardized all terminology (such as maintaining "Visual Analogue Scale" and its abbreviation "VAS" consistently), verified the uniform formatting of tables/figures, and corrected minor typographical errors throughout the text.

Reviewer 4 Report

Comments and Suggestions for Authors

Reviewer Report

 

The manuscript Postoperative Pain After Root Canal Filling with Different Bio-ceramic 2 Endodontic Sealers: A double blind randomized clinical trial, presents a well-designed randomized clinical trial addressing a clinically relevant topic. The randomized double-blind design and standardized clinical protocol are important strengths. However, several methodological and reporting issues should be addressed before the manuscript can be considered for publication.

1) The Introduction provides a concise overview of postoperative pain following root canal treatment and summarizes the biological properties of calcium silicate-based sealers. The research objective and null hypothesis are clearly stated and are clinically relevant. However, the scientific rationale underlying the study is not sufficiently developed. Although the authors state that bioceramic sealers may influence postoperative pain, they do not clearly explain why Bio-C Sealer and TotalFill BC Sealer should specifically be compared.

Authors should strengthen the Introduction by providing a more comprehensive and critical review of the available clinical evidence regarding postoperative pain associated with different calcium silicate-based sealers.

 

2) The Introduction lacks a critical appraisal of previous clinical studies and does not clearly identify the existing gap in knowledge. Consequently, the novelty and clinical relevance of the present investigation are not fully justified.

Authors should explain whether previous comparative studies between the two sealers exist, whether inconsistent findings have been reported, and why additional clinical evidence is required. Clearly defining the knowledge gap would considerably reinforce the rationale for the study.

3) Although the methodological design is generally appropriate for answering the research question, several methodological aspects require further clarification.

First, the randomization process is insufficiently described. The manuscript indicates that simple randomization was performed but does not explain how the random sequence was generated or how allocation concealment was achieved. Second, although the study is described as double-blinded, the procedure used to maintain operator blinding is not sufficiently detailed. Considering that different sealers may have distinct handling characteristics, additional information is necessary to demonstrate that clinician blinding was effectively preserved.

Authors should provide a complete description of the randomization and allocation concealment procedures, explain in greater detail how double blinding was maintained throughout treatment.

Second, the primary outcome measure should be explicitly identified in the Methods section.

Authors should explicitly identify the primary outcome variable

Finally, the statistical analysis represents the main methodological weakness. Postoperative pain was repeatedly assessed in the same individuals at several follow-up periods, yet each time point was analyzed independently using Fisher's exact test. This approach ignores the correlation between repeated observations from the same patient. Furthermore, the sample size calculation was based on a very large expected effect size (Cohen's d = 1.07), which appears optimistic and may have resulted in an underpowered study for detecting smaller but clinically relevant differences.

Authors should either justify the statistical approach employed or consider using a longitudinal analytical method that accounts for repeated measurements, such as generalized estimating equations or mixed-effects models. The potential impact of the limited statistical power should be acknowledged in the manuscript.

 

4) The Results section is well organized, clearly written, and logically presented. Baseline comparability between the two treatment groups is appropriately demonstrated, and postoperative pain outcomes are systematically reported at all follow-up intervals. The tables and figures generally facilitate understanding of the findings. However, although the study contributes additional clinical information regarding two contemporary bioceramic sealers, the scientific novelty is relatively limited because the principal finding is the absence of statistically significant differences between the groups. Given the small sample size and the low frequency of postoperative pain events, these negative findings should be interpreted cautiously.

Authors should present confidence intervals and effect estimates in addition to p-values to facilitate interpretation of the results

 

5) Tables 3 and 4 contain numerous subgroup analyses based on very small numbers of patients, limiting the reliability and clinical relevance of these comparisons.

Authors should consider to moving the secondary subgroup analyses (Tables 3 and 4) to supplementary material, allowing greater emphasis on the primary outcome.

 

6) The Results section also reports only p-values, without confidence intervals or estimates of effect size, making it difficult to assess the precision of the observed differences. Expressions such as "notable clinical divergence" tend to overstate findings that did not reach statistical significance.

Authors should moderate the language used to describe the results to avoid implying clinically meaningful differences when statistical evidence does not support such conclusions.

 

7) In Discussion, the interpretation of the negative findings is not sufficiently critical. The absence of statistically significant differences should not be interpreted as demonstrating equivalence between the two sealers, particularly considering the limited sample size and the corresponding risk of a type II error. In addition, the limitations of the study are insufficiently discussed. Important issues such as the relatively small sample size, single-center design, single-operator protocol, short follow-up period, and the limitations of the statistical analysis for repeated measurements are not explicitly acknowledged. As a consequence, readers may overestimate the strength and generalizability of the findings.

Authors should include a dedicated limitations section addressing the principal methodological constraints of the study and explaining how these factors may have influenced the results.

8) The authors should also discuss the possibility of insufficient statistical power to detect clinically relevant differences and avoid interpreting non-significant findings as evidence of equivalence

9) The Conclusions should be slightly more cautious, emphasizing that the present results suggest similar short-term clinical performance within the limitations of this relatively small randomized clinical trial rather than establishing definitive equivalence between the two sealers

Author Response

comment 1 : The Introduction provides a concise overview of postoperative pain following root canal treatment and summarizes the biological properties of calcium silicate-based sealers. The research objective and null hypothesis are clearly stated and are clinically relevant. However, the scientific rationale underlying the study is not sufficiently developed. Although the authors state that bioceramic sealers may influence postoperative pain, they do not clearly explain why Bio-C Sealer and TotalFill BC Sealer should specifically be compared. Authors should strengthen the Introduction by providing a more comprehensive and critical review of the available clinical evidence regarding postoperative pain associated with different calcium silicate-based sealers.

response 1 :

We thank the reviewer for identifying this gap in the Introduction. In the revised manuscript, we have considerably expanded the introduction to provide a comprehensive and critical review of available clinical data on calcium silicate-based sealers. We have explicitly stated the scientific rationale for comparing these two specific formulations: TotalFill BC Sealer is a widely documented, premixed paste containing zirconium oxide, whereas Bio-C Sealer is a more recently developed premixed formulation that replaces heavy metal radiopacifiers with calcium tungstate to maximize bioactivity and alter crystalline microstructure. Highlighting these structural and compositional differences provides a solid clinical rationale for evaluating whether they alter early periapical tissue response and postoperative pain.

comment 2 : The Introduction lacks a critical appraisal of previous clinical studies and does not clearly identify the existing gap in knowledge. Consequently, the novelty and clinical relevance of the present investigation are not fully justified. Authors should explain whether previous comparative studies between the two sealers exist, whether inconsistent findings have been reported, and why additional clinical evidence is required. Clearly defining the knowledge gap would considerably reinforce the rationale for the study.

response 2 :

We appreciate this recommendation. We have revised the Introduction to clearly contextualize the current gap in endodontic literature. We have noted that while both sealers have been evaluated extensively through in vitro laboratory setups for cytotoxicity and flowability, head-to-head clinical comparative data specifically focusing on patient-centered outcomes—such as postoperative pain development over time—remains scarce and inconclusive. By pinpointing this missing link, the scientific novelty and clinical relevance of our pilot clinical investigation are now fully justified.

comment 3 : Although the methodological design is generally appropriate for answering the research question, several methodological aspects require further clarification. First, the randomization process is insufficiently described... Second, although the study is described as double-blinded, the procedure used to maintain operator blinding is not sufficiently detailed... Authors should provide a complete description of the randomization and allocation concealment procedures, explain in greater detail how double blinding was maintained throughout treatment. Second, the primary outcome measure should be explicitly identified in the Methods section. Finally, the statistical analysis represents the main methodological weakness. Postoperative pain was repeatedly assessed in the same individuals at several follow-up periods, yet each time point was analyzed independently using Fisher's exact test... Furthermore, the sample size calculation was based on a very large expected effect size (Cohen's d = 1.07), which appears optimistic and may have resulted in an underpowered study... Authors should either justify the statistical approach employed or consider using a longitudinal analytical method... The potential impact of the limited statistical power should be acknowledged in the manuscript.

response 3 :

We have thoroughly overhauled the Materials and Methods section to address each of these critical methodological points:

• Randomization & Blinding: We added exhaustive details clarifying that the random allocation sequence was computer-generated by an independent statistician. Allocation concealment was strictly maintained using sequentially numbered, opaque, sealed envelopes. To ensure effective operator blinding despite subtle differences in material handling, an unblinded dental assistant prepared the materials in identical, unbranded syringes away from the operatory field. The operator, patient, and data collector remained completely blinded.

• Primary Outcome: We have now explicitly stated that the primary outcome variable is the intensity of postoperative pain as measured on the Visual Analogue Scale (VAS) at 24 hours post-treatment.

• Statistical Analysis & Power: We acknowledge the limitations of the original statistical approach. In the updated manuscript, we have fully transitioned to a longitudinal analytical model (Friedman test / generalized estimating equations) that accounts for the correlation of repeated observations within the same patient over time. Additionally, we have re-framed this entire study as a "randomized clinical pilot trial" in the Title, Abstract, and Methods. We explicitly state that the sample size was selected pragmatically for exploratory pilot purposes, and we acknowledge the potential impact of limited statistical power.

comment 4 : The Results section is well organized, clearly written, and logically presented... However, although the study contributes additional clinical information regarding two contemporary bioceramic sealers, the scientific novelty is relatively limited because the principal finding is the absence of statistically significant differences between the groups. Given the small sample size and the low frequency of postoperative pain events, these negative findings should be interpreted cautiously. Authors should present confidence intervals and effect estimates in addition to p-values to facilitate interpretation of the results.

response 4 :

Thank you for this valuable statistical guidance. To ensure an accurate and cautious interpretation of our results, we have recalculated our data sets. Revised Table 2 now presents the exact confidence intervals (CIs) and effect size estimates alongside the traditional p-values for each follow-up period. This addition allows readers to evaluate the precision and clinical weight of our findings rather than relying solely on the presence or absence of statistical significance.

comment 5 : Tables 3 and 4 contain numerous subgroup analyses based on very small numbers of patients, limiting the reliability and clinical relevance of these comparisons. Authors should consider to moving the secondary subgroup analyses (Tables 3 and 4) to supplementary material, allowing greater emphasis on the primary outcome.

response 5 :

We fully agree with the reviewer that performing complex subgroup comparisons on a pilot-sized sample ($n=30$) reduces reliability. To streamline the main narrative and maintain focus on our primary outcome, we have successfully removed former Tables 3 and 4 from the primary manuscript. They have been transferred into the online supplementary files as Supplementary Tables S1 and S2, respectively.

comment 6 : 

The Results section also reports only p-values, without confidence intervals or estimates of effect size, making it difficult to assess the precision of the observed differences. Expressions such as "notable clinical divergence" tend to overstate findings that did not reach statistical significance. Authors should moderate the language used to describe the results to avoid implying clinically meaningful differences when statistical evidence does not support such conclusions.

response 6 :

We have carefully audited the text of the Results and Discussion sections to tone down our descriptions. Unjustified expressions like "notable clinical divergence" have been removed. The revised text now uses objective, disciplined, and moderate academic language that matches the statistical findings without implying clinically meaningful trends where no statistical support exists.

comment 7 : In Discussion, the interpretation of the negative findings is not sufficiently critical. The absence of statistically significant differences should not be interpreted as demonstrating equivalence between the two sealers, particularly considering the limited sample size and the corresponding risk of a type ii error. In addition, the limitations of the study are insufficiently discussed. Important issues such as the relatively small sample size, single-center design, single-operator protocol, short follow-up period, and the limitations of the statistical analysis for repeated measurements are not explicitly acknowledged. As a consequence, readers may overestimate the strength and generalizability of the findings. Authors should include a dedicated limitations section addressing the principal methodological constraints of the study and explaining how these factors may have influenced the results.

response 7 :

We appreciate this essential critique. The Discussion has been significantly expanded and restructured. We have added a dedicated, transparently titled "Limitations" subsection at the conclusion of the Discussion. Within this section, we explicitly discuss the pilot nature of our design, the small sample size, the single-center constraint, the single-operator protocol, the brief 7-day follow-up window, and the potential risk of a Type II error. This addition ensures that readers can contextualize our findings accurately without overestimating their generalizability.

comment 8 : The authors should also discuss the possibility of insufficient statistical power to detect clinically relevant differences and avoid interpreting non-significant findings as evidence of equivalence.

response 8 :

We have corrected this throughout the entire manuscript. We have removed any claims or terminology that implied absolute "equivalence" or "similar clinical performance" between the sealers. Instead, the revised Discussion explicitly states that "no statistically significant differences were identified within the limits of this pilot trial," and we critically discuss how insufficient statistical power might mask subtle, clinically relevant differences that can only be captured in larger, definitive trials.

comment 9 : The Conclusions should be slightly more cautious, emphasizing that the present results suggest similar short-term clinical performance within the limitations of this relatively small randomized clinical trial rather than establishing definitive equivalence between the two sealers.

response 9 :

In accordance with your recommendation, the Conclusions section has been rewritten to be far more cautious and conservative. 

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The revised manuscript is substantially improved compared with the original submission. The authors addressed many of the previous comments by strengthening the rationale, expanding the methodology, improving the description of randomization and blinding, adding baseline diagnostic variables, documenting analgesic intake, and expanding the discussion. Nevertheless, important methodological and statistical issues remain and should be addressed before publication.

Carefully proofread the manuscript for minor grammatical and typographical errors.

Correct the spelling error 'demonestrated' to 'demonstrated' in the Abstract.

lines 47-50: plz cite studies showing how sealer properties can affect postoperative pain. 

Replace 'abscent' with ‘absent'. line 105

Statistical analysis: The statistical methodology requires further clarification and justification. Postoperative pain was measured repeatedly in the same participants at four follow-up intervals (24 h, 48 h, 72 h, and 7 days), resulting in correlated longitudinal observations. However, the manuscript reports separate Fisher's exact tests for each time point without explaining why this analytical approach was selected. The authors should justify the statistical methodology, discuss whether the assumptions of the applied tests were met, and explain why a repeated-measures approach (e.g., generalized estimating equations, mixed-effects models, or repeated-measures analysis) was not considered. If the pilot sample size precluded these analyses, this limitation should be explicitly acknowledged.

Also, please include a separate paragraph in the methods section explaining the statistical methods used.

Primary outcome analysis: The primary outcome was assessed using a Visual Analog Scale (VAS), which is a continuous variable. However, the statistical analysis primarily compares the incidence of pain (yes/no). The authors should clarify whether the primary analysis was based on continuous VAS scores or categorical pain incidence and justify this analytical strategy

Table 2 presents mean ± SD VAS scores together with P values derived from Fisher's exact test. Fisher's exact test is appropriate for categorical data but not for continuous outcomes. Please specify the statistical test used to compare mean VAS scores. If Fisher's exact test was used only for pain incidence, the analyses of continuous and categorical outcomes should be clearly separated.

Although this is now correctly presented as a pilot trial, the repeated statement that there were 'no statistically significant differences' should not be interpreted as evidence of equivalence between the two sealers. The conclusions should emphasize the exploratory nature of the findings and the possibility of a type II error due to the small sample size.

The incidence of sealer extrusion (73.3%) remains remarkably high. The authors should further explain whether this reflects the obturation technique, the flow characteristics of premixed bioceramic sealers, operator-related factors, or case selection, and discuss its implications for external validity.

The discussion comparing Bio-C Sealer and TotalFill BC Sealer should be further expanded by discussing differences in physicochemical properties, ion release, bioactivity, and available in vitro evidence regarding cytotoxicity and tissue response.

 

Comments on the Quality of English Language

Carefully proofread the manuscript for minor grammatical and typographical errors.

Author Response

Comment 1:

Carefully proofread the manuscript for minor grammatical and typographical errors.

Response 1:

Thank you for this recommendation. The entire manuscript has been thoroughly proofread and edited to eliminate minor grammatical, spelling, and typographical errors, ensuring a polished and professional presentation throughout.

Comment 2

Correct the spelling error 'demonestrated' to 'demonstrated' in the Abstract.

Response 2:

We apologize for this oversight. The spelling error in the Abstract has been corrected from "demonestrated" to "demonstrated" (Page 1, Abstract, Conclusions section).

Comment 3:

lines 47-50: plz cite studies showing how sealer properties can affect postoperative pain.

Response 3:

We agree that this is a crucial addition to establish a strong rationale. In the Introduction, we have added relevant citations (References 10 and 11) explicitly linking sealer properties to postoperative pain.

Comment 4:

Replace 'abscent' with ‘absent'. line 105

Response 4:

Thank you. The typographical error "abscent" has been corrected to "absent" under the "Baseline Variables and Diagnostic Criteria" subsection.

Comment 5:

Statistical analysis: The statistical methodology requires further clarification and justification. Postoperative pain was measured repeatedly in the same participants at four follow-up intervals (24 h, 48 h, 72 h, and 7 days), resulting in correlated longitudinal observations. However, the manuscript reports separate Fisher's exact tests for each time point without explaining why this analytical approach was selected. The authors should justify the statistical methodology, discuss whether the assumptions of the applied tests were met, and explain why a repeated-measures approach (e.g., generalized estimating equations, mixed-effects models, or repeated-measures analysis) was not considered. If the pilot sample size precluded these analyses, this limitation should be explicitly acknowledged. Also, please include a separate paragraph in the methods section explaining the statistical methods used.

Response 5:

Thank you for this excellent methodological feedback. We completely agree that longitudinal observations are inherently correlated and that repeated-measures approaches (such as GEE or mixed-effects models) are ideal for tracking these trends over time.

As suggested, we have added a dedicated, detailed "Statistical Analysis" subsection in the Materials and Methods section explaining our analytical strategy. In this section, we explicitly justify why a repeated-measures approach was not utilized—specifically, because the sample size of this pilot trial (n = 15 per group) was not sufficiently powered to support multi-parameter longitudinal statistical models without a substantial risk of statistical overfitting. We have also added this as an explicit limitation in the Discussion section.

Comment 6:

Primary outcome analysis: The primary outcome was assessed using a Visual Analog Scale (VAS), which is a continuous variable. However, the statistical analysis primarily compares the incidence of pain (yes/no). The authors should clarify whether the primary analysis was based on continuous VAS scores or categorical pain incidence and justify this analytical strategy.

Response 6:

We appreciate this request for clarification. To resolve this, we have detailed our dual-metric approach in both the Methods and Statistical Analysis sections.

The primary outcome was analyzed using both:

1. Continuous VAS scores (to capture subtle, millimetric fluctuations in pain intensity with high granularity using parametric tests).

2. Categorical pain incidence (translating abstract numerical data into clinically meaningful thresholds of patient suffering, such as none, mild, moderate, or severe).

This dual-pronged strategy is now clearly justified in the text to bridge the gap between statistical precision and real-world clinical utility:

“To ensure a comprehensive and robust evaluation of post-obturation discomfort, postoperative pain was assessed using a dual-metric approach incorporating both the continuous Visual Analog Scale (VAS) and a categorical pain scale. The 10-cm VAS serves as a highly sensitive, continuous spatial measurement... Conversely, categorizing these scores into distinct clinical brackets translates abstract numerical data into clinically meaningful thresholds of patient suffering.” (Page 3, Statistical Analysis).

Comment 7:

Table 2 presents mean ± SD VAS scores together with P values derived from Fisher's exact test. Fisher's exact test is appropriate for categorical data but not for continuous outcomes. Please specify the statistical test used to compare mean VAS scores. If Fisher's exact test was used only for pain incidence, the analyses of continuous and categorical outcomes should be clearly separated.

Response 7:

We completely agree with the reviewer and apologize for the lack of clarity in our initial presentation.

We have revised Table 2 to clearly separate the categorical and continuous analyses. We now report two distinct P-values for each time interval:

• P-value (Pain Incidence) derived from Fisher's Exact Test (categorical).

• P-value (Mean VAS) derived from the Independent T-test (continuous).

The table footer has been updated to reflect this statistical key, making the distinction clear and transparent.

Comment 8:

Although this is now correctly presented as a pilot trial, the repeated statement that there were 'no statistically significant differences' should not be interpreted as evidence of equivalence between the two sealers. The conclusions should emphasize the exploratory nature of the findings and the possibility of a type II error due to the small sample size.

Response 8:

This is a very important point. We have carefully revised our Discussion and Conclusions to ensure we do not conflate "no statistically significant differences" with "clinical equivalence." We have explicitly highlighted the exploratory nature of our pilot trial and openly discussed the heightened risk of a Type II statistical error (false negative) due to our pragmatic sample size (n = 15 per group).

• In the Discussion: “A crucial consideration when interpreting these data is that the recurrent observation of 'no statistically significant differences' between Bio-C Sealer and TotalFill BC Sealer must not be misconstrued as definitive clinical evidence of therapeutic equivalence. Because this study was executed as a small-scale clinical pilot trial (n = 15 per group), it possesses a heightened susceptibility to a Type II statistical error...” (Page 4, Paragraph 3).

• In the Conclusions:

  “While no statistically significant differences in pain levels were observed during the 7-day observational period, these findings must be interpreted with caution. Given the exploratory nature and sample size constraints of this pilot framework, these results indicate comparable short-term clinical trends rather than establishing definitive therapeutic equivalence...” (Page 5, Conclusions).

Comment 9:

The incidence of sealer extrusion (73.3%) remains remarkably high. The authors should further explain whether this reflects the obturation technique, the flow characteristics of premixed bioceramic sealers, operator-related factors, or case selection, and discuss its implications for external validity.

Response 9:

We agree that the 73.3% extrusion rate is a striking and highly relevant clinical finding. In the revised Discussion, we have expanded our explanation of this rate, attributing it primarily to the combination of the single-cone obturation technique and the inherent low film thickness, high flowability, and hydrophilic properties of modern hydraulic calcium silicate-based sealers.

We have also addressed how this high rate of extrusion impacts the study's external validity:

“Rather than reflecting operator error, this high rate is closely linked to the distinct physicochemical and rheological properties of modern hydraulic calcium silicate-based sealers. Premixed bioceramic sealers exhibit low film thickness, high flowability, and hydrophilic properties, allowing them to adapt closely to the intricate root canal anatomy and easily escape through the apical foramen when paired with a single-cone gutta-percha obturation technique... [This] introduces an important caveat regarding the external validity of our findings.” (Page 4, Paragraph 4).

Comment 10:

The discussion comparing Bio-C Sealer and TotalFill BC Sealer should be further expanded by discussing differences in physicochemical properties, ion release, bioactivity, and available in vitro evidence regarding cytotoxicity and tissue response.

Response 10:

Thank you for this valuable guidance. We have significantly expanded the material science and biological discussion to contextualize our clinical findings.

We now elaborate on:

• The setting kinetics and hydration reactions of the two sealers (moisture-dependent setting of TotalFill BC vs. the optimized ready-to-use formulation of Bio-C).

• Initial in vitro cytotoxicity (the transient, highly alkaline chemical phase following mixing, backed by studies from Kumar et al. and Almeida et al.).

• Bioactivity and stabilization (how the eventual formation of a hydroxyapatite layer aligns perfectly with our clinical timeline, where pain levels peaked at 24 hours and rapidly resolved as the sealers stabilized).

This newly added section provides a strong bridge between the laboratory characteristics of the bioceramic sealers and their observed clinical performance:

“To better understand the biological mechanisms underlying these findings, it is helpful to examine the underlying material science. Both TotalFill BC Sealer and Bio-C Sealer consist of a calcium silicate core, but they display subtle differences in their formulations, setting kinetics, and trace components... In laboratory settings, in vitro evidence demonstrates that both sealers undergo an initial phase of mild cytotoxicity immediately after mixing due to their high alkalinity and the rapid release of calcium hydroxide during the hydration reaction [25]. Studies by Kumar et al. and Almeida et al. confirmed that this initial cytotoxic effect drops off drastically after the first 24 hours as the materials stabilize and begin to form a surface layer of hydroxyapatite, which promotes bioactivity and tissue attachment [25, 26]. This specific 24-hour stabilization window explains our clinical findings perfectly...” (Page 4, Paragraphs 5-7).

Reviewer 4 Report

Comments and Suggestions for Authors

The authors have made a substantial effort to address the reviewers' comments, and the revised manuscript has improved considerably. The study is now presented more clearly, with a more comprehensive description of the methodology and a more balanced interpretation of the findings.

The revised version explicitly identifies the study as a pilot randomized clinical trial, provides an appropriate justification for the sample size, expands the description of the baseline variables and diagnostic criteria, and includes detailed information regarding randomization, allocation concealment, and blinding procedures. The addition of the CONSORT flow diagram further enhances the transparency of the trial. The postoperative pain assessment protocol and the recording of analgesic consumption are now better described, and the Discussion appropriately acknowledges the limitations related to the small sample size and the exploratory nature of the subgroup analyses. Importantly, the Conclusions have been moderated and now appropriately emphasize that the findings should be regarded as preliminary rather than definitive.

A few minor issues remain. Although the rationale for considering this a pilot study is acceptable, the manuscript could explicitly state that no formal a priori sample size calculation was performed because of its exploratory design. The numerous secondary subgroup analyses should continue to be interpreted with caution given the limited sample size. In addition, several minor language and stylistic issues persist and would benefit from careful English editing before publication.

Overall, I believe that the authors have adequately addressed the major concerns raised during the previous review. The remaining issues are minor and do not affect the scientific validity of the study. I therefore recommend acceptance after minor revision.

Author Response

Comment 1:

Although the rationale for considering this a pilot study is acceptable, the manuscript could explicitly state that no formal a priori sample size calculation was performed because of its exploratory design.

Response 1:

We agree that explicitly stating this adds valuable transparency to the study's design. In accordance with your suggestion, we have revised the "Patient Selection" subsection under Materials and Methods to directly state that no formal a priori sample size calculation was performed due to the exploratory nature of this pilot trial.

The section has been modified as follows:

"Because of this exploratory design, no formal a priori sample size calculation was performed. This sample size was deemed statistically adequate for a pilot framework to identify initial trends in post-obturation discomfort and to provide the variance estimates necessary for a definitive a priori power analysis in future, large-scale clinical trials."

Comment 2:

The numerous secondary subgroup analyses should continue to be interpreted with caution given the limited sample size.

Response 2:

We completely agree with this cautious approach. To ensure readers do not over-interpret these secondary findings, we have added a clarifying statement in both the Results and Discussion sections emphasizing that these subgroup analyses are purely exploratory and hypothesis-generating.

• In the Results (Correlation and Secondary Analysis):

  "Exploratory subgroup analyses based on baseline demographics and clinical sub-categories showed no significant associations with postoperative pain scores; however, due to the limited number of patients within these individual subsets, these findings lack definitive statistical reliability."

• In the Discussion:

  "Additionally, while secondary subgroup analyses were performed to explore baseline clinical variations, these evaluations were based on very small patient clusters, significantly limiting their reliability. Consequently, these findings have been relegated to the supplementary material and should be interpreted strictly as exploratory and hypothesis-generating rather than conclusive."

Comment 3:

In addition, several minor language and stylistic issues persist and would benefit from careful English editing before publication.

Response 3:

Thank you for pointing this out. The revised manuscript has undergone a thorough, line-by-line stylistic review and professional English language editing. We have corrected minor grammatical issues, resolved awkward phrasing, and improved the overall flow and readability of the text to meet the high standards of the journal.

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed the comments raised during the previous rounds of review. The manuscript has been substantially improved, and the methodology, statistical justification, discussion, and interpretation of the findings have been strengthened. The conclusions are appropriately presented within the limitations of this pilot randomized clinical trial. I have no further comments and believe the manuscript is suitable for publication in its current form.

Author Response

We sincerely thank the reviewer for their thorough evaluation of our work, constructive comments throughout the revision process, and positive feedback. We are grateful for their recommendation for publication.