Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Interesting work. 
(1) Nice to know that chitosan play a crucial role in this study. Why 2% chitosan solution was used?

(2) Why 1%, 3% or 4% chitosan was not tested in this study?

(3) Chiotsan is soluble in acetic acid. Will it have any outcome on the human studies if extrapolated?

(4) Why dog was chosen over other species. Need more clarity?

(5) Predominantly images are shown. Images can be explained in detail along with the instrumentation used to obtain these images. 

(6) Any comparative studies on similar material tested in dog and later used for human treatment can be included in the discussion section

(7) Stability of the chitosan based material and its shelf life as well as stability in the biological matrix for a certain period of time will be crucial. How authors will address this and extend it to other higher species can be captured in conclusion and future perspectives

(8) How the developed material will be better than any of the existing material used for treatment can be detailed. 

Author Response

Dear Editor and Reviewers,

We sincerely thank you for the careful evaluation of our manuscript and for the constructive comments and suggestions. We greatly appreciate the time and effort invested in improving the clarity, scientific rigor, and translational relevance of our work. We have revised the manuscript accordingly and addressed each comment point by point below. The revisions were made to strengthen the scientific rationale, clarify the methodology, and refine the interpretation of the findings where appropriate.

Comment 1. Nice to know that chitosan plays a crucial role in this study. Why was 2% chitosan solution was used?

Response: Thank you for this insightful comment. The selection of a 2% chitosan solution was based on its favorable balance between physicochemical properties and biological performance. Chitosan is a biocompatible and biodegradable natural polymer with low toxicity and excellent drug delivery characteristics, making it suitable for biomedical applications. Its free amine groups enable ionic interactions and crosslinking, which are essential for nanoparticle formation and structural stability. Importantly, the 2% concentration provides an optimal balance between viscosity, mechanical stability, ease of fabrication, and biocompatibility. Previous evidence has demonstrated that this concentration supports favorable cellular responses, particularly in periodontal ligament stem cells, indicating its suitability for regenerative applications.
Accordingly, the 2% concentration was selected as a clinically relevant and biologically favorable formulation. This rationale has been clarified in the manuscript, and References 24 and 25 have been added to support this justification, with subsequent references adjusted accordingly.

Location of revision:

• Materials and Methods, Section 2.4
• Page: 4
• Lines: 159–163

Added  A 2% chitosan NPs concentration was selected based on previous evidence demonstrating a favorable balance between antimicrobial activity, biocompatibility, and stem cell viability. Chitosan nanoparticles prepared by this method are typically characterized by nanoscale size and a positive surface charge, facilitating interaction with negatively charged bacterial cell walls.

Comment 2. Why 1%, 3% or 4% chitosan was not tested in this study?

Response: Thank you for this important comment. The present study was designed to evaluate the biological performance of chitosan nanoparticles using a single, well-established concentration rather than to perform a concentration-dependent comparison.

The 2% concentration was selected based on previous evidence demonstrating favorable biocompatibility and minimal cytotoxicity, particularly with respect to stem cell viability. It is also well recognized that the efficiency of chitosan as a chelating agent is influenced by multiple factors, including concentration, pH, and application conditions, and that increasing concentration can alter its physicochemical behavior and biological performance .

Therefore, introducing additional concentrations (e.g., 1%, 3%, and 4%) would have introduced multiple variables affecting viscosity, chelating efficiency, and nanoparticle stability, which was beyond the scope of the current study. Future studies may further investigate different concentrations to establish a dose–response relationship and optimize the formulation.

Location of revision:

• Discussion section
• Page: 11
• Paragraph: 4
• Lines: 486-488

Added  The present study evaluated a single concentration to standardize experimental conditions, while acknowledging that concentration-dependent effects may influence the physicochemical and biological behavior of chitosan.

Comment 3. Chitosan is soluble in acetic acid. Will it have any outcome on the human studies if extrapolated?

Response: Thank you for this important comment. Chitosan is dissolved in dilute acetic acid for solubilization; however, during nanoparticle preparation via ionotropic gelation, residual acetic acid is minimized through electrostatic crosslinking with tripolyphosphate. In addition, the irrigation protocol includes a final rinsing step, further reducing any residual acid. Chitosan is well known for its excellent biocompatibility and low toxicity, and the minimal acetic acid concentration used is not expected to cause adverse biological effects. Nevertheless, further in vivo studies are recommended to confirm clinical safety.

Location of revision: This technique is based on electrostatic interaction between the positively charged amino groups of chitosan and the negatively charged phosphate groups of TPP, resulting in nanoparticle formation,     and the added part is  while minimizing residual acetic acid through ionic crosslinking.

Location of revision

• Materials and Methods, Section 2.4
• Page: 4
• Paragraph: 1
• Lines: 153-154

Location of revision:

• Discussion section
• Page: 12
• Paragraph: 3
• Lines: 527-530

Added  In addition, although chitosan nanoparticles were prepared using dilute acetic acid, the preparation method and subsequent irrigation protocol are expected to minimize residual effects; however, further in vivo and clinical studies are needed to confirm the safety of this formulation in human applications.

Comment 4. Why dog was chosen over other species. Need more clarity? need to be added to the manuscript.

Response: Thank you for this valuable comment. Dogs were selected as an experimental model because their immature teeth exhibit anatomical and biological characteristics comparable to human immature permanent teeth. The physiological stages of root maturation and the inflammatory response to periapical infection closely mirror the conditions encountered in human pediatric patients. In addition, canine teeth demonstrate similar apical healing patterns, but over a shorter time frame due to their higher growth rate, allowing efficient evaluation of regenerative outcomes. Their relatively large tooth size facilitates endodontic procedures and provides adequate access for standardized experimental manipulation. Furthermore, the presence of multiple teeth within the same animal allows for adequate sample size while minimizing the number of animals required. The paragraph was edited and the required information was added.

Location of revision:

• Discussion section
• page 11
• lines 491– 501

Comment 5. Predominantly images are shown. Images can be explained in detail along with the instrumentation used to obtain these images.

Response: We appreciate this valuable suggestion. The imaging methodology has been clarified in the Materials and Methods section (Section 2.6). Specifically, histological sections were examined using a light microscope (Olympus BX51, Olympus Corp., Tokyo, Japan) at ×200 magnification, and representative images were captured using a digital camera system attached to the microscope.

Location of revision:

• Materials and Methods, Section 2.6
• Page 5
• first paragraph
• Lines 237–239

Comment 6. Any comparative studies on similar material tested in dog and later used for human treatment can be included in the discussion section.

Response: Thank you for this valuable suggestion. A statement has been added to the Discussion section to highlight the translational relevance of canine models in regenerative endodontics, including examples of biomaterials and procedures that have been successfully translated from animal studies to clinical human applications.

(Preclinical studies using canine models have historically played an important role in the development and validation of regenerative endodontic procedures and biomaterials prior to clinical application in humans. Several regenerative strategies, including revascularization protocols and bioactive materials such as MTA and scaffold-based approaches, were initially evaluated in animal models and subsequently translated into clinical practice with favorable outcomes. These findings support the relevance of the canine model as a reliable platform for assessing the biological performance and clinical potential of novel regenerative materials).

Location of revision:

• Discussion section
• Page: 11,12
• Last paragraph
• Lines: 502-509

Comment 7. Stability of the chitosan-based material and its shelf life as well as stability in the biological matrix for a certain period of time will be crucial. How authors will address this and extend it to other higher species can be captured in conclusion and future perspectives.

Response: Thank you for this valuable comment. The Conclusion section has been revised to address the importance of evaluating the physicochemical stability, shelf life, and behavior of chitosan nanoparticles within the biological environment, as well as the need for further validation in higher animal models and clinical human applications.

(Further studies with longer follow-up periods are required to evaluate the physicochemical stability, shelf life, and behavior of chitosan nanoparticles within the biological environment, as well as to validate their safety and efficacy in higher animal models and clinical human applications)

Location of revision:

• Conclusion section
• Page 12
• Conclusion paragraph
• Lines 554–557

Comment 8. How the developed material will be better than any of the existing material used for treatment can be detailed.

Response: Thank you for this valuable comment. A clarifying statement has been added to the Discussion section to highlight the potential advantages of chitosan nanoparticles compared with conventional irrigants, emphasizing their combined antimicrobial activity, enhanced penetration, biocompatibility, and ability to support extracellular matrix organization, thereby promoting a more favorable microenvironment for regenerative endodontic procedures.

(The improved performance of chitosan nanoparticles compared with conventional irrigants may be attributed to their combined antimicrobial activity, enhanced penetration, biocompatibility, and ability to support extracellular matrix organization, thereby creating a more favorable microenvironment for regenerative endodontic procedures).

Location of revision:

• Discussion section
• Page: 12
• Paragraph: 3
• Lines: 539-543

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents an in vivo evaluation of 2% chitosan nanoparticles as an irrigant in regenerative endodontic therapy. The topic is timely and relevant within the field of bioactive nanomaterials for dental regeneration.

The study is well-designed, clearly structured, and the histological and histomorphometry findings are coherent and support the authors’ conclusions. Overall, the work provides meaningful experimental evidence and contributes to the growing interest in biologically driven irrigation strategies.

Some clarifications and additions are suggested to further strengthen the manuscript.

While the preparation method is clearly described, the inclusion or brief discussion of key physicochemical parameters of the nanoparticles (e.g., particle size distribution, surface charge) would improve reproducibility and help contextualize the biological outcomes.

The rationale for selecting the 2% chitosan nanoparticle concentration could also be briefly clarified.

The results convincingly demonstrate improved tissue organization and collagen deposition. However, it may be appropriate to slightly moderate the terminology related to “regeneration,” emphasizing that the findings primarily reflect enhanced reparative tissue formation within the investigated time frame.

A brief clarification on whether samples derived from the same animal were treated as independent observations would further strengthen methodological transparency.

Finally, minor typographical and formatting inconsistencies (e.g., encoding artifacts such as “beĴer”) should be corrected, and figure labeling and readability (Figures 1–2) could be slightly improved.

The discussion could be further enriched by briefly contextualizing the findings within the broader framework of bioactive material design for tissue regeneration. In particular, the authors may consider highlighting how both material composition (e.g., incorporation of bioactive inorganic phases, https://doi.org/10.3390/polym12010037) and structural design (e.g., architected hybrid scaffolds, https://doi.org/10.1021/acsabm.5c00561) can synergistically influence antimicrobial performance, cell–material interactions, and overall tissue integration. This perspective would further support the rationale of using bioactive nanosystems to optimize the regenerative microenvironment. The manuscript is well conducted and suitable for publication after minor revisions aimed at improving clarity and contextualization.

Author Response

Comment 1: While the preparation method is clearly described, the inclusion or brief discussion of key physicochemical parameters of the nanoparticles (e.g., particle size distribution, surface charge) would improve reproducibility.

Response: We sincerely thank the reviewer for the positive and encouraging feedback. We appreciate the thoughtful evaluation of our work. Additionally, we thank the reviewer for this valuable suggestion. The manuscript has been revised to include key physicochemical characteristics of chitosan nanoparticles, including their nanoscale dimensions, positive surface charge, and high surface area-to-volume ratio, which are relevant to their biological interactions and antimicrobial performance.

Added  Chitosan NPs produced via ionic gelation possess nanoscale dimensions (1–100 nm) and a positive surface charge derived from protonated amino groups, while their high surface area-to-volume ratio and charge characteristics enhance their interaction with microbial cell walls and biological tissues, contributing to their antimicrobial and bioactive performance

Location of revision

• Materials and Methods, Section 2.4
• Page: 4
• Paragraph: 1
• Lines: 154-158

Comment 2. The rationale for selecting the 2% chitosan nanoparticle concentration could also be briefly clarified.

Response: Thank you for this insightful comment. The selection of a 2% chitosan solution was based on its favorable balance between physicochemical properties and biological performance. Chitosan is a biocompatible and biodegradable natural polymer with low toxicity and excellent drug delivery characteristics, making it highly suitable for biomedical applications. Its free amine groups enable ionic interactions and crosslinking, which are essential for nanoparticle formation and structural stability. Importantly, the 2% concentration provides an optimal balance between viscosity, mechanical stability, ease of fabrication, and biocompatibility. Previous evidence has demonstrated that this concentration maintains favorable cellular responses, particularly with respect to periodontal ligament stem cells, indicating its suitability for regenerative applications.

Accordingly, the 2% concentration was selected as a clinically relevant and biologically favorable formulation. This rationale has now been clarified in the Materials and Methods section (page 4, lines 159–163), and 2 Reference 24,25 has been added to support this justification and the other refferences adjusted accordingly.

Location of revision: A 2% chitosan NPs concentration was selected based on previous evidence demonstrating a favorable balance between antimicrobial activity, biocompatibility, and stem cell viability. Chitosan nanoparticles prepared by this method are typically characterized by nanoscale size and a positive surface charge, facilitating interaction with negatively charged bacterial cell walls.

Comment 3. The results convincingly demonstrate improved tissue organization and collagen deposition. However, it may be appropriate to slightly moderate the terminology related to “regeneration,” emphasizing that the findings primarily reflect enhanced reparative tissue formation within the investigated time frame.

 Response: Thank you for this insightful comment. We agree that the term “regeneration” should be interpreted cautiously within the context of the present experimental timeframe. Accordingly, the manuscript has been revised to moderate the terminology and to emphasize that the findings primarily reflect enhanced reparative tissue formation rather than complete functional pulp regeneration.

Revised Manuscript Text:

Added  Therefore, the present findings should be interpreted as indicative of enhanced reparative tissue formation rather than complete functional pulp regeneration. The higher collagen content in the chitosan group, when interpreted together with the histologic findings, supports the conclusion that chitosan NPs promoted more advanced connective tissue remodeling and a more organized regenerative response.

And more advanced pulp-like tissue regeneration changes to more advanced pulp-like tissue formation

Location of revision:

• Discussion section
• Page: 10, 6
• Paragraph: 4
• Lines: 430-435, 272

Comment 4. A brief clarification on whether samples derived from the same animal were treated as independent observations would further strengthen methodological transparency.

Response: Thank you for this important comment. A clarification has been added in the Materials and Methods section to indicate that, although multiple samples were obtained from the same animal, teeth were randomly allocated and treated as independent observations, in line with commonly adopted designs in similar preclinical studies.

Although multiple samples were obtained from the same animal, all teeth were randomly assigned to experimental groups and treated as independent observations, consistent with commonly used experimental designs in similar preclinical endodontic studies.

Location of revision:

• Material and methods section 2.3
• Page: 4
• Paragraph: 3
• Lines: 145-148

Comment 5. Finally, minor typographical and formatting inconsistencies (e.g., encoding artifacts such as “beĴer”) should be corrected, and figure labeling and readability (Figures 1–2) could be slightly improved.

Response: Thank you for this helpful comment. All typographical and formatting inconsistencies, including encoding artifacts, have been carefully reviewed and corrected throughout the manuscript. In addition, figure labeling and readability (Figures 1–2) have been improved by revising the figure legends to provide clearer and more descriptive explanations of the histological findings.

In figure 1

Group II displayed pulp-like tissue characterized by irregularly arranged collagen fibers, increased vascularization, and early predentin formation

 

In figure 2

Group II revealed more advanced pulp-like tissue with regularly arranged collagen fibers, increased vascularization, and the presence of odontoblast-like cells associated with predentin deposition

Location of revision:

• Results section
• Page: 7,8
• Paragraph: 1
• Lines: 319-320, 367-368

Comment 6. The discussion could be further enriched by briefly contextualizing the findings within the broader framework of bioactive material design for tissue regeneration...

Response: Thank you for this helpful suggestion. A brief contextualization has been added in the Discussion section to relate the present findings to previous studies demonstrating the favorable biological effects of chitosan-based irrigation systems on stem cell viability and proliferation.

 

Added  These findings are consistent with previous studies demonstrating that chitosan-based irrigation systems promote stem cell viability and proliferation while maintaining a favorable biological microenvironment for regenerative endodontic procedures. A reffernce was added to support the argument

Location of revision:

• Discussion section
• Page: 12
• Paragraph: 2
• Lines: 445-450

 

We are grateful for the reviewers’ thoughtful feedback, which has helped us improve the scientific clarity, methodological transparency, and translational framing of the manuscript. We believe the revisions have strengthened the paper substantially, and we remain open to any further suggestions that may enhance its quality and suitability for publication.

 

Sincerely,

Dr. Safwat Elwaseef, DDS, MSc, PhD.

University of Rochester, New York, USA

On behalf of all co-authors