PONE-D-24-55861Early cellular and molecular events of osteomucosal healing in the tooth extraction socketPLOS ONE

Dear Dr. Kim,

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

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Academic Editor

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Additional Editor Comments:

Dear Dr. Kim,

Thank you for submitting your manuscript to PLOS One. Your paper is undoubtedly interesting and raises important points that I believe will be highly appreciated in the field. However, the majority of the reviewers have raised some significant concerns about the paper that need to be addressed before it can be considered for publication.

My considerations focus on the Methods section, addressing specific points such as:

- The authors did not present the ARRIVE guidelines.

- The sample size was not justified.

- Details regarding histochemical staining need clarification—specifically, describe the TRAP immunohistochemistry.

- Some methodology is repeated in the Results section.

- Trichrome staining is not included in the Methods section. (Also, which type of Trichrome staining was used? Masson's?)

- Please explain how osteoclast coating was performed.

- Lastly, please include a clear discussion of the limitations of your study.

I look forward to receiving your revised manuscript.

Best regards,

Dr Henrique Hadad, DDS, PhD

Academic Editor - PLOS One

Reviewers' comments:

Reviewer #1:

This is a nice study with an easy-to-read flow, that gathers attention from the reader.

Minor questions and suggestions should be assessed by the authors:

1) When the authors cite MRONJ in the discussion panel, it should be discussed with another statement " The early presence of osteoclasts allows for bone resorption at the existing bone surface and triggers osteoclast-osteoblast coupling to stimulate new bone formation".

2) The authors should discuss the timing of socket cicatrization. It is interesting and not well addressed in the literature how soft tissue and hard tissue heal at different paces but a similar finish line.

3) This is a great study, with interesting results. Image 06 does not represent this study, but a grade school paperwork. The authors should present a professional Graphic for Image 06.

Reviewer #2:

Comments to the author

The manuscript is focused in evaluating epithelial regeneration, connective tissue proliferation and bone formation over the first week following tooth extraction in mice. The authors claim this is unprecedented data, however, the cascade of events occurring during socket healing is well documented. Maybe, their design (consecutive euthanasia over the week) might have added information, but the authors failed to make clear which gap was filled by their data.

Besides this limitation that should be addressed, some other major changes need to be done prior to re-consideration for publication, once the authors succeed in

ABASTRACT

Use the background to state the singularity of osteomucosal healing and exactly which information is been added by your research. “Early events” is vague.

INTRODUCTION

The rationale is currently based in distinct healing across tissues. Instead, the introduction should focus on the unknown events in osteomucosal healing and plausible crosstalk between the events occurring within the socket.

MATERIALS AND METHODS

Did you perform sample size calculation? Based on most literature of socket healing in mice, N=3 seems not sufficient to assure relevance.

You mention that both right and left first molars were extracted, which is different from the abstract. Which one is correct?

Describe how you defined the region of interest and the method of analysis for the microCT.

Mason’s Trichrome was only mentioned in the results and the information is lacking in the M&M.

The method of analysis for all outcome parameters was neglected or poorly described. An extensive revision on that is necessary.

RESULTS

The clinical analysis should focus on wound closure only, instead of describing tissues. Epithelium can be appreciated in the histological slides.

The word “matured” should be changed to proper histologic terminology.

The figures are shown in poor quality, hindering a proper appreciation of their representativeness. Additionally, they are presented in low magnification, which compromises a proper visualization of the tissue events described in the text.

The IHC is also shown in low magnification. I understand that the labeling is not confined to the cytosolic compartment since these proteins exert their function when secreted in the extracellular matrix. However, higher magnification images are necessary to identify what is antigen-antibody labeling and what is background labeling.

Discussion

Overall, the discussion is missing comprehensive understanding of the data shown by the authors. Also, the authors could explore the sequential (and overlapping) events trying to establish a spatial relevance of uneventful osteomucosal healing.

Conclusion

A proper conclusion based on the results should be drawn.

Reviewer #3:

The proposed paper details the first week of alveolar repair in daily analyses. A very interesting study that will certainly support studies regarding interferences in the natural course.

The objectives are clear and the methodology is consolidated in the literature in relation to alveolar repair.

It precisely details the role of osteoclasts in the early stages after tooth extraction, which is well explored in the discussion as possible indicators for studies of the mechanisms of MRONJ.

Based on the analysis of the study, I suggest some small changes:

1. There is a tendency in the results section to repeat some points from the methods, for example:

- “To investigate the kinetics of collagen formation and deposition following tooth extraction, we stained the sections with Trichrome, a maker of collagens in general…”

- “To investigate the kinetics of collagen formation and deposition following tooth extraction, we stained the sections with Trichrome, a maker of collagens in general..”

I suggest that these points be part of the method only and not repeated in the results section.

2. Regarding Figure 6: although it seems obvious that the markings on the horizontal axis refer to the 7 days studied, I suggest that the numbers of the days to which the markings refer be written. In the same way, markings be added to the vertical axis.

3. In the caption of Figure 1, I suggest removing "N = 6 tooth

extraction sites with 3 mice per group.", which refers to the method and is already very clear in the text.

Reviewer #4:

Kim et al. presented an original study investigating early cellular and molecular events in osteomucosal healing in a mouse model. This study provides a new understanding of collagen deposition, osteoclast activity, and remodeling. The results have clear clinical implications for MRONJ and dry sockets using advanced imaging and staining techniques. Overall, the manuscript provides valuable contributions to the understanding of osteomucosal healing. Its clarity and impact will be further enhanced by addressing the following suggestions.

Abstract: While the abstract is informative, it lacks brief details on the materials and methods used. Including this information would improve clarity and help readers understand how the results were obtained.

Suggestions for improvement:

1. Although the figures and results are robust, the discussion could be more explicit in linking the results to potential future studies on clinical implications, such as MRONJ or dry socket.

2. The narrative would be streamlined, and readability would be improved by reducing redundancy in the introduction and discussion.

3. This study did not use additional molecular methods, such as qRT-PCR for gene expression analysis, that could have deepened the findings.

4. Potential limitations, such as differences in healing mechanisms between mice and humans, should be clarified. This would better contextualize the findings.

Reviewer #5:

I was pleased to review this manuscript. The initial stages of healing in fresh sockets remain highly important and intriguing. However, the paper lacks adequate support from the existing literature, despite the availability of a substantial works on the topic that could better support the study.

I recommend major review based on the following comments:

Abstract: I suggest restructuring the abstract, as the study design is not well described, nor are the results and conclusion clearly presented. Additionally, consider adding keywords to improve the visibility of your study in database searches.

Introduction: The introduction lacks sufficient references to support the context and rationale of the study. While the aim is stated, there is no clear hypothesis. What did the authors expect to find or address within this gap?

Methods: Methodologically, this is essentially a result based on histomorphometric parameters, as you did not explore deeply into gene and protein expression in the samples. It is important to strengthen what is being presented. Alternatively, you could change the approach to frame it as a pilot study investigating the initial events of alveolar remodeling.

Did the authors perform a sample size calculation? Considering that the number of animals per time point is very low, with only three animals per period, this would not provide sufficient statistical confidence, even with the use of a split-mouth design. doi: 10.4103/0976-500X.119726

"Tooth-extraction mouse model" : What instrument was used for the atraumatic extraction? If your article is published, it must be reproducible, allowing other researchers to replicate the study. Additionally, others may draw inspiration from your work for future studies.

μCT Scan: The parameters evaluated are not described, nor are they presented in the results. I suggest using parameters from the literature, such as those described in the study by Viera (2015), as cited by the authors.

Histochemical Staining: Improve the description, there is no cited eosin staining with hematoxilin.

Immunohistochemical staining: Why did the authors use only OPG and RANKL markers? In the early stages, there are many other important markers that could have been investigated, such as osteopontin, osteocalcin, BSP, and Runx2.

Statistical Analysis: What do the authors mean by "To compare the group differences" if only one group is being analyzed at different time points? Why were so many significance factors used? Since you mentioned that the p-value is < 0.05, the other p-values shown are already within this range.

RESULTS: In general, the results are not well presented, especially in the description. They are confusing, and it is not possible to correlate them with what is being shown in the images, which also lack sufficient resolution for the message the author intends to convey to the readers. I suggest using images with higher magnification.

For example: "By day 3, roughened bone surfaces were observed in the tooth-extracted sockets, suggesting that bone remodeling had started through bone resorption. As a result of this bone remodeling, the sharp edge of the buccal wall margin of the extraction socket became rounded by day 4." This cannot be visualized properly. I suggest using arrows or asterisks to highlight what you would like to show in the images. Once again, the study cited in your references by Vieira is well illustrated in terms of the images.

Woven bone formation: You have access to a high-resolution 3D imaging tool, but it is not being used to its full potential. I suggest utilizing better evaluation parameters, as it was not described how the region of interest (ROI) was selected. Once again, I recommend referring to the studies by Vieira and Bouxsein (2010) DOI: 10.1002/jbmr.141 for better guidance.

Osteoclast formation: I am unable to visualize the osteoclasts present. While we know how the histological sections were made and their positioning, I suggest providing images with higher magnification specifically of the representative areas where these cell types need to be visualized.

Discussion: The discussion is very bold and poorly supported by the literature. Many statements are not backed by existing references. Additionally, it is confusing, as it moves back and forth on the same topic across several paragraphs. I suggest a thorough rereading and restructuring to improve clarity and coherence.

"Therefore, these observations indicate that new bone formation in the tooth-extraction socket occurs in two ways: 1) osteoclast-dependent bone formation from the existing bone surface via osteoclast-osteoblast coupling, and 2) osteoclast-independent de novo bone formation via intramembranous ossification." - How can you strongly affirm this if your sample size is low, and you have only evaluated histomorphometric parameters? Such imply may require more robust data, including gene and protein expression analysis, and a larger sample size to provide stronger evidence.

"The reason for this preferential presence of osteoclasts is presently unclear; however, it seems intuitive that nature has evolved to prevent osteoclasts from targeting the newly forming bone during the healing process to increase the efficiency of new bone formation." - If it is unclear, how can you suggest this information? Based on what evidence or reasoning is this claim being made? It is important to provide solid references or data to support such hypotheses, especially when the mechanism is not yet fully understood.

The conclusion is not clear, and the text ends abruptly with a paragraph that seems to suggest continuity, followed by another strong, unreferenced statement: "Unlike other parts of the body, the healing of the soft and hard tissues in the oral cavity often occurs simultaneously after a traumatic event such as tooth extraction." A conclusion needs to be well-defined and should summarize the key findings in a clear and coherent manner, avoiding unsupported or vague statements.

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

5. Review Comments to the Author

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

Reviewer #1: This is a nice study with an easy-to-read flow, that gathers attention from the reader.

Minor questions and suggestions should be assessed by the authors:

1) When the authors cite MRONJ in the discussion panel, it should be discussed with another statement " The early presence of osteoclasts allows for bone resorption at the existing bone surface and triggers osteoclast-osteoblast coupling to stimulate new bone formation".

2) The authors should discuss the timing of socket cicatrization. It is interesting and not well addressed in the literature how soft tissue and hard tissue heal at different paces but a similar finish line.

3) This is a great study, with interesting results. Image 06 does not represent this study, but a grade school paperwork. The authors should present a professional Graphic for Image 06.

Reviewer #2: Comments to the author

The manuscript is focused in evaluating epithelial regeneration, connective tissue proliferation and bone formation over the first week following tooth extraction in mice. The authors claim this is unprecedented data, however, the cascade of events occurring during socket healing is well documented. Maybe, their design (consecutive euthanasia over the week) might have added information, but the authors failed to make clear which gap was filled by their data.

Besides this limitation that should be addressed, some other major changes need to be done prior to re-consideration for publication, once the authors succeed in

ABASTRACT

Use the background to state the singularity of osteomucosal healing and exactly which information is been added by your research. “Early events” is vague.

INTRODUCTION

The rationale is currently based in distinct healing across tissues. Instead, the introduction should focus on the unknown events in osteomucosal healing and plausible crosstalk between the events occurring within the socket.

MATERIALS AND METHODS

Did you perform sample size calculation? Based on most literature of socket healing in mice, N=3 seems not sufficient to assure relevance.

You mention that both right and left first molars were extracted, which is different from the abstract. Which one is correct?

Describe how you defined the region of interest and the method of analysis for the microCT.

Mason’s Trichrome was only mentioned in the results and the information is lacking in the M&M.

The method of analysis for all outcome parameters was neglected or poorly described. An extensive revision on that is necessary.

RESULTS

The clinical analysis should focus on wound closure only, instead of describing tissues. Epithelium can be appreciated in the histological slides.

The word “matured” should be changed to proper histologic terminology.

The figures are shown in poor quality, hindering a proper appreciation of their representativeness. Additionally, they are presented in low magnification, which compromises a proper visualization of the tissue events described in the text.

The IHC is also shown in low magnification. I understand that the labeling is not confined to the cytosolic compartment since these proteins exert their function when secreted in the extracellular matrix. However, higher magnification images are necessary to identify what is antigen-antibody labeling and what is background labeling.

Discussion

Overall, the discussion is missing comprehensive understanding of the data shown by the authors. Also, the authors could explore the sequential (and overlapping) events trying to establish a spatial relevance of uneventful osteomucosal healing.

Conclusion

A proper conclusion based on the results should be drawn.

Reviewer #3: The proposed paper details the first week of alveolar repair in daily analyses. A very interesting study that will certainly support studies regarding interferences in the natural course.

The objectives are clear and the methodology is consolidated in the literature in relation to alveolar repair.

It precisely details the role of osteoclasts in the early stages after tooth extraction, which is well explored in the discussion as possible indicators for studies of the mechanisms of MRONJ.

Based on the analysis of the study, I suggest some small changes:

1. There is a tendency in the results section to repeat some points from the methods, for example:

- “To investigate the kinetics of collagen formation and deposition following tooth extraction, we stained the sections with Trichrome, a maker of collagens in general…”

- “To investigate the kinetics of collagen formation and deposition following tooth extraction, we stained the sections with Trichrome, a maker of collagens in general..”

I suggest that these points be part of the method only and not repeated in the results section.

2. Regarding Figure 6: although it seems obvious that the markings on the horizontal axis refer to the 7 days studied, I suggest that the numbers of the days to which the markings refer be written. In the same way, markings be added to the vertical axis.

3. In the caption of Figure 1, I suggest removing "N = 6 tooth

extraction sites with 3 mice per group.", which refers to the method and is already very clear in the text.

Reviewer #4: Kim et al. presented an original study investigating early cellular and molecular events in osteomucosal healing in a mouse model. This study provides a new understanding of collagen deposition, osteoclast activity, and remodeling. The results have clear clinical implications for MRONJ and dry sockets using advanced imaging and staining techniques. Overall, the manuscript provides valuable contributions to the understanding of osteomucosal healing. Its clarity and impact will be further enhanced by addressing the following suggestions.

Abstract: While the abstract is informative, it lacks brief details on the materials and methods used. Including this information would improve clarity and help readers understand how the results were obtained.

Suggestions for improvement:

1. Although the figures and results are robust, the discussion could be more explicit in linking the results to potential future studies on clinical implications, such as MRONJ or dry socket.

2. The narrative would be streamlined, and readability would be improved by reducing redundancy in the introduction and discussion.

3. This study did not use additional molecular methods, such as qRT-PCR for gene expression analysis, that could have deepened the findings.

4. Potential limitations, such as differences in healing mechanisms between mice and humans, should be clarified. This would better contextualize the findings.

Reviewer #5: I was pleased to review this manuscript. The initial stages of healing in fresh sockets remain highly important and intriguing. However, the paper lacks adequate support from the existing literature, despite the availability of a substantial works on the topic that could better support the study.

I recommend major review based on the following comments:

Abstract: I suggest restructuring the abstract, as the study design is not well described, nor are the results and conclusion clearly presented. Additionally, consider adding keywords to improve the visibility of your study in database searches.

Introduction: The introduction lacks sufficient references to support the context and rationale of the study. While the aim is stated, there is no clear hypothesis. What did the authors expect to find or address within this gap?

Methods: Methodologically, this is essentially a result based on histomorphometric parameters, as you did not explore deeply into gene and protein expression in the samples. It is important to strengthen what is being presented. Alternatively, you could change the approach to frame it as a pilot study investigating the initial events of alveolar remodeling.

Did the authors perform a sample size calculation? Considering that the number of animals per time point is very low, with only three animals per period, this would not provide sufficient statistical confidence, even with the use of a split-mouth design. doi: 10.4103/0976-500X.119726

"Tooth-extraction mouse model" : What instrument was used for the atraumatic extraction? If your article is published, it must be reproducible, allowing other researchers to replicate the study. Additionally, others may draw inspiration from your work for future studies.

μCT Scan: The parameters evaluated are not described, nor are they presented in the results. I suggest using parameters from the literature, such as those described in the study by Viera (2015), as cited by the authors.

Histochemical Staining: Improve the description, there is no cited eosin staining with hematoxilin.

Immunohistochemical staining: Why did the authors use only OPG and RANKL markers? In the early stages, there are many other important markers that could have been investigated, such as osteopontin, osteocalcin, BSP, and Runx2.

Statistical Analysis: What do the authors mean by "To compare the group differences" if only one group is being analyzed at different time points? Why were so many significance factors used? Since you mentioned that the p-value is < 0.05, the other p-values shown are already within this range.

RESULTS: In general, the results are not well presented, especially in the description. They are confusing, and it is not possible to correlate them with what is being shown in the images, which also lack sufficient resolution for the message the author intends to convey to the readers. I suggest using images with higher magnification.

For example: "By day 3, roughened bone surfaces were observed in the tooth-extracted sockets, suggesting that bone remodeling had started through bone resorption. As a result of this bone remodeling, the sharp edge of the buccal wall margin of the extraction socket became rounded by day 4." This cannot be visualized properly. I suggest using arrows or asterisks to highlight what you would like to show in the images. Once again, the study cited in your references by Vieira is well illustrated in terms of the images.

Woven bone formation: You have access to a high-resolution 3D imaging tool, but it is not being used to its full potential. I suggest utilizing better evaluation parameters, as it was not described how the region of interest (ROI) was selected. Once again, I recommend referring to the studies by Vieira and Bouxsein (2010) DOI: 10.1002/jbmr.141 for better guidance.

Osteoclast formation: I am unable to visualize the osteoclasts present. While we know how the histological sections were made and their positioning, I suggest providing images with higher magnification specifically of the representative areas where these cell types need to be visualized.

Discussion: The discussion is very bold and poorly supported by the literature. Many statements are not backed by existing references. Additionally, it is confusing, as it moves back and forth on the same topic across several paragraphs. I suggest a thorough rereading and restructuring to improve clarity and coherence.

"Therefore, these observations indicate that new bone formation in the tooth-extraction socket occurs in two ways: 1) osteoclast-dependent bone formation from the existing bone surface via osteoclast-osteoblast coupling, and 2) osteoclast-independent de novo bone formation via intramembranous ossification." - How can you strongly affirm this if your sample size is low, and you have only evaluated histomorphometric parameters? Such imply may require more robust data, including gene and protein expression analysis, and a larger sample size to provide stronger evidence.

"The reason for this preferential presence of osteoclasts is presently unclear; however, it seems intuitive that nature has evolved to prevent osteoclasts from targeting the newly forming bone during the healing process to increase the efficiency of new bone formation." - If it is unclear, how can you suggest this information? Based on what evidence or reasoning is this claim being made? It is important to provide solid references or data to support such hypotheses, especially when the mechanism is not yet fully understood.

The conclusion is not clear, and the text ends abruptly with a paragraph that seems to suggest continuity, followed by another strong, unreferenced statement: "Unlike other parts of the body, the healing of the soft and hard tissues in the oral cavity often occurs simultaneously after a traumatic event such as tooth extraction." A conclusion needs to be well-defined and should summarize the key findings in a clear and coherent manner, avoiding unsupported or vague statements.

**********

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Reviewer #1: Yes: Samuel Macedo Costa

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes: Avneesh Chopra

Reviewer #5: No

**********

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<div>PONE-D-24-55861R1Early cellular events of osteomucosal healing in the tooth extraction socketPLOS ONE

Dear Dr. Kim,

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

Please submit your revised manuscript by Jun 07 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Henrique Hadad, DDS, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments:

Dear Authors,

Thank you for your submission. I believe your manuscript presents an interesting contribution to the field. However, I remain concerned about the Methodology section and certain aspects of the Results.

Please find attached the reviewers' comments for your consideration. Both reviewers expressed concern that the manuscript does not adequately address the stated aim of the study, noting that it "does not provide sufficient depth to fully elucidate the regulatory mechanisms underlying socket healing."

I strongly encourage you to address these points thoroughly and revise your manuscript accordingly before resubmitting it for further evaluation.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #2: (No Response)

Reviewer #6: (No Response)

Reviewer #7: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

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

Reviewer #2: Yes

Reviewer #6: Partly

Reviewer #7: Partly

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

Reviewer #2: Yes

Reviewer #6: Yes

Reviewer #7: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #6: Yes

Reviewer #7: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #6: Yes

Reviewer #7: (No Response)

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6. Review Comments to the Author

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

Reviewer #2: The authors have implemented significant revisions to the manuscript, notably enhancing its quality. Among the previous comments provided by this reviewer, the authors have neglected to address one particular issue related to the methods of analysis for the histological and histochemical data. It is recommended that the authors include the detailed methods of analysis for each of the outcome measures evaluated, not limited to microCT.

Although the manuscript is written in formal and intelligible English, a comprehensive revision is necessary to resolve the grammatical inconsistencies that have emerged during the review process.

Reviewer #6: The authors set out to investigate the spatial and temporal changes in epithelial, connective and bone tissues, in addition to the presence of osteoclasts during the early stages of osteomucosal healing.

I have seen that this article has already been reviewed by extremely meticulous reviewers, which has certainly made it easier for the current reviewer to make his considerations.

First of all, I would like to point out that the main objective, which was to investigate spatial and temporal changes in epithelial, connective and bone tissues, was not fully achieved. Epithelial tissue was not included in either the methodology or the results. Information on epithelial proliferation and maturation of epithelial layers with keratin formation was not analysed. Immunohistochemistry for epithelial markers such as CKPool and EGFR would have enriched the study. In addition, temporal changes were only made between connective tissue and new bone formation.

Materials and Methods

The authors said: “Atraumatic extraction of the maxillary first molars (M1) was carried out”. However, rodent maxillary first molars have a variable number of roots and are very thin. This reviewer is curious to know whether root fractures were found. This is important information to clarify for the reader.

Immunohistochemical staining

It is important to describe the immunohistochemical evaluation parameters, including the cellular localisation of expression of the two antibodies evaluated. RANK-L is expressed at the cell membrane and in the cytoplasm. Do the authors take this location into account when scoring the samples? What about the anti-osteoprotegerin antibody? It is usually secreted.

Results

Figure 1(D) Day 3 - I would suggest revising the description of the histological findings. It is possible to see a proliferation of spindle cells at a higher magnification, and this data is important for the healing process.

Figure 1(D) Day 4 - I suggest replacement of the microphotograph from this day, as the histological aspects are worse in the healing process than on day 3. Do the basophilic areas suggest necrosis or the accumulation of polymorphonuclear neutrophils cells?

Figure 1(D) Day 5 - This photomicrograph shows the proliferative pattern of spindle cells. However, there are a lot of wood shavings remaining in the upper field, which could hinder the healing process. Do the authors have another image with no wood shavings to replace it?

Figure 1(D) Days 6 and 7 were good.

With regard to Figure 2, this reviewer did not see any temporal changes in the alveoli, with Mason's trichrome staining in terms of new bone formation, except on days 6 and 7.

On the other hand, PS Red staining, with and without polarised light, did not contribute to the analysis and actually exacerbated the visualisation of wood shavings within the alveoli on days 4 and 5.

The durability of this set of micrographs should be re-evaluated.

The authors describe this in the Results section: “Oral mucosal tissue closure following tooth extraction in mice”, “By day 1, the blood clot was replaced by a granulation tissue that seemingly consisted of three distinct layers: the residual coagulum layer in the apical portion, the loose provisional matrix layer in the middle of the socket, and the neo-epithelial layers in the coronal portion.”

In pathology, granulation tissue is a vascular connective tissue that forms in wounds during the healing process. It's a hallmark of wound healing and is characterised histologically by the presence and proliferation of fibroblasts, macrophages, keratinocytes, endothelial cells, new thin-walled capillaries and inflammatory cell infiltration of the extracellular matrix.

The presence of granulation tissue in a wound means that the inflammatory phase has passed and the proliferative phase has begun. It's important to remember that under normal conditions, wound healing and tissue repair occur in 4 stages: haemostasis, inflammatory stage, proliferative stage (when granulation tissue formation occurs) and remodelling stage.

Therefore, it is not possible to say that the blood clot has been replaced by granulation tissue on day 1.

Finally, this reviewer would like to know why the teeth in the tomographic and histological images have their roots in the lower part of the image and the crowns in the upper part, simulating teeth from the lower arch. Have the upper/maxillary first molars not been extracted? If so, the roots should be in the upper part of the images.

Please correct these words:

Such difference is largely owing to multiple layers of in-beween (in-between) anatomical structures such as muscle, fat, or fascia that separates soft and hard tissues.

…..analysis revealed that both collagen type I (yellow/organge (orange)/red) and III (green) followed a similar……

Reviewer #7: This revised manuscript investigates the early healing process in tooth extraction sockets. As the authors note, morphological analyses provide valuable insights into post-extraction healing. The selected methodologies are generally appropriate; however, the resolution and depth of the observations are limited, and as such, they fall short of offering substantial mechanistic insight into the healing process. Detailed comments are provided below.

1. Oral and Mucosal Tissue Closure Following Tooth Extraction in Mice (Figure 1)

The authors describe bone resorption mediated by osteoclasts; however, based on the current image resolution, osteoclasts cannot be clearly identified.

Figure 1D: The image size and resolution are insufficient to support the conclusions drawn in the results. Since these images are foundational to the study’s claims, they should be presented in higher resolution and at larger sizes to improve clarity and impact.

2. Collagen Deposition in the Tooth Extraction Sockets (Figure 2)

The rationale for this analysis, particularly the distinction between Type I and Type III collagen, needs to be explicitly stated. Additionally, a detailed explanation of the spatial distribution of these collagen types within the healing socket should be provided.

While Picrosirius Red staining under polarized light is commonly used to assess collagen types, it is not definitive. Therefore, confirmation via immunohistochemistry is necessary.

The image quality, especially under polarized microscopy, is not sufficient to clearly distinguish collagen types or their distribution. Larger, higher-resolution images are required to support the data.

3. Woven Bone Formation in the Tooth Extraction Sockets (Figure 3)

The purpose of this analysis should be clearly stated. In the discussion, the authors note the continuity between woven and pre-existing bone, and the woven bone is formed independently within the healing connective tissue. It is an important observation, and thus should be explicitly addressed here. Micro-CT 3D reconstruction could clarify whether distinct bone formation, such as appositional growth on existing bone surfaces versus de novo intramembranous ossification, is occurring. Additionally, further exploring these distinct processes associated with molecular regulation would greatly enhance the depth of the study.

In Panel A, the distobuccal (DB) socket appears to exhibit earlier woven bone formation compared to the mesial (M) socket. The authors should acknowledge this and analyze whether healing differs significantly between the two sites.

4. Osteoclast Formation in the Tooth Extraction Sockets (Figure 4)

To better differentiate between osteoclasts associated with newly formed versus pre-existing bone, magnified insets of these respective areas should be provided. This would allow clearer visualization of osteoclast localization relative to bone context.

5. Expression of RANKL and OPG in the Tooth Extraction Sockets (Figure 5)

Since RANKL is produced not only by osteoblasts but also by lymphocytes and stromal cells, the authors should carefully delineate the association of RANKL-positive staining with specific cell types and tissue structures.

To confirm the relationship between RANKL expression and osteoclast activity, double staining for RANKL and TRAP is recommended.

6. Overall Comments

The current dataset does not provide sufficient depth to fully elucidate the regulatory mechanisms underlying socket healing. Given the breadth of existing literature on bone wound healing, the authors should contextualize their findings with key molecular events known to occur during skeletal repair.

Moreover, tooth extraction results in disruption and torsion of collagen fibers from the periodontal ligament (PDL) that insert into the alveolar bone, likely causing structural changes to the bone surface. The mechanical load during tooth extraction may also affect osteocytes adjacent to the bone surface. Beyond the observed difference in the osteoclast distribution, the authors need to demonstrate the possible causes that could influence osteoclast recruitment and socket bone remodeling during the healing process. Investigating these structural and cellular dynamics could offer important insights into the interplay between immune responses, stromal cells, and osteoclastogenesis in the healing socket.

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

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #6: No

Reviewer #7: No

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Early cellular events of osteomucosal healing in the tooth extraction socket

PONE-D-24-55861R2

Dear Dr. Kim,

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

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Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #6: All comments have been addressed

Reviewer #7: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

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

Reviewer #6: Yes

Reviewer #7: Yes

**********

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

Reviewer #6: Yes

Reviewer #7: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #6: Yes

Reviewer #7: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #6: Yes

Reviewer #7: Yes

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6. Review Comments to the Author

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

Reviewer #6: After analyzing the updated version of the article, I found that it had been enhanced with more detailed and relevant information. Many suggestions made by different reviewers were considered and incorporated, resulting in a more substantial article. The current dataset may help clarify the mechanisms of dental alveolar healing, despite the lack of some analyses.

I believe that the manuscript meets the standards for publication of this prestigious scientific journal, but I would like to leave some comments for the authors.

I realize that I was unclear about the spatial and temporal aspects of changes in epithelial, connective, and bone tissues. I expected the authors to describe only the histological aspects of epithelial tissue during phases when collagen and bone tissue were deposited. This would provide readers with a spatial and temporal understanding of the relationship between these tissues in each analyzed phase. Besides reviewing the hematoxylin and eosin staining, no other methodology would be necessary.

Epithelial tissue is only mentioned on day four, when the authors wrote, "At the same time, epithelial layers began migrating toward the wound..." What about the other days? I believe it would be very interesting if the opportunity arises and there is enough time.

Reviewer #7: This revision addresses most of the reviewer’s concerns; however, the rationale and significance behind the respective evaluation of Type I and Type III collagens still require further clarification. In the current results and discussion, these two collagen types are collectively referred to simply as “collagens,” despite their known distinct roles in wound healing. Given that the authors specifically highlight Type I and Type III collagens using polarized light microscopy, it is important to explain the reasoning behind this approach and to interpret the findings accordingly. The discussion should elaborate on their respective spatial distributions, especially if distinct localization patterns were observed. A more detailed comparison of the similarities and differences between Type I and Type III collagens in the context of the healing process would enhance the depth and impact of this study.

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

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #6: No

Reviewer #7: No

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