PONE-D-22-18003Ternary regulation mechanism of Rhizoma drynariae total flavonoids on induced membrane formation and bone remodeling in Masquelet techniquePLOS ONE

Dear Dr. Li,

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 Sep 26 2022 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,

Selvaraj Vimalraj

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering.

3. Thank you for stating the following in your Competing Interests section:  

"The authors report no conflict of interest."

Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state ""The authors have declared that no competing interests exist."", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now 

 This information should be included in your cover letter; we will change the online submission form on your behalf.

4. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section.

Additional Editor Comments:

Carefully consider the reviewers' comments and suggested to submit a revised manuscript.

[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: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: 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

**********

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: Comments

1. What is the rationale behind choosing oral administration of RDTF rather than other modes of drug administration or even any implantation modes?

2. Did the author carry out experiments on the study of in vitro cyto-compatibility of the drug in any means?

3. Did the author go through literatures having similar work performed in any other animal models?

4. Kindly check the spelling for Histological in page 16 (Line 188), Quantitative Page 17 (Line 196), Page 18 (Line 217), Page 18 (Line 236) and change it wherever applicable.

5. Justify the reason for selecting these particular concentrations of the drug for animal experiment? Why the author did not try with series of concentrations of the drug but ended up with one particular concentration for HG, MG, LG in animal study? Please justify.

6. The author has performed experiments on animal model to evaluate the levels of proteins and other biomarkers but failed to work on the extraction of individual bio-active flavonoid and its role in protein expression at in vitro cellular level. How would you validate that the present study with whole tuber flavonoid extract of drynaria gives enough stuff on ternary regulation of bone defect in animal model?

7. It is validated that Rhizoma drynaria contains HCN and did the author work on any procedures to remove it before extracting the total flavonoids.

8. Also, did the author check the anti-oxidant activity of the compound using in vitro biological assays specially to study the effect of oxidative stress markers?

9. In page 10, (line 64-66) the sentence “After 4 to 8 weeks a membrane surrounding the spacer was formation, then the second stage surgery was operated, including removal of the spacer and implant the bone grafts (citation).” can be changed to “After 4 to 8 weeks, a membrane surrounding the spacer was formed and then the second stage surgery was operated to remove the spacer for implantation of bone grafts (Include citation) “

10. The author has claimed that the autografts implanted are rat coccyx. Justify how did the author manages post-operative pain for the animal, risk of fracture at the donor site.

11. Is there any quantified data on mesenchymal stem cell (MSCs) production in induced membranes of IFGs/ECG/IMG with respect to normal bone marrow of their respective groups to validate the osteo-angiogenic potential

12. Did the author perform any experiments with induction membranes retrieved from reconstructed CSD femur model to quantify angiogenic and osteogenic bio-markers

13. Did the author validate any results on soft callus regeneration during bone remodeling with PMMA like how it is done after bone graft repair? Since this method includes long treatment period, did the author experimented on early bone healing phases during PMMA post-surgery?

14. The author has claimed that the induction membrane serves as periosteum like matrix to help in cell proliferation, growth factor production and signaling but the expected study did not prove the pro-angiogenic ability of IMGs individually. The gene and protein expression studies as mentioned in Fig 3A and 3B for VEGFA and CD31 showed minimal effect. Justify why PECAM and VEGFA expressions are hindered in IMGs than other groups.

Reviewer #2: The manuscript titled on " Ternary regulation mechanism of Rhizoma drynariae total flavonoids on inducedmembrane formation and bone remodeling in Masquelet technique" is an interesting work done by author. However, there are few questions need to be addressed in this study.

1) what is the rationale for choosing Rhizoma drynariaetotal flavonoids(RDTF) ? There are several flavonoids currently used for treating bone defect. How different from other study ?

2) Bone remodeling involves the secretion of bone markers such as ALP, OC etc. Do an author checked the expression of these other than VEGF and PDGF?

3) section 2.4-2.7 contains lot of spelling mistake in the title. For example, instead of histological author wrote ological analysys. It needs to be corrected prior to publication.

Fig.4 legends are not visible, poor resolution. Need a better resolution. In the RT-PCR, author didn't explain how did normalization done. And what method did you use to calculate mRNA expresssion ?

Do an author checked any of the protein expression such as VEGF and PDGF?

**********

6. 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 #1: Yes: DR. DHARUNYA GOVINDARAJAN

Reviewer #2: Yes: Arumugam Balasubramanian

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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

Ternary regulation mechanism of Rhizoma drynariae total flavonoids on induced membrane formation and bone remodeling in Masquelet technique

PONE-D-22-18003R1

Dear Dr. Li,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Selvaraj Vimalraj

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

accept

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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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 #1: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: N/A

**********

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 #1: Yes

Reviewer #2: Yes

**********

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 #1: Yes

Reviewer #2: Yes

**********

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 #1: Kindly check the spelling and grammatical error and make changes wherever applicable. The answers are satisfying and so the manuscript can be forwarded for publication.

All the best.

Reviewer #2: The author clearly addressed all the comments raised in this manuscript. Therefore the manuscript can be accepted for publication.

**********

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 #1: Yes: DR. DHARUNYA GOVINDARAJAN

Reviewer #2: No

**********