PONE-D-20-38401

Endoscopic application of mussel-inspired phenolic chitosan as a hemostatic agent for gastrointestinal bleeding: a preclinical study in a heparinized pig model

PLOS ONE

Dear Dr. Lee,

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 Apr 17 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Panayiotis Maghsoudlou

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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. We note that you have provided ethics information within your manuscript. However, we ask that you also provide it in the metadata that you are asked to fill out when you resubmit. This is the section where you are prompted to fill in the title, abstract data availability statement, etc. One of these sections is labelled "Ethics Statement". You have currently written that the information has been included in the manuscript. Please change this to:

'This study was approved by the Institutional Animal Care and Use Committee of KNOTUS Co., Ltd. (KNOTUS IACUC, approval no.16-KE-200) and conducted in compliance with Good Laboratory Practice (GLP) regulations (21 CFR Part 58, US FDA). The animals were sedated by intramuscular injection with a mixture of tiletamine/zolazepam (Zoletil® 95 , Virbac,

5 Carros, France) and xylazine (Rompun® 96 , Bayer, Leverkusen, Germany). General anesthesia was maintained by mechanical ventilation with 1% to 2% isoflurane (Forane® 97 , Baxter, Deerfield, IL, USA) in oxygen. If humane endpoints were reached, such as significant weight loss exceeding 20% of initial baseline body weight, lethargy, pyrexia, hypothermia, or behavior that signals severe pain, the animals were sacrificed and excluded from this study. The animals were humanely euthanized by intravenous (IV) injection of suxamethonium chloride (Succicholine Inj® 50 mg/mL, Ilsung Pharmaceuticals, Seoul, Republic of Korea) at a dose of 0.1 mL/kg under deep anesthesia.'

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"This study was supported in part by a grant from the National R&D Program for Cancer

Control, the Ministry for Health and Welfare, Republic of Korea (HA16C0016 (1631060), H.L.)."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

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

"NO authors have competing interests."

We note that one or more of the authors are employed by a commercial company: InnoTherapy Inc..

4.1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

4.2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.  

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: This paper is generally well-written and provides a thorough assessment of the new catechol-chitosan wound patch technology. The paper thoroughly addresses both bleeding as well as wound healing, and the methods for the most part are well done. Thus, I have only a few critiques and requests for clarification:

1. It is unclear why an aPTT was run rather than a PT. While both reflect the effect of heparin on slowing coagulation, PT better reflects the ability of clot to form in tissue, while aPTT reflects the speed of clotting at artificial surfaces. Thus the aPTT is a good measure for what might occur close to the gauze or the CHI-C patch, the PT is a better reflection of the tendency of the injury to bleed.

2. The heparin was given as a bolus, rather than a drip, and thus the aPTT is falling over time, as acknowledged in the paper. Because of this, it is important to denote in what order the interventions were performed (APC, gauze, and CHI-C). Was it randomized or in a specific order? It should be clear if certain interventions faced a more challenging bleed. If so, that could potentially damage the findings for the hemostasis portion of the study.

3. Some of healing data is examined serially in one subject over multiple days. Thus, you should have a statistical method with repeated measures that incorporates a subject variable. From what I can tell, you haven’t done this. For continuous data, such as the % healing measurement, I would used a mixed model because it is better when you have some lost data (which I think you have from one pig). I am not as familiar with non-parametric statistics with repeated measures, so I recommend consulting a statistician to find the ideal repeated measures method for the ranked healing scores.

4. The discussion is a bit unfocused, particularly in its discussion of the healing results. These are scattered over several different paragraphs. Bringing them together and more thoroughly discussing these results in one place would improve the discussion. Thus, I recommend:

P1. In the first paragraph, summarize your findings that CHI-C and APC are the best at stopping bleeding, but that APC causes more tissue injury. Exhaustively discuss the differences in tissue injury here, rather than spread over the other paragraphs.

P2. Then talk about the other advantages you see with CHI-C - accessibility

P3. Then limitations.

Reviewer #2: Interesting study with promising results...

- Further details and clarifications about the CHI-C hemostatic agent preparation, characterization, properties. must be provided (substitution degree, sterilization, etc. ). If all these details are provided in a previously published manuscript, it should be clearly indicated in the text

- I believe that the follow-up endoscopy should have been blinded. Given that APC and CHI-C results are somewhat similar and that the study has other limitations specified by the authors, I would tone down the last sentence in the abstract by replacing outstanding by promising,

- Please clarify if histological analysis was blinded or non-blinded

- Specific comment about the Conclusions: The mention of clinical trials in the last sentence is premature in my opinion, more pre-clinical studies will have to be performed prior to initiate any clinical assessment and this sentence must be revised.

According to PLOS Data policy,

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.

Some data points are missing as its mostly summary statistics that are provided, it therefore seems that the authors didn't fully comply with this policy

Typos:

Sentence on line 171 is to be revised (was more severe or milder)

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

Endoscopic application of mussel-inspired phenolic chitosan as a hemostatic agent for gastrointestinal bleeding: a preclinical study in a heparinized pig model

PONE-D-20-38401R1

Dear Dr. Lee,

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,

Panayiotis Maghsoudlou

Academic Editor

PLOS ONE