Prevention of adhesions post-abdominal surgery: Assessing the safety and efficacy of Chitogel with Deferiprone in a rat model

Rajan Sundaresan Vediappan; Catherine Bennett; Clare Cooksley; John Finnie; Markus Trochsler; Ryan D. Quarrington; Claire F. Jones; Ahmed Bassiouni; Stephen Moratti; Alkis J. Psaltis; Guy Maddern; Sarah Vreugde; P. J. Wormald

doi:10.1371/journal.pone.0244503

Peer Review History

Original SubmissionMay 15, 2020
13 Jul 2020 Decision Letter - Aleksandar R. Zivkovic, Editor PONE-D-20-14448 Prevention of adhesions post-abdominal surgery: Assessing the safety and efficacy of Chitogel with Deferiprone in a Rat Model PLOS ONE Dear Dr. Wormald, 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 31.08.2020. 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. 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If the data are not a core part of the research study being presented, we ask that authors remove any references to these data." 4. In the Methods section, please provide the product number and any lot numbers of the Kaolin, Chitogel, deferipone, Gallium Protoporphyrin IX purchased from Sigma Aldrich and Frontier Scientific for your study. 5. 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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 [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 Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: I Don't Know Reviewer #3: 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: No Reviewer #3: 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 ******** 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: The authors describe a well planned and conducted animal study on an important topic. But there are some considerations and limitations which needs a major revision: Introduction: 1. The sentence:“The mortality rate due to postsurgical adhesions is almost 10%...” needs a revision, because this statement is not reasonable. After every abdominal operation, postsurgical adhesions occur in a variable intensity. 2. The statement: “Numerous strategies have been devised to prevent peritoneal adhesions, such as minimal access surgeries, hydro flotation, barrier agents such as anti-adherencehyaluronic acid/carboxymethylcellulose, regenerated and expanded oxidised cellulose 0.5% in ferric hyaluronate and chlorine dioxide (5). However, none of these strategies have been widely adopted due to poor efficacy or risk of adverse events” needs a revision as well, because it is widely established that minimal access e.g. laparoscopic surgery is able re recuse adhesions significantly compared to open surgery. Methods: 3. Why is Def-GaPP as an antibacterial agent only used in the abrasion and not in the enterotomy group? 4. The used adhesions score is very simple and reflects only adhesions quality but not the quantity. The “Diamond score” (Diamond MP, Linsky CB, Cunningham T, Constantine B, diZerega GS, DeCherney AH: A model for sidewall adhesions in the rabbit: reduction by an absorbable barrier. Microsurgery 1987, 8:197-200) is a well established and more detailed score which reflects in addition the adhesions quantity. 5. The statistical analysis needs a correction for multiple comparisons e.g. Bonferroni, because you are comparing more than two groups. Therefore, the significance level is not “traditional < 0.05”. The correction for multiple comparisons might result in non-significant differences. Results: 6. Please state especially all p-values more clearly within the text 7. Please provide a table with all results of the abrasions / abrasions and enterotomy model with all p-values Discussion: 8. Please enlarge the discussion with a hypothesis why “Chitogel with 1mM Def not only prevents adhesion formation but also promotes efficient healing of the enterotomy site”. Reviewer #2: This is a well written and interesting study in an experimental rat model about the adhesions prevention. The study is suitable for publication, well designed, data supports the conclusions. I recommend further technical examination to judge the appropriateness of the statistics. Unfortunately the complete dataset could not be shared. Reviewer #3: The purpose of the study was to investigate the safety and the efficacy of Chitogel with Deferiprone and/or antibacterial Gallium Protoporphyrin in different concentrations in order to prevent adhesion formations after abdominal surgery. The study, performed in an animal model, demonstrates that Chitogel with Deferiprone 1 mM constitutes an effective preventative anti-adhesion barrier after abdominal surgery. The results presented in the paper are interesting and the manuscript it certainly can be considered for publication, but in my opinion there are a some points which should clarified before considering the text suitable for publication. Firstly, the figures explaining histopathologic alteration gradind are unclear, aboveall because these are of very small size. In my opinion it would be better to prepare tables in order to have a lower magnification image that allows the identification of the portion of tissue examined and inserts within these figures for a proper cell recognition. This is impossible in the present form. The text is written in English which is correct for my knowledge, the references are correctly cited and updated. In conclusion, I think the manuscript can be considered suitable for publication after the necessary revision. [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. https://doi.org/10.1371/journal.pone.0244503.r001
Revision 1
10 Dec 2020 Author Response Dear Madam/Sir, Thank you for your comments and giving us an opportunity to clarify/correct. Kindly find below our response : Reviewer #1: Introduction: 1. The sentence: “The mortality rate due to postsurgical adhesions is almost 10%...” needs a revision, because this statement is not reasonable. After every abdominal operation, postsurgical adhesions occur in a variable intensity. Ans: We have rewritten the statement as follows “ Occurrence of adhesions after upper and lower abdominal surgery ranges from 67-93% [4, 5]. The mortality rate due to postsurgical adhesions can be high, especially among elderly [6]” 2. The statement: “Numerous strategies have been devised to prevent peritoneal adhesions, such as minimal access surgeries, hydro flotation, barrier agents such as anti-adherencehyaluronic acid/carboxymethylcellulose, regenerated and expanded oxidised cellulose 0.5% in ferric hyaluronate and chlorine dioxide (5). However, none of these strategies have been widely adopted due to poor efficacy or risk of adverse events” needs a revision as well, because it is widely established that minimal access e.g. laparoscopic surgery is able re recuse adhesions significantly compared to open surgery. This sentence is re-written and describing that minimal access surgery does not the same incidence of adhesions as open sugery. Methods: 3. Why is Def-GaPP as an antibacterial agent only used in the abrasion and not in the enterotomy group? Ans: We have explained this in line 425-433 in discussion: “In the enterotomy part of this study, the antimicrobial GaPP was omitted as no microorganisms were cultured from the abdominal cavity in the control animals at day 21. Without a positive bacteriological swab at day 21, we would have been unable to show any benefit of adding GaPP.” 4. The used adhesions score is very simple and reflects only adhesions quality but not the quantity. The “Diamond score” (Diamond MP, Linsky CB, Cunningham T, Constantine B, diZerega GS, DeCherney AH: A model for sidewall adhesions in the rabbit: reduction by an absorbable barrier. Microsurgery 1987, 8:197-200) is a well established and more detailed score which reflects in addition the adhesions quantity. Ans: The Diamond score is good for a definable surface model of abrasion in the abdominal cavity. In this study the surface areas of injury was confined to the caecal region and therefore the first part of the Diamond classification was not used. The number of adhesion was counted in the grading system we used in the macroscopic evaluation section. Tensile strength testing, which evaluated the quality of the or the bowel repair and was studied in detail and in a objective fashion in this study. 5. The statistical analysis needs a correction for multiple comparisons e.g. Bonferroni, because you are comparing more than two groups. Therefore, the significance level is not “traditional < 0.05”. The correction for multiple comparisons might result in non-significant differences. Ans: All statistics were performed using R statistical software (R Foundation for Statistical Computing, Vienna, Austria) through the Jupyter notebook interface. The R package "ordinal" was used for ordinal regression. The "clm" function was used to fit a Cumulative Link Model, with the semi-quantitative adhesion scores as the ordinal outcome variable. The means of the ordinal response (interpreted as a numeric value from 1 to the number of classes) were calculated and post-hoc pairwise contrasts for each pair of levels of the treatment variable were compared using the "emmeans" package,(cite emmeans) with p-value corrections for multiple comparisons applied using the False Discovery Rate (FDR) method. 6. Please state especially all p-values more clearly within the text Ans: Corrected as suggested. 7. Please provide a table with all results of the abrasions / abrasions and enterotomy model with all p-values. Ans: Supplementary Table 1 A, B & C Discussion: 8. Please enlarge the discussion with a hypothesis why “Chitogel with 1mM Def not only prevents adhesion formation but also promotes efficient healing of the enterotomy site”. Ans: The discussion is enlarged as suggested. Reviewer #2: This is a well written and interesting study in an experimental rat model about the adhesion’s prevention. The study is suitable for publication, well designed, data supports the conclusions. I recommend further technical examination to judge the appropriateness of the statistics. Unfortunately the complete dataset could not be shared. Ans: We will share a anonymised data set. Reviewer #3: The purpose of the study was to investigate the safety and the efficacy of Chitogel with Deferiprone and/or antibacterial Gallium Protoporphyrin in different concentrations in order to prevent adhesion formations after abdominal surgery. The study, performed in an animal model, demonstrates that Chitogel with Deferiprone 1 mM constitutes an effective preventative anti-adhesion barrier after abdominal surgery. The results presented in the paper are interesting and the manuscript it certainly can be considered for publication, but in my opinion, there are a some points which should clarified before considering the text suitable for publication. Firstly, the figures explaining histopathologic alteration grading are unclear, above all because these are of very small size. In my opinion it would be better to prepare tables in order to have a lower magnification image that allows the identification of the portion of tissue examined and inserts within these figures for a proper cell recognition. This is impossible in the present form. The text is written in English which is correct for my knowledge, the references are correctly cited and updated. In conclusion, I think the manuscript can be considered suitable for publication after the necessary revision. Ans: We have included a macroscopic picture inset, with A = adhesion Attachments Attachment Submitted filename: Revised Response to Reviwer .docx https://doi.org/10.1371/journal.pone.0244503.r002
11 Dec 2020 Decision Letter - Aleksandar R. Zivkovic, Editor Prevention of adhesions post-abdominal surgery: Assessing the safety and efficacy of Chitogel with Deferiprone in a Rat Model PONE-D-20-14448R1 Dear Dr. Wormald, 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, Aleksandar R. Zivkovic Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0244503.r003
Formally Accepted
26 Dec 2020 Acceptance Letter - Aleksandar R. Zivkovic, Editor PONE-D-20-14448R1 Prevention of adhesions post-abdominal surgery: Assessing the safety and efficacy of Chitogel with Deferiprone in a Rat Model Dear Dr. Wormald: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Aleksandar R. Zivkovic Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0244503.r004

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