Cell sources of inflammatory mediators present in bone marrow areas inside the meniscus

Francisco Airton Castro Rocha; Virgínia Claudia Carneiro Girão; Rodolfo de Melo Nunes; Ana Carolina Matias Dinelly Pinto; Bruno Vidal; João Eurico Fonseca

doi:10.1371/journal.pone.0226986

Peer Review History

Original SubmissionOctober 15, 2019
4 Dec 2019 Decision Letter - Oreste Gualillo, Editor PONE-D-19-28554 Cell sources of inflammatory mediators present in bone marrow areas inside the meniscus PLOS ONE Dear DR. Rocha, 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. We would appreciate receiving your revised manuscript by Jan 18 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines. Specifically, Please describe the post-operative care received by the animals, including the frequency of monitoring for the 21 or 49 days after the surgical procedure and the criteria used to assess animal health and well-being. In addition, please state specifcally in the Methods section of the manuscript that the mice were euthanized using an overdose of ketamine and xylazine Thank you for your attention to these requests. 3. To comply with PLOS ONE submission guidelines, in your Methods section, please provide additional information regarding your statistical analyses. For more information on PLOS ONE's expectations for statistical reporting, please see https://journals.plos.org/plosone/s/submission-guidelines.#loc-statistical-reporting. Additional Editor Comments: Dear Authors, Your paper has been reviewed by one reviewer and by myself as academic editor of the journal. There are some minor issues that should be improved in order to accept your article for publication in PLOSOne. Please, stress in the introduction the rationale of the study, as well as in the discussion. As editor, I am particularly concerned about the clinical implications of your results. I appreciate that you will discuss more these aspects in your discussion. Your paper will be reviewed again after these minor aspects are issued. Thank you for submit youyr results to PLOSONE Dr. Oreste Gualillo, PLOSONE Academic Editor. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know ****** 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 ****** 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 ******** 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 manuscript by Rocha et al aims to assess the cell sources of proinflammatory mediators in the meniscus of rodent animals at different ages, and to study the effect of OA in the presence of these mediators in the menisci. The paper is well designed and the experiments correctly carried out. However, some aspects need to be clarified: * The specific objective of this manuscript is only stated in the first sentence of the abstract. It needs to be also included in the paper, preferably at the end of the introduction/background section. Furthermore, authors should emphasize, also in the sentence in the abstract, the study is only focused on rodent meniscus. * Although macrophages have been already identified in this tissue in adult humans, the presence of this bone marrow areas inside the meniscus has been only described in animals. In my opinion, this and other apparently minor anatomical differences between rodents and humans could in some way be responsible for the lack of reproducibility of the experimental studies in comparison to the results in patients. Could authors comment on this? * Although the age of the animals is correctly indicated in the paper, authors might include the age of skeletal maturation in each specie. * Did authors assess whether OA increased the presence of bone marrow areas in mice, or the extension of these areas? ******** 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: Raquel Largo [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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0226986.r001
Revision 1
5 Dec 2019 Author Response Answers to Editor’s and Reviewer’s comments: PONE-D-19-28554 Cell sources of inflammatory mediators present in bone marrow areas inside the meniscus Comment: Journal Requirements: 1. When submitting your revision, we need you to address these additional requirements. 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Answer: Style was rechecked Comment: 2. At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines. Specifically, Please describe the post-operative care received by the animals, including the frequency of monitoring for the 21 or 49 days after the surgical procedure and the criteria used to assess animal health and well-being. In addition, please state specifcally in the Methods section of the manuscript that the mice were euthanized using an overdose of ketamine and xylazine Answers: Please see the modified methods section, as follows: Both the sham and operated groups of mice were evaluated every other day following the surgical procedure, during routine cleaning of the cages, checking for possible behavior alterations. Body weight, food and water consumption were checked weekly until the end of the protocol. Euthanasia of the animals was processed under terminal anesthesia (i.m. ketamine/xylazine overdose). Those subjected to the surgical protocol were euthanized also under terminal anesthesia (i.m. ketamine/xylazine overdose) 21 or 49 days after the surgical procedure and had their knee joints evaluated under histopathology. Comment: 3. To comply with PLOS ONE submission guidelines, in your Methods section, please provide additional information regarding your statistical analyses. For more information on PLOS ONE's expectations for statistical reporting, please see https://journals.plos.org/plosone/s/submission-guidelines.#loc-statistical-reporting. Answer: The name and version of the software package used for statistical analysis was included, as follows: Statistical analysis of the data was performed using SPSS Statistics for Windows, Version 16. Additional Editor Comments: Dear Authors, Your paper has been reviewed by one reviewer and by myself as academic editor of the journal. There are some minor issues that should be improved in order to accept your article for publication in PLOSOne. Please, stress in the introduction the rationale of the study, as well as in the discussion. As editor, I am particularly concerned about the clinical implications of your results. I appreciate that you will discuss more these aspects in your discussion. Your paper will be reviewed again after these minor aspects are issued. Thank you for submit youyr results to PLOSONE Dr. Oreste Gualillo, PLOSONE Academic Editor. Answer: Dear Dr. Gaulillo, We greatly appreciate your interest and comments. Being rheumatologists, we surely are also concerned about clinical implications. This was the reason to include this sentence in the original discussion section: In addition to contributing to the morphological detailing of the meniscal histology per se, our results have implications regarding the development of joint lesions....The occurrence of cells able to initiate and perpetuate an inflammatory reaction inside the meniscus, as those CD68+ macrophages, lead us to speculate that in addition to the mechanical derangement and joint instability represented by meniscal damage, cellular components of the meniscus may also trigger and/or potentiate inflammation inside joints following an insult. Trying to meet your request, we added another comments in the text, as follows: - rationale (end of introduction section): During the observation of knee samples from mice subjected to experimental OA following meniscotomy, we found defined bone marrow cavities in some surgically lesioned menisci (our unpublished data). Given the inflammatory potential of bone marrow derived cells, we decided to perform an exhaustive evaluation of menisci obtained from mice and rat knees. Our data revealed active osteoclasts... - clinical implication (end of discussion section): Although speculative, if those rodent changes are reproduced in humans, patients subjected to knee trauma or damage secondary to daily life activities who present inflammatory cells inside their meniscus may be more prone to develop rapid and severe OA changes as compared to those with meniscus displaying merely fibrocartilage containing fibroblasts and fibrochondrocytes. This possibility justifies a need to search for those changes in human menisci. We are currently pursuing this objective. Comment: Reviewer #1: The manuscript by Rocha et al aims to assess the cell sources of proinflammatory mediators in the meniscus of rodent animals at different ages, and to study the effect of OA in the presence of these mediators in the menisci. The paper is well designed and the experiments correctly carried out. However, some aspects need to be clarified: Comment: * The specific objective of this manuscript is only stated in the first sentence of the abstract. It needs to be also included in the paper, preferably at the end of the introduction/background section. Furthermore, authors should emphasize, also in the sentence in the abstract, the study is only focused on rodent meniscus. Answer: Thank you for this comment. We have introduced the proposed modifications, as follows: - Abstract: Purpose: to demonstrate the production of inflammatory mediators by cells located in bone marrow spaces inside rodent menisci. - rationale (end of introduction section): During the observation of knee samples from mice subjected to experimental OA following meniscotomy, we found defined bone marrow cavities in some surgically lesioned menisci (our unpublished data). Given the inflammatory potential of bone marrow derived cells, we decided to perform an exhaustive evaluation of menisci obtained from mice and rat knees. Our data revealed active osteoclasts... In order to meet an Editor’s request, we also included a speculative comment on the clinical implication. We believe this reinforces that we actually dealt with rodents, hopefully meeting your request. Please see: - clinical implication (end of discussion section): Although speculative, if those rodent changes are reproduced in humans, patients subjected to knee trauma or damage secondary to daily life activities who present inflammatory cells inside their meniscus may be more prone to develop rapid and severe OA changes as compared to those with meniscus displaying merely fibrocartilage containing fibroblasts and fibrochondrocytes. This possibility justifies a need to search for those changes in human menisci. We are currently pursuing this objective. Comment:* Although macrophages have been already identified in this tissue in adult humans, the presence of this bone marrow areas inside the meniscus has been only described in animals. In my opinion, this and other apparently minor anatomical differences between rodents and humans could in some way be responsible for the lack of reproducibility of the experimental studies in comparison to the results in patients. Could authors comment on this? Answer: Thank you for this comment. We believe there should not be great morphological (macro and micro) differences between human and rodent knees. This was the reason for our comment on the discussion, quoting prior studies: Given the phylogenetically conserved appearance of the knee anatomy and histology, regardless of being biped or quadruped animals [21], and the relevance of animal models to study OA pathogenesis.. Perhaps the main difficulty in reproducing data is due to mechanical impact (4 vs 2 stance gait), virtually no obesity in experimental animals, or other less obvious reasons. But this remains to be proven. There are some issues to get human data. Usually, human samples are just thrown away following meniscectomy in elderly patients; during arthroscopy, they are minced to tiny pieces. On the other hand, in the young patient, following trauma, surgeons preserve as much meniscus as possible. During arthroplasty, there is almost no meniscus left. The best chance would be to perform an active search using cadavers. We are pursuing this strategy. Comment:* Although the age of the animals is correctly indicated in the paper, authors might include the age of skeletal maturation in each specie. Answer: Thank you for this comment. Actually, our ethics committee limited the number of animals and we explained our intention to cover periods pre and post skeletal maturation. Please see the discussion section (another ref was included), as follows: The age range of the animals was based on an attempt to cover skeletal maturity in both mouse and rat species, estimated to be at 10 weeks and 3 months, respectively [21] Comment:* Did authors assess whether OA increased the presence of bone marrow areas in mice, or the extension of these areas? Answer: Thank you for this very relevant comment. We really wanted to do that. With the number of animals we got that was impossible. We can’t know in advance (prior to the start of experiments) which animals will present those areas. This means we would need more samples. We had asked to include 15 animals per OA group, based on a sample calculation (however, merely estimating) but our committee on animal experimentation did not allow that. ________________________________________ 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: Raquel Largo [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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: AnswerReviewers.docx https://doi.org/10.1371/journal.pone.0226986.r002
11 Dec 2019 Decision Letter - Oreste Gualillo, Editor Cell sources of inflammatory mediators present in bone marrow areas inside the meniscus PONE-D-19-28554R1 Dear Dr. Rocha, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Oreste Gualillo, PharmD, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): No further comments Reviewers' comments: https://doi.org/10.1371/journal.pone.0226986.r003
Formally Accepted
12 Dec 2019 Acceptance Letter - Oreste Gualillo, Editor PONE-D-19-28554R1 Cell sources of inflammatory mediators present in bone marrow areas inside the meniscus Dear Dr. Rocha: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr Oreste Gualillo Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0226986.r004

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