RETRACTED: Zoledronic acid generates a spatiotemporal effect to attenuate osteoarthritis by inhibiting potential Wnt5a-associated abnormal subchondral bone resorption

Dong Ding; Limei Wang; Jiangbo Yan; Yong Zhou; Gangning Feng; Long Ma; Yong Yang; Xiuying Pei; Qunhua Jin

doi:10.1371/journal.pone.0271485

Peer Review History

Original SubmissionMarch 21, 2022
11 May 2022 Decision Letter - Juha Tuukkanen, Editor PONE-D-22-08357Zoledronic acid generates a spatiotemporal effect to attenuate osteoarthritis by inhibiting Wnt5a-mediated abnormal bone resorption in subchondral bonePLOS ONE Dear Dr. Ding, 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 all the points raised during the review process. In the addition, for the ethics section, please describe the use of adeqate analgesics after the surgery. Please submit your revised manuscript by Jun 11 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. 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Reviewer #1: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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: No ****** 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: Ding et al show that zoledronic acid can halt OA progression. They further provide interesting data on bone remodelling and especially on osteoclast activity during early OA. This data is very interesting and very important. In addition, authors claim that effects are mediated by inhibiting wnt5a signaling. Associated analyses are performed well but I find it slightly difficult that this is only focused on wnt5a, thus I see that part of the wording should be reconsidered so that made claims are aligned with results. In general, manuscript is well written and the study design is mostly appropriate. There are some major methodological issues that also have to be improved. Major comments: 1. Wnt signaling is very important to bone physiology. Is seems to play role in bone development and mechanosensation. Furthermore, lorecivivint seems to be most potential to be accepted as first disease modifying osteoarthris drug. Lorecivivint also targets wnt signaling by inhibiting DYRK1A and CLK2. So how can you be sure that the halting effect for OA progression is mediated trough wnt5a signaling? Furthermore, I think last graph makes little sense. Thus, I would propose to make wnt5a as potential mechanism. For example, conclusion is ok but title indicates that it is wnt5a only. Furthermore, please see Wang, L., Fang, D., Xu, J. et al. Various pathways of zoledronic acid against osteoclasts and bone cancer metastasis: a brief review. BMC Cancer 20, 1059 (2020). https://doi.org/10.1186/s12885-020-07568-9 2. One big limitation of the current study is the young age of animals (juvenile OA) and this should be added in discussion. 3. Reference 19 cannot be correct. That is the editorial for the issue that introduced the grading system for human cartilage. Rats have specific grading system introduced few years later. Please correct. Gerwin N, Bendele AM, Glasson S, Carlson CS. The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the rat. Osteoarthritis Cartilage. 2010 Oct;18 Suppl 3:S24-34. doi: 10.1016/j.joca.2010.05.030. PMID: 20864021. 4. It is challenging to understand where bone morphometric analysis (microCT) has been conducted and it would be good to add explanatory visualization. 5. This data is important for the as typically OA animal models show bone loss and you have bone sclerosis at 12 weeks. However, it seems that the there is significant bone destruction in DMM group at 12 weeks and thus the BV/TV value alone is misleading. Please provide some cross-anatomy evaluation in addition to morphometric parameters. 6. In general N=3 is not sufficient for any statistical evaluation. Thus, the molecular biology part is also statistically weak. How come there is no deviation in controls in fig 6 F-I? 7. Please provide better quality (4 of the current ones are now yellow) and more (larger area) representative histological images in Fig 4 A. 8. Figure 8 aims to summarize the study but I find it weird. Please remove. Minor: 9. Alltough manuscript is mainly well written there are some poorly written parts like OA induction and should be rewritten with appropriate English. 10. Please do not call bone morphometric parameters indexes and give the full name for all acronyms (CD – connective density?). 11. 250 µA is not intensity but tube current (10W). Please follow “Bouxsein, M.L., Boyd, S.K., Christiansen, B.A., Guldberg, R.E., Jepsen, K.J. and Müller, R. (2010), Guidelines for assessment of bone microstructure in rodents using micro–computed tomography. J Bone Miner Res, 25: 1468-1486. https://doi.org/10.1002/jbmr.141” when reporting microCT methods and results. ****** 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: No [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.0271485.r001
Revision 1
18 May 2022 Author Response Dear editor, Thanks for the comments from you and peer reviewers about the manuscript (PONE-D-22-08357). I have consulted a large number of materials according to the references you provided, and carefully studied and analyzed your review comments. I think these comments are of great help to modify and improve the quality of my article. I have revised the manuscript according to these valuable opinions, and the following is my reply to the revised opinions. I ensure that my manuscript meets PLOS ONE's style requirements, including those for file naming. I provided the original uncropped and unadjusted images underlying all blot or gel results reported both in a submission’s figures and Supporting Information file described as “Details about some experiments”. I re-analyzed the data of qPCR and WB, and uploaded the updated data. All the original data can be queried in the following public database: https://doi.org/10.7910/DVN/ECQM07, Harvard Dataverse, V2. I used the PACE before submitting to check my figures and convert to the accepted formats. 1. “In the addition, for the ethics section, please describe the use of adequate analgesics after the surgery.” Answer: Thanks for your opinion, I described the pain management of experimental animals as required in the ethics and OA induction sections. Major comments: 1. “So how can you be sure that the halting effect for OA progression is mediated trough wnt5a signaling? Furthermore, I think last graph makes little sense. Thus, I would propose to make wnt5a as potential mechanism. For example, conclusion is ok but title indicates that it is wnt5a only. Furthermore, please see Wang, L., Fang, D., Xu, J. et al. a brief review. BMC Cancer 20, 1059 (2020). https://doi.org/10.1186/s12885-020-07568-9.” Answer: Thanks for reviewer’s comments, which are consistent with my initial idea. I gained a great deal of useful information from the review provided by the reviewer. In the present study, a possible correlation between abnormal subchondral bone resorption and Wnt5a was found in the early stage of OA in rats, and this possible relationship was preliminarily proved by the in vitro experiment of the primary BMMs. Therefore, I agree with the reviewer that the results of this study can only preliminarily show that Wnt5a is a possible mechanism related to early abnormal subchondral bone absorption of OA, but cannot completely clarify the specific mechanism related to Wnt5a. According to the suggestions of reviewers, I seriously reconsidered and modified some words in the title, results and conclusion so that made claims are aligned with results. In fact, another study we conduct is in progress, which is about the possible mechanism of Wnt5a mediating intercellular crosstalk and regulating bone remodeling in the subchondral bone microenvironment at the early stage of OA, and I hope it can be accepted by the journal in the later submission. 2. “One big limitation of the current study is the young age of animals (juvenile OA) and this should be added in discussion.” Answer: Thanks for reviewer’s comments. I agree with the reviewer's comments and add the discussion about this part in the manuscript. OA is a disease of adults, and growing animals, like children, seem to have a better capacity at managing joint damage. Moreover, in controlled experiments, the complexities of changing skeletal size and metabolism during growth can obscure how joints respond to injury and therapy. Thus, to obtain the most meaningful insights into human OA, it is essential to use skeletally mature animals whenever possible. There are many animal models that reproduce key aspects of human OA in terms of natural history, mechanisms, signs and symptoms. Yet, as not one animal model completely reproduces the signs and symptoms of human OA, we must remain mindful to use the most appropriate model to answer our scientific questions. As for the age of the OA model, I referred to and agreed with the literature recommended by reviewer, suggesting that rats older than 12 weeks should be used to establish OA model to simulate the development of human OA as much as possible. The 8-week-old male rats were uniformly used in this study. Although the study period was 12 weeks after modeling, the 20-week-old rats also had the longest possible course of OA. The weight of the 8-20-week-old rats was relatively stable, consistent with the statistical significance of normal distribution, and the hormone and metabolism levels were stable. In this way, the influence of stress, hormone, metabolism and other factors on the results was avoided. Rats in this age range may have a more reasonable metabolism of positive control drugs (ZOL). As the age of old SD rats increases, the incidence of cartilage spontaneous cysts increases, which is not conducive to the study of OA. Young rats can shorten the decalcification time, but too young age may be a deficiency of this study, and the possible influence of age factor and rat tissue maturity on the results is not fully considered. We will improve this in the later study. Thanks to reviewers for their important suggestions and opinions. 3. “Reference 19 cannot be correct.” Answer: Thanks for the reviewer's reminding, I corrected this mistake and modified the experimental method accordingly. 4. “It is challenging to understand where bone morphometric analysis (microCT) has been conducted and it would be good to add explanatory visualization.” Answer: Thank you for your advice. As requested, I added and improved the explanatory visualization in the method about “Micro-computer tomography (CT) analysis” as following: A portion (0.5 mm below the growth plate and 1 mm in Height, 3.5 mm ventrodorsal length) of the load-bearing region at the medial tibial plateau was identified as the region of interest (ROI). 5. “However, it seems that the there is significant bone destruction in DMM group at 12 weeks and thus the BV/TV value alone is misleading. Please provide some cross-anatomy evaluation in addition to morphometric parameters.” Answer: Thank you for your advice. In the revised manuscript, I provided the figures of Saffron O-fast green staining as required, which may be used as cross-anatomy evaluation. In addition, in our unpublished articles, general observation figures (SF.1) of rats’ knee joints at 4 and 8 weeks postoperative can be used as supporting documents as evidence of cross-anatomy evaluation. Supplement figure 1. General observation of rats’ knee joints at 4 and 8 weeks postoperative. Ideally, it will be possible to consider MRI and micro-CT as less invasive technologies for the gold standard of macroscopic and microscopic evaluation. However, due to limited conditions, our laboratory did not have micro-CT, and due to the impact of COVID-19, we could not transport live experimental animals to the laboratory in Shanghai for dynamic observation with micro-CT. Therefore, we had to take samples and fix the knee joints of rats at various time points. It is then sent to a micro-CT lab in Shanghai for testing. I described this in the discussion as one of the limitations of this study, and we will try our best to overcome this in the later study by using micro-CT to achieve non-invasive dynamic observation of the course of OA in rats. Thank you for your advice and understanding. 6.“In general N=3 is not sufficient for any statistical evaluation. Thus, the molecular biology part is also statistically weak. How come there is no deviation in controls in fig 7 F-I?” Answer: Thank you for your advice and reminder. In fact, 18 male rats were randomly divided into 3 groups, and 3 of the 6 rats in each group were randomly selected for morphological and molecular biological detection respectively. It may be that we wrongly believed that 3 rats randomly selected from each group could represent the situation of each group, which also met the principle of "Reduction" in animal experiments and reduced the sample size of animal experiments to the minimum. This may not completely meet the statistical requirements, I will pay attention to and improve this point in the future, thank you for reminding. No deviation in Figure 7 may be related to my mishandling of the data. I aimed to compare the difference between the DMM group and the ZOL group by the “fold of increase” in protein and mRNA compared with the sham group, and I wrongly quantified the level of the sham group as “1” in the three repeated experiments. I realized this error and represented the level of sham group in the three repeated experiments as the means ± standard deviation. 7. Please provide better quality (4 of the current ones are now yellow) and more (larger area) representative histological images in Fig 4 A. Answer: Thank you for your advice and reminder. According to the requirements of reviewer, I improved and replaced all the figures by the “PACE”. 8. Figure 8 aims to summarize the study but I find it weird. Please remove. Answer: Thank you for your advice and reminder. According to the requirements of reviewers, I removed the figure 8. 9. Although manuscript is mainly well written there are some poorly written parts like OA induction and should be rewritten with appropriate English. Answer: Thank you for your advice and reminder. According to the requirements of reviewers, I rewritten and improved the “OA induction” with appropriate English according to the steps shown in the following Supplement figure 2. Supplement figure 2: DMM induced OA in rats. 10. Please do not call bone morphometric parameters indexes and give the full name for all acronyms (CD – connective density?). Answer: Thank you for your advice and reminder. I have referred to the literature you provided and modified the description of trabecular bone morphometry according to the guidelines involved in the literature (The following table 2). 11. “250 µA is not intensity but tube current (10W). Please follow” Answer: Thank you for your advice and reminder. I have referred to the literature you provided and modified the description of key parameters for μCT scan acquisition following the guidelines involved in the literature (The following table 1). Thanks again to the editors and reviewers for their comments, which are of great help to modify and improve the quality of my article. Yours sincerely, Corresponding author: Dong Ding Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0271485.r002
5 Jul 2022 Decision Letter - Juha Tuukkanen, Editor Zoledronic acid generates a spatiotemporal effect to attenuate osteoarthritis by inhibiting potential Wnt5a-associated abnormal subchondral bone resorption PONE-D-22-08357R1 Dear Dr. Ding, 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, Juha Tuukkanen 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 #1: 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 #1: No ****** 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 ****** 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: The authors have revised the submitted manuscript. It seems that they have made the necessary changes. In general, I hope that you provide author reply and author action with all of your future revisions. There is an author reply in this revision but the reviewer must search for possible changes in the manuscript. This is highly time-consuming and frustrating. ****** 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: No ******** https://doi.org/10.1371/journal.pone.0271485.r003
Formally Accepted
19 Jul 2022 Acceptance Letter - Juha Tuukkanen, Editor PONE-D-22-08357R1 Zoledronic acid generates a spatiotemporal effect to attenuate osteoarthritis by inhibiting potential Wnt5a-associated abnormal subchondral bone resorption Dear Dr. Ding: 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. Juha Tuukkanen Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0271485.r004

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