Hyaluronic acid hydrogels support to generate integrated bone formation through endochondral ossification in vivo using mesenchymal stem cells

Shintaro Yamazaki; Ryoko Hirayama; Yayoi Ikeda; Sachiko Iseki; Tetsuya Yoda; Masa-Aki Ikeda

doi:10.1371/journal.pone.0281345

Peer Review History

Original SubmissionAugust 31, 2022
12 Sep 2022 Decision Letter - Gianpaolo Papaccio, Editor PONE-D-22-24282Hyaluronic acid hydrogels support to generate integrated bone formation through endochondral ossification in vivo using mesenchymal stem cellsPLOS ONE Dear Dr. Ikeda, 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. ============================== The paper is of interest pending some alterations and more data experiments as suggested by myself and by the reviewers.The Authors in their rebuttal letter must answer point o point to the all the criticisms raised by the reviewers. ============================== Please submit your revised manuscript by Oct 27 2022 11:59PM. 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We will update your Data Availability statement to reflect the information you provide in your cover letter. 7. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Additional Editor Comments: Albeit the paper is of interest some alterations are needed: q-RT PCR data must be confirmed by the relative protein expression in WB. In addition, Alizarin Red staining in the in vitro samples must be added. Some recent papers must be added to the References. [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: 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 ****** 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: In this study Authors aimed to compare the ability of HA and collagen hydrogels in supporting in vitro differentiation of MSC-based hypertrophic cartilage tissues for promoting endochondral bone formation in vivo. The paper is interesting and the experiments are well conducted. In any case, some changes are needed. Authors should update the bibliography: there are more recent papers published in the matter (Cells. 2021 Oct 26;10(11):2899. doi: 10.3390/cells10112899; Acta Biomater. 2021 Mar 15;123:364-378. doi: 10.1016/j.actbio.2020.12.056.) q-RT PCR data should be confirmed by the relative protein expression through WB. Moreover, it would be interesting to perform also Alizarin Red staining on in vitro samples. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Revision 1
26 Dec 2022 Author Response Response to Reviewer ＃1 1. Reviewer’s comment: q-RT PCR data must be confirmed by the relative protein expression in WB. Thank you very much for your critical comment. As suggested by the reviewer, we analyzed the relative protein expression of col1, col2, and ACAN by Western blot analysis (S1 Fig). First of all、it has been pointed out that the metabolic pathways and cellular structures change considerably when MSCs differentiate into chondrocytes, making it inappropriate to use typically used proteins as loading controls (such as GAPDH). Therefore, in this study, we used DNA content for normalizing sample quantities in Western blotting and the YWHAZ gene as a reference gene for q-RT PCR [35]. Consistent with the q-RT PCR data, undifferentiated MSCs expressed Col I but no or very low levels of ACAN and Col II. Although q-RT PCR analysis revealed that Col I, Col II, and ACAN transcript levels decreased from 3W to 5W, except for ACAN in the HA constructs, there was an increased accumulation of Col I, Col II, and ACAN proteins at 5W than 3W, indicating that these proteins continued to accumulate in the constructs, even though their mRNA expression declines during hypertrophic differentiation. Sox-9 and MMP13 expression was confirmed by immunohistological staining (S2 Fig), because they were difficult to detect by Western blot analysis. Sox-9expression was observed in cells with fibroblast-like morphology in the cartilage at the margins of the col constructs at 3W. However, no expression was found in the well-differentiated center of the col or HA constructs, which is consistent with the fact that Sox-9 expression peaks at 3 days after MSC induction of chondrogenic differentiation, and after 3 weeks, the expression levels drop to undetectable by Western blotting (Weissenberger et al. BMC Musculoskeletal Disorders (2020) 21:109). Consistent with the q-RT PCR data, MMP-13 protein was not detected at 3W for both constructs; MMP-13 expression at 5W was confined to the marginal area in the collagen constructs, whereas it was observed in the vicinity of chondrocytes throughout the HA construct. Finally, consistent with the q-RT PCR data, the expression of Col X and OCN proteins was detected at both 3W and 5W. Collectively, these results demonstrated that the differentiation markers of cartilage and hypertrophic cartilage are expressed in both HA and collagen constructs, although differences in mRNA and protein expression levels are observed. Raw images of Western blot are submitted as ‘S1_raw_images’. 2. Reviewer’s comment: Authors should update the bibliography. Thank you very much for your helpful comment. We have mentioned and cited the recent papers suggested by the reviewer in Introduction (Lines 62-64) [31,32]. 3. Reviewer’s comment: Alizarin Red staining in the in vitro samples must be added. As suggested by the reviewer, we have included the data showing Alizarin Red staining in the in vitro samples (Fig 2C). As seen with Von Kossa staining, strong staining was observed limited to the outer edges in both constructs. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0281345.r002
23 Jan 2023 Decision Letter - Gianpaolo Papaccio, Editor Hyaluronic acid hydrogels support to generate integrated bone formation through endochondral ossification in vivo using mesenchymal stem cells PONE-D-22-24282R1 Dear Dr. Ikeda, 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, Gianpaolo Papaccio, M.D., Ph.D. 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: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) ****** 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 Response) ****** 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: (No Response) ****** 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: (No Response) ****** 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.0281345.r003
Formally Accepted
25 Jan 2023 Acceptance Letter - Gianpaolo Papaccio, Editor PONE-D-22-24282R1 Hyaluronic acid hydrogels support to generate integrated bone formation through endochondral ossification in vivo using mesenchymal stem cells Dear Dr. Ikeda: 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 Prof. Gianpaolo Papaccio Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0281345.r004

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