Decellularized vascularized bone grafts as therapeutic solution for bone reconstruction: A mechanical evaluation

Ugo Heller; Robin Evrard; Benoit Lengelé; Thomas Schubert; Natacha Kadlub; Jean Boisson

doi:10.1371/journal.pone.0280193

Peer Review History

Original SubmissionJuly 11, 2022
10 Aug 2022 Decision Letter - Mohammad Azadi, Editor PONE-D-22-19559Decellularized vascularized bone grafts as therapeutic solution for bone reconstruction: A mechanical evaluationPLOS ONE Dear Dr. Heller, 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 Sep 24 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. 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Mohammad Azadi Academic Editor PLOS ONE Journal Requirements: 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. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: "The authors would like to thank the Fondation des Gueules Cassées for its help and support for this study." 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: "This work was supported by Fondation des Gueules Cassées (Application N°41-2020, France) granted by Dr. UH 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. 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: 1) Quantitative results should be added to the abstract. 2) Using 42 references in the introduction makes the novelty unclear. It is better that the innovation is highlighted in the introduction, compared to the literature review. 3) All formulations need references. 4) The discussion is poor. Obtained results must be compared to other results of other similar publications. 5) The scale bar should be added to Figures 2, 4, 5, 6, and 7. 6) It is better to add a table to introduce the samples. What was the repeatability of testing? The number of specimens could be added to this new table. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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: 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 ****** 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: 1. Both protocols were completed by rinsing with PBS. It neutralizes the reaction of previously used detergents, however, the protocol should be completed by rinsing with deionized or distilled water to minimize the presence of detergent microelements in the graft. Maybe you used it but didn't mention it. Please add or explain if you don't use it how you confirm the complete release of detergent elements from the graft. 2. There are no legends (explanation) on figures and tables, what makes them difficult to understand. 3. Why are the figure titles in the middle of the text and not next to the figures or separated? What does it mean on line 206 "a) Pull-out Screw Test (Fig 4)" I did not understand why it is written here. I think it is not necessary to use A and B as a test, just name it simply. 4. On line 339 (in the results) the abbreviation GLMM appears. I think you mean "General Linear Mixed Model", but it is not explained in the text. Please explain all abbreviations. Reviewer #2: PEER PAPER REVIEW INSTRUCTIONS/TEMPLATE Title: Biofunctionalization of 3D printed porous tantalum using Vancomycin-carboxymethyl chitosan composite coating to improve osteogenesis and anti-biofilm properties Authors: liu, tuozhou; Xiangya Journal / ID: am-2022-11715k Decision: Minor Revision Summary: In this study, the porcine forearms were used as the experimental sites and two methods (SDS; NaOH and H2O2) were adopted to decellularize them separately. The treated samples as well as the natural porcine bone were then subjected to biomechanical tests, specifically including compression, three-point bending, indentation and screw pull-out tests.The results showed similar performance from pull-out screw, compression, 3-point bending and indentation tests carried out on bones decellularized with the SDS protocol and native bones. Bones decellularized with the NaOH protocol showed different results from those obtained with the SDS protocol or native bones during the pull-out screw and 3-point bending tests. The other tests, compression and indentation, gave similar results for all their samples.This article shows that decellularization can have an effect on the biomechanical properties of natural bone and provides insights into which methods might reduce this effect. Critique: General Comments • Materials and Methods: After bone grafting at the site of bone defect, the biomechanical properties of the implant may be altered due to the in vivo biological environment. And this of the biomechanical performance of the decellularized sample in the organism precisely determines whether the defect site has a good osseointegration effect or not. Therefore, it is suggested that the authors may consider the inclusion of in vivo experiments. •Discussion In this article, Sodium Dodecyl Sulfate (SDS) and NaOH and H2O2 substitute based on the perfusion-decellularisation technique were chosen as the two variables of this experiment, so the interactions between these two variables and more relevant research are suggested to add into the discussion section, in order to make this manuscript more logical and complete. Specific Comments: •Abstract Line44: A full stop is missing in the last sentence, please add it. • Materials and Methods: Line 160-166:The operation steps of Protocol 2(D2) are not written in detail. Line 220:It is not stated exactly how many times the experiment was repeated. • Results： Line 387：Is there supposed to be a minus sign in front of 72.7. ****** 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 Reviewer #2: 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. 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Revision 1
14 Sep 2022 Author Response Dear Editor, Dear Reviewers, We thank you for having considered our paper entitled: Decellularized vascularized bone grafts as therapeutic solution for bone reconstruction: A mechanical evaluation. We would like to sincerely thank you for your relevant comments and questions that will significantly help us to enhance the quality of the article. In the following section, we respond to specific comments from reviewers. We also pointed in the article the main changes, that we colored according to one who induced it: Academic editor, Reviewer #1, Reviewer #2. We thank you for considering another time this reviewed manuscript. Academic editor, 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. We tried to ensure that our manuscript was as close as possible to the recommendations made by PLOS ONE. The article should now meet PLOS ONE's style requirements. 2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. Thank you for pointing out this lack. We have added this to our manuscript 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: "The authors would like to thank the Fondation des Gueules Cassées for its help and support for this study." 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: "This work was supported by Fondation des Gueules Cassées (Application N°41-2020, France) granted by Dr. UH The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." We apologize for this confusion. We have removed this part in Acknowledgments. The Funding Statement complete, now, your recommendations 4. 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 This was modified and added in the manuscript. 5. Quantitative results should be added to the abstract. We added quantitative results in the manuscript. 6. Using 42 references in the introduction makes the novelty unclear. It is better that the innovation is highlighted in the introduction, compared to the literature review. We have reduced the number of references in the introduction and try to express the novelty of our work by pointing out that this is the first time that this kind of mechanical comparison is performed. We also have tried to highlight the novelty of our paper in the introduction. 7. All formulations need references. Added in the manuscript. 8. The discussion is poor. Obtained results must be compared to other results of other similar publications. Thank you for your comment. We have added comparisons with the literature to enrich our discussion, but as it is the first time this kind of comparison is done the literature is poor on the subject. 9. The scale bar should be added to Figures 2, 4, 5, 6, and 7. Done. 10. It is better to add a table to introduce the samples. What was the repeatability of testing? The number of specimens could be added to this new table. Thank you for this relevant comment. We added a table to make sample allocations and repeatability easier for the reader to understand. Reviewer #1: 1. Both protocols were completed by rinsing with PBS. It neutralizes the reaction of previously used detergents; however, the protocol should be completed by rinsing with deionized or distilled water to minimize the presence of detergent microelements in the graft. Maybe you used it but didn't mention it. Please add or explain if you don't use it how you confirm the complete release of detergent elements from the graft. Thank you for bringing this to our attention. Indeed, we did wash with deionized water between each stage. We have added this point to the text (line 163 and line 170). 2. There are no legends (explanation) on figures and tables, what makes them difficult to understand. We have added legends to the figures and tables to make them more understandable to the reader. 3. Why are the figure titles in the middle of the text and not next to the figures or separated? We have tried to follow the publisher's recommendations. The figures are on a separate file and the titles and legends are in the manuscript at the request of the editor. We have uploaded a version of the manuscript with images within it for better readability. 4. What does it mean on line 206 "a) Pull-out Screw Test (Fig 4)" I did not understand why it is written here. I think it is not necessary to use A and B as a test, just name it simply. Done. 5. On line 339 (in the results) the abbreviation GLMM appears. I think you mean "General Linear Mixed Model", but it is not explained in the text. Please explain all abbreviations. Thank you for pointing that out. We corrected it in the text. Reviewer #2: 1. After bone grafting at the site of bone defect, the biomechanical properties of the implant may be altered due to the in vivo biological environment. And this of the biomechanical performance of the decellularized sample in the organism precisely determines whether the defect site has a good osseointegration effect or not. Therefore, it is suggested that the authors may consider the inclusion of in vivo experiments. Dear reviewer, thank you for this pertinent comment. We are currently planning an in vivo study on bone grafts decellularized by vascular injection. This study will be conducted over the coming year and will be the subject of an article on the histological, mechanical, and biological results. 2. In this article, Sodium Dodecyl Sulfate (SDS) and NaOH and H2O2 substitute based on the perfusion-decellularisation technique were chosen as the two variables of this experiment, so the interactions between these two variables and more relevant research are suggested to add into the discussion section, in order to make this manuscript more logical and complete. The SDS and NaOH protocols are currently the most promising in our research area. Most of the recent articles on decellularization focus on these methods of decellularization by varying the concentrations of these products. We have added a section in the discussion to try to explain our choice to the readers. 3. Line44: A full stop is missing in the last sentence, please add it. Done. 4. Line 160-166: The operation steps of Protocol 2(D2) are not written in detail. The detailed protocol is the subject of a specific article currently in redaction. 5. Line 220: It is not stated exactly how many times the experiment was repeated. This was missing from our article and was also pointed out by the editor. We have therefore added a paragraph to our article and a descriptive table. We hope to have made the information more understandable. 6. Line 387：Is there supposed to be a minus sign in front of 72.7. Thank you for the remark. We have corrected in the manuscript Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0280193.r002
3 Oct 2022 Decision Letter - Mohammad Azadi, Editor PONE-D-22-19559R1Decellularized vascularized bone grafts as therapeutic solution for bone reconstruction: A mechanical evaluationPLOS ONE Dear Dr. Heller, 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 Nov 17 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. 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Mohammad Azadi 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. Additional Editor Comments: 1) Figure 2 still has no scale bar. It must be mention on the image and not in the title. 2) All formulations need references, unless they were extracted by the authors. 3) The discussion is still poor. The authors do not need to find the exact topic! The results could be trendly compared to other similar results. These results are in the references that the authors have used them for a comparison in the literature. 4) Using some sentences about that there is no literature for the discussion is not proper and they must be removed or rewritten in a proper state. [Note: HTML markup is below. Please do not edit.] 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 #2: (No Response) ******** 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 #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: 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 #2: Yes ****** 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 #2: 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 #2: Based on perfusion-decellularized technique, this article compared the biomechanical differences between decellularized vascularized bone grafts prepared by different decellularization protocols and untreated samples. Overall, there are some flaws existing. I hope these suggestions will be helpful, and the article will meet the publication standards after revision. 1. This article proves that the decellularization method affects the mechanical results. However, the description of the decellularization method is vague. A detailed description is needed, such as the duration and frequency of each step, the end point of decellularization. These may also have some influence on the biomechanical properties. It is better to supplement this information. 2. Line 193: Fig 2. The results of histologic analysis before decellularization are blurred and unclear. In addition, it would be more convincing if the results of histological obtained by different decellularization methods were compared with those before decellularization. 3. Line 214: The annotations of Table 1 does not indicate the meaning of X properly. 4. The discussion of biomechanical differences between the two methods is not sufficient. In addition, Line 544-546, "NaOH and H2O2 cause demineralization of the cortical bone (57)". The presentation does not correspond to the original text of the literature. ****** 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 #2: 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.0280193.r003
Revision 2
15 Nov 2022 Author Response Dear Editor, Dear Reviewers, We thank you for having considered our paper entitled: Decellularized vascularized bone grafts as therapeutic solution for bone reconstruction: A mechanical evaluation. We would like to thank you for your comments and questions that will help us to enhance the quality of our article. In the following section, we respond to specific comments from reviewers. We also pointed in the article the main changes, that we colored according to one who induced it: Academic editor, Reviewer #1. We thank you for considering another time this reviewed manuscript. Academic editor, 1. Figure 2 still has no scale bar. It must be mention on the image and not in the title. We have changed figure 2 to a more readable figure with a scale bar included 2. All formulations need references unless they were extracted by the authors. Thank you for pointing out this lack. We have added this to our manuscript. However, we are not sure what you mean by "formulation". We understood that (1) we had to put the references of the products used: ex DNase I from bovine pancreas, Sigma- AldrichÒ, Darmstadt, Germany (2) we have also made a call in the text to the mathematical formulas (in the previous revision) ex L245 3. The discussion is still poor. The authors do not need to find the exact topic! The results could be trendly compared to other similar results. These results are in the references that the authors have used them for a comparison in the literature. We have tried to support our discussion by adding comparisons with the literature on the subject. 4. Using some sentences about that there is no literature for the discussion is not proper and they must be removed or rewritten in a proper state. We changed this part when we modified the discussion. 5. 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. We have checked all our references and have not found any retracted items. However, we have added a reference from Ulklejewski (n°58 in our article) which we found interesting to illustrate our discussion. Reviewer #1: 1. This article proves that the decellularization method affects the mechanical results. However, the description of the decellularization method is vague. A detailed description is needed, such as the duration and frequency of each step, the end point of decellularization. These may also have some influence on the biomechanical properties. It is better to supplement this information. Thank you for the notification. Decellularization via the H2O2 process is not fully described in our article. We currently do not have the rights to publish this method, which will be published shortly in a full article on the subject. This decellularization method has already been published in 2018 (Van Steenberghe and al, Annals of Vascular Surgery, 2018). We know that this is an important gap in our paper as the concentrations and infusion times can change our results. 2. Line 193: Fig 2. The results of histologic analysis before decellularization are blurred and unclear. In addition, it would be more convincing if the results of histological obtained by different decellularization methods were compared with those before decellularization. We have modified this figure with a more readable and clearer one. We have not added an image according to the type of decellularization (SDS vs H2O2) because the differences are too small in photographic level. 3. Line 214: The annotations of Table 1 does not indicate the meaning of X properly. We modified this part in the manuscript. 4. The discussion of biomechanical differences between the two methods is not sufficient. In addition, Line 544-546, "NaOH and H2O2 cause demineralization of the cortical bone (57)". The presentation does not correspond to the original text of the literature. Thank you for notifying us of this. We have modified the part concerning this reference and we have expanded the conclusion of this article to make it more precise. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0280193.r004
28 Nov 2022 Decision Letter - Mohammad Azadi, Editor PONE-D-22-19559R2Decellularized vascularized bone grafts as therapeutic solution for bone reconstruction: A mechanical evaluationPLOS ONE Dear Dr. Heller, 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 Jan 12 2023 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Mohammad Azadi 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. Additional Editor Comments: Before the acceptance of the revised article, a further revision should be done based on the following comments. 1) Formulations mean equations! The authors have 4 formulation for stress, strain, etc. All they need references. 2) The authors mentioned "We currently do not have the rights to publish this method, which will be published shortly in a full article on the subject. This decellularization method has already been published in 2018 (Van Steenberghe and al, Annals of Vascular Surgery, 2018).". All these description should be fully described in the text. Moreover, the future work could be mentioned at the end part of the conclusion. 3) No references could be found for 2022 and 2021! This issue is not acceptable for a scientific article, which references are not updated. 4) No separated part could be found for the conclusion! Moreover, this part should be rewritten one by one, in bullets, to show the novelty with quantitative data. 5) The standard should be mentioned for all testing types. 6) No quantitative results could be found in the abstract. 7) The quality of Figure 7 is not proper. [Note: HTML markup is below. Please do not edit.] [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.0280193.r005
Revision 3
21 Dec 2022 Author Response Dear Editor, We thank you for having considered our paper entitled: Decellularized vascularized bone grafts as therapeutic solution for bone reconstruction: A mechanical evaluation. We would like to thank you for your comments and questions that will help us to enhance the quality of our article. In the following section, we respond to specific comments from the Academic Editor. We also pointed in the article the main changes, that we colored in blue. We thank you for considering another time this reviewed manuscript. 1. Formulations mean equations! The authors have 4 formulations for stress, strain, etc. All they need references Sorry for this confusion. We added references to all the equations cited in our paper. 2. The authors mentioned "We currently do not have the rights to publish this method, which will be published shortly in a full article on the subject. This decellularization method has already been published in 2018 (Van Steenberghe and al, Annals of Vascular Surgery, 2018).". All these description should be fully described in the text. Moreover, the future work could be mentioned at the end part of the conclusion Thank you for this comment. We tried to describe the protocol with as much details as possible in the manuscript and we also added a sentence in the conclusion on the future work. 3. No references could be found for 2022 and 2021! This issue is not acceptable for a scientific article, which references are not updated. We have updated our references to include the latest articles on the subject. For this reason, we have added the following articles: - Parmaksiz M, Elçin AE, Elçin YM. Biomimetic 3D-Bone Tissue Model. Methods Mol Biol. 2021;2273:239‑50. - Wüthrich T, Lese I, Haberthür D, Zubler C, Hlushchuk R, Hewer E, et al. Development of Vascularized Nerve Scaffold Using Perfusion-Decellularization and Recellularization ». Materials Science & Engineering C, Materials for Biological Applications. déc 2020;117:111311. - Gerli MFM, Guyette JP, Evangelista-Leite D, Ghoshhajra BB, Ott HC. Perfusion decellularization of a human limb: A novel platform for composite tissue engineering and reconstructive surgery. PLoS One. 2018;13(1):e0191497. - Kurokawa S, Hashimoto Y, Funamoto S, Murata K, Yamashita A, Yamazaki K, et al. In vivo recellularization of xenogeneic vascular grafts decellularized with high hydrostatic pressure method in a porcine carotid arterial interpose model. PLOS ONE. 22 juill 2021;16(7):e0254160. - Fooladi S, Faramarz S, Dabiri S, Kajbafzadeh A, Nematollahi MH, Mehrabani M. An efficient strategy to recellularization of a rat aorta scaffold: an optimized decellularization, detergent removal, and Apelin-13 immobilization. Biomater Res. 22 sept 2022;26(1):46. - Seiffert N, Tang P, Keshi E, Reutzel-Selke A, Moosburner S, Everwien H, et al. In vitro recellularization of decellularized bovine carotid arteries using human endothelial colony forming cells. J Biol Eng. 21 avr 2021;15(1):15. - Wang X, Chan V, Corridon PR. Decellularized blood vessel development: Current state-of-the-art and future directions. Front Bioeng Biotechnol. 2022;10:951644. - Adil A, Xu M, Haykal S. Recellularization of Bioengineered Scaffolds for Vascular Composite Allotransplantation. Front Surg. 25 mai 2022;9:843677. 4. No separated part could be found for the conclusion! Moreover, this part should be rewritten one by one, in bullets, to show the novelty with quantitative data. We have added a conclusion with bullet points to our article. 5. The standard should be mentioned for all testing types. Apart from the indentation tests - to which we added the standard reference - the mechanical tests we performed are not part of any standard procedure because they were applied to non-standardized samples. Therefore, no standardization process could be applied, even if the lab is ISO9001 certified. 6. No quantitative results could be found in the abstract. We have changed our abstract to include more quantitative results 7. The quality of Figure 7 is not proper. We have used a new figure. We wish this one to be more suitable. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0280193.r006
22 Dec 2022 Decision Letter - Mohammad Azadi, Editor Decellularized vascularized bone grafts as therapeutic solution for bone reconstruction: A mechanical evaluation PONE-D-22-19559R3 Dear Dr. Heller, 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, Mohammad Azadi Academic Editor PLOS ONE Additional Editor Comments (optional): It is done. Reviewers' comments: https://doi.org/10.1371/journal.pone.0280193.r007
Formally Accepted
5 Jan 2023 Acceptance Letter - Mohammad Azadi, Editor PONE-D-22-19559R3 Decellularized vascularized bone grafts as therapeutic solution for bone reconstruction: A mechanical evaluation Dear Dr. Heller: 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. Mohammad Azadi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0280193.r008

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