Nerve regeneration using a Bio 3D conduit derived from umbilical cord–Derived mesenchymal stem cells in a rat sciatic nerve defect model

Terunobu Iwai; Ryosuke Ikeguchi; Tomoki Aoyama; Takashi Noguchi; Koichi Yoshimoto; Daichi Sakamoto; Kazuaki Fujita; Yudai Miyazaki; Shizuka Akieda; Tokiko Nagamura-Inoue; Fumitaka Nagamura; Koichi Nakayama; Shuichi Matsuda

doi:10.1371/journal.pone.0310711

Peer Review History

Original SubmissionMarch 2, 2024
7 May 2024 Decision Letter - Atsushi Asakura, Editor PONE-D-24-05750Nerve regeneration using a Bio 3D conduit derived from umbilical cord–derived mesenchymal stem cells in a rat sciatic nerve defect modelPLOS ONE Dear Dr. Ikeguchi, 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 Jun 21 2024 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. 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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. We notice that your supplementary figures are included in the manuscript file. Please remove them and upload them with the file type 'Supporting Information'. Please ensure that each Supporting Information file has a legend listed in the manuscript after the references list. [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: No Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No 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: No 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: No 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: In this study, the authors utilized a Kenzan-based approach to engineer 3D biological conduits made from umbilical cord-derived mesenchymal stem cells (UC-MSCs). The therapeutic efficacy of these engineered bio 3D conduits was evaluated in vivo using a rat model of sciatic nerve transection. This research presents an interesting application of Kenzan technology, but its impact could be further amplified by addressing several areas of concern: 1. The Abstract does not explain the fabrication process of the conduit. 2. The Introduction provides limited background information. MSCs have been previously used in artificial nerve conduits to enhance peripheral nerve regeneration. The study should clarify the key differences between its approach and those documented in existing literature, particularly addressing the limitations of MSC-incorporated conduits that this study overcomes. 3. The description of the current methods is overly simplistic, lacking the detail necessary to accurately replicate the results of this study. For example, the methodology for inducing MSC spheroid formation and the specific parameters utilized in the Kenzan technique should be thoroughly detailed. 4. The bio 3D conduits require comprehensive characterization. 5. Including a healthy control group in the pinprick and toe-spread tests, kinematic analysis, and electrophysiological studies would significantly enhance the manuscript's quality. 6. For the analyses of compound muscle action potential and nerve conduction velocity, presenting representative waveforms triggered by stimulation could improve the quality of the manuscript. 7. In Figure 3D, the weight of the tibialis anterior muscle in the affected limb should be normalized to that of the healthy contralateral limb for better objectivity. 8. Figures 5A-C should include labels for the colors and their corresponding targets. 9. A typographical error "Merage" was identified in Figure 5F. 10. The sequence of presenting data in the Results section is unconventional, discussing Figures 6A-6F, then Figure 7, followed by Figures 6G-6K. 11. The results from the plasma cytokine analysis should be incorporated into one of the figures in the manuscript. 12. The Discussion section is currently too brief and lacks depth. A more comprehensive analysis and interpretation of the findings are warranted. 13. The rationale for using two different strains of rats in a study that employs human-derived UC-MSCs is unclear. 14. The manuscript should clearly define the symbols used to denote statistical analysis results in the figure captions. 15. The manuscript would benefit from improvements in English language use and overall flow. Reviewer #2: This article is to research a bio 3D conduit derived from umbilical cord–derived mesenchymal stem cells for peripheral nerve injury. The authors seem to aim a clinical application of this conduit. It is very important and timely to develop the strategy to repair peripheral nerve injury by regenerative medicine, thus the authors’ concept is reasonable and scientific in this meaning. However, some major deficiencies are seen in this article. These are described below: Major points: 1. The authors should put a negative control group. For all meanings, this study completely lacks the negative control group, such as rats which were only undergone sciatic nerve cut (5mm gap). The reviewer could not assess all data whether a 3D conduit effect and an autografting effect, the differences of allo-reactive reaction or foreign body reaction and normal peripheral regeneration or not. 2. The authors should explain the reason why actual function (ex. Pinprick test/ toe-spread test, DT, even though AoA showed significance) did not show differences even though the regenerative histology showed differences. According to these data, the conclusion resulted that 3D conduit accelerate peripheral nerve injury histologically, however, did not affect functional recovery. Nobody will agree that 3D conduit should be used for clinical application. 3. Did Figure 1D and 4A show 3D conduit or sciatic nerve itself? Concerning Figure 1C, sciatic nerve seemed inside silicon tube (the small knots could be pointed out lower part of Figure 1D. Does it mean that the whitish sheath remained?) Was a 3D conduit absorbed after 8 weeks? Did the authors investigate HLA immunohistochemistry to investigate which tissue was regenerated? 4. The conclusion of xeno- or allo-transplantation immunity was premature. At first, where were infiltrative mononuclear cells in Figure 6G, H and I? The reviewer completely confused whether the infiltrative mononuclear cells existed or not in these figures. Secondly, why the authors only showed 7 post-operative day? Thirdly, why the authors investigated pan-T (CD3) only? The data must differ from infiltrated monocytes and pan-T cells. 5. Authors must show the citations if the authors stated that “UC-MSCs are known to have a greater paracrine effect than bone marrow–derived MSCs or adipose derived MSCs, and are presumed to be superior for nerve regeneration” in Discussion section. Minor point: 1. Figure 6K is poor. ****** 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. 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.0310711.r001
Revision 1
23 Jun 2024 Author Response Reviewer #1 Thank you very much for reviewing our manuscript. We agree with your comments and suggestions and have revised our manuscript accordingly. 1. The Abstract does not explain the fabrication process of the conduit. Thank you for your valuable comment. According to your suggestion, we added the sentence to describe the fabrication process of the conduit in the Abstract (line 37-38). 2. The Introduction provides limited background information. MSCs have been previously used in artificial nerve conduits to enhance peripheral nerve regeneration. The study should clarify the key differences between its approach and those documented in existing literature, particularly addressing the limitations of MSC-incorporated conduits that this study overcomes. Thank you for your valuable comment. According to your suggestion, we added the sentence to describe the key differences from our previous studies (line72-82). 3. The description of the current methods is overly simplistic, lacking the detail necessary to accurately replicate the results of this study. For example, the methodology for inducing MSC spheroid formation and the specific parameters utilized in the Kenzan technique should be thoroughly detailed. Thank you for your instruction. According to your instruction, we added the sentences to explain the methods (line103 -116). 4. The bio 3D conduits require comprehensive characterization. Thank you for your valuable comment. According to your suggestion, we added the sentence to describe the characterization of the Bio 3D conduits (line115-116). 5. Including a healthy control group in the pinprick and toe-spread tests, kinematic analysis, and electrophysiological studies would significantly enhance the manuscript's quality. Thank you for your instruction. In the pinprick test and toe-spread tests, the healthy group received a Grade 3. Regarding DT, the healthy control group has a value close to 0. Regarding AoA, the surgical treatment group takes a negative value, whereas the healthy control group takes a positive value. In electrophysiological studies, evaluation was performed based on the ratio of the affected side to the healthy side. According to your suggestion, we added the sentence to describe the control group in the Discussion and reference 14 (line39-403) . 6. For the analyses of compound muscle action potential and nerve conduction velocity, presenting representative waveforms triggered by stimulation could improve the quality of the manuscript. Thank you for your instruction. According to your instruction, we added the waveforms in Figure 2 7. In Figure 3D, the weight of the tibialis anterior muscle in the affected limb should be normalized to that of the healthy contralateral limb for better objectivity. Thank you for your valuable comment. According to your suggestion, we added the ratio of the wet muscle weight of the operated side to that of the heathy side. We also added the sentences to describe the methods and results (line 209-210) (line 326-329). We revised Figure 3. 8. Figures 5A-C should include labels for the colors and their corresponding targets. Thank you for your instruction. According to your instruction, we added the labels for the colors and their corresponding targets in Figure 5. We also revise the figure legend of Figure 5. 9. A typographical error "Merage" was identified in Figure 5F. We appreciate your pointing out the mistake. We replace "Merage" with “Merge” in Figure 5. 10. The sequence of presenting data in the Results section is unconventional, discussing Figures 6A-6F, then Figure 7, followed by Figures 6G-6K. Thank you for your instruction. According to your instruction, we put the order of Mononuclear cell infiltration ~ Immunohistochemistry to make it easier to understand and arrange the photos and graphs of Figure 6. 11. The results from the plasma cytokine analysis should be incorporated into one of the figures in the manuscript. Thank you for your instruction. According to your instruction, we have described cytokines in Figure 8. However, the editor recommend us upload them as Supporting Information. 12. The Discussion section is currently too brief and lacks depth. A more comprehensive analysis and interpretation of the findings are warranted. Thank you for your instruction. According to your instruction, we added the sentences in the Discussion section (line 394-412) (line 439-451). 13. The rationale for using two different strains of rats in a study that employs human-derived UC-MSCs is unclear　to evaluate acute graft rejection, in the second experiment, the Bio 3D group was compared with the autograft group and the allograft group (in which nerve segments harvested from Brown Norway rats were transplanted to Lewis rats) 1 week after surgery. Thank you for your instruction. According to your instruction, we added the sentences in the Discussion section to explain the reason of using two different strains of rats (line 439-445). 14. The manuscript should clearly define the symbols used to denote statistical analysis results in the figure captions. Thank you for your instruction. According to your instruction, we added replace the symbols with the statistical analysis results in figures. 15. The manuscript would benefit from improvements in English language use and overall flow. Thank you for your instruction. According to your instruction, we asked again the company of proofreading to improve English. Reviewer #2: Thank you very much for reviewing our manuscript. We agree with your comments and suggestions and have revised our manuscript accordingly. 1. The authors should put a negative control group. For all meanings, this study completely lacks the negative control group, such as rats which were only undergone sciatic nerve cut (5mm gap). The reviewer could not assess all data whether a 3D conduit effect and an autografting effect, the differences of allo-reactive reaction or foreign body reaction and normal peripheral regeneration or not. Thank you for your valuable comment. Scaffolds play an important role in nerve regeneration. The purpose of this study was to compare cross-linked materials for nerve regeneration evaluation, and a silicone group that has a luminal structure that prevents scar invasion but does not contain growth factors was used as a negative control. According to your suggestion, we added the sentences in conclusion as our study limitation and reference 15 (line 394-403). 2. The authors should explain the reason why actual function (ex. Pinprick test/ toe-spread test, DT, even though AoA showed significance) did not show differences even though the regenerative histology showed differences. According to these data, the conclusion resulted that 3D conduit accelerate peripheral nerve injury histologically, however, did not affect functional recovery. Nobody will agree that 3D conduit should be used for clinical application. Thank you for your instruction. According to your instruction, we added the explanations in Discussion (line 406-412). 3. Did Figure 1D and 4A show 3D conduit or sciatic nerve itself? Concerning Figure 1C, sciatic nerve seemed inside silicon tube (the small knots could be pointed out lower part of Figure 1D. Does it mean that the whitish sheath remained?) Was a 3D conduit absorbed after 8 weeks? Did the authors investigate HLA immunohistochemistry to investigate which tissue was regenerated? Thank you for your valuable comment. In Figure 1D, most of the nerve appears to be regenerated sciatic nerve. In Figure 4A, we replaced the figure to represent that most of the nerves appear to be regenerated in sciatic nerve and 3D conduit absorbed after 8 weeks. According to your comments, we add the sentences in Discussion (line 479-484). 4. The conclusion of xeno- or allo-transplantation immunity was premature. At first, where were infiltrative mononuclear cells in Figure 6G, H and I? The reviewer completely confused whether the infiltrative mononuclear cells existed or not in these figures. Secondly, why the authors only showed 7 post-operative day? Thirdly, why the authors investigated pan-T (CD3) only? The data must differ from infiltrated monocytes and pan-T cells. Thank you for your valuable comment. According to your comments, we added the arrow to show infiltrative mononuclear cells (Figure5). We also add the explanation in figure legend of Figure 5.　We also add the explanation in the Discussion section and reference 22 and 28 (line445 -451) (line 479-487). 5. Authors must show the citations if the authors stated that “UC-MSCs are known to have a greater paracrine effect than bone marrow–derived MSCs or adipose derived MSCs, and are presumed to be superior for nerve regeneration” in Discussion section. Thank you for your valuable comment. According to your suggestion, we added reference 15 (line 430). Minor point: 1. Figure 6K is poor. Thank you for your instruction. According to your instruction, we modified the figure (Figure 5D in revised version). Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0310711.r002
6 Sep 2024 Decision Letter - Atsushi Asakura, Editor Nerve regeneration using a Bio 3D conduit derived from umbilical cord–derived mesenchymal stem cells in a rat sciatic nerve defect model PONE-D-24-05750R1 Dear Dr. Ikeguchi, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Atsushi Asakura, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0310711.r003
Formally Accepted
14 Oct 2024 Acceptance Letter - Atsushi Asakura, Editor PONE-D-24-05750R1 PLOS ONE Dear Dr. Ikeguchi, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Atsushi Asakura Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0310711.r004

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