Single-cell RNA sequencing of healthy and diseased rat temporomandibular joint condyle cartilage

Sara Trbojevic; Xudong Dong; Robert Lafyatis; Michael S. Gold; Juan M. Taboas; Alejandro J. Almarza

doi:10.1371/journal.pone.0348175

Peer Review History

Original SubmissionOctober 1, 2025
9 Dec 2025 Decision Letter - Esmaiel Jabbari, Editor Dear Dr. Almarza, 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 23 2026 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.. 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.. 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.. 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. 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. 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Read more information on sharing protocols at . 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, Esmaiel Jabbari, PhD 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. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. [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? Reviewer #1: Yes Reviewer #2: No Reviewer #3: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: No Reviewer #3: 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.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: No Reviewer #3: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** Reviewer #1: 1. The authors state that cells from all n=8 animals per group were pooled for analysis to ensure sufficient cell yield. This is a significant limitation, as it precludes the assessment of biological variability between individuals and, crucially, any formal statistical testing for differences between the naïve and splinted conditions (e.g., using a mixed-model approach accounting for multiple biological replicates). This critical limitation must be explicitly stated and emphasized in the Discussion, and the conclusions should be framed with appropriate caution. We recommend explicitly mentioning this design choice and its implications for statistical inference in the main text (e.g., Methods or Results) and the Discussion. 2. Due to the lack of perfusion, immune cell populations were excluded from analysis as they were considered potentially circulatory. However, tissue-resident immune cells (e.g., macrophages in the synovium) play a key role in OA. This issue should be more thoroughly addressed by (1) explicitly stating this as a limitation in the Methods and (2) providing a more in-depth discussion of how this decision might impact the interpretation of the inflammatory microenvironment in OA. 3. The filtering criteria for "fibrocartilage cells" need clearer description. What specific marker genes or clustering results informed this selection? Furthermore, details of the trajectory inference, specifically the process and exact criteria for selecting the root nodes, should be elaborated upon to ensure the analysis is reproducible. 4. Given the identification of multiple cell types, leveraging the existing scRNA-seq data to infer potential intercellular communication changes in OA (using tools like NicheNet or CellChat) could provide deeper insights into the pathological mechanisms. This could be a valuable addition for future consideration if feasible. 5. The Discussion could briefly contextualize how the cell subsets and key pathways identified (e.g., cholesterol metabolism, NGF signaling) in this rat TMJOA model compare or contrast with known pathways in human TMJOA or peripheral joint OA, enhancing the potential translational relevance of the findings. Reviewer #2: The manuscript analyzed the unique cell populations present in healthy and OA-induced condylar cartilage of adult rats through single-cell RNA-sequencing. However, the analysis is too rough and need to do further and deeper experiments. 1. Figure 1 need add further experiments, such as S.O. staining, Micro-CT images and Quantitative analysis. 2. Single cell analysis figures can put together to be one figure. Further experiments such as Westernblot verification for OA Target Genes should be done. Reviewer #3: This study presents the first single-cell landscape of the rat TMJ condyle in both health and a mechanically induced OA model. The identification of "Pre-chondrocytes" (PreC) and "Chondrocyte Progenitors" (ProC) provides valuable insight into the unique cellular architecture of the mandibular condyle, particularly given its neural crest origin. The manuscript is generally well-written and the trajectory analysis offers a compelling hypothesis regarding lineage plasticity. However there are a number of major and minor concerns, outlined below: 1. The "ProC" Population and Developmental Context: The authors identify a "Chondrocyte Progenitor" (ProC) population marked by Ptn, Tnc, and Id4. In the trajectory analysis (Figure 4), ProC appears as a distinct root separate from the fibroblast lineage. Critique: Standard developmental models of the TMJ suggest that the superficial polymorphic/proliferative layer (often called the reserve zone) contains the progenitors for the underlying cartilage. This layer is usually continuous with the fibrous zone. Action: Please clarify: Does the ProC population correspond spatially to the "polymorphic layer" in histology? The RNAscope for Ptn (Figure 5B) shows expression in the proliferative layer. The authors should discuss whether the ProC and Fibroblast clusters represent a continuum of the superficial zone or distinct lineage restrictions 2. Clarification of "Inflammation" vs. "Immune Response": The authors excluded immune cells because the rats were not perfused. However, the results section extensively discusses inflammatory pathways in matrix cells (e.g., Fibroblasts expressing Matr3, PreC expressing S100a8). Critique: It is crucial to distinguish between intrinsic inflammatory signaling by chondrocytes (which drives catabolism) and extrinsic immune cell infiltration. Action: Please explicitly discuss that the "inflammatory response" observed is likely an autocrine/paracrine stress response of the matrix cells themselves, rather than immune infiltration, as the immune cells were bioinformatically removed. 3. Validation Quantification: The RNAscope images (Figure 5) are excellent for localization. However, to bolster the claims of cell identity, quantification (even semi-quantitative counting of positive cells in specific zones) would strengthen the link between the scRNA-seq clusters and the histological layers. Minor Revisions Abstract: The phrase "they are of neural architecture" is awkward. Consider revising to "they are of neural crest origin." Methods - Sample Pooling: Please clarify if the "pooling" involved barcoding individual animals before pooling (hashing) or if they were physically pooled into one suspension without individual tags. The text mentions "Cell Multiplexing Oligo (CMO)... to tag naïve and splinted cells", implying two pools (one per group), not individual animal tagging. This confirms n=1 per group. Please be precise here. Figure 7 (Pathways): The bar charts show "Activation z-score" and "-log(p-value)". Ensure the legend clarifies that "Positive" z-score means upregulated in the Splinted group relative to Control. Typos: : "The cartilage layers, appeared hypocellular..." (Remove comma). : "The transition is appears to be..." (Remove "is") ****** what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..--> Reviewer #1: No Reviewer #2: No Reviewer #3: Yes: Neal AnthwalNeal AnthwalNeal AnthwalNeal Anthwal ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. https://doi.org/10.1371/journal.pone.0348175.r001
Revision 1
19 Jan 2026 Author Response We would like to thank the reviewers for their thoughtful comments that have led to an improved manuscript revision. We specifically addressed each one, including clarifying methods and limitations. We have also added additional analyses, including cell-cell signaling analysis using CellChat, as well as a semiquantitative analysis of RNAScope results. All responses are copied below, but are also included in the Response to Reviewers file in this resubmission. Reviewer #1: 1. The authors state that cells from all n=8 animals per group were pooled for analysis to ensure sufficient cell yield. This is a significant limitation, as it precludes the assessment of biological variability between individuals and, crucially, any formal statistical testing for differences between the naïve and splinted conditions (e.g., using a mixed-model approach accounting for multiple biological replicates). This critical limitation must be explicitly stated and emphasized in the Discussion, and the conclusions should be framed with appropriate caution. We recommend explicitly mentioning this design choice and its implications for statistical inference in the main text (e.g., Methods or Results) and the Discussion. This limitation has now been addressed in both the methods section and the discussion to ensure transparency of the design and lack of statistical power/analyses. 2. Due to the lack of perfusion, immune cell populations were excluded from analysis as they were considered potentially circulatory. However, tissue-resident immune cells (e.g., macrophages in the synovium) play a key role in OA. This issue should be more thoroughly addressed by (1) explicitly stating this as a limitation in the Methods and (2) providing a more in-depth discussion of how this decision might impact the interpretation of the inflammatory microenvironment in OA. We have now included this limitation in the methods and further addressed it in the discussion by clarifying that while immune cell-specific response could not be accurately captured, tissue-matrix cell inflammatory signaling via cytokine, chemokine, and growth factor secretion was captured and interpreted as such. 3. The filtering criteria for "fibrocartilage cells" need clearer description. What specific marker genes or clustering results informed this selection? Furthermore, details of the trajectory inference, specifically the process and exact criteria for selecting the root nodes, should be elaborated upon to ensure the analysis is reproducible. This description was already present in the results section, but it has now been expanded upon in the methods section, describing specific genes and their expression levels. Briefly, high Dcn, moderate Col2a1, and low Prg4 was chosen as the fibroblast root node, as this is the most classic fibroblast signature. High Ptn and low Alpl was chosen as the chondrocyte progenitor root node, as this indicates the least mature chondrocyte gene expression. 4. Given the identification of multiple cell types, leveraging the existing scRNA-seq data to infer potential intercellular communication changes in OA (using tools like NicheNet or CellChat) could provide deeper insights into the pathological mechanisms. This could be a valuable addition for future consideration if feasible. CellChat communication analysis has been added, highlighting a decline in PreC signaling and a shift towards increased chondrocyte and ProC signaling. We also identified key pathways that were altered in our OA-induced model. 5. The Discussion could briefly contextualize how the cell subsets and key pathways identified (e.g., cholesterol metabolism, NGF signaling) in this rat TMJOA model compare or contrast with known pathways in human TMJOA or peripheral joint OA, enhancing the potential translational relevance of the findings. The suggested addition to the discussion has been added, providing links from the top altered pathways in our data to those that are well-established in human OA. It should be noted that the activation or inactivation of the pathway in our model may differ from that of human OA, likely due to the stage of disease development that we are modeling. However, the inter-species similarities remains an important addition. Reviewer #2: 1. Figure 1 need add further experiments, such as S.O. staining, Micro-CT images and Quantitative analysis. Micro-CT 3D reconstructions and sections of the control and splinted condyles have been added to figure 1, along with their acquisition methods. These images further highlight the extensive remodeling and corticalization changes in the bone due to splinting, which is supported by the single-cell data. 2. Single cell analysis figures can put together to be one figure. Further experiments such as Westernblot verification for OA Target Genes should be done. Some figures have now been combined, resulting in 5 figures rather than 9. To the second point, while we acknowledge the importance of protein-level expression validation, the primary objective of this study was to define the cell type specific transcriptomic profiles and how these profiles change in our splinted rat model. This body of work can then serve as a foundation for the development of more pointed, protein-level studies. However, we have added this as to the limitations in our discussion. Reviewer #3: 1. The "ProC" Population and Developmental Context: The authors identify a "Chondrocyte Progenitor" (ProC) population marked by Ptn, Tnc, and Id4. In the trajectory analysis (Figure 4), ProC appears as a distinct root separate from the fibroblast lineage. Critique: Standard developmental models of the TMJ suggest that the superficial polymorphic/proliferative layer (often called the reserve zone) contains the progenitors for the underlying cartilage. This layer is usually continuous with the fibrous zone. Action: Please clarify: Does the ProC population correspond spatially to the "polymorphic layer" in histology? The RNAscope for Ptn (Figure 5B) shows expression in the proliferative layer. The authors should discuss whether the ProC and Fibroblast clusters represent a continuum of the superficial zone or distinct lineage restrictions The trajectory analysis is interpreted as groups with similar transcriptional programming maturing along parallel paths simultaneously, with both PreC and ProC populations stemming from the proliferative zone. This clarification has been expanded upon and added to the discussion. 2. Clarification of "Inflammation" vs. "Immune Response": The authors excluded immune cells because the rats were not perfused. However, the results section extensively discusses inflammatory pathways in matrix cells (e.g., Fibroblasts expressing Matr3, PreC expressing S100a8). Critique: It is crucial to distinguish between intrinsic inflammatory signaling by chondrocytes (which drives catabolism) and extrinsic immune cell infiltration. Action: Please explicitly discuss that the "inflammatory response" observed is likely an autocrine/paracrine stress response of the matrix cells themselves, rather than immune infiltration, as the immune cells were bioinformatically removed. This comment has been addressed in another similar comment (review 1, #2). We have now included the exclusion of immune cells more explicitly in the discussion limitations, as well as a discussion on the role of matrix cells in the inflammatory response. 3. Validation Quantification: The RNAscope images (Figure 5) are excellent for localization. However, to bolster the claims of cell identity, quantification (even semi-quantitative counting of positive cells in specific zones) would strengthen the link between the scRNA-seq clusters and the histological layers. Semi-quantitative measurements of positive stain in each layer (reported as %area) have been added, including a bar graph of the results. However, with a measurement sample size of n=1 per stain, no statistics was added and it has been mentioned in the methods that these measurements are only to be viewed as additional supportive evidence for the qualitative claims of RNA localization. Minor Revisions Abstract: The phrase "they are of neural architecture" is awkward. Consider revising to "they are of neural crest origin." Methods - Sample Pooling: Please clarify if the "pooling" involved barcoding individual animals before pooling (hashing) or if they were physically pooled into one suspension without individual tags. The text mentions "Cell Multiplexing Oligo (CMO)... to tag naïve and splinted cells", implying two pools (one per group), not individual animal tagging. This confirms n=1 per group. Please be precise here. This has now been addressed and clarified in the methods. Figure 7 (Pathways): The bar charts show "Activation z-score" and "-log(p-value)". Ensure the legend clarifies that "Positive" z-score means upregulated in the Splinted group relative to Control. This clarification has been added to the figure caption. Typos: : "The cartilage layers, appeared hypocellular..." (Remove comma). : "The transition is appears to be..." (Remove "is") The mentioned errors have been fixed. Attachments Attachment Submitted filename: ReviewerResponse.docx https://doi.org/10.1371/journal.pone.0348175.r002
14 Apr 2026 Decision Letter - James Cray, Editor Single-cell RNA sequencing of healthy and diseased rat temporomandibular joint condyle cartilage PONE-D-25-53506R1 Dear Dr. Almarza, 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. 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For more information, please contact onepress@plos.org. Kind regards, James J Cray Jr., Ph.D. Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed Reviewer #3: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #3: I Don't Know ****** 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.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 #3: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #3: Yes ****** Reviewer #1: The authors have answered all of my questions of this article, and I am satisfied with the authors' responses. Reviewer #3: The Reviewers have answered the comments to a suffient standard that the article is now suitible for publication in PLOSOne ****** what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). 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 For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..--> Reviewer #1: No Reviewer #3: Yes: Neal AnthwalNeal AnthwalNeal AnthwalNeal Anthwal ******** https://doi.org/10.1371/journal.pone.0348175.r003
Formally Accepted
Acceptance Letter - James Cray, Editor PONE-D-25-53506R1 PLOS One Dear Dr. Almarza, 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 You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. 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. James J Cray Jr. Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0348175.r004

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