Review Reports - Platelet-Rich Plasma Enhances Adhesion and Short-Term Retention of Bone Marrow-Derived Mesenchymal Stromal Cells to Articular Cartilage

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The scientific paper "Platelet-rich plasma enhances adhesion and short-term retention of bone marrow-derived mesenchymal stem cells to articular cartilage aimed to improve the adhesion and retention abilities of MSCs to regenerate injured articular cartilage using PRP. After careful reading, I can make the following observations:

1) The similarity index, according to the iThenticate report, is at 42%, considered outside acceptable standards. Please reduce it by adjusting texts and phrases.

2) The abstract should be rewritten, expanding the methodology and results sections, and reducing the background information.

3) The keywords are copies of the manuscript title. I recommend modifying them to increase visibility in databases.

4) The first paragraph of the introduction is too long (lines 38-72). Please separate it into shorter, linked paragraphs.

5) In the last paragraph of the introduction, before the objective, clearly state the "gap" in the literature regarding the study conducted and its clinical justification.

6) Figures 2B, 3B and 4B needs to be enlarged. It is difficult to visualize and interpret in its current format.

7) Incorporate ethical aspects into the methodology as well.

8) The discussion is a weak point in the manuscript. It offers little comparison of the results obtained with recent literature (last 5 years). It should be expanded in length and quality.

9) In the conclusions, you should initially contextualize the objectives, then present the main findings and conclusions, as well as explain the importance and innovation, especially in clinical terms, for world science.

Author Response

Response to comments from the reviewers’ comments

Dear Editor,

We sincerely appreciate your time and the reviewer’s constructive comments on our manuscript (ID: cells-4274968). We have carefully revised the manuscript to address all questions and concerns raised during the review process.

We believe that these revisions have substantially improved the clarity and quality of the manuscript, and we hope that the revised version is now suitable for publication in Cells. Below, we provide a detailed, point-by-point response to each of the reviewer’s comments. For the reviewer’s convenience, all changes made during the revision process are highlighted in blue in the revised manuscript.

We would be pleased to respond to any further questions or suggestions you may have.

Reviewer #1: Dear Authors,

Response to Reviewer #1 – Comment 1

Comment:

1) The similarity index, according to the iThenticate report, is at 42%, considered outside acceptable standards. Please reduce it by adjusting texts and phrases.

Response:

1) Thank you for your valuable advice. We have lowered the similarity score by modifying the texts and phrases. Sentences corrected in the manuscript are highlighted in blue.

Response to Reviewer #1 – Comment 2

Comment:

2) The abstract should be rewritten, expanding the methodology and results sections, and reducing the background information.

Response:

2) Thank you for the valuable comment. We revised the abstract by reducing the background information and expanding the descriptions of the methodology and key findings to better reflect the design and outcomes of the study.

Response to Reviewer #1 – Comment 3

Comment:

3) The keywords are copies of the manuscript title. I recommend modifying them to increase visibility in databases.

Response:

3) Thank you for the helpful suggestion. We revised the keywords to avoid duplication with the manuscript title and to improve searchability and visibility in scientific databases.

Response to Reviewer #1 – Comment 4

Comment:

4) The first paragraph of the introduction is too long (lines 38-72). Please separate it into shorter, linked paragraphs.

Response:

4) Thank you for your valuable comment. We agree that the first paragraph of the Introduction was overly long and could affect readability. Accordingly, we revised the section by dividing the original paragraph into shorter, logically connected paragraphs to improve clarity and flow. Please check lines 37-62 of the first paragraph of the Introduction section.

Response to Reviewer #1 – Comment 5

Comment:

5) In the last paragraph of the introduction, before the objective, clearly state the "gap" in the literature regarding the study conducted and its clinical justification.

Response:

5) Thank you for your insightful comment. We agree that the knowledge “gap” and clinical relevance of the study should be more clearly emphasized in the final paragraph of the Introduction. Accordingly, we revised the paragraph to explicitly describe the limited understanding of how PRP affects the adhesion and short-term retention of MSCs on damaged cartilage, despite the widespread combined use of PRP and MSCs in clinical practice. We also clarified the clinical importance of improving early cell adhesion and retention for enhancing the therapeutic efficacy of cell-based cartilage repair. We added the following paragraph before the presentation of the objective in last paragraph of the Introduction.

Response to Reviewer #1 – Comment 6

Comment:

6) Figures 2B, 3B and 4B needs to be enlarged. It is difficult to visualize and interpret in its current format.

Response:

6) Thank you for your valuable comment. We agree that the images in Figures 2B, 3B, and 4B were difficult to visualize and interpret in the original format. Accordingly, we have enlarged these panels to improve clarity and readability.

Response to Reviewer #1 – Comment 7

Comment:

7) Incorporate ethical aspects into the methodology as well.

Response:

7) Thank you for your valuable comment. In response, we added the ethical approval and informed consent information to the Materials and Methods section (Section 2.3, Preparation of PRP). The following statement was included:

“All procedures involving human samples were conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of SMG-SNU Boramae Medical Center (No. 30-2019-124; approved on 5 December 2019). Written informed consent was obtained from all participants prior to sample collection.”

Response to Reviewer #1 – Comment 8

Comment:

8) The discussion is a weak point in the manuscript. It offers little comparison of the results obtained with recent literature (last 5 years). It should be expanded in length and quality.

Response:

8) Thank you for your insightful comment. We agree that the original Discussion section lacked sufficient comparison with recent literature and required further expansion. Accordingly, we revised the Discussion by incorporating additional recent studies regarding the effects of PRP on MSC proliferation, migration, regenerative activity, and cartilage repair. We also clarified the current knowledge gap by emphasizing that relatively few studies have investigated the effects of PRP on the early adhesion and retention of MSCs on damaged articular cartilage.

Response to Reviewer #1 – Comment 9

Comment:

Response:

9) Thank you very much for your valuable suggestion. According to your suggestion, we have modified the conclusion section.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

The manuscript entitled “Platelet-rich plasma enhances adhesion and short-term reten- 2 tion of bone marrow-derived mesenchymal stem cells to articular cartilage” shows the potential use of PRP in bone marrow MSCs in the regeneration of the articular cartilage. However, the manuscript requires further revisions before further processed. Below, you will find my comments for the submitted manuscript.

Before the abstract, there is a session indicting the Highlights. Is this session necessary, according to journal’s guidelines? If not please remove this session.
In the Introduction section, the authors have indicated regarding the cell therapies. However now all cellular therapies are belonging to a greater group referring as advanced therapy medicinal products (ATMPs). The authors should revise accordingly and also include the current legislation for their use.
In the introduction section-lines 43-44, Stem cell-based therapies are classified into three categories: pluripotent stem cells (PSCs), adult stem cells (ASCs), and cancer stem cells (CSCs). CSCs are not an established therapy. I totally do not understand the intended meaning here. The authors should revise this.
In the Introduction section- Totally the introduction needs revision. For example, lines 45-72 Nonstem cell-based therapies include various cells... tissues are essential for successful cell therapy. This paragraph is not relevant with the main scope of the manuscript. The scope of the study is not clear enough. In my opinion, the introduction section requires further improvement, by removing all the unnecessary parts and focused more on cartilage regeneration and how the MSCs along with PRP can assist in this process.
Materials and Methods section-Human MSCS and production of PRP does not include obtained informed consent from the patients according to declaration of Helsinki, as the journal’s guidelines indicating. Without formal informed consent the manuscript cannot be proceeded.
The authors have indicated the use of Mesenchymal Stem Cells (MSCs), however based on the recent criteria outlined by the ISCT, Mesenchymal Stromal Cells is the proper term to be used. If the authors use for their experiments Mesenchymal Stem Cells, appropriate tasks should be performed.
The basic criteria of MSCs outlined by the ISCT should be fulfilled (DOI: 1080/14653240600855905)
PRP quality results should also pe included. A proteomic analysis indicating the presence of the growth factors of PRP products prior application (Please check the following publications DOI: 10.3390/bioengineering8050053, DOI: 10.3390/bioengineering6030066 and DOI: 10.3390/bioengineering5010019, cited appropriately and including in discussion section as comparison)
Statistical analysis. The authors have used parametric statistical tests to show the significance. However, the data are non parametric therefore Mann Whitney U test and Kruskal Wallis should be performed instead of student’s t test and ANOVA
In Results section, no scale bars have been added to images. Please add the original magnification and scale bar in the figure legend. The scale bars should be added also to each figures.
The authors have used only in vitro models to support their results, however it is necessary an animal model to be applied.
The discussion should be accordingly revised, to be more precise, include the above results and perform comparisons with similar works in the field.

Comments on the Quality of English Language

The English is fine, only minor corrections are required.

Author Response

Response to comments from the reviewers’ comments

Dear Editor,

We would be pleased to respond to any further questions or suggestions you may have.

Reviewer #2: Dear Authors,

The manuscript entitled “Platelet-rich plasma enhances adhesion and short-term retention of bone marrow-derived mesenchymal stem cells to articular cartilage” shows the potential use of PRP in bone marrow MSCs in the regeneration of the articular cartilage. However, the manuscript requires further revisions before further processed. Below, you will find my comments for the submitted manuscript:

Response to Reviewer #2 – Comment 1

Comment:

1) Before the abstract, there is a session indicting the Highlights. Is this session necessary, according to journal’s guidelines? If not please remove this session.

Response:

1) Thank you for your valuable comment. We checked the journal guidelines and confirmed that a separate “Highlights” section is not required for submission to Cells. Accordingly, we removed the Highlights section from the revised manuscript.

Response to Reviewer #2 – Comment 2

Comment:

2) In the Introduction section, the authors have indicated regarding the cell therapies. However now all cellular therapies are belonging to a greater group referring as advanced therapy medicinal products (ATMPs). The authors should revise accordingly and also include the current legislation for their use.

Response:

2) We appreciate the reviewer’s comment. In response, we revised the Introduction to acknowledge that cell-based therapies are currently classified within the broader category of advanced therapy medicinal products (ATMPs) and briefly described the regulatory framework established for their clinical application.

Response to Reviewer #2 – Comment 3

Comment:

3) In the introduction section-lines 43-44, Stem cell-based therapies are classified into three categories: pluripotent stem cells (PSCs), adult stem cells (ASCs), and cancer stem cells (CSCs). CSCs are not an established therapy. I totally do not understand the intended meaning here. The authors should revise this.

Response:

3) Thank you for your valuable comment. We agree that the previous wording could be misleading because cancer stem cells (CSCs) are not currently considered an established therapeutic category in stem cell-based therapy. Therefore, we have revised the relevant content.

Response to Reviewer #2 – Comment 4

Comment:

4) In the Introduction section- Totally the introduction needs revision. For example, lines 45-72 Nonstem cell-based therapies include various cells... tissues are essential for successful cell therapy. This paragraph is not relevant with the main scope of the manuscript. The scope of the study is not clear enough. In my opinion, the introduction section requires further improvement, by removing all the unnecessary parts and focused more on cartilage regeneration and how the MSCs along with PRP can assist in this process.

Response:

4) Thank you for your insightful comment. We agree that the original Introduction section contained several broad descriptions of general cell therapy that were not sufficiently focused on the main scope of the present study. Accordingly, we substantially revised and streamlined the Introduction by removing less relevant content and placing greater emphasis on cartilage regeneration, MSC engraftment, and the potential role of PRP in enhancing MSC adhesion and retention on damaged articular cartilage. These revisions were made to improve the clarity, focus, and overall flow of the Introduction section.

Response to Reviewer #2 – Comment 5

Comment:

5) Materials and Methods section-Human MSCS and production of PRP does not include obtained informed consent from the patients according to declaration of Helsinki, as the journal’s guidelines indicating. Without formal informed consent the manuscript cannot be proceeded.

Response:

5) Thank you for your important comment. In response, we added a statement regarding ethical approval and informed consent to the Materials and Methods section (Section 2.3, Preparation of PRP). The revised manuscript now clearly states that all procedures involving human samples were conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of SMG-SNU Boramae Medical Center (No. 30-2019-124; approved on 5 December 2019). We also clarified that written informed consent was obtained from all participants prior to sample collection.

Response to Reviewer #2 – Comment 6

Comment:

6) The authors have indicated the use of Mesenchymal Stem Cells (MSCs), however based on the recent criteria outlined by the ISCT, Mesenchymal Stromal Cells is the proper term to be used. If the authors use for their experiments Mesenchymal Stem Cells, appropriate tasks should be performed.

Response:

6) Thank you for your valuable comment. We agree that, according to the current ISCT recommendations, the term “mesenchymal stromal cells” is more appropriate unless additional functional stemness assays are performed. Accordingly, we revised the terminology throughout the manuscript from “mesenchymal stem cells” to “mesenchymal stromal cells (MSCs)” where appropriate.

Response to Reviewer #2 – Comment 7

Comment:

7) The basic criteria of MSCs outlined by the ISCT should be fulfilled (DOI: 1080/14653240600855905)

Response:

7) Thank you for your valuable comment. In the present study, the BM-MSCs used were previously characterized by flow cytometric analysis of MSC-associated surface markers (CD73, CD90, and CD105 positive; CD11a, CD34, and CD45 negative) and trilineage differentiation potential, including adipogenic, osteogenic, and chondrogenic differentiation, as reported in our previous study, which has now been cited in the revised manuscript. In addition, the terminology throughout the manuscript was revised from “mesenchymal stem cells” to “mesenchymal stromal cells (MSCs)” in accordance with current ISCT recommendations.

Materials and Methods

2.1 Mesenchymal stromal cells (MSCs)

The cells used in this study were previously characterized by flow cytometric analysis of MSC-associated surface markers (CD73, CD90, and CD105 positive; CD11a, CD34, and CD45 negative) and trilineage differentiation potential, including adipogenic, os-teogenic, and chondrogenic differentiation, as described in our previous study [28].

Response to Reviewer #2 – Comment 8

Comment:

8) PRP quality results should also pe included. A proteomic analysis indicating the presence of the growth factors of PRP products prior application (Please check the following publications DOI: 10.3390/bioengineering8050053, DOI: 10.3390/bioengineering6030066 and DOI: 10.3390/bioengineering5010019, cited appropriately and including in discussion section as comparison).

Response:

8) Thank you for your valuable comment. In the present study, the PRP preparation protocol and its quality characteristics were based on our previously published plateletpheresis-derived leukocyte-poor PRP (LP-PRP) system, which included detailed analyses of platelet concentration, red blood cell depletion, and white blood cell reduction. In our previous study, platelet counts increased approximately 6-fold after plateletpheresis, whereas red and white blood cell counts were markedly reduced, demonstrating the reproducibility and leukocyte-poor characteristics of the PRP preparation method. These previous characterization data have now been cited in the revised manuscript. The following paragraph has been added to 2.3 Preparation of PRP in the Materials and Methods section.

Materials and Methods

2.3 Preparation of PRP

The PRP preparation protocol used in this study was based on our previously published leukocyte-poor PRP (LP-PRP) system generated by plateletpheresis, which demonstrated standardized platelet enrichment with minimal red and white blood cell contamination [29].

We agree that comprehensive proteomic analysis of PRP-derived growth factors could provide additional mechanistic insights into PRP-mediated biological effects. However, the primary objective of the present study was to investigate the effects of PRP pretreatment on MSC adhesion and short-term retention rather than to characterize the molecular composition of PRP itself. Nevertheless, we added the suggested references and expanded the Discussion section to compare our findings with previous reports describing the biological composition and regenerative properties of PRP products.

Discussion

Previous studies investigating platelet gel and fibrin gel derived from cord blood demonstrated the presence of regenerative signaling molecules, including TGF-β1, PDGF, VEGF, FGF, ICAM-1, and VCAM-1, which are associated with cell activation, migration, adhesion, and tissue regeneration [41]. Proteomic analyses have further shown that platelet-derived biologics contain diverse signaling molecules, receptors, and adhesion-related proteins involved in wound healing and extracellular matrix (ECM) interactions [41]. These findings suggest that the enhanced short-term retention observed in the present study may not be solely attributable to fibrin formation, but also to bioactive molecules within PRP that modulate early cell–matrix and cell–cartilage interactions.

Previous studies have emphasized that platelet concentration and contamination with leukocytes or red blood cells may substantially influence the biological activity and regenerative potential of PRP products, highlighting the importance of standardization and characterization of PRP preparations [49].

Response to Reviewer #2 – Comment 9

Comment:

9) Statistical analysis. The authors have used parametric statistical tests to show the significance. However, the data are non parametric therefore Mann Whitney U test and Kruskal Wallis should be performed instead of student’s t test and ANOVA.

Response:

9) Thank you for your valuable comment. In response, we carefully re-evaluated the statistical analyses according to the sample size and distribution characteristics of each dataset. For the in vitro experiments (BM-MSCs: n = 12 per group per time point; chondrocytes: n = 6 per group per time point), normality testing was performed prior to analysis, and parametric statistical methods were retained because the data satisfied the assumptions for normal distribution. Therefore, Student’s t-test and one-way ANOVA were considered appropriate for the in vitro datasets.

However, for the ex vivo experiments, which involved smaller sample sizes (n = 3 per group), nonparametric statistical methods were applied as recommended by the reviewer. Accordingly, the ex vivo datasets were reanalyzed using Mann–Whitney U and Kruskal–Wallis tests. After reanalysis, statistically significant differences were confirmed between the control and PRP-pretreated 30 min groups at each cartilage lesion grade (G0–G3). In addition, comparisons between the G0 control group and PRP-pretreated 30 min groups at different lesion grades also demonstrated significant differences. The Materials and Methods section, figure legends, Results, and figure were revised accordingly.

Response to Reviewer #2 – Comment 10

Comment:

10) In Results section, no scale bars have been added to images. Please add the original magnification and scale bar in the figure legend. The scale bars should be added also to each figures.

Response:

10) Thank you for your valuable comment. In response, scale bars were added to all microscopic images in the revised figures. In addition, the original magnifications and corresponding scale bar information have been included in the figure legends to improve image interpretation and clarity.

Response to Reviewer #2 – Comment 11

Comment:

11) The authors have used only in vitro models to support their results, however it is necessary an animal model to be applied.

Response:

11) Thank you for your important comment. We agree that in vivo animal studies are important for evaluating the therapeutic relevance and biological effects of PRP-mediated MSC adhesion and retention under physiological conditions. However, the primary objective of the present study was to investigate the early adhesion and short-term retention effects of PRP on MSCs under controlled in vitro and ex vivo experimental conditions. Therefore, in vivo animal experiments were beyond the scope of the current study.

To address this limitation, we have clarified in the revised Discussion section that the present findings are limited to in vitro and ex vivo models and should not be interpreted as direct evidence of in vivo homing or engraftment. We also emphasized that further studies using appropriate animal models are necessary to validate the therapeutic efficacy and biological mechanisms of PRP-enhanced MSC retention in cartilage repair.

Response to Reviewer #2 – Comment 12

Comment:

12) The discussion should be accordingly revised, to be more precise, include the above results and perform comparisons with similar works in the field.

Response:

12) Thank you for your crucial comment. In response, the Discussion section was extensively revised to improve precision, scientific interpretation, and comparison with related studies in the field. We expanded the discussion of the present findings regarding PRP-enhanced adhesion and short-term retention of BM-MSCs and incorporated additional comparisons with recent studies investigating the biological and regenerative effects of PRP on MSC function and cartilage repair.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

If the manuscript is to be considered further in its present form, I would suggest the following revisions:

The abstract should include quantitative results rather than being purely descriptive.
The introduction requires editing for clarity and focus; in particular, the opening paragraph on general principles of cell therapy could be streamlined.
The Materials and Methods section need clarification in several areas, including the number of experimental repeats and how PRP from different donors was used across experiments.
Some sections of the Results appear repetitive and should either be clarified or consolidated.
The images in Figures 2 and 3 need improvement in clarity, and the associated histograms appear blurred.
The discussion should present a more balanced view of the clinical application of PRP.
A section discussing the immunogenic properties of cartilage, particularly in the context of autologous versus allogeneic MSCs and PRP, would strengthen the discussion.
The authors should include a paragraph outlining future work, particularly experiments that could elucidate the mechanisms by which PRP enhances cell adhesion.
The limitations section should be expanded, especially with respect to sample size and number of experimental repeats.
The conclusion should provide a practical perspective on how PRP could be used alongside MSCs for the treatment of cartilage lesions.

Comments on the Quality of English Language

Please see my line by line comments below

30 The abstract is largely qualitative. Please include quantitative data (e.g., percentage differences in adhesion) to better support the conclusions.

39–47 This section assumes limited reader familiarity with cell therapy. Consider reducing and focusing this paragraph, as it is overly general for the target audience.

72 The sentence ending on this line is redundant and repeats points made earlier. Please remove or revise.

79 This statement requires 2–3 supporting references.

111, 127 Please clarify what is meant by a “3:1 ratio.”

114 This refers to cartilage rather than chondrocytes. Please specify the source of the cartilage.

132 You state that PRP has n = 3. Does this correspond to three different donors? If so, platelet concentrations are likely to vary between donors. Please clarify how this variability was handled. Additionally, throughout the Results you refer generically to “PRP”—it is unclear whether this represents pooled samples or individual donors. This must be clearly described in the Materials and Methods.

153 Please specify whether these cells are MSCs.

173 Only one cartilage donor appears to have been used. This is a limitation and should be explicitly acknowledged and discussed.

Figure 1 The histology images lack clarity. Higher magnification micrographs are recommended.

190 Please indicate how many times the measurements were performed and how many observers were involved.

192 This sentence is unclear and should be revised for clarity.

193 Please specify that the cartilage surface was exposed.

210 How long were the samples incubated? It appears that plugs were removed, leaving attached cells on the dish—please confirm and clarify this in the text.

Figures 2 and 3

Panel A images are unclear; consider using higher-quality or colour images.
The histograms are low resolution and appear blurred.

Results section (general comment)
The Results section is currently confusing and requires restructuring for clarity.

Section 3.1 describes the effects of PRP on adhesion and short-term retention of both chondrocytes and MSCs, with reference to Figures 2 and 3.
Section 3.2 (BM-derived MSCs): The subheading is too general and should be more specific. The data presented (e.g., Fig. 2B) appear to overlap with Section 3.1—please avoid repetition.
Section 3.3 (Chondrocytes): Similarly, this subheading should be more specific. The data (e.g., Fig. 3B) also appear repetitive of Section 3.1.

Please ensure that Sections 3.1–3.3 are clearly differentiated and that data are not duplicated across sections.

319 Please indicate the number of experimental repeats. If cartilage plugs were derived from a single donor (N = 1), this must be clearly stated, along with the number of repeat experiments performed using that donor.

322 Figure 2A appears to describe PRP effects on BM-MSC adhesion and short-term retention, rather than the ex vivo study. Please clarify.

352 Consider using a term other than “pleiotropic,” as this typically refers to a single gene or factor with multiple effects. PRP contains multiple components, each of which may contribute to the observed effects.

358 Please provide a more balanced discussion of PRP, including evidence that PRP treatment (e.g., in tendinopathy) does not always yield beneficial outcomes. Supporting references should be included.

379 Please elaborate on how this could be implemented in practice.

Author Response

Response to comments from the reviewers’ comments

Dear Editor,

We would be pleased to respond to any further questions or suggestions you may have.

Reviewer #3: Dear Authors,

If the manuscript is to be considered further in its present form, I would suggest the following revisions:

Response to Reviewer #3 – Comment 1

Comment:

1) The abstract should include quantitative results rather than being purely descriptive.

Response:

1) Thank you for this valuable suggestion. The Abstract has been revised to include representative quantitative results, including fold increases in BM-MSC attachment, statistical significance values, and ex vivo lesion grade-dependent enhancement of adhesion and short-term retention following PRP pretreatment. These revisions provide a more data-driven summary of the main findings.

Revised Abstract

Mesenchymal stromal cell (MSC) adhesion and retention at sites of cartilage degeneration are critical for improving cartilage repair. This study investigated whether platelet-rich plasma (PRP) enhances the adhesion and short-term retention of bone marrow-derived MSCs (BM-MSCs) and chondrocytes under in vitro and ex vivo conditions. BM-MSCs and chondrocytes were treated with PRP or pretreated with PRP for 10 or 30 min, and cell adhesion to collagen-coated surfaces was evaluated using a cell viability assay. Ex vivo adhesion and short-term retention of BM-MSCs on osteochondral discs with varying lesion severity were assessed by fluorescence imaging analysis. PRP significantly enhanced the adhesion of both BM-MSCs and chondrocytes in a time-dependent manner, with the 30 min PRP pretreatment group showing the greatest effect. After 30 min of incubation, BM-MSC attachment increased by 129.1-fold in the 30 min PRP pretreatment group compared with that in the control group at 2.5 min (P < 0.001), whereas chondrocyte attachment was also markedly increased following PRP pretreatment. PRP pretreatment significantly enhanced BM-MSC attachment compared with PRP treatment alone at 20 and 30 min of incubation (both P < 0.001). In ex vivo experiments, adhesion and short-term retention increased significantly with increasing lesion severity from G1 to G3 (P < 0.05 and P < 0.01, respectively). In G2 and G3 lesions, PRP pretreatment for 30 min significantly enhanced BM-MSC adhesion and short-term retention compared with the control group (both P < 0.01). These findings suggest that PRP improves the early adhesion and retention of MSCs on damaged cartilage and may enhance the efficacy of cell-based cartilage repair strategies.

Keywords: mesenchymal stromal cells; cartilage regeneration; osteochondral defect; extracellular matrix; cell engraftment; regenerative medicine; osteoarthritis

Response to Reviewer #3 – Comment 2

Comment:

2) The introduction requires editing for clarity and focus; in particular, the opening paragraph on general principles of cell therapy could be streamlined.

Response:

2) Thank you for this helpful suggestion. We revised the opening paragraph of the Introduction to improve clarity and focus by streamlining the general background on cell therapy. While maintaining the description of ATMPs, the Introduction was reorganized to focus more directly on MSC engraftment, adhesion, and retention, which are central to the aim of the present study. Redundant explanations regarding stem cell classifications and homing-related mechanisms were reduced for conciseness and readability.

Introduction

Cell therapy refers to the administration of autologous or allogeneic cellular components for therapeutic purposes and is widely applied in regenerative medicine, immunotherapy, and cancer treatment [1]. These cellular therapies are broadly categorized as advanced therapy medicinal products (ATMPs), which include somatic cell therapies and tissue-engineered products regulated under established regulatory frameworks. Among stem cell–based approaches, mesenchymal stromal cells (MSCs) have attracted considerable attention because of their regenerative and immunomodulatory properties [2]. However, the therapeutic efficacy of transplanted MSCs is often limited by poor homing, engraftment, and survival within injured tissues [3]. Anoikis is a form of programmed cell death that is triggered in anchorage-dependent cells when they lose attachment to the surrounding extracellular matrix (ECM) [4]. In addition, culture-expanded MSCs frequently exhibit reduced expression of chemokine receptors associated with tissue homing, resulting in lower engraftment efficiency [5,6]. Therefore, improving MSC adhesion and retention within damaged tissues is considered an important strategy for enhancing the therapeutic efficacy of cell-based therapies. Integrins mediate MSC adhesion to ECM proteins such as collagen and fibronectin and play critical roles in cell migration, survival, and cartilage regeneration [7-9].

Response to Reviewer #3 – Comment 3

Comment:

3) The Materials and Methods section need clarification in several areas, including the number of experimental repeats and how PRP from different donors was used across experiments.

Response:

3) Thank you for this important comment. We revised the Materials and Methods section to clarify the number of experimental repeats and the use of donor-derived PRP. Specifically, we added that PRP was independently prepared from three healthy donors and that each donor-derived PRP preparation was used in independent biological experiments. In addition, we clarified that quantitative data are presented as the mean ± SD from three independent experiments in figure legends.

Materials and Methods

2.3 Preparation of PRP

Platelet-rich plasma (PRP; n = 3) was prepared from three independent healthy donors using a plateletpheresis system equipped with a leukoreduction set (COBE Spectra LRS Turbo, Caridian BCT, Lakewood, CO, USA) and a standard collection pro-gram consisting of a 90 min double-needle procedure [29]. Each donor-derived PRP preparation was used in independent biological experiments.

Figure legends

Quantitative results are presented as the mean ± SD from three independent biological experiments.

Response to Reviewer #3 – Comment 4

Comment:

4) Some sections of the Results appear repetitive and should either be clarified or consolidated.

Response:

4) Thank you for this helpful comment.

In response to this suggestion, we revised several parts of the Results section to improve clarity and reduce repetitive descriptions. In particular, the BM-MSC and chondrocyte adhesion sections were reorganized to avoid repetitive sentence patterns and redundant comparative expressions. We also streamlined the presentation of time-dependent changes while retaining the key fold-change values and statistical outcomes necessary to support the robustness of the findings.

Results

3.1. PRP improves the adhesion and short-term retention abilities of cells in vitro

BM-MSC attachment increased over time and was further augmented by PRP treatment (Figure 2A). After 30 min of incubation, attachment increased by 50.8-fold in the control group, 72.3-fold in the PRP-treated group, and 110.5-fold and 129.1-fold in the groups pretreated with PRP for 10 and 30 min, respectively, compared with the control group at 2.5 min (all P < 0.001). No significant difference was observed between the 10 and 30 min pretreatment groups at 20 and 30 min of incubation, suggesting a plateau effect in attachment capacity (Figure 2B).

Chondrocyte attachment also increased in a time-dependent manner was further enhanced by PRP treatment (Figure 3A). Relative to the control group at 2.5 min, attachment after 30 min increased by 286.3-, 464.8-, 564-, and 910.3-fold in the control, PRP-treated, 10 min pretreatment, and 30 min pretreatment groups, respectively (P < 0.01 for all groups except the 30 min pretreatment group, P < 0.001). Among the treatment groups, the 30 min pretreatment group showed the highest attachment capacity and remained significantly higher than the 10 min pretreatment group at 30 min of incubation (P < 0.05). A plateau in attachment was observed after 30 min of incubation (Figure 3B).

Response to Reviewer #3 – Comment 5

Comment:

5) The images in Figures 2 and 3 need improvement in clarity, and the associated histograms appear blurred.

Response:

5) Thank you for your helpful suggestion. We revised Figures 2 and 3 to improve overall clarity and readability.

Response to Reviewer #3 – Comment 6

Comment:

6) The discussion should present a more balanced view of the clinical application of PRP.

Response:

6) Thank you for this valuable comment. We revised the Discussion section to provide a more balanced perspective regarding the clinical application of PRP. Specifically, we added discussion regarding the variability in PRP composition and preparation methods, the inconsistent clinical outcomes, and the need for caution when translating experimental findings into clinical applications.

Discussion

The limitations of this study are that the results were limited to in vitro and ex vivo models; therefore, further studies are needed to determine whether the same effects are observed in preclinical and clinical settings. Although PRP has shown promising therapeutic potential in musculoskeletal disorders, clinical outcomes have not always been consistent across studies. Variability in platelet concentration, leukocyte content, donor-related factors, and preparation protocols may substantially influence the biological activity and therapeutic efficacy of PRP products. Therefore, caution is required when translating experimental findings into clinical applications, and standardized PRP preparation protocols should be established.

Response to Reviewer #3 – Comment 7

Comment:

7) A section discussing the immunogenic properties of cartilage, particularly in the context of autologous versus allogeneic MSCs and PRP, would strengthen the discussion.

Response:

7) We appreciate the reviewer’s valuable comment. A discussion regarding the immunogenic characteristics of cartilage and the potential differences between autologous and allogeneic MSC/PRP applications has been added to the revised Discussion section. The revised manuscript also addresses the relatively immune-privileged nature of cartilage, the low immunogenicity of MSCs, and the need for further studies evaluating immunological responses in clinical settings.

Discussion

PRP can be derived from autologous or allogeneic sources and can be prepared from either platelets or plasma. Articular cartilage is generally considered an immune-privileged tissue because of its avascular nature and low cellularity, which may partially support the use of allogeneic cell-based therapies in cartilage regeneration. In particular, MSCs exhibit relatively low immunogenicity and possess immunomodulatory properties, making allogeneic MSC applications clinically attractive. However, immune responses may still occur depending on donor-recipient compatibility, inflammatory conditions, and the persistence of engrafted cells. Similarly, PRP products may differ immunologically according to whether they are prepared from autologous or allogeneic sources, as variations in donor-derived bioactive factors and plasma proteins could influence therapeutic responses. In the present study, leukocyte-poor (LP) PRP and BM-MSCs were evaluated under controlled experimental conditions; however, further studies are required to investigate the immunological interactions and therapeutic efficacy of autologous versus allogeneic combinations in vivo and in clinical settings.

Response to Reviewer #3 – Comment 8

Comment:

8) The authors should include a paragraph outlining future work, particularly experiments that could elucidate the mechanisms by which PRP enhances cell adhesion.

Response:

8) We appreciate the reviewer’s insightful suggestion. In response, we added a new paragraph to the revised Discussion section describing future studies aimed at elucidating the mechanisms underlying PRP-enhanced MSC adhesion and short-term retention. Specifically, we discussed the need for further investigations into adhesion-related molecules and focal adhesion-associated signaling pathways, including integrins, ICAM-1, and VCAM-1, as well as omics-based approaches to identify key bioactive factors within PRP that may contribute to enhanced cell adhesion and retention.

Discussion

The present study did not directly evaluate changes in chemotactic signaling pathways or adhesion-related molecule expression following PRP pretreatment; therefore, the proposed mechanisms remain speculative and require further investigation. Future studies should focus on elucidating the molecular mechanisms underlying PRP-enhanced MSC adhesion and short-term retention. In particular, investigations of adhesion-related molecules, including integrins, ICAM-1, VCAM-1, and focal adhesion-associated signaling pathways, may provide further insight into the mechanisms regulating early cell attachment to damaged cartilage. Furthermore, transcriptomic or proteomic approaches may help identify key bioactive factors within PRP that contribute to enhanced MSC adhesion and retention.

Response to Reviewer #3 – Comment 9

Comment:

9) The limitations section should be expanded, especially with respect to sample size and number of experimental repeats.

Response:

9) We appreciate the reviewer’s valuable comment. In response, we expanded the limitations section to explicitly address the relatively small sample size and limited number of experimental repeats. We also clarified that all in vitro experiments were performed using three independent biological replicates and emphasized the need for future studies with larger sample sizes and additional repetitions to further validate the reproducibility and translational applicability of the findings.

Discussion

In addition, the relatively small sample size and limited number of experimental repeats may restrict the statistical robustness and generalizability of the findings. Although all experiments were performed using three independent biological replicates and demonstrated consistent trends, further studies with larger sample sizes and additional repetitions are warranted to validate the reproducibility of the present findings.

Response to Reviewer #3 – Comment 10

Comment:

10) The conclusion should provide a practical perspective on how PRP could be used alongside MSCs for the treatment of cartilage lesions.

Response:

10) We appreciate the reviewer’s insightful comment. In response, we revised the Conclusion section to provide a more practical and translational perspective regarding the potential combined use of PRP and MSCs for cartilage repair. Specifically, we added statements describing how PRP may function as a supportive biological environment that enhances early MSC retention at cartilage lesion sites following intra-articular administration, thereby potentially improving the therapeutic efficacy of MSC-based therapies.

Conclusion

From a practical perspective, PRP may serve as a supportive biological environment for MSC-based therapies by enhancing early cell retention at cartilage lesion sites following intra-articular administration. Such an approach may help improve the local persistence and therapeutic efficacy of transplanted MSCs in osteoarthritis and cartilage degeneration. Although further studies are required to confirm these effects under in vivo conditions, the combined use of PRP and MSCs may represent a promising strategy for cartilage repair and regenerative treatment.

Reviewer #3: The Quality of English Language

Please see my line by line comments below:

Response to Reviewer #3 – Comment 1

Comment:

1) 30 The abstract is largely qualitative. Please include quantitative data (e.g., percentage differences in adhesion) to better support the conclusions.

Response:

1) Thank you for this valuable comment.

To strengthen the abstract and provide clearer support for the conclusions, quantitative outcome data have been added to the Results section of the Abstract, including representative percentage increases and statistical significance values for BM-MSC adhesion and short-term retention following PRP treatment/pretreatment.

Revised Abstract

Mesenchymal stromal cell (MSC) adhesion and retention at sites of cartilage degeneration are critical for improving cartilage repair. This study investigated whether platelet-rich plasma (PRP) enhances the adhesion and short-term retention of bone marrow-derived MSCs (BM-MSCs) and chondrocytes under in vitro and ex vivo conditions. BM-MSCs and chondrocytes were treated with PRP or pretreated with PRP for 10 or 30 min, and cell adhesion to collagen-coated surfaces was evaluated using a cell viability assay. Ex vivo adhesion and short-term retention of BM-MSCs on osteochondral discs with varying lesion severity were assessed by fluorescence imaging analysis. PRP significantly enhanced the adhesion of both BM-MSCs and chondrocytes in a time-dependent manner, with the 30 min PRP pretreatment group showing the greatest effect. BM-MSC attachment in the 30 min PRP pretreatment group was significantly higher than that in the untreated control group after 30 min of incubation (P < 0.001), whereas chondrocyte attachment was also significantly increased following PRP pretreatment. In addition, PRP pretreatment significantly enhanced BM-MSC attachment compared with PRP treatment alone at 20 and 30 min of incubation (both P < 0.001). In ex vivo experiments, adhesion and short-term retention increased significantly with increasing lesion severity from G1 to G3 (P < 0.05 and P < 0.01, respectively). In G2 and G3 lesions, PRP pretreatment for 30 min significantly enhanced BM-MSC adhesion and short-term retention compared with the control group (both P < 0.01). These findings suggest that PRP may improve the early adhesion and retention of MSCs on damaged cartilage and support the potential use of PRP as a biological adjunct for MSC-based cartilage repair strategies.

Response to Reviewer #3 – Comment 2

Comment:

2) 39–47 This section assumes limited reader familiarity with cell therapy. Consider reducing and focusing this paragraph, as it is overly general for the target audience.

Response:

2) Thank you for this helpful suggestion.

The introductory paragraph (Lines 39–47) was shortened and refocused to better suit the target readership by removing overly general background information and emphasizing the relevance of MSC-based therapies to the present study.

Introduction

These cellular therapies are broadly categorized as advanced therapy medicinal products (ATMPs), which include somatic cell therapies and tissue-engineered products regulated under established regulatory frameworks. Among stem cell–based approaches, mesenchymal stromal cells (MSCs) have attracted considerable attention because of their regenerative and immunomodulatory properties [2].

Response to Reviewer #3 – Comment 3

Comment:

3) 72 The sentence ending on this line is redundant and repeats points made earlier. Please remove or revise.

Response:

3) Thank you for the helpful comment. The redundant sentence was revised to improve conciseness and reduce repetition within the Introduction section.

Introduction

Therefore, improving MSC adhesion and retention within damaged tissues may improve the therapeutic efficacy of MSC-based therapies.

Response to Reviewer #3 – Comment 4

Comment:

4) 79 This statement requires 2–3 supporting references.

Response:

4) Thank you for your helpful comment. We have integrated related sentences and added references in Introduction section.

Introduction

Moreover, PRP has the capacity to restore or regenerate damaged vascular tissues and is therefore used in the treatment of conditions such as arthritis, chronic lower back pain, chronic pelvic pain, and ligament injuries of the shoulder or knee, functioning as a stimulus to enhance tissue repair in prolotherapy and skin regeneration. PRP promotes tissue healing and regeneration in various musculoskeletal tissues, including bone, cartilage, tendons, ligaments, and muscles [15-20].

Response to Reviewer #3 – Comment 5

Comment:

5) 111, 127 Please clarify what is meant by a “3:1 ratio.”

Response:

5) Thank you for the comment. The description of the “1:3 ratio” was clarified by specifying that it refers to the split ratio used during cell passaging/subculture. You can find the following sentence in the Materials and Methods section.

Materials and Methods

When cells reached 80% confluence, they were passaged at a split ratio of 1:3.

Response to Reviewer #3 – Comment 6

Comment:

6) 114 This refers to cartilage rather than chondrocytes. Please specify the source of the cartilage.

Response:

6) Thank you for pointing this out. The sentence was revised to clarify that the cartilage tissue, rather than isolated chondrocytes, was minced during the chondrocyte isolation procedure. You can find the following sentence in the Materials and Methods section.

Materials and Methods

2.2 Chondrocytes

Cartilage tissue was washed with phosphate-buffered saline (PBS) without calcium and magnesium, and minced into small pieces for chondrocyte isolation.

Response to Reviewer #3 – Comment 7

Comment:

7) 132 You state that PRP has n = 3. Does this correspond to three different donors? If so, platelet concentrations are likely to vary between donors. Please clarify how this variability was handled. Additionally, throughout the Results you refer generically to “PRP”—it is unclear whether this represents pooled samples or individual donors. This must be clearly described in the Materials and Methods.

Response:

7) Thank you for this important comment.

The Materials and Methods section has been revised to clarify that PRP was obtained from three independent healthy donors and that PRP samples were not pooled. Each donor-derived PRP preparation was used in independent biological experiments. In addition, all PRP preparations were adjusted to the same target platelet concentration prior to use in order to minimize donor-to-donor variability. You can find the following sentence in the Materials and Methods section.

Materials and Methods

2.3 Preparation of PRP

PRP samples from different donors were not pooled, and each donor-derived PRP preparation was used in independent biological experiments.

2.4 Characterization of PRP

To minimize donor-to-donor variability, all PRP preparations were adjusted to the same target platelet concentration prior to use in experiments.

Response to Reviewer #3 – Comment 8

Comment:

8) 153 Please specify whether these cells are MSCs.

Response:

8) Thank you for the comment.

The sentence was revised to clarify that the term “cells” refers to both BM-MSCs and chondrocytes used in the in vitro adhesion assay. You can find the following sentence in the Materials and Methods section.

Materials and Methods

2.5 In vitro cell adhesion assay

BM-MSCs and chondrocytes were suspended in saline (control group) or treated with PRP before seeding onto plates.

Response to Reviewer #3 – Comment 9

Comment:

9) 173 Only one cartilage donor appears to have been used. This is a limitation and should be explicitly acknowledged and discussed.

Response:

9) Thank you for this important comment.

We acknowledge that the ex vivo osteochondral explant experiments were performed using cartilage obtained from a single donor, which may limit the generalizability of the findings because donor-specific tissue characteristics could influence cell adhesion and retention. This limitation has now been explicitly stated in the Materials and Methods and further discussed in the Limitations section of the manuscript.

Materials and Methods

2.6 Osteochondral disc preparation

The proximal tibial fragment used for the ex vivo experiments was derived from a single patient who underwent knee joint replacement surgery.

Discussion

Furthermore, the ex vivo osteochondral explant experiments were performed using carti-lage obtained from a single donor, and donor-specific factors such as cartilage quality, age, and osteoarthritic severity may have influenced the observed cell adhesion and retention results. Therefore, further studies using cartilage samples from multiple donors are need-ed to confirm the reproducibility and generalizability of the present findings.

Response to Reviewer #3 – Comment 10

Comment:

10) Figure 1 The histology images lack clarity. Higher magnification micrographs are recommended.

Response:

10) We appreciate the reviewer’s comment. Although additional histological sections that could be re-captured at higher magnification were not obtained, the image quality of Figure 1 was improved by enhancing resolution and contrast and adding a scale bar.

Response to Reviewer #3 – Comment 11

Comment:

11) 190 Please indicate how many times the measurements were performed and how many observers were involved.

Response:

11) Thank you for this important comment. We have clarified the number of experimental repeats and observers in the revised manuscript. All measurements were performed using three independent biological replicates, and image analyses were conducted by two independent blinded observers to minimize observational bias. You can find the following sentence in the Materials and Methods section.

Materials and Methods

2.8 Statistical analysis

All experiments were performed using three independent biological replicates. Quantitative image analyses were conducted by two independent observers blinded to the experimental groups.

Response to Reviewer #3 – Comment 12

Comment:

12) 192 This sentence is unclear and should be revised for clarity.

Response:

12) Thank you for this helpful comment. The sentence has been revised for clarity to better describe the positioning and embedding of the osteochondral plugs in agarose gel. The revised sentence now reads:

“Osteochondral plugs were placed in 35-mm culture dishes and embedded in 2% agarose gel, leaving the cartilage surface exposed upward.”

Response to Reviewer #3 – Comment 13

Comment:

13) 193 Please specify that the cartilage surface was exposed.

Response:

13) Thank you for this helpful comment. We have revised the sentence to clarify that the cartilage surface of the osteochondral plugs was left exposed during embedding in agarose gel. The revised sentence now reads:

“Osteochondral plugs were placed in 35-mm culture dishes and embedded in 2% agarose gel, leaving the cartilage surface exposed upward.”

Response to Reviewer #3 – Comment 14

Comment:

14) 210 How long were the samples incubated? It appears that plugs were removed, leaving attached cells on the dish—please confirm and clarify this in the text.

Response:

14) Thank you for this helpful comment. The incubation duration was described in the previous sentence; however, we agree that the subsequent procedure was not sufficiently clear. Therefore, we revised the text to clarify that the osteochondral plugs were washed to remove non-adherent cells, transferred to a 96-well plate, and imaged to evaluate cells attached to the osteochondral plug surface.

The revised text now reads:

Cells were then added to the culture dish containing the plugs at a concentration of 1.0 × 10⁴ cells/mm² and incubated for 15 min at 37°C in a humidified 5% CO₂ incubator. After incubation, the osteochondral plugs were washed four times to remove non-adherent cells and transferred to a 96-well plate with the cartilage surface facing downward. Fluorescent images of cells attached to the osteochondral plug surface were captured using a confocal microscope (Carl Zeiss, Jena, Germany). Fluorescence-labeled cells were quantified using a Multilabel Plate Reader Victor3 (PerkinElmer, Waltham, MA, USA) (Figure 4B).

Response to Reviewer #3 – Comment 15

Comment:

15) Figures 2 and 3

Panel A images are unclear; consider using higher-quality or colour images.

The histograms are low resolution and appear blurred.

Response:

15) Thank you for this helpful comment. We revised Figures 2 and 3 to improve image quality and readability. Scale bars were added to the representative images in Panels A, and the resolution and clarity of the histograms were improved to reduce blurring in the revised figures.

Response to Reviewer #3 – Comment 16

Comment:

16) Results section (general comment)

The Results section is currently confusing and requires restructuring for clarity. Please ensure that Sections 3.1–3.3 are clearly differentiated and that data are not duplicated across sections.

Comment:

16-1) Section 3.1 describes the effects of PRP on adhesion and short-term retention of both chondrocytes and MSCs, with reference to Figures 2 and 3.

Response:

16-1) Thank you for this helpful comment. We revised the title and introductory sentence of Section 3.1 to more clearly indicate that the section describes the effects of PRP on the adhesion and short-term retention of both BM-MSCs and chondrocytes, corresponding to Figures 2 and 3.

The revised section title and the first sentence are as follows.

“3.1. PRP promotes the in vitro adhesion and short-term retention of BM-MSCs and chondrocytes”

PRP enhanced the attachment of both BM-MSCs and chondrocytes in a time-dependent manner.

Comment:

16-2) Section 3.2 (BM-derived MSCs): The subheading is too general and should be more specific. The data presented (e.g., Fig. 2B) appear to overlap with Section 3.1—please avoid repetition.

Response:

16-2) Thank you for this helpful comment. We agree that the previous subheading was too general and that parts of the description overlapped with the results presented in Section 3.1. Therefore, we revised the subheading to more specifically describe the BM-MSC attachment experiment and condensed the text to avoid repetitive descriptions of the data shown in Figure 2B while retaining the key findings.

The revised subheading now reads:

“3.2 Time-dependent effects of PRP pretreatment on BM-MSC attachment”

Comment:

16-3) Section 3.3 (Chondrocytes): Similarly, this subheading should be more specific. The data (e.g., Fig. 3B) also appear repetitive of Section 3.1.

Response:

16-3) Thank you for this helpful comment. We agree that the previous subheading was overly general and that portions of the description repeated findings already summarized in Section 3.1. Therefore, we revised the subheading to more specifically reflect the chondrocyte attachment experiment and condensed the text to avoid repetitive descriptions of the data presented in Figure 3B while retaining the key findings.

The revised subheading now reads:

“3.3 Time-dependent effects of PRP pretreatment on chondrocyte attachment”

Response to Reviewer #3 – Comment 17

Comment:

17) 319 Please indicate the number of experimental repeats. If cartilage plugs were derived from a single donor (N = 1), this must be clearly stated, along with the number of repeat experiments performed using that donor.

Response:

17) Thank you for this important comment. We agree that the number of donors and experimental repeats should be more clearly described. Therefore, we revised the manuscript to clarify that the ex vivo osteochondral plug experiments were performed using cartilage obtained from a single donor and that the experiments were independently repeated three times.

The revised sentence now reads:

“Ex vivo osteochondral plug experiments were performed using cartilage obtained from a single donor, and the experiments were independently repeated three times.”

In addition, we included this limitation in the Discussion section to acknowledge the potential influence of donor-specific factors on the observed results.

Response to Reviewer #3 – Comment 18

Comment:

18) 322 Figure 2A appears to describe PRP effects on BM-MSC adhesion and short-term retention, rather than the ex vivo study. Please clarify.

Response:

18) Thank you for this helpful comment. We agree that the previous sentence incorrectly referenced Figure 2A and may have caused confusion regarding the ex vivo osteochondral plug experiment. We revised the text to clarify that the representative fluorescent images of BM-MSC attachment in the ex vivo model are presented in Figure 4A.

The revised sentence now reads:

“Representative histological characteristics of articular cartilage according to injury grade are shown in Figure 1A. Representative fluorescent images of BM-MSC attachment to osteochondral plugs in the ex vivo model are shown in Figure 4A.”

Response to Reviewer #3 – Comment 19

Comment:

19) 352 Consider using a term other than “pleiotropic,” as this typically refers to a single gene or factor with multiple effects. PRP contains multiple components, each of which may contribute to the observed effects.

Response:

19) Thank you for this helpful comment. We agree that the term “pleiotropic” may not be the most appropriate descriptor for PRP, as PRP contains multiple bioactive components that may contribute to the observed effects. Therefore, we revised the sentence to use a more accurate expression.

The revised sentence now reads:

“A previous study demonstrated that allogeneic pure PRP exerted multiple biological effects on tenocytes depending on the inflammatory state.”

Response to Reviewer #3 – Comment 20

Comment:

20) 358 Please provide a more balanced discussion of PRP, including evidence that PRP treatment (e.g., in tendinopathy) does not always yield beneficial outcomes. Supporting references should be included.

Response:

20) Thank you for this helpful comment. We agree that a more balanced discussion regarding the therapeutic effects of PRP is warranted. Therefore, we revised the Discussion section to acknowledge that PRP treatment does not always produce consistently beneficial clinical outcomes, particularly in tendinopathy. We also added supporting references from systematic reviews and meta-analyses discussing the variable efficacy of PRP treatment and the potential influence of PRP heterogeneity on therapeutic outcomes.

Response to Reviewer #3 – Comment 21

Comment:

21) 379 Please elaborate on how this could be implemented in practice.

Response:

21) Thank you for this helpful comment. We agree that the potential mechanisms underlying PRP-enhanced MSC adhesion and retention should be discussed in greater detail. Therefore, we expanded the Discussion section to include specific examples of future experimental approaches that could help elucidate the underlying molecular mechanisms. These additions include analyses of adhesion-related molecules (e.g., integrins, ICAM-1, and VCAM-1), focal adhesion-associated signaling pathways, and transcriptomic/proteomic approaches to identify key bioactive factors involved in PRP-mediated enhancement of MSC adhesion and retention.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have correctly responded to all my comments. I consider the article ready for publication in its current form.