Sequential Application of Autologous Platelet Rich Plasma and Muscle-Derived Mesenchymal Stem Cells for Acute Tendon Injuries in Horses: Early Clinical and Ultrasonographic Outcomes in a Randomized, Double-Blind Controlled Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

Very nice and original research work. Very interesting initial results. I have some general comments and suggestions on how to improve the manuscript.

The clinical question of whether autologous mdMSCs provide added benefit after standardized PRP in acute tendon/ligament injuries—is clearly articulated and relevant to current equine orthobiologic use. The rationale for combining an immediate biologic (PRP) with a delayed autologous cell therapy is conceptually sound and aligns with real‑world clinical decision‑making. However, the study conflates two distinct questions: (1) whether mdMSCs are effective versus placebo after PRP pretreatment, and (2) whether a second mdMSC injection adds benefit, without fully controlling for confounders such as natural healing over time, non‑standardized rehabilitation, and heterogeneous lesion types. The focus on early (up to 16 weeks) outcomes is understandable, but the discussion frequently extrapolates to recurrence risk and long‑term performance despite the absence of long‑term data in this cohort.

-The age distribution (mean 13 ± 5 years) is relatively wide; the authors should comment on how age might influence tendon healing and whether younger versus older horses differed in response, or experienced versus unexperienced horses.

-Owners and investigators were blinded, but at T2 a second injection of mdMSCs was administered “at the investigator’s discretion” introducing potential performance and selection bias even if treatment allocation remained blinded, and it could be discussed.

-PRP preparation is described using a specific commercial kit and standardized centrifugation, but no quantitative data are provided on platelet or leukocyte counts, nor on intra‑ or inter‑horse variability. This limits the ability to compare with existing PRP literature and to confirm that “leukocyte‑reduced” PRP was consistently obtained. Is it possible to add more specific quantitative data on the counts of PRP?

-The muscle microbiopsy and culture process is described in reasonable detail, including GMP conditions and phenotypic characterization. What are the costs of this approach versus standard autologous MSC culture from bone marrow? is it applicable on a clinical daily basis or it remains too costly? Why the triceps muscle specifically?

-Why there is no explicit description of the final cell product beyond dose (10⁷ cells in 1 mL Cryostor CS5®): no viability at the time of injection, passage number, specific surface marker profile, or functional assays (e.g., trilineage differentiation, immunomodulatory assays), even though “phenotypic characterization” is mentioned. Also the use of Cryostor CS5® for cell suspension in both treatment and placebo is appropriate, but the authors should specify whether cells were thawed immediately before injection and whether a recovery/wash step was performed to remove cryoprotectant.

-The discussion rightly highlights immunologic concerns with allogeneic MSCs and positions autologous mdMSCs as an immunologically safer alternative, but the current trial does not actually measure immune responses (e.g., alloantibody formation, lymphocyte activation), so such claims should be framed more cautiously as theoretical advantages supported by external literature.​

Lines 1–4: The title accurately reflects the design and interventions but does not indicate that the trial focuses on early outcomes; consider adding “early clinical and ultrasonographic outcomes” or similar to align expectations.

​Lines 11–30: The abstract is generally clear, but it omits the fact that analyses for specific mdMSC efficacy exclude PRP responders and are per‑protocol; this should be specified to transparently describe the analyzed population.

​Lines 21–25: The statement “18 horses from both groups received at T2 a second injection of mdMSC” in the abstract could be misread as a pre‑planned, randomized second phase; clarifying that this was at investigator discretion and not randomized would avoid misunderstanding.

Line 218: Phenylbutazone

Line 56: define what MHC-mismatched means

Lines 65–73: The therapeutic “dilemma” and sequential strategy are well framed, but the introduction could benefit from a brief mention of existing clinical data on autologous muscle‑derived MSCs in horses (beyond the methodology paper) to justify the choice of tissue source.

Lines 98–106: It would be helpful to specify whether ultrasonographic evaluations were performed by the same operator at each site, whether intra‑ and inter‑observer variability were assessed, and how images were reviewed.

Lines 139–141: Clarify whether cells were fresh or cryopreserved at the time of injection, the viability threshold required for release, and whether any dose adjustments were made if the yield differed from the target 10⁷ cells.

Lines 145–146: Since rehabilitation was “gradual” and “as tolerated,” please describe the recommended exercise protocol (e.g., box rest vs hand‑walking vs trotting at specified weeks) and whether adherence was monitored, as this is a major determinant of outcome.

Lines 231–237: The assertion that a second injection “further reinforces tissue remodeling and consolidation” should be toned down or more clearly qualified, given the absence of a non‑re‑injected comparison group over the same interval and the small sample size.

 

Author Response

Response to Reviewer 1

We thank the reviewer for the careful and constructive evaluation of our manuscript and for the positive assessment of the study. We appreciate the reviewer’s insightful comments and suggestions, which helped us improve the clarity and transparency of the manuscript.

In response to these comments, several clarifications and minor revisions have been made throughout the manuscript. In particular, we have:

• clarified the analyzed population in the abstract,
• added a definition of MHC-mismatched,
• provided additional details regarding ultrasonographic assessments,
• included quantitative characterization of the PRP preparation,
• clarified the preparation and quality control criteria of the mdMSC product,
• described the recommended rehabilitation protocol, and
• revised the discussion to provide a more cautious interpretation of the second injection findings.

All corresponding modifications have been incorporated into the revised manuscript. Detailed responses to each comment are provided below.

1/ Reviewer comment:
Concern regarding the interpretation of treatment effects given that all horses initially received PRP and that some animals may have improved before evaluation of the randomized treatment.

Answer:

We thank the reviewer for this relevant comment. As correctly pointed out, all horses received PRP at inclusion as part of the standardized protocol, and the study was conducted under double-blind conditions. Consequently, early clinical improvement attributable to PRP could only be identified after completion of the study and unblinding. In a subset of horses, lesion regression after PRP alone was sufficiently marked that further evaluation of a potential treatment effect became difficult. We have added a short clarification in the material and method (study design) and in the discussion section to better explain this point and to acknowledge its implications for interpretation of the results.

 

2/ Reviewer comment:

Why there is no explicit description of the final cell product beyond dose (10⁷ cells in 1 mL Cryostor CS5®): no viability at the time of injection, passage number, specific surface marker profile, or functional assays (e.g., trilineage differentiation, immunomodulatory assays), even though “phenotypic characterization” is mentioned. Also the use of Cryostor CS5® for cell suspension in both treatment and placebo is appropriate, but the authors should specify whether cells were thawed immediately before injection and whether a recovery/wash step was performed to remove cryoprotectant.

Answer:

We add the release criteria in the material and method section and a precision about injection. Cells were thawed immediately before injection.

 

3/ Reviewer Comment

The muscle microbiopsy and culture process is described in reasonable detail, including GMP conditions and phenotypic characterization. What are the costs of this approach versus standard autologous MSC culture from bone marrow? is it applicable on a clinical daily basis or it remains too costly? Why the triceps muscle specifically?

Answer

The costs are similar to a culture from bone marrow. The main advantage is a much less invasive technique as a bone marrow aspiration. The triceps brachii muscle is used for the facility but could be replaced by another one

 

 

4/ Reviewer Comment

PRP preparation is described using a specific commercial kit and standardized centrifugation, but no quantitative data are provided on platelet or leukocyte counts, nor on intra‑ or inter‑horse variability. This limits the ability to compare with existing PRP literature and to confirm that “leukocyte‑reduced” PRP was consistently obtained. Is it possible to add more specific quantitative data on the counts of PRP?

Answer

Quantitative characterization of the PRP preparation was available and has now been added to the Materials and Methods section. The PRP kit contains a specific gel that has the particularity of removing erythrocytes and granulocytes, but mononuclear blood cells (< 1.10³/µl) and platelets (300-400.10³/µl) can be easily resuspended after PPP removal.

5/ Reviewer comment:
The average age of the horses is 13 ± 5 years, which means that some horses were probably quite old. It might be interesting to discuss whether age or training experience could influence the response to treatment.

Response:
The age distribution of the study population reflects the typical population of client-owned sport horses presenting with tendon or suspensory ligament injuries. The study was not powered to perform subgroup analyses according to age or training level. We have added a short comment in the Discussion section acknowledging that age-related differences in healing capacity may influence treatment response and should be addressed in future studies

6. Reviewer comment:
   The discussion rightly highlights immunologic concerns with allogeneic MSCs and positions autologous mdMSCs as an immunologically safer alternative, but the current trial does not actually measure immune responses (e.g., alloantibody formation, lymphocyte activation), so such claims should be framed more cautiously as theoretical advantages supported by external literature.

Answer:

 We agree that the present study did not include direct assessment of immune responses such as alloantibody formation or lymphocyte activation. The corresponding statements in the Discussion have therefore been rephrased to present the immunological advantages of autologous cells more cautiously, as a theoretical advantage supported by previous literature rather than a finding demonstrated in the current trial.

Other Reviewer comments:

Lines 1–4: The title accurately reflects the design and interventions but does not indicate that the trial focuses on early outcomes; consider adding “early clinical and ultrasonographic outcomes” or similar to align expectations.

Response:
We thank the reviewer for this helpful suggestion. The title has been revised to better reflect the focus on early outcomes

Lines 11–30: The abstract is generally clear, but it omits the fact that analyses for specific mdMSC efficacy exclude PRP responders and are per protocol; this should be specified to transparently describe the analyzed population.

Response:
To clarify the analyzed population, the abstract has been revised to specify that the evaluation of the specific efficacy of mdMSCs was performed in the per-protocol population excluding horses showing early clinical response after PRP. This clarification has been added to the Methods section of the abstract.

Lines 21–25: The statement “18 horses from both groups received at T2 a second injection of mdMSC” in the abstract could be misread as a pre-planned, randomized second phase; clarifying that this was at investigator discretion and not randomized would avoid misunderstanding.

Response:
We thank the reviewer for this helpful comment. The abstract has been revised to clarify that the second injection administered at T2 was not part of a randomized phase but was performed at the investigator’s discretion. This clarification has been added to avoid potential misunderstanding

Line 56: define what MHC-mismatched means.

Response:
A brief definition of “MHC-mismatched” has now been added in the Introduction for clarity.

Lines 65–73: The therapeutic “dilemma” and sequential strategy are well framed, but the introduction could benefit from a brief mention of existing clinical data on autologous muscle-derived MSCs in horses (beyond the methodology paper) to justify the choice of tissue source.

Response:
We thank the reviewer for this helpful suggestion. A brief statement has been added in the Introduction to acknowledge the existing experimental and preliminary clinical data supporting the use of skeletal muscle as a source of autologous mesenchymal stem/stromal cells in horses. This addition provides further context for the choice of tissue source used in the present study.

Lines 65–73: The therapeutic “dilemma” and sequential strategy are well framed, but the introduction could benefit from a brief mention of existing clinical data on autologous muscle-derived MSCs in horses (beyond the methodology paper) to justify the choice of tissue source.

Response:
A brief statement has been added in the Introduction to acknowledge the existing experimental and preliminary clinical data supporting the use of skeletal muscle as a source of autologous mesenchymal stem/stromal cells in horses. This addition provides further context for the choice of tissue source used in the present study.

Lines 98–106: It would be helpful to specify whether ultrasonographic evaluations were performed by the same operator at each site, whether intra- and inter-observer variability were assessed, and how images were reviewed.

Response:

In order to minimize operator-related variability, each horse was examined throughout the study by the same investigator. Ultrasonographic examinations were performed according to a standardized protocol by experienced clinicians. This clarification has now been added in the Materials and Methods section.

Lines 139–141: Clarify whether cells were fresh or cryopreserved at the time of injection, the viability threshold required for release, and whether any dose adjustments were made if the yield differed from the target 10⁷ cells.

Response:

 The manuscript has been clarified to indicate that the mdMSCs were cryopreserved after expansion and thawed immediately prior to administration. Cell viability was assessed as part of product release testing, and only preparations meeting predefined quality criteria were used. The target dose of 10⁷ cells was administered without dose adjustment

Lines 145–146: Since rehabilitation was “gradual” and “as tolerated,” please describe the recommended exercise protocol (e.g., box rest vs hand walking vs trotting at specified weeks) and whether adherence was monitored, as this is a major determinant of outcome.

Response:
We thank the reviewer for this important comment. A standardized rehabilitation program was recommended after treatment, including an initial period of restricted activity followed by progressive hand walking and gradual reintroduction of exercise according to clinical evolution. Because this was a clinical field study involving client-owned horses, adherence to the rehabilitation program was monitored during follow-up visits but could not be strictly standardized in all cases. This clarification has now been added in the Materials and Methods section

Lines 231–237: The assertion that a second injection “further reinforces tissue remodeling and consolidation” should be toned down or more clearly qualified, given the absence of a non re-injected comparison group over the same interval and the small sample size.

Response:
We thank the reviewer for this helpful comment. We agree that the interpretation of the second injection effect should be presented more cautiously given the absence of a non-reinjected comparison group and the limited sample size. The corresponding sentence in the Discussion has therefore been revised to provide a more cautious interpretation of these findings

Reviewer 2 Report

Comments and Suggestions for Authors

dear authors the topic is interesting and actual but in my opinion this work needs some changes to be considered for publication; the M&M section need to be expanded to better understand the position and severity of the lesion togheter with a accurate description of the ultrasonographic finding. Results have also something to be changed, below you can find more precise comments

line 29: i do not think that incomplete healing is a correct definition, many of them reoccur because the poor quality of the scar tissue

line 101: superficial OR deep, moreover i would describe better which is the anatomical district you were considering for the study: the metacarpus? metatarsus? pastern? front or hind legs?

line 161-162: followed by gradual return to controlled exercise; i think you need to expand this topic, it is of paramount importance to know which exercise horses would have done in the period of the study and if it was standardized for all the patients. it's to me a key point to validate your results

line 172-176 i would have more informations about the ultrasonographic appearence and the severity of the lesions and their distribution between the two groups; in a not huge number of cases like your group it is important to help the reader understand your results

line 196: ultrasonographic parameters, you never described them in M&M and this is very important to me maybe a table would be useful in the M&M

line 234: fenylbutazone

line 239: i would define better the concept of 'did not show sufficient early improvement' erlier in the paper

Author Response

Response to Reviewer 2

We thank the reviewer for the careful evaluation of our manuscript and for the constructive comments and suggestions. We appreciate the reviewer’s interest in the topic and the detailed remarks aimed at improving the clarity of the manuscript.

In response to these comments, several clarifications and additions have been made, particularly in the Materials and Methods and Discussion sections. These revisions include:

• clarification of the anatomical location of the lesions included in the study,
• improved description of the ultrasonographic evaluation and lesion severity parameters,
• clarification of the rehabilitation program recommended during the study period, and
• refinement of certain statements in the Introduction and Discussion to improve scientific accuracy.

These modifications were intended to improve the transparency of the study design and the characterization of the study population while maintaining the original structure of the manuscript.

Detailed responses to each comment are provided below.

1/Reviewer comment:
Dear authors, the topic is interesting and actual but in my opinion this work needs some changes to be considered for publication; the M&M section needs to be expanded to better understand the position and severity of the lesion together with an accurate description of the ultrasonographic findings.

Response:
We thank the reviewer for this constructive comment. To improve the clarity of the Materials and Methods section, we have expanded the description of lesion localization and ultrasonographic evaluation. Additional information has been included regarding the anatomical location of the lesions and the ultrasonographic parameters used to assess lesion severity (including fiber alignment score, echogenicity, and lesion size). We have also clarified that ultrasonographic examinations were performed according to a standardized protocol by experienced clinicians, with each horse being evaluated throughout the study by the same investigator.

These additions aim to provide a clearer description of lesion characterization and imaging assessment.

2/ Reviewer comment:
Line 29: I do not think that incomplete healing is a correct definition, many of them reoccur because of the poor quality of the scar tissue.

Response:
We thank the reviewer for this helpful comment and agree that recurrence of tendon injuries is often related to the formation of mechanically inferior scar tissue rather than strictly incomplete healing. The sentence in the Introduction has been revised accordingly.

3/ Reviewer comment:
Line 101: superficial OR deep; moreover, I would describe better which anatomical district you were considering for the study: the metacarpus? metatarsus? pastern? front or hind legs?

Response:
The description of lesion localization has been clarified in the Materials and Methods section to better specify the anatomical structures and regions considered in the study. In particular, we now indicate that lesions involved the superficial or deep digital flexor tendons and suspensory ligament branches, and we specify the anatomical districts examined.

4/ Reviewer comment:
Line 161–162: followed by gradual return to controlled exercise; I think you need to expand this topic. It is of paramount importance to know which exercise horses would have done in the period of the study and if it was standardized for all the patients. It is, to me, a key point to validate your results.

Response:
We thank the reviewer for this important comment and fully agree that rehabilitation is a key determinant of tendon healing. The manuscript has been revised to provide a clearer description of the recommended rehabilitation program following treatment. All horses followed a controlled exercise program that included an initial period of restricted activity, followed by progressive hand walking and gradual reintroduction of exercise according to clinical evolution and ultrasonographic findings.

Because this was a clinical study involving client-owned horses managed by different practitioners, rehabilitation protocols followed these general guidelines but could not be strictly standardized in every case. This clarification has been added to the Materials and Methods section. In addition, a short statement has been included in the Discussion section acknowledging that variability in rehabilitation may have contributed to some variability in clinical outcomes.

5/ Reviewer comment:
Line 172–176: I would have more information about the ultrasonographic appearance and the severity of the lesions and their distribution between the two groups; in a not huge number of cases like your group it is important to help the reader understand your results.

Response:

 Additional clarification has been added to the manuscript to better describe the ultrasonographic characteristics and severity of the lesions at inclusion. Lesion assessment included fiber alignment score (FAS), echogenicity, and percentage of lesion area relative to the tendon cross-sectional area, which were used to characterize lesion severity. We have also clarified that the distribution and baseline severity of lesions were comparable between the two groups. These details have been added to improve the reader’s understanding of the study population.

6/ Reviewer comment:
Line 196: ultrasonographic parameters; you never described them in M&M and this is very important to me. Maybe a table would be useful in the M&M.

Response:
We thank the reviewer for this comment. The ultrasonographic parameters used to characterize lesion severity are already described in the Materials and Methods section, including the fiber alignment score (FAS), echogenicity, and the percentage of lesion area relative to the tendon cross-sectional area. These parameters were used to assess lesion severity and to monitor structural changes during follow-up examinations. We have clarified this point in the revised manuscript to improve readability.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

dear authors the apper has improved and all comments have been addressed