Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an interesting study evaluating the biological effects of platelet-rich plasma (PRP) on the subacromial bursa and torn supraspinatus tendon in a randomized design. The authors report that preoperative leukocyte-poor PRP (LP-PRP) injection improved the histological characteristics of the supraspinatus tendon and was associated with biologically relevant reductions in selected bursal gene expression.

However, only 16 patients were recruited for this study. The authors should clarify the rationale for this sample size. Was a power analysis performed for the primary endpoint? If so, it should be clearly described in the Methods section, including the assumptions used (effect size, alpha level, and power). If not, this limitation should be explicitly acknowledged and justified.
In addition, the Discussion section would benefit from a broader contextualization of the findings.

The authors should expand on the different therapeutic approaches for subacromial bursa pathology and torn supraspinatus tendon, particularly in the setting of rotator cuff tendinopathy. It would be important to cite and discuss studies comparing PRP with other treatment modalities (e.g., corticosteroid injections, physical therapy, surgical management, or other biologic therapies), as well as the clinical outcomes reported in those studies.

Finally, the limitations of the study should be discussed more thoroughly. In particular, the small sample size, potential selection bias, short follow-up (if applicable), lack of clinical outcome correlation (if applicable), and any methodological constraints should be clearly acknowledged.

Overall, this study addresses a relevant and timely topic, but further clarification and expansion of the discussion would strengthen the manuscript.

Author Response

Comment: However, only 16 patients were recruited for this study. The authors should clarify the rationale for this sample size. Was a power analysis performed for the primary endpoint? If so, it should be clearly described in the Methods section, including the assumptions used (effect size, alpha level, and power). If not, this limitation should be explicitly acknowledged and justified.

Reply: Thank you for your comment. We have added information about the power analysis for total modified Movin score as primary endpoint to the Methods section, including the assumed α level, desired power and detectable effect size (lines 448-454).

 

Comment: The authors should expand on the different therapeutic approaches for subacromial bursa pathology and torn supraspinatus tendon, particularly in the setting of rotator cuff tendinopathy. It would be important to cite and discuss studies comparing PRP with other treatment modalities (e.g., corticosteroid injections, physical therapy, surgical management, or other biologic therapies), as well as the clinical outcomes reported in those studies.

Reply: Thank you for your comment. Three paragraphs discussing the different treatment modalities and their clinical outcomes on rotator cuff tear and subacromial bursitis have been added in the Discussion. (lines 272-302)

 

Comment: Finally, the limitations of the study should be discussed more thoroughly. In particular, the small sample size, potential selection bias, short follow-up (if applicable), lack of clinical outcome correlation (if applicable), and any methodological constraints should be clearly acknowledged.

Reply: Thank you for your comment. The limitation section has been modified according to your suggestion and includes more information about limitations related to the small sample size, lack of clinical outcome and methodological constraints (lines 304-311, 315-317). The follow-up has not been discussed, as there was no evaluation of the clinical outcome.

Reviewer 2 Report

Comments and Suggestions for Authors

Executive Summary: Strengths and Weaknesses

This manuscript presents an in vivo, single-blinded randomized controlled trial evaluating the preoperative biological effects of leukocyte-poor platelet-rich plasma (LP-PRP) subacromial injection on supraspinatus tendon histomorphology and subacromial bursa cell gene expression in patients with full-thickness degenerative rotator cuff tears. The study is original in its design, combining both histological and molecular endpoints in the same patient cohort, and addresses a genuinely underexplored question in orthopedic biology.

1.1 Strengths

The most compelling strength of this work lies in its originality: to the best of the reviewer's knowledge, no prior study has simultaneously examined the in vivo effect of LP-PRP on both supraspinatus tendon histology and subacromial bursa cell gene expression in the setting of degenerative rotator cuff tear. The choice of the modified Movin score (Chen modification) as the primary histological outcome is methodologically sound, as this instrument evaluates six distinct tendon tissue characteristics and has been validated in the literature. The use of hematoxylin and eosin staining alongside transmission electron microscopy provides a complementary multi-resolution assessment of tendon architecture. The molecular analysis via quantitative real-time PCR (qRT-PCR) with rigorously defined primer sequences (Table 4) for seven target genes – COL1A1, COL2A1, COL3A1, MMP3, MMP13, IL-1β, and IL-6 – reflects a well-considered panel relevant to tendon pathobiology. The blinding of the pathologist who scored the Movin parameter is a noteworthy design strength. The PICO framework application and the clear Ethics Committee approval documentation further reinforce the methodological rigor. The statistical approach employing the ΔCt method for relative gene expression, with log₂ transformation for parametric analysis, is appropriate. The predefined biological significance threshold of ≥ 1 log₂ unit (≥ 2-fold change) is a reasonable strategy to identify potentially relevant molecular changes in the context of an underpowered study.

1.2 Weaknesses

The principal weakness of this manuscript is the extremely limited sample size (n = 8 per group, total N = 16), which substantially constrains statistical power and generalizability. The power analysis justification provided at page 8, line 275 – "at least eight pairs were required according to the power analysis based on experimental studies" – is circular, opaque, and fails to satisfy standard reporting requirements (alpha level, expected effect size, desired power). This deficiency is compounded by unequal sample sizes within the gene expression analysis (as few as n = 4 in the control group for IL-1β), which the manuscript does not justify adequately.

A second critical gap is the complete absence of inter-rater reliability statistics for the Movin score assessment. Although the pathologist is described as working blindly, no intraclass correlation coefficient (ICC) or Cohen's kappa is reported between raters or between repeated assessments, which undermines the reproducibility of the primary histological outcome.

Device specifications are systematically absent throughout the methods section. The ultrasound machine used for guided injection, the optical microscope, and the transmission electron microscope are not identified with brand name, model, country of manufacture, or technical parameters (e.g., magnification range, light source specifications). This represents a critical reproducibility deficiency. Furthermore, the PRP preparation kit (TriCell PRP M Blood Separation Kit, REV-MED) lacks key specifications: the centrifugation protocol is provided (5 min at 3200 rpm) but the resulting platelet concentration, leukocyte count, and growth factor content of the PRP preparation are not reported, making it impossible to characterize or reproduce the biological agent administered.

Figure 4, presenting gene expression data, displays only group mean lines (horizontal bars) without standard deviation or standard error bars, violating standard graphical reporting requirements for continuous data. The absence of error bars prevents visual assessment of data spread and statistical uncertainty. Similarly, although Table 2 provides individual modified Movin scores (a notable transparency strength), the summary statistics in the text could be supplemented with standard deviations.

The English writing is generally clear and scientifically appropriate; however, a typographical error was identified at page 7, line 240: "suggesting that it can may serve as a useful biological adjunct" – the double modal "can may" is grammatically incorrect and should read "suggesting that it may serve as a useful biological adjunct". Additional minor language concerns are addressed in Section 5.

Finally, the study is described as "single-blinded" (page 8, lines 271–273), with blinding applied only to the pathologist at assessment. Patients in the control group received no injection at all, which introduces a procedural asymmetry that may affect patient perception and behavior in the weeks following the non-intervention. A sham injection with normal saline would have strengthened the allocation concealment and reduced performance bias.

 

 

 

2. Detailed Section-by-Section Critique with Page and Line References

2.1 Abstract (Page 1, Lines 19–36)

The abstract is well-structured and conveys the essential elements of the study. However, the sample size per group (n = 8) is reported without confidence intervals around the key effect estimates. The fold-change values reported for COL2, MMP3, and MMP13 (0.23, 0.24, and 0.26, respectively) are meaningful, but the absence of any confidence interval around these fold-change estimates is a limitation that should be acknowledged in the abstract itself. The authors should additionally clarify the distinction between statistical non-significance and biological relevance in the abstract, as the current phrasing risks misleading readers into overinterpreting the molecular findings.

It is noted that the decimal separator "p=0.002" is correctly written using the period (".") as decimal separator throughout the abstract, which is appropriate.

2.2 Introduction (Page 1–2, Lines 38–96)

The introduction provides an adequate, though not exhaustive, review of rotator cuff anatomy, supraspinatus tendon biology, subacromial bursa function, and the rationale for PRP. The progression from anatomical context to molecular pathophysiology to therapeutic hypothesis is logical. However, the introduction would benefit from a dedicated paragraph framing the current landscape of biologics in musculoskeletal tendon disorders, as recent evidence suggests that orthobiologics including PRP present divergent outcomes depending on formulation (LR-PRP vs LP-PRP), concentration, and delivery route. In this context, the authors are encouraged to integrate recent evidence on orthobiologics in tendon treatment to provide a more comprehensive backdrop for their specific hypothesis.

Suggested reference for introduction (page 1–2, lines 75–82): Vascellari A, Demeco A, Vittadini F, et al. Orthobiologics Injection Therapies in the Treatment of Muscle and Tendon Disorders in Athletes: Fact or Fake? Muscles Ligaments Tendons J. 2024;14(2):239–246. This narrative review provides a critical appraisal of the evidence base for orthobiologics including PRP in tendon and muscle disorders, and would strengthen the rationale for LP-PRP injection in the current study.

2.3 Results – Demographics (Page 2–3, Lines 100–106)

Table 1 presents patient demographics adequately. The groups are well-balanced across age, sex, laterality, smoking status, comorbidities, and tear classification. The statistical tests used for group comparison are appropriate: independent t-test for continuous variables and chi-square for categorical ones. P-values are reported correctly with the decimal point separator. However, the age data is presented as "mean (range)" without standard deviation. Since parametric testing was applied (independent t-test, p = 0.767), the standard deviation should be reported alongside the mean to allow verification of the distributional assumptions. The reviewers recommend reporting age as mean ± SD.

2.4 Results – Optical Microscopy and Modified Movin Score (Pages 2–4, Lines 107–134)

The primary outcome results are clearly reported and the raw individual scores in Table 2 are commendably transparent, allowing independent verification. The reviewer confirms the arithmetic: the mean total Movin score in the PRP group sums to 52/8 = 6.50, and in the control group to 97/8 = 12.13 (reported as 12.1), which are consistent. The p-value of 0.002 for the between-group difference in total score, derived from an independent-sample t-test, is plausible given the magnitude of separation. However, with n = 8 per group, the assumption of normality underlying the t-test may not be satisfied, and a non-parametric Mann-Whitney U test should either be used or justified as unnecessary through reference to normality testing (e.g., Shapiro-Wilk test).

A critical deficiency is the absence of inter-rater reliability reporting for the Movin score assessment. The modified Movin score involves inherently subjective qualitative grading of histological parameters. The manuscript states that "the pathologist inspected each sample blindly" (page 8, line 282), but no intra-rater or inter-rater reliability statistics (ICC, Cohen's kappa, or weighted kappa) are provided. This is a standard requirement for any study employing a semi-quantitative scoring instrument and constitutes a reproducibility gap. At a minimum, the authors should provide Cohen's weighted kappa or ICC for the total Movin score based on repeat readings by the same pathologist or assessment by two independent pathologists.

 

2.5 Results – Electron Microscopy (Pages 4–6, Lines 135–155)

The qualitative electron microscopy data are described coherently and the micrographs (Figures 2 and 3) are of acceptable resolution. However, the device used for electron microscopy is not specified anywhere in the manuscript: brand, model, accelerating voltage, magnification calibration method, and country of manufacture are all absent. This information is mandatory for scientific reproducibility. The reviewer specifically requests that the following technical details be added to the methods section (Section 4.7, page 10, lines 335–340): the transmission electron microscope brand (e.g., JEOL, FEI/Thermo Fisher, Zeiss), model number, accelerating voltage (kV), country of manufacture, and a validation or calibration reference.

Additionally, Figures 2 and 3 present scale bars in micrometers (μm) at each panel, which is appropriate. However, the panel-level magnification inconsistency noted in the figure legends (x6000, x10000, x12000 in Figure 2; x6000, x10000, x30000 in Figure 3) should be standardized where possible, or the rationale for selecting different magnifications per sample should be explained.

2.6 Results – Gene Expression (Pages 6–7, Lines 157–188)

This is the section requiring the most substantial revision. Several critical issues are identified. First, the unequal sample sizes represent a significant analytical problem. For COL1, COL2, COL3, and MMP13, n = 7 per group was analyzed; for MMP3 and IL6, n = 6 per group; and most critically, for IL-1β, n = 6 (PRP group) and only n = 4 (control group). The reasons for sample exclusion are not provided and should be clearly stated. The loss of half the control group samples for IL-1β analysis renders any comparative conclusion for this gene biologically and statistically unreliable, and the authors should either exclude IL-1β from comparative analysis or explicitly acknowledge this as a major limitation.

Second, Figure 4 presents gene expression data as individual data points with only horizontal mean lines. The absence of error bars (standard deviation, standard error, or 95% confidence intervals) is a critical graphical deficiency. For a figure in a peer-reviewed molecular journal, error representation is mandatory. The authors must add error bars (at minimum, standard deviation bars) to all panels of Figure 4.

Third, applying parametric statistical testing (independent-sample t-test) to gene expression data with n = 4–7 per group without demonstrating normality is questionable. Given the small and unequal sample sizes, the Mann-Whitney U test would be more appropriate, or the authors must demonstrate normality using the Shapiro-Wilk test and report the test statistics and p-values.

Fourth, the biological significance threshold of ≥ 1 log₂ unit is a reasonable convention but is post-hoc and not pre-registered, which should be acknowledged in the limitations. While the authors justify it in the methods section, the lack of pre-registration with a recognized registry for this threshold raises concerns about potential inflation of biological significance.

Suggested reference for gene expression methodology context (page 6–7, lines 160–175): Saremi H, Heidari B, Sharifi A, Khanlarzadeh E. Arthroscopic Biceps Tenodesis: Midterm Clinical Results of a New Anchor Suture Technique in Patients with Single-Row Rotator Cuff Repair. Muscles Ligaments Tendons J. 2023;13(2):259–266. This original study involving rotator cuff surgery outcome assessment demonstrates the methodological framework for reporting biological outcomes in rotator cuff intervention studies, and provides relevant context for the current molecular findings.

2.7 Discussion (Pages 7–8, Lines 191–262)

The discussion is generally well-structured, and the authors appropriately contextualise their histological findings within the existing literature on PRP and tendon Movin scoring. The comparison with animal model studies (Yüksel et al., Genc et al.) is reasonable given the limited human in vivo literature. However, the discussion of gene expression findings is speculative in several passages and would benefit from greater restraint. The assertion that "reduced MMP expression may enhance the potential for tendon repair" (page 8, lines 227–228) is plausible but is presented without acknowledging that MMPs also play essential roles in matrix remodeling during the healing cascade, and their blanket suppression may not be uniformly beneficial.

The discussion also fails to adequately address the clinical significance threshold for the modified Movin score. What constitutes a minimal clinically important difference (MCID) on this instrument? Without this anchoring, the between-group difference of 6.5 vs. 12.1 points, though statistically significant, cannot be placed in a clinical context.

Suggested reference for clinical relevance framing of tendon biology (page 7–8, lines 213–230): Kahraman Y. A Critical Review on Tendon Structure and Load Remodeling. Muscles Ligaments Tendons J. 2023;13(2):218–227. This narrative review comprehensively addresses tendon extracellular matrix dynamics, including collagen remodeling and MMP regulation, providing a critical theoretical framework for interpreting the molecular findings observed in the current study.

 

2.8 Materials and Methods – Study Design (Page 8, Lines 271–283)

The single-blinded design with blinding restricted to the pathologist is acknowledged as a limitation, which is appropriate. However, the randomization method is described only as "one-by-one distribution of sixteen consecutive eligible patients into two groups" (page 8, lines 272–274), which lacks specificity. Was this simple alternation, block randomization, or concealed allocation? The method of allocation concealment is not described, raising the risk of selection bias. The CONSORT guidelines for reporting randomized trials require explicit description of the sequence generation method, allocation concealment mechanism, and implementation of randomization. This information must be added.

The power calculation is stated at page 8, line 275: "at least eight pairs were required according to the power analysis based on experimental studies". This is entirely insufficient. The authors must report: the primary outcome used for the power calculation (modified Movin score or fold-change in a specific gene), the expected effect size and its source (which experimental studies?), the assumed standard deviation, the alpha level (typically 0.05), and the desired power (typically 0.80 or 0.90). Without these elements, the adequacy of the sample size cannot be evaluated.

2.9 Materials and Methods – PRP Preparation (Page 9, Lines 299–306)

The PRP preparation section identifies the commercial kit used (TriCell PRP M Blood Separation Kit, REV-MED) but fails to provide the country of manufacture/sale, the centrifuge brand, model, and rotor type, the resulting platelet concentration (fold-concentration above baseline), the leukocyte count in the final preparation (critical for confirming LP-PRP classification), and the growth factor content (e.g., PDGF-AB, TGF-β1, VEGF). Without platelet count verification, the classification of the preparation as "leukocyte-poor" cannot be independently verified, and the biological agent administered is not reproducibly characterized. At minimum, mean platelet concentration and leukocyte count in the PRP preparations should be reported.

Suggested reference for orthobiologics characterization standards (page 9, lines 299–306): Vascellari A, Demeco A, Vittadini F, et al. (already cited above). Additionally: Bergamin F, Civera M, Rodriguez Reinoso M, Burgio V, Grimaldo Ruiz O, Surace C. Worldwide Incidence and Surgical Costs of Tendon Injuries: A Systematic Review and Meta-Analysis. Muscles Ligaments Tendons J. 2023;13(1):31–45. This systematic review of tendon injuries provides epidemiological context for the clinical burden addressed by this study and supports the public health relevance of developing effective PRP protocols.

2.10 Materials and Methods – Device Specifications

The following devices are used in this study but lack mandatory technical specifications:

• Ultrasound machine (page 9, line 310): The injection is described as "administered under ultrasound guidance" but no brand, model, transducer type (linear array), frequency range (MHz), country of manufacture, or validation reference is provided. Required additions: manufacturer, model, transducer frequency (e.g., 7–15 MHz linear), country of origin.
• Optical microscope (page 10, line 333): The light microscopy section references staining and magnification (×100, ×400) but no microscope brand, model, camera, or image acquisition system is specified. Required additions: microscope brand (e.g., Nikon, Olympus, Leica), model, digital camera model, image analysis software.
• Transmission electron microscope (page 10, lines 335–340): Brand, model, accelerating voltage (kV), and country of manufacture must be specified.
• Centrifuge (page 9, lines 303–305): Brand, model, rotor type, temperature during centrifugation, and country of manufacture should be added.
• PCR system (page 11, lines 356–362): The qRT-PCR instrument brand, model, country of manufacture, and cycling conditions (thermal profile) are not reported. These are essential for reproducibility.

2.11 Materials and Methods – Statistical Analysis (Page 11, Lines 371–388)

The use of IBM SPSS Statistics Version 25.0 (Armonk, NY, USA: IBM Corp., 2017) is appropriately cited. The independent-samples t-test and chi-square test are described correctly. However, as noted above, the normality assumption for the t-test with n = 4–8 subjects should be tested and reported. The authors should add: (a) Shapiro-Wilk test results for the primary outcome variables in each group; (b) justification for parametric testing with these sample sizes; (c) exact p-values for all comparisons in Table 3 rather than the thresholded notation currently used; (d) the power calculation parameters as described in Section 2.8 of this review. The ΔCt formula presented at line 378 is correctly stated: ΔCt = Ct_gene − Ct_β-actin. The use of the 2^(−ΔCt) transformation and subsequent log₂ conversion is methodologically appropriate.

2.12 Test-Retest Reliability

This is an original article employing a semi-quantitative scoring instrument (modified Movin score) as the primary outcome measure. According to fundamental measurement standards, any study utilizing an observer-dependent scoring system must demonstrate test-retest reliability, either through intra-rater ICC (same pathologist, repeated at ≥ 2 weeks interval) or inter-rater reliability (two independent pathologists). The current manuscript provides no such data. The authors should add a reliability sub-study or cite an existing validation study demonstrating the inter-rater reliability of the Chen-modified Movin score specifically. If this cannot be done retrospectively, the limitation must be prominently acknowledged and the potential impact on results discussed.

 

 

 

3. Statistical Verification

The reviewer performed an independent verification of key statistical values reported in the manuscript.

3.1 Modified Movin Score (Table 2)

PRP group individual total scores: 9, 4, 3, 5, 9, 9, 9, 4. Sum = 52. Mean = 52/8 = 6.500. This matches the reported value of 6.5 (range 3–9). ✓

Control group individual total scores: 14, 8, 9, 15, 14, 8, 15, 14. Sum = 97. Mean = 97/8 = 12.125 ≈ 12.1 (range 8–15). ✓

The between-group difference (12.125 − 6.500 = 5.625) is substantial in absolute terms. The reported p-value of 0.002 is consistent with this magnitude of separation at n = 8 per group.

Sub-category verification: Fiber structure: PRP = (2+1+1+1+2+2+2+1)/8 = 12/8 = 1.50; Control = (3+2+2+3+3+2+3+3)/8 = 21/8 = 2.625 ≈ 2.6 ✓. Fiber arrangement: same calculation yields identical values ✓. Nuclear rounding: PRP = (2+1+1+2+2+2+2+2)/8 = 14/8 = 1.75 ≈ 1.8 ✓; Control = (3+2+2+3+3+2+3+3)/8 = 21/8 = 2.625 ≈ 2.6 ✓. All arithmetic checks are consistent with reported values.

3.2 Gene Expression Data (Table 3)

The fold-change values are calculated as 2^(ΔΔCt) where ΔΔCt = ΔCt_PRP − ΔCt_Control. For COL2: PRP mean = −8.24, Control mean = −6.12, ΔΔCt = −8.24 − (−6.12) = −2.12. Fold change = 2^(−2.12) = 0.229 ≈ 0.23 ✓. For MMP3: ΔΔCt = −7.46 − (−5.43) = −2.03. Fold change = 2^(−2.03) = 0.243 ≈ 0.24 ✓. For MMP13: ΔΔCt = −6.85 − (−4.89) = −1.96. Fold change = 2^(−1.96) = 0.259 ≈ 0.26 ✓. All fold-change arithmetic is consistent with the reported values.

However, the reviewer notes a critical concern: the sample sizes for MMP3 and IL6 (n = 6 per group) and especially IL-1β (n = 6 PRP, n = 4 control) are insufficient for any meaningful parametric comparison. For IL-1β with n = 4 controls and assumed high variability (SD = 1.84 log₂ units), the detectable effect size at α = 0.05 and power = 0.80 would require approximately 20+ subjects per group according to standard power tables. The statement in the manuscript that "the discrepancy between statistical and biological significance likely reflects sample size limitations" (page 6, lines 171–172) is acknowledged, but the consequence for the IL-1β and MMP3 comparisons specifically is underappreciated.

Furthermore, the COL1 fold change is reported as 0.51× with a Δlog₂ of −0.96, which falls just below the predefined biological significance threshold of 1 log₂ unit. This near-threshold finding should be discussed, particularly given that COL1 upregulation is a key marker of tendon healing quality.

3.3 Decimal Notation Verification

Throughout the manuscript, decimal separators are consistently written as periods (".") rather than commas (","), which is correct for international scientific publications. No violation of this standard was detected in the main text, tables, or figure legends. All p-values in Tables 1, 2, and 3 use the period as decimal separator and are presented with three decimal places, which is appropriate.

 

 

 

4. Quality Assessment of Tables and Figures

4.1 Table 1 – Patient Demographic Data

Table 1 is clearly structured and the data are presented in an accessible format. The comparisons between groups are appropriate. The main deficiency is the presentation of age as "mean (range)" without standard deviation, as noted above. Adding SD to the age column would allow readers to assess whether the parametric t-test was appropriate.

4.2 Table 2 – Modified Movin Scores

This table is a particular strength of the manuscript: the presentation of individual patient scores alongside group means allows full transparency and independent verification. The column structure is logical. The only recommendation is to add a summary row with mean ± SD for each category to facilitate cross-group comparison without requiring the reader to perform arithmetic manually.

4.3 Table 3 – Gene Expression Comparison

Table 3 is informationally rich but has several deficiencies. The "NoP" column (number of patients) immediately reveals the unequal sample sizes, which is transparent. However, the p-values are provided only to three decimal places in some cases (e.g., IL-1β p = 0.605), and the standard deviations are provided in log₂ units, which may be unfamiliar to clinically oriented readers. The authors should consider adding a column for 95% confidence intervals of the fold change and explicitly flagging cells where sample size imbalance (n = 4 vs. n = 6) renders the comparison particularly uncertain.

4.4 Figures 1, 2, 3 – Microscopy Images

The histological photomicrographs in Figure 1 (H&E, ×100 and ×400) and the electron micrographs in Figures 2 and 3 are of adequate quality for peer-review purposes. Scale bars are present in all panels, which is appropriate. The magnification labels in the figure legends are consistent with the scale bars shown. However, the legend for Figure 3 describes panel D at "x30000" magnification, while the scale bar reads 1 μm – this is consistent and appears correct, but the reviewer recommends verifying that this was not an inadvertent transcription error during manuscript preparation.

4.5 Figure 4 – Gene Expression Scatter Plot

This figure presents a critical deficiency: no error bars are shown. Only horizontal lines indicating group means are plotted against individual data points. For a molecular science journal, the absence of error bars (standard deviation or standard error) violates standard graphical reporting norms. The scatter plot format is generally appropriate for small n, but the mean representation must be accompanied by a measure of dispersion. The authors must add error bars (at minimum ± SD) to all panels of Figure 4. Additionally, the x-axis labels (PRP, Ctr) are small and may benefit from larger font size, and the panels could benefit from individual p-value annotations consistent with Table 3.

 

 

 

5. English Language Quality

The overall English quality of this manuscript is acceptable and the scientific writing is generally clear. However, the following specific corrections are recommended:

• Page 7, line 240: "suggesting that it can may serve as a useful biological adjunct" → CORRECTION: "suggesting that it may serve as a useful biological adjunct" (double modal verb error).
• Page 1, line 33: "The preoperative LP-PRP injection improved supraspinatus tendon histological characteristics" → This is an overstatement in the abstract; consider "was associated with improved" to more accurately reflect the observational nature of the between-group comparison.
• Page 8, line 251: "the number of patients enrolled in each group was relatively small" → The qualifier "relatively" is vague; prefer "limited" or "small" without qualification, followed by the power analysis context.
• Page 9, line 301: "TriCell PRP M Blood Separation Kit (REV-MED)" → Please provide the country of the manufacturer (REV-MED) for completeness.
• Page 11, line 371: "IBMSPSS" should be "IBM SPSS" (missing space between IBM and SPSS).
• Throughout: The manuscript inconsistently uses "Rotator Cuff" with capitalization mid-sentence (page 1 line 38, etc.). Maintain consistent capitalization: "rotator cuff" in running text, reserved for proper noun usage at sentence beginnings only.

 

 

 

6. Additional Critical Considerations

6.1 CONSORT Compliance

As a randomized controlled trial, this manuscript should include or reference a CONSORT flow diagram showing the number of patients screened, eligible, randomized, allocated, receiving intervention, followed up, analyzed, and excluded at each stage with reasons. No CONSORT flow diagram is present in this manuscript. While the authors describe the allocation process in the text, the absence of this standardized reporting element is a significant deficiency for a clinical trial article in a molecular science journal.

6.2 Trial Registration

The manuscript should identify whether this randomized controlled trial was prospectively registered with a clinical trials registry (e.g., ClinicalTrials.gov, WHO ICTRP, or national equivalents). Trial registration is required by the ICMJE for randomized trials. The Ethics Committee approval is noted (Protocol No. 15043 – 27/09/2023 and IRB Protocol 11604/26-07-2023), and there is a reference to a prior publication of the first 20 patients [44], but no trial registration number is provided. If not registered, the authors must acknowledge this as a limitation and explain why registration was not performed.

 

6.3 Reference Citation Accuracy

The references are generally cited in appropriate contexts. Reference [44] (Pitsilos et al., IJMS 2024) is cited correctly as the prior publication of the first 20 patients. References [45] (DeOrio and Cofield classification) and [46] (Fuchs fatty infiltration classification) are cited at their first methodological use. No obviously inappropriate citations were detected. However, the reviewer notes that several important recent contributions to the field of tendon biology and rotator cuff repair are absent from the reference list, as highlighted in the section-by-section critique above.

 

 

 

8. Prioritized Action Items for Authors

8.1 Critical (Must address before re-submission)

• Add complete power calculation parameters (alpha, beta, effect size, SD source) to Section 4.1 [Page 8, line 275]
• Add inter-rater reliability statistics (ICC or Cohen's kappa) for the modified Movin score [Page 10, Section 4.8]
• Add error bars (± SD) to all panels of Figure 4 [Page 7, Figure 4]
• Specify all device technical details (ultrasound, microscopes, centrifuge, PCR system) [Sections 4.3–4.9]
• Report platelet count and leukocyte count in PRP preparations [Page 9, Section 4.3]
• Provide CONSORT flow diagram as supplementary material
• State clinical trial registration number or explicitly justify absence of registration

8.2 High Priority

• Provide justification for unequal sample sizes in gene expression analysis, particularly n=4 for IL-1β control group [Table 3]
• Either add normality test results (Shapiro-Wilk) or switch to Mann-Whitney U tests for all continuous comparisons [Section 4.10]
• Add SD to age data in Table 1
• Describe randomization sequence generation and allocation concealment mechanism [Page 8, lines 272–274]

8.3 Moderate Priority

• Correct typographical error: "can may serve" → "may serve" [Page 7, line 240]
• Add qRT-PCR thermal cycling conditions to Section 4.9
• Discuss the minimal clinically important difference (MCID) for the modified Movin score in the discussion
• Integrate suggested references as detailed in Section 2 of this review

 

 

 

 Concluding Remarks

This manuscript presents a genuinely novel and scientifically valuable investigation into the in vivo biological effects of LP-PRP on human rotator cuff tissue. The simultaneous assessment of supraspinatus tendon histomorphology and subacromial bursa cell gene expression constitutes a meaningful contribution to the PRP literature. The histological findings, showing improved modified Movin score with lower inflammation and better fiber organization in PRP-treated tendons, are supported by internally consistent data. The molecular findings, while not reaching statistical significance, suggest biologically relevant trends in COL2, MMP3, and MMP13 expression that warrant further investigation in adequately powered studies.

However, as detailed in this review, the manuscript requires substantial revision before it can be considered for publication. The critical requirements center on the adequacy of power reporting, the completeness of device specifications, the reliability of the primary scoring instrument, and the quality of graphical data presentation. The authors are encouraged to address these points comprehensively and to consider the suggested references as tools to strengthen both the scientific framing and methodological transparency of the revised manuscript.

Author Response

Comment: The abstract is well-structured and conveys the essential elements of the study. However, the sample size per group (n = 8) is reported without confidence intervals around the key effect estimates. The fold-change values reported for COL2, MMP3, and MMP13 (0.23, 0.24, and 0.26, respectively) are meaningful, but the absence of any confidence interval around these fold-change estimates is a limitation that should be acknowledged in the abstract itself. The authors should additionally clarify the distinction between statistical non-significance and biological relevance in the abstract, as the current phrasing risks misleading readers into overinterpreting the molecular findings.

Reply: Thank you for your comment. The Abstract was revised to include the confidence intervals and to clarify the difference between statistical non-significance and biological relevance (lines 27-28, 34-37).

 

Comment: However, the introduction would benefit from a dedicated paragraph framing the current landscape of biologics in musculoskeletal tendon disorders, as recent evidence suggests that orthobiologics including PRP present divergent outcomes depending on formulation (LR-PRP vs LP-PRP), concentration, and delivery route. In this context, the authors are encouraged to integrate recent evidence on orthobiologics in tendon treatment to provide a more comprehensive backdrop for their specific hypothesis.

Reply: Thank you for your comment. A paragraph discussing the orthobiologics used for rotator cuff tendinopathy or tear and subacromial bursitis has been added in the Introduction (lines 78-89)

 

Comment: Suggested reference for introduction (page 1–2, lines 75–82): Vascellari A, Demeco A, Vittadini F, et al. Orthobiologics Injection Therapies in the Treatment of Muscle and Tendon Disorders in Athletes: Fact or Fake? Muscles Ligaments Tendons J. 2024;14(2):239–246. This narrative review provides a critical appraisal of the evidence base for orthobiologics including PRP in tendon and muscle disorders, and would strengthen the rationale for LP-PRP injection in the current study.

Reply: Thank you for your comment. This article has been cited (line 83).

 

Comment: However, the age data is presented as "mean (range)" without standard deviation. Since parametric testing was applied (independent t-test, p = 0.767), the standard deviation should be reported alongside the mean to allow verification of the distributional assumptions. The reviewers recommend reporting age as mean ± SD.

Reply: Thank you for your comment. Standard deviation of age in the two groups was added in Table 1.

 

Comment: However, with n = 8 per group, the assumption of normality underlying the t-test may not be satisfied, and a non-parametric Mann-Whitney U test should either be used or justified as unnecessary through reference to normality testing (e.g., Shapiro-Wilk test).

Reply: Thank you for your comment. The statistical analysis was revised using the Mann-Whitney U test (line 461). Standard deviation was added in the Results and p-value was changed (lines 142-149).

 

Comment: A critical deficiency is the absence of inter-rater reliability reporting for the Movin score assessment. The modified Movin score involves inherently subjective qualitative grading of histological parameters. The manuscript states that "the pathologist inspected each sample blindly" (page 8, line 282), but no intra-rater or inter-rater reliability statistics (ICC, Cohen's kappa, or weighted kappa) are provided. This is a standard requirement for any study employing a semi-quantitative scoring instrument and constitutes a reproducibility gap. At a minimum, the authors should provide Cohen's weighted kappa or ICC for the total Movin score based on repeat readings by the same pathologist or assessment by two independent pathologists.

Reply: Thank you for your comment. Respectfully, two rater initially evaluated the tendon samples and decided on the modified Moving score, blinded to group allocation. If disagreed, a third rater decided on the value of each parameter and that score was used for the statistical analysis. Thus, the intra-rater reliability was not evaluated in this study. Relative comment was added in the Methods section (lines 419-421).

 

Comment: However, the device used for electron microscopy is not specified anywhere in the manuscript: brand, model, accelerating voltage, magnification calibration method, and country of manufacture are all absent. This information is mandatory for scientific reproducibility. The reviewer specifically requests that the following technical details be added to the methods section (Section 4.7, page 10, lines 335–340): the transmission electron microscope brand (e.g., JEOL, FEI/Thermo Fisher, Zeiss), model number, accelerating voltage (kV), country of manufacture, and a validation or calibration reference.

Reply: Thank you for your comment. Information about the electron microscope used were added in the Methods section (lines 411, ).

 

Comment: However, the panel-level magnification inconsistency noted in the figure legends (x6000, x10000, x12000 in Figure 2; x6000, x10000, x30000 in Figure 3) should be standardized where possible, or the rationale for selecting different magnifications per sample should be explained.

Reply: Thank you for your comment. The representative images were selected based on image quality and their ability to clearly illustrate the relevant histological features described in the text. Different magnifications were used to best visualize specific structural details.

 

Comment: First, the unequal sample sizes represent a significant analytical problem. For COL1, COL2, COL3, and MMP13, n = 7 per group was analyzed; for MMP3 and IL6, n = 6 per group; and most critically, for IL-1β, n = 6 (PRP group) and only n = 4 (control group). The reasons for sample exclusion are not provided and should be clearly stated. The loss of half the control group samples for IL-1β analysis renders any comparative conclusion for this gene biologically and statistically unreliable, and the authors should either exclude IL-1β from comparative analysis or explicitly acknowledge this as a major limitation.

Reply: Thank you for your comment. A relative comment has been added to limitations (lines 306-308)

 

 

Comment: Second, Figure 4 presents gene expression data as individual data points with only horizontal mean lines. The absence of error bars (standard deviation, standard error, or 95% confidence intervals) is a critical graphical deficiency. For a figure in a peer-reviewed molecular journal, error representation is mandatory. The authors must add error bars (at minimum, standard deviation bars) to all panels of Figure 4.

Reply: Thank you for your comment. Standard deviation bars have been added to Figure 4.

 

Comment: Third, applying parametric statistical testing (independent-sample t-test) to gene expression data with n = 4–7 per group without demonstrating normality is questionable. Given the small and unequal sample sizes, the Mann-Whitney U test would be more appropriate, or the authors must demonstrate normality using the Shapiro-Wilk test and report the test statistics and p-values.

Reply: Thank you for your comment. Details of the statistical analysis were added to Methods (lines 462-464). The Welch t-test had been already used, but not reported, thus the p-values of the comparison of gene expression has not changed.

 

Comment: Fourth, the biological significance threshold of ≥ 1 log₂ unit is a reasonable convention but is post-hoc and not pre-registered, which should be acknowledged in the limitations. While the authors justify it in the methods section, the lack of pre-registration with a recognized registry for this threshold raises concerns about potential inflation of biological significance.

Reply: Thank you for your comment. The biological significance threshold of ≥ 1 log₂ is commonly used in literature to access the effect of different treatment modalities on gene expression. A relative comment was added in limitations (lines 308-311).

 

Comment: Suggested reference for gene expression methodology context (page 6–7, lines 160–175): Saremi H, Heidari B, Sharifi A, Khanlarzadeh E. Arthroscopic Biceps Tenodesis: Midterm Clinical Results of a New Anchor Suture Technique in Patients with Single-Row Rotator Cuff Repair. Muscles Ligaments Tendons J. 2023;13(2):259–266. This original study involving rotator cuff surgery outcome assessment demonstrates the methodological framework for reporting biological outcomes in rotator cuff intervention studies, and provides relevant context for the current molecular findings.

Reply: Thank you for your comment. Respectfully, the context of this manuscript is irrelevant to biological outcomes, as only clinical results were evaluated.

 

Comment: However, the discussion of gene expression findings is speculative in several passages and would benefit from greater restraint. The assertion that "reduced MMP expression may enhance the potential for tendon repair" (page 8, lines 227–228) is plausible but is presented without acknowledging that MMPs also play essential roles in matrix remodeling during the healing cascade, and their blanket suppression may not be uniformly beneficial.

Reply: Thank you for your comment. This statement has been added in Discussion (lines 244-250)  

 

Comment: The discussion also fails to adequately address the clinical significance threshold for the modified Movin score. What constitutes a minimal clinically important difference (MCID) on this instrument? Without this anchoring, the between-group difference of 6.5 vs. 12.1 points, though statistically significant, cannot be placed in a clinical context.

Reply: Thank you for your comment. The modified Movin score is a histopathologic grading instrument and, to our knowledge, does not have a validated minimal clinically important difference (MCID).

 

Comment: Suggested reference for clinical relevance framing of tendon biology (page 7–8, lines 213–230): Kahraman Y. A Critical Review on Tendon Structure and Load Remodeling. Muscles Ligaments Tendons J. 2023;13(2):218–227. This narrative review comprehensively addresses tendon extracellular matrix dynamics, including collagen remodeling and MMP regulation, providing a critical theoretical framework for interpreting the molecular findings observed in the current study.

Reply: Thank you for your comment. Respectfully, the context of this manuscript is irrelevant to collagen and MMP regulation and changes.

 

Comment: However, the randomization method is described only as "one-by-one distribution of sixteen consecutive eligible patients into two groups" (page 8, lines 272–274), which lacks specificity. Was this simple alternation, block randomization, or concealed allocation? The method of allocation concealment is not described, raising the risk of selection bias. The CONSORT guidelines for reporting randomized trials require explicit description of the sequence generation method, allocation concealment mechanism, and implementation of randomization. This information must be added.

Reply: Thank you for your comment. Participants were allocated by alternating assignment of consecutive eligible patients (quasi-random). Allocation was not concealed. This information has been added in Methods (line 332).

 

Comment: The authors must report: the primary outcome used for the power calculation (modified Movin score or fold-change in a specific gene), the expected effect size and its source (which experimental studies?), the assumed standard deviation, the alpha level (typically 0.05), and the desired power (typically 0.80 or 0.90). Without these elements, the adequacy of the sample size cannot be evaluated.

Reply: Thank you for your comment. The subsection “Sample size” was added to include all these information (lines 448-454).

 

Comment: The PRP preparation section identifies the commercial kit used (TriCell PRP M Blood Separation Kit, REV-MED) but fails to provide the country of manufacture/sale, the centrifuge brand, model, and rotor type, the resulting platelet concentration (fold-concentration above baseline), the leukocyte count in the final preparation (critical for confirming LP-PRP classification), and the growth factor content (e.g., PDGF-AB, TGF-β1, VEGF). Without platelet count verification, the classification of the preparation as "leukocyte-poor" cannot be independently verified, and the biological agent administered is not reproducibly characterized. At minimum, mean platelet concentration and leukocyte count in the PRP preparations should be reported.

Reply: Thank you for your comment. Information about TriCell PRP kit has been added in Methods (lines 372-373) and platelet and leukocyte concentration in the final product was added in Results (lines 119-121).

 

Comment: Suggested reference for orthobiologics characterization standards (page 9, lines 299–306): Vascellari A, Demeco A, Vittadini F, et al. (already cited above). Additionally: Bergamin F, Civera M, Rodriguez Reinoso M, Burgio V, Grimaldo Ruiz O, Surace C. Worldwide Incidence and Surgical Costs of Tendon Injuries: A Systematic Review and Meta-Analysis. Muscles Ligaments Tendons J. 2023;13(1):31–45. This systematic review of tendon injuries provides epidemiological context for the clinical burden addressed by this study and supports the public health relevance of developing effective PRP protocols.

Reply: Thank you for your comment. This article has been cited to justify the socioeconomic impact of rotator cuff pathology (line 79).

 

Comment: The following devices are used in this study but lack mandatory technical specifications: Ultrasound machine, Required additions: manufacturer, model, transducer frequency (e.g., 7–15 MHz linear), country of origin.; Optical microscope: Required additions: microscope brand (e.g., Nikon, Olympus, Leica), model, digital camera model, image analysis software; Transmission electron microscope: Brand, model, accelerating voltage (kV), and country of manufacture must be specified. Centrifuge: Brand, model, rotor type, temperature during centrifugation, and country of manufacture should be added. PCR system: The qRT-PCR instrument brand, model, country of manufacture, and cycling conditions (thermal profile) are not reported. These are essential for reproducibility.

Reply: Thank you for your comment. The respective details have been added in Methods (lines 369, 378, 402, 411)

 

Comment: The authors should add a reliability sub-study or cite an existing validation study demonstrating the inter-rater reliability of the Chen-modified Movin score specifically. If this cannot be done retrospectively, the limitation must be prominently acknowledged and the potential impact on results discussed.

Reply: Thank you for your comment. In this study, two rater evaluated the tendon samples under the optical microscopy. A third rater solved any disagreement. Whole numbers of the modified Movin score were used, and not the mean score of the two rater. Thus, interobserver reliability was not investigated.

 

Comment: Furthermore, the COL1 fold change is reported as 0.51× with a Δlog₂ of −0.96, which falls just below the predefined biological significance threshold of 1 log₂ unit. This near-threshold finding should be discussed, particularly given that COL1 upregulation is a key marker of tendon healing quality.

Reply: Thank you for your comment. A relative comment has been added in the Results (lines 193-194)

 

Comment: Table 1 is clearly structured and the data are presented in an accessible format. The comparisons between groups are appropriate. The main deficiency is the presentation of age as "mean (range)" without standard deviation, as noted above. Adding SD to the age column would allow readers to assess whether the parametric t-test was appropriate.

Reply: Thank you for your comment. Standard deviation of age has been added in Table 1.

 

Comment: 4.2 Table 2 – Modified Movin Scores. This table is a particular strength of the manuscript: the presentation of individual patient scores alongside group means allows full transparency and independent verification. The column structure is logical. The only recommendation is to add a summary row with mean ± SD for each category to facilitate cross-group comparison without requiring the reader to perform arithmetic manually.

Reply: Thank you for your comment. Standard deviation of each category od modified Movin score has been added in the text in Results (lines 142-148)

 

Comment: Table 3 is informationally rich but has several deficiencies. The "NoP" column (number of patients) immediately reveals the unequal sample sizes, which is transparent. However, the p-values are provided only to three decimal places in some cases (e.g., IL-1β p = 0.605), and the standard deviations are provided in log₂ units, which may be unfamiliar to clinically oriented readers. The authors should consider adding a column for 95% confidence intervals of the fold change and explicitly flagging cells where sample size imbalance (n = 4 vs. n = 6) renders the comparison particularly uncertain.

Reply: Thank you for your comment. A column with 95% confidence intervals of the fold change has been added in Table 3.

 

Comment: The magnification labels in the figure legends are consistent with the scale bars shown. However, the legend for Figure 3 describes panel D at "x30000" magnification, while the scale bar reads 1 μm – this is consistent and appears correct, but the reviewer recommends verifying that this was not an inadvertent transcription error during manuscript preparation.

Reply: Thank you for your comment. The description is correct.

 

Comment: 4.5 Figure 4 – Gene Expression Scatter Plot. This figure presents a critical deficiency: no error bars are shown. Only horizontal lines indicating group means are plotted against individual data points. For a molecular science journal, the absence of error bars (standard deviation or standard error) violates standard graphical reporting norms. The scatter plot format is generally appropriate for small n, but the mean representation must be accompanied by a measure of dispersion. The authors must add error bars (at minimum ± SD) to all panels of Figure 4. Additionally, the x-axis labels (PRP, Ctr) are small and may benefit from larger font size, and the panels could benefit from individual p-value annotations consistent with Table 3.

Reply: Thank you for your comment. The plot has been modified to include error bars for standard deviation.

 

Comment: However, the following specific corrections are recommended:

• Page 7, line 240: "suggesting that it can may serve as a useful biological adjunct" → CORRECTION: "suggesting that it may serve as a useful biological adjunct" (double modal verb error).
• Page 1, line 33: "The preoperative LP-PRP injection improved supraspinatus tendon histological characteristics" → This is an overstatement in the abstract; consider "was associated with improved" to more accurately reflect the observational nature of the between-group comparison.
• Page 8, line 251: "the number of patients enrolled in each group was relatively small" → The qualifier "relatively" is vague; prefer "limited" or "small" without qualification, followed by the power analysis context.
• Page 9, line 301: "TriCell PRP M Blood Separation Kit (REV-MED)" → Please provide the country of the manufacturer (REV-MED) for completeness.
• Page 11, line 371: "IBMSPSS" should be "IBM SPSS" (missing space between IBM and SPSS).
• Throughout: The manuscript inconsistently uses "Rotator Cuff" with capitalization mid-sentence (page 1 line 38, etc.). Maintain consistent capitalization: "rotator cuff" in running text, reserved for proper noun usage at sentence beginnings only.

 Reply: Thank you for your comment. Changes have been applied according to your suggestions. Respectfully, a few of them could not been identified in the text.

Comment: As a randomized controlled trial, this manuscript should include or reference a CONSORT flow diagram showing the number of patients screened, eligible, randomized, allocated, receiving intervention, followed up, analyzed, and excluded at each stage with reasons. No CONSORT flow diagram is present in this manuscript.

Reply: Thank you for your comment. The CONSORT diagram has been added (Figure 5) (lines 341-347)

 

Comment: The manuscript should identify whether this randomized controlled trial was prospectively registered with a clinical trials registry (e.g., ClinicalTrials.gov, WHO ICTRP, or national equivalents). If not registered, the authors must acknowledge this as a limitation and explain why registration was not performed.

Reply: Thank you for your comment. The ClinicalTrials.gov ID number has been added in Methods (line 324).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Most of revisions have been fulfilled.

Author Response

Comment: Most of revisions have been fulfilled.

 

Reply: Thank you for your statement.