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  • Kornelia Kuźnik-Trocha 1,*,
  • Katarzyna Winsz-Szczotka 2 and
  • Katarzyna Komosińska-Vassev 1
  • et al.

Reviewer 1: Anonymous Reviewer 2: Anonymous Reviewer 3: Anonymous

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Circulating osteoarticular biomarkers play a crucial role in juvenile idiopathic arthritis (JIA) by providing valuable insights into the disease's activity, progression, and response to therapy.  Biomarkers can help identify subclinical joint inflammation before structural damage occurs, enabling earlier diagnosis and intervention. Levels of specific biomarkers reflect ongoing inflammation and joint involvement, helping clinicians assess disease activity in real-time.

Certain biomarkers are associated with disease prognosis, aiding in predicting which patients may experience more aggressive disease or remission. Changes in circulating biomarkers can indicate how well a patient is responding to treatment, allowing for personalized adjustments. Biomarkers shed light on the underlying mechanisms of joint inflammation and destruction, guiding the development of targeted therapies. Reliable biomarkers can supplement or replace invasive assessments like joint biopsies or frequent imaging, reducing patient discomfort.

Therefore, the study of Dr. Kuznik-Trocha at al. is very important.

Comments

  1. Title should be rephrased: Circulating aggrecan, biglycan, and decorin as biomarkers of osteoarticular alterations in JIA.
  2. Line 47 vs line 52: All the repeats should be corrected throughout the text.
  3. Figures 1-3 should be modified: Fig 1 should contain fragments from Fig1a, fig 1c and Fig2.
  4. Fig 2 should contain fragments from Fig1b and Fig3.
  5. It is not clear why ROC curve for decorin is different on Fig2 and Fig 3? This should be corrected.
  6. All the Figures and Tables should be placed next to the text which describes them. For example, the reference on Table 4 (line 152) and Table 4 (line 231).
  7. Lines 226-230 and Table 4: The authors should not repeat the data from the Table in the text. The data should be referred exactly; there should not be any discrepances. Non-statistically significant data should be indicated as n.s. All the typos should be corrected.
  8. Tables 4 and 5 should be entitled as “Spearman rank correlations between the concentrations…”
  9. Discussion should be shortened. All the descriptions of Aggrecan, Biglycan, and Decorin structures should be moved to the Introduction section. All the repeats should be corrected.
  10. Line 351: the reference is required at the end of this sentence.
  11. Lines 362-367: The rationale for this hypothesis should be presented.
  12. Line 390: How the authors can explain the coexistence of compensatory anabolic response (line 364) with elevated ROS activity (line 390)?
  13. Lines 422-451: There should be presented a clear scheme how the patients were treated.
  14. Lines 440-442: this sentence is not clear. It should be clarified.

  

Comments on the Quality of English Language
  1. Lines 440-442: this sentence is not clear. It should be clarified.

Author Response

Answer to the Reviewer's comment

We would like to thank the Reviewer for the evaluation of our article. We are grateful for the important comments, which we addressed in the revised manuscript. The text of our manuscript (in each individual part, changes made in red) has been modified, so as to facilitate its understanding and make it acceptable for publication.

Comments

  1. Title should be rephrased: Circulating aggrecan, biglycan, and decorin as biomarkers of osteoarticular alterations in JIA.

ANSWER:

Thank you for your valuable suggestion. The title has been revised according to the recommendation and now reads: “Circulating Aggrecan, Biglycan, And Decorin As Biomarkers Of Osteoarticular Alterations In Juvenile Idiopathic Arthritis (Preliminary Study).”

Additionally, as recommended by the second reviewer, a description of the preliminary study has been added to the manuscript.

 

  1. Line 47 vs line 52: All the repeats should be corrected throughout the text

ANSWER:

Thank you for noting this issue. The repeated expressions identified in these lines have been corrected, and the manuscript has been carefully reviewed to remove other redundant or repetitive statements.

 

  1. Figures 1-3 should be modified: Fig 1 should contain fragments from Fig1a, fig 1c and Fig2.

ANSWER:

Thank you for this helpful suggestion regarding Figures 1–3. We have revised the figures to improve their clarity and readability. However, due to technical limitations, we were unable to combine the elements from Figures 1a, 1c, and 2 into a single composite figure as proposed. Instead, the figures have been reorganized and reformatted in a clearer and more coherent manner. Additionally, each figure now appears in the section where the corresponding proteoglycan is discussed, which further enhances readability and helps guide the reader through the results.  All revised figures are clearly marked in the manuscript. All modified figures have been marked in red to clearly indicate the changes introduced.

 

  1. Fig 2 should contain fragments from Fig1b and Fig3.

ANSWER:

Thank you for this recommendation. Although merging the elements into one composite figure was not technically feasible, the figures have been reorganized and visually improved. Each figure now appears together with the proteoglycan it refers to, ensuring improved readability and structural consistency. All modified figures have been marked in red to clearly indicate the changes introduced.

 

  1. It is not clear why ROC curve for decorin is different on Fig2 and Fig 3? This should be corrected.

ANSWER:

Thank you for pointing out the inconsistency regarding the ROC curves for decorin presented in Figures 2 and 3. The two ROC curves originally differed because they assessed two distinct diagnostic contexts:

- Figure 2 assessed the ability of decorin to discriminate between patients with JIA (TB) and healthy controls (HC), whereas

- Figure 3 evaluated the usefulness of decorin in monitoring treatment response, comparing patients before therapy (T0) and after biological treatment (T24).

Thus, the ROC curves were based on different study groups.

To ensure clarity, we consolidated both decorin-related ROC analyses into one unified figure (new Figure 6), placed within the decorin Results subsection. The tables and figure order were reorganized accordingly.

 

  1. All the Figures and Tables should be placed next to the text which describes them. For example, the reference on Table 4 (line 152) and Table 4 (line 231).

ANSWER:

Thank you for highlighting this. The entire manuscript was reviewed to ensure that every figure and table is located directly adjacent to its first mention in the text. Additionally, a previously incorrect table reference in line 152 (now line 138) has been corrected from “Table 4” to “Table 1.”

 

  1. Lines 226-230 and Table 4: The authors should not repeat the data from the Table in the text. The data should be referred exactly; there should not be any discrepances. Non-statistically significant data should be indicated as n.s. All the typos should be corrected.

ANSWER:

Thank you for this important comment. We understand that numerical results should generally not be repeated in the main text. Only statistically significant correlation coefficients (R values) are mentioned descriptively to aid interpretation. Furthermore, to avoid repeating the data in both the text and the table and to ensure full consistency of the presented results, we have removed Tables 2 and 3, and their results are now described only in the text.

All non-significant values have been marked as NS, and the entire section was carefully reviewed to remove typographical errors.

 

  1. Tables 4 and 5 should be entitled as “Spearman rank correlations between the concentrations…”.

ANSWER:

We thank the Reviewer for this suggestion. The titles of the tables presenting correlation analyses have been revised accordingly. In the final version they appear as Table 2 and Table 3, following updated numbering.

  1. Discussion should be shortened. All the descriptions of Aggrecan, Biglycan, and Decorin structures should be moved to the Introduction section. All the repeats should be corrected.

ANSWER:

Thank you for this valuable comment. The Discussion section has been substantially shortened and streamlined. Descriptions of proteoglycan structure and function were condensed and integrated into a concise background paragraph, avoiding redundancy while maintaining conceptual clarity. The Introduction was not expanded, as another Reviewer requested that this section remain brief.

Additionally, all repeated statements were removed. All revisions made in the Discussion section have been marked in red.

  1. Line 351: the reference is required at the end of this sentence.

ANSWER:

We thank the Reviewer for this remark. An appropriate reference has now been added at the end of the corresponding sentence (line 348 in the revised manuscript).

 

  1. Lines 362-367: The rationale for this hypothesis should be presented.

ANSWER:

We appreciate the Reviewer’s insightful comment. In the revised manuscript, we have expanded the explanation supporting this hypothesis (lines 359-377). The rationale now includes:

„ Although changes in plasma aggrecan concentrations also occur in children with JIA receiving biological therapy, these changes were observed only after 2 years of treatment. The pattern observed in our cohort — elevated aggrecan levels in untreated JIA, stability during the first 18 months of etanercept treatment, followed by a sharp and significant increase after 24 months — is consistent with a two-phase model of cartilage response. During the first phase (0–18 months), biological therapy suppresses inflammation and halts ECM degradation but does not yet activate anabolic pathways. Only after prolonged clinical remission under TNF blockade — reflected by a marked improvement in JADAS-27 scores — do aggrecan concentrations increase sharply, exceeding levels observed in healthy children. This delayed overshoot is biologically plausible: once the inflammatory environment is durably stabilized, chondrocytes can shift from a catabolic or survival mode to a reparative phenotype, initiating anabolic programs and producing proteoglycans at rates higher than those required for physiological homeostasis [Liu, S.; Deng, Z.; Chen, K.; Jian, S.; Zhou, F.; Yang, Y.; Fu, Z.; Xie, H.; Xiong, J.; Zhu, W. Cartilage tissue engineering: From proinflammatory and anti‑inflammatory cytokines to osteoarthritis treatments (Review). Mol Med Rep. 2022, 25(3), 99. https://doi:10.3892/mmr.2022.12615]. Such enhanced synthesis likely reflects ECM remodeling during the restoration of cartilage integrity. This interpretation is consistent with recent evidence showing that chondrocytes retain the capacity to re-enter regenerative, anabolic states under sustained anti-inflammatory conditions [Varela-Eirin, M.; Díaz-Flores, L.; González-García, C.; Fonseca, E.  Chondrocytes revisited: Functional heterogeneity, plasticity, and their role in cartilage repair. Biomedicines 2024, 12(6), 1367. https://doi.org/10.3390/biomedicines12061367]. Thus, the marked late increase in plasma aggrecan is best interpreted not as a pathological change but as evidence of a delayed, compensatory anabolic response during cartilage repair. Further studies are needed to validate this hypothesis.”

  1. Line 390: How the authors can explain the coexistence of compensatory anabolic response (line 364) with elevated ROS activity (line 390)?

ANSWER:

We thank the Reviewer for this important comment. In our study, the observed increase in plasma aggrecan concentration after 24 months of etanercept therapy likely reflects the activation of cartilage repair and ECM-remodeling processes following long-term suppression of inflammation. In contrast, AOPP levels—although slightly higher than baseline—did not significantly differ from those of healthy controls and did not increase during treatment. Therefore, the data do not indicate markedly elevated oxidative stress but rather the persistence of a mild, subclinical level of oxidative activity, which is commonly described in JIA due to chronic immune dysregulation.

 

Importantly, moderate or low-grade oxidative activity does not preclude the initiation of tissue repair. Experimental and clinical studies show that regenerative processes may begin even in the presence of residual oxidative stress, provided that it remains within a non-damaging range. Thus, the coexistence of an anabolic response with mildly increased AOPP levels is biologically plausible. We clarified this interpretation in the revised manuscript.

 

“In contrast, plasma AOPP concentrations did not change after 24 months of etanercept therapy and remained only slightly higher than baseline values, with no significant differences relative to healthy children. Because AOPP reflects the severity of oxidative stress [35], these findings suggest that a mild residual level of oxidative activity may persist in patients with JIA despite clinical improvement and inhibition of excessive cartilage degradation during treatment.” Lines 395-400.

 

  1. Lines 422-451: There should be presented a clear scheme how the patients were treated.

ANSWER:

Thank you for this valuable comment. In response, we have added a clear and comprehensive graphical treatment scheme for the patients, which is now presented in the revised manuscript as Figure 7 (line 436). The flowchart summarizes all treatment steps and therapeutic decisions applied in the study cohort. We believe that this addition improves the clarity and transparency of the methodology.

 

  1. Lines 440-442: this sentence is not clear. It should be clarified.

ANSWER:

We thank the Reviewer for pointing this out. The sentence has been rewritten for full clarity. The updated version now clearly states that etanercept was initiated together with methotrexate, sulfasalazine, and prednisone, and that sulfasalazine and prednisone were discontinued after 3 months of effective therapy, while etanercept and methotrexate were continued.

„In all patients, etanercept was administered together with methotrexate, sulfasalazine, and prednisone at treatment initiation. After 3 months of effective therapy, sulfasalazine and prednisone were discontinued, while etanercept and methotrexate were continued for 24 months.” Lines 457-460.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

It was a pleasure reviewing your valuable manuscript. The work offers a novel approach by comparing three key proteoglycans and their longitudinal changes during different therapeutic phases. However, I would like to offer some suggestions for improvement:

Although the introduction is well-founded, I would suggest restructuring it beyond a single long paragraph. Furthermore, the specific hypothesis and/or objectives are not clearly defined.

The findings regarding decorin and biglycan as biomarkers are promising, but the evidence is preliminary. Therefore, the study title should state that it is a preliminary study.

The figures need higher resolution and size to facilitate their understanding and reading.

 

Author Response

Answer to the Reviewer's comment:

Thank you very much for your positive assessment of our manuscript and for the constructive suggestions that helped us improve the clarity and quality of the work.

Comments

  1. Although the introduction is well-founded, I would suggest restructuring it beyond a single long paragraph. Furthermore, the specific hypothesis and/or objectives are not clearly defined.

ANSWER:

We appreciate the Reviewer’s recommendation to restructure the Introduction. In the revised manuscript, the Introduction has been divided into shorter, thematically focused paragraphs to improve both readability and logical flow. Additionally, we have explicitly stated the study objectives and the underlying hypothesis at the end of the Introduction section.

„Given the central role of PGs in ECM integrity, their degradation and subsequent release into the bloodstream may provide insight into ongoing osteoarticular damage. Therefore, the aim of this study was to evaluate the diagnostic utility of circulating levels of aggrecan, decorin, and biglycan as potential biomarkers of tissue destruction and/or indicators of therapeutic response in children with JIA treated with the biologic drug etanercept (ETA). The diagnostic and monitoring value of these proteoglycans was assessed using ROC curve analyses. In addition, we measured levels of matrix metalloproteinase-12 (MMP-12) and advanced oxidation protein products (AOPP) as biochemical markers associated with PG degradation. Finally, we investigated the relationships between AGC, DCN, and BGN levels and the concentrations of MMP-12 and AOPP in the blood of children with JIA. This allowed us to evaluate the associations between PG levels and MMP-12/AOPP concentrations and to better understand the mechanisms underlying tissue damage and oxidative stress in JIA” (lines 69-81).

 

  1. The findings regarding decorin and biglycan as biomarkers are promising, but the evidence is preliminary. Therefore, the study title should state that it is a preliminary study.

ANSWER:

Thank you for highlighting that the biomarker findings—particularly those concerning decorin and biglycan— should be interpreted as preliminary. In response, we have revised the manuscript title to reflect this:

“Circulating Aggrecan, Biglycan, And Decorin As Biomarkers Of Osteoarticular Alterations In Juvenile Idiopathic Arthritis (Preliminary Study)”

This change ensures that the exploratory nature of the work is clearly communicated.

 

  1. The figures need higher resolution and size to facilitate their understanding and reading.

ANSWER:

We agree that improvements to figure quality enhance the clarity and accessibility of the results. Accordingly, all figures have been updated and adjusted in size to enhance readability and comprehension. Furthermore, the figures were reorganized and redesigned in alignment with the suggestions provided by the other Reviewer, ensuring a more coherent presentation.

We appreciate the Reviewer’s thoughtful comments and believe that the implemented revisions have substantially strengthened the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

The paper evaluates the potential role of aggrecan, biglycan, and decorin as biomarkers of diagnosis and disease activity in juvenile idiopathic arthritis.

Lines 29–32. This sentence seems too complicated. Would it be possible to simplify it?

Lines 32–35. It is difficult to draw such conclusions based on the results you present. Could you explain and elaborate on this issue?

The introduction could benefit from being split into smaller paragraphs. Moreover, it is not clear to me why such an extensive description is needed in the context of the clinical relevance of the study. Please explain.

Please be more specific regarding the aim of the study; it would be helpful if you could explain your reasoning step by step.

How do you explain the increase in AGC levels over time?

Please change “gender” to “sex.”

The relatively high prevalence of ANA in the study cohort is somewhat interesting.

What about the treatment pattern? Please provide the specific number of patients.

Why were other key JIA-related cytokines not assessed in this study? Potential biomarkers could correlate with known biomarkers, which represents another limitation of the study.

Please explain how the conclusions are supported by the study results.

Methods are well described including statistics.

Author Response

Answer to the Reviewer's comment:

 

Comments

  1. Lines 29–32. This sentence seems too complicated. Would it be possible to simplify it?

ANSWER:

Thank you for pointing out that the sentence in lines 29–32 was overly complex. We have simplified the wording to improve clarity. The revised text now reads as follows:

“In contrast, biglycan levels were similar to those in healthy controls but dropped during etanercept therapy. Decorin levels were initially high in untreated and DMARD-treated patients and returned to normal after 24 months of biologic treatment.” Lines 30-33.

We hope this modification meets the Reviewer’s expectations.

 

  1. Lines 32–35. It is difficult to draw such conclusions based on the results you present. Could you explain and elaborate on this issue?.

ANSWER:

Thank you for your valuable remark. We agree that the conclusions in lines 32–35 required clearer justification Although the original abstract summarised the final interpretation, we have now expanded and clarified the reasoning in the revised manuscript. We incorporated the ROC curve analysis to strengthen the evidence supporting the diagnostic and monitoring potential of decorin and biglycan. The revised sentence now reads:

“After considering both these findings and the ROC analysis, it can be concluded that decorin appears to be a promising biomarker for the diagnosis and monitoring of etanercept therapy in JIA, whereas biglycan seems to be a useful biochemical marker for assessing the effectiveness of ETA treatment.” Lines 33-37.

We trust that this expanded explanation adequately addresses the Reviewer’s concern.

 

  1. The introduction could benefit from being split into smaller paragraphs. Moreover, it is not clear to me why such an extensive description is needed in the context of the clinical relevance of the study. Please explain.

ANSWER:

Thank you very much for this insightful comment. We agree that the original Introduction was overly long and dense. In the revised manuscript, we have divided it into shorter, thematically focused paragraphs to improve readability and logical flow. The detailed description of proteoglycans (aggrecan, decorin, and biglycan) has been retained because it provides essential background for understanding their biological roles in the extracellular matrix, their involvement in tissue integrity, and the mechanisms leading to their release into the bloodstream. This context is directly relevant to the clinical rationale of our study, as it supports the investigation of these molecules as potential biomarkers for disease activity and therapeutic response in JIA.

 

  1. Please be more specific regarding the aim of the study; it would be helpful if you could explain your reasoning step by step.

ANSWER:

Thank you for this important suggestion. We agree that the study aim benefits from a clearer, stepwise rationale. We have revised the manuscript to make the logic explicit. We have now clearly stated the study aims in a dedicated paragraph at the end of the Introduction:

„Given the central role of PGs in ECM integrity, their degradation and subsequent release into the bloodstream may provide insight into ongoing osteoarticular damage. Therefore, the aim of this study was to evaluate the diagnostic utility of circulating levels of aggrecan, decorin, and biglycan as potential biomarkers of tissue destruction and/or indicators of therapeutic response in children with JIA treated with the biologic drug etanercept (ETA). The diagnostic and monitoring value of these proteoglycans was assessed using ROC curve analyses. In addition, we measured levels of matrix metalloproteinase-12 (MMP-12) and advanced oxidation protein products (AOPP) as biochemical markers associated with PG degradation. Finally, we investigated the relationships between AGC, DCN, and BGN levels and the concentrations of MMP-12 and AOPP in the blood of children with JIA. This allowed us to evaluate the associations between PG levels and MMP-12/AOPP concentrations and to better understand the mechanisms underlying tissue damage and oxidative stress in JIA” (lines 69-81).

We trust that this explanation clarifies the study aim and the rationale connecting our hypotheses and methodological approach.

 

  1. How do you explain the increase in AGC levels over time?

ANSWER:

Thank you for this insightful question. We have expanded the manuscript to provide a detailed explanation of the observed increase in aggrecan (AGC) levels, which now appears in lines 359–377 of the revised version. In this section, we describe a two-phase model of cartilage response.

 

Specifically, we explain that AGC levels remain stable during the first 18 months of etanercept therapy, a period during which inflammation is suppressed and ECM degradation is halted, but anabolic pathways in chondrocytes are not yet fully activated. After 24 months of sustained clinical remission, AGC levels rise sharply and exceed those found in healthy controls. This delayed increase is biologically plausible and reflects a shift from a catabolic/survival phenotype toward an anabolic, reparative chondrocyte phenotype once the inflammatory environment has been durably stabilized. We support this interpretation with recent literature indicating that chondrocytes can regain anabolic and regenerative activity under prolonged anti-inflammatory conditions.

Thus, we interpret the late rise in AGC not as a pathological signal but as evidence of compensatory ECM remodeling during cartilage repair. We note in the revised text that further studies are required to confirm this hypothesis.

 

  1. Please change “gender” to “sex.”.

ANSWER:

Thank you for this comment. We have corrected the terminology accordingly and replaced all instances of “gender” with “sex” throughout the manuscript. All modifications have been marked in red in the revised version.

 

  1. The relatively high prevalence of ANA in the study cohort is somewhat interesting.

ANSWER:

Thank you for this observation. We agree that the relatively high prevalence of ANA positivity in our cohort is noteworthy. ANA positivity is commonly observed in children with oligoarticular and polyarticular forms of JIA, which are well represented in our study group. This distribution of JIA subtypes likely contributes to the higher ANA frequency. Moreover, our cohort reflects the typical clinical population treated in our center, where ANA-positive patients are diagnosed more frequently.

 

  1. What about the treatment pattern? Please provide the specific number of patients.

ANSWER:

Thank you for this comment. In the revised manuscript, we have added a detailed treatment scheme, which is now presented in Figure 7 (line 436). We also clarified in the text that this treatment pattern refers to all 34 patients included in the study. All additions have been marked in red in the revised version (lines 434-435).

 

  1. Why were other key JIA-related cytokines not assessed in this study? Potential biomarkers could correlate with known biomarkers, which represents another limitation of the study.

ANSWER:

Thank you for this important comment. We agree that including additional cytokines would have strengthened the study. However, cytokine profiling was not part of the original project design and was limited by financial constraints. The study focused primarily on ECM-related markers (PGs, MMP-12, AOPP). We fully acknowledge that the lack of cytokine data restricts our ability to assess relationships between PGs and established immuno-inflammatory pathways in JIA. This limitation has now been added to the Limitations section, with the new text marked in red.

"In addition, cytokine profiling was not performed, which prevented the assessment of correlations between proteoglycans and established inflammatory biomarkers." (lines 409-411).

 

  1. Please explain how the conclusions are supported by the study results.

ANSWER:

Thank you for this important comment. We have clarified in the revised manuscript how the study conclusions are directly supported by the presented results. Specifically, the designation of decorin as the most promising biomarker for both diagnosis and monitoring is based on its significantly elevated baseline levels, their normalization during etanercept therapy, and the strong diagnostic and monitoring performance demonstrated by ROC analyses (high AUC values, good sensitivity and specificity). Similarly, the conclusion regarding biglycan is supported by its stable baseline values, followed by a clear decline during treatment, together with ROC results indicating its usefulness as a marker of treatment effectiveness. We have revised the concluding section to more explicitly link these findings with the final interpretation.

„ Of the proteoglycans analyzed, decorin showed the strongest combined diagnostic and monitoring potential, as evidenced by its markedly elevated baseline levels, their normalization during etanercept therapy, and the robust diagnostic performance demonstrated by ROC analysis. Biglycan, characterized by a systematic decrease in concentration during treatment and a clear ability to distinguish clinical improvement in ROC analysis, appears to be the most reliable indicator of therapeutic response. These results highlight decorin and biglycan as promising biomarkers in JIA patients treated with etanercept, although confirmation in larger cohorts is necessary.” (lines 536-543).

 

  1. Methods are well described including statistics.

ANSWER:

We thank the Reviewer for this positive assessment and we are pleased that the methodology and statistical analyses were found to be clearly described.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Comments

  1. Tables 2,3: For non-significant correlations with P>0.05 the authors should no show R-values. They should be removed.
  2. Lines 338, 339, 341: References are required at the end of these sentences.

Author Response

Dear Reviewer,

Thank you for your valuable comments.

Regarding Tables 2 and 3, we agree that R-values should not be presented for non-significant correlations (P > 0.05). We have removed these R-values as recommended.

For lines 338, 339, and 341, we have added the appropriate references at the end of each sentence to ensure proper citation and clarity.

We appreciate your careful review and helpful suggestions.

Best regards,

Kornelia Kuźnik-Trocha

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you very much for following the suggestions. I believe the manuscript has improved considerably.

Only one minor suggestion. Regarding the title, instead of "...(Preliminary Study)" I would suggest replacing it with "... - A Preliminary Study."

Best regards.

Author Response

Dear Reviewer,

Thank you very much for your positive assessment and for noting the improvements in our revised manuscript. We appreciate your additional suggestion regarding the title. We agree with your recommendation to replace “(Preliminary Study)” with “– A Preliminary Study” and have updated the title accordingly.

Thank you once again for your constructive feedback and valuable guidance.

Best regards,

Kornelia Kuźnik-Trocha

Reviewer 3 Report

Comments and Suggestions for Authors

The paper has been much improved. The authors have addressed all comments, and the treatment scheme is valuable.

Author Response

Dear Reviewer,

We sincerely thank the Reviewer for the positive evaluation of our revised manuscript. We are pleased to hear that the paper has been significantly improved and that all comments have been satisfactorily addressed. We also appreciate the acknowledgment that the proposed treatment scheme is valuable. Your constructive feedback has greatly contributed to strengthening the quality and clarity of our work.

Best regards,

Kornelia Kuźnik-Trocha