Novel Combination of Icariin and Bone Xenograft Promotes the Expression of Collagen Fibers, BMP-2, and HIF-1α During Wound Healing in Wistar Rats

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

Thank you for submitting your manuscript to the prestigious Journal of Molecular Pathology. The paper is overall interesting and fits the scope of the journal. I hope that my remarks and suggestions will be useful in order to increase the quality of the manuscript.

General Aspects and Considerations

This manuscript explores the biological effects of combining icariin with a bovine xenograft on early extraction socket healing in a Wistar rat model. By focusing on collagen fiber maturation and the immunoexpression of BMP-2 and HIF-1α at early healing time points (Days 7 and 14), the study addresses a topic of clear relevance to molecular pathology, bone biology, and regenerative dentistry.

The experimental rationale is well supported by current literature, and the results consistently indicate that the icariin-enhanced xenograft group demonstrates improved early healing responses compared to xenograft alone and untreated controls. The manuscript is generally well structured, the findings are biologically plausible, and the discussion integrates the results with known osteogenic and hypoxia-related pathways.

Major Comments

1. The manuscript reports quantitative values for collagen fibers, BMP-2, and HIF-1α expression; however, additional methodological detail would enhance transparency and reproducibility, which is essential in this type of studies.
2. The use of one-way ANOVA is acceptable, but, I consider that the manuscript would benefit from a brief justification of this approach because of the two time points. Clarifying that day 7 and day 14 represent independent cohorts would help readers interpret the data structure more clearly.
3. It would be helpful to elaborate the explination regarding that HIF-1α serves as an indirect marker of angiogenic signaling rather than a direct measure of angiogenesis, which would slightly temper the interpretation without diminishing the significance of the findings.
4. Please rephrase the conclusions section in such a way that will emphasise the practical/clinical impact of your results and what benefit will provide in optimizing current protocols.

Minor Issues

1. The manuscript requires technical editing. E.g. The title should respect the font and the style of MDPI publishing. Capital letters are not required.
2. Authorship: academic titles should be removed.
3. Professional e-mail is recommended for the corresponding author.
4. Figure 1 should be re-organized in such a way that it would be readable. The details incorporated are not clearly visible.
5. The number and date of the ethical approval is missing. One copy should be e-mailed to the assistant editor as well.
6. Authors contribution should be corrected according to the MDPI style and international authorship criteria.ț
7. References are not properly cited in the text. The references should be included in [ ] and inserted before “.”

From my point of view, the paper could be considered for publishing after revising the above-mentioned aspects.

Author Response

Response to Reviewer's comments:  

 

Reviewer 1 (Major comment)

 

1. Reviewer Comment 1: The manuscript reports quantitative values for collagen fibers, BMP-2, and HIF-1α expression; however, additional methodological detail would enhance transparency and reproducibility, which is essential in this type of studies.

 

Response:

We thank the reviewer for this important comment. The Methods section has been revised to include additional methodological details for the quantitative assessment of collagen fibers, BMP-2, and HIF-1α expression. We have clarified image acquisition parameters, analysis procedures, and data quantification methods to enhance transparency and reproducibility. These revisions have been incorporated into the revised manuscript (L123-128).

 

Digital images were captured at ×400 magnification from standardized 3 regions of interest within the extraction socket. For collagen fiber assessment, color thresholding was applied to isolate positively stained areas scoring, which were expressed as a percentage of the total area. BMP-2 and HIF-1α immunoreactivity were quantified as the positively stained area relative to the total field. All measurements were performed twice by two histologist observer.

 

2. Reviewer Comment 2: The use of one-way ANOVA is acceptable, but I consider that the manuscript would benefit from a brief justification of this approach because of the two time points. Clarifying that day 7 and day 14 represent independent cohorts would help readers interpret the data structure more clearly.

Response:

We thank the reviewer for this valuable suggestion. We have now clarified the rationale for using one-way ANOVA in the Methods section. Specifically, we state that the day 7 and day 14 observations were obtained from independent cohorts of animals, rather than repeated measurements from the same subjects. Therefore, comparisons were performed within each time point using one-way ANOVA. Because histological evaluation required animal sacrifice, different animals were used at day 7 and day 14. A post-test only control group design was used in this study. This clarification has been added to the revised manuscript (L86-88).

 

3. Reviewer Comment 3: It would be helpful to elaborate the explanation regarding that HIF-1α serves as an indirect marker of angiogenic signaling rather than a direct measure of angiogenesis, which would slightly temper the interpretation without diminishing the significance of the findings.

Response:

We thank the reviewer for this insightful comment. We have revised the manuscript to clarify that HIF-1α functions as an upstream regulator and indirect marker of angiogenic signaling rather than a direct measure of angiogenesis. We now explicitly state that increased HIF-1α expression reflects hypoxia-induced activation of pro-angiogenic pathways, such as VEGF signaling, rather than the formation of new blood vessels. This clarification has been incorporated into the Discussion section to provide a more balanced interpretation of the findings without altering their biological significance (L237-240).

 

4. Reviewer Comment 4: Please rephrase the conclusions section in such a way that will emphasise the practical/clinical impact of your results and what benefit will provide in optimizing current protocols.

Response:

We thank the reviewer for this constructive suggestion. The Conclusions section has been revised to better emphasize the practical and potential clinical implications of our findings. Specifically, we now highlight how the observed effects may contribute to optimizing post-extraction socket management strategies and inform the refinement of current regenerative protocols. This revision has been incorporated into the Conclusions section of the revised manuscript (L332-334).

 

 

Reviewer 1 (Minor Issues)

 

5. Reviewer comment 1: The manuscript requires technical editing. E.g. The title should respect the font and the style of MDPI publishing. Capital letters are not required.

Response:

We thank the reviewer for this comment. The manuscript has undergone technical editing to ensure compliance with MDPI formatting and style guidelines. Specifically, the title has been revised to follow MDPI style, and unnecessary capitalization has been removed. These changes have been implemented throughout the manuscript. (L1-2)

 

6. Reviewer comment 2: Authorship: academic titles should be removed.

Response:

We thank the reviewer for this comment. Academic titles have been removed from the authorship information to comply with the journal’s authorship guidelines.

 

7. Reviewer comment 3: Professional e-mail is recommended for the corresponding author.

Response:

We thank the reviewer for this comment. Professional email already exists in the authorship information.

 

8. Reviewer comment 4: Figure 1 should be reorganized in such a way that it would be readable. The details incorporated are not clearly visible.

Response:

We thank the reviewer for this helpful comment. Figure 1 has been reorganized to improve readability, with adjustments to clarify its explanation. (L178-179)

 

9. Reviewer comment 5: The number and date of the ethical approval is missing. One copy should be e-mailed to the assistant editor as well.

Response:

We thank the reviewer for this comment. The ethical approval number and date have now been added to the manuscript in the Ethics Statement section. Ethic  No: 1132/HRECC.FODM/XI/2025 (L337)

 

10. Reviewer comment 6: Authors contribution should be corrected according to the MDPI style and international authorship criteria.

Response:

We thank the reviewer for this important comment. The Author Contributions section has been revised to comply with MDPI formatting requirements and international authorship criteria. Each author’s specific contribution has now been clearly defined in accordance with these guidelines. (L340-342)

 

11. Reviewer comment 7: References are not properly cited in the text. The references should be included in [ ] and inserted before punctuation.

Response:

We thank the reviewer for this comment. All in-text citations have been corrected to comply with MDPI referencing style, with references now presented in square brackets [ ] and placed before punctuation throughout the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Introduction
1.The Introduction would benefit from a clearer articulation of the clinical relevance of early socket healing in implant dentistry, particularly linking early molecular events (collagen maturation, BMP-2, HIF-1α) to long-term ridge preservation and implant stability.

2.The novelty of combining icariin with a bovine xenograft should be more explicitly highlighted by contrasting this approach with existing strategies such as rhBMP-2–loaded grafts, emphasizing differences in safety, cost, and biological modulation.

Materials and Methods
3.More detailed justification for the selected icariin concentration (10 µM) is recommended, including whether dose–response data or pilot experiments were considered to optimize biological efficacy without cytotoxicity.

4.Quantitative methods for histomorphometric assessment of collagen fibers, BMP-2, and HIF-1α should be described in greater detail, including image analysis software, thresholding criteria, and intra-observer reliability.

Results

5.The Results section would benefit from clearer separation between descriptive histological observations and statistically analyzed quantitative outcomes to improve readability and scientific rigor.

6. The temporal changes in HIF-1α expression between Day 7 and Day 14 warrant a more explicit description in the Results section, as this dynamic trend is central to the study’s hypothesis.

Discussion

7.The Discussion would be strengthened by proposing specific future studies, such as long-term micro-CT analysis or biomechanical testing, to validate the functional relevance of the observed early healing enhancement.

Comments on the Quality of English Language

The English language throughout the manuscript needs improvement for clarity and readability.

Author Response

Response to Reviewer's comments:  

 

Reviewer 2

 

 

1. Reviewer comment 1: The Introduction would benefit from a clearer articulation of the clinical relevance of early socket healing in implant dentistry, particularly linking early molecular events (collagen maturation, BMP-2, HIF-1α) to long-term ridge preservation and implant stability.

Response:

We thank the reviewer for this insightful comment. The Introduction has been revised to more clearly emphasize the clinical relevance of early socket healing in implant dentistry. Specifically, we have strengthened the link between early molecular events, including collagen maturation, BMP-2 expression, and HIF-1α–mediated angiogenic signaling, and their potential roles in long-term alveolar ridge preservation and implant stability. These clarifications have been incorporated into the revised Introduction section (L40-46).

 

 

2. Reviewer comment 2: The novelty of combining icariin with a bovine xenograft should be more explicitly highlighted by contrasting this approach with existing strategies such as rhBMP-2–loaded grafts, emphasizing differences in safety, cost, and biological modulation.

 

Response:

We thank the reviewer for this insightful comment. The manuscript has been revised to more explicitly highlight the novelty of combining icariin with a bovine xenograft. In the Introduction sections, we now contrast this approach with established strategies such as rhBMP-2–loaded grafts, emphasizing differences in safety profiles, cost considerations, and mechanisms of biological modulation. These revisions clarify the unique contribution of the present study while maintaining a balanced interpretation of the findings (L68-72).

 

 

3. Reviewer comment 3: A more detailed justification for the selected icariin concentration (10 µM) is recommended, including whether dose–response data or pilot experiments were used to optimize biological efficacy without cytotoxicity.

Response:

Thank you for this insightful suggestion. The icariin concentration of 10 µM was selected based on preliminary pilot experiments and previous reports demonstrating favorable osteogenic and angiogenic effects without cytotoxicity. Higher concentrations did not provide additional biological benefit, whereas lower concentrations showed reduced efficacy. (L108-111)

4. Reviewer comment 4: Quantitative methods for histomorphometric assessment of collagen fibers, BMP-2, and HIF-1α should be described in greater detail, including image analysis software, thresholding criteria, and intra-observer reliability.

Response:

We thank the reviewer for this important comment. The Methods section has been revised to provide a more detailed description of the quantitative histomorphometric analysis. Specifically, we have clarified the thresholding criteria applied for quantification, and the procedures employed to ensure intra-observer reliability. These additions improve the transparency and reproducibility of the quantitative assessments (L121-126).

 

5. Reviewer comment 5: The Results section would benefit from clearer separation between descriptive histological observations and statistically analyzed quantitative outcomes to improve readability and scientific rigor.

Response:

We thank the reviewer for this valuable comment. The Results section has been reorganized to clearly separate descriptive histological observations from statistically analyzed quantitative outcomes. Starting from the boxplot graph for the entire treatment group, then followed by a quantitative data table and finally an immunohistochemical images. (L174-204).

 

6. Reviewer comment 6:

The temporal changes in HIF-1α expression between Day 7 and Day 14 warrant a more explicit description in the Results section, as this dynamic trend is central to the study’s hypothesis.

Response:

We thank the reviewer for this insightful comment. The Results section has been revised to more explicitly describe the temporal changes in HIF-1α expression between Day 7 and Day 14. We now highlight the direction and magnitude of these changes within and between experimental groups, emphasizing their relevance to the study hypothesis. These clarifications have been incorporated into the revised Results section (L186-189).

 

7. Reviewer comment 7:

The Discussion would be strengthened by proposing specific future studies, such as long-term micro-CT analysis or biomechanical testing, to validate the functional relevance of the observed early healing enhancement.

 

Response:

We thank the reviewer for this valuable suggestion. The Discussion section has been revised to propose specific future studies to validate the functional relevance of the observed early-healing enhancement. In particular, we now suggest long-term micro-CT analysis to assess bone volume and architecture, as well as biomechanical testing to evaluate the mechanical competence of the regenerated tissue. These additions have been incorporated into the revised Discussion section (L320-323).

Reviewer 3 Report

Comments and Suggestions for Authors

In relation to the paper mentioned above, it is an interesting experimental investigation performed in rats. The manuscript is well written, and each section entails an adequate structure and content. I recommend it for publication, although clear explanation about draw backs, limitations and potential inconvenient must be deeply described.

This experimental study explores a new approach to face post-extraction bone defects. Consequently, a clear description of potential inconvenient, side effects and negative consequences are mandatory to be named and described, specially focused on the human beings.

In addition, it must be better described how the doses and the method of application would be related to the real scenario of human beings, involving any kind of precaution on this new use.

I would also suggest to extent the limitation paragraph situated at the end of the discussion to better explain the limitations of the current study, and how these results can be translated to the human beings, but recognizing the limitation of an animal study.

 

 

 

Author Response

Response to Reviewer's comments:  

 

Reviewer 3

 

 

1. Reviewer comment 1: In relation to the paper mentioned above, it is an interesting experimental investigation performed in rats. The manuscript is well written, and each section entails an adequate structure and content. I recommend it for publication, although clear explanation about drawbacks, limitations, and potential inconveniences must be deeply described.

Response:

We sincerely thank the reviewer for the positive evaluation of our work and for the recommendation for publication. In response to this valuable suggestion, we have expanded the Discussion section to more clearly and thoroughly describe the drawbacks, limitations, and potential inconveniences of the present study. Specifically, we now address the experimental nature of the rat model, the focus on early healing time points, the absence of long-term functional assessments, and the translational limitations to clinical practice. These additions have been incorporated into the revised Discussion section (L294-310).

 

2. Reviewer comment 2: This experimental study explores a new approach to face post-extraction bone defects. Consequently, a clear description of potential inconvenient, side effects and negative consequences are mandatory to be named and described, specially focused on the human beings.

Response:

We thank the reviewer for this important and thoughtful comment. The Discussion section has been expanded to clearly describe potential inconveniences, side effects, and negative consequences associated with the proposed approach, with particular emphasis on considerations relevant to human application. These additions provide a balanced and ethically responsible interpretation of the findings and have been incorporated into the revised Discussion section (L305-309).

 

Despite the promising experimental findings, several potential inconveniences and risks should be considered before clinical translation. Icariin, although derived from natural sources, may exhibit dose-dependent biological effects, and its safety profile in humans requires careful evaluation. The use of bovine-derived xenografts also raises considerations related to immunogenicity, disease transmission, and patient acceptance. Furthermore, modulation of early osteogenic and angiogenic signaling may have unintended effects if not appropriately controlled. Therefore, comprehensive toxicological assessments, long-term safety studies, and controlled clinical trials are required before this approach can be considered for human application.

 

3. Reviewer comment 3: In addition, it must be better described how the doses and the method of application would be related to the real scenario of human beings, involving any kind of precaution on this new use.

Response:

We thank the reviewer for this important comment. The method section has been expanded to clarify how the experimental dose and method of application used in this study relate to potential human clinical scenarios. We now explicitly address the limitations of dose extrapolation from animal models, the differences between experimental and clinical delivery methods, and the precautions that would be required for any future human application. These additions provide a more responsible translational perspective and have been incorporated into the revised Discussion section (L297-301).

 

The dosing regimen and method of application used in this experimental study were designed for localized delivery within a rat extraction socket and cannot be directly extrapolated to human clinical practice. Differences in socket size, healing dynamics, and metabolic responses necessitate careful dose-scaling and formulation optimization. In a potential human scenario, localized delivery systems with controlled release would be required to avoid systemic exposure and unintended biological effects.

 

4. Reviewer comment 4: I would also suggest to extend the limitation paragraph situated at the end of the discussion to better explain the limitations of the current study, and how these results can be translated to the human beings, but recognizing the limitation of an animal study.

Response:

We thank the reviewer for this important and constructive suggestion. The limitations paragraph at the end of the Discussion section has been expanded to more clearly describe the limitations of the current animal study and to cautiously address the potential translational relevance to human application. We explicitly acknowledge the constraints of the rat model, the focus on early healing time points, and the challenges of extrapolating these findings to clinical practice. These revisions have been incorporated into the revised Discussion section (L294-303).

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript titled “Novel combination of icariin and bone xenograft promotes the expression of collagen fibers, BMP‑2, and HIF‑1α during wound healing in Wistar rats” addresses a clear and relevant research question by investigating the combined effect of icariin and xenografts on early extraction wound healing. The study is academically significant due to its systematic evaluation of early healing markers—collagen matrix, BMP‑2, and HIF‑1α—and its integrated investigation of osteogenesis–angiogenesis coupling, a concept previously examined in isolation. Nevertheless, the absence of quantitative and functional data to substantiate the proposed mechanisms, limitations in control group design, and overly simplistic statistical analyses constrain the strength and scope of the conclusions. The reviewer therefore provides several comments to enhance the manuscript’s rigor and suitability for publication in the Journal of Molecular Pathology.

 

1. The rationale for setting icariin at 10 µM was presented, but there is no information on the concentration locally maintained and released in the xenograft (loading efficiency, release kinetics). This represents a significant limitation for interpreting biological effects.
2. To demonstrate the combined effect of xenograft and icariin, a group receiving icariin alone is necessary. With the current design, it is difficult to distinguish between synergistic effects and simple additive effects.
3. In Group IX, HIF-1α expression decreased on Day 14 compared to Day 7. However, this cannot be interpreted as “vascular maturation” since vascular markers such as VEGF, CD31, and endomucin were not presented.
4. The use of one-way ANOVA rather than two-way ANOVA, which simultaneously accounts for both time (Day 7 vs Day 14) and treatment group, limits the statistical interpretation. The authors should conduct the two-way ANOVA analysis to interpret the correlation between time and treatment.
5. Increased BMP-2 expression alone is insufficient functional evidence to conclusively determine the osteoinductive properties of xenograft + icariin. Also, the mechanism linking whether icariin increases collagen synthesis via BMP-2 or through an independent effect has not been experimentally demonstrated.
6. There is insufficient discussion regarding the safety of icariin in clinical circumstance, including potential side effects, dosage ranges, and regulatory issues when applied topically.

 

 

Author Response

Response to Reviewer's comments:  

 

Reviewer 4

 

 

Reviewer comment 1: The rationale for setting icariin at 10 µM was presented, but there is no information on the concentration locally maintained and released in the xenograft (loading efficiency, release kinetics). This represents a significant limitation for interpreting biological effects.

Response:

We thank the reviewer for this important and insightful comment. We agree that information regarding loading efficiency and release kinetics would provide a more complete interpretation of the biological effects. However, these analyses were beyond the scope of the present study. To address this limitation, we have explicitly acknowledged the absence of loading efficiency and release kinetics data in the Discussion section and clarified that the observed biological effects reflect the combined influence of the icariin–xenograft system rather than a precisely quantified local icariin concentration (L297-304).

 

A limitation of the present study is the lack of direct characterization of icariin loading efficiency and release kinetics from the xenograft. Therefore, the exact local concentration of icariin maintained within the extraction socket over time remains unknown. Consequently, the observed biological effects should be interpreted as the result of icariin–xenograft system rather than a precisely controlled dose-dependent response.

 

Reviewer comment 2: To demonstrate the combined effect of xenograft and icariin, a group receiving icariin alone is necessary. With the current design, it is difficult to distinguish between synergistic effects and simple additive effects.

Response:

We thank the reviewer for this important and well-founded comment. We agree that inclusion of an icariin-only group would allow a more precise distinction between synergistic and additive effects. However, the primary objective of the present study was to evaluate the biological response to the combined icariin–xenograft approach as a proof-of-concept strategy for socket healing. We have now explicitly acknowledged the absence of an icariin-only group as a limitation in the Discussion section and clarified that the observed effects should be interpreted as combined effects rather than definitive synergistic interactions. We also identify inclusion of an icariin-only group as an important direction for future studies (L303-307).

 

Reviewer comment 3: In Group IX, HIF-1α expression decreased on Day 14 compared to Day 7. However, this cannot be interpreted as “vascular maturation” since vascular markers such as VEGF, CD31, and endomucin were not presented.

Response:

We thank the reviewer for this important and well-founded comment. We agree that decreased HIF-1α expression alone cannot be interpreted as evidence of vascular maturation in the absence of direct vascular markers such as VEGF, CD31, or endomucin. Accordingly, we have revised the Results and Discussion sections to remove the term “vascular maturation” and to clarify that the observed decrease in HIF-1α expression at Day 14 should be interpreted as hypoxia-responsive signaling rather than direct evidence of vascular maturation. This correction provides a more accurate and cautious interpretation of the findings (L235-238).

 

Reviewer comment 4: The use of one-way ANOVA rather than two-way ANOVA, which simultaneously accounts for both time (Day 7 vs Day 14) and treatment group, limits the statistical interpretation. The authors should conduct the two-way ANOVA analysis to interpret the correlation between time and treatment.

Response:

We thank the reviewer for this valuable comment. We agree that two-way ANOVA is a powerful approach for evaluating the interaction between time and treatment. However, in the present study, Day 7 and Day 14 represent independent animal cohorts rather than repeated or longitudinal measurements. As such, the study was not designed to assess interaction effects between time and treatment within the same subjects. Therefore, one-way ANOVA was applied separately at each time point. We have clarified this rationale more explicitly in the Methods and Discussion sections and acknowledged this limitation in the revised manuscript (L86-88).

 

Reviewer comment 5: Increased BMP-2 expression alone is insufficient functional evidence to conclusively determine the osteoinductive properties of xenograft + icariin. Also, the mechanism linking whether icariin increases collagen synthesis via BMP-2 or through an independent effect has not been experimentally demonstrated.

Response:

We thank the reviewer for this important and well-founded comment. We agree that increased BMP-2 expression alone does not constitute definitive functional evidence of osteoinduction. Accordingly, we have revised the manuscript to avoid overinterpretation of BMP-2 expression as direct proof of osteoinductive activity. The Results and Discussion sections now clarify that BMP-2 is presented as an osteogenic-related molecular marker rather than a functional endpoint. (L 228-231).

Although increased BMP-2 expression suggests activation of osteogenic-related signaling pathways, it does not provide direct functional evidence of osteoinduction. Therefore, these findings should be interpreted as indicative of molecular modulation rather than definitive proof of osteoinductive capacity.

 

Reviewer comment 6: There is insufficient discussion regarding the safety of icariin in clinical circumstance, including potential side effects, dosage ranges, and regulatory issues when applied topically.

 

Response:

We thank the reviewer for this important and constructive comment. The Discussion section has been expanded to more thoroughly address the safety considerations of icariin in potential clinical applications. Specifically, we now discuss reported safety profiles and potential side effects, considerations regarding dosage ranges, and regulatory aspects related to topical application. These additions provide a more balanced and clinically relevant perspective and have been incorporated into the revised Discussion section (L305-309).

 

Although icariin is a naturally derived flavonoid, its clinical safety profile, particularly for topical application in oral tissues, requires careful consideration. Reported studies suggest that icariin exhibits low cytotoxicity within certain concentration ranges; however, dose-dependent effects and potential local or systemic side effects cannot be excluded. Moreover, the optimal therapeutic window for topical delivery remains undefined. From a regulatory perspective, the use of icariin as a bioactive adjunct in dental applications would require comprehensive toxicological evaluation, standardized formulation, and approval by relevant regulatory authorities before clinical translation.

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have diligently addressed all the points raised by the reviewer, and therefore, I approve the manuscript for publication.

Comments on the Quality of English Language

The English language throughout the manuscript needs improvement for clarity and readability.

Author Response

Reviewer 2 Comments:

1. The authors have diligently addressed all the points raised by the reviewer, and therefore, I approve the manuscript for publication.

Response:
We sincerely thank the reviewer for the positive evaluation and for approving our manuscript for publication. We greatly appreciate your time, insightful comments, and constructive feedback, which have significantly improved the quality and clarity of our work.

 

2. The English language throughout the manuscript needs improvement for clarity and readability.

Response:
Thank you for your suggestion. The manuscript has been carefully edited to improve clarity and readability. Specifically, we refined sentence structure, improved word choice, corrected grammar and punctuation, and ensured consistency in terminology across all sections. These revisions have been incorporated into the updated manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript has been thoroughly revised in response to the main reviewer comments. However, some results and discussions still contain overinterpretation regarding the ‘synergistic effect’ and ‘vascular maturation,’. Therefore, the authors should correct this term and re-submit for the publication in JMP. 

Author Response

Reviewer 4 Comments:

This manuscript has been thoroughly revised in response to the main reviewer comments. However, some results and discussions still contain overinterpretation regarding the ‘synergistic effect’ and ‘vascular maturation,’. Therefore, the authors should correct this term and re-submit for the publication in JMP. 

Response:
Thank you for your careful re-evaluation and constructive comments. We acknowledge that the previous version of the manuscript may have contained overinterpretation regarding the terms “synergistic effect” and “vascular maturation.” In response, we have revised the Results and Discussion sections to ensure that our conclusions remain strictly supported by the presented histological and immunohistochemical findings.

Specifically, we replaced “synergistic effect” with more appropriate terms such as “enhanced effect,” “improved outcome” (L174, L247-250, L331-332). In addition, we revised “vascular maturation” to “increased angiogenesis-related response” or “enhanced expression of HIF-1α,” (L240-243, L273-274, L292-293).

All relevant statements have been corrected throughout the manuscript, and the conclusions have been modified accordingly to avoid overstatement.

 

Round 3

Reviewer 4 Report

Comments and Suggestions for Authors

The reviewer has no further comments on the revised manuscript.