Comparative Evaluation of Decellularized Human Amniotic Membrane and Wharton’s Jelly in a Rat Model of Myocardial Infarction: Experimental Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study investigated the regeneration potential of decellularized human amniotic membrane  and Wharton’s jelly in a rat model after acute myocardial infarction. The good number of rats (33) underwent left anterior descending coronary artery ligation and were randomized into three groups: control, WJ, and HAM. In 30 days, echocardiographic, histopathological, and immunohistochemical assessments were performed. No significant differences in ventricular function were observed among groups. HAM-treated group showed a significant reduction in myocardial fibrosis compared with control (p = 0.009), suggesting attenuation of post-infarction remodeling. As a results the results indicate that decellularized HAM may act as a structural modulator of myocardial remodeling.

The study is well-designed and the proper methods  of heart investigation was used (Echo, histochemistry, infarction area estimation, etc.).  Also the limitations were correctly described. 

Suprisingly the immunohistochemical analysis showed no change between the groups. Generally the study has a strict logic and has some perspectives in the future. I agree with the Study results but tell me why you did not study TnI/T and CF шт the serum?  That will explain the degee of cardiac necrosis.

 

Author Response

 Comments - I agree with the Study results but tell me why you did not study TnI/T and CF шт the serum?  That will explain the degee of cardiac necrosis.

Thanks for commenting

Response -  

Response - Response - We sincerely thank the reviewer for the careful evaluation of our manuscript and for recognizing the quality of the experimental design, methodology, and the potential of decellularized human amniotic membrane (HAM) as a structural modulator of myocardial remodeling. We fully agree that serum cardiac biomarkers such as cTnI/cTnT and CK-MB could provide complementary information regarding the initial extent of myocardial necrosis after infarction induction.

However, our study was specifically designed to evaluate late cardiac remodeling and the regenerative potential of decellularized biomaterials at 30 days post-infarction, focusing on functional, histological, and immunohistochemical outcomes. Therefore, we prioritized analyses related to fibrosis, tissue architecture, and local regenerative response. In addition, troponin and CK-MB measurements are most relevant during the acute phase of myocardial injury, especially within the first hours after coronary ligation, whereas our analyses were performed only at a late chronic time point. We also considered the limited blood volume available in Wistar rats and prioritized the analyses previously established in the approved animal ethics protocol. Nevertheless, we acknowledge that the inclusion of early serum cardiac biomarkers could strengthen future studies by allowing a better correlation between initial necrosis and subsequent ventricular remodeling. This limitation and future perspective were added to the revised manuscript.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript investigates the structural and functional effects of implanting cell-free, decellularized human amniotic membrane (HAM) and Wharton’s jelly (WJ) patches into a subacute rat model of acute myocardial infarction (AMI) presenting with established left ventricular dysfunction (LVEF ≤45%). Over a 30-day post-implantation period, the authors track cardiac function via transthoracic echocardiography and evaluate structural remodeling using histopathological and immunohistochemical assays.

Comments for author File: Comments.pdf

Author Response

 

We sincerely thank the reviewer for the careful, insightful, and constructive comments, which significantly contributed to improving the scientific quality, clarity, and overall rigor of our manuscript.

REVIEWER 2

Response - We sincerely thank the reviewer for the careful reading and constructive comments. Each observation was taken seriously and genuinely contributed to improving the manuscript.

Comment 1 – Differences in delivery protocols between groups

The reviewer raised a very pertinent point: the control group received intramyocardial saline injections, whereas the WJ and HAM groups received epicardially sutured biomaterial patches. He questions whether the benefits observed in the HAM group might simply derive from the mechanical stabilization provided by the suturing process, rather than from the intrinsic properties of the biomaterial.

We fully agree. Epicardial implantation of a sutured patch may, on its own, provide passive mechanical support to the infarcted ventricular wall and this may have contributed to the results independently of the biochemical properties of the scaffold. In the revised manuscript, we expanded the Discussion to explicitly acknowledge that the reduction in fibrosis and infarct area observed in the HAM group likely reflects a combination of biomechanical reinforcement, epicardial constraint, and preserved extracellular matrix bioactivity. We also clarified that the absence of a sham-patch or suture-only control group prevents us from precisely isolating the biological contribution of the HAM scaffold from the mechanical effects of the surgical procedure itself. This limitation was added to the manuscript.

Comment 2 – Patch thickness characterization

Thank you for this important observation: Section 2.2 describes a thickness of 30 µm, while Section 2.5 mentions 80–120 µm. He also correctly noted that Wharton's jelly is not an amniotic membrane. We clarified: the 30 µm refers to the average thickness of the individual decellularized membrane layer, measured histologically immediately after processing. The 80–120 µm corresponds to the thickness of the hydrated patches prepared for implantation naturally greater due to hydration and surgical handling. Regarding terminology, we revised the text to clearly distinguish HAM from Wharton's jelly-derived matrices, avoiding any mistaken reference to the latter as an "amniotic membrane."

Comment 3 – Post-hoc exclusion of animals with small infarcts

Thank you for this important observation the exclusion of two animals after histological analysis may introduce selection bias. This is a legitimate concern. We clarified that the exclusion was not based on treatment response or histological improvement, but rather on the absence of transmural infarction compatible with the established experimental model identified only upon detailed histopathological evaluation, despite these animals having initially met the echocardiographic inclusion criteria at Day 7. To ensure transparency, we made this explicit in the revised manuscript and acknowledged in the Discussion that exclusions after randomization represent a potential source of bias.

Comment 4 – Decellularization validation

We acknowledge the reviewer's concern the detergent concentrations used were lower than those of conventional protocols and that no quantitative validation of decellularization was performed. This is fair criticism. The protocol was intentionally developed using low detergent concentrations to preserve the ultrastructural integrity and bioactive composition of the tissues as a deliberate methodological choice. However, we acknowledge that methods such as residual dsDNA quantification and DAPI staining would have provided more robust confirmation of decellularization efficiency. In the revised manuscript, we address this limitation directly and highlight that future studies should incorporate standardized molecular validation assays to better characterize the decellularization process and potential residual immunogenicity.

Comment 5 – Inconsistency in animal numbers

Thank you for your comments, the abstract mentions 23 rats randomized, while the Results section describes 70 rats undergoing coronary ligation, with 23 surviving to the end of the study. After verification, we corrected the abstract to accurately reflect the experimental flow: 70 animals underwent infarction induction; 36 met the inclusion criteria and were randomized into experimental groups; 23 survived to the end of the 30-day follow-up and were included in the final analysis. The text was standardized throughout the manuscript.

Comment 6 – Incorrect group description in Section 3

We appreciate your feedback regarding the inaccurate description of the experimental groups. This was a writing error. The sentence was corrected to accurately describe the three groups: Saline Control Group, Wharton's Jelly Patch Group, and Human Amniotic Membrane Patch Group.

Comment 7 – Statistical inconsistencies in Tables 1 and 2

Thank you by your comments, we thoroughly reviewed all tables. The implausible values resulted from formatting errors during manuscript preparation. Decimal notation was standardized throughout, and all statistical values were verified directly against the original SPSS outputs.

Comment 8 – P-value discrepancy for collagen I and III analysis

Thank you for your comments, the text reports p = 0.74, while Table 3 presents p = 0.074. After direct verification against the SPSS outputs, we confirmed that the correct value is p = 0.074. The text was corrected accordingly.

Comment 9 – Incorrect table citation in the infarct area analysis

Thank you for your comments, the text refers to Table 3 instead of Table 4. This was a typographical error. The citation was corrected in the revised manuscript.

Comment 10 – Incorrect standard deviation in Table 6 (Factor VIII)

Thank you for your comments, the value "1.01 ± 50.76" is clearly incorrect. Confirmed this was a typographical error. The correct value is "1.01 ± 0.76," consistent with the text and the original analysis. Table 6 was corrected accordingly.

Comment 11 – Discrepancy in alpha-actin and CD31 p-values

Thank you for your comments, the p-values reported in the text and in Table 6 do not match. The data were re-verified, and the manuscript was corrected to ensure full consistency between the text and the table.

Comment 12 – CD68 value discrepancy

Thank you for your comments, the difference between the CD68 value in the text and that in Table 6. This was a typographical error introduced during manuscript editing. The values were corrected and standardized throughout the manuscript based on the original statistical dataset.

We once again thank the reviewer for the thorough and detailed reading. The comments were invaluable in improving the clarity, transparency, and scientific rigor of this work.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for considering all the comments and submitting the revised manuscript, all of the concerns were addressed and modified accordingly