Efficacy of Supercritical Fluid Decellularized Porcine Acellular Dermal Matrix in the Post-Repair of Full-Thickness Abdominal Wall Defects in the Rabbit Hernia Model
, Yun-Ju Chen 3, Srinivasan Periasamy 3, Ko-Chung Yen 3 and Dar-Jen Hsieh 3,*Round 1
Reviewer 1 Report
The present manuscript can be accepted after some corrections.
Title.
The manuscript title must be changed. After revision of section 2.1, the decellularization was performed by means of CO2 – ethanol. Therefore, I suggest: “Efficacy of supercritical fluid (carbon dioxide – ethanol) decellularized porcine ….”
Similarity analysis.
Make some corrections. The manuscript has 38% of similarity according to Turnitin software. The most important are: https://pubmed.ncbi.nlm.nih.gov/32132871/, https://doi.org/10.1016/j.mtla.2019.100576, https://www.mdpi.com/1996-1944/14/11/2790/html, https://medsci.org/v17p0354.pdf, https://www.liebertpub.com/doi/10.1089/ten.tec.2017.0167, https://journals.lww.com/plasreconsurg/Abstract/2011/06000/Human_versus_Non_Cross_Linked_Porcine_Acellular.21.aspx, https://onlinelibrary.wiley.com/doi/10.1002/term.3252
Abbreviations are not explained: DNA, DAPI, EPM, ECM, scCO2 or SCCO2, IHC,
Abstract.
I suggest to change the order for some sentences: “The loss of abdominal wall integrity may occur after trauma, infection and herniation. Hernia repair remains to be one of the most recurrent general surgery procedures done. Acellular dermal matrix (ADM) was manufactured to repair the full-thickness abdominal wall defects and hernia. The ADM was …..”
Abstract is not chronologically clear and induce doubts. Correct it. “Manufacture” and “created” are too repetitive. Confirm the following: First, the porcine skin was subject to SCCO2; The ADM was manufactured from porcine skin (how?). The ADM was characterized.
Give quantitative results instead of “complete decellularization”, “excellent performance”, “good biocompatibility and excellent mechanical strength”
Introduction.
Line 71. “The scCO2 technology can be employed to eradicate fat and cellular components while retaining the intact ECM without impairment.” Which kind of compounds in fat are removed? The same question for cellular components, this last is too general.
Line 75. Include the critical temperature.
Lines 71-72 and lines 77-78 are related, combine them in one sentence.
Conclude about literature insights: what are the disadvantages of SCCO2 for this topic? Besides, how efficient is SCCO2 against the other decellularization techniques.
Section 2.1
Some questions and confirmation of sentences are required about the brief description for supercritical extraction:
1. Was the fat layer with polyethene gauze arranged in a tissue holder loaded into the vessel? or was the fat layer loaded into the vessel?
3. Was the extraction performed in static or dynamic mode?
4. I do not understand 10 ml of ethanol? the same for 75% for ethanol, mass volume, mole basis.
5. What was the rate for CO2 and ethanol?
6. Where there several experiments at different conditions? or the reported ranges were used in different periods? If experiments were performed at different conditions, these will affect the final product. It seemed that there was a unique systematic experiment performed at different conditions in function of time. Clarify.
Detail the discussion for figure 2.
Discussion.
Provide quantitative comparison instead of qualitative.
Author Response
Response to the reviewer's comment
Reviewer # 1
General comment: The present manuscript can be accepted after some corrections.
Response: We thank the reviewer for the comments.
Comment 1: Title. The manuscript title must be changed. After revision of section 2.1, the decellularization was performed by means of CO2 – ethanol. Therefore, I suggest: “Efficacy of supercritical fluid (carbon dioxide – ethanol) decellularized porcine ….”
Response: We have modified the title.
Revision: Page No. 1, Line No. 2.
Comment 2: Similarity analysis. Make some corrections. The manuscript has 38% of similarity according to Turnitin software. The most important are: https://pubmed.ncbi.nlm.nih.gov/32132871/, https://doi.org/10.1016/j.mtla.2019.100576, https://www.mdpi.com/1996-1944/14/11/2790/html, https://medsci.org/v17p0354.pdf, https://www.liebertpub.com/doi/10.1089/ten.tec.2017.0167, https://journals.lww.com/plasreconsurg/Abstract/2011/06000/Human_versus_Non_Cross_Linked_Porcine_Acellular.21.aspx, https://onlinelibrary.wiley.com/doi/10.1002/term.3252
Response: We have made corrections in the manuscript text. We are aware of the similarities of the text. However, most of the links given in the similarity analysis were our previous publications or as a coauthor, in which we performed the supercritical carbon dioxide and wrote that part of those manuscripts. Therefore, the text regarding the supercritical carbon dioxide, scaffold and acellular dermal matrix was shown in the similarity analysis.
Revision: Highlighted in red colour text.
Comment 3: Abbreviations are not explained: DNA, DAPI, EPM, ECM, scCO2 or SCCO2, IHC,
Response: We have explained the abbreviations as suggested by the reviewer.
Revision: Highlighted in red colour text.
Comment 4: Abstract. I suggest to change the order for some sentences: “The loss of abdominal wall integrity may occur after trauma, infection and herniation. Hernia repair remains to be one of the most recurrent general surgery procedures done. Acellular dermal matrix (ADM) was manufactured to repair the full-thickness abdominal wall defects and hernia. The ADM was …..”
Response: We have changed the order and modified the sentence, as suggested by the reviewer.
Revision: Page No. 1, Line No. 11-14.
Comment 5: Abstract is not chronologically clear and induce doubts. Correct it. “Manufacture” and “created” are too repetitive. Confirm the following: First, the porcine skin was subject to SCCO2; The ADM was manufactured from porcine skin (how?). The ADM was characterized.
Response: We have changed the chronological order and modified the abstract, as suggested by the reviewer.
Revision: Page No. 1, Line No. 14-16.
Comment 6: Give quantitative results instead of “complete decellularization”, “excellent performance”, “good biocompatibility and excellent mechanical strength”
Response: We have given the quantitative results and modified the text, as suggested by the reviewer. However, for the decellularization hematoxylin and eosin (H&E) staining, 4,6-Diamidino-2-phenylindole, dihydrochloride (DAPI) staining, residual deoxyribonucleic acid (DNA) contents and alpha-galactosidase, was assessed the complete decellularization of ADM. Most of the techniques to evaluate decellularization is staining procedures, based on the presence of the nucleus in H&E and DAPI staining routine decellularization confirmation (Wu et al., 2021; Chou et al., 2020; Lee et al., 2021) is done.
Revision: Page No. 1, Line No. 23-28.
Comment 7: Introduction. Line 71. “The scCO2 technology can be employed to eradicate fat and cellular components while retaining the intact ECM without impairment.” Which kind of compounds in fat are removed? The same question for cellular components, this last is too general.
Response: We have modified the text in the Introduction part. The scCO2 technology was found to break the cells by the penetration of supercritical carbon dioxide. The broken cells were washed off by the cosolvent ethanol used in the process. Fat cells and ethanol-soluble fats were removed. The intracellular protein and the functional cellular proteins of cellular proteins were removed (Wu et al., 2021; Chou et al., 2020; Lee et al., 2021).
Revision: Page No. 2, Line No. 76- 79.
Comment 8: Line 75. Include the critical temperature.
Response: We have included the critical temperature.
Revision: Page No. 2, Line No. 82.
Comment 9: Lines 71-72 and lines 77-78 are related, combine them in one sentence.
Response: We have combined the related into one sentence.
Revision: Page No. 2, Line No. 76- 79.
Comment 10: Conclude about literature insights: what are the disadvantages of SCCO2 for this topic? Besides, how efficient is SCCO2 against the other decellularization techniques.
Response: We have included the efficiency of SCCO2 against the other decellularization techniques. However, regarding the disadvantages of SCCO2 for this topic, until now there are no know disadvantages. Because the traditional use of SCCO2 is to collect the extract with cosolvent ethanol, but with the current technology we discard the ethanol cosolvent extract and cleaned-off, and the remaining scaffold of the skin which is ADM.
Revision: Page No. 2, Line No. 70-74; 84-86.
Comment 11: Section 2.1. Some questions and confirmation of sentences are required about the brief description for supercritical extraction:
-Was the fat layer with polyethene gauze arranged in a tissue holder loaded into the vessel? or was the fat layer loaded into the vessel?
Response: We have modified the text by making it into three sentences. “The fat layer of the hide was trimmed off and discarded. The remaining part hide was washed with phosphate-buffered saline (PBS). The hide was delicately sliced to 0.4–2 mm in thickness. The sliced hide was rolled along with polyethene gauze arranged in a tissue holder”.
-Was the extraction performed in static or dynamic mode?
Response: We have added the mode of operation of SCCO2 system in the text.
-I do not understand 10 ml of ethanol? the same for 75% for ethanol, mass volume, mole basis.
Response: It’s a typo, we have corrected that in the text. It is “cosolvent filled to 10 % volume of the vessel with 75% ethanol”.
-What was the rate for CO2 and ethanol?
Response: We have added the flow rate in the manuscript. The flow rate of carbon dioxide is 0.3 litres per minute (LPM). The cosolvent ethanol is 10 % volume of the vessel with 75% ethanol. The principle of the SCCO2 in this experiment is to discard the ethanol cosolvent extract and cleaned-off, and the remaining scaffold of the skin which is ADM.
Revision: Page No. 3, Line No. 105, 106.
-Where there several experiments at different conditions? or the reported ranges were used in different periods? If experiments were performed at different conditions, these will affect the final product. It seemed that there was a unique systematic experiment performed at different conditions in function of time. Clarify.
Response: The experimental condition is the same thought out the whole study. We have
revised the text in 2.1.
Revision: Page No. 3, Line No. 99-109.
Comment 12: Detail the discussion for figure 2.
Response: We have detailed the result and discussion section of Figure 2.
Revision: Page No. 5, Line No.217-222; Page No. 9, Line No. 319-323.
Comment 13: Discussion. Provide quantitative comparison instead of qualitative.
Response: We have provided a detailed quantitative comparison in the result section, it will redundant if we again do the quantitative comparison in the discussion part.
References
Wu CC, Tarng YW, Hsu DZ, Srinivasan P, Yeh YC, Lai YP, Hsieh DJ. Supercritical carbon dioxide decellularized porcine cartilage graft with PRP attenuated OA progression and regenerated articular cartilage in ACLT-induced OA rats. J Tissue Eng Regen Med. 2021 Dec;15(12):1118-1130.
Chou PR, Lin YN, Wu SH, Lin SD, Srinivasan P, Hsieh DJ, Huang SH. Supercritical Carbon Dioxide-decellularized Porcine Acellular Dermal Matrix combined with Autologous Adipose-derived Stem Cells: Its Role in Accelerated Diabetic Wound Healing. Int J Med Sci. 2020 Feb 4;17(3):354-367.
Lee SS, Wu YC, Huang SH, Chen YC, Srinivasan P, Hsieh DJ, Yeh YC, Lai YP, Lin YN. A novel 3D histotypic cartilage construct engineered by supercritical carbon dioxide decellularized porcine nasal cartilage graft and chondrocytes exhibited chondrogenic capability in vitro. Int J Med Sci. 2021 Mar 25;18(10):2217-2227.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Dear Authors,
I read with great enthusiasm your paper and enjoyed it!
but there were few things to address:
1. Line 37, 38, 39 – not concordant with citation, phrase is not quite understandable
2. Line 42 - ECM abbreviations stand for what? (I know what it is, but you should name it first when it appears in text)
3. Line 77 – what has to do with our text at line 77/or our paper the reference no 18?
4. Line 322,323 – why is justified to extrapolate in humans? We all know humans are different from other mammals when injured, we are older, sicker, and usually these implants are used in an infected environment, which was not the case in these rabbits, so you must be more cautious with your sentences.
5. Lines 337 to 347 – English or text should be revised as I don’t understand your ideas.
6. You have no biases discussed in this study, and no weak points – you should address that
7. You didn’t mention if these mesh of yours had any adhesions with the abdominal organs…
8. Final advice – be more cautious, does not mean that your work is not important, but it is a start, and the only way you can say something about humans is by doing a study on humans.
Author Response
Response to the reviewer's comment
Reviewer # 2
General comment: I read with great enthusiasm your paper and enjoyed it!
Response: We thank the reviewer for the appreciation and encouraging comments.
Comment 1: Lines 37, 38, 39 – not concordant with citation, phrase is not quite understandable
Response: We have modified the text according to the citation for consistency and the phrase was modified for comprehension.
Revision: Page No. 1, Line No. 40- 42.
Comment 2: Line 42 - ECM abbreviations stand for what? (I know what it is, but you should name it first when it appears in text)
Response: We have explained the abbreviations for ECM as extracellular matrix.
Revision: Page No. 2, Line No. 45.
Comment 3: Line 77 – what has to do with our text at line 77/or our paper the reference no 18?
Response: We have corrected and modified the text. The reference is a typo. We have changed reference No. 18.
Revision: Page No. 2, Line No. 82-83. Reference -Page No. 12, Line No. 452-454.
Comment 4: Line 322,323 – why is justified to extrapolate in humans? We all know humans are different from other mammals when injured, we are older, sicker, and usually these implants are used in an infected environment, which was not the case in these rabbits, so you must be more cautious with your sentences.
Response: As suggested by the reviewer we have slightly modified the sentence. We are aware and understand rabbits and humans are not equal. However, to test the medical device biomaterial as guided by ISO 10993-6: 2016, New Zealand White Rabbit is a species suitable for any implantation experiments related to biomaterial medical devices. In addition, rabbit models are the best animal model to verify the performance of medical device products such as ADM by comparing the commercially available surgical mesh (Pascual et al., 2013). We consider this study a preclinical study before performing clinical trials in humans.
Revision: Page No. 10, Line No. 336-337.
Comment 5: Lines 337 to 347 – English or text should be revised as I don’t understand your ideas.
Response: We have revised the manuscript for English and text, as suggested by the reviewer.
Revision: Page No. 10, Line No. 351-358.
Comment 6: You have no biases discussed in this study, and no weak points – you should address that
Response: We have addressed the advantage of the ADM in the discussion part as suggested by the reviewer.
Revision: Page No. 11, Line No. 405-408.
Comment 7: You didn’t mention if these mesh of yours had any adhesions with the abdominal organs…
Response: We have mentioned in the discussion part that our ADM did not have any adhesions to the abdominal organs.
Revision: Page No. 10, Line No. 383-384.
Comment 8: Final advice – be more cautious, does not mean that your work is not important, but it is a start, and the only way you can say something about humans is by doing a study on humans.
Response: We understand the caution of the reviewer. We have modified the text accordingly. We performed the experiment for medical device biomaterial ADM as guided by ISO 10993-6: 2016 as a preclinical study in anticipation of continuing human clinical trials. We have been working on supercritical carbon dioxide extraction technology to manufacture medical devices for human use. We have been approved by the US FDA for two of our medical devices that were developed using supercritical carbon dioxide extraction technology. Therefore, we anticipate the results of our current experiment can be extrapolated to humans.
Revision: Page No. 10, Line No. 336-337.
Reference
ISO 10993. Biological evaluation of medical devices - Part 6: Tests for local effects after implantation ISO 10993 (2016).
Pascual G, Sotomayor S, Rodríguez M, Bayon Y, Bellón JM. Behaviour of a New Composite Mesh for the Repair of Full-Thickness Abdominal Wall Defects in a Rabbit Model. PLoS One. 2013;8(11).
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Al corrections were completed
