Evaluation of Full Thickness Wounds Following Application of a Visco-Liquid Hemostat in a Swine Model

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors should consider the followings:

Methodology of how the "POSS gel polymer" was made should be supplemented. The authors should clarify why saline was used as the negative control, and whether any comparable vehicle control from the vehicle base making POSS, should be used as negative control instead.

Positive control of wound healing agent is missing in this research work. The authors should justify why this is missing.

In situ zymography (and/or the functional enzymatic based assay) of the interested MMPx markers should be performed to truely assess the MMPx/TIMPx activities.

As for "MMP-1, MMP-2, MMP-3, and MMP13", the authors should clarify whether the Elisa were measuring both the pro-form and/or active forms of the captioned MMPx, and whether the % specificity covered swine? Dynamic range of the validated concentration of each analytes should be provided, together with which validated method the manufacturer were based.

In histological grading, the authors should clarify and supplement the experience and qualifications required for the examiners. 

Triage system of the situation, where discrepant scoring from the examiners was found, should be in place and specified in the methodology.

In Figure 3 to 5 and figure 8, the authors should state whether the comparison found as statistical significance, or stating NS (not significant) in the figues.

As for Figure 6, the authors should clarify the distance of the scale bar.

 The authors may consider to supplement IHC slide for staining the relevant key inflammatory markers of interest, to cross-check with other results.

The sectioning should require a professional and trained lab technician, not to create artifacts, which hinder the analyses, i.e. Figure 6A and 6B showed poor skills in sectioning and/or other treatments procedures.

Trichrome staining should photograph and record within a close period of time, the authors should stately clear in the methodology, whether the micrographs were captured within the standard periods.

The authors should provide rationales why they choose day 3 and day 7 for the wound assessment.  

"JL holds patents and is affiliated with and has financial interest in the company that designs/manufactures/sells the material used in this study." The author JL should justify and clarify the amount and types of financial support (in proportion to this research work), of the company.

Methodology of the assay for TIMP-1 and TIMP-2 were missing in this article.

Comments on the Quality of English Language

Moderate level of english checking is required.

Author Response

We appreciate the time and effort the reviewer has put into our manuscript. 

Methodology of how the "POSS gel polymer" was made should be supplemented. The authors should clarify why saline was used as the negative control, and whether any comparable vehicle control from the vehicle base making POSS, should be used as negative control instead.
Added We thank the reviewer. In assessing the reviewers comments we does not appear that we did a through job in defining the objective which was to determine the effects of using a hemostatic agent in the wound and the outcome in the acute phase of healing.
Normal sterile saline is regarded as the most appropriate and preferred solution to be placed into the wound because it is a nontoxic, isotonic solution that does not damage healing tissues.
Positive control of wound healing agent is missing in this research work. The authors should justify why this is missing
The goal was to show that if the hemostat POSS if left in a wound bed would not be detrimental to wound healing. We were not trying to show that POSS was superior to an antibiotic gel that would be placed in the wound.
In situ zymography (and/or the functional enzymatic based assay) of the interested MMPx markers should be performed to truely assess the MMPx/TIMPx activities.
We agree that Zymography is a great method to study extracellular matrix-degrading enzymes, such as matrix metalloproteinases (MMPs), in tissue extracts. We understand that only the active forms of MMPs, and not the proMMPs or TIMP-MMP complexes are capable of catalysis. Thus, only the active MMPs can play roles in physiology or disease pathology. Zymography would allow for enzyme-based degradation of the substrates and analyze their enzymatic activities. In zymography, MMP activity is visualized by substrate conversion and detection of the reaction product or disappearance of the substrate.The most widely used method is gelatin zymography, which detects the gelatinases MMP-2 and MMP-9. This method discriminates proMMPs, however it does distinguish between active unregulated MMPs and TIMP-complexed MMPs because under the denaturing conditions, the TIMP-MMP non-covalent complexes dissociate and appear as active MMP bands. In addition, a recent publication by Mayland Chang (2023) (Matrix metalloproteinase profiling and their roles in disease RSC Adv. 2023 Feb 14; 13(9): 6304–6316.. doi: 10.1039/d2ra07005g) suggest that additional proteomic and immunoprecipitation techniques would also be needed. Also, because this is not a disease but normal tissue healing, we believe that reporting concentrations by using ELISA are just as reasonable.
As for "MMP-1, MMP-2, MMP-3, and MMP13", the authors should clarify whether the Elisa were measuring both the pro-form and/or active forms of the captioned MMPx, and whether the % specificity covered swine? Dynamic range of the validated concentration of each analytes should be provided, together with which validated method the manufacturer were based.
Added
We agree that we should have provided more information in the methods section to address the sensitivity of the assays and the species cross reactivity.
In histological grading, the authors should clarify and supplement the experience and qualifications required for the examiners.
Triage system of the situation, where discrepant scoring from the examiners was found, should be in place and specified in the methodology.
More information for this was placed within the methods section.
In Figure 3 to 5 and figure 8, the authors should state whether the comparison found as statistical significance, or stating NS (not significant) in the figues.
ADDED
As for Figure 6, the authors should clarify the distance of the scale bar.
We have updated all pictures with scale bars at the same magnifications
The authors may consider to supplement IHC slide for staining the relevant key inflammatory markers of interest, to cross-check with other results.
Thank you for the suggestion. We have submitted a second grant with additives to the POSS material and will definitely use the suggestion in that study
The sectioning should require a professional and trained lab technician, not to create artifacts, which hinder the analyses, i.e. Figure 6A and 6B showed poor skills in sectioning and/or other treatments procedures.
The biopsies were sent to our University’s core histology center and were processed, sectioned, and stained by a certified histotechnologist and not processed in our lab. I think that the reviewer may have misinterpreted the slide. The slide contains both normal and injured tissue response. We have provided better pictures of the similar regions to make this clear
Trichrome staining should photograph and record within a close period of time, the authors should stately clear in the methodology, whether the micrographs were captured within the standard periods.
This has been updated in the methods section along with H&E staining
The authors should provide rationales why they choose day 3 and day 7 for the wound assessment.
We want to thank the reviewer for this comment. We have added the following comment
According to the literature, full thickness wounds in pigs are approximately 50% closed within 8 days and three distinct phases of wound healing: the inflammatory phase with a slight reduction in wound size, significant wound closure after day 7, and re-epithelialization for wound coverage with a slow slope (Kuo, 2022). that can be compared for differences between treatment groups.
Kuo, TY., Huang, CC., Shieh, SJ. et al. Skin wound healing assessment via an optimized wound array model in miniature pigs. Sci Rep 12, 445 (2022). https://doi.org/10.1038/s41598-021-03855-y
"JL holds patents and is affiliated with and has financial interest in the company that designs/manufactures/sells the material used in this study." The author JL should justify and clarify the amount and types of financial support (in proportion to this research work), of the company.
J.L. is the PI of the grant and the funding for the project was supported by the grant funding. J.L. was involved in the planning phase and experimental phase, but was not involved in the data analysis phase.
Methodology of the assay for TIMP-1 and TIMP-2 were missing in this article.
Added
Comments on the Quality of English Language
Moderate level of English checking is required. The draft was reviewed a second time for grammar structure 

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript by Tucci and colleagues describes in vivo studies using a visco-liquid polymer containing polyhedral oligomeric silsesquioxane (POSS) applied to a wound.  The goal of this study is to show that this material hemostat has improved healing as well as expression of metalloproteinases and inhibitors of metalloproteinases in these treated wounds compared to a saline control.  The authors conclude that the POSS treated wounds had improved healing, i.e., better keratinocyte migration but all other parameters assessed were marginal or not statistically significant.  Overall, this manuscript has some redeeming aspects and adds to the literature, but their conclusions are only marginally supported by their data for an improved hemostatic gel.  There are concerns regarding the lack of information on the polymer, experimental design, controls used and the histologic images, which are unclear and not entirely supportive of the conclusions offered.   This manuscript needs major revisions. 

Specific Comments:

1.      Although this is now being seen more commonly in the literature (albeit, quite incorrect in its usage), please do not start sentences with abbreviations or numbers, for example page 1, line 27, “MMP-13…”, page 5, line 176, “MMP 1…”, page 6, line 198, “TIMP-1…” and others, please check all in the manuscript and correct all.  Page 2, line 67-68 has some words missing or unclear meaning, “…high…?” and “...environment, long…?” 

2.      There are significant concerns regarding Materials and Methods as well as experimental design.  First, there is nothing in the Methods section about the polymer chemistry, how it is prepared and in what solution, from what source, concentrations used, rationale, etc.  The use of a saline control may not be appropriate since the “gel” without POSS should be the appropriate control, again what is the gel?  Secondly, a positive control material should be used, there are many gels on the market for comparison, how do the authors know that their gel is comparable?  Third, the number of animals used is three, which is a small number and the authors have only looked at brief time points, three and seven days, so the study is not comprehensive and needs to have a longer time point for appropriate assessment of the healing response.

3.      There are significant concerns regarding the data and the conclusions offered in this manuscript.  Most of the data is not significant or marginally significant for wound changes (Figures 1 and 2), Decorin (Figure 3), MMPs (Figure 4) and TIMPs (Figure 5) levels. The data shown in Figure 6, the histologic images with H&E, are not of the same magnifications when comparing Figures 6A vs. 6B and the details of these images are poorly described in the Results section on page 6.  The figures are not clearly labelled in each as these photographs, as there are three to the right of the low magnification image and it is not clear where they were derived from.  Figure 6C does not show any foreign body giant cells and the blue arrow appears to point to a blood vessel, not an encapsulation. Is this an area of the hypodermis?

4.      Figure 7 images are not of the same magnification for controls vs. the test material.  The speculation on Page 7, lines 232-234, that there is “…more ordered mature collagen…” is not proven by using a Trichrome stain.  Use of Picrosirius Red staining with polarizing light could show differences between Type I and Type III collagen or use of immunostaining would be more confirmative. 

5.      There are many hemostatic and wound healing gels, hydrogels, chitosan gels, nanoparticle gels that have been used and reported in the literature, even recent studies on POSS gels.  The authors provide little information in the Discussion section on POSS and its use as a wound gel or for other applications, especially recent work and few references are included in this manuscript.   

Comments on the Quality of English Language

Minor comments are indicated in reviewer Comment #1

Author Response

We thank the reviewer for the effort and time dedicated to our manuscript.

Although this is now being seen more commonly in the literature (albeit, quite incorrect in its usage), please do not start sentences with abbreviations or numbers, for example page 1, line 27, “MMP-13…”, page 5, line 176, “MMP 1…”, page 6, line 198, “TIMP-1…” and others, please check all in the manuscript and correct all. Page 2, line 67-68 has some words missing or unclear meaning, “…high…?” and “...environment, long…?”
We have made adjustments as suggested.
2. There are significant concerns regarding Materials and Methods as well as experimental design. First, there is nothing in the Methods section about the polymer chemistry, how it is prepared and in what solution, from what source, concentrations used, rationale, etc. The use of a saline control may not be appropriate since the “gel” without POSS should be the appropriate control, again what is the gel? Secondly, a positive control material should be used, there are many gels on the market for comparison, how do the authors know that their gel is comparable? Third, the number of animals used is three, which is a small number and the authors have only looked at brief time points, three and seven days, so the study is not comprehensive and needs to have a longer time point for appropriate assessment of the healing response.
WE have made significant changes in the manuscript and methods to try to get the point across of evaluating a hemostat in a wound bed and evaluating changes that can occur in the acute phase of healing
3. There are significant concerns regarding the data and the conclusions offered in this manuscript. Most of the data is not significant or marginally significant for wound changes (Figures 1 and 2), Decorin (Figure 3), MMPs (Figure 4) and TIMPs (Figure 5) levels. The data shown in Figure 6, the histologic images with H&E, are not of the same magnifications when comparing Figures 6A vs. 6B and the details of these images are poorly described in the Results section on page 6. The figures are not clearly labelled in each as these photographs, as there are three to the right of the low magnification image and it is not clear where they were derived from. Figure 6C does not show any foreign body giant cells and the blue arrow appears to point to a blood vessel, not an encapsulation. Is this an area of the hypodermis?
We appreciate the comments and have made an effort in this draft to make sure that all images are explained more thoroughly.
4. Figure 7 images are not of the same magnification for controls vs. the test material. The speculation on Page 7, lines 232-234, that there is “…more ordered mature collagen…” is not proven by using a Trichrome stain. Use of Picrosirius Red staining with polarizing light could show differences between Type I and Type III collagen or use of immunostaining would be more confirmative.
We agree with the reviewer and have removed “more ordered collagen” We also have provided better images for clarity and comparison
5. There are many hemostatic and wound healing gels, hydrogels, chitosan gels, nanoparticle gels that have been used and reported in the literature, even recent studies on POSS gels. The authors provide little information in the Discussion section on POSS and its use as a wound gel or for other applications, especially recent work and few references are included in this manuscript.
Our primary goal was to assess if the hemostatic POSS if left in a wound bed would deter the healing response. What we found was that it improved healing. The material was placed once within the wound bed and the wounds were followed over time. We have made this concept clear in the manuscript.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

This is a revised manuscript and the authors have made appreciable changes, added new content and have improved this manuscript.  The data is still rather marginal ad not impressive that is why it is rated for low originality/novelty as well as low overall merit.  However it does add to the literature in this field, so it is acceptable for publication.