Bioactive Factors Isolated and Purified from Bovine Colostrum Can Restore Extracellular Matrix Under Degradation by Metalloproteinases

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this work, the authors tested the effects of a mixture of biomolecules extracted and purified from cow colostrum on restoring the mechanical properties of the ECM. The authors claimed that collagen contraction significantly improved in samples treated with a 2% colostrum mixture compared to untreated ones. Further analysis revealed that the treatment reduced the expression of a specific enzyme (metalloproteinase-2) and increased collagen production by fibroblasts. Finally, the authors concluded that these molecules from cow colostrum help restore the ECM’s structure and function, aiding wound healing by activating specific cell signals through integrins. It is a good effort by the authors to address this issue, but the current set of results presented in this paper is insufficient to fully support these claims. The authors need to repeat most of their experiments with proper replicates and provide more detailed explanations. Even though this is a communication, some additional explanation is still required. 

Please find detailed comments below:

1. 'The aim of our research is to demonstrate that bioactive factors purified from bovine colostrum are like molecules normally present in the extracellular matrix.' Since the ECM is complex and consists of many factors, the authors should conduct a thorough study or cite standard papers that claim bovine colostrum contains similar factors to those in the ECM.

2. 'In all experiments, we used a 2% colostrum derivative mixture because, in our unpublished data, we have seen that this corresponds to the concentration of growth factors and cytokines normally present in the extracellular matrix.' In this case, please provide sufficient experimental evidence to support this claim.

3. 'The Human Dermal Fibroblasts (HDF) (106-05A, Merck) were grown in Dulbecco’s Modified Eagle Medium (DMEM). The medium was supplemented with 10% fetal bovine serum. The medium was replaced every 2 days, starting when the cells reached 80% confluence. Cultures were maintained at 37°C in a humidified environment containing 5% CO2.' Please explain why the authors decided not to use antibiotics in their culture medium and how they maintained aseptic conditions without them.

4. The authors have cited this paper (PMID: 12601036) for the gel contraction assay, but it would be better to explain how they conducted the assay in their own setting and how they analyzed the data. This will improve readability for the audience.

5. In all experimental methods, the authors have not mentioned any details about replicates or how they chose those replicates. It is crucial to include this information in the methods section under each experimental method. If the authors used any kits, it is important to mention the kit details and catalog numbers.

6. Table 1 is poorly represented. Please provide more details about the experiment, such as the type of ELISA, sensitivity, antibody dilution, assay conditions, signal detection method, analysis, false positives, etc.

7. Figures 1, 2, and 3 legends lack details like the number of replicates per experiment and standard deviations. Without these details, it is difficult to interpret the results and draw any conclusions. Additionally, there is no detailed explanation/rationale provided for why these experiments were conducted.

8. Figure 4 lacks figure legend.

9. '9. Our hypothesis is that the mol-20 ecules extracted and purified from bovine colostrum restore the ECM environment qualitative and quantitative characteristics, thus guaranteeing through a mechanical action the restoration of the wound due to the transduction of the signal activated by the integrins. Authors need to explain their hypothesis in simple scientific terms and please avoid using terms like "guaranteeing". 

Comments on the Quality of English Language

Writing can be improved. Authors need to be careful while using certain words. I have highlighted them in my comments. It is always good to use scientific terms and direct words in scientific writing.

Author Response

Dear Editor,

we have responded to all your requests and comments. We hope that you now find our manuscript suitable for publication.

    All Authors

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The mechanism of wound healing depends on the mechanobiology behind the fibroblast-mediated ECM remodeling process during the inflammatory phase of tissue regeneration. In this study, the authors postulated that bioactive molecules from bovine colostrum can be used to help restore structure and function of damaged tissues. To test this hypothesis, the authors conducted a series of experiments to determine the collagen contraction after CDM treatment along with the quantification of MMP-2. The manuscript can be improved by addressing the following limitations.

• What is the motivation behind purifying bioactive factors from bovine colostrum? In other words, why was bovine colostrum chosen as the source of bioactive factors used in this study? Since bioactive factors from bovine colostrum are chosen in this study and are used to inform the experiments, it’s necessary to consider how to apply this information to human data. 
• Please carefully review the manuscript to correct any grammatical or syntax issues. For example, In lines 36-37, the sentence: “The ECM is a dynamic structure, constantly undergoing a remodeling process, an important mechanism whereby cell differentiation can be regulated wound repair.” The authors can correct the grammatical error in this sentence. The sentence should be: “The ECM is a dynamic structure, which constantly undergoes remodeling, an important mechanism whereby cell differentiation regulates wound repair.” Another example is sentence in line 127 that states: “But on cells can act mechanical forces exerted from ECM if as tissues are deformed, such as in wounds.” 
• It is not too clear on how the collagen contraction test was done from Section 2.3 of the manuscript. If would be beneficial to the readers if the authors were to include more information and additional details on how the test was done in addition to citing the previous literature.
• Why was 2% CDM chosen as the treatment condition? What happens if the CDM composition is less than 2%? What happens if the CDM composition is more than 2%?

• The authors state that the treatment of bioactive molecules derived from CDM help restore the chemical/physical ECM environment along with the anisotropic characteristics of the damaged tissue. However, more experiments need to be done in order to verify this. For example, this manuscript can be improved by the inclusion of immunostaining data of ECM markers and F-actin to showcase the anistropic characteristics of damaged tissue after CDM treatment. In addition, it would be beneficial for the authors to include an in-vitro wound healing model (since the paper focuses on the utilization of bioactive molecules for wound healing purposes), such as performing a scraping test and looking into the migration of fibroblasts before and after CDM treatment. 
• Does the treatment of CDM increases fibroblast proliferation compared to the control? What does control fibroblasts mean in this study? Does it mean that the fibroblasts are not treated with CDM?

Comments on the Quality of English Language

Minor corrections in terms of grammar and sentence structure can be made.

Author Response

Dear Editor,

we have responded to all your requests and comments. We hope that you now find our manuscript suitable for publication.

    All Authors

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

In this work, the effects of bovine colostrum on collagen production, collagen contraction, and MMP-2 levels were analyzed. The idea and motivation of the study was good, but the manuscript needs further improvements. Please address the comments below:

1.     Abstract: “Our hypothesis is that the molecules extracted and purified from bovine colostrum restore the ECM environment qualitative and quantitative characteristics…”. This statement needs refining to delineate the qualitative and quantitative characteristics of ECM. The authors could provide some examples of these characteristics of collagen.

2.     The authors mentioned “collagen gel” in Figure 1 caption. Other than this, there is no mention of collagen gel in the manuscript. On which surface were the fibroblasts cultured? Were they seeded on a collagen gel or on tissue culture plate? How much was the gel thickness? What was its concentration? How many cells were seeded? How did they treat the fibroblasts with 2% colostrum derivative mixture? How were the cells harvested back from the collagen matrix? Please mention the experimental methods in detail.

3.     The authors need to have a negative control without cells and with colostrum for collagen production test. How did they delineate the collagen generated by the cells from the collagen matrix used?

4.     The authors need to provide a better explanation for Figure 4. Please give reference for negative correlation between MMP-2 and collagen contraction as shown here.

5.     The two pages after Figure 4 only discuss the literature, there is no discussion with respect to the results reported here. Please discuss only relevant literature and connect the discussion with the results found in this work.

 

6.     The hypothesis given by the authors is too bold. They are measuring collagen production, MMP-2 levels, and collagen contraction and these can only indicate that wound healing might be better. They cannot guarantee that.

Comments on the Quality of English Language

ok

Author Response

Dear Editor,

we have responded to all your requests and comments. We hope that you now find our manuscript suitable for publication.

    All Authors

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors The authors have a significant effort to improve the quality of this work, but there are few more points authors needs to work on or provide enough explanations

Please find details comments below 

Major issue:   1. Authors replied that "All the information requested is not part of this work, but we are publishing it (the paper is currently under review in the journal Growth Factors, Taylor & Francis), but I can provide the link to the pre-prints: https://www.preprints.org/manuscript/202307.1291/v1"   My concern is that this table is still under review in another journal. I recommend that the authors perform these experiments for this paper separately.

2. For collagen contraction assay and for scratch wound assay, authors must include all the representatives images in the manuscript.   3. Authors claimed "In this paper, we demonstrated that biomolecules extracted and purified of bovine colostrum can restore chemical/physical ECM environment, reestablish the mechanical properties of ECM and the anisotropy characteristics of the damaged tissue. This process activates transduction of the signal by the integrins and reactivates the ability of the fibroblast to synthesize new collagen, inducing the autologous mechanisms of repair and remodeling of damaged connective tissue"   The experimental evidence suggests reduced Pro-MMP2 expression (Figure 3), increased collagen deposition (Figure 2), and increased gel contraction (Figure 1B).

- My concern is why the authors have not checked MT1-MMP or other collagenases.
- Can the authors comment on which integrins are involved in the conclusion section?   Minor issue:   - Please readjust the scales for all the figures so that error bars are visible to the authors.  Comments on the Quality of English Language

Moderate editing is required.

Author Response

The authors have a significant effort to improve the quality of this work, but there are few more points authors needs to work on or provide enough explanations

Please find details comments below 

Major issue:   1. Authors replied that "All the information requested is not part of this work, but we are publishing it (the paper is currently under review in the journal Growth Factors, Taylor & Francis), but I can provide the link to the pre-prints: https://www.preprints.org/manuscript/202307.1291/v1"   My concern is that this table is still under review in another journal. I recommend that the authors perform these experiments for this paper separately.

 

The manuscript has already had excellent reviews and will therefore be published shortly. In any case, the analyzes requested were not part of this research.

2. For collagen contraction assay and for scratch wound assay, authors must include all the representatives images in the manuscript.  

 

Unfortunately we don't have pictures

 

3. Authors claimed "In this paper, we demonstrated that biomolecules extracted and purified of bovine colostrum can restore chemical/physical ECM environment, reestablish the mechanical properties of ECM and the anisotropy characteristics of the damaged tissue. This process activates transduction of the signal by the integrins and reactivates the ability of the fibroblast to synthesize new collagen, inducing the autologous mechanisms of repair and remodeling of damaged connective tissue"   The experimental evidence suggests reduced Pro-MMP2 expression (Figure 3), increased collagen deposition (Figure 2), and increased gel contraction (Figure 1B).
   - My concern is why the authors have not checked MT1-MMP or other collagenases.
   - Can the authors comment on which integrins are involved in the conclusion section?  

 

These analyzes were not foreseen in our research. Perhaps in the future we could extend our analysis to these factors too.

 

Minor issue:   - Please readjust the scales for all the figures so that error bars are visible to the authors. 

DONE

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors addressed my comments and have edited the manuscript accordingly. I have no further comments. 

Author Response

The authors addressed my comments and have edited the manuscript accordingly. I have no further comments. 

We thank the reviewer for the valuable suggestions that allowed us to improve our manuscript

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors addressed the comments, and the manuscript is recommended for publication. But I request the authors to include the images that they obtained with optical microscope for area measurement for the Scratch Assay in the supplementary data.

In addition, I checked the iThenticate Report and I suggest re-phrasing of sections 2.2, 2.3, 2.4, and 2.5. There is a lot of overlap in section 3, I would say the authors should rephrase that as well including Figure 1 caption. 

Author Response

The authors addressed the comments, and the manuscript is recommended for publication. But I request the authors to include the images that they obtained with optical microscope for area measurement for the Scratch Assay in the supplementary data.

We thank the reviewer for the valuable suggestions that allowed us to improve our manuscript

In addition, I checked the iThenticate Report and I suggest re-phrasing of sections 2.2, 2.3, 2.4, and 2.5. There is a lot of overlap in section 3, I would say the authors should rephrase that as well including Figure 1 caption. 

As required, we have enriched the content, added additional figures  and eliminated the similarity rate of this article.

 

Author Response File: Author Response.pdf