PONE-D-24-00754In vitro experimental conditions and tools can influence the safety and biocompatibility results of antimicrobial electrospun biomaterials for wound healingPLOS ONE

Dear Dr. Kogermann,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================Reviewer 1:This is a review of PONE-D-24-00754, a manuscript comparing in vitro cytotoxicity testing methods of electrospun polycaprolactone and polyethylene oxide membranes with chloramphenicol. This is a very well described, technically sound article describing different cytotoxicity testing methods. This manuscript is useful for those performing similar cytotoxicity testing as it describes a clear framework for thinking through these important in vitro experiments. I support the publication of this paper after the following comments are addressed.

Abstract

I would edit the sentence containing “true biocompatibility” to mention in vitro as one could argue that “true biocompatibility” requires animal testing.

Intro

This is only a suggestion, but the first paragraph could be shortened and collapsed into the second paragraph. It is likely that the readers of this manuscript have relevant background in biomaterials to not need the definition of biomaterials or drug delivery systems.

In the fourth paragraph, I would mention the purpose of the microscopy-based methods (LIVE/DEAD, etc) to give better context.

Methods

Information should be added about how the samples were or were not sanitized/sterilized.

Any washing of the samples after preparation, considering the solvents used, should be mentioned.

The authors should mention why these two particular cells were selected.

Results and Discussion

The figures are well-constructed and clear, congrats to the authors. However, Figures 6 and 7 should say “conjugated” – typo.

This is only a suggestion, but the authors could add information to their discussion about destructive vs. non-destructive testing methods as it relates to cytotoxicity testing.

The author should mention the degradation kinetics of these membranes as it relates to leaching monomers, etc. and differences herein between direct vs. indirect.

Another topic, only a suggestion, is discussion of differences between MTS, MTT, resazurin, etc.

Conclusion

Satisfactory, thank you authors. Reviewer 2: The manuscript titled “in vitro experimental conditions and tools can influence the safety and biocompatibility results of antimicrobial electrospun biomaterials for wound healing” is well written and highlights the necessity of investigating biocompatibility using different cell types. This is important not only for electrospun fibers but also for other nanomaterials and biomaterials for biological and biomedical applications. The manuscript does need major revision prior to acceptance.

1. The title says “antimicrobial electrospun biomaterials” while discussing the cytotoxicity of PCL/PEO electrospun fiber pristine and loaded with Chloramphenicol for wound healing applications but no antimicrobial or wound healing assessments were performed.

Introduction:

1. Line 36, “various novel biomaterials” include some examples and potential applications.

2. Line 38, What does “controversial reports can be found” mean?

3. Line 42, “The major mutual characteristic to be termed as a …………. Should be prepared for a specific purpose 1-4”, the statement is complex. Please simplify the statement.

4. Line 47, “a high surface area for efficient drug release”, high surface area provide high drug or molecule loading capacity not the release.

5. Line 52, “Providing enough oxygen and moisture to …” this does provide optimal conditions for bacterial growth as well.

6. Line 69, “with test material” not tested material.

7. Line 75-78, “after an exhaustive literature …….. (MTT, MTS) has been used.” That is true. But MTT/MTS are used for cell metabolic activity assessment and when standardized with cell count supports cell viability/toxicity assessment as well. But in general, MTT/MTS assays are used in conjunction with fluorescence or other biological assessment procedures.

Results:

1. Line 99-104, “SEM images and size analysis revealed ……….” 0.94 ± 1.56 micron for pristine and 1.03 ± 1.30 micron for CAM loaded ES fiber. That is a big standard deviation as big or bigger than measured average diameter suggesting non homogenous diameter as reported by authors. Based on this statement, how the authors can rely on their biological analysis. This large variation in ES fiber diameter questions all analyses and investigations.

2. This variation in diameter can significantly alter the physical and mechanical properties of ES fibers, not reported in manuscript.

3. Line 106, raw material’s batch variability has been reported and known but the trend can be investigated or speculated with couple of batches.

4. Line 162, as both the cell types have different size and morphology, the confluency in the well or on well surface will be different with time even after same seeding cell density because cell might have different proliferation rate.

5. Line 187, if the authors believed the different passage number can affect the behavior which is well known for primary cells. The authors should have repeated with study with same P number.

6. Line 226, metabolic activity can be a measure of comparable number cells but more metabolically active or more cells with lesser metabolic activity based on if cells like the surface or not.

7. Line 242, ES fibers are known to be porous which can adsorb MTS reagent leading to false signal.

8. Line 248, with different cells size and non-uniform ES fiber diameters, the cell distribution over the ES Fiber mat will be irregular making the any type of biological assessment a big challenge.

9. Line 253, “a fold increase in MTS activity” this is not clear.

10. Line 285, the ES fiber mat may not be cytotoxic but the biocompatibility or likeability for fiber mat for both cell types might be different, leading to modulating cell attachment, cell morphology, and or cell metabolic activity. Also, as reported by authors the ES fiber diameter was not homogenous which can tailor the ES fiber or fiber mat interaction with cells.

11. Line 300, what are different physical, mechanical, and chemical characterization techniques. Authors should have included the physical, mechanical, and chemical assessment for ES fiber pristine and CAM loaded.

12. Line 302, the biological investigation is not limited to toxicity/cytotoxicity and inflammation assay. Assays such as ROS regeneration, DNA damage, DNA/cell number quantification, proliferation assay, attachment assay, different pathway studies are equally important.

13. How about the degradation rate or degradability of ES PEO/PCL fiber with or without CAM.

Conclusion:

The ES fiber under investigation are suggested to be antimicrobial so, the antimicrobial property should be assessed with at least some of the bacterial species causing wound infection such as Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecalis, and Acinetobacter baumannii. Please submit your revised manuscript by May 04 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Isha Mutreja

Academic Editor

PLOS ONE

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Estonian Research Council grant number PRG1507 (Karin Kogermann)

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a review of PONE-D-24-00754, a manuscript comparing in vitro cytotoxicity testing methods of electrospun polycaprolactone and polyethylene oxide membranes with chloramphenicol. This is a very well described, technically sound article describing different cytotoxicity testing methods. This manuscript is useful for those performing similar cytotoxicity testing as it describes a clear framework for thinking through these important in vitro experiments. I support the publication of this paper after the following quite minor comments are addressed.

Abstract

I would edit the sentence containing “true biocompatibility” to mention in vitro as one could argue that “true biocompatibility” requires animal testing.

Intro

This is only a suggestion, but the first paragraph could be shortened and collapsed into the second paragraph. It is likely that the readers of this manuscript have relevant background in biomaterials to not need the definition of biomaterials or drug delivery systems.

In the fourth paragraph, I would mention the purpose of the microscopy-based methods (LIVE/DEAD, etc) to give better context.

Methods

Information should be added about how the samples were or were not sanitized/sterilized.

Any washing of the samples after preparation, considering the solvents used, should be mentioned.

The authors should mention why these two particular cells were selected.

Results and Discussion

The figures are well-constructed and clear, congrats to the authors. However, Figures 6 and 7 should say “conjugated” – typo.

This is only a suggestion, but the authors could add information to their discussion about destructive vs. non-destructive testing methods as it relates to cytotoxicity testing.

The author should mention the degradation kinetics of these membranes as it relates to leaching monomers, etc. and differences herein between direct vs. indirect.

Another topic, only a suggestion, is discussion of differences between MTS, MTT, resazurin, etc.

Conclusion

Satisfactory, thank you authors.

Reviewer #2: The manuscript titled “in vitro experimental conditions and tools can influence the safety and biocompatibility results of antimicrobial electrospun biomaterials for wound healing” is well written and highlights the necessity of investigating biocompatibility using different cell types. This is important not only for electrospun fibers but also for other nanomaterials and biomaterials for biological and biomedical applications. The manuscript does need major revision prior to acceptance.

1. The title says “antimicrobial electrospun biomaterials” while discussing the cytotoxicity of PCL/PEO electrospun fiber pristine and loaded with Chloramphenicol for wound healing applications but no antimicrobial or wound healing assessments were performed.

Introduction:

1. Line 36, “various novel biomaterials” include some examples and potential applications.

2. Line 38, What does “controversial reports can be found” mean?

3. Line 42, “The major mutual characteristic to be termed as a …………. Should be prepared for a specific purpose 1-4”, the statement is complex. Please simplify the statement.

4. Line 47, “a high surface area for efficient drug release”, high surface area provide high drug or molecule loading capacity not the release.

5. Line 52, “Providing enough oxygen and moisture to …” this does provide optimal conditions for bacterial growth as well.

6. Line 69, “with test material” not tested material.

7. Line 75-78, “after an exhaustive literature …….. (MTT, MTS) has been used.” That is true. But MTT/MTS are used for cell metabolic activity assessment and when standardized with cell count supports cell viability/toxicity assessment as well. But in general, MTT/MTS assays are used in conjunction with fluorescence or other biological assessment procedures.

Results:

1. Line 99-104, “SEM images and size analysis revealed ……….” 0.94 ± 1.56 micron for pristine and 1.03 ± 1.30 micron for CAM loaded ES fiber. That is a big standard deviation as big or bigger than measured average diameter suggesting non homogenous diameter as reported by authors. Based on this statement, how the authors can rely on their biological analysis. This large variation in ES fiber diameter questions all analyses and investigations.

2. This variation in diameter can significantly alter the physical and mechanical properties of ES fibers, not reported in manuscript.

3. Line 106, raw material’s batch variability has been reported and known but the trend can be investigated or speculated with couple of batches.

4. Line 162, as both the cell types have different size and morphology, the confluency in the well or on well surface will be different with time even after same seeding cell density because cell might have different proliferation rate.

5. Line 187, if the authors believed the different passage number can affect the behavior which is well known for primary cells. The authors should have repeated with study with same P number.

6. Line 226, metabolic activity can be a measure of comparable number cells but more metabolically active or more cells with lesser metabolic activity based on if cells like the surface or not.

7. Line 242, ES fibers are known to be porous which can adsorb MTS reagent leading to false signal.

8. Line 248, with different cells size and non-uniform ES fiber diameters, the cell distribution over the ES Fiber mat will be irregular making the any type of biological assessment a big challenge.

9. Line 253, “a fold increase in MTS activity” this is not clear.

10. Line 285, the ES fiber mat may not be cytotoxic but the biocompatibility or likeability for fiber mat for both cell types might be different, leading to modulating cell attachment, cell morphology, and or cell metabolic activity. Also, as reported by authors the ES fiber diameter was not homogenous which can tailor the ES fiber or fiber mat interaction with cells.

11. Line 300, what are different physical, mechanical, and chemical characterization techniques. Authors should have included the physical, mechanical, and chemical assessment for ES fiber pristine and CAM loaded.

12. Line 302, the biological investigation is not limited to toxicity/cytotoxicity and inflammation assay. Assays such as ROS regeneration, DNA damage, DNA/cell number quantification, proliferation assay, attachment assay, different pathway studies are equally important.

13. How about the degradation rate or degradability of ES PEO/PCL fiber with or without CAM.

Conclusion:

The ES fiber under investigation are suggested to be antimicrobial so, the antimicrobial property should be assessed with at least some of the bacterial species causing wound infection such as Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecalis, and Acinetobacter baumannii.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

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In vitro experimental conditions and tools can influence the safety and biocompatibility results of antimicrobial electrospun biomaterials for wound healing

PONE-D-24-00754R1

Dear Dr. Kogermann,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Isha Mutreja

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have responded in details to my questions and suggestions for improvement. I have gone through in detail the response to me and have skimmed the other reviewer response. I have no further comments and want to to congratulate the authors on a very clear and technically sound paper.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

**********