Cytotoxicity and UV Light Absorption in Biopolymeric Membranes from Native Vegetation of Mexico

Round 1

Reviewer 1 Report

This work is related with natural materials intended in future as biomaterials. However in my opinion work must be corrected. Here are some important, in my opinion, problems related with this article:

• The introduction is not focused on the topic of the work. It must be reconstructed. It must be shorter. In current form it (first 168 lines) contains elementary, school-grade information that does not really introduce the reader into the issue. The style is also pure.
• The aim is not well-constructed, because authors suggest that materials may be used as “ the sustenance for a possible application of the materials as dosing controllers, mainly with agents that can be related to tissue regeneration. “ If these are the authors' intentions, it will be good to mention this in the introduction, but probably not as the aim, because no real research in this direction was conducted. Interpretation of results also does not indicate such applications  to, in fact. This may be a far-reaching goal, but not the purpose of this particular job.
• There is no statistical analysis of the results. It should be performed, primarily for cytotoxicity tests and adsorption of ultraviolet radiation.
• ESEM results should be described in more details. “ESEM micrographs of the longitudinal sections of the LBMs show the adhesion” – I think that this sentence is not entirely accurate.
• Reading a discussion, I feel unsatisfied because there is little .... discussion. It would be nice to read a bit deeper consideration of the results obtained.
• Conclusions should be more focused on the results achieved in this work - they are too lengthy.

Author Response

Ms. Luksamon Pratumma
Assistant Editor

First of all, Thanks a lot. According to the final suggestions for the submission named:

Cytotoxicity and UV light absorption in biopolymeric membranes from native vegetation of Mexico

by Mayela García de Alva Magos, Jesús Santa-Olalla Tapia, Miguel Ángel Ramos López, Jessica Molina Maturano, Antonio Jiménez Aparicio, Brenda Camacho Díaz, Luz Arcelia García Serrano

We hope that the answers are agree with the ones requested by the journal, otherwise. We attended all the suggestion with yellow highlights in the adjunted document. I have tried respond soon as posible. Please feel free to contact me.

1.Query

The introduction does not focus on the topic of work. It must be rebuilt. It should be shorter. In its current form, (first 168 lines) contains primary, grade-level information that does not really introduce the reader to the topic. The style is also pure. The objective is not well constructed, because the authors suggest that the materials can be used as "the support for a possible application of the materials as dosage controllers, mainly with agents that may be related to tissue regeneration." If these are the Intentions of the authors, it will be good to mention this in the introduction, but probably not as the goal, because no real research was done in this direction. Interpretation of the results does not indicate such applications, in fact. This may be a goal of long-range, but not the purpose of this particular job.

Answer

The introduction was improved, restructured and summarized from line 34 to line 125 (page 1-3). It was done to framing the objective of the work.

2.Query

There is no statistical analysis of the results. It should be performed mainly for cytotoxicity tests and adsorption of ultraviolet radiation.

Answer

Statistical analysis was incorporated and discussed for cytotoxicity (lines 319-340) and ultraviolet radiation absorption (lines 404-411) tests.

3.Query

ESEM results should be described in more detail. "ESEM micrographs of longitudinal sections of LBMs show adhesion." I think this sentence is not entirely accurate. Reading a discussion, I am dissatisfied because there is little discussion. It would be good to read a little more in depth the results obtained.

Answer

ESEM results were described with more detail, eliminating phrase that was out of place (lines 474-497)

4.Query

The conclusions should focus more on the results achieved in this work: they are too long.

Answer

The conclusions was improved, restructured and summarized from 572-584.

Author Response File: Author Response.pdf

Reviewer 2 Report

Please see attached file.

Comments for author File: Comments.pdf

Author Response

June 26, 2020

Ms. Luksamon Pratumma
Assistant Editor

First of all, Thanks a lot. According to the final suggestions for the submission named:

Cytotoxicity and UV light absorption in biopolymeric membranes from native vegetation of Mexico

by Mayela García de Alva Magos, Jesús Santa-Olalla Tapia, Miguel Ángel Ramos López, Jessica Molina Maturano, Antonio Jiménez Aparicio, Brenda Camacho Díaz, Luz Arcelia García Serrano

We hope that the answers are agree with the ones requested by the journal, otherwise. We attended all the suggestion with yellow highlights in the adjunted document. I have tried respond soon as posible. Please feel free to contact me.

1.Query

Long and rather disjointed introduction, which does not clearly provide the motivation and context as well as the potential of the authors' approach.

Answer

The introduction was improved, restructured and summarized. It was highlighting the motivation and potential of the native vegetation as a source of functional ingredients, from line 34 to line 125 (page 1-3).

2.Query

Materials and Methods 2.1: Please provide full details of the process and / or appropriate citations so that the work can be reproduced.

Answer

It was added more details about of the methods used from lines128-140.

3.Query

Precision details such as: grinding was performed in an industrial blender for 3 minutes. (lines 141-149).

Answer

Precision details were added such as: that the material from which the mucilage was extracted was from the leaves, in addition to milling was carried out in an industrial blender for 3 minutes. (lines150-161).

Precision details such as the patent number and name of the method of extraction of agave fructans were added. Mention is made of the spray drying time was 24 hours, until the moisture content was 5% (lines 162-168).

5.Query

Why was the content of mucilage and fructans changed with the fiber content?

Ratio of the two components?

Answer

Precision details such as: that Aloe Barbadensis Miller was used due to its properties as a binder, amulifier and viscosity were added. It stands out that fructans were added for their technological functions: stabilizer, sweetener, humectant, gelling agent, among others. The warm-up was for 10 minutes. (lines 169-180)

6.Query

Materials and methods, 2.3.1: provide the appropriate cell names: normal primary dermal fibroblasts; Human, neonatal (HDFn) and complete information on the culture medium, such as serum content, antibiotics, etc. How were the samples sterilized?

Answer

The appropriate cell name was mentioned, the information about the culture medium was added. The sterilization method was described and clarified that the test carried out in triplicate (lines183-194).

7.Query

Materials and methods, 2.3.1: lines 253-5 this sentence seems out of place, presumably the cells were passed and then plated at 10k cells / cm2 in 24-well plates.

Answer

This sentence is deleted. 253-255

8.Query

Materials and methods, 2.3.1: lines 255 what does "MBL" mean? Fully describe TransWell inserts (brand, pore size). How were the samples sterilized?

Answer

Corrected abbreviation that was over: MBL

9.Query

Materials and methods, 2.3.2: This section needs to accurately describe the experiment: what did the cells adhere to: plastic material, with samples in cross-pits or in the samples? Transwells were mentioned in 2.3.1, but cell seeding was provided as 10k cells / well and culture for 4 days, here are 19k cells / well and 24 hours, which is correct? Was this an indirect contact trial?

Answer

Details were added on the adhesion of the membranes in indirect contact by the support of the transwell insert in the cell culture medium and of the development of this process (lines 198-207).

10.Query

Materials and methods, 2.3.4: What is the origin of equation 1? It provides strange results, for example, when the OD of the test is half of the controls (50% viability), the equation results in 0.02 CP. Furthermore, to facilitate interpretation and comparison with other data, it would be preferable to calculate feasibility, as a percentage of the control, as done in ISO10993. In addition, line 287 mentions fluorescence, when line 278 indicates that the assay was colorimetric. Please clarify (here and line 303 as well).

Answer

Added reference of the origin of equation 1 and it is changed by the calculation of the viability, as a percentage of the control, as it is done in ISO10993. In addition, the term fluorescence homogenization is done throughout the article (lines 227-229).

11.Query

Viability calculation

Answer

The % viability was calculated (lines 240-248).

12.Query

Materials and methods, 2.4.4: What was the material of the ATR prism? What was the resolution and number of scans averaged? How were the samples prepared, for example, dried, pressed, etc.

Answer

We Added the ATR prism material: Zinc and selenium, resolution and number of scans averaged. In addition to the tellades of the sample preparation, which were dried, sterilized and mounted on the sample plate of the team (lines 259-265).

13.Query

Materials and Methods, 2.4.6: Properly cite the ImageJ distribution used and provide full names and citations, as appropriate, of the supplements used, to allow reproducibility of analysis.

Answer

Changed quote from ImageJ software used and provide full names and quotes (lines 274-316).

14.Query

Results, 3.1: Fig. 2: the micrographs are so small that they are useless. Please provide larger images in Supplemental Information. The number of passages is irrelevant here, to be clear, the data presented represents the average of 3 separate experiments. And do the error bars indicate SD?

Answer

The graph of the images was separated, the size of the micrographs was changed, the number of passages was eliminated and it was clarified that the data presented represent the average of 3 evaluations, indicating that the error bars indicate the SD. The micrographs were changed to the qualitative part. The micrographs were separated from the graph, and linked with the micrographs and statistical analysis (lines 318-326).

15.Query

Results, 3.3: Fig. 4: What is FT and FTS? Presumably these are related to FC and FL previously used? What is "ST.A.B." and "ST Of-I"? The legend is rare.

Answer

Abbreviation correction was performed in Fig. 4  and legend is added in figure describing the elements (line 378).

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The article has been revised sufficiently to make it suitable for publication.

Author Response

Reviewer 1 

Thank you for your recommendations. We improve the errors in the writing of the introduction to highlight the objective of the paper.
We used yellow highlights to the changes in the text.


Author Response File: Author Response.pdf

Reviewer 2 Report

The Authors response does have not include answers/replies to comments regarding the Discussion, which is essentially unchanged. From the original review:

• Discussion, 4.1.2: lines 460-462 saying “all membranes … are acceptable according to ISO” is not really supported, because at least one sample was below the 70% viability threshold set by the ISO. Further without a positive control it cannot be certain that the assay was sufficiently sensitive (sample size, media volume, etc.). Finally, to really evaluate from the point of view of tissue engineering applications direct contact and or adhesion tests would be appropriate, so the authors should here instead underscore that this a preliminary in vitro screening.
• Discussion, 4.1.2: lines 467-471 these lines seem to discuss aspects relating to cell adhesion to the materials, while the methods and results seem to rather indicate an indirect contact assay. This makes this paragraph rather misleading. Please clarify.
• Discussion, 4.1.3: lines 481-482 again reference to direct contact, while the methods and results seem to rather indicate an indirect contact assay. This makes this paragraph rather misleading. Please clarify.
• Discussion, 4.1.3: lines 484 here it is stated that there was “appreciable cytotoxic effect” which does not square well with the statements in 4.1.2. I would recommend merging the discussion of both quantitative and qualitative observations together, since the results are from the same samples and assays—as I understand.
• Discussion 4.3: lines 510-514: this paragraph is unclear, how can these membranes cross cell membranes? Why would this even be desired? Why does the variety of functional groups “suggest their use … for human topical use”? Please clarify with citations.
• Discussion 4.4: In discussing the results of UV absorption some reference to the literature would be useful in order to help interpret the results—are they sufficient to offer protection to a wound while it is healing? (I presume this is the goal.) How do these materials compare to other wound dressings, eg. cellulose.
• Discussion 4.5: It would be interesting if the Authors’ could relate these measured parameters to their other results, for example one could expect that high roughness could reduce transmission of UV light via increased light scattering/reflection. Likewise, some context from the literature would be useful.

The Authors have not clarified the issue of a positive control, to ensure their cell culture study is sufficiently sensitive.

It's still not clear how the viability assay was performed. Lines 221-2 mention absorbance first, then fluorescence, while equation 1 uses OD a measure of absorbance. If it was a fluorescence assay, then please provide excitation and emission wavelengths. If it was an absorbance assay, please make this clear.

I am not able to locate the ImageJ plugins: "GCM Texture" and "DDBC". Please ensure the plugin names are correct and that the plugins are properly cited.

Author Response

First of all, thank you for your comments and suggestions. We hope that the answers were agree with your requests. We responded in the best possible way to all their suggestions. We used yellow highlights to the changes in the document.


1.Query

4.1.2: lines 460-462 saying “all membranes … are acceptable according to ISO” is not really supported, because at least one sample was below the 70% viability threshold set by the ISO. Further without a positive control it cannot be certain that the assay was sufficiently sensitive (sample size, media volume, etc.). Finally, to really evaluate from the point of view of tissue engineering applications direct contact and or adhesion tests would be appropriate, so the authors should here instead underscore that this a preliminary in vitro screening.

Answer

Lines 445-452

It was clarified that not all membranes were within viability, with samples declared to be below the 70% viability threshold established by ISO.

Lines 202-204

We highlight that there was a positive control group that consisted of cells in culture medium, with normal growth.

Lines 451-452

We declared that this a preliminary in vitro evaluation, as suggested.

2.Query

Discussion, 4.1.2: lines 467-471 these lines seem to discuss aspects relating to cell adhesion to the materials, while the methods and results seem to rather indicate an indirect contact assay. This makes this paragraph rather misleading. Please clarify.

Answer

Lines 469-473

It is clarified that the contact of the membrane and the culture medium was from the insert, that is indirect contact.

3.Query

Discussion, 4.1.3: lines 481-482 again reference to direct contact, while the methods and results seem to rather indicate an indirect contact assay. This makes this paragraph rather misleading. Please clarify.

Answer

Clarified that the contact of the membrane and the culture medium was from the insert, that is indirect contact.

4.Query

Discussion, 4.1.3: lines 484 here it is stated that there was “appreciable cytotoxic effect” which does not square well with the statements in 4.1.2. I would recommend merging the discussion of both quantitative and qualitative observations together, since the results are from the same samples and assays—as I understand.

Answer

Observation statements were unified

Line 445

The discussion of both quantitative and qualitative observations it was merged, as suggested.

5.Query

Discussion 4.3: lines 510-514: this paragraph is unclear, how can these membranes cross cell membranes? Why would this even be desired? Why does the variety of functional groups “suggest their use … for human topical use”? Please clarify with citations.

Answer

Lines 506—510

The paragraph regarding the crosslinking in the conformation of the membranes and why the variety of functional groups suggests as an alternative for human topical use was modified. References added.

6.Query

Discussion 4.4: In discussing the results of UV absorption some reference to the literature would be useful in order to help interpret the results are they sufficient to offer protection to a wound while it is healing? (I presume this is the goal.) How do these materials compare to other wound dressings, eg. cellulose.

Answer

Lines 547-551

Reference and interpretation of results was added.

7.Query

Discussion 4.5: It would be interesting if the Authors’ could relate these measured parameters to their other results, for example one could expect that high roughness could reduce transmission of UV light via increased light scattering/reflection. Likewise, some context from the literature would be useful.

Answer

Lines 567-573

The parameters were related to other results, reference from literature was added.

8.Query

It's still not clear how the viability assay was performed. Lines 221-2 mention absorbance first, then fluorescence, while equation 1 uses OD a measure of absorbance. If it was a fluorescence assay, then please provide excitation and emission wavelengths. If it was an absorbance assay, please make this clear.

Answer

Lines 221-223

The wording on the viability test by absorbance was modified.

9.Query
I am not able to locate the ImageJ plugins: "GCM Texture" and "DDBC". Please ensure the plugin names are correct and that the plugins are properly cited.

Answer

Lines 306-311

Fixed ImageJ plugin names and abbreviations

Author Response File: Author Response.pdf