Hair Pores Caused by Surfactants via the Cell Membrane Complex and a Prevention Strategy through the Use of Cuticle Sealing

Round 1

Reviewer 1 Report

Dear Authors,

I write you in regard to your manuscript entitled Hair Pores Caused by Surfactants via Cell Membrane Complex and Prevention Strategy through Cuticle Sealing. The issue worked on the text was interesting and it has merit to be investigated, however, it was not clear what would be the contribution to the field.

- Abstract was too confusing. Please, try write it in a manner to valorize the research and to allow the readers to understand what was done and the main findings.

- Introduction brought well-established data about hair and shaft properties. Please, try to update it with less known data to differentiate this manuscript from others.

- Experiential section must be revised and structured in subitems. 

- In Results, there was not clarity enough to understand that the images of fibers had pores or other types of damage.

- Overall, this manuscript was difficult to read and to follow the Authors findings and innovation. It must be deeply revised to highlight the research novelty. 

Author Response

Response to Reviewer #1

In spite of previous English editing by a native speaker, we admit there have still been a lot of errors. For more accurate editing, as the reviewer pointed out, the entire manuscript has been further revised by the professional English Editing Services at this stage. We believe that almost all spelling and grammatical errors have been corrected.

1. Abstract was too confusing. Please, try write it in a manner to valorize the research and to allow the readers to understand what was done and the main findings.

Response

We agree with the lack of the content of original Abstract. We have revised the overall abstract, and specifically, the issue pointed out has been modified as follows.

Line 9

Abstract: In this study, we discovered that washing hair with surfactants causes a decrease in the internal density of the hair, and we propose a cuticle-sealing strategy to inhibit this phenomenon. This phenomenon was revealed based on optical analyses such as optical microscopy, scanning electron microscopy (SEM), drop shape analysis, atomic force microscopy (AFM), and single hair analysis. Repeated treatment with surfactants creates areas of low density within the hair. Additionally, treatment with low-molecular-weight materials resulted in replenishment of the internal density of the hair. It has been shown that the more severe the degree of cuticle lifting, the more the internal density of the hair is reduced by surfactants. In addition, the study confirmed that a decrease in internal density could be prevented by sealing the cell membrane complex (CMC), and it was suggested that this reduced internal density may reflect the pore structure of hair. This study investigates the mass transfer phenomenon that occurs in hair and proposes a strategy to maintain hair homeostasis.

2. Introduction brought well-established data about hair and shaft properties. Please, try to update it with less known data to differentiate this manuscript from others.

Response

We are very grateful for this constructive review, and we have supplemented references to the Introduction section to introduce less known background : Surfactants cause loss of proteins inside the hair, porous-like structure by heat or oxidation. Our results found that washing hair with surfactants easily causes pores. We have described our differentiation in more detail.

Line 56

Pores form inside hair due to oxidative damage or heat, and as the damage becomes more severe, the volume and size of the pores increase [15]. These internal pores are also observed in SEM images [9,16] and Raman spectral images [15]. Meanwhile, in the case of optical microscopy, pores inside hair can be easily discovered using a simple method because light is scattered due to changes in the refractive index of the medium. When light is shined on the hair under a microscope in reflection mode, the pores inside the hair appear bright due to scattering [17,18], and when light is shined through the microscope in transmission mode, the pores inside the hair appear dark [11,19]. It is interpreted that when the vacuoles that form the medulla are damaged, the internal pores form a porous-like structure [11].

Line 66

Additionally, hair protein loss occurs when hair is washed with surfactants at room tem-perature, and to a lesser extent, protein loss also occurs when hair is washed with water [21]. Until now, it was thought that pores occur due to oxidative damage, heat, disease, etc. [11], but this study revealed for the first time that pores occur when hair is washed with a surfactant.

3. Experimental section must be revised and structured in subitems.

Response

Organizing experimental section into subitems is a really good idea. We divided it into a total of 9 subsections.

2.1. Materials

2.2. Analysis of the internal density of hair

2.3. Treatment of hair and surface modification

2.4. Hair gloss measurement

2.5. SEM

2.6. AFM

2.7. Contact angle measurement

2.8. Color measurement

2.9. The degree of cuticle lifting

4. In Results, there was not clarity enough to understand that the images of fibers had pores or other types of damage.

Response

We also agree with the reviewer’s comment. In Figure 1(c), we have added SEM images of a cross-section of hair with the pores directly visible.

After washing the hair for 10 seconds, we could see that the black area in the middle of the hair became larger. Although we used only 9 images in Figure 1 and 36 images of hair in Figures 3 and 5, in reality we observed numerous hairs. Interestingly, we found that the black areas did not change when we washed hair more than 4-6 times. Conversely, treatment with low-molecular-weight materials resulted in decrease of black area.

It is difficult to estimate what these black areas are. So, we changed “pore” to “black area” in the Results section.

This black area has been reported in several papers. In some papers (ex. Cosmetics, 2019, 6, 43), black areas appear bright when measured in reflection mode. We also obtained bright images of the inside of the hair. However, we used black mode because black is easier to see. Several papers report that dark area is pores inside the damaged hair. Pores can appear to form when lipids, proteins, and water escape. This information was added in the Introduction.

Line 342

Owing to methodological limitations, the components lost in the pores are unknown.

Line 368

Areas presumed to be pores are created by surfactants inside the hair.

Line 379

In the future, additional research will need to be conducted to identify the components re-leased from inside hair.

5. Overall, this manuscript was difficult to read and to follow the Authors findings and innovation. It must be deeply revised to highlight the research novelty.

Response

We are very grateful to the reviewer for constructive criticism. We have thoroughly revised the article again, including those mentioned by the reviewer.

We think that this difficulty is due to the lack of our explanation for pores inside hair. Based on your comments, we distinguished between cleansing damage and previous thermal/chemical damage of hair in Introduction.

We replaced “pore” with “reduced internal density” in the Abstract and “black areas” in the Results section. From the Discussion section, we cited existing references and carefully suggested that these black areas can be pores.

We've written about the limitations of our research that we cannot check out the internal components directly, and we're currently tracking what changes occur in the black area depending on the physical properties of the surfactant. Based on your comments, we’ve written that future studies should analyze which components are extracted.

We would really appreciate it if the reviewer would judge this positively.

Author Response File: Author Response.docx

Reviewer 2 Report

It is a well written article, with enough experiments performed for proving the preventive  action of sealing substances through Cell Membrane Complex.

Probably, some critical things are repeated regrading the methods used but it is unavoidable. I accept it in present form, although there are some printing and English grammar mistakes, which have to be corrected.

Probably, some critical things are repeated regrading the methods used but it is unavoidable. I accept it in present form, although there are some printing and English grammar mistakes, which have to be corrected.

Author Response

Response to Reviewer #2

We are very grateful to the reviewer for understanding the conclusions of our research.

1. Method part

Response

It is our carelessness not to mention these contents in the Materials and Methods section. We have revised the overall Materials and Methods section.

2. English grammar mistakes

Response

In spite of previous English editing by a native speaker, we admit there have still been a lot of errors. For more accurate editing, as the reviewer pointed out, the entire manuscript has been further revised by the professional English Editing Services at this stage. We believe that almost all spelling and grammatical errors have been corrected.

Author Response File: Author Response.docx

Reviewer 3 Report

The purpose of this article is to address the damage caused by repeated washing on hair due to surfactants. Although the topic is currently of interest, it is still scientifically underdeveloped. Nevertheless, there are several considerations to be made.

The article is difficult to understand due to its messy and confusing content, particularly in the Results section. Additionally, the Materials and Methods section lacks sufficient detail and clarity. It is unclear how many locks the experiments were conducted on, and the magnification used to obtain the optical microscope and SEM images is not specified. Moreover, in line 114, the article mentions a home-built device, but there is not enough information on the measuring principle.

The unit of measurement of the presumed pores analyzed is unclear, although Figure 1 states "hair pore size" (see line 132).

Furthermore, the graph in Figure 1 does not show the name of the axes.

In line 151, it is stated that the pores in hair are caused by a loss of components. However, it is not clear what kind of components are being referred to - lipids or proteins? Additionally, it is difficult to determine this based on the short contact time with the surfactant. The article also mentions CMC components, but lipid extraction from hair typically requires organic solvents and high temperatures. Therefore, it is hard to believe that washing alone could cause such damage.

I also recommend conducting the experiment with contact times more similar to those of an in vivo wash.

From lines 153 to 158 they talk about filling the hair with amino acids, please specify how this filling took place in the Materials and Methods section. Furthermore, in lines 181-183, the authors state that the passage of substances into the hair occurs through the CMC, how do the authors explain that an aa such as Phe cannot pass even though it is more lipophilic than Tyr, which moreover has a higher molecular weight, contrary to what is stated in line 169.

Specify how the pictures in Figure 3b were obtained.

In section 3.3 there are several unspecified abbreviations (ATC, CSA, AFM)

Line 215: on what basis is this stated?

In my opinion, the article serves as a valuable source of inspiration. However, there is a substantial scope for improvement in terms of the order in which the contents are presented. Additionally, the methods used in the study need to be elaborated upon with a scientific basis, which is only provided for the analysis of color and brightness - secondary outcomes of the study.

I suggest making corrections in the references, some of which have DOIs, while others do not. Additionally, providing more specific details on the instruments and solvents used would be helpful.

Please ensure to pay close attention to proper punctuation usage and carefully review the concepts to ensure that the language is clear and easy to understand. Corrections of any spelling, grammar, and punctuation errors are also important for a more fluent and polished final effect.

Author Response

Response to Reviewer #3

We are very grateful to the reviewer for understanding the conclusions of our research and for constructive criticism. We have thoroughly revised the article again, including those mentioned by the reviewer. Furthermore, this article has been further revised by the professional English Editing Services at this stage. As a result, we believe that almost all spelling and grammatical errors have been corrected. We will try our best to answer the comments of the reviewer as follows.

1. It is unclear how many locks the experiments were conducted on, and the magnification used to obtain the optical microscope and SEM images is not specified.

Response

It is our carelessness not to mention these contents in the Materials and Methods section. It has been added as follows.

Line 99

The hair was immersed in 10 % SLES aqueous solution for 10 seconds and then rinsed in running water for 30 seconds (n=9).

Line 105

During one experiment, as shown in Figure 3 and Figure 5, 12 hair fibers were prepared and tested. Twelve images of different parts of one hair were obtained, and a total of three experiments were performed. To coat the hair with CSA, ATC, or polyethyleneimine, the hair was left in 5 % targeting polymer solution for 15 min, lightly rinsed with water, and dried naturally (n = 36).

Line 124

The cuticle edge portion of a total of three hairs was scanned in contact mode.

We added scale bars to the SEM images.

2. In line 114, the article mentions a home-built device, but there is not enough information.

Response

The equipment that measures cuticle lifting was manufactured by IK Lab, a venture company at Korea University of Technology. Prof. Ik Hyun Lee made this device while he was a researcher at MIT, and his paper was published here: IEEE Access (2019), 7, 91809.

We purchased this device. So home-built was incorrect. The issue pointed out has been modified as follows.

Line 139

To quantify the degree of cuticle lifting, a single hair analyzer for damage assessment (IKLab Inc., Korea) was used. This is a custom-made commercial device that can determine the degree of cuticle lifting based on the degree of light reflection, and detailed information is provided elsewhere [23].

3. The unit of measurement of the presumed pores analyzed is unclear, although Figure 1 states “hair pore size” (see line 132).

Response

We are very grateful for this constructive review, and we have modified the description to prevent any further confusion.

In Figure 1(c), we have added SEM images of a cross-section of hair with the pores directly visible. We replaced “pore” with “reduced internal density” in the abstract and “black areas” in the results section. In the discussion section, we cited existing references and carefully suggested that these black areas can be pores.

4. The graph in Figure 1 does not show the name of the axes.

Response

We are very grateful for the specific indication of our mistake. And this part was changed to Area_black/Area_total.

5. It is not clear what kind of components are being referred to – lipids or proteins? Additionally, it is difficult to determine this based on the short contact time with the surfactant. The article also mentions CMC components but lipid extraction from hair typically requires organic solvents and high temperatures. Therefore, it is hard to believe that washing alone could cause such damage.

Response

We also agree with the reviewer’s comment that lipid (lost component) was our hypothesis. After washing the hair for 10 seconds, we could see that the black area in the middle of the hair became larger. Although we used only 9 images in Figure 1 and 36 images of hair in Figures 3 and 5, in reality we observed numerous hairs. Interestingly, we found that the black areas did not change when we washed hair more than 4-6 times. Conversely, treatment with low-molecular-weight materials resulted in decrease of black area.

It is difficult to estimate what these black areas are. So, as mentioned above, we changed “pore” to “black area” in the Results section.

This black area has been reported in several papers. In some papers (ex. Cosmetics, 2019, 6, 43), black areas appear bright when measured in reflection mode. We also obtained bright images of the inside of the hair. However, we used black mode because black is easier to see. Several papers report that dark area is pores inside the damaged hair. Pores can appear to form when lipids, proteins, and water escape. This information was added in the introduction. We would really appreciate it if the reviewer would judge this positively.

Line 333

Owing to methodological limitations, the components lost in the pores are unknown.

Line 359

Areas presumed to be pores are created by surfactants inside the hair.

Line 370

In the future, additional research will need to be conducted to identify the components re-leased from inside hair.

6. I also recommend conducting the experiment with contact times more similar to those of an in vivo wash.

Response

It is a really good idea to keep hair sample wash time consistent with in vivo wash time. But the important thing in this experiment is to measure hair at the same position before and after washing hair. So we used a sample holder. Since a single strand of hair was used at the holder, the time the hair is in contact with the surfactant is different from the actual contact time. We added these sentences in Discussion.

Line 309

Because strands of hair stick together, it takes less time for the surfactants to reach each individual hair than the total time it takes for the surfactants to lather the entire hair. In this experiment, the hair had to be kept in the same position before and after washing, so one strand of hair was fixed to the sample holder and then washed using SLES. Therefore, the hair washing time was set to 10 seconds, which is shorter than the in vivo wash time.

7. From lines 153 to 158 they talk about filling the hair with amino acids, please specify how this filling took place in the Materials and Methods section.

Response

We have described experimental method in more detail.

Line 101

To check amino acid penetration, the hair was soaked in 1% amino acid solution for 15 minutes. Afterwards, the hair was lightly wiped with a paper towel and dried naturally.

Line 92

To ensure that the focus of the hair images captured under the microscope was uniform, we created and tested a sample holder that could capture images while the hair was pulled tightly. Using the sample holder, images were captured at the same location, even after washing, and the location was rechecked based on the original porous point to secure the same location.

8. How do the authors explain that an aa such as Phe cannot pass even though it is more lipophilic than Tyr, which moreover has a higher molecular weight, contrary to what is stated in line 169.

Response

We really appreciate the reviewer for pointing out this. As described in the Introduction section, it is well known that CMC consists of beta and delta layer. The beta layer is composed of lipids, but the composition of the delta layer is unknown. We believe that internal components move through the delta layer of CMC, but further research is needed on this aspect. This is because it is difficult for water to move through the beta layer, making it difficult to become an intercellular pathway. This was chosen with reference to a book [Robbins, C.R. Chemical and Physical Behavior of Human Hair]. Since this book has cited 1756 times on the basis of Google Scholars, we judged the contents in this book to be reliable.

In introduction, this sentence “CMC plays a role in structurally supplying the medulla through lipids.” has been deleted because it is unclear.

It has been added as follows. Once again, we sincerely appreciate the criticism for giving us a sharp indication of the wrong interpretation.

Line 40

Because it is difficult to separate the delta layer in the CMC, the composition of the delta layer is still unclear, but it is known to contain protein [1].

Line 335

However, the fact that hair is also filled with glycine means that the pores inside the hair are spaces where not only lipids but also other substances can enter. Phenylalanine is more lipophilic than tyrosine, and its inability to fill the inside of the hair appears to be due to its inability to pass through the delta layer of the CMC, which is thought to be made of protein (its composition is unknown).

9. Specify how the pictures in Figure 3b were obtained.

Response

We agree with the lack of the content in Material section, and we have added a brief statement of the experiment.

Line 89

When calculating the black area, the black area at the edge of the hair was not counted because it is an area where light does not reach the detector.

Line 105

During one experiment, as shown in Figure 3 and Figure 5, 12 hair fibers were prepared and tested. Twelve images of different parts of one hair were obtained, and a total of three experiments were performed. To coat the hair with CSA, ATC, or polyethyleneimine, the hair was left in 5 % targeting polymer solution for 15 min, lightly rinsed with water, and dried naturally (n = 36).

10. In section 3.3 there are several unspecified abbreviations (ATC, CSA, AFM)

Response

The abbreviations were defined in this paper upon first use. Most abbreviation was defined in the Materials and Methods sections. However, since ATC and CSA are not general academic abbreviations, they are mentioned again in the text and figures to aid understanding.

11. Line 215: on what basis is this stated?

“Figure 4(a) shows that hair cuticle height was reduced by up to 110 nm after sealing.”

Response

We have very much considered how much the height of hair cuticle changed after sealing. After sealing the hair cuticle, the topography of hair cuticle was measured with atomic force microscope (AFM). The vertical resolution of AFM can be up to 0.1 nm. A graph of the height difference is added in Figure 4(a). The difference was calculated to be 80 nm, excluding the two parts that protrude as contour lines, but the maximum height difference was corrected to 110 nm for objective consideration.

12. The methods used in this study need to be elaborated upon with a scientific basis, which is only provided for the analysis of color and brightness - secondary outcomes of the study.

Response

We are really grateful for pointing out the problem. We acknowledge that our study has shortcomings. Based on your comments, we’ve written that future studies should analyze which components are extracted. We've written about the limitations of our research that we cannot check out the internal components directly, and we're currently tracking what changes occur in the black area depending on the physical properties of the surfactant.

Based on your opinion, we have added SEM images that actually show the inside of the hair. We have also supplemented references to the Discussion section to reinforce the scientific background in depth. Single hair analyzer for damage assessment that estimated cuticle lifting is the one that calculating figures with a physically scientific background. The topographic result scanned the same point of hair with atomic force microscopy is a very high level of scientific achievement.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear Authors,

Thank you for your kind and comprehensive reply. A final revision could eliminate types along the text.

Author Response

We are very grateful to the reviewer for understanding the conclusions of our research and constructive criticism.

After reading the Reviewer's comments, it appears that there are no suggestions for modifications. Please let us know if there are any additional changes you need to make.

Sincerely,

Authors

Author Response File: Author Response.docx

Reviewer 3 Report

The authors made the required corrections, improved the comprehensibility of the study and the presentation of the data.

Author Response

We are very grateful to the reviewer for understanding the conclusions of our research and constructive criticism.

We revised the Discussion and Conclusions based on Reviewer’s opinion that the conclusions could be further improved. The expression that the surfactant penetrates directly into the hair has been deleted.

Line 359

Before)

In this study, three methods were used to prevent surfactant penetration. First, the cuticle was sealed to prevent the surfactants from entering the CMC.

After)

In this study, three methods were used to prevent the reduction of the internal density of hair caused by surfactants. First, the cuticle was sealed to prevent direct contact between the surfactants and CMC.

Line 370

Before)

It was confirmed that, in terms of the pores, the low internal density region inside the hair could be filled with low-molecular-weight molecules.

After)

It was confirmed that, in terms of the pores, the low internal density region inside the hair could be filled with low-molecular-weight amino acids.

Line 378

Before)

This implies that, when the surfactants penetrate the hair, the ingredients inside the hair leak out through the CMC, and cuticle-sealing technology is important for hair damage repair.

After)

This implies that the ingredients inside the hair leak out through the CMC, and cuticle-sealing technology is important for hair damage repair.

We believe that this revision will help further clarify our conclusions.

After reading the Reviewer's comments, it appears that there are no suggestions for modifications. Please let us know if there are any additional changes you need to make.

Sincerely,

Authors

Author Response File: Author Response.docx