Study of the Repair Action and Mechanisms of a Moisturizing Cream on an SLS-Damaged Skin Model Using Two-Photon Microscopy

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study is well-designed with appropriate comparators and thorough analyses. It contributes to the field of skin care and cosmetics. I do not see any major issues that need to be addressed or fixed in this article, as it is already of high quality. However, I have the following comments that should be addressed.  

Comments

The abbreviation ORC is used throughout the paper, and I’m guessing it stands for “original repair concentrating,” mentioned as the name of the commercial cream on line 86. It would be helpful for the reader to clarify this abbreviation.

The cream in the study contains 52 ingredients. Assuming that the ingredients are listed in descending order of concentration, the ceramides (Ceramide NP, NS, and AP) are likely present in relatively small amounts, as they appear towards the end of the list. This suggests their combined concentration is quite low, likely less than 1%. Considering this, I strongly recommend toning down any suggestions about observed improvements due to the presence of ceramides. This would require a test involving formulations with and without ceramides, in my opinion. Similarly, the potential beneficial effects of lactobacillus/soybean ferment extract and bacillus ferment are difficult to prove without a test involving formulations with and without these ingredients.

T/C values are provided without clear reporting of the TEWL values. It is recommended to add the TEWL values corresponding to the Corneometer values presented in Fig. 3 (maybe in SI).

In Fig. 5 it is not totally clear if the 10 images of the top row goes from outside (i.e., 0 µm) to 20 ) to 20 µm, and so on for the following rows beneath. Please clarify.

In the conclusion, line 375-378, it is concluded that the cream’s repair mechanisms are confirmed by the TPM results, showing beneficial epidermal regeneration and collagen synthesis. However, it is not clear from Fig. 7 that the collagen synthesis (or SHG signal) is enhanced following ORC treatment. This issue should be clarified.

Author Response

Response to Reviewer Comments

Comment: The study is well-designed with appropriate comparators and thorough analyses. It contributes to the field of skin care and cosmetics. I do not see any major issues that need to be addressed or fixed in this article, as it is already of high quality. However, I have the following comments that should be addressed.

Response: We would like to express our sincere gratitude for your thorough review of our manuscript and your encouraging feedback. We have carefully revised the manuscript in accordance with your suggestions. Reviewer comments are presented in italics, and our responses follow in standard font.

 

Comment 1: The abbreviation ORC is used throughout the paper, and I’m guessing it stands for “original repair concentrating,” mentioned as the name of the commercial cream on line 86. It would be helpful for the reader to clarify this abbreviation.

Response 1: We thank the reviewer for pointing this out. The abbreviation “ORC” refers to the test product, Proya Advanced Original Repair Concentrating Essence Cream. We have updated the manuscript to define “ORC” explicitly at its first mention in Section 2.1 (“Materials and Methods”) and have ensured consistent usage throughout the text.

 

Comment 2: The cream in the study contains 52 ingredients. Assuming that the ingredients are listed in descending order of concentration, the ceramides (Ceramide NP, NS, and AP) are likely present in relatively small amounts, as they appear towards the end of the list. This suggests their combined concentration is quite low, likely less than 1%. Considering this, I strongly recommend toning down any suggestions about observed improvements due to the presence of ceramides. This would require a test involving formulations with and without ceramides, in my opinion. Similarly, the potential beneficial effects of lactobacillus/soybean ferment extract and bacillus ferment are difficult to prove without a test involving formulations with and without these ingredients.

Response 2: We appreciate the reviewer’s thoughtful comment. The suggestion is well taken. We would like to clarify that the suppliers of the key ingredients (Ceramide NP, NS, AP, Lactobacillus/soybean ferment extract, and Bacillus ferment) have already conducted controlled studies comparing formulations with and without these ingredients, and have demonstrated their functional efficacy. Furthermore, published literature has reported the skin-repairing and anti-inflammatory benefits of these ingredients, as referenced in Lines 63–71 and 380–391 of our manuscript.

To better reflect this, we have added the following sentence to Lines 71–74:

“The benefit of the cream is mainly attributed to the essential ingredients of ceramide NP, ceramide NS, ceramide AP, lactobacillus/soybean ferment extract, and bacillus ferment, as well as the synergistic interplay of these components with other ingredients in the formulation.”

This revision acknowledges the reviewer’s concern and emphasizes the importance of the overall formula rather than attributing efficacy solely to any single ingredient.

 

Comment 3: T/C values are provided without clear reporting of the TEWL values. It is recommended to add the TEWL values corresponding to the Corneometer values presented in Fig. 3 (maybe in SI).

Response 3: Thank you for this suggestion. We have added the TEWL (transepidermal water loss) values corresponding to the Corneometer data in Figure 3 as a new dataset in the Supplementary Materials (Data S7). A reference to this dataset has also been included in Section 3.2.3.2.

Specifically, we have added the following sentence (Lines 299–300):

“Changes in TEWL values were shown in Date S7. After using the cream for 168 hours, the TEWL value decreased significantly compared with the NC group. ”

We believe this addition provides greater transparency and addresses the comment.

 

Comment 4: In Fig. 5 it is not totally clear if the 10 images of the top row goes from outside (i.e., 0 µm) to 20 ) to 20 µm, and so on for the following rows beneath. Please clarify.

Response 4: We apologize for the lack of clarity in the original figure. To improve readability, we have revised Figure 5 to provide a more intuitive and informative visualization. The figure now clearly displays representative TPM images captured at 2 µm intervals, ranging from the stratum corneum (0 µm) to a depth of 120 µm in the dermis, totaling 60 sequential images.

We have also updated the figure legend to include the following clarification:

“TPEF and SHG images were acquired at 2 µm intervals from the skin surface (0 µm) to a depth of 120 µm, totaling 60 images.”

 

Comment 5: In the conclusion, line 375-378, it is concluded that the cream’s repair mechanisms are confirmed by the TPM results, showing beneficial epidermal regeneration and collagen synthesis. However, it is not clear from Fig. 7 that the collagen synthesis (or SHG signal) is enhanced following ORC treatment. This issue should be clarified.

Response 5: Thank you for raising this important point. As correctly observed, Figure 8 (formerly Figure 7) does not show a significant quantitative increase in SHG signal intensity, which would indicate collagen synthesis. We have therefore revised the conclusion to ensure alignment with the actual findings.

The revised sentence (Lines 415–418) now reads:

“The use of TPM not only enabled real-time, high-resolution imaging of the skin’s repair process but also provided insights into the cream’s reparative mechanisms, confirming its benefits in epidermal regeneration and DEJ repair.”

This revision removes reference to collagen synthesis and more accurately reflects the data obtained through TPM imaging.

Reviewer 2 Report

Comments and Suggestions for Authors

A manuscript titled “Study of the Repair Action and Mechanisms of a Moisturizing Cream on SLS- Damaged Skin Model Using Two-Photon Microscopy”. The study indicates that TPM is a revolutionary cosmetic dermatology tool that can analyze skincare products' impact on skin structure and function in real time. A sodium lauryl sulfate (SLS)-induced skin damage model and two-photon microscopy (TPM) are used to test a new moisturizing cream. The cream dramatically increased the expression of barrier proteins including filaggrin (FLG), loricrin (LOR), and transglutaminase 1 (TGM1) in SLS-stimulated 3D skin models and clinical situations, while decreased inflammatory markers like IL-1α, TNF-α, and PGE2. TPM showed that the cream increased epidermal thickness and DEJI, indicating deep skin healing and barrier functioning. Patients with sensitive skin and post-IPL therapy showed considerable improvement in symptoms after using the cream in clinical studies. The work is well-written and organized. However, the positive control for each assay must be documented. The concise protocol references should be supplied. Additional methodological details of 2.2 in vitro and clinical studies must be well documented. The manufacturer's name and lot number must be documented.

Author Response

Response to Reviewer Comments

Comment: A manuscript titled “Study of the Repair Action and Mechanisms of a Moisturizing Cream on SLS- Damaged Skin Model Using Two-Photon Microscopy”. The study indicates that TPM is a revolutionary cosmetic dermatology tool that can analyze skincare products' impact on skin structure and function in real time. A sodium lauryl sulfate (SLS)-induced skin damage model and two-photon microscopy (TPM) are used to test a new moisturizing cream. The cream dramatically increased the expression of barrier proteins including filaggrin (FLG), loricrin (LOR), and transglutaminase 1 (TGM1) in SLS-stimulated 3D skin models and clinical situations, while decreased inflammatory markers like IL-1α, TNF-α, and PGE2. TPM showed that the cream increased epidermal thickness and DEJI, indicating deep skin healing and barrier functioning. Patients with sensitive skin and post-IPL therapy showed considerable improvement in symptoms after using the cream in clinical studies. The work is well-written and organized.

Response: We sincerely thank the reviewer for their thoughtful reading of our manuscript and for the encouraging feedback regarding the study’s structure and contribution. We have carefully addressed each of the following comments. Reviewer comments are presented in italics, and our responses are in standard font.

 

Comment 1: However, the positive control for each assay must be documented.

Response 1: Thank you for pointing out this important omission. The positive control group (PC) in the in vitro experiments was treated with 0.01% dexamethasone solution (C₂₂H₂₉FO₅, MW: 392.46, purity > 98%, Sigma, USA, Batch No.: 220119) following SLS stimulation. We have now included this information in Section 2.2 of the main text and in Data S2 of the Supplementary Materials. In addition, a description of the positive control has been added to the legend of Figure 1 for clarity.

 

Comment 2: The concise protocol references should be supplied.

Response 2: We appreciate this valuable suggestion. We have updated the methodological descriptions with relevant standardized protocol references to support our in vitro studies. The following sources are now cited in Section 2.2.1:

26. Masri, S.; Fauzi, M.B.; Rajab, N.F. In vitro 3D skin culture and its sustainability in toxicology: a narrative review. Artif. Cells Nanomed. Biotechnol. 2024, 52(1), 476–499.
27. Wang, R.; Yan, S.; Ma, X. The pivotal role of Bifida Ferment Lysate on reinforcing the skin barrier function and maintaining homeostasis of skin defenses in vitro. J. Cosmet. Dermatol. 2023, 22(12), 3427–3435.
28. Quan, Q.; Weng, D.; Li, X. Analysis of drug efficacy for inflammatory skin on an organ-chip system. Front. Bioeng. Biotechnol. 2022, 10, 939629.

These references enhance the methodological rigor and reproducibility of our experimental procedures.

 

Comment 3: Additional methodological details of 2.2 in vitro and clinical studies must be well documented.

Response 3: Thank you for emphasizing the importance of methodological transparency. In response, we have significantly expanded Section 2.2 to include detailed experimental parameters and step-by-step procedures for both the in vitro 3D model and the clinical studies (see Rows 103-129). Furthermore, the complete protocols for each experiment are now comprehensively documented in the Supplementary Materials:

• Data S2: SLS-stimulated 3D epidermal skin model
• Data S3: Four-week clinical study
• Data S4: Post-IPL treatment study

We believe these additions provide a clear and replicable methodological framework.

 

Comment 4: The manufacturer's name and lot number must be documented.

Response 4: Thank you for this valuable recommendation. We have now thoroughly documented the manufacturers, locations, and lot numbers for all reagents and materials used in our experiments. These details can be found in:

• Section 2.2.1: The SLS-stimulated 3D epidermal skin model (Rows 103–118)
• Supplementary Materials: Data S2

We hope this level of documentation meets the standards of experimental transparency.

Reviewer 3 Report

Comments and Suggestions for Authors

This paper shows how the effectiveness of a cosmetic cream can be evaluated in different settings. It not only details various approaches, such as clinical studies, 3D epidermal skin model, but also introduces the new method of TPM and its possibilities in cosmetic dermatology. Providing the opportunity to find out, not only the results of the cream's action, but also the mechanism by which it achieves its effects. It is excellently written, methodologically precise, excellently structured. Great job! 

Author Response

Response to Reviewer Comments

Comment: This paper shows how the effectiveness of a cosmetic cream can be evaluated in different settings. It not only details various approaches, such as clinical studies, 3D epidermal skin model, but also introduces the new method of TPM and its possibilities in cosmetic dermatology. Providing the opportunity to find out, not only the results of the cream's action, but also the mechanism by which it achieves its effects. It is excellently written, methodologically precise, excellently structured. Great job! 

Response: We sincerely appreciate your generous and encouraging feedback on our work. Your recognition of the study’s methodological rigor, the multi-faceted research approach, and the potential of two-photon microscopy (TPM) in advancing cosmetic dermatology is truly motivating. We also share your enthusiasm for the broader applications of integrative methodologies in skin research.

Thank you again for your thoughtful review and for highlighting the strengths of our manuscript. Your support serves as a strong encouragement for us to continue pursuing innovation in this field.

Reviewer 4 Report

Comments and Suggestions for Authors

In general, the paper is not very consistent with regard to the use of MPT. There are not many explicit advantages to using this type of technique other than the morphological study highlighted in the paper. Little is shown about chemical composition results or how this type of technique can help researchers obtain better results. It seems to me that the technique has been explored little or not at all and does not deserve the prominence it receives in the title. Another point is that I don't understand whether the molecular results of other tests have already been presented in other publications or only here. The excessive use of abbreviations makes the paper tiresome to evaluate and many figures need better layout. I think the paper should rethink the title and clarify it:
1- are all the results original and never published before?
2- improve images.
3- convince that the use of MPT is really advantageous, from what I was shown, I saw no real advantage or novelty.
4- improve the writing, reducing the use of abbreviations and better explaining the techniques and study groups at each stage.

Below I have more requests for the authors, please answer them all.

1) what ORC group means? Please detailed better your abbreviations.

2) Figure 2 - too low quality, please improve it

3) What T/C means? Please include in the figure legend.

4) Figure 3 - too low quality, please improve it.

5) Figure 4 - too low quality, please improve it.

6) Figure 5 is too confuse, no information can be addressed. Please improve how you present it.

7) Did the authors applied a very high technique just to access epidermal thickness? What is the advantage of this technique in face of others such as vivascope?

8) Why the authors did not measured other parameters? In my point of view, the applicant of TPM is a little underated in this paper. 

9) What DEJI means? Please explore this value and explain better.

10) Figure 8 - The observation here may be limited due to the fact that the area selected for the analysis. How can we guarantee that the area selected is the most appropriate? Are there any evaluations available for the stratum corneum, which is the area most affected by surfactants?

11) Which MOLECULAR levels were evaluated using TPM? I just can see morphological aspects. Please explicit this point and rethink the first part of your discussion.

12) lines 333-335- how TPM and the microscopy were correlated? Please explicit this finding.

13) lines 354-356 - however, where did the authors evaluated something at the cellular level? The MPT results are related with the morphological aspect. Please clarify.

Author Response

Response to Reviewer Comments

Comment: In general, the paper is not very consistent with regard to the use of MPT. There are not many explicit advantages to using this type of technique other than the morphological study highlighted in the paper. Little is shown about chemical composition results or how this type of technique can help researchers obtain better results. It seems to me that the technique has been explored little or not at all and does not deserve the prominence it receives in the title. Another point is that I don't understand whether the molecular results of other tests have already been presented in other publications or only here. The excessive use of abbreviations makes the paper tiresome to evaluate and many figures need better layout. I think the paper should rethink the title and clarify it.

Response: We sincerely thank the reviewer for the detailed evaluation of our manuscript and your constructive feedback. In response, we have carefully revised the manuscript to address all the concerns raised. Reviewer comments are italicized, and our responses are provided in standard font.

 

Comment 1: are all the results original and never published before?

Response 1: Thank you for highlighting the importance of originality. We confirm that all results presented in this manuscript are entirely novel and have not been published elsewhere. This includes all datasets and their interpretations—covering clinical data, the 3D epidermal skin model results, and two-photon microscopy (TPM) analyses.

 

Comment 2: improve images.

Response 2: We apologize for the suboptimal image quality in the original submission. All figures have now been re-exported at 600 dpi to ensure significantly improved clarity and resolution.

 

Comment 3: convince that the use of MPT is really advantageous, from what I was shown, I saw no real advantage or novelty.

Response 3: Thank you for this important comment. To address this, we have added the following clarification to Lines 52-59:

“In this study, TPM was used as an advanced imaging tool to explore the tissue repair mechanisms of the moisturizing cream, particularly through the quantitative analysis of epidermal thickness and DEJ undulation. A key innovation of this study is the introduction of the Dermal-Epidermal Junction Index (DEJI), a novel 3D metric that quantifies DEJ undulation. TPM’s ability to capture depth-resolved two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) signals enabled us to compute DEJI.”

We believe this highlights the added value and novelty of TPM in our context.

 

Comment 4: improve the writing, reducing the use of abbreviations and better explaining the techniques and study groups at each stage.

Response 4:  We appreciate this suggestion. To improve clarity:

1. All abbreviations are now defined upon first use (e.g., sodium lauryl sulfate (SLS), two-photon microscopy (TPM)).
2. A Glossary of Abbreviations has been added at the end of the manuscript (Lines 449–464).
3. Section 2.2 has been expanded to include detailed methodological explanations for both in vitro and clinical studies (Lines 103–129).
4. Full protocols are available in the Supplementary Materials (Data S2, S3, and S4).

We hope these changes enhance readability and transparency.

 

Comment 5: what ORC group means? Please detailed better your abbreviations.

Response 5: Thank you for pointing this out. “ORC” refers to the test product—Proya Advanced Original Repair Concentrating Essence Cream. We have now explicitly defined “ORC” at its first mention in Section 2.1 and ensured consistent usage throughout the manuscript.

 

Comment 6: Figure 2 - too low quality, please improve it

Response 6: Thank you. Figure 2 has been re-exported at 600 dpi to enhance clarity and resolution.

 

Comment 7: What T/C means? Please include in the figure legend.

Response 7: Thank you for the suggestion. We have defined the T/C value in Section 3.2.2 (Lines 262–265) as the ratio of TEWL to stratum corneum moisture content, representing the efficiency of the skin’s water barrier. We also added explanatory notes to the legends of Figures 2 and 4 (Lines 278–279 and 313–314, respectively).

 

Comment 8: Figure 3 - too low quality, please improve it.

Response 8: We appreciate this comment. Figure 3 has been re-exported at 600 dpi, and we have also merged Figures 3A and 3B into a single image to improve clarity and facilitate comparison. Improvement values are now clearly shown in the figure.

 

Comment 9: Figure 4 - too low quality, please improve it.

Response 9: Thank you. Figure 4 has also been re-exported at 600 dpi, and as with Figure 3, we have combined panels 4A and 4B into one figure to improve clarity. Improvement data are included in the updated figure.

 

Comment 10: Figure 5 is too confuse, no information can be addressed. Please improve how you present it.

Response 10: We apologize for the confusion caused by the original figure. We have revised Figure 5 to provide a clearer and more intuitive layout. The revised version includes TPM images captured at 2 µm intervals from the stratum corneum (0 µm) down to the dermis (120 µm), totaling 60 images. We have also updated the legend with the following clarification:

“TPEF and SHG images were acquired at 2 µm intervals from the skin surface (0 µm) to a depth of 120 µm, totaling 60 images.”

 

Comment 11: Did the authors applied a very high technique just to access epidermal thickness? What is the advantage of this technique in face of others such as vivascope?

Response 11: Thank you for this thoughtful question. While reflectance confocal microscopy (RCM) such as Vivascope® is indeed a valuable tool for superficial skin imaging, its resolution diminishes significantly at greater depths. In contrast:

1. TPM provides superior deep-tissue imaging, maintaining high resolution up to 120 µm, allowing us to observe both epidermal regeneration and DEJ repair.
2. We introduce a novel quantitative metric, the Dermal-Epidermal Junction Index (DEJI), enabled by TPM’s depth-resolved SHG/TPEF signal acquisition. This metric is not possible to compute with RCM.

In summary, TPM offers unique advantages in visualizing three-dimensional tissue repair, justifying its selection for our study. We hope this encourages further exploration of TPM in skin research.

 

Comment 12: Why the authors did not measured other parameters? In my point of view, the applicant of TPM is a little underated in this paper.

Response 12: Thank you for raising this point. In this study, we focused on TPM-derived structural metrics—epidermal thickness and DEJI—as indicators of skin repair. Molecular-level parameters (e.g., IL-1α, TNF-α, PGE2, FLG, LOR, TGM1) were assessed using ELISA and immunofluorescence in the 3D skin model (Figure 1), to complement the TPM-based morphological observations.

We agree that TPM offers further potential, such as quantifying SHG/TPEF signal intensities (e.g., for collagen or NAD(P)H metabolic activity). We plan to include these aspects in future studies.

 

Comment 13: What DEJI means? Please explore this value and explain better.

Response 13: Thank you for the comment. We have revised our explanation of DEJI as follows:

“The Dermal-Epidermal Junction Index (DEJI) is a novel metric introduced in this study, defined as the ratio of the actual surface area of the DEJ to its projected area along the skin surface’s normal axis. A higher DEJI value indicates greater DEJ undulation, which enhances dermal-epidermal nutrient exchange and mechanical resilience.”

The calculation method is detailed in Section 2.3.3 (Lines 168–169).

 

Comment 14: Figure 8 - The observation here may be limited due to the fact that the area selected for the analysis. How can we guarantee that the area selected is the most appropriate? Are there any evaluations available for the stratum corneum, which is the area most affected by surfactants?

Response 14: We understand the reviewer’s concern. To ensure representativeness, we followed a standardized imaging protocol within a fixed 18 mm² test area on the inner forearm. For each subject (n = 22), three non-overlapping points were randomly selected to minimize sampling bias. Results were then processed statistically. Figure 8 presents a representative case.

Regarding the stratum corneum, we assessed its hydration using Corneometer® and barrier function using Tewameter® (TEWL). These surface-level biophysical data, combined with structural data from TPM, offer a comprehensive view of the cream’s reparative effects.

 

Comment 15: Which MOLECULAR levels were evaluated using TPM? I just can see morphological aspects. Please explicit this point and rethink the first part of your discussion.

Response 15: We appreciate this observation. Although TPM can detect molecular autofluorescence (e.g., NAD(P)H), our study focused on structural parameters, such as epidermal thickness and DEJ morphology, to reflect tissue-level repair.

To avoid misunderstanding, we have revised Section 4 (Lines 354–358) as follows:

“This study confirms the efficacy of a novel moisturizing cream in repairing SLS-induced skin damage, with improvements observed in skin hydration, barrier function, and structural integrity. Using TPM, we gained detailed insight into the skin repair process—specifically, changes in epidermal thickness and DEJ undulation—that are difficult to capture using traditional imaging techniques.”

We have also corrected the earlier sentence at Line 393 from

“TPM offers the advantage of visualizing cellular-level details…”

to

“TPM offers the advantage of visualizing structural and morphological details…”

 

Comment 16: lines 333-335- how TPM and the microscopy were correlated? Please explicit this finding.

Response 16: Thank you for this important point. Our H&E staining (Figure 1C) revealed loosening of epidermal structure after SLS stimulation. TPM imaging (Figure 6) subsequently showed that epidermal thickness increased significantly following treatment with the cream (8.5 µm at 168 h vs. 2.9 µm in the NC group).

This increase in thickness correlates with the re-densification of epidermal layers observed in histology, suggesting recovery of keratinocyte proliferation and differentiation. We have clarified this in the Discussion (Lines 371–373):

“The change from loose to dense tissue structure shown by H&E and the increase in epidermal thickness shown by TPM both reflect the cream’s effect on repairing the epidermal barrier.”

 

Comment 17: lines 354-356 - however, where did the authors evaluated something at the cellular level? The MPT results are related with the morphological aspect. Please clarify.

Response 17: We thank the reviewer for catching this inconsistency. Indeed, our TPM analyses were morphological in nature. Cellular-level findings (e.g., inflammatory cytokines and barrier proteins) were evaluated separately via ELISA and immunofluorescence in 3D models (Figure 1).

To reflect this, we revised the sentence in Section 4 (Lines 354–358) and corrected Line 393 as follows:

• Original: “TPM offers the advantage of visualizing cellular-level details…”
• Revised: “TPM offers the advantage of visualizing structural and morphological details within the skin without requiring invasive biopsies.”

This ensures accurate representation of the method’s capabilities within our study context.

 

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

Thanks for the corrections, but many points in the article still need to be improved, especially what DEJI means, how it is measured and how to avoid evaluation problems. In my opinion, it is still not very clear how we can use the DEJI without it being just a random selection of the most convenient areas for evaluation. All my comments are in bold.

I do not agree with this statment:

Thank you for this thoughtful question. While reflectance confocal microscopy (RCM) such as Vivascope® is indeed a valuable tool for superficial skin imaging, its resolution diminishes significantly at greater depths. In contrast:

1. TPM provides superior deep-tissue imaging, maintaining high resolution up to 120 µm, allowing us to observe both epidermal regeneration and DEJ repair. Did it? From my experience, LSM can reach until 250µm while TPM mostly can reach until 100-120µm. It is true that TPM presents better resolution but the statment regarding LSM is not true. Please rethink the necessity of your work and the wording of the present statment.
2. We introduce a novel quantitative metric, the Dermal-Epidermal Junction Index (DEJI), enabled by TPM’s depth-resolved SHG/TPEF signal acquisition. This metric is not possible to compute with RCM. This should be better addressed. Readers can be confused about what this DEJI means, which parameters were evaluated? How did you calculate that? Please be more visual, direct and explict. Is this just due to the ondulation? This can be really problematic since the are which is accessed is very limited. How can the authors be sure that the evaluation was not just in a specific area?

This study confirms the efficacy of a novel moisturizing cream in repairing SLS-induced skin damage, with improvements observed in skin hydration, barrier function, and structural integrity. Using TPM, we gained detailed insight into the skin repair process—specifically, changes in epidermal thickness and DEJ undulation—that are difficult to capture using traditional imaging techniques.

Why did the authors find it convenient to divide TEWL by Corneometer? I've never seen this type of evaluation in the literature and to be honest, although they are parameters related to skin barrier and hydration, they are hardly correlated, since barrier function doesn't only depend on hydration and vice versa. I strongly recommend that the authors present the TEWL and hydration data separately (hydration is already in figure 3 and TEWL in figure S7 should be moved to the original text). Without separate and clear TEWL data, it is difficult to assess the possible damage to the skin barrier and consequently the improvement from moisturizer application described by the authors. In my opinion, as the authors did not find a good solution to reduce TEWL quickly, they simply divided the measurements and this has no practical value or at least I don't know about it. Please rethink the whole work from the separate TEWL and SH values and/or present references which support this evaluation. 

All the images regarding corneometer should present the results as "A.U." instead of "C.U.". Please correct also in the text. 

Author Response

Comment: Thanks for the corrections, but many points in the article still need to be improved, especially what DEJI means, how it is measured and how to avoid evaluation problems. In my opinion, it is still not very clear how we can use the DEJI without it being just a random selection of the most convenient areas for evaluation. All my comments are in bold.

Response: We sincerely thank you for your insightful and constructive feedback. We have carefully revised the manuscript to address your concerns, particularly regarding the definition, measurement methodology, and evaluation reliability of the DEJI. We have clarified how DEJI is calculated, detailed the procedures implemented to avoid selection bias, and ensured that these improvements are clearly presented in the revised version. We are truly grateful for your valuable suggestions, which have significantly helped us enhance the clarity and scientific rigor of the manuscript.

 

 

Comment 1: “TPM provides superior deep-tissue imaging, maintaining high resolution up to 120 µm, allowing us to observe both epidermal regeneration and DEJ repair.” Did it? From my experience, LSM can reach until 250µm while TPM mostly can reach until 100-120µm. It is true that TPM presents better resolution but the statment regarding LSM is not true. Please rethink the necessity of your work and the wording of the present statment.

Response 1: We sincerely thank the reviewer for this valuable comment and completely agree with your observation. Indeed, laser scanning confocal microscopy (LSM) offers strong depth penetration capabilities, reaching up to approximately 250 µm, whereas two-photon microscopy (TPM) typically achieves penetration depths of around 100–120 µm.

Nevertheless, we believe that TPM provides unique insights in our study, particularly by enabling the novel quantitative evaluation of the dermal-epidermal junction through the DEJI metric. We appreciate this important point, and we hope to further explore and directly compare the capabilities of LSM and TPM in future studies.

 

 

Comment 2: “We introduce a novel quantitative metric, the Dermal-Epidermal Junction Index (DEJI), enabled by TPM’s depth-resolved SHG/TPEF signal acquisition. This metric is not possible to compute with RCM. This should be better addressed.” Readers can be confused about what this DEJI means, which parameters were evaluated? How did you calculate that? Please be more visual, direct and explict. Is this just due to the ondulation? This can be really problematic since the are which is accessed is very limited. How can the authors be sure that the evaluation was not just in a specific area?

Response 2: We sincerely thank the reviewer for the insightful and constructive comments. Following your suggestions, we have carefully revised the Methods, Results, and Supplementary Data sections to provide a clearer, more detailed explanation of the DEJI concept, its calculation method, and the measures we adopted to ensure reproducibility and minimize evaluation bias.

 

1) Definition clarification and software algorithm (Section 2.3.3 Instrumental Measurement):

We have revised the relevant paragraph from:

“The DEJ can be precisely delineated based on the transition from the appearance of collagen to the disappearance of columnar or cubic basal cells. The epidermal thickness is defined as the mean distance from the skin’s upper surface to the DEJ. The DEJI is calculated as the ratio of the actual surface area of the DEJ to its projected area along the normal direction of the skin surface. Image J software was used to process the images captured by the TPM.”

to:

“The DEJ can be precisely delineated based on the transition from the appearance of collagen to the disappearance of columnar or cubic basal cells. Epidermal thickness is defined as the mean distance from the skin’s upper surface to the DEJ. The DEJI is defined as the ratio of the actual 3D surface area of the DEJ to its 2D projected area along the skin surface normal. A DEJI value ≥1 is expected: a completely flat DEJ yields a DEJI of 1, whereas greater undulation results in higher DEJI values. A higher DEJI thus indicates a larger contact area between the epidermis and dermis. The DEJI was computed using a dedicated computer software algorithm, based on the SHG signal from collagen fibers, which allowed clear 3D delineation of the DEJ surface. Our algorithm was adapted from previously published studies [29,30]. More details of the algorithm are provided in the Supplementary Data.”

 

2) Formula inclusion:

We have now explicitly provided the DEJI calculation formula in the manuscript (Line 180):

DEJI = Actual Surface Area of the DEJ / Projected Flat Surface Area

 

3) Measurement method refinement (Section 3.2.3.5 The Morphology of the DEJ):

To make the boundary identification process clearer, we expanded the Results section description to:

“The DEJ boundary was determined by identifying the transition zone between SHG signals (red) and TPEF signals (green). The interface between strong SHG and dominant TPEF signals was designated as the DEJ (Figure 9).” (Lines 356–358)

 

4) Standardization of sampling areas (Section 2.3.2 Methods):

To address concerns about potential selection bias, we detailed our sampling strategy as follows:

“For each test site, three non-overlapping fields of view (140 × 140 μm each) were randomly selected for imaging. The DEJI was calculated for each field, and the mean of the three measurements was used for analysis to minimize variability and avoid area-specific bias.” (Lines 158–162)

In addition, we analyzed the data across 22 subjects, and baseline consistency (as shown in Table 1) further supports the robustness of the TPM measurements over time.

 

5) Supplementary data on computer software algorithm:

We provided a detailed description of the DEJI computation in the Supplementary Data S7, based on published methodologies.

In brief, the workflow included: (i) global OTSU thresholding to identify collagen areas in SHG image stacks; (ii) 3×3 Gaussian and median filtering to reduce noise; (iii) depth map generation to reconstruct the DEJ surface; (iv) triangular mesh formation and surface area calculation using Heron’s formula. The DEJI was then normalized against the projected flat surface area.

 

Furthermore, we added a supplementary video to visually illustrate the DEJI calculation process to enhance clarity and reader understanding.

 

Once again, we are grateful to the reviewer for raising these important points, which allowed us to significantly improve the scientific rigor, transparency, and readability of our manuscript.

 

 

Comment 3: Why did the authors find it convenient to divide TEWL by Corneometer? I've never seen this type of evaluation in the literature and to be honest, although they are parameters related to skin barrier and hydration, they are hardly correlated, since barrier function doesn't only depend on hydration and vice versa. I strongly recommend that the authors present the TEWL and hydration data separately (hydration is already in figure 3 and TEWL in figure S7 should be moved to the original text). Without separate and clear TEWL data, it is difficult to assess the possible damage to the skin barrier and consequently the improvement from moisturizer application described by the authors. In my opinion, as the authors did not find a good solution to reduce TEWL quickly, they simply divided the measurements and this has no practical value or at least I don't know about it. Please rethink the whole work from the separate TEWL and SH values and/or present references which support this evaluation.

Response 3: We thank the reviewer for this thoughtful and important comment. We fully agree that TEWL and stratum corneum hydration are distinct physiological parameters, and that their relationship should be interpreted with caution.

 

We have now presented TEWL and hydration data separately in the revised manuscript, as recommended. The TEWL data, previously in the Supplementary Materials (Figure S3), has now been moved into the main text as Figure 4. The manuscript has been revised accordingly to clearly report the changes in TEWL values before and after SLS-induced damage and after cream application. (Lines 273-284, 309-319)

 

Regarding the use of the T/C value, we acknowledge the reviewer’s concerns and have clarified the rationale and literature basis for including this composite indicator in the revised manuscript  (Lines 275-279). Specifically, the T/C value is derived from the ratio of TEWL to hydration (stratum corneum moisture content), and has been described in prior research as a secondary parameter reflecting the efficiency of water retention relative to available moisture content in the stratum corneum. We have now included a citation to support this approach:

 

“Yuan, C.; Wang, X.M.; Tan, Y.M. Short-term repairing function of moisture cream on lactic acid stingers. Chin. J. Aesthet. Med. 2011, 20, 1121–1123.”

 

In that study, the T/C ratio was proposed as a non-invasive composite metric that more comprehensively reflects the functional integrity of the skin's water barrier. While TEWL reflects water loss and hydration indicates water content, their ratio provides an estimate of the relative water retention efficiency per unit of moisture—i.e., how well the stratum corneum retains water when hydrated. This index has been used as a supplementary parameter rather than a replacement for individual TEWL or hydration values.

 

In our study, the T/C value was reported alongside TEWL and hydration data to provide an additional dimension to interpret skin barrier repair. We greatly appreciate your recommendation, which has helped us improve the clarity and robustness of our data presentation.

 

 

Comment 4: All the images regarding corneometer should present the results as "A.U." instead of "C.U.". Please correct also in the text. 

Response 4: Thank you very much for your careful review and valuable suggestion.

We fully understand the convention of using “A.U.” (Arbitrary Units) in scientific reporting. However, in our study, we used a Corneometer CM825 (Courage & Khazaka, Germany), and according to the manufacturer’s documentation and common usage in related technical reports, the Corneometer output is often expressed as “C.U.” (Corneometer Units) to distinguish it from other arbitrary unit measurements.

Therefore, to maintain consistency with the device’s standard terminology and to ensure clarity regarding the specific measurement method used, we have retained “C.U.” in the manuscript.

That said, if the editorial office or reviewer prefers a unified terminology for the journal style, we are more than happy to revise all instances of “C.U.” to “A.U.” accordingly. We sincerely appreciate your attention to this detail and your support in improving the quality of our manuscript.