Quantification of Microstructural Changes in the Dermis of Elderly Women Using Morphometric Indices of the Skin Surface

Round 1

Reviewer 1 Report

I find this study to be designed and performed very well, and the presentation clear and complete. There are only a few points I would like to point out for the authors to correct before it is ready to be published.

lines 171-172: "A characteristic length scale (Lc), averaging
the sum of the equivalent-diameter and perimeter of the polygons, was also calculated." - The provided description is not sufficiently detailed (e.g., the equivalent diemter is not defined). In addition, it appears incorrect, since the average value of Lc in Tbl. 1 (1.64 mm) is lower than the Mean perimeter (4.04 mm). Please correct and clarify, possibly by providing the exact expresison.

line 185:  "The false-colour overlay image, with green representing collagen fibres and red representing elastin fibres ," - Exactly the opposite seems to be true. (see the caption to Fig. 2)

Figure 3 and Tbl. 2: In this study, you essentially treat the MPM-derived parameter REC as the ground truth, against which the morphological parameters are evaluated. It would be benefitial to the readers if this was reflected in the organization of Fig. 3 and Tbl. 2 (where REC is now presented at a "random" positin between the other values.

Figure 3: I would find it very informative if actual (not absolute) values were presented here. In this way, readers would also see which correlations are positive and which are negative, without this obstructing the study protocol in any way. (The stronger red and blue colors would then indicate strong positive and negative correlations, respectively).

line 273: "

line 283: "So far, all methods agreed to reveal that relative content of collagen decreases with intrinsic ageing based on diminished collagen synthesis and elastin degradation being slower than synthesis, leads to an accumulation of partially degraded fibres." - This sentence is grammatically unsound and the meaning can not be extracted. Please rephrase, possibly in two sentences.

Author Response

Q1 : lines 171-172: "A characteristic length scale (Lc), averaging the sum of the equivalent-diameter and perimeter of the polygons, was also calculated." – The provided description is not sufficiently detailed (e.g., the equivalent diemter is not defined). In addition, it appears incorrect, since the average value of Lc in Tbl. 1 (1.64 mm) is lower than the Mean perimeter (4.04 mm). Please correct and clarify, possibly by providing the exact expresison.

R1: The characteristic length aims to represent, in a single parameter, the size and shape of polygons. The exact expression is :

Correction:

“A characteristic length scale (Lc), averaging the sum of the equivalent-diameter () and equivalent-side of the polygons (), was also calculated."

Q2 : line 185: "The false-colour overlay image, with green representing collagen fibres and red representing elastin fibres ," - Exactly the opposite seems to be true. (see the caption to Fig. 2)

Answer 2:

R2: Thank you for this remark. This is indeed a mistake.

Correction:

"The false-colour overlay image, with red representing collagen fibres and green representing elastin fibres”

Figure 3 and Tbl. 2: In this study, you essentially treat the MPM-derived parameter REC as the ground truth, against which the morphological parameters are evaluated. It would be benefitial to the readers if this was reflected in the organization of Fig. 3 and Tbl. 2 (where REC is now presented at a "random" positin between the other values.

R2: Thank you very much for this remark. Table 2 and Figure 3 have been modified accordingly. See answer to the next point.

Q3 : Figure 3: I would find it very informative if actual (not absolute) values were presented here. In this way, readers would also see which correlations are positive and which are negative, without this obstructing the study protocol in any way. (The stronger red and blue colors would then indicate strong positive and negative correlations, respectively).

R3: Figure 3 has been corrected to reflect the actual values of the correlation coefficients. The legend now reads:
"Heatmap of correlation coefficients (r) between all data (skin Elastin-to-Collagen Ratio, skin replica segmentation, roughness and demographic). The stronger red and blue colors would then indicate strong positive and negative correlations, respectively."

Q5 : line 273: "in particular the presence of the collagen signal in the AF channel was carefully considered." - Please explain more clearly (why, how ...)

R5: Thank you for giving us the opportunity to expand on this point.
As explained in Materials and Methods, we used k-Means clustering to automatically determine gray level thresholds. In the first channel, the collagen signal was unambiguously separated from the background signal (k=2). In the second channel, mainly composed of the elastin signal, a part of the collagen signal was also present. A specific algorithm was therefore developed, also based on k-Means clustering but with k=3 to separate the contributions from collagen and elastin and was applied on several projections (top, center, bottom of the image stacks). This allowed the determination of centroid values for the three clusters labeled as background, elastin and collagen.

Correction :
“The Elastin-to-Collagen ratio was determined on an entire 3D image. The imperfections of the acquisition signal related to the presence of the collagen signal in the AF channel were corrected. The background signal and collagen traces in the AF signal were removed using the k-Means clustering method (k=3), so that the elastin signal could be properly separated.”

Q6 : line 283: "So far, all methods agreed to reveal that relative content of collagen decreases with intrinsic ageing based on diminished collagen synthesis and elastin degradation being slower than synthesis, leads to an accumulation of partially degraded fibres." - This sentence is grammatically unsound and the meaning can not be extracted. Please rephrase, possibly in two sentences.

R6: Thank you for your comment. The sentence has been rewritten.

Correction :
“To date, all methods agreed to reveal that the relative collagen content decreases with intrinsic ageing. This can be attributed to a decrease in collagen synthesis; or/and to an accumulation of partially degraded elastin fibers.”

Reviewer 2 Report

It seems to be very interesting if we could predict about the architecture of skin only by its outline surface, or we could predict biological age of patients only by looking/ measuring the skin.

The study looks as first/preliminary  reasearches and should be continued.

Author Response

Dear reviewer,

Thank you very much for your comments. We will proceed as you requested.

Sincerely,

The authors

Reviewer 3 Report

A work with a topic of great interest. At the introduction, a brief review of  methods currently used for skin research should be introduced. 

Author Response

Dear Reviewer,

Thank you very much for your comment. -We understand tou request and we added a short description of the other methods used to explore the skin and relates the finding to skin aging:

Line 65 "Several methods, such as two-photon microscopy, multiphoton microscopy and multiphoton laser scanning tomography, have been used to explore skin microstructure and to relate it to ageing (2-10)."

Sincerely,

The authors

Reviewer 4 Report

1. The discussion should be separated from the study limitations and  conclusions, please make:

4. Discussion 5. Study limitations and 6. Conclusions 

2. The references should be written like that, please adapt them to the MDPI style:

Luo, Y.; Li, J.; Wu, W. Nugget quality prediction of resistance spot welding on aluminium alloy based on structure borne acoustic emission signals. Sci. Technol. Weld. Join. 2013, 18, 301–306. 

3. Please change in all manuscript, the references should be written in the text like that. Please adapt it to the MDPI style [1].

[1-4].

Author Response

Question 1:  The discussion should be separated from the study limitations and  conclusions, please make: 4. Discussion 5. Study limitations and 6. Conclusions 

R1: Thank you for your comment Corrections made accordingly.

Question 2: The references should be written like that, please adapt them to the MDPI style: Luo, Y.; Li, J.; Wu, W. Nugget quality prediction of resistance spot welding on aluminium alloy based on structure borne acoustic emission signals. Sci. Technol. Weld. Join. 2013, 18, 301–306. 

R2: Thank you for your comment Corrections made accordingly.

Reviewer 5 Report

The study of skin-aging is a very interesting topic. This work had proposed “the characteristic length” to characterize microstructure changes of the dermis, it is very interesting, especially in the evaluation of cosmetics. However, this reviewer doesn’t recommend its publication considering following point:

The design of the current experiment cannot achieve its subject- “provide the biological age of the patient”, because the authors choose those females over 60 years old and suffering from osteoporotic”, different peoples with age changing from young to elder should be tested. For current work, to correlate the morphometric indices of the skin surface and bone density may be meaningful for the diagnosis of osteoporotic.

Author Response

Thank you very much for your review.

Author Response File: Author Response.pdf

Reviewer 6 Report

In the present article, the authors suggest a new marker the characteristic length to describe the biological age of the skin. This marker is important since the biological age is not directly proportional to the chronological age due to various intrinsic and extrinsic factors. The study is based on the analysis of skin obtained from women over 60 years old and the authors have described the limitations of their study.

The study is interesting the significant for designing studies for pharmaceutical and cosmetic products etc.

I only have the following minor points

11. Lines 184-188 not 3a but 2a

22.  Line 185 it is written that green collagen fibers and red elastin, but it is the opposite as shown and indicated in Figure 2.

33. It is not clear what are the low and high Lc values depicted on Table 2. Line 206-206 the authors define the low and high Lc based on the characteristic length, but on Table 2 low and high Lc appears to be a value based on elastin-collagen ration, watershed segmentation and average roughness.

44. Line 328 the characteristic length is written as specific length, please use the same term throughout the manuscript

Author Response

Question 1:  Lines 184-188 not 3a but 2a.

Thank you very much for your remark. Corrections made in the revised manuscript. "Collagen and elastin fibres could easily be identified in the SHG and AF images, re-spectively. In the low REC sample, a dense collagen network with thick intertwined bundles is visible (2a) and elastin fibres appeared as thin distinct bundles (2b). "

Question 2: Line 185 it is written that green collagen fibers and red elastin, but it is the opposite as shown and indicated in Figure 2.

R2: Thank you for this remark. This is indeed a mistake. Correction: "The false-colour overlay image, with red representing collagen fibres and green representing elastin fibres”

R3. It is not clear what are the low and high LC values depicted on Table 2. Line 206-206 the authors define the low and high Lc based on the characteristic length, but on table 2 low and high Lc appears to be a value based on elastin-collagen ratio, watershed segmentation and average roughness.

R3: Thank you for your comment. The sentence line 206 has been clarified. Correction : “…samples were grouped according to the characteristic length values to perform statistical analysis: samples with characteristic length below the median value were assigned into a « low_Lc » group; samples with characteristic length above the median value into a « high_Lc » group. The detailed results of statistical analysis between the two groups are summarized in Table 2. The Elastin-to-Collagen ratio was significantly higher in the « high_Lc » group, whereas no significant differences were found between the two groups with respect to patient data…”

Round 2

Reviewer 5 Report

I don't think the experimental design is proper to achieve the object the authors claimed.