Molecular Basis for Mechanical Properties of ECMs: Proposed Role of Fibrillar Collagen and Proteoglycans in Tissue Biomechanics

Round 1

Reviewer 1 Report

In the present manuscript, “Molecular Basis for Mechanical Properties of ECMs: Role of Fibrillar Collagen and Proteoglycans in Tissue Biomechanics”, the Authors use a non-invasive approach, the vibrational optical coherence tomography, to measure the skin elastic energy storage and propose a model describing the skin collagen fibers organization. Authors also claim that the mechanical properties of the skin change with age. Although the idea behind this work might be interesting, the data presented are insufficient to warrant a scientific publication.

Major points:

-as the method is non-invasive, why the study is based on only three skin samples?

-the three skin samples analyzed are from individuals of very different age (from 25 to 71 years of age). In addition, Authors never show the data obtained by each single skin sample but always a unique graph with no standard deviations. To make a solid study, authors should investigate more samples of similar age and then compare the skin mechano-physical properties of different aged samples. Only in this way they can assert and claim that ageing “leads to down regulation of mechano-transduction and tissue metabolism leading to tissue atrophy” (as indicated in the abstract).

-no experimental evidence show that the decellularized human skin mainly contains collagen type I.

-A better explanation of the data indicated in Fig. 5 should be provided. It is not clear to this Reviewer why the Authors have chosen these particular conditions (0 and 90 degrees) and why they refer to a variation from about 2.2 to 3.5 MPa (which is the unit of measurement of the elastic modulus) even though in the graph it is represented the weighted displacement (measured in µm).

-Authors should discuss the result and the implication of the “shift of the dermal collagen peak from about 90 Hz (perpendicular) to 110 Hz (parallel)” that is completely missing.

-Fig.10 Authors should clarify how they extrapolate the depicted model. It is not clear from which data it is based on.

Minor points:

-more references should be included in the first part of the introduction, not only book chapters s written by the first author himself.

Author Response

1#-as the method is non-invasive, why the study is based on only three skin samples?
-the three skin samples analysed are from individuals of very different age (from 25 to 71
years of,age).
We have added data published previously based on VOCT measurements on human skin
from over 20 individuals illustrating that the modulus of skin in vivo is 2.34 +/- 0.691 MPa
which is statistically lower than the modulus on stretched skin reported in revised Table 2
at angles of 0 and 67.5 degrees. The increased modulus measured after stretching is due to
alignment of the collagen fibers with the stretch loading direction.
In addition, Authors never show the data obtained by each single skin sample but always a
unique graph with no standard deviations.
Figure 4 shows the standard deviation of the modulus values.
To make a solid study, authors should investigate more samples of similar age and then
compare the skin mechano-physical properties of different aged samples.
The modulus of skin in vivo without stretching is not significantly dependent on age under
physiologic conditions and as previously published and also based on the data of Table 2. It
only changes on stretching but depends on the orientation of the loading.
Only in this way they can assert,and claim that ageing “leads to down regulation of
mechano-transduction and tissue metabolism leading to tissue atrophy” (as indicated in the
abstract).-no experimental evidence shows that the decellularized human skin mainly
contains collagen type
We have revised the manuscript to more clearly state that we hypothesize that ageing
“leads to down regulation of mechano-transduction and tissue metabolism leading to
tissue atrophy”. This is a hypothesis based on the literature studies that show changes in
cellular and tissue behavior with age.
I.
-A better explanation of the data indicated in Fig. 5 should be provided.
We have revised the explanation of Figure 5. These two angles were chosen to show the
raw weighted displacement versus frequency data that was used with equation (1) to
calculate the change in modulus.
2
It is not clear to this Reviewer why the Authors have chosen these particular conditions (0
and 90 degrees) and why they refer to a variation from about 2.2 to 3.5 MPa (which is the
unit of measurement of the elastic modulus) even though in the graph it is represented the
weighted displacement (measured in μm).
Figure 5 is used to calculate the resonant frequency of the dermal collagen at different
angles of stretching. The resonant frequency of the skin increased from 90 to 110 Hz and
using equation (1) this converts to changes in modulus.
-Authors should discuss the result and the implication of the “shift of the dermal collagen
peak from about 90 Hz (perpendicular) to 110 Hz (parallel)” that is completely missing.
We have added a discussion of the significance of the shift in the collagen dermal peak to
the text.
-
Minor points:
-more references should be included in the first part of the introduction, not only book
chapters s written by the first author himself.
We have added additional references.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors measured the frequency dependence of deformation of tissue components within skin samples taken from three individuals. I believe the study focused on an important phenomena as the mechanical contribution of proteoglycans has been somehow neglected in the past. However, several improvements are suggested to make the paper more beneficial to the Journal readers:

-I do not believe that the last sentence of the abstract is justified by your data. You did not study these phenomena such as UV light exposure, aging, or tissue atrophy.

-Part of the terminolog yused in the Figure 1 is not compatible with the official International Terms for Human Histology (Terminologia Histologica). The authors should use either Latin terms or their English equivalents, namely: Endotende (Lat) or Endotendon (Eng), Epitendo / Epitendon.

-I was missing a clearly formulated and testable working hypothesis.

-2.1. Subjects: Listing the ages of the three individuals would be much more informative than providing the age range. I am completely missing crucial information for repeatability of your measurements, such as: Which parts of arm and leg were the skin smaples taken from? What was the time interval betwen removing the samples and the measurement? What was the size of the samples? What was the number of samples? Number of measurements? Temperature, Humidity? What device was used (manufacturer etc.).

-Table 1: Do all the data refer to human tissues? Or were these data from other species as well?

-3. Results. In the first paragraph, you write about decellularization. Were your samples decellularized? If so, what was the protocol? If not, why is this mentioned in the Results?

-Many graphs are missing units.

-Table 2: What was the hypothesis tested by the Student T-test? Whar were the p-values? 

-Provide all the primary data along with your manuscript (preferably as an e-supplement), not just the statistical analysis.

-I believe the Results should be more clearly separated from the Discussion and the interpretation of your data.For example, the text on page 13 below the Fig. 8 - how is this justified by your measurements? In the Results, provide your results only. The rest goes to the Discussion.

-Figure 6: At what frequency was this done?

-Font size in Figure 8 an 10 is too small to be readable in the printed form.

-Section 4.1 - provide reference to the Langer's work zou are referring to.

-The references 6, 12, and 27 have an issue with the typesetting.

-It would be really useful if you could formulate one or more hypotheses generated by your work. I believe that sharing your insight with other researchers might help them to focus on further work in this field.

Author Response

The authors measured the frequency dependence of deformation of tissue components
within skin samples taken from three individuals. I believe the study focused on an
important phenomenon as the mechanical contribution of proteoglycans has been
somehow neglected in the past. However, several improvements are suggested to make
the paper more beneficial to the Journal readers:
-I do not believe that the last sentence of the abstract is justified by your data. You did not
study these phenomena such as UV light exposure, aging, or tissue atrophy.
We have added that we hyporthesize to the sentence.
-Part of the terminology used in the Figure 1 is not compatible with the official
International Termsfor Human Histology (Terminologia Histologica). The authors should use
either Latin terms or their English equivalents, namely: Endotende (Lat) or Endotendon
(Eng), Epitendo / Epitendon.
We have changed the terminology in Figure 1 to epitendon.
-I was missing a clearly formulated and testable working hypothesis.
We have added to the manuscript that we hypothesize that the modulus of skin is
dependent both on the direction of the collagen fibers in the dermis as well as the cellcollagen
interactions that are present that oppose the stress in the collagen fibers.
-2.1. Subjects: Listing the ages of the three individuals would be much more informative
than providing the age range.
We list the ages of the three subjects in the methods.
I am completely missing crucial information for repeatability of your measurements, such
as: Which parts of arm and leg were the skin samples taken from? What was the time
interval between removing the samples and the measurement? What was the size of
the,samples? What was the number of samples? Number of measurements? Temperature,
Humidity?
2
These studies were all done in vivo. Therefore, the samples were not removed from the
subject. We have added additional details to the methods that have been published many
times in the references.
What device was used (manufacturer etc.).
-Table 1: Do all the data refer to human tissues? Or were these data from other species as
well?
All the measurements were made in vivo on human tissue as indicated in the methods.
-3. Results. In the first paragraph, you write about decellularization. Were your samples
decellularized? If so, what was the protocol? If not, why is this mentioned in the Results?
-Many graphs are missing units.
Only the data on decellularized dermis was obtained in vitro. All other data is from in vivo
studies. We have this information to the methods. We have added refernces to the
decellularization that were published previously.
-Table 2: What was the hypothesis tested by the Student T-test? What were the p-values?
-Provide all the primary data along with your manuscript (preferably as an e-supplement),
not just the statistical analysis.
We tested whether the modulus values at different angles at a strain of 5% were
significantly different than the unstretched skin. We have added the p vaues to the revised
manuscript in Table 2.
-I believe the Results should be more clearly separated from the Discussion and the
interpretation of your data. For example, the text on page 13 below the Fig. 8 - how is this
justified by your measurements? In the Results, provide your results only. The rest goes to
the Discussion.
Figures 8, 9 and 10 and the associated text have been moved into the Discussion section.
-Figure 6: At what frequency was this done?
As discussed in the text, the energy stored is related to the elastic modulus of the collagen
fibers and the strain. The modulus of the collagen fibers depends on the resonant
3
frequency via equation (1) and varies depending on the strain. Therefore the resonant
frequency varies from 90 to 110 Hz as discussed in Figure 5.
-Font size in Figure 8 and 10 is too small to be readable in the printed form.
We have increased the font sizes in Figs 8 and 110.
-Section 4.1 - provide reference to the Langer's work Zou are referring to.
A reference is added in the revised manuscript to Langer’s lines and the orientation of the
collagen fibers in skin from previous review paper.
-The references 6, 12, and 27 have an issue with the typesetting.
These references are corrected in the revised manuscript.
-It would be really useful if you could formulate one or more hypotheses generated by your
work. I believe that sharing your insight with other researchers might help them to focus on
further work in this field.
We have added several hypotheses to the revised manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The present version of the manuscript is even more difficult to understand.

The answer to the Reviewer’s request of showing the single data of the skin samples from each analyzed individual (2 males and 1 female) has been the addition of 20 individuals as controls who were previously published. This does not address the issue as no single data have been shown. In line with this, the sample size in Table 2 is totally unclear. Most of the Authors’ responses are incomplete and almost speculative, so they don’t clarify the Reviewer’s concerns.

This Reviewer still believes that the quality of the data and the manner they are presented is insufficient to warrant a scientific publication.

Author Response

All data is now shown in the supplement as indicated in my email. We have no way of answering the other comments since they are not specific questions.

Reviewer 2 Report

From my point of view, the authors improved the manuscript significantly. However, two important points raised during the first round of review were not addressed, namely:

-Provide all the primary data along with your manuscript (preferably as an e-supplement), not just the statistical analysis. No primary data were provided or uploaded to some repository. This point was not addressed by the authors at all.

-Many graphs are missing units. Indeed, even the revised version is lacking units in graphs in Figures 3, 5, 6, 7. I do not believe it would be a sign of good scientific practice to publish graphs without declaring the units of variables on both x- and y-axes.

Author Response

All Primary data has been added to the supplement.

All graphs have units added.