Engineered GO-Silk Fibroin-Based Hydrogel for the Promotion of Collagen Synthesis in Full-Thickness Skin Defect

Round 1

Reviewer 1 Report

1. Please consider using appropriate linking words to improve the coherence of the text. Additionally, it is recommended that the authors revise and polish the entire article to correct grammar mistakes.

 

2. Since it has been mentioned in the text that "GO combined with chitosan can induce spontaneous differentiation of myoblasts" and "each of the components of our hydrogels individually promote healing of skin wounds", is it necessary to verify if the effect of DSF/Cs/GO hydrogels is superior to Cs/GO hydrogel?

 

3. The authors conducted research on the biocompatibility of the material, but further investigation and supplementation may be necessary for the physicochemical properties in order to apply the material in production and clinical settings.

 

 

4. Background descriptions for hydrogel wound healing can be strengthened by citing 10.1016/j.carbpol.2020.117213; 10.1016/j.carbpol.2020.116585 and what are the advantages of the current work compared to published articles?

 

5. The results contained too much information. Using a table format may make the results clearer and more concise. Figure 4 can be optimized by adjusting its color scheme, scale, and arrangement to better display data and visual effects. What’s more a brief explanation of the figure can be provided in the annotation.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 2 Report

Line 118: Please add the cocoon to solvent ration (g/ml or % (w/v).

Line 119: Please specify the PEG used in terms of Molar Mass and the concentration during dialysis.

Line 126: Chapter 2.1 ends with a fibroin solution and here the authors start with a porous material. Please include generation of the porous material, probably made by freeze-drying, however, this needs to be defined. In addition how much porous silk fibroin was dissolved (concentration missing).

Line 129: Concentration of acetic acid is missing.

Line 131: Source of graphene oxide is missing.

Line 133: In what ratio were the different solutions mixed to form the blend, please specify.

Line 162: What is the reason for the chosen wound diameter? Please explain.

Line 190: For animal studies, it is important to calculate the minimal sample size to detect an effect if the effect actually exists (statistical power). The statistical power is defined as the probability of correctly rejecting a false null hypothesis. This is completely missing and it is not explained how the sample size was calculated. In addition, usually a dropout rate of 1 to 2 animals is taken into consideration when calculating the necessary animal number. Furthermore, the information how many animals died or survived during the study is missing.

Line 196: The authors confirmed the preparation of GO. Was the GO synthesized by the authors or why is this part important? If the GO was synthesized by the authors this step needs to be explained in the methods section.

Line 205: The authors stated a proliferation supporting activity of the hydrogels but the MTS assay shows only viability. Proliferation could be measured if two different timepoints are measured and an increase of viable cells could be detected. However, the statement of proliferation support need to be changed. In addition, the  viability of fibroblast (Fibr) shows ca 0.55 OD and the hydrogels are below 0.25 OD. This is almost 50% reduction of viability after 24 h. According the European standard ISO 10993 only 20 % reduction of viability is classified as cytocompatible. Furthermore the contrast of the cell pictures is pure and cells are hardly visible.

Line 213: This is confusing because in line 144 the authors stated that they seeded the cells on pre-coated plates. What was done exactly, precoating and seeding or seeding and hydrogel coverage?

Line 220: The support of proliferation by the hydrogels is not shown by the results!

Line 244: The authors state a significant difference in coherence of the direction of collagen fibers but there is no significance shown in the according figure (*p<0,05).

Line 251: The authors state “cases of complete epithelization were observed”. How many? Maybe a table would be good summarizing such observations per group.

The complete in vivo results part need to be revised. It is very difficult for the reader to follow the descriptions of the results. Thus, it is necessary to indicate the corresponding illustrations to which the statements refer, so that the reader does not have to search for a long time.

Line 335: Neither for the statement of proliferation support nor for cytocompatibility the authors show sufficient experimental data.

Line 370 – 377: Contraction is fine for rodents, but what does this mean for wound healing in humans?

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The manuscript was carefully revised according to the reviewer's suggestions and provide detailed explanations. I recommend it to publish.

Author Response

Dear Reviewer,

thanks a lot for consideration of our manuscript 

 

Reviewer 2 Report

Most of the points have been explained and implemented by the authors. However, one very important aspect, namely the in vitro analysis, is still not sufficiently clarified. Although the authors explain the function of the MTS assay correctly, the MTS test does not determine proliferation but only the vitality of the cells at the time of the assay by determination of the metabolic activity of the mitochondria of the cells. Thus, if a small number of cells is highly metabolically active compared to a high number of cells with low metabolic activity, the MTS results could be the same.  Therefore, proliferation can only be determined by culturing equal numbers of cells per test material for different incubation times (e.g. for 12 and 24 h) and then comparing the viability values of both time points. If the vitality after 24 hours is higher than after 12 hours, then the authors might speak about proliferation. Although the authors have included in the method section that the assay was performed after 1 h and after 24 h, they only show one column per sample in Fig. 2.B. The results of the vitality assays at both time points are not shown. Thus, no proliferation can be derived from this!

Furthermore, the OD values shown in the new Fig 2 B differ from the original Fig 2. How can this be explained? Why are the values of the samples DSF/Cs, DSF/Cs/GO and Cs now much higher compared to the "fibroblasts"?

In addition, it is not clear what was compared for the significance calculation. The authors describe in line 255-257 "compared to control (of ordinary cell culture medium)" but in the discussion (line 388 - 390) it can be read out that the "fibroblasts" column is the control! 

In summary, it should be noted that neither the proliferation of the cells could be proven nor the question of cytocompatibility was addressed.

Author Response

Thank you very much for this comment.

We have made changes to the manuscript

Metabolic activity directly correlates with the number of cells, which increases with the increase in the number of proliferating cells (increase in the number of mitochondria). The MTS test has been used in many studies to assess proliferation and viability. The MTS test has become the gold standard for proliferation and cell viability studies (https://doi.org/10.1016/j.canlet.2010.04.023).

In all comparison groups, the same number of cells of the same type (human fibroblasts) were used "96 well plates with pre-coated with hydrogels DSF/Cs or DSF/Cs/GO or Cs in a volume of 50 μl per well, 10,000 cells per well in 200 μl of aMEM + 10% FBS medium". Cells with both high and low metabolic activity simultaneously enter the wells. Cells were incubated in equal numbers on the material for an equal amount of time (24 hours). After one hour of incubation, the studied samples showed complete uniformity. They did not differ from each other, which may indicate the absence of an acute toxic reaction of cells in vitro and the same metabolic activity.

As previously mentioned, the in vitro component of the research endeavor underwent a redesign, resulting in the emergence of novel data points.

The study's authors believe that the MTS test data indicates in vitro cell proliferation. Of course, we will consider your comments in future studies, in particularly using the Real Time Cell Analysis xCelligence.

 

Round 3

Reviewer 2 Report

My last comments have been implemented satisfactorily and, from my point of view, there is no longer any obstacle to publication.