Effects of Blood-Derived Products on Cellular Senescence and Inflammatory Response: A Study on Skin Rejuvenation
, Markus Pasztorek 3, Olga Kuten-Pella 1,4, Karina Kramer 1, Christoph Bauer 1, Zsombor Lacza 4,5,6 and Stefan Nehrer 1Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript by Kuhnel et al. shows interesting results on the influence of blood-derived products on human dermal cells and their senescence. However, it is difficult to read and understand the sections Materials and Methods, and Results. I managed to read the manuscript until the section Discussion and I recommend to improve the text and figures for further reading.
Some the most important changes that have to be made you can find below. The text of the paper should be verified by the English native speaker to make it easier to understand.
Abstract. It is necessary to write full names of all blood derivatives instead of abbreviations. Any reader cannot decipher BP, HAS or CPRP since those appear for the first time.
Materials and Methods. Please expand the abbreviation FCS in section 2.1 and shortly explain that the abbreviation will be in use to the end of the text. It is further stated as the normal growth medium but the abbreviation does not fit the long description and can be confusing. Probably you supplemented the medium with 10% Fetal Calf Serum but it is not stated clearly.
Expand PFA abbreviation for the first time in 2.4 section. Please decide on the full name of the fixative: formaldehyde (FA) or paraformaldehyde (PFA). Those are different. FA is PFA dissolved in a solvent, whereas PFA is a white powder.
Line 175. 5 mM potassium ferrocyanide appears twice, one by one.
Line 272. What does “und” stand for?
Please modify chapter 2.9. RNA extraction and qPCR.
GAPDH, COL1 and p21 – what kind of method did you apple to analyse expression of those genes? In case of p16 the analysis “was performed with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories) in a reaction total volume of 10 µl (…) fold change was calculated by delta delta Ct method with reference genes (…)”. It is clear and I think that expression of other genes mentioned above were measured in the same manner. However, it is not clearly stated in the manuscript chapter. One might suppose that classic gel electrophoresis was applied to define gene expression.
Results. Descriptions of Figures are quite complicated to understand and they do not correspond to those in the text. It is difficult to read because of lack commas or dots in sentences. Figure 1, line 326: blue and red areas are not horizontal but vertical. Lines 327 and 328 contain the same information about the p21 expression. What does (e) mean in the line 328?
Replace gamma and beta with Greek letters γ and β, respectively when H2AX and Gal are mentioned.
Figure 2. There are not scale bars in the microscope images.
Line 338 send the reader to upper right of Figure 1b but there is nothing about histone staining.
Figure 4 c and d. The number in the upper part of the Y axis are not clearly displayed. Please remove every other number on the Y scale.
Line 383. The results of CASP 3/7 activities are not shown in Figure 4.
In chapter 3.4.1. the authors mentioned about CuSO4 treated cells. However, there is nothing about such treatment in the Materials and Methods section. What was the motivation to treat the cells with CuSO4? It should be clearly explained and appropriate references must be provided.
Line 401 and 407, respectively. There are not Figures 6a and 6c.
In my opinion, the paragraph starting on line 407 is incomplete. The sentence on line 409 ends abruptly and is meaningless.
Figure 7a and c. It is better to use less 0 digits. They might be replaced with the description [number x1000]. Again, please remove every other number on the upper part of the Y scale. The description of Figure 7 is incomprehensible. Please read it carefully.
Line 418. The results of cell morphology are not shown in Figure 7. Moreover, the authors presents cell size not the morphology – cubic, fibric, oval etc. cell shapes.
Figure 8a and b present the same results. Please use one figure – a or b to indicate changes in cell size. It also concerns Figure 8c and d.
Line 430. The results of p21 expression are not shown in Figure 8.
Figure 9a and b present the same results. Please use one figure – a or b to indicate changes in p21 expression. It also concerns Figure 9c and d, e and f, g and h.
Line 461. The results of IL-6 (and IL-8) concentration are not shown in Figure 9.
Figure 10a and b present the same results. Please use one figure – a or b to indicate changes in IL-6 and -8 concentrations. Thus it concerns Figure 10c and d, e and f, g and h.
Comments on the Quality of English LanguageSee my comments above.
Author Response
Dear reviewer,
Thank you for improving the manuscript and your valuable input.
I have overworked the whole text and put some data in a supplemental file.
Please find the reply to your comments in the following file.
Best regards Harald Kühnel
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsReview of:
Effect of Blood-Derived Products on Senescence Associated Secretory Phenotype in an Etoposide Induced Senescence Human Dermal HDF Model
Comments to the Authors
General remarks:
All Figures need to be reworked to be more accurate and to explain and clarify the experiments made. The text mentions days but the graphs are in hours. This makes it really hard to evaluate the data. Also, the legends need to mention the number of replicates for each graph.
What is the reasoning between the different length of treatments throughout the manuscript?
Better proof reading is necessary, the figure numbers do not match the text!
The results section does not contain any conclusion for each experiment and no reasoning why those experiments have been performed.
Specific remarks:
Abstract:
A lot of abbreviations need to be spelled out: BP, XTT, HAS, CPRP.
Line 35: Those publications need to be updated, being from 2011 and 2016 is not from “recent” years.
Line 66: This is the first mentioning of STAT1.
Line 125: Which cell line was used, please add the catalogue number.
Figure 1: The authors say that the yellowish area in 1a represents 20 doublings, but it goes from 0 to 20. Either the authors rephrase that or put a line at at PD = 20. The black line with x is not clear. It would be better to stop the x-axis at the end of this line. Is it 50 + 21? That way the blue and red vertical lines (called horizontal in line 326!!) are better resolved (they should span 2 and 3 days respectively?). Please add the key of the labels (Y, RS, ES) used in 1b into the legend of the table. Ehat is the RS control? 1c: I don’t understand the FCS vs “growing” comparisons. This need to be explained better. Is this the quantification from the western in 1b? How has p16 in 1d being measured? What is rge, ETO? What is time 0? What is the blue area? What is the black vertical line at -3 days? Please add statistical analysis to this line graph. Are these timepoints at 8 and 15 days, respectively?
Line 338: is it Fig. 1b or 2b?
Figure 2: Please add a scale bar to each panel. The “growing” cells are much denser. A better control would be untreated cells at the same density as the treated ones. Please add statistics for these observations to test for significance.
Figure 3: Please spell out ETO? The lines are very hard to identify. Easier to see colors and a lighter background may be better. It looks like all the cells in the blue area have been treated with etoposide. I don’t see a significant difference between 25 and 50 uM etoposide. Please add statistical analysis to these line graphs.
Figure 4: Can the better growth be due to treatment with human instead of bovine serum? Fig. 4b, please move the legend away from the red area. All samples should be labeled as “growing”. Why is there no error bar? Again, I don’t see a significant difference between 25 and 50 uM etoposide. Please add statistical analysis to these line graphs.
Line 385: Please correct condensation to concentration
Figure 5: Is it 24h or 48 h of etoposide treatment? This is not clear in lines 380 and 385. There are too many error bars. Just compare the HAS treatments to FCS and to CPRP. It looks like CPRP is not significant different to FCS control. In contrast, to what the authors state in lines 378 to 384. Only 10% HAS is significant different from FCS and CPRP. Why is there no statistics in 1c and 1d? Please add.
Figure 6: What is the reason to use CuSO4? The effect of this salt needs to be evaluated at some point in this manuscript. Please add experimental details to the legend. Please ad representative photographs of the SA-b-Gal-stained cells. What is “repl. senescent”?
Line 401, 407: please correct to Fig. 7a and 7c, respectively.
Figure 7: There is no conclusion to Fig. 7, How is the %vitality measured.
Line 413: Please correct to (c).
Line 416 to 425: please correct to Fig. 8.
Figure 8: In a and c all error bars overlap but in b and c there is significance. How come? Where is the “growing” curve in a and b?
Line 429 to 452: please correct to Fig. 9.
Figure 9: It is not clear which panel shows expression data at which time point (early, late, day 8 day 15?). Please label the panels and the legend better. Also, the x axes show hours but the text is in days. Please synchronize this. Nothing seems to be significant to the FCS control. There is no conclusion to Fig. 9. Where is the “growing” curve in a, c, e, and g?
Line 457 to 476: please correct to Fig. 10.
Figure 10: Where is the “growing” curve in a, c, e, and g? Nothing seems to be significant vs the FCS control.
Comments on the Quality of English LanguageSome sentences are hard to understand, please have a native speaker read the manuscript.
Author Response
Dear Reviewer,
In general, I overworked the whole manuscript to make it clearer. The treatment conditions should be better understandable because I added a treatment figure for better understanding. Number of replicates were stated in the legends. Reasoning of experiments was added.
Thank you for your valuable input to improve my text.
I hope I fit the requirements now. Please find the details in word file.
Best regards Harald Kühnel
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThis research explores the impact of blood-derived products (BP), such as citrate-platelet-rich plasma and hyperacute serum, on senescence-associated secretory phenotype in etoposide-induced senescence in human dermal fibroblasts. Two treatments with BP were administered, early and late, and evaluated through various assays. Results indicate slightly positive effects on skin rejuvenation, with improved collagen expression and healing-associated inflammatory markers. The study emphasizes the complexity of treating aging skin with BP, highlighting no observed detrimental effects and overall positive outcomes.
The manuscript would benefit from the following revisions:
Major revisions
1. Ensure inclusion of a statement confirming approval by the appropriate institutional/national research ethics committee, adhering to the Declaration of Helsinki's ethical standards, and specifying the committee's name. Also, certify that the study involved informed consent from all participants.
2. Address the inconclusive nature of Figure 1b for western blotting, where the molecular ladder is too bright compared to blurry bands of senescent control and etoposide-induced senescent cells. Consider repeating the experiment or providing a clearer image.
Minor Revisions
1. Condense and refocus the introduction section, eliminating irrelevant content to streamline the manuscript.
2. Specify details for HDF cells, including the cell line number, batch information, and recent authentication method.
3. Expand the protocol for treatments (2.8.2.-5) to provide more detail on the number of replicates used for statistical analysis.
4. For RNA extraction and qPCR (2.9), include information on how cell differentiation was confirmed.
5. Clarify the method used for determining cell morphology in 3.4.2, and correct the reference to figures (7a,b) as it should be Figure 8.
6. Correct figure references throughout the text, ensuring consistency between text and figures. Address discrepancies such as missing figures (e.g., Figure 8e) and confusion between IL-6 concentration in supernatant and pPCR of mRNA expression of p21 (Figure 9).
Author Response
Dear Reviewer,
Thank you for your valuable input. I improved the text according to your advice. Please find detailed reply in the document.
Best regards Harald Kühnel
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsIn this paper, the authors investigated the effect of blood-derived products on the senescence of human dermal HDFs. While the paper could be of interest, there are some issues that should be addressed before considering it suitable for publication.
A recent study outlined an algorithm for correctly discriminating among cycling, stressed, quiescent, and senescent cells. This involved evaluating the expression of Ki67 (a marker of cycling cells), pRPS6 (an active protein synthesis marker proposed to distinguish senescent from quiescent cells), and senescence-associated beta-galactosidase (SA-β-gal) activity. The Ki67 (−) pRPS6 (+) SA-β-gal (+) cells were identified as senescent cells (DOI: 10.3390/ijms22063102). The authors are encouraged to refer to this algorithm to better characterize cellular senescence, specifically identifying senescent cells as Ki67 (−) SA-β-gal (+) cells.
The reliability of SA-β-gal activity as an indicator is questioned due to its non-specific nature, as it is not exclusive to cell senescence. Some studies have indicated that factors other than cell senescence can increase SA-β-gal activity (DOI: 10.1016/j.exger.2005.07.011). Therefore, the authors need to specify in the methods section how they quantified the SA-β-gal experiments. Counting SA-β-gal-positive cells (blue) could be the preferred quantification method. To comprehensively assess cellular senescence, the authors should analyze the expression of key proteins involved in the onset of senescence and cell cycle exit, including p53, p21, p16, RB1, and RB2. This analysis is crucial for a thorough definition of cellular senescence (doi: 10.1016/j.tcb.2018.02.001).
The authors claimed to have analyzed the effect of blood-derived products on SASP. However, it appears they evaluated only some factors involved in the modulation of inflammation, which are part of SASP content.
The authors claimed to have analyzed the effect of BP on SASP. However, they evaluated only some factors involved in the modulation of inflammation, which are part of the SASP content. The SASP contains many more factors, and the authors should provide further clarification on this issue (see, for example, doi: 10.1186/s12964-023-01280-4).
Author Response
Dear Reviewer,
Thank you for your valuable input. I improved the text according to your advice. Please find detailed reply in the document.
Best regards Harald Kühnel
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors revised the manuscript extensively, while the introduction and the results are improved the discussion is not acceptable in the current form. As it stands most of the discussion is about the methods (pro and cons of beta-gal staining and other methods to quantify senescence etc.) used and not a discussion about the results of using BP in rejuvenation of fibroblasts in context of known literature.
Line 61: excretion to secretion
Figure 3d: Please add the original picture of the total western blot as supplement.
Figure 4: still no scale bars!
Figure 5: Are the bars significantly different from each other? If not please state that in the text/legend. Please add panels with BetaGal staining as in figure 4.
Line 374: What is "replicative senescence"?
Figure 6c: The control experiment should be 10d FCS treatment and not growing cells on day 1. This means all the the treatments are not significant different. Please remove bar with growing cells on day 1.
Line 401: Is this significant?
Figure 8 and 9: The author should focus on the BP treatments being significantly different from the FCS control treatment.
Line 438: You need to discuss this.
Lines 449-450: What are those numbers? pValues?
Line 467: Why was size measured? Please explain.
Line 478-479: This should go to material and methods.
Lines 490-494 are repeated in lines 503-507.
Comments on the Quality of English Language
Please have a native English speaker go over the manuscript
Author Response
Dear Reviewer,
I improved my text following your kind advice, I hope it fits your requirements.
I added a file where you can see my changes, additionally I marked the changes in green in final text.
Best regards Harald Kühnel
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsKindly provide a full image of Western blot in good quality which clearly shows molecular ladder and your samples.
Please provide information on the authentication method used to ensure the research was indeed conducted on the HDF cells.
Author Response
Dear reviewer,
I added a full image of the westernblot in the supplemental section, as well as the Information I have about my cells.
Thank you very much for your valuable input.
best regards Harald Kühnel
Author Response File: 
Author Response.docx
Reviewer 4 Report
Comments and Suggestions for Authorsnone
Author Response
Dear Reviewer,
thank you for your feedback.
best regards Harald Kühnel
Round 3
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors clarified their experimental approach and expanded somewhat their discussion, although it still lacks a thorough reflection of their results of using BP in rejuvenation and senescence of fibroblasts in context of known literature. Rather than discussing various ways of measuring senescence.
Fig 5: Is this bar graph the quantification of the Fig4b? If not can the authors add the panels with exemplary cell culture images of the evaluated cells?
Line 568: "Studies have shown that certain signals such as transforming growth factor beta 1"... , please add references for those studies
Please have a native English speaker go over the manuscript
Author Response
Dear Reviewer,
please find the comments in attached file
best regards Harald
Author Response File: Author Response.pdf
