Supramolecular Hydrogel-Wrapped Gingival Mesenchymal Stem Cells in Cutaneous Radiation Injury

Round 1

Reviewer 1 Report

The study of Nie and colleagues showed the potential of gingival derived-MSC (GMSC) associated with a hydrogel in reducing skin injury in an in vivo model of cutaneous radiation injury. To investigate the mechanism that generates the improvement in vivo, the authors investigated the effect of GMSC-conditioned medium in vitro and explore its potential. However, this idea (of the use of CM) is not clear from the start. The manuscript is interesting, showing potential applications of gingival derived-MSC, but some points need to be improved to make the manuscript clearer.

1 - The abstract is confusing. The initial introduction and the objectives of the study are not clear. Please rewrite it. Some critical points are:

- In the first sentence there are many repetitions of “skin” and “radiation” words.

- What did the authors mean by “without carcinogenicity than other stem cells” Are the cells less carcinogenic than other MSCs or compared with pluripotent stem cells?

- Is it "...prevents rapid spread of cells" (line 20)?

- Line 26: “…and reduce inflammatory response”. Which experiment demonstrates this effect?

2 – About Introduction section:

- Please include a reference for the sentence in lines 44-46.

- The MSCs are characterized by being multipotent and not pluripotent. Please review.

- The effects of MSC treatment cited by the authors for skin trauma are largely related to the factors secreted by MSC. This paracrine potential could be described in the Introduction section since the authors will explore this in the experiments.

- Please confirm the reference in line 65. Is it [27] or [26]?

- I suggest that the authors update the introduction since a great part of the references were published more than 3 years ago.

- In lines 67-68 the authors present the hydrogel and mention its use only for tendon regeneration. It would be interesting to increase the references and justification of the use of hydrogels with cells, citing other examples, e.g., for wound healing. Also, it is unclear why the authors choose to work with pY-Gel. According to the studies cited by the authors, this hydrogel was used as an adjuvant for vaccines and to produce antibodies. Why use this hydrogel with GMSC? Would this be different from other peptide hydrogels? 

3 – The Material and methods section needs to be improved. Many details are missing that greatly impair the understanding of the manuscript and would make it difficult to reproduce the work.

- Were the GMSCs previously characterized based on ISCT criteria? If so, please include the information or the reference.

- What is the cell culture medium used for each cell line?

- The authors used a conditioned medium from GMSC. However, there is no information about how it was collected (methodology, number of donors, others). This information should be included.

- In the description of the “2.11 Transwell migration assay” it was indicated that CM was used as a “supplement”. What was the concentration/ratio? Is it the same for all in vitro assays?

- Information is lacking in the hydrogel description. If it is based on another study, please include the citation. Also, what is the concentration of the hydrogel used in the assays?

 - Why did the authors choose to treat the cells with CM first and then irradiate them? Would this be comparable to the in vivo model? How long was the CM treatment before irradiation?

- It would be interesting to include information on how radiation exposure was decided for each in vitro assay.

4 – In the results section, some information can be included to improve understanding.

- I couldn’t find the Supplementary figures. Please, include.

- Considering that the GMSCs were encapsulated in the hydrogel, did their behavior (e.g., proliferation rate, viability) remain the same as in the standard culture condition? Is the hydrogel cytotoxic at some concentration?

- In lines 237-238 authors state “We observed that the pY-Gel prevented the rapid diffusion of GMSCs from the injection sites.” What experiment demonstrates this? Please, include.

- Please, check all the figures and figure legends. The information described in the legends is, sometimes, different from what is seen in the figures.

- Why were some in vitro experiments performed with radiation exposure of 4 Gy and others of 8Gy (e.g. Figure 6)? This raises my concern since to be able to relate and compare the results it is necessary that they are all based on the same parameters.

- In figure 3 the authors show the results of two migration assays, each one from a different cell. Were the two assays performed with both cell types?

- It would be interesting to include an experiment to verify the cell death rate (necrosis or apoptosis) of GMSC treated and non-treated cells after radiation exposure.

- Figure 4G-H and Figure 5D-H are not described in the results. Please include.

- In figure 5, it would be very helpful and interesting to quantify the Western Blot results. Also, in Figure 5A, the authors showed a time-dependent protein level of EGFR, STAT, and others. Then, they choose to work with 6h in other assays. Why 6h?

- The authors explore the activation of EGFR and STAT3 in the in vitro and in vivo assays, but it would not be possible to say with certainty that STAT3 is being activated only by EGFR. This must be changed throughout the text. Furthermore, in the siEGFR experiments, it would be interesting to show if there is an alteration in the phosphorylated STAT3.

- Did the authors characterize de CM? Considering that, in vivo, the GMSC were encapsulated in a hydrogel and that 3D microenvironment could change the secretory profile of cells, it would be interesting to perform a CM characterization and verify if the secretome of GMSC were similar in standard condition and in pY-Gel.

5 – The Discussion could be improved. There are few discussions and comparisons with the literature. In addition, it would be interesting to explore the perspectives of the study.

- The sentence in lines 493-495 should be revised. The authors state that “the pY-Gel self-assembling peptide hydrogels for GMSCs intervention may be the best option for the treatment of radiation-induced skin injury”; however, the authors only tested the pY-Gel and did not compare it with other hydrogels. Please review this affirmation.

- In addition, the authors showed the CM’s positive effects. Would it be possible to use the hydrogel with the conditioned medium instead of the cells? This is hardly explored in the discussion. 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

In the study entitled "Supramolecular hydrogel-wrapped gingival mesenchymal stem cells in cutaneous radiation injury", Shasha Nie and colleagues present interesting results for a therapeutic alternative to skin damage induced by radiation. Though that, some aspects need to be clarified and the text improved to consider the manuscript for publication. 

Major revisions

1.       Supplementary figures are not available, and this make it difficult to review the manuscript. Please include them.

2.       Using the in vivo model, mice were treated with pY-Gel+GMSCs 24h after radiation exposition to evaluate its effect on skin damage. When in vitro experiments were performed, cells were first incubated with conditioned media from GMSCs and then exposed to IR. Why did the authors decide to apply this experimental desing? It seems counter-intuitive, considering that these experiments seek to validate what was observed when mice were treated after radiation injury. Please, justify.

3.    Taking into account that Y-Gel can be used as vaccine adjuvant and optimize immune responses, did authors considered a possible effect of Y-Gel and Y-Gel-GMSCs on the immune response of mice when radiation injuries were treated? I strongly suggest to authors to evaluate the extent of immune response elicited by Y-Gel and Y-Gel-GMSCs in treated mice.

Minor revisions

1.       In general, English language doesn’t need major corrections, but the abstract section needs to be reviewed. As examples, in the first sentence, the word “radiation” is repeated 4 times; and in several cases, capital letters are employed after a comma.

2.       Authors describe MSCs in general and GMSCs as pluripotent, while these cells present a multipotent capacity. Please, correct.  

3.   In introduction (lines 49-52), authors describe the regenerative potential of MSCs, without mentioning the role of the secretome in these stem cells effects. I suggest including this, considering that conditioned media was used to validate the regenerative effect seen in vivo.

4.   In scratch-wound healing assay and trans-well migration assay (sections 2.10 and 2.11) authors do not specify whether the treatment with CM was before or after radiation exposition. Please, clarify. Also, in trans-well assay, authors incubated cells with complete fresh medium supplemented with or without GMSCs-CM. In the case of supplementation, which dose of CM was used in combination with fresh media?

5. Please include in Materials and Methods section a more detailed explanation of hydrogel preparation and GMSCs encapsulation. In lines 237-238, authors say that pY-Gel prevented the diffusion of GMSCs from the injection sites. How authors measure this? Please, clarify.

6.       Are HaCat cells of human or murine origin? Please, specify.

7.  How authors collected the conditioned media from GMSCs? Which conditions were employed (confluence, culture media, incubation time, etc.)? Please detail this issue in Materials and Methods section.

8. The results represent how many biological replicates? This is not specified in the manuscript.

9. In legend of figure 1, is described the measurement of cytokines expression by qPCR (E and F) but these figures are not included nor commented in the text. Please explain or correct it.

10.    I suggest to authors to include in the introduction of results in section 3.3, some lines explaining the use of GMSCs-CM and its rationale. Example:

“Recent studies have shown that MSCs can secrete various pro-proliferation, anti-in-flammatory, and pro-angiogenic factors or chemokines to promote the survival and pro-liferation of skin cells, thereby accelerating wound repair [16-18].” TO EVALUATE IF THE GMSC’s EFFECT SEEN IN VIVO WAS RELATED TO FACTORS SECRETED FROM THE CELLS, WE DECIDED TO PERFORM IN VITRO ASSAYS USING GMSCs-CM. FIRST, “we measured the proliferation capacity of skin cells using the CCK-8 assay.”

11.    In figure 4 are included the analysis of cyclin A expression by western blot (Fig. 4G) and CCNA mRNA level by qPCR (Fig. 4H), but these results are not commented in the text. Please correct it.

12.    In figure 5, figures 5D-H are not mentioned in the text. Also, what is the difference between fig. 5B and C? Please, clarify. In the text, when the western blot to assess the expression of EGFR-STAT3 pathway in skin tissue is cited, authors cited as figure 5D, while in the figure, it corresponds to Fig 5I. Please correct all these concerns about figure 5.

13.    The experimental design for treatment with CM and exposition to IR in cells with EGFR and STAT3 knocked-down, is not clear enough. Please detail more in the Materials and Methods section.

14.    In the legend of figure 6, please exclude the phrase "(E) Quantitative analysis of the migration rates in (D)" (line 436).

15.    Different IR doses were employed in different experiments. As examples, 4 and 8 Gy were used in proliferation assays (figure 3A-D); for colony formation were used 0, 1, 2 and 4 Gy (figure E-F); 8 Gy were used to evaluate the effect of IR on EGFR and STAT3 activation, while 4 and 8 Gy to test CM effect (figure 5). It would be interesting to describe the doses used in each experiment in the Materials and Methods section, and the reason for choosing each dose for each experiment.

16.    In the discussion section (lines 493-495) authors state that “These results indicate that the pY-Gel self-assembling peptide hydrogels for GMSCs intervention may be the best option for the treatment of radiation-induced skin injury.” Although the results represent a promising approach to treat IR-induced injuries, I suggest to the authors consider change the statement to “…may be a promising/good option for the treatment…”. To be considered “the best option”, a comparative study with other treatments should be carried out. Also, I strongly suggest to authors to include a final paragraph in the discussion section with concluding remarks to close the manuscript.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

1. Picture 1 shows from the fifth day, but it seems that the changes after radiation occurred on the 15th day, which is not clearly expressed in the description.

2. Please check your data, because the magnification of NO-IR in Figure 2 A, B, E seems to be different from others (the microscopic details, the nucleus and interstitial tissue are not clear);  in addition, the enlarged area should be marked. Is there a better picture of NO-IR, PBS and pyGEl in FIG. 2B, where the entire subcutaneous tissue is missing?

3. Both Figures 3I and 4A lack scale bars.

4. There is no description to clarify the EGFR-STAT3 pathway, but WB involves many indicators. I suggest improving more detailed content and adding a simple schematic diagram.

5. Repair of DNA damage and EGFR-STAT3 pathway appear to be two separate validation components that are not linked in a scientifically logical manner.

6. The references are too old, only 7 out of the 50 have been published in recent five years. In fact, there has been much research progress on this series in recent years, and it is suggested to update the references.

7. The "...and reduce the inflammatory response" mentioned in the Abstract is not well represented in the experimental design.

8. Inflammatory cascade reaction is actually a characteristic manifestation of radiation skin injury, but this article does not explicitly explain or design special experiments for "inflammation" and the differences between radiation wounds and ordinary wounds.

Author Response

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Reviewer 4 Report

The manuscript entitled “Supramolecular hydrogel-wrapped Gingival mesenchymal stem cells in Cutaneous radiation injury”, deals with a topic of great interest in the field of molecular radiobiology, in particular for possible clinical applications in order to avoid the adverse effects of radiation therapy (RT).

The authors showed the use of GMSCs and the application of a molecular hydrogel to evaluate the therapeutic effects on radiation-induced skin injury in mice. In addition, the authors investigated the putative molecular mechanisms that regulate the response to damage induced by ionizing radiation (IR) in cellular experiments.

The manuscript is overall well written, provides some novelty to advance the field of radiobiology, the experimental design and methodological approach is good, supported by statistical analysis.

In my opinion the manuscript needs some improvement in order to be published, such as minor revisions, listed below:

- Cell cultures: 1) the authors should explain the different use of the two chosen cell lines to perform the in vitro cell experiments, HaCaT and HFF, keratinocytes and fibroblasts, considering the important role of the two cell types in epidermal homeostasis of the skin; 2) Why did the authors perform the clonogenic assay on the HACaT cells and not on the HFF cells as well?; 3) the authors should justify the use of the HaCaT cells for the scratch wound assay and the use of the HFF cells for the transwell assay; 4) Moreover, the authors should justify using only the HaCaT cells for the other experiments conducted (Western blot, immunofluorescence, cell cycle, RNAi ...)

- Use of the IR doses delivered in cell experiments: the authors should specify in the text in each mat and met section which IR doses they used for each cell experiment performed (proliferation assay, colony formation assay…etc).

- In order to better show the molecular mechanisms involved in the response to the IR-induced damage studied, the authors should add a summary figure of the main protein factors analyzed (EGFR, STAT et al…) and involved in the pathways activated following radiation skin injury.

- In the discussion section, the authors should explain what possible strategies to translate the use of the GMSCs and molecular hydrogel system for applications in clinical practice and whether clinical trials have already begun. In the latter case the authors should cite the supporting bibliography.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors addressed and answered all the questions mentioned, including details and clarifying several points. The manuscript showed great improvement. However, there are still some points to be clarified or corrected:

1- Not all abbreviations are described in the abstract (e.g. MSC and CM). Please include.

2-Line 37: Is it mechanically or mechanistically?

3- The legend of figure 1 is still wrong. (A) is the experimental scheme, (B) are the representative images of lesions over time, (C) the skin damage score, and (D) hind limb extension analysis. Please correct.

4- Regarding GMSC characterization, the authors cited two references in the response. However, are these the same cells used in the study? If cells were received from Beijing Taisheng Biotechnology, did the company itself characterize them? Is this published? If so, it would be interesting to include this reference. Or mention that the cells were previously characterized and showed the characteristics of mesenchymal stem cells.

5- The authors included a description of the collection of the conditioned medium, which was collected with FBS. But two assays cite a serum-free conditioned medium. Why the difference? Would it not be possible to use the conditioned medium without FBS for all assays?

6- In the answer referring to the radiation exposure used for the in vitro assays, the authors affirm that they were based on previous studies. Please include this information in the manuscript (in the material and methods section or before starting to describe the in vitro results). For example, include a sentence like “The irradiation doses used in the assays were established based on previous studies [references]’’ after the new sentence in lines 353-355, and before starting to discuss the proliferation result.

7- Please, in Supplementary Table 1, include all the primers used (it is missing primers from IL6, IL1b, TNFa).

8- Figures S1F and S1G are representative images of pY-gel. What is the difference? G is polymerized? Please, explain in the figure legend.

9- Since the authors do not show that GMSC remains longer in the wound when encapsulated in the hydrogel (compared to the GMSC alone), the sentence: “We observed that the pY-Gel prevented the rapid diffusion of GMSCs from the injection sites.”, lines 278-279 should be removed. Thus, it is not possible to say with certainty that the cells remain longer at the site of injury.

10- In lines 421-426 the authors present the result of cyclin A expression in HACAT cells. They state that "... the level of cyclin A protein in HaCaT cells after exposure to IR was significantly higher than that in the non-irradiated cells…" (lines 422-423). However, the difference between NO and irradiated (4Gy or 8Gy) is not clear in the Western Blot (Figure 4G) and is not statistically significant in the PCR (Figure 4H), so it is not possible to confirm that they are significantly higher; the difference seems to be only related to GMSC-CM. Please review this sentence.

11- As suggested, the authors quantified the Western Blot results from Figure 5, but they did not include this neither in Figure 5 nor in any supplementary figure. Since it could improve the interpretation of data, I suggest including the quantification graphs (in the manuscript or as a supplementary figure).

12- The discussion has improved a lot. But some of the new sentences are a bit confusing:

a) In lines 556-558 the authors are talking about how wound healing is a dynamic process and that “MSC play a crucial role in wound healing”. But it is not clear if the authors are talking about resident MSC or therapy with MSC. If it is about cell therapy, change the sentence to make it clearer. Example: "MSC therapy can influence each of these stages of tissue repair, improving wound healing."

b) Sentence in lines 613-616 is confusing. It has been reported that WHAT increases keratinocyte migration and proliferation? Please, review.

c) The paragraph from lines 628-640 could be restructured, since there are some repetitive sentences.

Author Response

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Reviewer 2 Report

After the first round of corrections, Shasha Nie and co-authors improved the manuscript significantly, responding to suggestions and making clearer several aspects in the text. However, there are still some points that need to be reviewed.

1. Abstract, lines 24-27: the phrase is confusing. Consider to replace with: "This study investigated the therapeutic efficacy of Nap-GDFDFpDY (pY-Gel) self-assembled peptide hydrogel-encapsulated GMSCs to treat 137Cs-γ-radiation-induced skin wounds in mice." 

In line 39, replace "...EGFR/STAT3 signaling pathway may involve in..." by "...EGFR/STAT3 signaling pathway may be involved in...".

2. In Point 3 from my previous minor revisions, I suggested to include in the introduction some lines about the paracrine effects of MSCs. Please, revise that phrase to make more sense.

3. As suggested, authors included the ratio of conditioned medium:fresh medium in methodology. I suggest to describe this along the materials and methos section in a more detailed way. Example: "These cells were treated with fresh medium supplemented with GMSCs-CM, in the ratio 1:1."

4. In scratch-wound healing assay, authors said that serum-free conditioned medium was used in combination with fresh medium. While in transwell assay, cells were suspended in serum free medium, and then complemented with complete fresh medium supplemented or not with GMSCs-CM. Please clarify which conditions were employed in each case, and the reasons for. 

5. In section 2.14, lines 262-264, I suggest to modify the phrase to: "Forty eight hours after transfection, cells were treated with fresh medium supplemented with or without GMSCs-CM, in the ratio 1: 1, and immediately exposed to IR, 4Gy and 8Gy.

6. In relation to the response 5 from the minor revisions: although the authors described hydrogel preparation, authors did not measure whether cells diffuse from the hydrogel. Therefore, I suggest to removing the last sentence of section 3.1: "We observed that the pY-Gel prevented the rapid diffusion of GMSCs from the injection sites.". 

7. In lines 317-318, authors say that inflammatory response is reduced in skin tissue and cells. But considering that only IL6, IL1-beta and TNF-a were measured (and at mRNA level) I suggest to re-write the sentence in this way: "In addition, we have verified in skin tissue and cells that the expression of IL6, IL1-beta and TNF-a was reduced with treatment, suggesting that inflammatory response is decreased (Figure S2)."

8. Discussion: review the sentence among lines 632-635 and consider the following modifications: "Our study suggest that GMSCs-CM can promote skin cell proliferation and migration by activating EGFR/STAT3 signaling pathway in vitro; while pY-Gel supramolecular hydrogel associated to GMSCs can promote the damage repair of radioactive skin tissue in vivo."

The following two sentences: "Furthermore, it has been reported that GMSCs secrete various growth factors and chemokines for promoting wound healing [24, 29, 66]" (lines 635-636), and "Increasing evidence supports that GMSCs contribute to tissue repair and regeneration through paracrine effects [67]" (lines 636-638) are repetitive and can be condensed in only one phrase.

Author Response

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Reviewer 4 Report

The authors have fully addressed all suggestions provided to improve the manuscript. In my opinion, the revised manuscript is now suitable for publication in present form.