MIC-1 Antlerogenic Stem Cells Homogenate from Cervus elaphus Accelerate Corneal Burn Reepithelization in Rabbits

Round 1

Reviewer 1 Report

 This work evaluated the effect of deer antler stem cells on healing of superficial and deep rabbit corneal wounds. It seems that the effect to deep damage is better than the medicine Solcoseryl. This is very interesting and worthy of further investigation. However, the authors should provide more scientific and clear results.

 

1. Is there any research using other stem cells, especially rabbit stem cells? How is the effect？
2. To explain the importance of using ASC，I think there should be at least one kind of cell as a control，rather than vehicle.
3. Only the histological results of the cornea after injury, not results of the healing process, or even a normal cornea

Comments for author File: Comments.pdf

Author Response

Dear editor,

The authors would like to thank the reviewers for all useful and helpful comments on

our manuscript. All comments have been taken into account and the paper has been

revised accordingly.

Further to the revisions made in the manuscript, the authors believe that the whole

paper has been improved and we are happy to forward it back to you for your

consideration and for the publication of manuscript to the “Applied Sciences”.

Please find attached a revised version of our manuscript, as well as a rebuttal letter

with an itemized, point-by-point response to the editor’s and reviewers’ comments.

Reviewer 1

1. Is there any research using other stem cells, especially rabbit stem cells? How is the effect？
2. To explain the importance of using ASC，I think there should be at least one kind of cell as a control，rather than vehicle.
3. Only the histological results of the cornea after injury, not results of the healing process, or even a normal cornea

 

 

1 There are many papers describing rabbit stem cells.

Rabbit stem cell research has been conducted on the eyeball.

In an alkali burn corneal wound healing model, it was investigated whether systemically transplanted mesenchymal stem cells (MSCs) could nest and implant into tissue to promote corneal wound healing. At 1-month follow-up, it was found that systemically transplanted MSCs could implant into the injured cornea to accelerate wound healing through differentiation, proliferation and synergy with hematopoietic stem cells.(Ye, Yao et al. 2006)

 

The effect of cultured human adipose tissue-derived stem cells on the regeneration of the rabbit cornea after alkaline chemical burn was studied, using human adipose tissue-derived stem cells as the source material. Immediately after the chemical burn, the experimental eye received a single subconjunctival injection of a stem cell suspension. Transplantation of cultured human adipose tissue-derived stem cells as a treatment for chemical corneal burn was found to promote cell renewal and help repair damage to the rabbit cornea. (Lin, Lai et al. 2013)

Human limbal niche cells (LNC) or bone marrow derived mesenchymal stem cells (BMMSC) were used to prevent limbal stem cell deficiency (LSCD) in an alkali burn rabbit model and their results were compared. In contrast to the control group, the severity of LSCD in both the LNC and BMMSC transplantation groups was dramatically reduced as shown by smaller epithelial cell defects, decreased fluorescein sodium staining, decreased neovascularization and decreased goblet cell density. Interestingly, the LNC group was shown to more effectively prevent LSCD than the BMMSC group.(Li, Zhang et al. 2018)

Kobayashi, Kan et al. developed automated cell culture systems to achieve mass production of transplantable cell sheets. Using the robotic system, transplantable corneal epithelial cell sheets were successfully produced in cell cassettes. The manufactured cell sheets were then transplanted onto the ocular surface in a rabbit corneal epithelial stem cell deficiency model. Within a week after transplantation, the normal corneal epithelium was successfully regenerated. This automated cell culture system would be useful for industrialising tissue engineering products for regenerative medicine.(Kobayashi, Kan et al. 2013)

Due to the ethical and religious problems associated with the use of embryonic stem cells and the limited plasticity of adult stem cells, an alternative source of cells may be fetal stem cells derived from extra-embryonic tissue, which are highly proliferative, cultured in vitro and have interesting immunogenic characteristics. Mesenchymal progenitor cells derived from rabbit amniotic membrane were studied. These cells adhered to culture plates and showed high proliferative capacity with a fibroblast-like morphology. The cells showed a positive response to markers of the cytoskeleton, mesenchymal stem cells and proliferation, pluripotency and hematopoietic precursor stem cells. These cells had the ability to induce differentiation into osteogenic, adipogenic and chondrogenic lineages. Furthermore, when the cells were injected into nude mice, we did not observe tumour formation. It was concluded that multipotent mesenchymal stem cells can be obtained from rabbit amniotic membrane for possible use in future cell therapy applications .(Borghesi, Mario et al. 2017)

 

Chinese scientists have revealed that they have transferred cell nuclei from human cells into nucleus-free rabbit oocytes (Nature, 27 September). They hope to use the resulting embryos to generate ES cells (rabbit embryonic stem cell lines) for regenerative medicine research. Chen Xigu (Sun Yat-Sen University of Medical Sciences, Guangzhou, Taiwan) removed chromosomes from rabbit oocytes and replaced them with nuclei from a seven-year-old boy's skin cells. In some of the 100 or so successful nuclear transfers, the embryo developed to the morula stage - a compact ball of cells that forms after about three days of embryonic development. Before ES cells can be isolated from human embryos, they must develop beyond this stage to form a blastocyst. (McKay 2001)

 

An example of the similarity between rabbits and humans is the similar airway anatomy and response to inflammatory mediators. The inflammatory responses exhibited by the rabbit model, particularly in asthma, are comparable to those in humans. The allergic rabbit model has been widely used in drug screening tests, and this model and humans appear to be sensitive to similar drugs. The rabbit has been shown to serve as a good platform for cell delivery for stem cell-based lung disease therapy.(Kamaruzaman, Kardia et al. 2013)

Honda et al. demonstrated the similarity of ES cells (rabbit embryonic stem cells) between rabbits and humans. These cell lines can be used directly using gene targeting techniques or in combination with induced pluripotent stem cells. ES cells are a suitable model for studying human transplantation therapy and disease treatment.(Honda 2013)

 

Human induced pluripotent stem (iPS) cells may create a new field of promising regenerative medicine. Rabbit iPS cells were the first human-type iPS cells generated from a small species of laboratory animal. Using lentiviral vectors, four human reprogramming genes (c-MYC, KLF4, SOX2 and OCT3/4) were successfully introduced into adult rabbit liver and stomach cells. The resulting rabbit iPS cells closely resembled human iPS cells. Using in vitro differentiation conditions, the rabbit iPS cells readily differentiated into ectoderm, mesoderm and endoderm. The rabbit iPS cells met all the requirements for a fully reprogrammed state, showing high similarity to their embryonic stem cell counterparts. The rabbit model should allow comparison of iPS cells and embryonic stem cells to evaluate their application in cell-based regenerative medicine in humans. (Honda, Hirose et al. 2010)

2. Thank you for this comment. The results we have described are preliminary studies. So far we have used Solcoseryl as a commercially available drug. In the future we will think about extending the control group on another known stem cell line to compare their effects.
3. For the supplementary materials we include photographs of the normal cornea and of the healing process.

 

Reviewer 2 Report

The study by Dziewiszek et al, is an interesting finding where they have use the MIC1 (antler stem cell) homogenate to treat corneal burn. There are significant concern that authors need to address to make this article significant.

Same line of study has been done by Keilbowicz et al, 2021. In the comprehensive study Keilbowicz et al showed the use of ASC in alleviating the corneal burn in rabbits. Authors have not referred to that important paper in this article.

Major concers:

Title:

1) The authors have used MIC1 homogenate for all there experiment but then the title says antler stem cells (ASC) accelerate corneal burn reepithelization in rabbits. Which is not correct and truly doesn’t reflect the study.

Introduction:

2) The introduction provides enough background to the reader but fails to signify why you need antler stem cells to treat corneal burn when you have so many other stem cell therapies to treat corneal burn.

3) Overall the Introduction lacks references for many statements. Please add references for line 58-61, line 61-67, line 69-70, line 76-78. Please include 1-2 sentences for studies with references that have used corneal reepithelization to alleviate corneal injury.

4) Please mention2-3 key stem cell markers for AP & PP with reference (line 47-48). Please omit the word dermis from line 57 as dermis is different from stroma in morphology & cell function.

Material & Methods:

5) Detail method for cell isolation should be mentioned here instead of referring to another paper. Was the MIC1 cell line purchased, gifted or prepared in-house. If in-house please detail the procedure. Although authors have explained the detail procedure in the patent document. “Patent document is not part of this article. It is a separate entity and this article should stand out itself. Also, authors can’t expect a researcher reading this article to refer to the patent doc”. So, please kindly include all the relevant information in the article itself.

6) In line 97 what is this fluid, please mention the media used and report the number of cells per ml instead.

7) Line 114 says one eye and line 115 says erosion on both eye. Please correct its confusing. Same for line 121-122. Please mention catalog no. for alcaine and n-heptanol (line 115& 117). Also mention the reference 5 (Chung) in this paragraph.

8) What was the vehicle for superficial and deep model, line 136.

9) Please mention all the groups in a table form. It’s very confusing to make out from the method section. Might be easier for the reader to understand.

10) Please provide a reference for the corneal damage scaling. In Scale 3& 4 if opacity is 50% what does completely slightly haze means. The scale is not very clear. Also authors have used the term “translucency” in line 208. It should be explained/mentioned in the scale if you prefer to use that.

Results:

11) Fig-1- x& y-axis label missing in the figure. Legend doesn’t specify number of rabbits or number of times the experiment was done. Please include all the relevant information in the figure legend. Please make the same changes for fig.2 & 3.

12) Please provide the eye images which might be very insightful.  Comparative images for the treated vs control group for both the superficial and deep damage model.

13) Line 196-203 can be moved to the discussion. Rather authors might explain their findings here (Fig-1).  For superficial damage- authors should provide the transparency data (corneal haze) graph. Did the authors measured any blood vessel penetration during the course of the study?

14) For the deep damage model, authors have used MIC1 homogenate gel. But then line 213, how did they observed regeneration of ASC. This is very confusing and please clarify. The words “statistically insignificant” can be changed to “moderate”, if the author’s thinks right.

15) Para, line 217-221 can be moved to discussion and authors might want to discuss their result in details here (fig2 & 3). Again please include eye images for deep model and corneal surface area graph for the deep damage.

16) Author’s mentioned fluorescein test in the methods. Please include comparative images between the treated and control group for both superficial and deep damage model.  

17) Please provide images to support the statements in line 239-245 and same for 246-250. A comparative histology images between the treated vs control group will be very meaningful.

18) Again the table 1, line 250 is missing in the article. The author can’t expect reader to refer their patent document. So, please include it in the main article.

19) Fig-4 is not very meaningful. Scale bar is missing. Please explain each panel in the legend. What b, c, d, e & f is showing. Put arrow to indicate the changes and the layers.

Discussion:

20) Line 282-287, lacks reference. Please use scientific words for “very good experience”, line 296. Line 339 is a repeat.

Author Response

Reviewer 2

The study by Dziewiszek et al, is an interesting finding where they have use the MIC1 (antler stem cell) homogenate to treat corneal burn. There are significant concern that authors need to address to make this article significant.

Same line of study has been done by Keilbowicz et al, 2021. In the comprehensive study Keilbowicz et al showed the use of ASC in alleviating the corneal burn in rabbits. Authors have not referred to that important paper in this article.

Thank you for suggestion. Abovementioned paper and appropriate information was added to the  section “Discussion”.

 

Major concers:           

Title:

The authors have used MIC1 homogenate for all there experiment but then the title says antler stem cells (ASC) accelerate corneal burn reepithelization in rabbits. Which is not correct and truly doesn’t reflect the study.

Antlerogenic stem cells from Cervus elaphus accelerate corneal burn reepithelization in rabbits

 

According to Reviewer’s suggestion, title was changed into “MIC-1 antlerogenic stem cells homogenate from Cervus elaphus accelerate corneal burn reepithelization in rabbits”

Introduction:

2) The introduction provides enough background to the reader but fails to signify why you need antler stem cells to treat corneal burn when you have so many other stem cell therapies to treat corneal burn.

3) Overall the Introduction lacks references for many statements. Please add references for line 58-61, line 61-67, line 69-70, line 76-78. Please include 1-2 sentences for studies with references that have used corneal reepithelization to alleviate corneal injury.

4) Please mention2-3 key stem cell markers for AP & PP with reference (line 47-48). Please omit the word dermis from line 57 as dermis is different from stroma in morphology & cell function.

Material & Methods:

5) Detail method for cell isolation should be mentioned here instead of referring to another paper. Was the MIC1 cell line purchased, gifted or prepared in-house. If in-house please detail the procedure. Although authors have explained the detail procedure in the patent document. “Patent document is not part of this article. It is a separate entity and this article should stand out itself. Also, authors can’t expect a researcher reading this article to refer to the patent doc”. So, please kindly include all the relevant information in the article itself.

The MIC1 cell line was isolated from antlers at the Wroclaw Medical University.

From the terminal lateral sections of growing antlers of red deer (Cervus Elaphus), in the period of most intensive growth, sections with an average thickness of 0.5 cm and with an area of 1 to 2 cm², in the amount of 2-3 grams, were taken sterile, under anaesthesia conducted with a remote injection system (during grooming procedures in the Wrocław Zoological Garden).  The collected cuttings were mechanically minced to obtain microscopic fragments of about 100 - 900 micrometres in size. Half of the fragmented tissue was left for microscopic and electron-microscopic examination.  Proliferating cells were isolated from the fragmented antlers to be cultured, using the migration phenomenon. The isolated cells were placed in culture bottles. Medium was used as growth fluid: SmGM-2 SingleQuots by CAMBREX, supplemented with L-glutamine at 1 mM/mL, penicillin at 100 UI /mL, streptomycin at 0.1 mg/mL by Merck. The cells were placed in a greenhouse where they grew under standard conditions at 5% CO₂ and +37^oC.  The line was run for four months with a yield of approximately 5 million cells per week. The cultured cell line was frozen and placed in liquid nitrogen dishes.

For the experiment, it is thawed and then further cultured in the same way for the time needed to produce the necessary number of stem cells.  After obtaining mono-layers in culture bottles, the cells are trypsinised, centrifuged and washed.

 

6) In line 97 what is this fluid, please mention the media used and report the number of cells per ml instead.

As a fluid, following mixture was used: SmGM-2 SingleQuots by CAMBREX, supplemented with L-glutamine at 1 mM/mL, penicillin at 100 UI /mL, streptomycin at 0.1 mg/mL.

The number of cells is now described in manuscript as  is expressed on one mL

Appropriate information was added to the manuscript.

7) Line 114 says one eye and line 115 says erosion on both eye. Please correct its confusing. Same for line 121-122. Please mention catalog no. for alcaine and n-heptanol (line 115& 117). Also mention the reference 5 (Chung) in this paragraph.

n-Heptanol (former Sigma-Aldrich, now Merck) was used for experiment. Catalog nr for n-heptanol was 8206240100. NaOH was purchased in UQF (Poland). Alcaine, chemically proxymetacaine hydrochloride, 5 mg/mL) was purchased from former Alcon (now Phizer). We corrected the chemical name in manuscript;

8) What was the vehicle for superficial and deep model, line 136.

In superficial denaturation model sodium hyaluronate (commercially available as Hyal-Drop Multi, , containing 2,4 mg/mL  of polymer Bausch & Lomb) was  used as a vehuculum.

In deep denaturation model carbomer (Vidisic gel, 2 mg/g, Bausch & Lomb) was used as vehiculum.

 Appropriate information was added to the manuscript.

 

9) Please mention all the groups in a table form. It’s very confusing to make out from the method section. Might be easier for the reader to understand.

10) Please provide a reference for the corneal damage scaling. In Scale 3& 4 if opacity is 50% what does completely slightly haze means. The scale is not very clear. Also authors have used the term “translucency” in line 208. It should be explained/mentioned in the scale if you prefer to use that.

Corneal opacity after alkali burn in rabbits was graded with a slight modification of protocol of Gupta N et al. (Gupta, Kalaivani et al. 2011), based on the area of the haze (Figure 2).

We added appropriate Table to the manuscript.

Grading of corneal clarity on the basis of corneal haze

Grade	Ocular details
1	Cornea completely clear
2	Iris details visible
3	Pupillary margin visible, iris details not visible
4	Pupillary margin not visible
5	Cornea completely opaque

 

In line 208 we have changed the term ”translucency” into “clarity”.

Results:

11) Fig-1- x& y-axis label missing in the figure. Legend doesn’t specify number of rabbits or number of times the experiment was done. Please include all the relevant information in the figure legend. Please make the same changes for fig.2 & 3.

Done according to reviewer’s suggestion

12) Please provide the eye images which might be very insightful.  Comparative images for the treated vs control group for both the superficial and deep damage model.

The images has been added into Supplementary materials

13) Line 196-203 can be moved to the discussion. Rather authors might explain their findings here (Fig-1).  For superficial damage- authors should provide the transparency data (corneal haze) graph. Did the authors measured any blood vessel penetration during the course of the study?

In the superficial damage model, no permanent changes in corneal transparency were observed during corneal epithelial damage and after healing. All tested corneas were completely healed, without the presence of haze. This experimental model was chosen not to cause such corneal haze.

During the described experiments: superficial model and deep model, we did not note any blood vessel penetration in any eye. We believe that this was due to the relatively small area of the lesion (3 mm diameter circle). Comparing, for example, the study by Kielbowicz et al. in 2020, these authors performed a 6mm lesion, so with twice the surface area. During the corneal lesion, the blotting paper disc was placed on the cornea paracentrally, away from the corneal stroma, which we believe further resulted in the lack of accrual of new vessels.

The tissue paper soaked in the corneal damaging substance was placed above the corneal stroma, i.e. at the junction of the cornea and the conjunctiva. In these rabbits, we observed each time a proliferation of conjunctival vessels at the site of damage. However, we did not include this information in the article because there were no differences between the study groups.

 

14) For the deep damage model, authors have used MIC1 homogenate gel. But then line 213, how did they observed regeneration of ASC. This is very confusing and please clarify. The words “statistically insignificant” can be changed to “moderate”, if the author’s thinks right.

Thank you for pointing out this issue. It was a mistake during translation. We changed it in manuscript.

15) Para, line 217-221 can be moved to discussion and authors might want to discuss their result in details here (fig2 & 3). Again please include eye images for deep model and corneal surface area graph for the deep damage. According to the Reviewer’s suggestion, we provided to  Supplementary materials (S.1-3)  pictures of superficial corneal damage in both groups

16) Author’s mentioned fluorescein test in the methods. Please include comparative images between the treated and control group for both superficial and deep damage model.  

Due to chemical properties of fluorescein (instability in basic conditions) we were not able to dye  the injuries of cornea caused by sodium hydroxide. According to the Reviewer’s suggestion, we provided to  Supplementary materials (S.1-3)  pictures of superficial corneal damage in both groups.

17) Please provide images to support the statements in line 239-245 and same for 246-250. A comparative histology images between the treated vs control group will be very meaningful.

Appropriate images with histological examination  has been added into Supplementary material S.4

18) Again the table 1, line 250 is missing in the article. The author can’t expect reader to refer their patent document. So, please include it in the main article.

Table was added as Table nr 2

19) Fig-4 is not very meaningful. Scale bar is missing. Please explain each panel in the legend. What b, c, d, e & f is showing. Put arrow to indicate the changes and the layers.

Appropriate images with histological examination  has been added into Supplementary material S.4

 

Discussion:

20) Line 282-287, lacks reference. Please use scientific words for “very good experience”, line 296. Line 339 is a repeat.

 

 

1. Hamill CE, Bozorg S, Chang H-YP, et al. Corneal alkali burns: a review of the literature and proposed protocol for evaluation and treatment. International ophthalmology clinics. 2013;53(4):185-194.
2. Gupta N, Kalaivani M, Tandon R. Comparison of prognostic value of Roper Hall and Dua classification systems in acute ocular burns. British journal of ophthalmology. 2011;95(2):194-198.
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10. Saghizadeh M, Kramerov AA, Svendsen CN, Ljubimov AV. Concise review: stem cells for corneal wound healing. Stem Cells. 2017;35(10):2105-2114.
11. Kiełbowicz M, Kuropka P, Cegielski M, et al. Influence of antlerogenic stem cells on the healing of lesions in the corneal epithelium and corneal stroma in rabbits. Polish journal of veterinary sciences. Jun 2020;23(2):281-290.
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Author Response File: Author Response.docx

Reviewer 3 Report

MIC-1 antlerogenic stem cells homogenate from Cervus elaphus accelerate corneal burn reepithelization in rabbits

 

The manuscript “MIC-1 antlerogenic stem cells homogenate from Cervus elaphus accelerate corneal burn reepithelization in rabbits“ evaluates the effect of deer antler stem cells homogenate from Cervus elaphus on corneal wound healing after superficial and deeper damage.

This study is well designed and illustrated.

However, a few points should be taken into account:

Section 2.  Material and Methods

 Page 3, Line 111: What do the authors mean by “The cells were placed in a greenhouse”? It is like an incubator?

Page 3, Line 114: At which passages the cells were used?

Page 3, Line 124: Was the homogenate filtered or not? Authors should do a western blot and/or qPCR to study the cell extract obtained.

Page 3, Line 129: In addition to the slit-lamp examination, optical coherence microscopy and in vivo confocal microscopy should be performed. It is important in this study to evaluate the epithelial layers before and after treatments.

 Page 4, Line 151: Please give more information about Solcoseryl. Is it commonly used in ophthalmology to promote healing? Why did the authors choose this one and not another?

Page 4, Line 161: Authors used as vehicle sodium hyaluronate in drops solution in superficial denaturation model and carbomer in gel for deep denaturation model, Why? Can you explain, please?

Page 4, Line 177: Table 1, Can the authors add the number of rabbits used in each case?

Page 5, Line 181: The histological study is not well designed. The histological examination would be performed a few hours after denaturation, in order to have an overview of the damage caused in the epithelial layers.

 

 

Section 3. Results

Page 5, Line 204: the damage caused with n-heptanol did not extend to the limbal niche (3mm diameter disc), we expect here that limbal epithelial stem cells will replace the damaged epithelium. Discuss this point.

Page 6, Line 241: Switch this paragraph to the discussion and discuss it deeply.

Page 7 Figure 3: correct Solkoseryl.

Page 8, Line 259: It is important here to have a histological examination on day 0, and just after the superficial damage, it is difficult to conclude, we do not know what is the number of damaged epithelial layers.

Page 8, Line 265: «  Where the healing of the corneal endothelial cells after alcohol burn was studied” did the author mean “… healing of the corneal epithelial cells….)? It is better to discuss it in the discussion section.

Page 8, Line 273: “the phenomenon of corneal changing after chemical burn…” Switch it and discuss it deeply in the discussion section.

Page 11, Line 293, Figure 4: If A, B, and C images correspond to the superficial damages of the corneal epithelium, C cannot be an image of a Solcoseryl treatment. Please correct.

Page 9, Figure 4: Can the Authors change the orientation of the images for better understanding? Cornea oriented correctly, epithelium in the top and endothelium in the bottom?

 

Section 4. Discussion

The discussion could be improved by comparing it with studies that used homogenate to see their therapeutic effects. How to improve the process? what are the constituents of the homogenate responsible for this effect? discuss the effect of rabbit limbal stem cells added to the contribution of the homogenate to improve corneal healing.

Comments for author File: Comments.pdf

Author Response

Dear editor,

The authors would like to thank the reviewers for all useful and helpful comments on

our manuscript. All comments have been taken into account and the paper has been

revised accordingly.

Further to the revisions made in the manuscript, the authors believe that the whole

paper has been improved and we are happy to forward it back to you for your

consideration and for the publication of manuscript to the “Applied Sciences”.

Please find attached a revised version of our manuscript, as well as a rebuttal letter

with an itemized, point-by-point response to the editor’s and reviewers’ comments.

 

 

Section 2.  Material and Methods

 Page 3, Line 111: What do the authors mean by “The cells were placed in a greenhouse”? It is like an incubator?

Corrected

Page 3, Line 114: At which passages the cells were used?

We used the cells after eight passages.

Page 3, Line 124: Was the homogenate filtered or not? Authors should do a western blot and/or qPCR to study the cell extract obtained.

The homogenate has not been filtered. External administration does not require the maximum size of the components. The composition of the preparation is the cell membrane and the cell interior. Filtration deprived the preparation of activity.

Unfortunately, we did not have the opportunity to perform the qPCR studies.

Page 3, Line 129: In addition to the slit-lamp examination, optical coherence microscopy and in vivo confocal microscopy should be performed. It is important in this study to evaluate the epithelial layers before and after treatments.

Thank you for suggestion. We haven’t got possibility to optical coherence microscopy and confocal microscopy.

 Page 4, Line 151: Please give more information about Solcoseryl. Is it commonly used in ophthalmology to promote healing? Why did the authors choose this one and not another?

Solcoseryl is widely used in wound healing. Due to availability, well-established effectivity and our practice (we use it for human application in corneal damages) we decided to use Solcoseryl. We mentioned it in manuscript.

 

Page 4, Line 161: Authors used as vehicle sodium hyaluronate in drops solution in superficial denaturation model and carbomer in gel for deep denaturation model, Why? Can you explain, please?

Sodium hyaluronate increased radically the viscosity of eye-drops and time of action. The same was in case of carbomer. As the gel's retention time in the conjunctival sac is much longer than that of eye drops, we compared the same drug forms with each other. Choosing the right substrate prolongs the duration of action of the drug in the conjunctival sac.

We compared eye-drops as well as gels to enriched one with ASC. Due to the fact, that time of presence of gel is longer in conjunctival sac in comparison to eye-drops.

Page 4, Line 177: Table 1, Can the authors add the number of rabbits used in each case?

Done.

Page 5, Line 181: The histological study is not well designed. The histological examination would be performed a few hours after denaturation, in order to have an overview of the damage caused in the epithelial layers.

Thank you for your comments; we take this into consideration during planning the next experiments.

From our experience as ophthalmologists after n-heptanol denaturation (as well as after acid burns) the key moment is the time, when the substance is in the coniunctival sac or on the corneal surface. Rapid removal of the conjunctival sac often completely stops tissue damage. The damage does not enlarge after washing out. In our opinion, in the group with n-heptanol, the time of histological study is not important. But we agree in groups after NaOH damage. The alcali burn does not stop after washing out the conjunctival sac. Alcali penetrates deep into the tissues and the whole damage is observed after a longer time. Returning to your comment, there will probably be differences between histological findings when it will be performed just after the denaturation, 6, 12, 24, or 48 hours later. Surely, we will think about this in the next experiment.

 

Section 3. Results

Page 5, Line 204: the damage caused with n-heptanol did not extend to the limbal niche (3mm diameter disc), we expect here that limbal epithelial stem cells will replace the damaged epithelium. Discuss this point.

In fact, our use of a small 3mm diameter disc applied centrally to the cornea did not cause damage to the limbal niche. In the superficial damage model, we observed complete healing of the epithelium with no permanent changes in corneal translucency and no penetration of the conjunctival vessels. This experimental model was chosen not to cause such a corneal haze.

We know that damage to the limbal niche around the periphery of the cornea causes a complete lack of corneal translucency. Patients with such damage must have a transplant not only of the cornea itself, but also of the corneal stroma so that the translucency can be maintained.

 

In our study, we also performed lesions (also with a 3 mm disc) on the corneal stroma, that is, at the junction between the cornea and the conjunctiva. In these rabbits, we observed corneal epithelium healing and proliferation of conjunctival vessels at the site of the lesion each time. However, we did not include this information in the article because there were no differences between the study groups.

 

Page 6, Line 241: Switch this paragraph to the discussion and discuss it deeply.

According to the other Reviewer, we wrote it in Results section. We also discussed it deeply.

Page 7 Figure 3: correct Solkoseryl.

Corrected

Page 8, Line 259: It is important here to have a histological examination on day 0, and just after the superficial damage, it is difficult to conclude, we do not know what is the number of damaged epithelial layers.

Thank you for your comments, we will take this into account in the next study.

Page 8, Line 265: «  Where the healing of the corneal endothelial cells after alcohol burn was studied” did the author mean “… healing of the corneal epithelial cells….)? It is better to discuss it in the discussion section.

In fact, we change into ‘corneal epithelial cells’.  According to the other reviewer, we wrote it in the Results section.

Page 8, Line 273: “the phenomenon of corneal changing after chemical burn…” Switch it and discuss it deeply in the discussion section.

According to the other reviewer, we wrote it in the Results section.

Page 11, Line 293, Figure 4: If A, B, and C images correspond to the superficial damages of the corneal epithelium, C cannot be an image of a Solcoseryl treatment. Please correct.

Corrected.

Page 9, Figure 4: Can the Authors change the orientation of the images for better understanding? Cornea oriented correctly, epithelium in the top and endothelium in the bottom?

Corrected.

 

Section 4. Discussion

The discussion could be improved by comparing it with studies that used homogenate to see their therapeutic effects. How to improve the process? what are the constituents of the homogenate responsible for this effect? discuss the effect of rabbit limbal stem cells added to the contribution of the homogenate to improve corneal healing.

 

We have included it in the discussion.

 

Antler stem cells (ASC) effectively accelerated the rate of wound closure and improved the quality of wound healing of the skin in rats, which may be due to the transformation of wound skin fibroblasts into their fetal counterparts. Therefore, they could potentially be developed as a new therapy for scarless wound healing.(Rong, Chu et al. 2019)

In a rat model of CCl4-induced liver fibrosis, ASCs were shown to effectively alleviate liver fibrosis and inhibit the activation of HSCs of liver stellate cells. In the future, ASCs may serve as a new source of stem cells for the treatment of liver fibrosis in the clinic.(Rong, Yang et al. 2020)

 

 

Dong et al. identify key pathways, molecular / cellular functions, and top-down regulators involved in mammalian organ regeneration. Antler regeneration is known to be initiated and sustained by neuron-derived stem cells in different states of activation. Therefore, antler stem cells can be used as a model to study the proteins and pathways involved in maintaining the stem cell niche and their activation and differentiation during organ formation. In this study, the MSC markers CD73, CD90 and CD105 were examined within the antler tip. The central role of stem cell activation in the development of this mammalian organ was confirmed by the localisation of MSC markers within the antler growth centre. The highest number of unique proteins (87) was found in the growth center. Antler stem cell activation has been implicated in multiple biological processes and signalling pathways, such as Hippo and canonical Wnt signalling.(Dong, Haines et al. 2020)

 

 

Li et al. identified proteins that were differentially expressed between antler stem cells and somatic cells (facial periosteum). They found activation of several molecular pathways (PI3K/Akt, ERK/MAPK, p38 MAPK, etc.) during proliferation. The transcription factors POU5F1, SOX2, NANOG and MYC, which are key markers of embryonic stem cells, were also expressed. The expression of these proteins was confirmed in both cultured cells and fresh tissues. Therefore, the molecular pathways and transcription factors identified in the study are common to embryonic and adult stem cells. However, the expression of embryonic stem cell transcription factors would suggest that antler stem cells are potentially an intermediate stem cell type between embryonic cells and more specialised tissue-specific stem cells, such as those residing in muscle, fat, or derived from the hematopoietic system. (Li, Harper et al. 2012)

 

Recent studies have shown that rapid elongation of the main bundle and antler branches is a controlled form of tumor growth, regulated by the TP73 and ADAMTS18 genes. Osteoclastogenesis, as well as osteogenic and chondrogenic differentiation, are also involved in this process. As a unique stem cell-based organ regeneration process in mammals, deer antlers provide a first-class model system to study regeneration mechanisms in mammalian tissues. Novel ASCs could provide cell-based therapies for regenerative medicine and bone remodelling for clinical applications. (Dąbrowska, Kiełbowicz et al. 2016, Feleke, Bennett et al. 2021)

 

Rabbit limbal stem cells (LSCs) were studied in an animal model of limbal stem cell deficiency (LSCD). During in vitro culture, LSCs underwent epithelial-mesenchymal transformation. Mesenchymal LSCs were shown to be a highly proliferative population and capable of restoring the corneal epithelium in rabbits. On the 90th day after transplantation of mesenchymal-like LSCs to rabbits with previously formed LSCD, normal corneal epithelium was restored, vascularization was absent, and goblet cells were absent. In the control group, opacity and neovascularization of the stroma was observed.(Selver, Durak et al. 2016)

Li et al. showed that human limbal niche cells or bone marrow-derived mesenchymal stem cells can also be used to prevent limbal stem cell deficiency (LSCD) in an alkali burn rabbit model. Human limbal niche cells are a more potent source than bone marrow-derived mesenchymal stem cells in preventing LSCD in the rabbit alkali burn model.(Li, Zhang et al. 2018)

 

 

 

Dąbrowska, N., Z. Kiełbowicz, W. Nowacki, J. Bajzert, P. Reichert, J. Bieżyński, J. Zebrowski, K. Haczkiewicz and M. Cegielski (2016). "Antlerogenic stem cells: molecular features and potential in rabbit bone regeneration." Connect Tissue Res 57(6): 539-554.

Dong, Z., S. Haines and D. Coates (2020). "Proteomic Profiling of Stem Cell Tissues during Regeneration of Deer Antler: A Model of Mammalian Organ Regeneration." Journal of Proteome Research 19(4): 1760-1775.

Feleke, M., S. Bennett, J. Chen, X. Hu, D. Williams and J. Xu (2021). "New physiological insights into the phenomena of deer antler: A unique model for skeletal tissue regeneration." J Orthop Translat 27: 57-66.

Li, C., A. Harper, J. Puddick, W. Wang and C. McMahon (2012). "Proteomes and Signalling Pathways of Antler Stem Cells." PLOS ONE 7(1): e30026.

Li, G., Y. Zhang, S. Cai, M. Sun, J. Wang, S. Li, X. Li, S. Tighe, S. Chen, H. Xie and Y. Zhu (2018). "Human limbal niche cells are a powerful regenerative source for the prevention of limbal stem cell deficiency in a rabbit model." Scientific Reports 8(1): 6566.

Rong, X., W. Chu, H. Zhang, Y. Wang, X. Qi, G. Zhang, Y. Wang and C. Li (2019). "Antler stem cell-conditioned medium stimulates regenerative wound healing in rats." Stem Cell Research & Therapy 10(1): 326.

Rong, X., Y. Yang, G. Zhang, H. Zhang, C. Li and Y. Wang (2020). "Antler stem cells as a novel stem cell source for reducing liver fibrosis." Cell and Tissue Research 379(1): 195-206.

Selver, O. B., I. Durak, M. Gürdal, K. Baysal, H. Ates, Z. Ozbek, Z. Wang, A. Wu and J. M. Wolosin (2016). "Corneal recovery in a rabbit limbal stem cell deficiency model by autologous grafts of tertiary outgrowths from cultivated limbal biopsy explants." Molecular vision 22: 138-149.

 

Author Response File: Author Response.docx

Reviewer 4 Report

  The authors  showed the usefulness of  antlerogenic stem cells from deer in corneal burn reepithelization in rabbits in the present article. The effect is clear, but there are many flaws in the presentation. 

Comments 

1. The authors should describe the difference between the antlerogenic stem cells and other stem cells such as mesenchymal stem cells,  embryonic stem cells, and so on in terms of the efficacy.
2. The authors comment on the human use of antlerogenic stem cells.
3. The authors indicate the position of Fig. 3.
4. The authors require extensive English editing. 

Author Response

Dear editor,

The authors would like to thank the reviewers for all useful and helpful comments on

our manuscript. All comments have been taken into account and the paper has been

revised accordingly.

Further to the revisions made in the manuscript, the authors believe that the whole

paper has been improved and we are happy to forward it back to you for your

consideration and for the publication of manuscript to the “Applied Sciences”.

Please find attached a revised version of our manuscript, as well as a rebuttal letter

with an itemized, point-by-point response to the editor’s and reviewers’ comments.

 

1. The authors should describe the difference between the antlerogenic stem cells and other stem cells such as mesenchymal stem cells, embryonic stem cells, and so on in terms of the efficacy.

 

 

Antler stem cells express both classic mesenchymal stem cells (MSCs) and embryonic stem cells (ESCs) and are capable of differentiate into multiple cell types in vitro. Thus, ASCs were defined as MSCs, but with partial ESC attributes.(Zhang, Ke et al. 2021).

For reviewer:

In our experiment, we did not compare ASC with MSC or ESC, thus we cannot conclude the advantage of any of them.

 

 

 

1. The authors comment on the human use of antlerogenic stem cells.

According to the best of our knowledge, ACS has been introduced on the market as active compounds in anti-aging cosmetics (Revitacell, Biocervin). There were also some attempts to produce medical products. We have no precise information on the commercial uses of ASC in treatment.

 

1. The authors indicate the position of Fig. 3.

Corrected.

1. The authors require extensive English editing. 

The English edition will be done by the Editorial Services of Applied Science.

 

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Although so many stem cell research contents are provided, none of them has been cited by in the paper. If ASC can't outperform other cells, what's the usefulness of this study?

The display of the picture results is very poor.

My suggestion is to wait until a systematic study is done before publishing.

Author Response

Dear editor,

The authors would like to thank the reviewers for all useful and helpful comments on

our manuscript. All comments have been taken into account and the paper has been

revised accordingly.

Further to the revisions made in the manuscript, the authors believe that the whole

paper has been improved and we are happy to forward it back to you for your

consideration and for the publication of manuscript to the “Applied Sciences”.

Please find attached a revised version of our manuscript, as well as a rebuttal letter

with an itemized, point-by-point response to the editor’s and reviewers’ comments.

 

Although so many stem cell research contents are provided, none of them has been cited by in the paper. If ASC can't outperform other cells, what's the usefulness of this study?

The use of ASC cells has many advantages over human stem cells. These include the absence of ethical concerns for patients and the absence of the need to obtain the relevant bioethical approvals. Unlike mesenchymal progenitor cells derived from rabbit amniotic membrane or rabbit nucleus-free oocytes, ASC lineage culture is not associated with the need to kill animals.  The continuous acquisition of material and its constant homogeneity are also an important aspect.

For the reviewer:

We have not performed studies comparing the efficacy of other stem cells with ASCs. However, this study should be considered very preliminary, proving the feasibility of ASC applications in the treatment of corneal wounds.

 

The display of the picture results is very poor.

We have pointed out this issue with editor, separated pictures (as jpg) could be send to improve their quality.

My suggestion is to wait until a systematic study is done before publishing.

 

The results presented in the manuscript are closed and separated parts of our research. At that moment we are not able to perform other experiments in that field (it required huge financial support, acceptance of ethical comity, etc.). Due to this fact, we are afraid that we cannot improve this manuscript with other results.

 

 

Reviewer 2 Report

Authors have addressed all the concerns raised earlier and have improved the manuscript considerably.

Can the authors label the Y & X-axis in the graph itself (Fig1,2 &3).

Author Response

Dear editor,

The authors would like to thank the reviewers for all useful and helpful comments on

our manuscript. All comments have been taken into account and the paper has been

revised accordingly.

Further to the revisions made in the manuscript, the authors believe that the whole

paper has been improved and we are happy to forward it back to you for your

consideration and for the publication of manuscript to the “Applied Sciences”.

Please find attached a revised version of our manuscript, as well as a rebuttal letter

with an itemized, point-by-point response to the editor’s and reviewers’ comments.

 

 

Can the authors label the Y & X-axis in the graph itself (Fig1,2 &3).

Done, according to Reviewer’s suggestion

Author Response File: Author Response.docx

Reviewer 3 Report

Authors take into account all the comments, and revised the manuscript accordingly. Thank's for that.

Reviewer 4 Report

The authors edited the manuscript according to the reviewer's comments. It is improved compared with the previous one.