Impact of Zinc Oxide on the Development of Aspergillus-Induced Maxillary Sinusitis Rabbit Model

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors This paper investigated Aspergillus-induced sinusitis in Rabbit models and the role of zinc oxide in this process. The experimental rabbits were divided into four groups, receiving PBS, ASP, ZO, and ASP with ZO. Results showed that ASP significantly increased inflammation, cytokine production, and biofilm formation in the sinus mucosa. However, adding zinc oxide with ASP hardly made any difference in the inflammatory responses. The conclusion of this work does not support its assumption, and I do not deem it a fit for publication at this stage. To help the authors to improve the quality of this work, I suggest the author address the following issues:

 

1. Introduction to the role of zinc oxide as the endodontic sealer that promotes fungal survival and proliferation was insufficient. I suggest the authors expand this part in the introduction as a separate paragraph and include more references.
2. It is unclear how ZO was prepared in the experimental section. Is it simple dispersion in PBS?
3. What are the other ways to increase the inflammatory responses with the presence of ZO?
4. More discussion on the formation of the biofilms in Figure 6 should be included. Higher magnification images of C and D to show any differences should be included.

Author Response

 Thank you very much for the attention given to our manuscript. We appreciate the time and effort you have invested to review our paper that has positively improved the quality of the paper. We have modified the manuscript according to the suggestions provided by the editor and reviewers.

We have attempted to address all the issues/comments from all the reviewers. Please find below point-by-point comments to your suggestions and comments.

 

1. Introduction to the role of zinc oxide as the endodontic sealer that promotes fungal survival and proliferation was insufficient. I suggest the authors expand this part in the introduction as a separate paragraph and include more references.

Answer) Immunologic role of ZO on development of fungal sinusitis was mentioned in introduction as a separate paragraph. Line 70-74 have been revised to mention about role of ZO and reference 12 and 13 have been incorporated as
‘The fungus ball contains various metallic components from different endodontic sealers, including ZO, and ZO can induce fungal growth in the maxillary sinus [8,11,12]. However, when the Aspergillus fumigatus were cultured with ZO on primary nasal epithelial cell, ZO did not influence on A. fumigatus-induced nasal epithelial cells activation and fungal bio-film formation [13].’ 

 

2. It is unclear how ZO was prepared in the experimental section. Is it simple dispersion in PBS?

Answer) Yes, ZO was dispersed in PBS as you mentioned.
        It was described in 2.2 subtitle, ZO preparation as
        ‘ZO was dispersed in PBS at a concentration of 5 mg/mL and stored at ­70 °C until        
        further use in experiments.’

 

3. What are the other ways to increase the inflammatory responses with the presence of ZO?

Answer) The possible role of ZO on the inflammatory response in sinonasal mucosa was mention in Discussion line 367-371, and Ref 25 was added as
‘ZO could increase the amount of fungi in contact with epithelial cells by acting as a nidus for fungal growth and interfering with the mucociliary movement of epithelial cells, thereby prolonging the contact time between the fungi and the epithelial cells. This leads to the maintenance and exacerbation of the inflammatory responses in the sinonasal mucosa [11,25]’..

 

4. More discussion on the formation of the biofilms in Figure 6 should be included. Higher magnification images of C and D to show any differences should be included.

Answer) Fig 6 was changed. We added The description of LIVE/DEAD BacLight staining has been added on the Methods and Results sections, and the differences in confocal  microscopic findings due to ZO was mentioned in Line 293-295 as
‘Although, A. fumigatus with ZO groups revealed thicker green clusters formation, no sig-nificant difference could be objectively demonstrated between the two groups (Figure 6).’

 

We wish to thank you for the effort and time. Your inputs have significantly improved the quality of the paper. We look forward to your kind consideration.

Thank you,

Reviewer 2 Report

Comments and Suggestions for Authors

It is meaningful to check ZO with ASP in an in vivo model. However, this paper needs further improvement to make it more convincing.

1. What is the source of zinc oxide (where did the authors purchase?)
2. What is the size of zinc oxide in this case and how did it get mixed with PBS?
3. Is there any in vitro data for this study? Why try in vivo directly?
4. If ZO could promote fungal proliferation, would it be better to check the minimal dosage of ZO that could promote the fungal proliferation? Direct culture of ASP with ZO on an agar plate would be the first item to check at the beginning before conducting in vivo experiment?
5. For figures, there is a term of “other groups”. For example, in figure 1, “† p < 0.05 compared with other experimental groups.” Please identify what “other” means.
6. Figure 5, why the conclusion is no such significant influence between ASP and ASP+ZO? How to tell based on the number shown in the figure? The 12 week result shows a larger difference in between and could it be expected to have a larger discrepancy at longer time?
7. Figure 6, if there are no fungal cells in A and B, why there is staining color in A and B? Please note what each color means in the caption. 

Author Response

Thank you very much for the attention given to our manuscript. We appreciate the time and effort you have invested to review our paper that has positively improved the quality of the paper. We have modified the manuscript according to the suggestions provided by the editor and reviewers.

We have attempted to address all the issues/comments from all the reviewers. Please find below point-by-point comments to your suggestions and comments.

1. What is the source of zinc oxide (where did the authors purchase?

2. What is the size of zinc oxide in this case and how did it get mixed with PBS?

Answers for queries 1 &2)

 New subsection, Methods 2.1 Reagents has been added.
 Information about ZO has also been included in session 2.1 & 2.2. As,
ZO was obtained from Avention (AV-2014571, 99.9%, 10-20 nm/spherical, Incheon, South Korea). And
ZO was dispersed in PBS at a concentration of 5 mg/mL and stored at ­-70°C until further use in experiments.

 

3. Is there any in vitro data for this study? Why try in vivo directly?

Answer) We performed this study based on the previous in vitro  experiment.
We conducted an in vitro study on ZO and development of A. fumigatus biofilm and published a paper (New Ref 13).
It was mentioned in Introduction Line 70-74 and Ref 13 was added as
‘The fungus ball contains various metallic components from different endodontic sealers, including ZO, and ZO can induce fungal growth in the maxillary sinus [8,11,12]. However, when the Aspergillus fumigatus were cultured with ZO on primary nasal epithelial cell, ZO did not influence on A. fumigatus-induced nasal epithelial cells activation and fungal biofilm formation [13].’

13. Geum, S.Y.; Park, J.W.; Park, H.J.; Ye, M.K.; Shin, S.H. In vitro studies on the role of zinc oxide in the development of Aspergillus fumigatus biofilm on nasal epithelial cells. Korean J Otorhinolaryngol-Head Neck Surg 2022, 65, 684-691, doi:10.3342/kjorl-hns.2022.00479.

 

4. If ZO could promote fungal proliferation, would it be better to check the minimal dosage of ZO that could promote the fungal proliferation? Direct culture of ASP with ZO on an agar plate would be the first item to check at the beginning before conducting in vivo experiment?

Answer) Metal ion enhance fungal growth in different culture media (agar, liquid, and soil) (Ref 10) at very low concentration of zinc (5 ug/mL).
And other study analyzed metal component in fungus ball, collected during endoscopic sinus surgery. The concentration of ZO in the fungus ball was about 3.2 mg/g (Ref 11).

In this study, because a significant portion of intramaxillary inoculated ZO could be removed by mucociliary clearance, we used a concentration of 5 mg/100uL of ZO. 

 

5. For figures, there is a term of “other groups”. For example, in figure 1, “† p < 0.05 compared with other experimental groups.” Please identify what “other” means.

Answer) Thank you for your precise feedback.
       To improve the reader’s understanding and clarity, we changed them as
        ‘ † p < 0.05 compared with PBS+ZO or ASP groups’ in Fig 1 to 6. 

 

6. Figure 5, why the conclusion is no such significant influence between ASP and ASP+ZO? How to tell based on the number shown in the figure? The 12 week result shows a larger difference in between and could it be expected to have a larger discrepancy at longer time?

Answer) Thank you for your careful comments.
   To stress the effect of intramaxillary ZO on histologic change of sinonasal      mucosa, in Discussion Line 398-403 was added as,
‘Generally, it takes several years for a fungus ball to develop after endodontic treatment [11]. Although intramaxillary ZO did not influence the expression of inflammatory cytokines and their transcription factors, ZO did enhance histological change in sinonasal mucosa, such as epithelial and stromal thickness and the number of mucus-producing cells, in a time-dependent manner. These histological changes suggest that extending the experimental period may allow ZO to also impact mucosal inflammation.’

 

7. Figure 6, if there are no fungal cells in A and B, why there is staining color in A and B? Please note what each color means in the caption. 

Answer) LIVE/DEAD BacLight staining utilize mixtures of SYTO 9 green-fluorescent nucleic acid stain and propidium iodide, the red-fluorescent nucleic acid stain. SYTO 9 stain with intact membrane and PI stain in damaged cells.  

In Method (Line 182-183,  LIVE/DEAD BacLight staining mixture at room temperature in darkness for 15 min as reported [14]. Viable cell and fungi stained green, while damaged cells and fungi stained red. Biofilms were observed with confocal scanning laser microscopy. ) and in Results (Line289-295 as, Fungal biofilms were observed as strong green fluorescent displayed ............ could be objectively demonstrated between the two groups (Figure 6).), we tried to explain about the LIVE/DEAD BacLight staining and the results and Fig 6 was change with high resolution pictures.

 

We wish to thank you for the effort and time. Your inputs have significantly improved the quality of the paper. We look forward to your kind consideration.

Thank you,

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for submitting your article for review. I appreciate the work you have done, but I have a few comments and would like you to address the following points:

1.      In the introduction, the authors should address the research niche. There is a lack of information on what studies have been done so far, what solutions are currently used, what the limitations of these known solutions and studies are, and how the authors' research impacts the scientific aspect of this field.

2.      Please expand the introduction and include additional literature references.

3.      Is the preparation described in the subsection "2.1. Aspergillus fumigatus conidia preparation" based on standards? From the literature? Such studies should follow standard protocols. Please explain why these conditions were chosen.

4.      There is a lack of a separate subsection detailing the origin of the reagents—company, purity. This should be compiled into one subsection to facilitate the replication of the presented studies by readers.

5.      Please provide the standard used when performing these studies: 2.3. Measurement of cytokines in sinus mucosa; 2.4. Measurement of transcription factors in sinus mucosa; 2.5. Histological analysis of sinus mucosa; 2.6. Biofilm detection in sinus mucosa.

6.      Why was ANOVA not chosen for statistical analysis?

7.      Figure 1 should be split into two separate figures—it is currently very difficult to read the results and variables.

8.      Add a legend explaining 4wk Intra and 12wk Intra (remove this from the figure description).

9.      Please remove theoretical considerations from the discussion and move them to the introduction. The discussion should only address the results and compare them with those obtained by other researchers. This way, you can verify your data in comparison with the results obtained by other authors. – The entire discussion is incorrect – please revise this section. Compare your results with those of other authors.

10.  Conclusions should also be improved. They should not include theories or references to the literature. Conclusions should be based on the findings from your research. They should be brief, concrete, and include numerical results. That is, you should provide the obtained values numerically – this supports the citation of the publication.

Thank you.

Comments on the Quality of English Language

Sample language errors:

·         "nasal secretion" should be "nasal secretions."

·         "normal healthy volunteers" should be "both CRS patients and healthy volunteers."

·         "presented about" should be "reported."

·         "associated airway fungal disease" should be "associated with airway fungal diseases."

·         "coexisted" should be "coexist."

·         "healthy mucosal" should be "healthy mucosa."

The text needs comprehensive language correction. The large number of corrections makes it difficult to indicate them all. The text should be reviewed by a native speaker.

Author Response

Thank you very much for the attention given to our manuscript. We appreciate the time and effort you have invested to review our paper that has positively improved the quality of the paper. We have modified the manuscript according to the suggestions provided by the editor and reviewers.

We have attempted to address all the issues/comments from all the reviewers. Please find below point-by-point comments to your suggestions and comments.

 

1. In the introduction, the authors should address the research niche. There is a lack of information on what studies have been done so far, what solutions are currently used, what the limitations of these known solutions and studies are, and how the authors' research impacts the scientific aspect of this field.

  Answer) Thank you for your precise feedback.
Zinc oxide has been known as an important factor in the development of fungal sinusitis, especially fungus ball. However, experimental evidence has not been presented.
Authors conducted in vitro study to investigate the effect of ZO on A. fumigatus biofilm formation and published a paper (New Ref 13). In this study we aimed to extend these in vitro results to in vivo studies, to determine whether intramaxillary ZO would develop or exacerbate the fungi-induced inflammatory response in sinus mucosa, or ZO would have no impact on the fungi-induced inflammation.

To improve the quality of Introduction, we separated second paragraph and added information on the immunopathologic role of ZO and previous in vitro study results, as well as Ref 12 & 13.
The purpose of the study was also revised as
‘In this study, the authors conducted experiments to determine whether the interaction between intramaxillary ZO and inhaled A. fumigatus could induce or exacerbate inflammatory response in the sinus mucosa and led to the development of fungal sinusitis or ZO could not impact on the influence A. fumigatus-induced sinus mucosal inflammation

 

2. Please expand the introduction and include additional literature references.

  Answer) As mentioned above, Ref 11 and 12 were added.

 

3. Is the preparation described in the subsection "2.1. Aspergillus fumigatus conidia preparation" based on standards? From the literature? Such studies should follow standard protocols. Please explain why these conditions were chosen.

  Answer) The preparation of fungal conidia was carried out according to the methods described in previous study and New Ref 14 was added.
And it was mentioned in Line 98-99 as,
‘Fungal conidia were collected according to a previously described method [14]. Briefly, conidia ….’

 

4. There is a lack of a separate subsection detailing the origin of the reagents—company, purity. This should be compiled into one subsection to facilitate the replication of the presented studies by readers.

  Answer) As recommended. Subsection ‘2.1. Reagents’ was added.

 

5. Please provide the standard used when performing these studies: 2.3. Measurement of cytokines in sinus mucosa; 2.4. Measurement of transcription factors in sinus mucosa; 2.5. Histological analysis of sinus mucosa; 2.6. Biofilm detection in sinus mucosa.

  Answer) All experimental results were compared and analyzed against those of the negative control group (Intramaxillary PBS).
If we have not fully understood the reviewer’s comments, could you please provide a more detailed explanation of the standard?
We will do our best to revise and improve accordingly.

 

6. Why was ANOVA not chosen for statistical analysis?

  Answer) We used ANOVA, to clarify ‘ANOVA’ was added in Line 189.

 

7. Figure 1 should be split into two separate figures—it is currently very difficult to read the results and variables.

  Answer) Thank you for your careful comments.

          Fig 1 was divided into Fig 1 & Fig 2 as recommended.

 

8. Add a legend explaining 4wk Intra and 12wk Intra (remove this from the figure description).

  Answer) Thank you for your precise feedback.

  4wk Intra and 12wk intra was changed to 4 weeks and 12 weeks as recommended in all figures.

 

9. Please remove theoretical considerations from the discussion and move them to the introduction. The discussion should only address the results and compare them with those obtained by other researchers. This way, you can verify your data in comparison with the results obtained by other authors. – The entire discussion is incorrect – please revise this section. Compare your results with those of other authors.

  Answer) In Discussion, some theoretical parts were moved to the Introduction, and many parts were rewritten and compared to other studies. Such as

 Line 329-332 (In contrast to a previous study, ……..),
Line 338-339 (A, fumigatus conidia contain……..),
Line 344-349 (However, the addition of ZO did not……..),
Line 367-371 (ZO could increase the amount of fungi…….),
Line 392-393 (Dufour et al. suggested that………),
and Line 398-403 (Generally, it takes several years for ……….).

 

10. Conclusions should also be improved. They should not include theories or references to the literature. Conclusions should be based on the findings from your research. They should be brief, concrete, and include numerical results. That is, you should provide the obtained values numerically – this supports the citation of the publication.

  Answer) Conclusion part was rewritten as recommended.

  

Comments on the Quality of English Language

Sample language errors:

• "nasal secretion" should be "nasal secretions."
• "normal healthy volunteers" should be "both CRS patients and healthy volunteers."
• "presented about" should be "reported."
• "associated airway fungal disease" should be "associated with airway fungal diseases."
• "coexisted" should be "coexist."
• "healthy mucosal" should be "healthy mucosa."

The text needs comprehensive language correction. The large number of corrections makes it difficult to indicate them all. The text should be reviewed by a native speaker.

 

Answer) Changed as recommended.
I received help from a company that specializes in editing papers (attach file).

 

We wish to thank you for the effort and time. Your inputs have significantly improved the quality of the paper. We look forward to your kind consideration.

Thank you,

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I thank the authors for their efforts in addressing my previous comments. The quality of this work has improved, and I think it can be published on CIMB now. 

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks for the revision from the authors. I am good with the revision and it is ready for publication.