Measurement of the Level of Nitric Oxide in Exhaled Air in Patients Using Acrylic Complete Dentures and with Oral Pathologies

Round 1

Reviewer 1 Report

First of all congratulatios to the authors for their work and for the subject of their paper that has not been studied yet in the literature( I teach Complete Denture in my University since 25 years).

I consider that the Abstract and the Introduction are well written, following the rules.

Regarding the Materials and Method, it demonstrates validity and reliability. All the measurements are very well described , also the statistical analysis is well performed.

The Discussion follow all the requirements of this section, and keeping in mind that the subject is not a studied one, I consider them well written.

Overall I appreciate that the paper should be published, with some minor revisions, in English language especially.

 

Author Response

Please see the attachement.

Author Response File: Author Response.doc

Reviewer 2 Report

The authors aim at demonstrating that the measurement of nitric oxide (NO) in the exhaled air of patients suffering from inflammation of the oral cavity can be a suitable diagnostic tool. The purpose of the study is very interesting and can stimulate the scientific community at large, not only in the dentistry field but could be also valuable for molecular biologists and clinicians in general. However, the manuscript needs major revisions and substantial changes at every level, having not an appropriate background description and experimental design, and results appear poorly discussed.

General comments

The authors should carefully revise the English usage of the manuscript, especially as regard grammar and spelling.

Please carefully check about acronyms. Some acronyms are not made explicit and some others are reported continuously in both forms. As an example, iNOS (inducible nitric oxide synthase) is always reported in the double form (acronym and full length) in the entire manuscript as in lines 89 and 100. Others are not made explicit at all, as for NADPH oxidase.

Please carefully check about names of bacteria and fungi. As an example, in line 63 is Candida and Turulopsis spp.

Introduction

Generally speaking, the introduction is too long and does not present the appropriate background supporting the main aim of the study, such as molecular mechanisms underlying NO-related cell functions in the oral cavity and cell responses towards nitrosative stress in details. Moreover, it should be more specific, especially when reporting experimental studies. References should be updated with more recent studies on the topic of oral cavity inflammation. Finally, the aim of the work should be clearly reported.

Lines 32-34: gingivitis, mucositis and periodontitis are described by the author as system diseases. Please carefully check this statement. Maybe the authors would like to say that substances or secondary metabolites released by bacteria stimulate cells in the oral cavity to produce cytokines which lead to local disease such as gingivitis, mucositis and periodontitis? And that the chronic inflammation can lead to systemic diseases such as endocarditis (P. gingivalis), as an example? Please check the literature, here some examples: Ninomiya M et al. Clin Oral Investig. 2020 Feb;24(2):833-840. doi: 10.1007/s00784-019-02973-2; Patrakka O et al. J Am Heart Assoc. 2019 Jun 4;8(11):e012330. doi: 10.1161/JAHA.119.012330; Kriebel K et al. Front Microbiol. 2018 Jan 30;9:53. doi: 10.3389/fmicb.2018.00053.

Lines 35-42: if the aim of the study is to demonstrate that nitric oxide is a diagnostic tool for the oral cavity inflammation in patients using acrylic complete dentures, the detailed description of the formation of the periodontal pockets during dental plaque apposition is an information not required. What should be highlighted instead, are bacterial species involved in NO-related pathway of inflammation, substances released like LPS and LTA which can stimulate NF-kB that in turn transcripts for the iNOS and interactions of methacrylates with cells of the oral cavity. For references see: Schweikl H et al. Dent Mater. 2021 Jan;37(1):175-190. doi: 10.1016/j.dental.2020.10.029; Barbadoro P et al. Nitric Oxide. 2021 Jan 1;106:66-71. doi: 10.1016/j.niox.2020.11.002.

Lines 67-72: Again, authors made a parallelism between systemic diseases and inflammations in the oral cavity. Moreover, they only describe the advantages of measuring NO in the exhaled air. Are there any limitations for this diagnostic technique? Why is it less common as a diagnostic test also for pathologies where the participation of NO is well established?

Lines 72-74: As stated in the next paragraphs, NO is produced by three isoforms, so nitric oxide synthases (NOSs) should be written instead of NOS.

Lines 74-78: Firstly, the authors should report that what they are describing is the mechanism of formation of reactive nitrogen species (RNS), so the readers can be aware of that. The RNS obtained from the reaction between NO and superoxide anion (O2•−) is peroxynitrite (ONOO^-), so please check “peroxynitrate”.

Line 86: Please check on this sentence. NO is mostly produced by the eNOS in endothelial cells but, as stated above, the nNOS isoform is physiologically present as well. Thus, NO represents a neurotransmitter in the central nervous system as well.

Line 90: What isoform of COX?

Line 98: Please check IF-beta.

Lines 95-100: Please carefully check this statement. Firstly, authors should more specifically describe inflammation dynamic in the oral cavity, where gingival fibroblasts and pulp cells are the main player in initiating cell responses against cariogenic bacteria. Secondly, cells in the site of inflammation like fibroblasts are stimulated by bacteria, viruses and parasites and secrete pro-inflammatory mediators for the recruitment of other cells in the site of inflammation, like cells of the immune system, like white cells of the blood. Circulating monocytes are one of these cells, which infiltrate in damaged tissues and differentiate into macrophages.

Materials and Methods

I have some concerns about the recruitment of patients for this study. The three groups are not homogeneous as regard the age, as an example. The authors compared 100 patients aged 25-65 and 100 patients aged 25-85. Patients aged 66-85 should be excluded from the analysis or included also in the first group. Next, the number of patients in the control group is not consistent. I can agree in a smaller number of patients as control, but 30 subjects against 100 is not appropriate. Why the age of patients in the control group is not declared? Finally, the exclusion criteria should be improved or better described. What does it mean that subjects with general somatic diseases or that use medicines for chronic diseases are excluded from the study? As an example, this means that people affected by diabetes, which is one of the major co-morbidity associated with the increase of parodontitis, are not included?

Discussion

In general, the discussion is redundant with the result section and does not correlates data presented with explanation by the authors.

Why the measurement of NO in exhaled air is not commonly used as a diagnostic tool for inflammation? Could be that the presence of NO is not a selective marker because could be increase also in other pathologies like tumors? From data collected, can the authors conclude that the NO measurement can be more advantageous for the diagnosis of particular pathologies of the oral cavity with respect to others?

Lines 389-390: The authors correlate patients with type 3 stomatitis and bad denture hygiene with the highest amount of NO measured. Can the author speculate about an explanation for that data correlation?

Lines 395-403: Not relevant for the discussion of a paper with the aim of demonstrating that NO could be a suitable tool for the diagnosis of inflammation in patients using acrylic complete dentures. What is the effect of acrylics and resin monomers in the tissues of the oral cavity as regard NO production?

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The paper has now reached the adequate standards to be accepted for publication. However moderate English changes are required, mainly in the introduction section. 

 

Author Response

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Author Response File: Author Response.docx