PONE-D-20-02327

Ethanol binge drinking exposure affects alveolar bone quality and aggravates bone loss in experimentally-induced periodontitis

PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: No

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript by Frazão and colleagues explores the potential role of ethanol consumption on alveolar bone loss. The study focused on the use of a rats to investigate whether ethanol exposure per se was sufficient to induce dental changes. It is an interesting and important question, and the group used a standard, well-accepted method for inducing periodontal tissue breakdown by placing ligatures around the molars. Four experimental groups were described: a “control” group, an ethanol exposure group in which rats are given ethanol for 3 days then off for 4, for a total of 28 days; another group that received ethanol and ligatures, and a fourth group that has ligatures and ethanol exposure as well.

If I understand the experimental groups correctly, those in G1 are not exposed to ethanol (the control group). Animals in G2 are exposed to ethanol (“Ethanol group (EtOH, G2) received ethanol binge drinking protocol characterized by 30% w/v ingestion…”). Animals in the G3 and G4 groups were also exposed to ethanol for 14 days (“On the 14th day of ethanol exposure protocol, animals from experimental periodontitis (G3) group and experimental periodontitis + ethanol (G4) group were submitted to… ligature-induced periodontal disease by insertion of cotton ligatures”). Consequently, there is no group that received ligatures to induce periodontitis) but did not receive ethanol. Therein lies my major concern with this work: without this group, it is impossible to separate the effects of the ligatures from the effects of ethanol exposure. Consequently, the data do not support the conclusion made by the authors, that binge-drinking decreases alveolar bone quality. (I would prefer that a measurable value be substituted since “bone quality” is a vague and non-quantitative descriptor). Other questions and concerns are outlined below, but in its current form the data do not support the conclusions drawn.

Animals in G4, which had both received ligatures and were exposed to ethanol presented a higher area of exposed root in comparation to “ethanol per se protocol and controls”. I don’t understand this result. If I go by the figure, the dark purple bar representing the G4 group is higher than the light purple G1 bar e.g., there is greater root exposure in G4 compared to G1 but that is easily understood by the fact that there is a ligature in the G4 group; it is expected that the ligature causes gingival recession.

The results in Figure 3 are described as follows: “animals submitted to binge drinking protocol plus experimental periodontitis presented an increase in alveolar bone loss compared to ethanol per se protocol.” The appropriate control, however, is the group that had ligatures but no ethanol exposure; this group does not exist, as far as I can tell.

In Figure 4, it appears that G3 and G4 exhibit an equivalent e.g., non-significant difference in BV/TV, trabecular number, and trabecular thickness. If I understand the groups correctly, that means that whether the animal is exposed to ethanol or not, the effect on alveolar bone loss is equivalent. Thus, I cannot see how

Reviewer #2: Dear Author, congratulations on a well designed study. It is well written and well performed. However, I am missing some more parameters from this study. Since you already did microCT, I think it would be benefical for this study to measure the distance between the cemento-enamel junction and the alveolar bone crest (CEJ-AC) at 6 different points (mesio-buccal, buccal, distobuccal, disto-lingual, lingual and mesio-lingual), noy just the four points that you have chosen, particular when you see the large different in alveolar bone promixal and distal. Please see Virto L, , et al. Melatonin expression in periodontitis and obesity: An experimental in-vivo investigation. J Periodont Res. 2018;53:825–831. on how to do it. It does not take long time, and would give you valuable information on the bone loss similar to how you would evaluated with a periodontal probe in the clinic. It would be beneficial for this study to include histology

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Reviewer #1: No

Reviewer #2: No

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Ethanol binge drinking exposure affects alveolar bone quality and aggravates bone loss in experimentally-induced periodontitis

PONE-D-20-02327R1

Dear Dr. Lima,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Timothy Damron

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: no more comments need for this round and I hereby accept the manuscript for publication. Thank you for address the comments made

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Reviewer #2: No