Complications of Teeth Affected by Molar-Incisor Malformation and Pathogenesis According to Microbiome Analysis

Round 1

Reviewer 1 Report

The aim of the present case report and in vitro study was to clinically present a case of MIM and, additionally, provide the pathogenesis of the complications that occur, by microbiologically analyzing the samples.

The clinical report is not new, however, the microbiological evaluation of the complications represents the original part of the study.

Although the topic is very interesting and current, the paper lacks accuracy in presentation and should be wholly revised as suggested below:

- Title should be edited as “Complication of teeth affected by Molar-Incisor Malformation and considerable pathogenesis according to the microbiome analysis”.

- Abstract should be edited and presented in a clearer fashion. Moreover, the aim of the study should be provided in past tense.

- Authors should better describe the following sentence at the end of the Introduction section “In this study, we analyzed the oral microbiomes inside and outside of the MIM teeth…”: what does it mean “inside and outside”? Moreover, the aim of the study should be provided in past tense.

- Clinical and radiographical images should be chronologically presented according to the quotation within the text. In its current form, is quite hard to precisely follow the clinical case.

- 2.3. Sampling section should be wholly revised. Particularly, precise description of MIM and non-MIM teeth, obtaining of oral microbiome samples and description of groups distribution should be provided.

- 2.5 section should be presented in past tense.

- Figure #3 should be more properly quoted within the Results section/paragraph 3.1 Clinical information.

- Within the Discussion section, Authors should avoid to repeat info already reported within Results section. Moreover, the first part of the same section (that is too lengthy) should be presented in a clearer fashion, resuming the basic information and comparing them with the scientific literature.

It would be interesting, in view of the obtained results, to discuss if an endodontic approach of MIM teeth could be considered a valid alternative to teeth extraction.

According to this reviewer’s consideration, the present manuscript needs extensive revision, therefore rejection of the present manuscript is strongly recommended. However, resubmission of a revised version of the paper is firmly suggested.

Author Response

Reviewer 1

The aim of the present case report and in vitro study was to clinically present a case of MIM and, additionally, provide the pathogenesis of the complications that occur, by microbiologically analyzing the samples.

The clinical report is not new, however, the microbiological evaluation of the complications represents the original part of the study.

Although the topic is very interesting and current, the paper lacks accuracy in presentation and should be wholly revised as suggested below:

- Title should be edited as “Complication of teeth affected by Molar-Incisor Malformation and considerable pathogenesis according to the microbiome analysis”.

=> Thank you for your comments. We edited the title.

- Abstract should be edited and presented in a clearer fashion. Moreover, the aim of the study should be provided in past tense.

=> We corrected as you recommended.

=> The aim of this study was to report the clinical process of MIM teeth and to investigate the pathogenesis according to the microbiome analysis.

- Authors should better describe the following sentence at the end of the Introduction section “In this study, we analyzed the oral microbiomes inside and outside of the MIM teeth…”: what does it mean “inside and outside”? Moreover, the aim of the study should be provided in past tense.

=> We corrected as you recommended.

- Clinical and radiographical images should be chronologically presented according to the quotation within the text. In its current form, is quite hard to precisely follow the clinical case.

=> We revised it in chronological order.

- 2.3. Sampling section should be wholly revised. Particularly, precise description of MIM and non-MIM teeth, obtaining of oral microbiome samples and description of groups distribution should be provided.

=> We revised 2.3 sampling.

- 2.5 section should be presented in past tense.

=> Yes, we corrected it.

- Figure #3 should be more properly quoted within the Results section/paragraph 3.1 Clinical information.

=> Thank you. We corrected it.

- Within the Discussion section, Authors should avoid to repeat info already reported within Results section. Moreover, the first part of the same section (that is too lengthy) should be presented in a clearer fashion, resuming the basic information and comparing them with the scientific literature.

=> We deleted that paragraph.

It would be interesting, in view of the obtained results, to discuss if an endodontic approach of MIM teeth could be considered a valid alternative to teeth extraction.

=> We wrote about the endodontic treatment in MIM teeth in introduction.

According to this reviewer’s consideration, the present manuscript needs extensive revision, therefore rejection of the present manuscript is strongly recommended. However, resubmission of a revised version of the paper is firmly suggested.

• We fully agree with your opinion. As clinic dentists, we have made various efforts to investigate and to find a way to treat it. Thank you for your comment.

Author Response File: Author Response.docx

Reviewer 2 Report

Summary- the conclusion should answer direct to the aim of the study. Please adjust.

Introduction- NGS will help as a tool in making differential diagnosis' Please make aim more clear (in this and previous section).

Discussion- please start with the last sentence of the first paragraph. Others belong to the introduction. 

Please check text for minor English spelling mistakes.

Author Response

Reviewer 2

Summary- the conclusion should answer direct to the aim of the study. Please adjust.

• Thank you. We correct it as you guided.

Introduction- NGS will help as a tool in making differential diagnosis' Please make aim more clear (in this and previous section).

• Thank you. We revised the aim as you and reviewer 1 guided.

Discussion- please start with the last sentence of the first paragraph. Others belong to the introduction. 

• Thank you. Reviewer 1 recommended to delete the first paragraph and followed his comment.

Please check text for minor English spelling mistakes.

• Thank you. We re-checked the English grammar. After all reviews have been completed, we will send it to an expert to check the grammar again.

Author Response File: Author Response.docx

Reviewer 3 Report

Dear authors,

thanks for having provided this manuscript.

What globally emerges from the deep analysis of the manuscript is that english needs to be revised. Other criticisms that raised are here underlined:

1. At the end of introduction it must be reported that the article is a simple case report;
2. Line 73: Birth at 9 months can be considered premature?
3. Line 106: what are complicated MIM?
4. It has to be cleared if groups are three or two.
5. Line 119-121: why isolation was not organized?
6. When endodontic files have been used for sampling in the root canal the tip area was put in contact with the pulp chamber. It should be considered as a bias;
7. If you used metagenomics every information about bacterial species (i.e. Aggregatibacter Actinomycetemcomitans) cannot be given since that technique cannot retrieve such type of results.
8. The attempt of finding a correlation between complications of MIM and the oral microbiome are not worthy as probably its main causes are to be found in the dental structure deficiency deeply connected to this rare condition.

Regards

Author Response

Reviewer 3

Dear authors,

thanks for having provided this manuscript.

What globally emerges from the deep analysis of the manuscript is that english needs to be revised.

• Thank you. We re-checked the English grammar. After all reviews have been completed, I will send it to an expert to check the grammar again.

Other criticisms that raised are here underlined:

1. At the end of introduction it must be reported that the article is a simple case report;

• Yes, we corrected the aim.

1. Line 73: Birth at 9 months can be considered premature?

• Thank you. Premature birth refers to delivery that occurs before 37 weeks form the date of conception. The mother said that the child was born in 9 months as premature birth, because the child was delivered before 37 weeks.

1. Line 106: what are complicated MIM?

• We revised the sentence. Originally, complicated MIM meant MIM with complication, such as periodontal abscess or fistula.

1. It has to be cleared if groups are three or two.

• We are so sorry for not writing clearly. There are two groups; endo group and perio group. We tried to investigate the origin of complication.

1. Line 119-121: why isolation was not organized?

• We are very sorry that we did not understand the exact meaning of this comment. In the study, isolation was performed according to the situation to prevent contamination of the sample.

1. When endodontic files have been used for sampling in the root canal the tip area was put in contact with the pulp chamber. It should be considered as a bias;

• We agree with your opinion. We were concerned about that and were careful not to touch the pulp chamber when removing the file.

1. If you used metagenomics every information about bacterial species (i.e. Aggregatibacter Actinomycetemcomitans) cannot be given since that technique cannot retrieve such type of results.
2. The attempt of finding a correlation between complications of MIM and the oral microbiome are not worthy as probably its main causes are to be found in the dental structure deficiency deeply connected to this rare condition.

• We answer question 7 and 8 together. Your point is right. There were several reasons for choosing NGS. We could identify aa, if aa was dominant or a novel species, if there was a novel species associated the complication of MIM. Also, we had been using NGS for the past researches, we do not think of any other methods other than NGS. Please note that we have tried to approach a new type of tooth anomaly, MIM from various aspects.

Reviewer 4 Report

Summary: The manuscript highlights the therapeutic interventions used to manage a case of a child with molar-incisor malformation (MIM). The authors also analyzed the oral microbiome associated with teeth involved in MIM. It is significant to report this case due to the scarcity of cases published on this topic particularly with microbial genetic analysis, which might give further insights on how to approach patients with a similar clinical scenario. Major reviews and edits are necessary before proceeding with a decision on the final version of the edited manuscript.

Broad and specific comments:

1- In the manuscript title, please remove the extra word "molar" and minimize Molar-Incisor to maintain consistency throughout the manuscript.

2- Abstract:

• English language grammatical errors were noted that may interfere with the scientific accuracy of the manuscript. Kindly revise the entire manuscript for such errors. Few examples include lines 16 and 18 MIM should be spelled out as molar-incisor malformed teeth, molars, etc. as MIM stands for molar-incisor malformation according to the authors. Another suggestion would be to say teeth affected with MIM.
• The perio group and endo group are not defined and summary of the methodology is largely lacking.
• The abstract should be re-written in a concise, scientific manner.

3- Main text (Introduction):

• MIM has been mistaken for molar-incisor hypomineralization (MIH) until recently, kindly describe the similarities and differences between the two clinical entities. Why is this a case of MIM and not MIH?
• Lines 46-48 would be more suitable to be included in the discussion section.
• Line 53, what are other factors that can alter the course of dentoalveolar infections other than the resident microbiome?
• Lines 59-60 kindly clarify inside vs outside of the molar-incisor malformed teeth.

4- Main text (Materials and Methods):

• The authors described that some extracted molars showed abundant granulation tissue and pulp necrosis, also vitality testing was incompletely performed, were any of the teeth involved with MIM tested, and if so, did any suggest pulp necrosis prior to radiographic examination? Additionally, was the granulation tissue submitted for histopathologic examination to confirm the nature of it?
• Indicate that the numbering system used for teeth nomenclature is the FDI two-digits notation.
• It is not clearly stated in the manuscript whether the perio group samples were collected before tooth extraction. Please include that clearly.
• For the endo group, a suitable control may be a sound extracted third molar or an extracted supernumerary tooth from another patient. Do the authors think it is an important control to include? Why would it be included, or why not such control is not included?

5- Main text (Results):

• Results of figure 5B do not match the legend of the figure, which one is the correct result? Is it at 5% frequency or 1% frequency, and is it all samples (endo and perio groups) or only perio samples?
• The authors did not conclude any results by comparing the average operational taxonomical units of perio group 1 samples with Nor-1 control, and that of the perio group 2 samples and Nor-2. Please address that in the results and the discussion sections.

6- Main text (Discussion):

• Can the authors elaborate on the relevance of absence of Aggregatibacter actinomyces in the MIM samples analyzed? How would the authors explain the lack of the periodontal stability to teeth involved in MIM and the increased susceptibility to infection, is it purely a structural anomaly? Or does the microbiome analyzed play a potential role in the pathogenesis of this condition?
• It seems that preterm birth strongly correlates with MIM, do the authors suggest a potential explanation to this observation?
• Line 263: “If the plaque was incorrect, the disease-related bacteria may not be accurately identified.” What does “If the plaque was incorrect” mean?

Author Response

Reviewer 4

Summary: The manuscript highlights the therapeutic interventions used to manage a case of a child with molar-incisor malformation (MIM). The authors also analyzed the oral microbiome associated with teeth involved in MIM. It is significant to report this case due to the scarcity of cases published on this topic particularly with microbial genetic analysis, which might give further insights on how to approach patients with a similar clinical scenario. Major reviews and edits are necessary before proceeding with a decision on the final version of the edited manuscript.

• Thank you for your comment.

Broad and specific comments:

1- In the manuscript title, please remove the extra word "molar" and minimize Molar-Incisor to maintain consistency throughout the manuscript.

• We removed the word “molar”.

2- Abstract:

• English language grammatical errors were noted that may interfere with the scientific accuracy of the manuscript. Kindly revise the entire manuscript for such errors. Few examples include lines 16 and 18 MIM should be spelled out as molar-incisor malformed teeth, molars, etc. as MIM stands for molar-incisor malformation according to the authors. Another suggestion would be to say teeth affected with MIM.
• We corrected the manuscript as you guided.
• The perio group and endo group are not defined and summary of the methodology is largely lacking.
• We corrected the manuscript as you guided.
• The abstract should be re-written in a concise, scientific manner.
• We corrected the manuscript as you guided.

3- Main text (Introduction):

• MIM has been mistaken for molar-incisor hypomineralization (MIH) until recently, kindly describe the similarities and differences between the two clinical entities. Why is this a case of MIM and not MIH?
• We added in the manuscript about the MIM and MIH.
• Lines 46-48 would be more suitable to be included in the discussion section.
• Thank you for your advice. We moved it to the discussion.
• Line 53, what are other factors that can alter the course of dentoalveolar infections other than the resident microbiome?
• In general periodontial disease, age, tobacco, diabetes,etc. affect it, but in localized periodontal disease at young age, tooth morphology and certain bacteria affect it. There was no research on micobiome around MIM, so we decided to study it.
• Lines 59-60 kindly clarify inside vs outside of the molar-incisor malformed teeth.
• Thank you for your advice.

4- Main text (Materials and Methods):

• The authors described that some extracted molars showed abundant granulation tissue and pulp necrosis, also vitality testing was incompletely performed, were any of the teeth involved with MIM tested, and if so, did any suggest pulp necrosis prior to radiographic examination? Additionally, was the granulation tissue submitted for histopathologic examination to confirm the nature of it?

Thanks for the good point. Pulp necrosis could be convinced if the apical radiolucent was assured as in the mandibular left first molar in Figure 2. However, for other teeth, the apical radiolucent properties were not certain. Even when a radiographic radiolucency is appeared, it was confused whether the cause was the perio or pulp. So we decided to investigate it. Biopsy was not performed for the granulation tissue. We will proceed in the next study.

• Indicate that the numbering system used for teeth nomenclature is the FDI two-digits notation.
• Thank you. We added that information.
• It is not clearly stated in the manuscript whether the perio group samples were collected before tooth extraction. Please include that clearly.
• We collected the perio group sample before extraction.
• For the endo group, a suitable control may be a sound extracted third molar or an extracted supernumerary tooth from another patient. Do the authors think it is an important control to include? Why would it be included, or why not such control is not included?

• Thanks for the good point. Since the composition of oral microbes differs from person to person, non-MIM were set as the control group. This advice will be reflected in the next study.

5- Main text (Results):

• Results of figure 5B do not match the legend of the figure, which one is the correct result? Is it at 5% frequency or 1% frequency, and is it all samples (endo and perio groups) or only perio samples?
• Sorry for the confusion. (B) is for all samples seen at 5%. This is because too many genus appear when set to 1%. (C) is 1% of the genus of the endo group. This is because there were very few bacterial species in the endo group.
• The authors did not conclude any results by comparing the average operational taxonomical units of perio group 1 samples with Nor-1 control, and that of the perio group 2 samples and Nor-2. Please address that in the results and the discussion sections.
• Thank you. We added that information.

6- Main text (Discussion):

• Can the authors elaborate on the relevance of absence of Aggregatibacter actinomyces in the MIM samples analyzed? How would the authors explain the lack of the periodontal stability to teeth involved in MIM and the increased susceptibility to infection, is it purely a structural anomaly? Or does the microbiome analyzed play a potential role in the pathogenesis of this condition?
• Thanks for the good point. It is like starting this study to find out. It was difficult to determine whether the cause of serious complications was pulp, periodontal, or complex. In addition, since aa, a bacteria, is widely known as the cause of severe periodontitis at a young age, evidence was needed whether it was the same or different. In this MIM periodontal group, which showed severe periodontitis, more bacteria were detected in general periodontitis not aa, so we concluded that it is periodontitis caused by tooth structure rather than a specific bacteria.
• It seems that preterm birth strongly correlates with MIM, do the authors suggest a potential explanation to this observation?

MIM has been shown to be associated with brain-related damage in the neonatal period, including premature birth. It is thought that epigenetic damage appeared in the relationship between the cranial nerve and hard tissue formation, but the exact mechanism needs to be explored further.

• Line 263: “If the plaque was incorrect, the disease-related bacteria may not be accurately identified.” What does “If the plaque was incorrect” mean?

• We tried to show that sampling is important. It has been modified.

Reviewer 5 Report

The article titled ‘Complication of Molar-Incisor malformation molar and considerable pathogenesis according to the microbiome analysis’ focuses on the case studies of MIM molar teeth and their survival treatments or extraction manifestations. The article is well structured and scientifically sound. Minor Grammatical errors needed to be corrected and proof read. 

Author Response

Reviewer 5

The article titled ‘Complication of Molar-Incisor malformation molar and considerable pathogenesis according to the microbiome analysis’ focuses on the case studies of MIM molar teeth and their survival treatments or extraction manifestations. The article is well structured and scientifically sound. Minor Grammatical errors needed to be corrected and proof read. 

• Thank you for your kindness about our poor manuscript. We will do our best to do better research in the future. After all reviews are over, we will send it to the expert for the correction of English grammar.

Round 2

Reviewer 1 Report

All the suggested revisions have been undertaken. Publication of the present manuscript is recommended pending grammar editing.

Author Response

Thank you so much for your help. 

Reviewer 3 Report

Dear authors,

  unfortunately you did not succeed in answering to the most crucial problems that I underlined in my first round of revision (isolation only partially described, justification for the meta genomics use very vague and inconsistent, same applies for the efforts of characterizing the oral microbiome in a rare condition as MIM).

Moreover when a reviewer suggests a language revision it is expected that such revision is conducted as soon as possible as such element contributes to the final evaluation.

I confirm my previous opinion of rejection.

Regards

Author Response

• We are very sorry for not responding properly to your review. In particular, even though we could do a language revision quickly, we  were too relaxed to watch the situation and try to do what we thought. We are really sorry for taking your time.

Reviewer 4 Report

Summary of the revised manuscript: The authors addressed most of the suggestions in a scientific manner, although some English language grammatical errors were still noted. Few other comments were added that need to be addressed clearly and concisely.

Broad and specific comments:

1- The abstract is still lacking brief description on how the microbiome analysis was performed, please emphasize on the methods in the abstract.

2- Main text (introduction):

Add a sentence about how MIM has been mistaken for molar-incisor hypomineralization (MIH) until recently before talking about the similarities and differences (correct difference to plural form (i.e. differences))

Lines 54-55: rewrite the sentence in a more scientific manner "NGS is a kind of molecular technique used for identifying microorganism and to possibly read more sequences faster and more economically." For example, is NGS has more broad uses than identifying microorganisms.

3- Main text (Materials and Methods):

Lines 102-103: remove "as one sample" because it is confusing to the reader.

4- Main text (Results):

Figure 2 legend: it is a fistula not fistular

Figure 5 and lines 214 and 215 are still confusing to me, perhaps using labels on the A, B, and C of the figure would help and make the results more clear (e.g. types of samples analyzed, etc..).

5- Main text (Discussion):

Elaborate on the following: How would the authors explain the lack of the periodontal stability to teeth involved in MIM and the increased susceptibility to infection? Is it purely (100%) a structural anomaly? Or does the microbiome analyzed play a potential role in the pathogenesis of this condition? Any theory the authors can include in the manuscript?

It seems that preterm birth strongly correlates with MIM, do the authors suggest a potential explanation to this observation? Your answer "MIM has been shown to be associated with brain-related damage in the neonatal period, including premature birth. It is thought that epigenetic damage appeared in the relationship between the cranial nerve and hard tissue formation, but the exact mechanism needs to be explored further." Add a sentence elaborating on the yet unexplored role of epigenetics, that with the appropriate citation.

Author Response

Reviewer 4

Summary for the revised manuscript: The authors addressed most of the suggestions in a scientific manner, although some English language grammatical errors were still noted. Few other comments were added that need to be addressed clearly and concisely.

• Thank you. We checked the English language in the revised manuscript.

Broad and specific comments:

1- The abstract is still lacking brief description on how the microbiome analysis was performed, please emphasize on the methods in the abstract.

• We apologize for the unclear description and added more details on the method to the Abstract.

2-Main text (introduction):

Add a sentence about how MIM has been mistaken for molar-incisor hypomineralization (MIH) until recently before talking about the similarities and differences (correct difference to plural form (i.e. differences))

• Thank you for your advice. We corrected the manuscript.

Lines 54-55: rewrite the sentence in a more scientific manner “NGS is a kind of molecular technique used for identifying microorganism and to possibly read more sequences faster and more economically.” For example, is NGS has more broad used than identifying microorganisms.

• Thank you for your advice. We corrected the manuscript.

3- Main text (Materials and methods):
Lines 102-103: it is a fistula not fistular

• Thank you for your advice. We corrected the manuscript.

Figure 5 and lines 214 and 215 are still confusing to me, perhaps using labels on the A, B, and C of the figure would help and make the results more clear (e.g. types of samples analyzed, etc..).

• Thank you for your advice. The editor also pointed out that problem. Therefore, we corrected Figure 5 and the manuscript.

5- Main text (Discussion):

 Elaborate on the following: How would the authors explain the lack of the periodontal stability to teeth involved in MIM and the increased susceptibility to infection? Is it purely (100%) a structural anomaly? Or does the microbiome analyzed play a potential role in the pathogenesis of this condition? Any theory the authors can include in the manuscript?

• Thank you for focusing on that important point. Determining what causes severe periodontitis at a young age was the most important motive for starting the study. The probable causes were tooth morphology, microbes such as Aggregatibacter actinomyces (A.a) causing juvenile aggressive periodontitis, and periodontitis due to pulp necrosis. Tooth morphology was already identified in previous studies. In this microbial analysis, we revealed the main microorganisms that could be found in general periodontitis, which did not include microorganisms such as A.a, and thus, juvenile periodontitis was excluded. In addition, the clinical examination confirmed that pulp necrosis and periodontitis occurred independently.
• During the course of the study, we found out that the classification of periodontitis was revised in 2018, and it was confirmed to belong to the category called localized tooth-related periodontitis. So, we came to a conclusion about the pathogenesis of MIM.
• Our explanation may still be lacking. If you or the other reviewers are still confused or feel our explanations and logic are lacking, we apologize. However, if you communicate these concerns, we will improve the manuscript further. Thank you very much for your help.

It seems that preterm birth strongly correlates with MIM, do the authors suggest a potential explanation to this observation: Your answer “MIM has been shown to be associated with brain-related damage in the neonatal period, including premature birth. It is thought that epigenetic damage appeared in the relationship between the cranial nerve and hard tissue formation, but the exact mechanism needs to be explore further.” Add a sentence elaborating on the yet unexplored role of epigenetics, that with the appropriate citation.

• Thank you. We explained the unknown cause by citing the most recently published review paper on MIM (Vargo et al., 2020). A sentence indicating that an epigenetic association was the opinion of the authors was added to the manuscript.

Round 3

Reviewer 3 Report

Dear authors,

I do not understand how can I change my opinion if you simply admit you were not able to answer to my questions and concerns.

I must confirm my judgment of general linguistic and methodological weaknesses.

Regards

Author Response

We are very sorry for not matching your criteria. It was never on purpose. As an pediatric clinical dentist, not a microbiologist, there are many shortcomings about the methodology and analysis. We are sorry, but if you tell us what`s wrong one more time, we will do our best. 

 In addition, it was submitted by revising the English language. 

Best regards,

Reviewer 4 Report

No further comments or suggestions.

Author Response

Thank you for your help. 

With best regards,