Review Reports - A Bioactive Enamel Sealer Can Protect Enamel during Orthodontic Treatment: An In Vitro Study

Round 1

Reviewer 1 Report

Dear Author,

The article may be useful for a potential reader if necessary additions/changes are made.
Minor remarks are marked directly in the text,
the others are listed in the attached file.

Comments for author File: Comments.pdf

Author Response

The revision is available for the honorable reviewer in the attached file.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors fabricated the bioactive resin sealer using bioglass that prevented enamel erosion by 1 % citric acid. The experiment itself is carried out with careful treatment, but I think the approach is quite common and also old. Therefore not so much valuable information will be included in the draft. I cannot recommend the publication of this manuscript.

The authors showed the degree of conversion by the FTIR analysis with not the single but the multiple reflection ATR. Is it true?
The authors applied the sealer near the orthodontic brackets, but it seems to me that it’s quite difficult to attach the sealed surface to the ATR crystal. The authors should clearly show where they coated the enamel surface using the bioactive sealer.
The experiment lacks a suitable control group that prevents enamel erosion. Because the degree of conversion of resin sealer is quite low compared to the commercially available dental products, I cannot evaluate whether the bioactive resin sealer is a unique product that prevents enamel erosion.
The method to calculate the degree of protection is missing.
I cannot understand the blue and orange bar in Figure 7.
The discussion is not based on the results of this study. Especially, the author must show evidence why the bioglass prevented the enamel erosion.

Author Response

The revision is available for the honorable reviewer in the attached file.

Author Response File: Author Response.pdf

Reviewer 3 Report

The author presents an in vitro study describing the protective role of a bioactive sealer with 45S5 bioglass on enamel around orthodontic brackets. The sealer formulation and degree of conversation evaluation are described. The application is performed in extracted human teeth, which are exposed to an acidic challenge and the protection capacity of the sealer is evaluated.

Title: I suggest “in vitro” to be added to the study title, since the reference to orthodontic treatment may mislead the readers about the manuscript content.

Lines 11: I suggest the author refer to the protective effect regarding erosion.

Line 16: please refer to what type of control was used.

Keywords: please remove the numbers and the word keyword.

Figure 1: please refer to what type of control was used.

Lines 81-82: the author refers to “Inter examiner calibration was conducted before actual recording of the obtained results”. Since the study only has one author, why the reference to inter examiner calibration?

Section 2.1: what was the maximum time the teeth were stored before being used for experiments?

I suggest the methods section be rearranged and sections 2.3 and 2.5 to be presented first (sealer production and degree of conversation evaluation) and after the remaining sections referring to teeth application and evaluation.

Line 113: I suggest referring to a negative control.

Section 2.8: the author refers to “no signs of erosion were considered 100% protection for enamel”. All the other situations were considered as non-protection and attributed a 0% score?

Line 164: the author refers to figure 2D. Please correct it.

Section 3.1 and figures 4 and 5: in the text, the author refers to figure 4 as the bioactive glass group and figure 5 as the resin group. However, figures 4 and 5 refer to the opposite way. Please correct it.

Line 177: I believe the author meant p > 0.05 since it refers to a non-significant statistically difference.

Figure 7: I suggest the figure be presented as a table instead since it would present the precise values. Also, please refer to the meaning of a and b letters.

Results section: I suggest section 3.2 to be presented first, following the same order of the methods section.

Do you think the acidic explosion model mimics the oral cavity exposure? In the oral cavity, the presence of saliva with its tampon characteristics and remineralization capacity attenuates the acidic challenges the enamel encounters.

The plagiarism detector found plagiarism issues, regarding a published manuscript from the same author. Please correct it. See, for instance, lines 84-90, 163-167, 172-174.

Author Response

The revision is available for the honorable reviewer in the attached file.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The second version of the paper is a poor improvement, and a substantial revision is needed to make this manuscript suitable for publication.

The originality of this study is still unclear. The author must explain why she did the research and point out the problem of the current study in the Introduction part.
The positive control group using fluoride agents is not suitable for this study. Because commercially available agents didn’t show a positive effect in this study. This means the research design is incorrect for the erosion challenge. Also, the author must explain why the degree of conversion is relatively low compared to the commercially available product.
The author replaced Figure 7 with the table and showed the protection ratio before and after the erosion challenge. The table shows the protection ratios before the erosion challenge is 100 %, but I cannot find that result from Figure 7.
The author discussed the mechanisms of the enamel protection by the bioglass are SiO₂ layer. However, EDS analysis didn’t show such evidence.
If calcium and phosphate ions from bioglass have the potential to decrease the erosion potential, the author should show its evidence.

Author Response

Honorable reviewer (2) report

The originality of this study is still unclear. The author must explain why she did the research and point out the problem of the current study in the Introduction part.

Response of the author

I wish to thank the honorable reviewer for the valuable scientific comment. The following part was added to the Introduction section.

Previous research showed the remineralization capacity of commercially available bonding systems doped with 10% > bioactive glass[1], other reports demonstrated the capability of the fluoride containing bioactive glasses resins 5%> as orthodontic primers[2], moreover bioactive acidic (resin free) pastes were utilized as orthodontic sealers[3]. However, in the current study a (fluoride-free) bioactive glass-resin blend having a 30% weight fillers load was tested, moreover, a customized resin blend was utilized to determine the interaction of the 45S5 bioglass with each component. Doping commercially available adhesive systems may pose some difficulties in determining the exact interaction of the added bioactive glasses to their components because many of the commercially available adhesive systems’ exact weight percentages are not fully described by the manufacturers.

2. The positive control group using fluoride agents is not suitable for this study. Because commercially available agents didn’t show a positive effect in this study. This means the research design is incorrect for the erosion challenge. Also, the author must explain why the degree of conversion is relatively low compared to the commercially available product.

Response of the author

I wish to thank the honorable reviewer for the valuable scientific comment The positive control group was added in which the Opalseal group ( Opalseal is a commercially available orthodontic enamel sealer) was added.

Regarding the degree of conversion values obtained in the current study, I added the following paragraph to the manuscript.

However, these values for degree of conversion are relatively low compared to the obtained degree of conversion values of the commercially available resin sealers, which may be attributed to the inhibitory effect exerted by the 45S5 bioactive glass on the BIS-GMA based resins [4] which may be improved if different resin systems were utilized[4], moreover, the degree of conversion test was conducted in the current study at room temperature (20^oc) and not exposed to any post cure temperatures[4].

3. The author replaced Figure 7 with the table and showed the protection ratio before and after the erosion challenge. The table shows the protection ratios before the erosion challenge is 100 %, but I cannot find that result from Figure 7.

Response of the author

I wish to thank the honorable reviewer for the valuable scientific comment. The other honorable two reviewers recommended the change of figure 7 into table and recommended simplifying the terms used in describing this experiment, accordingly I have replaced figure 7 with the table and used the term percentage of intact enamel to simplify the terms used. The explanation for the new terms used and the description of the experiment is currently available in the table legends and in the body of the manuscript

4. The author discussed the mechanisms of the enamel protection by the bioglass are SiO₂ layer. However, EDS analysis didn’t show such evidence.

Response of the author

I wish to thank the honorable reviewer for the valuable scientific comment. The figure legends of the bioactive res

The aforementioned hypothesis can be supported by the EDS analysis conducted in the current study which showed that average weight percentage of multiple areas of the bioactive resin (specially areas showing projection of fillers from the resin matrix) were 36 % (with detectable amount of phosphorus and calcium) while for the Opalseal group no signs of fillers projection were noted and the silica average weight % was approximately 9%. It worth mentioning here that, Opal Seal is an orthodontic enamel sealer which is 38% filled with glass ionomer and nanofillers, while the bioactive resin sealer examined in the current study is 35% filled with 45S5 glass.

5. If calcium and phosphate ions from bioglass have the potential to decrease the erosion potential, the author should show its evidence.

Response of the author

I wish to thank the honorable reviewer for the valuable scientific comment.

The following part was added to the discussion section.

the EDS analysis conducted in the current study which showed that average weight percentage of multiple areas of the bioactive resin (specially areas showing projection of fillers from the resin matrix) were 36 % (with detectable amount of phosphorus and calcium) while for the Opalseal group no signs of fillers projection were noted and the silica average weight % was approximately 9%.

Previous research confirmed that upon contact of the 45S5 bioglass with fluids it immediately starts its bioactive cycle by forming the silica rich layer and the calcium phosphate rich layer. Some of the evidence for this mechanism can be found in the following articles:

[1] S.M. Lee, K.H. Yoo, S.Y. Yoon, I.R. Kim, B.S. Park, W.S. Son, C.C. Ko, S.A. Son, Y.I. Kim, Enamel Anti-Demineralization Effect of Orthodontic Adhesive Containing Bioactive Glass and Graphene Oxide: An In-Vitro Study, Materials (Basel) 11(9) (2018) DOI: 10.3390/ma11091728.

[2] S.M. Lee, I.R. Kim, B.S. Park, D.J. Lee, C.C. Ko, W.S. Son, Y.I. Kim, Remineralization Property of an Orthodontic Primer Containing a Bioactive Glass with Silver and Zinc, Materials (Basel) 10(11) (2017) DOI: 10.3390/ma10111253.

[3] A.S. Bakry, M.A. Abbassy, H.F. Alharkan, S. Basuhail, K. Al-Ghamdi, R. Hill, A novel fluoride containing bioactive glass paste is capable of re-mineralizing early caries lesions, Materials 11(9) (2018) 1636.

[4] M. Par, N. Spanovic, T.T. Taubock, T. Attin, Z. Tarle, Degree of conversion of experimental resin composites containing bioactive glass 45S5: the effect of post-cure heating, Sci Rep 9(1) (2019) 17245 DOI: 10.1038/s41598-019-54035-y.

[5] A. Bakry, H. Takahashi, M. Otsuki, A. Sadr, K. Yamashita, J. Tagami, CO2 laser improves 45S5 bioglass interaction with dentin, Journal of dental research 90(2) (2011) 246-250.

[6] A. Bakry, H. Takahashi, M. Otsuki, J. Tagami, Evaluation of new treatment for incipient enamel demineralization using 45S5 bioglass, Dental Materials 30(3) (2014) 314-320.

[7] A.S. Bakry, Y. Tamura, M. Otsuki, S. Kasugai, K. Ohya, J. Tagami, Cytotoxicity of 45S5 bioglass paste used for dentine hypersensitivity treatment, Journal of dentistry 39(9) (2011) 599-603.

[8] E.M. Carvalho, P.V.C. Ferreira, M.F. Gutierrez, R.F. Sampaio, C.N. Carvalho, A.S. Menezes, A.D. Loguercio, J. Bauer, Development and characterization of self-etching adhesives doped with 45S5 and niobophosphate bioactive glasses: Physicochemical, mechanical, bioactivity and interface properties, Dent Mater 37(6) (2021) 1030-1045 DOI: 10.1016/j.dental.2021.03.004.

[9] X. Lu, J. Kolzow, R.R. Chen, J. Du, Effect of solution condition on hydroxyapatite formation in evaluating bioactivity of B2O3 containing 45S5 bioactive glasses, Bioact Mater 4 (2019) 207-214 DOI: 10.1016/j.bioactmat.2019.05.002.

[10] B.R. Spirandeli, R.G. Ribas, S.S. Amaral, E.F. Martins, E. Esposito, L.M.R. Vasconcellos, T.M.B. Campos, G.P. Thim, E.S. Triches, Incorporation of 45S5 bioglass via sol-gel in beta-TCP scaffolds: Bioactivity and antimicrobial activity evaluation, Mater Sci Eng C Mater Biol Appl 131 (2021) 112453 DOI: 10.1016/j.msec.2021.112453.

Reviewer 3 Report

The author presents an in vitro study describing the protective role of a bioactive sealer with 45S5 bioglass on enamel around orthodontic brackets. The performed modifications improved the manuscript quality. However, I still have some improvement suggestions.

Lines 11: I suggest referring to “experimental bioactive enamel resin sealer”.

Figure 1: in the figure, I suggest changing from “control application” to “untreated group” or “untreated enamel surface group”.

Lines 108-109: please add how the randomization process was performed.

Line 191: refer to figures 4A and 4B

Line 192: refer to figures 4C and 4D

Lines 194-195: refer to figures 5A and 5B when referring to before exposure to the acidic challenge and to figures 5C and 5D when referring to “was mostly disintegrated by the action of the acidic challenge”.

Lines 196-198: refer to figures 6A and 6B when referring to before and figures 6C and 6D when referring to after exposure to the acidic challenge.

Lines 198-200: refer to figures 7A and 7B when referring to before and figures 7C and 7D when referring to after exposure to the acidic challenge.

Section 2.8, lines 177-179: the author only attributed 100% (if no signs of erosion exist) and 0% values (if signs of erosion exist)? No other values were considered?

Table 2: present the meaning of the abbreviations on the table caption.

Author Response

Honorable reviewer (3) report

Lines 11: I suggest referring to “experimental bioactive enamel resin sealer”

Response of author

I wish to thank the honorable reviewer for the valuable scientific comment. This study aimed at evaluating the protective effect of experimental bioactive enamel resin sealer on enamel adjacent to orthodontic brackets against erosion.

Figure 1: in the figure, I suggest changing from “control application” to “untreated group” or “untreated enamel surface group”.

Response of author

I wish to thank the honorable reviewer for the valuable scientific comment. The figure was modified.

Lines 108-109: please add how the randomization process was performed.

Response of author

I wish to thank the honorable reviewer for the valuable scientific comment. Each sample was placed in a sealed vial. Numbering of the vials were done by a colleague not participating in the research. The numbers of the vials were randomized using an excel program

Line 191: refer to figures 4A and 4B

Response of author

I wish to thank the honorable reviewer for the valuable scientific comment. The line was modified as follows:

The enamel surface that was not treated by any bioactive glass (Control) before exposure to the erosion challenge revealed a smooth surface (Figures 4A, 4B).

Line 192: refer to figures 4C and 4D

Response of author

I wish to thank the honorable reviewer for the valuable scientific comment. The line was modified as follows:

The (Control) group (Figures 4C, 4D) (enamel exposed to the erosion) revealed an enamel surface in which the enamel prisms’ borders were clearly observed

Lines 194-195: refer to figures 5A and 5B when referring to before exposure to the acidic challenge and to figures 5C and 5D when referring to “was mostly disintegrated by the action of the acidic challenge”.

Response of author

I wish to thank the honorable reviewer for the valuable scientific comment. The line was modified as follows:

RS group having resin treated specimens showed the coverage of the whole surface by a homogenous layer of resin (Figures 5A, B) before exposure to the acid challenge, however the resin layer was mostly disintegrated by the action of the acid challenge (Figures 5C, 5D).

Lines 196-198: refer to figures 6A and 6B when referring to before and figures 6C and 6D when referring to after exposure to the acidic challenge.

Response of author

I wish to thank the honorable reviewer for the valuable scientific comment. The line was modified as follows:

bioactive glass resin showed a homogenous layer of particles of bioglass embedded in resin matrix (Figures 6A, 6B) before erosion challenge and after exposure to the acid challenge (Figures 6C, 6D).

Lines 198-200: refer to figures 7A and 7B when referring to before and figures 7C and 7D when referring to after exposure to the acidic challenge.

Response of author

I wish to thank the honorable reviewer for the valuable scientific comment. The line was modified as follows:

The enamel surfaces in the (fluoride group) before exposure to the erosion (Figures 7A, 7B) showed a smooth enamel surface, however after exposure to erosion the borders of the enamel prisms were clearly observed (Figures 7C, 7D).

Section 2.8, lines 177-179: the author only attributed 100% (if no signs of erosion exist) and 0% values (if signs of erosion exist)? No other values were considered ?

Response of author

I wish to thank the honorable reviewer for the valuable scientific comment. No other values are considered. I apologize for any unclear points regarding the method of evaluating the protected enamel area in my first version of the manuscript and I hope the current version of the manuscript is clear.

Table 2: present the meaning of the abbreviations on the table caption.

Response of author

I wish to thank the honorable reviewer for the valuable scientific comment. The table was modified.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

See attachment

Comments for author File: Comments.pdf

Author Response

Results is there something other than SEMs to look at? Or should I be looking a the small boxes in the

SEMs?

I thank the honorable reviewer for the valuable comment. The SEM pictures shows the morphological features of the enamel next to the orthodontic brackets and its response to the erosive attack, while the small boxes are representative areas of the samples that were chemically characterized by electron-dispersive-spectroscopy. All samples were examined by SEM and EDS and representative samples were demonstrated in the results section

On page 8 there is a figure, below the figure is 3.2 Degree of Conversion and then there is Figure 7

Percentage of Protection. This is presentation is confusing to the reader.

I am extremely sorry for this confusion. The degree of protection (figure 7) was placed directly after the (figure 6) that describes the morphological features of enamel next to the enamel brackets.

Discussion is fine except for numerous grammatical errors and spacing

I am extremely sorry for these mistakes. The whole text was reviewed by English native speaker

Conclusion: what are the limitations of the study?

I thank the honorable reviewer for the valuable comment. The following part was added to the manuscript. Lines 341-345.

The limitations of this study include its invitro setting, thus invivo studies should be carried out to confirm the obtained results, moreover detailed chemical characterization of the tested bioactive material should be done after long storage durations to examine chemical changes of the bioactive material. Additionally, the interface formed between the resin sealer and the enamel sealer should be investigated.

References: I read the Guidelines for Authors for this journal and the instructions are very vague

regarding formatting references. I assume they will not accept one author’s name followed y et al.

Only the first letter of the first word in the title is capitalized (reference #7) You have several references

in which the title is italicized (reference #28 -30).

Most commonly the journal title is followed by year, volume, issue number and page number and a DOI

if one is available. For reference #1 it should be Dent Mater 2012;28(10):1089-87.

I am extremely sorry for this mistake. The whole references were reformatted.

The entire manuscript will need to be rewritten to conform to proper English. For example, I rewrote

the abstract: see below.

Manufacturers’ names and location are not typically placed in the abstract.

Teeth were not bonded to the brackets, rather the brackets were bonded to the teeth.

The results that you stated were the same as the Conclusion. You will need to add some data for the

results.

I thank the honorable reviewer for the valuable comment. The abstract was modified according to the reviewer’s instructions.

The English language was revised in the whole manuscript.

The following abstract was added to the text.

Background: The aim of this study was to evaluate the protective effect of a bioactive enamel resin sealer adjacent to orthodontic brackets. Methods: Orthodontic brackets (n=40) were bonded to freshly extracted sound maxillary premolars using Transbond Plus Color Change adhesive (3M Unitek, USA). Experimental groups (n=10) consisted of a resin sealer with bioactive glass (35% by weight), resin sealer without bioactive glass, fluoride, and no treatment (control). All specimens received 18-minute acidic challenge from 1% citric acid. Enamel surfaces surrounding the ortho-dontic brackets were examined by SEM and electron-dispersive-spectroscopy (EDS). Wilcoxon Signed-Rank test was used to compare the enamel surfaces covered by the sealers before/after the acid challenge. Attenuated total reflectance Fourier transform infrared spectroscopy was used to detect the degree of resin conversion. Results: Percentage of bioactive resin sealer coverage to enamel after erosion was 100% which was significantly more than the other groups p<0.05. De-gree of conversion for the bioactive and the control resins were 42.4%+ 3.6 and 48.57% +5 respec-tively. Conclusion: the bioactive resin sealer resisted the acidic challenge significantly more than the experimental and control groups.

Author Response File: Author Response.docx

Reviewer 2 Report

The study is interesting and clear, but there is no novelty compared to what is already in the literature and what has been written by the authors themselves.
In the introduction and throughout the paper the authors should pay more attention to how the references have been placed. Often they are not in the right place in the sentence or are repeated several times in the same sentence or the punctuation is wrong. In general there are various typing errors.
In the materials and methods the statistical analysis is badly presented. It is not clearly explained what the coverage percentages are. It would also be interesting to compare the results of the different materials and not just the before and after the acid exposure of the different groups.
In the discussion a detailed comparison should be made with the previous study by the same author (9) in order to present differences and possible innovations.
The limitations of in vitro should be discussed and not just mentioned in the conclusions.

Author Response

Comments of the honorable reviewer (2)	Response of the author
The study is interesting and clear, but there is no novelty compared to what is already in the literature and what has been written by the authors themselves.	I wish to thank the honorable reviewer for the valuable comments. Lines 349-353 were added to elaborate this point. “The current technique of applying the 45S5 bioglass has some advantages over previous techniques [7, 9, 20, 21] that utilized an acidic paste based on 45S5 bioglass particles, as the current technique is fast and easier to apply and there is no need to wait for 24 hours [7, 9, 20, 21] to allow formation of an “Interaction layer” that can protect the enamel surface..”
In the introduction and throughout the paper the authors should pay more attention to how the references have been placed. Often they are not in the right place in the sentence or are repeated several times in the same sentence or the punctuation is wrong. In general there are various typing errors.	I apologize for these mistakes. The whole manuscript was revised.
In the materials and methods the statistical analysis is badly presented. It is not clearly explained what the coverage percentages are. It would also be interesting to compare the results of the different materials and not just the before and after the acid exposure of the different groups.	I apologize for these mistakes. The statistical analysis was rewritten again. To compare the efficacy of using the 45S5 bioglass powder; a resin blend free of any bioglass was tested. *Statistical analysis* The percentages of enamel protection exerted by the tested agents were compared using the Wilcoxon Signed-Rank test[28] before/after erosion; no signs of erosion for enamel surrounding the orthodontic brackets (After the erosive challenge) were considered 100% protection for enamel. Differences were considered statistically significant at the level of 0.05. Means and standard deviation of the degree of conversion for the experimental resins were obtained. Software utilized was SPSS (v24, IBM, Armonk, USA).
In the discussion a detailed comparison should be made with the previous study by the same author (9) in order to present differences and possible innovations.	I wish to thank the honorable reviewer for the valuable comments. The current technique of applying the 45S5 bioglass has some advantages over previous techniques [7, 9, 20, 21] that utilized an acidic paste based on 45S5 bioglass particles, as the current technique is fast and easier to apply and there is no need to wait for 24 hours [7, 9, 20, 21] to allow formation of an “Interaction layer” that can protect the enamel surface.
The limitations of in vitro should be discussed and not just mentioned in the conclusions.	I wish to thank the honorable reviewer for the valuable comments. The following paragraph was added. Lines 354-358 The limitations of this study include the in vitro setting of the experiment, thus in vivo studies should be carried out to confirm the obtained results. Moreover, detailed chemical characterization of the tested bioactive material should be done after long storage durations to examine chemical changes of the bioactive material. Additionally, the interface formed between the resin sealer and the enamel sealer should be investigated.

Author Response File: Author Response.docx

Reviewer 3 Report

please provide more information on 45S5 bioactive glass for the reader, expand nanomaterials with new papers (Nanomaterials 2020;10 (8), 1466).

methodology, add a section on sample size calculation

add more detail to figure 1, what medium did you use for the erosive challenge? would be good to add a consort type diagram as well showing study samples, interventions, final outcome,,

provide more information on the Synthesis of bioactive orthodontic sealer, how exactly did you turn this into a sealer, more info on 'co-monomers resin blend' please, any preliminary study on this specific formulation, did you get this from another study, add reference then

add a few sentences on the purpose of FT-IR/ATR analysis for the utilized resin systems, also add more info to the legend of figure 2, what does it show for reader

expand Statistical analysis,

what is EDS, spell out for reader

figure 7 , add footnote for variables showing on the graph

Author Response

Honorable reviewer (3) comments	Response to the honorable reviewer
please provide more information on 45S5 bioactive glass for the reader, expand nanomaterials with new papers (Nanomaterials 2020;10 (8), 1466).	We wish to thank the honorable reviewer for the valuable comments. The reference was added. Lines 53-61 were added 45S5 bioactive glass was introduced in the late 60s of the 20th century by professor Larry Hench [18] as a bioactive material that improved the biocompatibility of artificial prothesis implanted in the patients’ bodies. The unique property of this material is attributed to its ability to produce a rich layer of calcium and phosphate that is capable of bonding to hard and soft biologic tissues [18]. Recently, a technique for ap-plying 45S5 bioglass proved its capability for treating enamel and dentin lesions [7, 19-21]. Moreover, this application technique showed good biocompatibility of the 45S5 bioglass to pulp cells [22] and proved its resistant to the brushing abrasion [23]. Additionally, previous research showed promising results of incorporating 45S5 in resin blends [24].
methodology, add a section on sample size calculation	We wish to thank the honorable reviewer for the valuable comments. The following lines were modified. Lines 90-93 The number of specimens in each group was selected according to previous published articles and according to the threshold for significance which was set at 0.05 and means and standard deviation observed in a pilot study and the power of test which was set at 80%.
add more detail to figure 1, what medium did you use for the erosive challenge? would be good to add a consort type diagram as well showing study samples, interventions, final outcome,,	We wish to thank the honorable reviewer for the valuable comments. Figure 1 was modified
provide more information on the Synthesis of bioactive orthodontic sealer, how exactly did you turn this into a sealer, more info on 'co-monomers resin blend' please, any preliminary study on this specific formulation, did you get this from another study, add reference then	We wish to thank the honorable reviewer for the valuable comments. The resin blend mixture with the bioactive glass is innovative and there is a pending US patent application describing the same resin blend mixed with a different bioactive glass. The following lines were added to describe the detailed synthesis of the resin blend. Lines 108-121 The 45S5 Bioactive glass powder was produced by melting 46.1 mol% SiO2, 26.9 mol% CaO, 24.4 mol% Na2O and 2.5 mol% P2O5 in a platinum crucible at 10 ◦C/min up to 1,100 ◦C and maintained for 1 h. Subsequently the temperature was raised to 1450 ◦C (10 ◦C/min) and maintained for further 30 min. The glass melt was rapidly quenched in water (~20 ◦C), dehydrated in absolute ethanol for 3 h, dried overnight in a furnace at 110 ◦C, milled and finally sieved (~20 μm). All components of the resin blend were purchased from Sigma-Aldrich (St. Louis, MO, USA). The bisphenol A-glycidyl methacrylate, Triethylene glycol dimethacrylate, Ethyl 4-dimethylaminobenzoate and Camphor quinone were added according to the designated percentages Table 1 in a sealed dark container and mixed together using a magnetic stirrer (Cimarec, A.J.cope, UK) for 2 minutes. The 45S5 bioglass was added gradually to the resin blend under stirring until it was fully incorporated in the resin blend. The whole mixture was ultrasonicated in ultrasonic bath for two minutes. The control resin blend was prepared from the same chemicals without adding the 45S5 bioglass.
add a few sentences on the purpose of FT-IR/ATR analysis for the utilized resin systems, also add more info to the legend of figure 2, what does it show for reader	We wish to thank the honorable reviewer for the valuable comments. Lines 138-140 were added Degree of conversion of the experimental resins from the monomer state to the polymer state was detected using the FTIR/ATR technique figure 1 to determine the suitability of the experimental resin to be used in a clinical setting. Figure legend of figure 2 was modified Showing the FTIR/ATR peaks selected for degree of conversion experiment. The ratios of the absorbance peak corresponding to the aliphatic carbon-carbon double bond (1637 cm⁻¹peak height) with that of the internal standard (1720 cm⁻¹ peak height.) were obtained to detect the degree of conversion
expand Statistical analysis	We wish to thank the honorable reviewer for the valuable comments. The percentages of enamel protection exerted by the tested agents were compared using the Wilcoxon Signed-Rank test[28] before/after erosion; no signs of erosion for enamel surrounding the orthodontic brackets (After the erosive challenge) was considered 100% protection for enamel. Differences were considered statistically significant at the level of 0.05. Means and standard deviation of the degree of conversion for the experimental resins were obtained. Software utilized was SPSS (v24, IBM, Armonk, USA).
what is EDS, spell out for reader	*electron-dispersive-spectroscopy*
figure 7 , add footnote for variables showing on the graph	We wish to thank the honorable reviewer for the valuable comments. Figure 7. % Degree of protection of the enamel surface. Similar letters show no statistical significance p<0.05. (Control; untreated enamel surface), (BGRS, enamel surface covered with bioactive resin sealer), (RS; enamel surface covered with resin sealer), (Fluoride; enamel surface treated with fluoride)

Author Response File: Author Response.docx

Reviewer 4 Report

Materials and methods can be improved . It's not clear where and when the sealer is applied to the teeth. The description of the methods is not clear, and the design in table 1, too.

Author Response

Honorable reviewer (2) comments

Response to the honorable reviewer

Materials and methods can be improved . It's not clear where and when the sealer is applied to the teeth.

I thank the honorable reviewer for the valuable comments

Tested Material Application.

All specimens were covered with a nail varnish except for a treatment window of 2 mm surrounding the orthodontic brackets which was left uncovered. The 40 bonded specimens were divided into four equal groups; the first group (BGRS) had a bioactive glass sealer applied to their surfaces, second group (RS) teeth had resin sealer (Same resin composition of the bioactive sealer but devoid of bioactive glass) applied, third group (Fluoride) had fluoride (Nupro® Acidulated Phosphate Fluoride, Dentsply, Germany.), and the (Control) group had nothing applied on enamel

The description of the methods is not clear and the design in table 1, too.

I thank the honorable reviewer for the valuable comments

Table 1 was modified and the experimental resin synthesis section was rewritten

The 45S5 Bioactive glass powder was produced by melting 46.1 mol% SiO2, 26.9 mol% CaO, 24.4 mol% Na2O and 2.5 mol% P2O5 in a platinum crucible at 10 ◦C/min up to 1,100 ◦C and maintained for 1 h. Subsequently the temperature was raised to 1450 ◦C (10 ◦C/min) and maintained for further 30 min. The glass melt was rapidly quenched in water (~20 ◦C), dehydrated in absolute ethanol for 3 h, dried overnight in a furnace at 110 ◦C, milled and finally sieved (~20 μm). All components of the resin blend were purchased from Sigma-Aldrich (St. Louis, MO, USA). The bisphenol A-glycidyl methacrylate, Triethylene glycol dimethacrylate, Ethyl 4-dimethylaminobenzoate and Camphor quinone were added according to the designated percentages Table 1 in a sealed dark container and mixed together using a magnetic stirrer (Cimarec, A.J.cope, UK) for 2 minutes. The 45S5 bioglass was added gradually to the resin blend under stirring until it was fully incorporated in the resin blend. The whole mixture was ultrasonicated in ultrasonic bath for two minutes. The control resin blend was prepared from the same chemicals without adding the 45S5 bioglass.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Please see attachment which is a markup of the submission identifying grammatical errors. Please make the changes and comment regarding if a limitation of the study is not silanating the bioactive glass particles.

Comments for author File: Comments.pdf

Author Response

I wish to thank the honorable reviewer for the valuable comments. All of the manuscript was revised and the modified areas were written in red font.

The Limitations part was modified as advised by the honorable reviewer.

Reviewer 2 Report

I would like to thank the authors for making the changes I requested, which adequately reflect what I had pointed out.

However, I still do not find in this study an important scientific novelty compared to previous published works.

Author Response

I wish to thank the honorable reviewer for his valuable time. Previous research showed that mixing the 45S5 bioglass with phosphoric acid produces a paste capable of remineralizing enamel and dentin. The aforementioned technique needs the protection of the paste by a certain carrier that should keep it in place for 24 hours, however, in the current study the resin blend fortified by 45S5 bioglass was able to attach to the surface of enamel and dentin without any special carrier and produced the same protective effect of using the 45S5 bioactive paste.

Reviewer 3 Report

thank you for the revision

abstract,

add the name of your primer you used, add the composition of your

bioactive glass, add a sentence on 'degree of resin conversion.' its importance

please revise the following

Introduction,

page 2, line 49-50, correct reference citation (15-17) , not (15,17) and correct the text', as well as other strategies, including the application of probiotics, polyols, adhesives with nanomaterials such as Silver or titanium, and resin infiltration [15-17].'

figure 1, you need to make the figure wider and in the footnote spell out 'FTIR' and 'SEM-EDS', also add more detail on the bonding with trasbond and transbond plus, as well as add the following (summarise it)'The 40 bonded specimens were divided into four equal groups (n=10); the first group (BGRS) had a bioactive glass sealer applied to their surfaces (how ? explain, with a brush, how did you standardise it and made it reproducible ??), second group (RS) teeth had resin sealer (Same resin composition of the bioactive sealer but devoid of bioactive glass) applied, the third group (Fluoride) had fluoride (Nupro® Acidulated Phosphate Fluoride, Dentsply, Germany.), and the (Control) group had nothing applied on enamel.'

Author Response