The in vitro assessment of rheological properties and dentin remineralization of saliva substitutes containing propolis and aloe vera extracts

Saliva substitutes with enhanced dentin remineralization properties were expected to help manage caries progression in patients with xerostomia. This in vitro study examined the rheological properties and remineralization action of experimental saliva substitutes containing propolis extract and aloe vera extract on demineralized dentin. Four experimental saliva substitutes were formulated with varying concentrations of propolis extract (P) and aloe vera extract (A) were prepared. A commercial saliva substitute (Biotene Oral Rinse) was used as a commercial comparison. The rheological properties and viscosity of these materials were measured using a strain-controlled rheometer (n = 3). The remineralizing actions of saliva substitutes on demineralized dentin after 2 weeks were determined using ATR-FTIR and SEM-EDX (n = 8). The results were expressed as a percentage increase in the mineral-to-matrix ratio. Biotene demonstrated a significantly higher viscosity (13.5 mPa·s) than experimental saliva substitutes (p<0.05). The addition of extracts increased the viscosity of the saliva substitutes from 4.7 mPa·s to 5.2 mPa·s. All formulations showed minimal shear thinning behavior, which was the viscoelastic properties of natural saliva. The formulation containing 5 wt% of propolis exhibited the highest increase in the median mineral-to-matrix ratio (25.48%). The SEM-EDX analysis revealed substantial mineral precipitation in demineralized dentin, especially in formulations with 5 wt% or 2.5 wt% of propolis. The effect of the aloe vera extract was minimal. The addition of propolis and aloe vera extracts increased the viscosity of saliva substitutes. the addition of propolis for 2.5 or 5 wt% to saliva substitutes increased mineral apatite precipitation and tubule occlusion. To conclude, the saliva substitute containing propolis extract demonstrated superior remineralizing actions compared with those containing only aloe vera extract.


Answer
After careful consideration, we agree with the reviewer regarding the lack of supporting evidence to substantiate the claim about the limitations of commercially available saliva substitutes.Consequently, we have removed this statement from the manuscript to ensure all information presented is evidence-based and accurately referenced.

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Comment 4 Furthermore, some given information is not referenced.

Answer
We sincerely thank the reviewer for this helpful suggestion.To clarify the methodology, we have now included a brief summary of the artificial saliva preparation process in the Materials and Methods section of our manuscript.Overall, the information in this section is very condensed, even though you have to summarize the steps performed, all the information should be stated to ensure reproducibility.

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We sincerely thank the reviewer for emphasizing the importance of detailed methodological descriptions to ensure reproducibility.In response, we have expanded the descriptions in the methods section, detailing each step to clarify the step for the reader.

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Lines 165-172 Comment 7 234: The aim of the study is already stated on the introduction section and it should not be here again.Some statements also require references.The text also would benefit from a discussion with more studies addressed to discuss the findings of the present study.

Answer
We appreciate your observations regarding the redundancy and referencing within our manuscript.We have removed the repeated mention of the study's aim as suggested.Additionally, we have enriched the discussion section by incorporating more references that support and help elucidate our findings.
However, we must note that there is a limited number of original research studies that have conducted similar or relevant experiments, which constrains our ability to make extensive comparisons

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We appreciate the reviewer for highlighting the need to correct our citation source.We acknowledge this oversight and have accordingly updated the citation to accurately reflect that the data regarding the increase in the elderly population comes from the WHO's Ageing and Health report, as recommended.

Comment kindly include group 6 distilled water in Table 2 of your manuscript?
This addition will ensure completeness and clarity in presenting the experimental groups and their respective treatments.

Answer
Thank you for your suggestion to enhance the clarity and completeness of our presentation of experimental groups.We have added Group 6, which utilizes deionized water, to Table 2 as recommended.This update ensures that all treatment groups are accurately and comprehensively represented.

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Table 2 Comment Could you please provide clarification on why the sample temperature was maintained at 25°C rather than at 37°C, which is the normal temperature in the oral cavity?This deviation from physiological conditions warrants explanation to ensure the relevance and applicability of your experimental setup to real-clinical scenarios.

Answer
We sincerely thank the reviewer for this insightful question.The decision to maintain the sample temperature at 25°C, rather than the physiological norm of 37°C, was made due to equipment limitation.Specifically, while the strain-controlled rheometer can adjust for temperature, the compact-DDM,

Answer
We appreciate the reviewer's query regarding the orientation of tooth sectioning in our study.The decision to cut the crowns horizontally was primarily to minimize the risk of including the pulp space, which could complicate the handling and analysis of the specimens.This horizontal orientation facilitates the production of dentin discs, enhancing handling and ease of manipulation for FITR assessment and surface examination

Comment from reviewer 2
with SEM-EDX.While this method limits our ability to observe the depth of occlusion along the dentinal tubules, it aligns with our study's primary focus, which is not on depth measurement but rather on the presence and extent of tubule occlusion Revision -

Answer
We appreciate the reviewer's concern regarding the potential for crack formation in our samples due to the processes of dehydration and rehydration.During our experiments, no visible cracks were observed in the specimens.This may be due to the conditions under which the cycles of demineralization and remineralization were conducted, where the specimens likely remained sufficiently moist.We acknowledge the importance of this aspect for the integrity of the findings and will consider further monitoring and reporting on these conditions in future studies to ensure the robustness of our results.
Revision -Comment 1.I kindly note that the graph indicates a significant shift in the phosphate peak, which appears to be unreported.Additionally, in such cases, it is preferable to measure the area under the curve rather than focusing solely on peak height for a more comprehensive analysis.
2. Moreover, there are other important parameters worth considering to better understand the effects of propolis and aloe vera.These include the carbonate to phosphate ratio,

Comment from reviewer 2
collagen cross-linking, and the degree of crystallinity of dentin.
It is evident from the chart that these parameters are also influenced to a significant extent.Integrating these aspects into your analysis would enhance the depth of understanding of your study outcomes.
Answer 1.We thank the reviewer for this insightful comment.We were also analyzed the data using area under peak or peak height which showed the similar trend.Additionally, we acknowledge the significant shift in the phosphate peak after demineralization/remineralization and have now reported this observation in the materials and methods section, as well as discussed the possible reasons behind this finding in the discussion part.
2. We appreciate the suggestion to examine additional parameters such as the carbonate to phosphate ratio, collagen cross-linking, and the degree of crystallinity of apatite using XRD.While the current study focused primarily on screening remineralizing actions to aid future clinical trials or product optimizations, we recognize the value of these suggestions and have included a discussion on these future research directions in the manuscript.-The EDX analysis was primarily used to characterize the surface precipitates observed on the dentin samples.We acknowledge the need to indicate the location of reading which is now labelled in the figures to clarify the context and enhance the understanding of our findings.

Revision
talking about the reference exposed at the end of the paragraph, after each phrase it should be exposed.Multiple in-text citations to the same work over a large section of text can be visually jarring and it is true that they are not entirely necessary.However, the rule of thumb is to cite the very first sentence, make it clear you are still talking about the same work in your subsequent sentences (for example, "The study noted that..."), and then confirm you are still talking about the work by including another citation at the end (if this has continued for several sentences).If you have a simple follow-on sentence in which it is still clear that you are talking about the same work, you do not need the reference in the second sentence.If at any point you think it might not be clear in the sentence that you are still referring to the same work, include another in-text citation, otherwise, it will appear as an information without a proper citation.AnswerWe appreciate the invaluable suggestions from the reviewer regarding citation practices.We have reviewed our citation style and revised the manuscript accordingly to ensure clear attribution of sources throughout The prevalence of dental caries, particularly root caries, among the elderly is undoubtedly a significant oral health concern.the reviewer's insights, which have highlighted the need for a broader geographic perspective in our study.In response, we have updated the manuscript to include more recent data on the prevalence of root caries in developing countries.essential aspects to elucidate in your manuscript.Could you kindly include detailed explanations of their chemical compositions and mechanisms of action?This information willenhance the understanding of their therapeutic potential and contribute to the comprehensiveness of your study.Comment from reviewer 2AnswerThank you for emphasizing the importance of detailing the chemical compositions and mechanisms of action of propolis and aloe vera extracts.We recognize the significance of these details in understanding the therapeutic potential of these substances.Accordingly, we have expanded the relevant sections of the manuscript to include information on the typical chemical constituents of propolis and aloe vera, as well as their proposed therapeutic mechanisms based on current scientific understanding.Revision Lines 69-78, 82-83 Comment 4 While the introduction of your manuscript provides an overview of the techniques utilized to assess rheological characteristics, such as strain-controlled rheometer and compact-DDM method, it lacks an exploration of potential limitations or challenges associated with these techniques.For instance, it would be beneficial to address concerns regarding the accuracy, consistency, and comparability of measurements obtained using the compact-DDM method, particularly in laboratory settings.Please consider further examination of these aspects to provide a comprehensive understanding of the methodology employed in your study.AnswerThank you for your insightful comments.We have revised the introduction part to include the potential limitations and challenges associated with the compact-DDM method as suggested by the reviewer.highlighting the importance of addressing individual variability in responses to experimental saliva substitutes.We agree that understanding this variability is crucial for interpreting the broader implications of our study.Accordingly, we have revised the introduction to include findings from the recent systematic reviews that discuss the outcomes in how patients with xerostomia respond to different saliva substitutes.
used in the experiments, does not yet have temperature control functions.This limitation and its potential impact on the results have been duly acknowledged and discussed in the manuscript to ensure clarity regarding the experimental conditions and their applicability.
the manuscript and included protocol for cleaning of samples and FTIR study in the materials and method section Revision Lines 167-173, Lines 179-194 Comment I kindly inquire whether the phenomenon of crack formation in the samples due to the process of dehydration and rehydration has been observed in your study.Understanding any potential effects of these processes on sample integrity would contribute to the robustness of your findings.
to thank for the comment and included our compact-DDM limitation at higher shear rate in the discussion section.including the role of specific components in propolis and aloe vera extracts, would provide valuable insights for clinical applications.Your attention to these aspects would enrich the