Review Reports - Clinical Roles of Nanoparticles in Orthodontic Bonding Materials

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review explores the transformative role of nanoparticles in orthodontic bonding materials, addressing the critical issue of enamel demineralization and biofilm formation associated with fixed orthodontic appliances. Key findings reveal that various nanoparticles—including silver (AgNPs), titanium dioxide (TiO₂), zinc oxide (ZnO), amorphous calcium phosphate (ACP), nano-calcium fluoride (nCaF₂), mesoporous bioactive glass (MBGNs), and ZrO₂–TiO₂ nanocomposites—impart distinct functional benefits to bonding agents. AgNPs exhibit potent broad-spectrum antibacterial activity (inhibiting over 90% of Streptococcus mutans at 0.3–0.5 wt%), though they pose aesthetic limitations (discoloration) and cytotoxic risks at higher concentrations. Nanoparticle-enhanced bonding materials bridge the gap between mechanical durability and biological protection, promising to reduce WSL incidence and improve patient outcomes in fixed orthodontic therapy, while underscoring the need for personalized formulation based on patient risk profiles (e.g., caries susceptibility, esthetic demands) and standardized testing protocols. This work can be accepted after the following revisions:

The review adopts a narrative approach for synthesizing literature rather than a systematic review with predefined inclusion/exclusion criteria (e.g., no registration of a review protocol, lack of quantitative meta-analysis for key outcomes like shear bond strength or antibacterial efficacy across studies). This raises questions about whether potential publication bias or selective reporting of positive results has been adequately addressed, and whether the conclusions fully reflect the breadth of available evidence on nanoparticle-enhanced orthodontic bonding materials.
While the review categorizes nanoparticles by type and lists their mechanisms, it lacks explicit standardization of critical experimental parameters—such as nanoparticle size, surface modification (e.g., functionalization with curcumin or silane), and matrix composition (e.g., resin monomers used in adhesives)—across the cited studies. This inconsistency makes cross-study comparisons of antibacterial efficacy, ion release kinetics, and shear bond strength challenging; how can future research establish unified testing protocols to ensure reproducible and comparable results?
The following studies should be also discussed in the Introduction part, to further improve the discussion about anisotropic nanoparticles for biological application. J. T. Lovegrove, B. Kent, S. Förster, C. J. Garvey,M.H. Stenzel, Exploration 2023, 20220075. https://doi.org/10.1002/EXP.20220075; S. Theivendran, S. Lazarev, C. Yu, Exploration 2023, 3, 20220086. https://doi.org/10.1002/EXP.20220086; Y.He, S. Zhang, Y. She, Z.Liu, Y. Zhu, Q. Cheng, X. Ji, Exploration 2024,20230164. https://doi.org/10.1002/EXP.20230164; The Innovation Medicine 1(1), 100015, https://doi.org/10.59717/j.xinn-med.2023.100015.
The discussion of nanoparticle toxicity and biocompatibility relies heavily on in vitro studies and rodent models (e.g., AgNP accumulation in gingival tissue of rodents) but provides limited data from long-term in vivo clinical trials in humans. Given the dynamic and complex oral microenvironment (e.g., salivary enzyme activity, mechanical stress), what evidence gaps exist regarding the long-term systemic or localized biological effects (e.g., chronic inflammation, bioaccumulation) of nanoparticle-reinforced adhesives in human patients?
The review proposes personalized nanoparticle selection based on patient risk profiles (e.g., high caries risk, esthetic demands) but fails to define explicit, measurable criteria for these risk categories (e.g., no thresholds for plaque scores, salivary flow rates, or WSL severity to classify "high-risk" patients). Additionally, it does not specify optimal nanoparticle concentrations or synergistic formulation ratios (e.g., ACP-quaternary ammonium compound blends) for each profile. How can this framework be operationalized in clinical practice without clear, evidence-based guidelines for risk stratification and material dosing?
The conclusion emphasizes the potential of "smart" stimuli-responsive adhesives (e.g., pH-sensitive ion release, self-healing polymers) but provides no critical analysis of the technical feasibility or current developmental status of these innovations (e.g., no discussion of challenges in scaling up synthesis, ensuring long-term stability of responsive mechanisms, or integrating them with existing orthodontic bonding protocols). What key technical barriers need to be addressed to translate these conceptual "future directions" into clinically viable products?

Comments for author File: Comments.pdf

Author Response

Comment 1

“The review adopts a narrative approach for synthesizing literature rather than a systematic review with predefined inclusion/exclusion criteria (e.g., no registration of a review protocol, lack of quantitative meta-analysis for key outcomes like shear bond strength or antibacterial efficacy across studies). This raises questions about whether potential publication bias or selective reporting of positive results has been adequately addressed, and whether the conclusions fully reflect the breadth of available evidence on nanoparticle-enhanced orthodontic bonding materials.”

Response 1:
We thank the reviewer for this important comment and agree that the present work is a narrative rather than a fully systematic review. Our primary goal was to provide a clinically oriented overview that integrates mechanistic, preclinical, and early clinical data on nanoparticle-enhanced orthodontic bonding materials, including toxicity and regulatory aspects, which vary greatly in methodology and outcomes. This heterogeneity (e.g., in nanoparticle type, size, loading, surface functionalization, adhesive formulation, SBS test protocol, and antibacterial assay conditions) makes formal pooling of data and registration of a systematic review protocol challenging at this stage.

To address the reviewer’s concern, we have:

Clarified the review design in the “Materials and Methods” section, explicitly stating that this is a narrative review, without protocol registration, and that no formal risk-of-bias or meta-analytic procedures were performed because of the high degree of heterogeneity among studies (Lines 225-255).
Added a paragraph in the Discussion explicitly acknowledging the risk of publication bias and selective reporting (e.g., preferential publication of positive antibacterial or SBS findings) and stating that our conclusions should be interpreted with this limitation in mind (Lines 580-590).
More clearly referenced existing meta-analyses that have attempted to quantitatively synthesize the effects of nanoparticles or antimicrobial additives on orthodontic adhesives, emphasizing that our article complements these works by focusing on clinical translation, toxicity, regulatory classification and personalized risk-based material selection (Lines 134-153).

Comment 2

“While the review categorizes nanoparticles by type and lists their mechanisms, it lacks explicit standardization of critical experimental parameters—such as nanoparticle size, surface modification (e.g., functionalization with curcumin or silane), and matrix composition (e.g., resin monomers used in adhesives)—across the cited studies. This inconsistency makes cross-study comparisons of antibacterial efficacy, ion release kinetics, and shear bond strength challenging; how can future research establish unified testing protocols to ensure reproducible and comparable results?”

Response 2:
We agree that inconsistent experimental parameters across studies represent a major barrier to comparing outcomes such as antibacterial efficacy, ion release, and shear bond strength. In the revised manuscript, we expanded Table 1 and Table 2 by adding the parameters of “Representative particle size” (Table 1/ Line 383) and “Highest Level of Data/ Study Design” (Table 2/ Line 420).

We have expanded the Discussion to:

Explicitly highlight the lack of standardization in nanoparticle characterization (size, morphology, surface charge, and functionalization), adhesive formulation (monomer systems, filler loading), and testing conditions (thermocycling protocols, cross-head speed, bracket type, enamel preparation, biofilm models) (Lines 591-607).
Propose key elements of unified testing protocols, including:

Adherence to relevant ISO/ADA standards for bond-strength tests and biocompatibility, where applicable;
Defined test conditions for SBS (e.g., storage medium, thermocycling parameters, cross-head speed, bracket type) and antibacterial assays (bacterial strains, inoculum, incubation time, growth medium, and whether saliva or biofilm models are used) (Lines 627-630).

Emphasize that such harmonization would facilitate future meta-analyses and regulatory assessment of nanoparticle-enhanced adhesives (Lines 630-631).

Comment 3:

“The following studies should be also discussed in the Introduction part, to further improve the discussion about anisotropic nanoparticles for biological application. J. T. Lovegrove, B. Kent, S. Förster, C. J. Garvey, M.H. Stenzel, Exploration 2023, 20220075. https://doi.org/10.1002/EXP.20220075; S. Theivendran, S. Lazarev, C. Yu, Exploration 2023, 3, 20220086. https://doi.org/10.1002/EXP.20220086; Y. He, S. Zhang, Y. She, Z. Liu, Y. Zhu, Q. Cheng, X. Ji, Exploration 2024, 20230164. https://doi.org/10.1002/EXP.20230164; The Innovation Medicine 1(1), 100015, https://doi.org/10.59717/j.xinn-med.2023.100015.”

Response 3:
We appreciate this helpful suggestion. We have now incorporated a short paragraph in the Introduction summarizing the broader nanomedicine literature on anisotropic and cell membrane–coated nanoparticles for biological applications, including the suggested studies. These works highlight how nanoparticle shape, orientation under flow, and biomimetic surface engineering influence circulation, biodistribution, and targeting performance in vivo, which is highly relevant to the future design of orthodontic bonding systems (e.g., anisotropic fillers for controlled orientation at the bracket–enamel interface and improved interaction with the biofilm or pellicle) (Lines 122-129).

Comment 4:
“The discussion of nanoparticle toxicity and biocompatibility relies heavily on in vitro studies and rodent models (e.g., AgNP accumulation in gingival tissue of rodents) but provides limited data from long-term in vivo clinical trials in humans. Given the dynamic and complex oral microenvironment (e.g., salivary enzyme activity, mechanical stress), what evidence gaps exist regarding the long-term systemic or localized biological effects (e.g., chronic inflammation, bioaccumulation) of nanoparticle-reinforced adhesives in human patients?”

Response 4:
We appreciate this valuable comment and agree that the current evidence base is dominated by in vitro work and short-term animal studies, with very limited data from long-term clinical investigations in humans. In the revised manuscript the gaps or the barriers are discussed at many points in the text:

Lines 475-486: “Unlike biodegradable calcium phosphate particles, metallic NPs lack natural degrada-tion pathways in vivo, necessitating cautious dosing and careful monitoring of expo-sure duration [54]. Moreover, toxicological thresholds vary depending on the nanopar-ticle type. Several commercial products illustrate these principles. NanoCare Plus (TiO₂-based), Clinpro XT Varnish (3M, containing nCaF₂), and NovaMin (bioactive glass in desensitizing pastes) have been introduced into dental practice and evaluated under current FDA and CE frameworks [56,57]. Despite promising advances, long-term clinical validation remains essential, alongside continued collaboration be-tween material scientists, clinicians, and regulatory bodies to ensure safety and efficacy. The regulatory classification and guidance standards relevant to these nanoparti-cles are summarized in Table S1 while the cytotoxicity thresholds and biocompatibility of each nanoparticle are demonstrated in Table S2.”
Discussion Section

Comment 5:
The review proposes personalized nanoparticle selection… How can this framework be operationalized in clinical practice without clear, evidence-based guidelines for risk stratification and material dosing?

Response 5:
We thank the reviewer for this important observation. Our intention was to present the personalized nanoparticle selection concept as a theoretical framework, not as a ready-to-use clinical guideline. At present, there are insufficient data to define evidence-based cutoffs for caries risk (e.g., plaque indices, salivary flow, WSL severity) or to recommend specific nanoparticle concentrations and combination ratios for each risk category. In the revised manuscript, we now clarify that clinicians should, for the time being, rely on existing, validated caries-risk assessment tools and manufacturer-recommended nanoparticle loadings, and that dose–response and formulation studies are needed before formal protocols can be established (Lines 500-505).

Comment 6:
The conclusion emphasizes “smart” stimuli-responsive adhesives but offers no critical analysis of feasibility or developmental challenges. What key technical barriers must be addressed to translate these concepts into clinically viable products?

Response 6:
We appreciate the reviewer’s insightful comment. The discussion of smart, stimuli-responsive adhesives was intended to outline emerging research directions rather than imply that these technologies are ready for clinical integration. We fully acknowledge that several key technical challenges remain before such systems can become clinically viable. These include achieving consistent large-scale synthesis of functionalized nanoparticles, ensuring the long-term stability and reliability of responsive mechanisms in the highly variable oral environment, and integrating these materials into current bonding procedures without compromising polymerization, handling properties, or bond strength. As these approaches are still in early experimental stages, their translation will depend on future materials-engineering innovations and robust in vivo validation (Lines 644-660).

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, Dear authors,
Reading your study left me with the conclusion that the study design is set properly with thefollowing drawback: in Materials and Methods you included „Relevant review articles, in vitro and in vivo studies, and clinical trials“ (row 125), but during your review there is no differentiation and mention of this between different studies that are evaluated. I consider this relevant due to the fact that there is great difference, That you mentioned as well in the discussion, between the conclusions that can be made based on the findings of in vitro and in vivo studies. Please include this in your review as well. Also I have attached a Word document of minor errors that should be corrected.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Some expresions could be improved, resulting in more clear and straightforward explanations.

Author Response

Comment 1:
In Materials and Methods you included “Relevant review articles, in vitro and in vivo studies, and clinical trials”, but during your review there is no differentiation and mention of this between different studies that are evaluated… Please include this in your review as well.

Response 1:
We thank the reviewer for this thoughtful comment and agree that differentiating between in vitro, in vivo, and clinical evidence is important, especially given the different strengths and limitations of these study types, which we also highlight in the Discussion. To address this, we have added brief clarifications at key points in the manuscript to make the level of evidence more transparent without substantially lengthening the text. Specifically, in the Materials and Methods section we now explicitly state that the majority of available data are derived from in vitro and in vivo studies, with relatively few clinical trials (Lines 247-255).

Response to Reviewer 2 – Minor Comments

We thank the reviewer for the careful reading and useful suggestions. We have made the following corrections:

Repetition / wording (demineralization, biofilm-retentive surfaces)
– Rephrased sentences to remove redundancy and improve clarity.
Citation style ([3], [4] → [3,4])
– Standardized all in-text citations to the merged format throughout the manuscript.
Grammar (acid exposure sentence)
– Corrected to: “Fixed orthodontic appliances increase the time during which teeth are exposed to acid by making it harder for saliva to wash away the acid and protect the enamel surfaces.”
Salivary flow explanation
– Simplified and clarified to: “Also, the flow of saliva can change due to insufficient oral hygiene measures of the person undergoing orthodontic treatment.”
Materials and Methods phrasing (“Priority was given to…”)
– Corrected to a single, clear sentence describing the inclusion criteria.
Redundant/awkward phrases (“Each type has its own benefits…”, “Collectively, All together…”, “with TiO₂ have 40% less bacterial adhesion…”)
– All rephrased for grammar and style.
ZnO “safety” clarification
– Clarified as “antimicrobial efficacy, low cytotoxicity, and minimal risk of discoloration.”
ACP + QAC and Figure 1
– Removed the claim that ACP systems are “ideal” and the direct link to Figure 1.
– New text refers to ACP+QAC as potentially advantageous and directs readers to Table 1, Figure 1, and Table 2 for comparative data.
Sentence on Table 2
– Rewritten as: “An overview of the nanoparticles discussed in this section and their comparative antibacterial efficacy, shear bond strength, esthetic impact, and cytotoxicity is presented in Table 2.”
AgNPs drawback sentence (“However, Also…”)
– Corrected to: “However, AgNPs can cause adhesives to change color, especially in esthetically critical areas, which is a significant drawback.”
Differentiation of in vitro, in vivo, and clinical studies
– Added in Materials and Methods: that most data are from in vitro and in vivo studies, with fewer clinical trials.
– Emphasized in the Discussion that many conclusions are mainly supported by preclinical evidence.

Descriptions of tables and figures
– Added brief clarifying sentences in the text for Table 1, Table 2, Figure 1, and Figure 2 to state what each presents

Reviewer 3 Report

Comments and Suggestions for Authors

Lines 118–132: Describe the methodology in more detail, specifying the inclusion and exclusion criteria, the search period, the number of articles selected, and whether any guidelines such as PRISMA were followed.
Lines 49–84: Rewrite the introduction in a way that clearly demonstrates its connection to the proposed objectives.
The risk of WSL is repeated in lines 56–61 and again in lines 85–116.
Include information on particle size and levels of evidence in the tables.
Discuss the methodological differences, limitations, and variability in more detail.
Write the conclusion in a way that reflects its relationship to the study's objectives and future recommendations

Author Response

Comment 1:
Lines 118–132: Describe the methodology in more detail, specifying the inclusion and exclusion criteria, the search period, the number of articles selected, and whether any guidelines such as PRISMA were followed.

Response 1:
We thank the reviewer for this comment. The revised manuscript clarifies that this is a narrative review without a registered protocol or PRISMA framework. We have specified the databases searched (PubMed, Google Scholar, ScienceDirect), the key search terms used, and the inclusion and exclusion criteria applied. As this was a narrative synthesis of a heterogeneous body of literature, no formal PRISMA process or quantitative study count was implemented (Lines 226-235).

Comment 2:

Lines 49–84: Rewrite the introduction in a way that clearly demonstrates its connection to the proposed objectives.

Response 2:

We thank the reviewer for this suggestion. We have now improved the end of the introduction by adding a clearer linking sentence and a concise statement of the review’s purpose. The revised text explicitly connects the clinical problem with the aims of the review and clarifies that the objective is to summarize current evidence supporting the development of more effective and biologically protective orthodontic bonding systems (Introduction Section and Lines 151-153).

Comment 3:

The risk of WSL is repeated in lines 56–61 and again in lines 85–116.

Response 3:

We thank the reviewer for this observation. The repeated statements regarding the risk of WSL have now been streamlined. The earlier section retains the general background explanation, while the later section focuses only on specific mechanisms and epidemiological data to avoid redundancy. This revision removes overlap while preserving essential information.

Comment 4:

“Include information on particle size and levels of evidence in the tables.”

Response 4:

Thank you for this valuable suggestion. We have now revised both Table 1 and Table 2 to include the requested information.

Particle size added:
For each nanoparticle type, we extracted the representative particle size (in nm) from the original materials characterization data reported in the studies cited in the review. This information has been inserted as a new column titled “Representative particle size (nm)” in Table 1 and “Representative particle size (nm)” in Table 2. Where the orthodontic study did not provide a precise mean size, we have indicated this as “NR (not reported)”.
Level of evidence added:
Table 2 has been expanded with an additional column titled “Highest level of evidence”, categorizing each nanoparticle according to the strongest study design available (e.g., systematic review/meta-analysis, randomized clinical trial, in vivo split-mouth design, in vitro study). A footnote has been added to clarify the classification criteria.
Manuscript text updated:
We have added a short statement in the Methods section explaining how particle sizes and levels of evidence were extracted and classified.

Comment 5:
“Discuss the methodological differences, limitations, and variability in more detail.”

Response 5:
Thank you for this comment. We agree with the importance of clarifying methodological variability. We would like to note that the manuscript includes a detailed discussion of these aspects.

Specifically, in the Discussion, we highlight that most available studies differ in their experimental conditions, including variations in nanoparticle type, concentration, surface characteristics, resin matrix composition, and testing protocols. We also note that antibacterial studies use different bacterial models, incubation conditions, and outcome measures, which limits cross-study comparability. In addition, we already acknowledge that the majority of the evidence comes from short-term in-vitro experiments, and that factors such as saliva, patient behavior, enamel conditions, and clinical variability may influence outcomes and cannot be fully replicated in laboratory settings.

Furthermore, we discuss broader limitations of the current evidence base, including the scarcity of long-term or clinical data, the potential for publication bias, and the challenges of extrapolating laboratory findings to real-world orthodontic practice.

Comment 6:

“Write the conclusion in a way that reflects its relationship to the study’s objectives and future recommendations.”

Response 6:

Thank you for this valuable suggestion. The conclusion has been written to summarise the main findings in direct relation to the stated objectives of the review—namely, to compare the different nanoparticles used in orthodontic adhesives, evaluate their antibacterial and mechanical performance, and examine their potential clinical applicability. The conclusion also outlines key future recommendations by emphasizing the need for standardized methodologies, long-term durability assessments, and well-designed clinical trials to validate the promising in-vitro results. These components together link the review’s objectives with its final statements (Lines 644-660).

Reviewer 4 Report

Comments and Suggestions for Authors

Comments to authors

Dear authors,

Thank you for submitting your manuscript entitled “Clinical Roles of Nanoparticles in Orthodontic Bonding Materials” for possible publication. The topic is relevant, considering the increasing interest in nanotechnology and its potential applications in orthodontic bonding systems. However, the manuscript requires substantial revisions before publication. My detailed comments are provided below.

- The abstract is overly long, repetitive, and does not succinctly reflect the main findings of the review. Please reduce the length and focus on the essential information only.

- The objective stated in the abstract should be rewritten to clearly and concisely present the aim of the review. Currently, the wording is unclear and lacks fluency.

- The abstract should better distinguish between evidence derived from in vitro, in vivo, and clinical studies.

- The Conclusion section of the Abstract is vague and does not reflect the limitations or the true level of evidence available.

- In the Introduction section, several sentences are redundant and include unnecessary background information. Please condense this section to avoid repetition.

- The Introduction section lacks a clear justification for why a review on this topic is needed at this time.

- Some claims (e.g., prevalence of white spot lesions) should be supported with updated references.

- The Materials and Methods section needs attention. The methodology is insufficiently described. Even for a narrative review, it is important to specify the databases used, key terms searched, period covered, and inclusion/exclusion criteria.

- Currently, the statement “an overview has been presented” is not acceptable and lacks transparency.

- Please ensure that the search strategy used in each of the scientific databases (e.g., PubMed, Scopus, Web of Science, etc.) is reported.

- The process of article selection and screening is not described. Please clarify how many articles were identified, screened, and included. The lack of methodological details makes the review non-reproducible and significantly weakens the manuscript.

- The manuscript is excessively descriptive and repeats the same information (e.g., antibacterial effects, remineralization potential, mechanical properties) across multiple sections. A clear reorganization is necessary.

- The authors should categorize the nanoparticles in a more logical way (e.g., antibacterial particles, remineralizing particles, mechanical enhancers) to improve clarity. A Table should be presented to show the readers the results of the included papers.

- Please ensure that all acronyms (e.g., ACP, nHA, QAC, MBGN) are defined once and used consistently throughout the text.

- Several mechanistic descriptions are repeated multiple times and should be condensed.

- Distinguish clearly between laboratory results and clinical relevance. Many of the studies cited are in vitro and must be presented as such. No strong statements can be taken from these references.

- The text occasionally overstates the performance of nanoparticles without sufficient evidence. A more critical and balanced perspective is required. Please be self-critic and a realistic impression from this technology.

- Figures (especially Figure 1 and Figure 2) are poorly integrated into the manuscript. Their relevance should be explained in the text.

- Figure 2 caption appears incomplete and must be corrected.

- Table formatting and reference citations within tables require standardization and consistency.

- The quality and resolution of some figures are poor and should be improved to ensure readability.

- The Discussion section lacks structure and does not provide a critical interpretation of the findings. It mostly repeats information presented earlier.

- The authors dedicate significant space to summarizing other studies without adequate comparison to the main themes of their own review.

- A paragraph clearly summarizing the main findings of the review is missing and should be added at the beginning of the Discussion section.

- The limitations of nanoparticle-based orthodontic materials (e.g., cytotoxicity, changes in resin viscosity, polymerization interference, long-term stability) are only superficially addressed. A deeper, more self-critical analysis is necessary.

- There are many limitations from a narrative review that were not addressed in this manuscript. Please, include than in the Discussion.

- Please add a paragraph on the clinical relevance, emphasizing which nanoparticles have actual clinical evidence and which remain purely experimental.

- Recommendations for future research should be included.

- The conclusion is overly optimistic and not aligned with the actual level of evidence.

Since most studies are in vitro and highly heterogeneous, the authors should adopt a more moderate and cautious tone.

- Please avoid making strong statements about clinical application without robust clinical data.

- A balanced summary that acknowledges the limitations of current research is necessary in both Discussion and Conclusion sections.

- The manuscript has numerous issues with grammar, syntax, and repetition. A thorough English language revision is strongly recommended.

- Several paragraphs are very long and would benefit from being divided for better readability.

- Consistency in terminology (nanoparticles vs. nanocomposites vs. NPs) should be maintained.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The manuscript has numerous issues with grammar, syntax, and repetition. A thorough English language revision is strongly recommended.

Author Response

Comment 1: The abstract is overly long, repetitive, and does not succinctly reflect the main findings of the review. Please reduce the length and focus on the essential information only. The objective stated in the abstract should be rewritten to clearly and concisely present the aim of the review. Currently, the wording is unclear and lacks fluency. The abstract should be better distinguished between evidence derived from in vitro, in vivo, and clinical studies. The Conclusion section of the Abstract is vague and does not reflect the limitations or the true level of evidence available.

Response 1: We thank the Reviewer for this helpful comment. The abstract has been substantially shortened to reduce repetition and to focus on the essential information. We have rewritten the objective to clearly and concisely state that this is a narrative review summarizing in vitro, in vivo, and clinical evidence on nanoparticle-modified orthodontic bonding materials, with emphasis on antimicrobial, remineralizing, and mechanical outcomes and their clinical relevance. The results section of the abstract now explicitly distinguishes between in vitro, in vivo, and clinical studies, noting that the current evidence base is dominated by in vitro data, with relatively few animal and human investigations. The conclusion has been revised to more accurately reflect the limitations and the true level of evidence, highlighting the predominance of pre-clinical findings, the small sample sizes and short follow-up of existing clinical studies, and the need for well-designed long-term clinical trials before routine clinical adoption can be recommended (Lines 26-47).

Comment 2:

In the Introduction section, several sentences are redundant and include unnecessary background information. Please condense this section to avoid repetition.

Response 2: We thank the Reviewer for this valuable observation. The Introduction has been substantially revised to remove redundancies and unnecessary background while preserving all essential content and references. Specifically, we merged overlapping descriptions of fixed appliance–related demineralization, streamlined the explanation of white spot lesions and their epidemiology, and eliminated repeated mentions of salivary clearance, microbial imbalance, and lesion progression. The discussion of nanoparticles was also condensed to avoid reiteration of mechanisms presented later in the manuscript. The section on anisotropic and biomimetic nanoparticles has been shortened and integrated more succinctly as suggested.

The revised Introduction now presents a clearer, more concise flow:
(1) the clinical problem and limitations of conventional bonding materials,
(2) WSL development and epidemiology,
(3) the rationale for nanoparticle incorporation, and
(4) emerging nanotechnology concepts relevant to future orthodontic applications.

No references were removed, and all citations requested by previous reviewers have been retained (Lines 72-153).

Comment 3:

The Introduction section lacks clear justification for why a review on this topic is needed at this time.

Response 3: We thank the Reviewer for this insightful comment. To address this, we have added explicit justification in the Introduction explaining why a review on nanoparticle-enhanced orthodontic bonding materials is timely and necessary. The revised text highlights that although research in this field has expanded rapidly in recent years, the evidence remains dispersed across numerous in vitro, in vivo, and limited clinical studies, with considerable heterogeneity in nanoparticle types, concentrations, and evaluation protocols. Additionally, no current review synthesizes the most recent advances in antimicrobial and remineralizing nanoparticles, emerging nanotechnology concepts (e.g., anisotropic and biomimetic nanoparticles), and their potential implications for clinical orthodontics. Given the high prevalence of white spot lesions and the rising interest in bioactive bonding materials, a comprehensive and updated evaluation of the available evidence is needed.

Comment 4:

Some claims (e.g., prevalence of white spot lesions) should be supported with updated references.

Response 4:

We thank the Reviewer for this important observation. In response, we have added several updated, recently published references to strengthen statements on the prevalence and clinical significance of white spot lesions in orthodontic patients. Specifically, new 2023–2025 epidemiological and systematic review sources have been incorporated, including recent meta-analytic data reporting current prevalence and incidence rates of WSLs in patients undergoing fixed-appliance therapy, as well as updated longitudinal findings on lesion progression. These references have been inserted alongside the existing citations within the Introduction to provide a more contemporary and comprehensive evidence base.

Comment 5:

The Materials and Methods section needs attention. The methodology is insufficiently described. Even for a narrative review, it is important to specify the databases used, key terms searched, period covered, and inclusion/exclusion criteria.

Response 5:

We thank the Reviewer for this important comment. The Materials and Methods (Lines 225-255) section has now been clarified and expanded to provide a more transparent description of the narrative review methodology.

Comment 6:

Currently, the statement “an overview has been presented” is not acceptable and lacks transparency.

Response 6:

We thank the Reviewer for pointing this out. The vague statement has been removed from the manuscript to improve clarity and transparency. The revised text now directly describes the content without relying on general phrases.

Comment 7:

Please ensure that the search strategy used in each of the scientific databases (e.g., PubMed, Scopus, Web of Science, etc.) is reported.

Response 7:

Thank you for this important suggestion. We have now added a detailed Appendix presenting the full search strategies for all databases used. Appendix A (Table A1) includes the exact search strings, Boolean operators, filters applied, document types, language limits, and the final search date for PubMed, Scopus, Web of Science, ScienceDirect, and Google Scholar. In the Materials and Methods section, we have added a corresponding sentence directing readers to this appendix for full transparency.

Comment 8:

The process of article selection and screening is not described. Please clarify how many articles were identified, screened, and included. The lack of methodological details makes the review non-reproducible and significantly weakens the manuscript.

Response 8:

We thank the Reviewer for this important comment. We have now added a clear description of the article identification and screening process to strengthen methodological transparency and reproducibility. The revised Materials and Methods section reports that the initial search retrieved 378 records, of which 346 remained after removal of duplicates. Titles and abstracts were screened, followed by full-text assessment, resulting in 73 studies that met the inclusion criteria and were therefore included in the narrative synthesis.

These numbers are now explicitly reported in the Methods, and a summary of the screening steps has been added to Appendix A for clarity.

Comment 9:

The manuscript is excessively descriptive and repeats the same information (e.g., antibacterial effects, remineralization potential, mechanical properties) across multiple sections. A clear reorganization is necessary.

Response 9:

We thank the Reviewer for this constructive comment. In response, the manuscript has been reorganized and streamlined to minimize repetition and improve readability. Descriptions of antibacterial effects, remineralization potential, and mechanical properties that previously appeared in multiple sections have been consolidated and are now presented in a more structured way, grouped by nanoparticle type and level of evidence. Overlapping or redundant statements have been removed, and each subsection now focuses on distinct aspects to avoid reiterating the same information.

Comment 10:

The authors should categorize the nanoparticles in a more logical way (e.g., antibacterial particles, remineralizing particles, mechanical enhancers) to improve clarity. A Table should be presented to show the readers the results of the papers included.

Response 10:

We thank the Reviewer for this helpful suggestion. In response, the tables in the manuscript have been fully reorganized to present the nanoparticles in a clearer and more logical structure based on their dominant functional roles. The revised tables now group materials into antibacterial nanoparticles (e.g., AgNPs, ZnO, TiO₂), remineralizing/ion-releasing nanoparticles (ACP/NACP, nCaF₂, MBGNs), and mechanically reinforcing systems (e.g., ZrO₂–TiO₂ nanocomposites). For each nanoparticle category, the updated tables (Table 1,2) now include:

the mechanism of action,
primary benefits,
representative particle size, and
key references from the studies included in the review.

Comment 11:

Please ensure that all acronyms (e.g., ACP, nHA, QAC, MBGN) are defined once and used consistently throughout the text.

Response 11:

We thank the Reviewer for this helpful comment. All acronyms in the manuscript— including ACP, nHA, QAC, MBGN, and others—have now been checked, defined at first mention, and used consistently throughout the text, tables, and figure captions. Instances of repeated or undefined acronyms have been corrected to ensure clarity and uniformity across the manuscript.

Comment 12:

Several mechanistic descriptions are repeated multiple times and should be condensed.

Response 12:

We thank the Reviewer for this observation. The manuscript has been revised to remove repeated mechanistic descriptions and to condense overlapping explanations of antibacterial, remineralizing, and mechanical reinforcement pathways. Mechanisms are now presented once within each nanoparticle category, and subsequent sections refer to the primary explanation to avoid redundancy. This revision improves clarity, reduces repetition, and streamlines the overall presentation.

Comment 13:

Distinguish clearly between laboratory results and clinical relevance. Many of the studies cited are in vitro and must be presented as such. No strong statements can be taken from these references.

Response 13:

We thank the Reviewer for this important observation. We have carefully revised the manuscript to clearly differentiate between in vitro, in vivo, and clinical evidence throughout the text. All statements derived from laboratory studies are now explicitly labeled as in vitro findings, and we have removed or rephrased any wording that implied clinical effectiveness where only preclinical data exists. Likewise, the Discussion and Conclusion sections have been modified to avoid overinterpretation and to emphasize that most available evidence remains laboratory-based, with only limited clinical studies published to date. Clinical relevance is now discussed cautiously and only when supported by actual in vivo or human data.

Comment 14:

The text occasionally overstates the performance of nanoparticles without sufficient evidence. A more critical and balanced perspective is required. Please be self-critic and a realistic impression from this technology.

Response 14:

We thank the Reviewer for this important comment. The manuscript has been carefully revised to avoid overstating the performance of nanoparticle-based materials and to provide a more critical and balanced perspective. Specifically, we have:

Rephrased sentences that previously suggested clinical effectiveness when only in vitro or limited in vivo data are available, explicitly labeling such findings as laboratory or preclinical results.
Softened claims regarding caries prevention, white spot lesion reduction, and long-term durability, and now emphasize that these outcomes remain largely hypothetical until supported by robust clinical trials.
Expanded the Discussion and Conclusion to highlight key limitations, including the predominance of in vitro evidence, variability in particle size and concentration, short follow-up periods, potential cytotoxicity and esthetic issues, and the lack of standardized protocols and regulatory guidance.

These changes provide a more self-critical and realistic appraisal of the current state of nanoparticle technology in orthodontic bonding materials.

Comment 15:

Figures (especially Figure 1 and Figure 2) are poorly integrated into the manuscript. Their relevance should be explained in the text.

Response 15:

We thank the Reviewer for this helpful comment. We have revised the manuscript to improve the integration of Figure 1 and Figure 2 by explicitly referencing each figure at the appropriate point in the text and explaining its purpose and relevance.

For Figure 1, we now clarify in the Introduction/early Results section that it illustrates the clinical problem (biofilm accumulation and the pathway toward white spot lesion formation) and serves to contextualize why bioactive orthodontic bonding materials are being explored.

For Figure 2, we added explanatory text in the section on nanoparticle mechanisms to indicate that the figure visualizes the main antimicrobial, remineralizing, and mechanical reinforcement pathways associated with different nanoparticle systems. This makes the figure a functional summary aiding reader comprehension rather than a standalone graphic.

Comment 16:

Figure 2 caption appears incomplete and must be corrected.

Response:

Thank you for noting this. The caption for Figure 2 has been fully revised to provide a complete and accurate description of the schematic, including the distribution of nanoparticles within the adhesive layer, their pH-responsive ion release, and their localized antibacterial action at the bracket–enamel interface (Lines 449-453).

Comment 17:

Table formatting and reference citations within tables require standardization and consistency.

Response 17:

We appreciate the Reviewer’s observation. All tables have now been reformatted for consistency, including uniform column structure, standardized terminology, and harmonized reference formatting. Citation style within tables has been aligned with the manuscript’s main reference format to ensure clarity and readability.

Comment 18:

The quality and resolution of some figures are poor and should be improved to ensure readability.

Response 18:

The figures identified as low resolution have been replaced with higher-quality versions to ensure clear visualization of all elements. Line thickness, labels, and contrast were also adjusted to optimize readability in both digital and print formats.

Comment 19:

The Discussion section lacks structure and does not provide a critical interpretation of the findings. It mostly repeats information presented earlier.

Response 19:

We thank the Reviewer for this valuable feedback. The Discussion has been substantially restructured to avoid repetition and to provide a more analytical, critical assessment of the evidence. The revised section now:

synthesizes themes across nanoparticle categories,
highlights strengths, limitations, and inconsistencies in the available data,
clearly separates in vitro, in vivo, and clinical findings,
discusses practical implications and feasibility, and
outlines gaps and priorities for future research.

Comment 20:

The authors dedicate significant space to summarizing other studies without adequate comparison to the main themes of their own review.

Response 20:

We thank the Reviewer for this observation. The revised manuscript now limits purely descriptive summaries and instead focuses on integrating study findings into the central themes of the review. Each subsection has been edited to emphasize comparative insights—such as differences between nanoparticle types, variability in study outcomes, and alignment with clinical needs—rather than recounting study details. This revision strengthens the coherence of the review and improves the relevance of the included evidence.

Comment 21:

A paragraph clearly summarizing the main findings of the review is missing and should be added at the beginning of the Discussion section.

Response:

We thank the Reviewer for this helpful suggestion. In response, we have added a concise summary paragraph at the beginning of the Discussion section that synthesizes the main findings of the review across nanoparticle categories, distinguishes between in vitro, in vivo, and clinical evidence, and contextualizes the overall level of support for nanoparticle-enhanced orthodontic adhesives. This provides a clearer transition from the results to the critical analysis presented in the Discussion (Lines 534-546).

Comment 22:

The limitations of nanoparticle-based orthodontic materials (e.g., cytotoxicity, changes in resin viscosity, polymerization interference, long-term stability) are only superficially addressed. A deeper, more self-critical analysis is necessary.

Response 22:

We thank the Reviewer for this important comment. The Discussion section has been expanded to provide a more critical and detailed evaluation of the limitations associated with nanoparticle-based orthodontic materials. Specifically, we now address:

the potential cytotoxicity of certain nanoparticle systems and the variability in biocompatibility depending on particle size, concentration, and surface chemistry;
changes in resin viscosity and handling properties that may occur when nanoparticle loading increases;
the possibility of polymerization inhibition or altered curing depth, particularly with photocatalytic or opaque particles;
long-term stability concerns, including nanoparticle agglomeration, reduced ion-release capacity over time, discoloration risks, and degradation of mechanical properties;
the lack of standardized testing protocols, which complicates comparison across studies.

Comment 23:

There are many limitations from a narrative review that were not addressed in this manuscript. Please, include them in the Discussion.

Response 23:

We appreciate the Reviewer’s observation. A dedicated paragraph has been added to the Discussion to explicitly address the inherent limitations of a narrative review. This includes acknowledgement of:

the absence of a fully systematic search protocol;
potential selection bias despite broad database coverage;
heterogeneity in study designs, nanoparticle formulations, and testing methods that prevents direct comparison or meta-analysis;
reliance on descriptive synthesis rather than quantitative effect estimation;
the predominance of in vitro evidence, limiting the strength of clinical inference;
publication bias favoring positive findings.

These additions clarify the methodological constraints of our review and provide appropriate context for interpreting the conclusions.

Comment 24:

Please add a paragraph on the clinical relevance, emphasizing which nanoparticles have actual clinical evidence and which remain purely experimental.

Response 24:

We thank the Reviewer for this valuable suggestion. A new paragraph has been added to the Discussion explicitly addressing the clinical relevance of nanoparticle-enhanced orthodontic materials. This paragraph clarifies which nanoparticle systems have limited clinical or in vivo evidence, and which are still supported exclusively by in vitro studies. It also emphasizes that most formulations remain experimental and that current human studies are preliminary, short-term, and insufficient to guide routine clinical adoption (Lines 546-560).

Comment 25:

Recommendations for future research should be included.

Response 25:
We thank the Reviewer for this suggestion. The Discussion has been expanded to include explicit recommendations for future research. In particular, we now emphasize the need for: (i) standardized in-vitro protocols for SBS and antibacterial testing across nanoparticle systems; (ii) long-term durability and ion-release studies under conditions that better simulate the dynamic oral environment; (iii) comprehensive in vivo and toxicological investigations addressing chronic low-level nanoparticle release, bioaccumulation, and systemic exposure; and (iv) well-designed randomized clinical trials with long-term follow-up to evaluate white-spot lesion incidence, caries outcomes, bond failure rates, patient satisfaction, and cost–benefit ratios. We also highlight future directions such as stimuli-responsive “smart” adhesives and self-healing polymers, as well as integration with digital dentistry and AI-guided risk stratification.

Comment 26:

The conclusion is overly optimistic and not aligned with the actual level of evidence.

Response:
We appreciate this important comment. The Conclusion has been rewritten to align more closely with the current level of evidence. The revised text now explicitly states that most data on nanoparticle-enhanced orthodontic bonding materials are derived from short-term in-vitro studies with heterogeneous methodologies, that robust clinical evidence remains scarce, and that existing formulations should be regarded as experimental. Rather than presenting nanoparticles as a “transformative” or established solution, the Conclusion now frames them as promising but unproven technologies that require further clinical validation.

Comment 27:

Since most studies are in vitro and highly heterogeneous, the authors should adopt a more moderate and cautious tone.

Response 27:
We agree with the Reviewer and have carefully revised the manuscript to adopt a more moderate and cautious tone. Throughout the Results, Discussion, and Conclusion, statements originally suggesting clinical effectiveness have been rephrased to indicate when findings are based on in-vitro, in-situ, in vivo, or limited clinical data. We also explicitly acknowledge the substantial heterogeneity in nanoparticle types, particle sizes, concentrations, resin matrices, and testing conditions, and we now stress that this variability precludes strong comparative or quantitative conclusions.

Comment 28:

Please avoid making strong statements about clinical application without robust clinical data.

Response 28:
Thank you for highlighting this issue. All strong statements implying established clinical benefits have been removed or softened. Where potential clinical applications are discussed (e.g., caries-risk–based selection frameworks, use in high-risk or esthetically demanding cases), we now explicitly state that these concepts are conceptual and exploratory, not prescriptive, and that they depend on evidence from future clinical trials. The revised text repeatedly emphasizes that most nanoparticle-containing orthodontic materials remain experimental and cannot yet be recommended for routine use.

Comment 29:

A balanced summary that acknowledges the limitations of current research is necessary in both Discussion and Conclusion sections.

Response 29:
We appreciate this comment. A balanced summary has been added at the beginning of the Discussion, synthesizing the main findings while explicitly highlighting the limitations of the current evidence base, including the predominance of in-vitro studies, heterogeneity of methodologies, limited long-term biocompatibility data, and lack of standardized protocols. The Conclusion has likewise been revised to reiterate these constraints and to clearly state that, although laboratory results are encouraging, the true clinical benefit and long-term safety of nanoparticle-modified orthodontic materials remain uncertain.

Comment 30:

The manuscript has numerous issues with grammar, syntax, and repetition. A thorough English language revision is strongly recommended.

Response 30:
We thank the Reviewer for this observation. The entire manuscript has undergone careful English-language editing. We corrected grammatical and syntactic errors, simplified overly long or complex sentences, and removed redundant wording. Sections that previously repeated mechanistic descriptions or study outcomes were consolidated, and transitions between paragraphs were improved to enhance clarity and readability.

Comment 31:

Several paragraphs are very long and would benefit from being divided for better readability.

Response 31:
We agree with the Reviewer. Long paragraphs in the Introduction, Results, Discussion, and Nanotoxicity sections have been divided into shorter, thematically focused units. Each new paragraph now addresses a specific topic (e.g., WSL epidemiology, antibacterial mechanisms, remineralizing nanoparticles, toxicity and regulation), improving readability while preserving content.

Comment 32:

Consistency in terminology (nanoparticles vs. nanocomposites vs. NPs) should be maintained.

Response 32:
We thank the Reviewer for pointing this out. Terminology has been standardized throughout the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have solved all my concerns and this manuscript should be accepted.

Author Response

We thank the reviewer for the suggestions for improvement and the encouraging words

Reviewer 4 Report

Comments and Suggestions for Authors

I acknowledge that the authors have made several modifications and that the paper has improved compared to the previous submission. However, despite these revisions, my main concerns remain unresolved. In my opinion, the manuscript still presents fundamental limitations that prevent it from reaching the scientific rigor and clarity required for publication in this journal.

Author Response

We thank the Reviewer for his careful re-evaluation of the manuscript and for his continued critical assessment. We respectfully acknowledge the reviewer’s concerns; however, we would like to emphasize that the present work is designed as a narrative review intended to synthesize and contextualize a heterogeneous and predominantly preclinical body of literature. In response to earlier comments, we have substantially revised the manuscript to improve clarity, structure, and scientific focus, strengthened the critical appraisal of limitations, clearly distinguished between in vitro and clinical evidence, and avoided overinterpretation of findings. We have also enhanced the discussion of methodological heterogeneity, explicitly highlighted the investigational status of nanoparticle-enhanced materials, and reinforced the need for standardized protocols and high-quality clinical trials. While we recognize the inherent limitations of the available evidence base, we believe the revised manuscript now provides a balanced, transparent, and clinically relevant synthesis that meets the journal’s standards for narrative reviews and offers value to both researchers and clinicians in this emerging field.