Histological Tissue Response to Calcium Silicate-Based Cements Assessed in Human Tooth Culture Models: A Systematic Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewer Comments,

This manuscript described a systematic review summarizing the outcomes of calcium silicate–based cements applied in ex vivo culture models using extracted teeth. Although the topic partially covered with the scope of the Journal of Functional Biomaterials, several important points should be addressed to strengthen the scientific rigor, interpretability, and relevance of the review.

-Conceptual framework and limitations of the ex vivo model

In the Introduction, the authors mentioned that this review focuses on models based primarily on Reference 21. However, the inherent limitations of this experimental model are not sufficiently discussed, which is essential for readers to correctly interpret the summarized findings.

-In particular, this ex vivo tooth culture model is isolated from systemic circulation, including vascular and immune systems, and therefore does not fully reflect the clinical condition. This limitation should be explicitly discussed. Moreover, it remains unclear whether there are studies that have performed detailed analyses of hard tissue formation (e.g., tertiary dentin) generated in this model. If such analyses are lacking, this should also be acknowledged.

I strongly recommend that the authors clearly describe:

• What can and cannot be evaluated in vitro,
• What can and cannot be evaluated in vivo, and
• What can and cannot be evaluated ex vivo,

and discuss how these experimental systems complement each other when assessing pulp responses to calcium silicate–based materials. This discussion would be highly appropriate in the Limitations section of the Discussion.

-Graphical abstract

To improve readability and conceptual understanding, the authors are encouraged to add a graphical abstract illustrating the experimental models (in vitro, ex vivo, in vivo), their relative complexity, and the types of outcomes assessed in the reviewed studies.

-Risk-of-bias assessment

The manuscript states that risk-of-bias assessments were conducted; however, the results should be presented more clearly. I understand that risk of bias (RoB) assessment in ex vivo studies, unlike human clinical trials (RCTs) and animal experiments (in vivo), often does not have a definitive international standard tool, but SYRCLE's Risk of Bias tool may be applicable to ex vivo experimental systems.

Be sure to evaluate the following points.

・Sample collection/preprocessing bias:

Timing of tissue collection, type of anesthesia, etc.

・Sample storage period, transportation conditions, etc.

・Uniformity of culture conditions:

Were the cultures performed in the same environment?

・Blinding (evaluator bias):

・Data deficiencies (attrition bias):

This may not be listed.

-Future perspectives: molecular and spatial analyses

In the Discussion, the authors describe the ex vivo model as an intermediate approach between in vitro and in vivo systems. In this context, I suggest expanding the discussion to include RNA sequencing–based comprehensive analyses.

Currently, most studies rely on immunohistochemical evaluation of predefined, well-known proteins. Given the intermediate nature of the ex vivo model, it may allow experimental approaches closer to in vitro systems, including unbiased transcriptomic analyses. Furthermore, the potential application of spatial molecular techniques should be discussed, as they could provide spatially resolved insights into pulp responses.

This model may also be useful for evaluating novel pulp-capping or regenerative materials, and this broader applicability could be explicitly mentioned.

Relevant reference:

-doi: 10.3390/jcm809144-T-Three-dimensional evaluation using tissue clearing

The authors may also consider discussing the future application of tissue-clearing techniques combined with 3D analysis in ex vivo tooth models. Such approaches could further advance this experimental system by enabling volumetric and spatial assessment of pulp responses and hard tissue formation.

Relevant reference: doi: 10.1177/0022034519844510

 

The mentioned references represent pioneering work in this field; however, if more recent or methodologically appropriate studies are available, replacing them would be acceptable.

 

-Need for multimodal evaluation

While the current review focuses primarily on histopathological outcomes, the authors should emphasize the importance of multimodal assessment to improve experimental robustness in this exvivo model. Importantly, the presence of calcification alone does not necessarily indicate successful vital pulp therapy (VPT).

Markers such as osteopontin are associated with mineralization, but additional evaluation of inflammatory and immune-related factors would provide a more comprehensive understanding of pulp responses. If such secondary outcomes are reported in the included studies, the authors are encouraged to consider adding a dedicated Results subsection to summarize these findings.

-Experimental protocol consistency

In the Results section, I recommend that the authors examine whether the included studies followed comparable experimental protocols. If common methodological features can be identified, presenting a generalized experimental protocol as supplementary material would be highly valuable. Readers interested in this research area would likely appreciate such a reference framework.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Comments 1: This manuscript described a systematic review summarizing the outcomes of calcium silicate–based cements applied in ex vivo culture models using extracted teeth. Although the topic partially covered with the scope of the Journal of Functional Biomaterials, several important points should be addressed to strengthen the scientific rigor, interpretability, and relevance of the review. -Conceptual framework and limitations of the ex vivo model In the Introduction, the authors mentioned that this review focuses on models based primarily on Reference 21. However, the inherent limitations of this experimental model are not sufficiently discussed, which is essential for readers to correctly interpret the summarized findings. -In particular, this ex vivo tooth culture model is isolated from systemic circulation, including vascular and immune systems, and therefore does not fully reflect the clinical condition. This limitation should be explicitly discussed. Moreover, it remains unclear whether there are studies that have performed detailed analyses of hard tissue formation (e.g., tertiary dentin) generated in this model. If such analyses are lacking, this should also be acknowledged. I strongly recommend that the authors clearly describe: What can and cannot be evaluated in vitro, What can and cannot be evaluated in vivo, and What can and cannot be evaluated ex vivo, and discuss how these experimental systems complement each other when assessing pulp responses to calcium silicate–based materials. This discussion would be highly appropriate in the Limitations section of the Discussion.

Response 1: We sincerely thank you for this insightful and constructive comment. In response, we have substantially expanded the Discussion (Limitations section) to more clearly describe the inherent limitations of the ex vivo extracted-tooth culture model (e.g., absence of systemic circulation, vascular perfusion and immune system contribution), and how these constraints may influence the interpretation of inflammatory and reparative outcomes, including hard tissue formation. In addition, as recommended, we included a concise conceptual overview of what can and cannot be assessed using in vitro, ex vivo and in vivo approaches, emphasizing how these experimental systems complement each other when evaluating pulp responses to calcium silicate–based biomaterials and vital pulp therapy procedures. Lines 470-510.

Comments 2: To improve readability and conceptual understanding, the authors are encouraged to add a graphical abstract illustrating the experimental models (in vitro, ex vivo, in vivo), their relative complexity, and the types of outcomes assessed in the reviewed studies.

Response 2: We sincerely thank the Reviewer for this valuable suggestion. We agree that graphical elements can substantially improve readability and conceptual understanding. The primary aim of our review, however, is to specifically analyze the histological and immunohistochemical responses obtained using the intact tooth ex vivo culture model. For this reason, the scope of the manuscript is intentionally focused on this experimental system and the tissue-level outcomes derived from it. Although other experimental approaches (in vitro and in vivo) are discussed in the Discussion section to contextualize the model and its limitations, they are not the central subject of the review.   In line with the Reviewer’s recommendation, we have now included a graphical abstract. This visual summary is designed to reflect the core focus of the manuscript, namely the ex vivo intact tooth culture model, the histological features evaluated, and the types of calcium silicate–based materials assessed, together with the associated tissue responses observed in the reviewed studies. We believe this graphical abstract improves clarity while remaining consistent with the defined scope and objectives of our work.   Comments 3: The manuscript states that risk-of-bias assessments were conducted; however, the results should be presented more clearly. I understand that risk of bias (RoB) assessment in ex vivo studies, unlike human clinical trials (RCTs) and animal experiments (in vivo), often does not have a definitive international standard tool, but SYRCLE's Risk of Bias tool may be applicable to ex vivo experimental systems. Be sure to evaluate the following points. ・Sample collection/preprocessing bias: Timing of tissue collection, type of anesthesia, etc. ・Sample storage period, transportation conditions, etc. ・Uniformity of culture conditions: Were the cultures performed in the same environment? ・Blinding (evaluator bias): ・Data deficiencies (attrition bias): This may not be listed.   Response 3: We thank you for this important comment regarding the clarity of the risk-of-bias (RoB) assessment. As described in Section 2.7 (Methodology), risk of bias in the included studies was evaluated using the QUIN tool (Quality Assessment Tool for In Vitro Studies), which we considered appropriate for non-clinical experimental designs such as ex vivo tooth culture models. The assessment domains covered aspects related to sample handling, experimental standardization, outcome evaluation, and reporting quality.   For full transparency, the detailed scoring for each item and for each included study is provided in the Supplementary Material, specifically in Tables S2 and S3, where the complete RoB evaluation can be found. We have ensured that this information is clearly indicated in the manuscript to facilitate interpretation.   We appreciate the suggestion, which helped us emphasize the availability and location of these methodological details.   Comments 4: -Future perspectives: molecular and spatial analyses   In the Discussion, the authors describe the ex vivo model as an intermediate approach between in vitro and in vivo systems. In this context, I suggest expanding the discussion to include RNA sequencing–based comprehensive analyses.   Currently, most studies rely on immunohistochemical evaluation of predefined, well-known proteins. Given the intermediate nature of the ex vivo model, it may allow experimental approaches closer to in vitro systems, including unbiased transcriptomic analyses. Furthermore, the potential application of spatial molecular techniques should be discussed, as they could provide spatially resolved insights into pulp responses.   This model may also be useful for evaluating novel pulp-capping or regenerative materials, and this broader applicability could be explicitly mentioned.   Relevant reference:   -doi: 10.3390/jcm809144-T-Three-dimensional evaluation using tissue clearing   The authors may also consider discussing the future application of tissue-clearing techniques combined with 3D analysis in ex vivo tooth models. Such approaches could further advance this experimental system by enabling volumetric and spatial assessment of pulp responses and hard tissue formation.   Relevant reference: doi: 10.1177/0022034519844510   The mentioned references represent pioneering work in this field; however, if more recent or methodologically appropriate studies are available, replacing them would be acceptable.   Response 4: We sincerely thank the Reviewer for this constructive and forward-looking suggestion. In response, we have expanded the Discussion to include a dedicated section on future perspectives. In this revised text, we address the potential integration of advanced molecular and spatial analytical techniques within the intact tooth ex vivo culture model. Specifically, we now discuss the relevance of unbiased transcriptomic approaches, spatially resolved molecular analyses, and tissue-clearing methods combined with three-dimensional imaging, as these strategies could provide deeper and spatially contextualized insight into pulp tissue responses and hard tissue formation. Lines 518-537   Comments 5: -Need for multimodal evaluation   While the current review focuses primarily on histopathological outcomes, the authors should emphasize the importance of multimodal assessment to improve experimental robustness in this exvivo model. Importantly, the presence of calcification alone does not necessarily indicate successful vital pulp therapy (VPT).   Markers such as osteopontin are associated with mineralization, but additional evaluation of inflammatory and immune-related factors would provide a more comprehensive understanding of pulp responses. If such secondary outcomes are reported in the included studies, the authors are encouraged to consider adding a dedicated Results subsection to summarize these findings.   Response 5: We sincerely thank the Reviewer for this valuable comment highlighting the importance of multimodal assessment and the need for a broader evaluation of pulp responses beyond mineralization alone. We agree that the presence of calcified tissue does not necessarily equate to successful vital pulp therapy and that assessment of inflammatory and immune-related factors would provide a more comprehensive understanding of pulp tissue reactions. However, the primary aim of this review was to synthesize the outcomes reported in studies using the intact tooth ex vivo culture model, and the currently available literature within this experimental framework is predominantly focused on histological and immunohistochemical parameters related to tissue organization, cell differentiation, and hard tissue formation.   Among the included studies, the reported outcomes are largely limited to morphological assessment, identification of odontoblast-like cells, mineralized foci formation, and expression of differentiation-associated markers. Consistent and systematic evaluation of inflammatory mediators, immune-related markers, or broader immunobiological responses is generally not provided in these works.   Comments 6: -Experimental protocol consistency   In the Results section, I recommend that the authors examine whether the included studies followed comparable experimental protocols. If common methodological features can be identified, presenting a generalized experimental protocol as supplementary material would be highly valuable. Readers interested in this research area would likely appreciate such a reference framework.   Response 6: We sincerely thank you for this constructive suggestion, which we believe adds practical value to the manuscript. In response, we have prepared a generalized experimental protocol for the whole-tooth ex vivo culture model and included it as Supplementary Material. This reference framework is primarily based on the original model described by Téclès et al., which established the core methodological principles of intact tooth organ culture. In addition, the proposed protocol incorporates minor variations and methodological nuances identified across the studies included in this review, such as differences in sample handling, culture duration, and outcome assessment approaches. We believe that providing this synthesized protocol will help readers better understand the shared experimental basis of the model while acknowledging the methodological heterogeneity present in the current literature.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

I’ve really enjoyed spending time with your systematic review on how pulp tissue reacts to calcium-silicate cements in whole-tooth cultures. The work is solid, the topic is timely, and you’ve clearly put care into every section. I feel the paper is almost ready to go, and with just a few gentle tweaks it could shine even brighter.

1. Semi-quantitative touch  
Because the studies vary so much, a full meta-analysis is off the table, yet readers would still love a clearer snapshot of the numbers. A simple table or chart that shows, for instance, how often each material led to mineralization or how consistent the outcomes were could add weight to your narrative and make comparisons easier at a glance.

2. Bringing it back to the clinic  
You’ve got great histology data, so let’s spell out what it means for the dentist holding the handpiece. A short paragraph that links the microscopic findings to everyday choices—like which cement to pick, what sort of reparative dentin we can realistically expect, and how this fits into modern vital pulp therapy—will help bridge lab and chairside worlds.

3. A word on generalizability  
Most of the included teeth came from young donors with healthy, immature third molars. That is perfectly valid for the model, yet mature teeth or inflamed pulps might behave differently. Expanding your limitations section to touch on age, baseline inflammation, and how these factors could sway outcomes will give readers a fair sense of where the evidence does or doesn’t reach.

4. Reading the risk of bias  
You have already laid out the QUIN scores nicely. A brief comment on how a “mostly moderate” risk of bias should temper our confidence—without undermining the entire message—would round out the methods story and keep everything transparent.

5. Tidying up the discussion  
A few sentences repeat the point that hydraulic calcium-silicate cements tend to come out on top. Trimming those echoes will tighten the flow while leaving your core conclusion intact.

With these small refinements, I’m confident your manuscript will become an even more valuable guide for anyone interested in bioactive pulp-capping materials.

Warm regards,

Comments on the Quality of English Language

Your English already comes across clearly it just needs a quick touch up to make it flow a little more smoothly and trim the small spots where ideas repeat

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Comments 1:

1. Semi-quantitative touch  

Because the studies vary so much, a full meta-analysis is off the table, yet readers would still love a clearer snapshot of the numbers. A simple table or chart that shows, for instance, how often each material led to mineralization or how consistent the outcomes were could add weight to your narrative and make comparisons easier at a glance.

 

 

Response 1: We thank you for this helpful suggestion. We agree that numerical summaries can enhance readability when methodological conditions allow meaningful comparison.

Some of the included studies reported numerical or semi-quantitative findings related to mineralized tissue formation. However, these outcomes were assessed using substantially different scoring systems, morphometric approaches, definitions of mineralization, and evaluation time points. Reported parameters ranged from semi-quantitative histological scores to percentages of teeth exhibiting mineralized foci, morphometric measurements of mineralized areas, and other non-equivalent indicators of hard tissue formation. Because these measures were obtained under different culture durations and experimental conditions, direct comparison or pooled frequency-based synthesis across studies would not be scientifically reliable and could potentially lead to misleading interpretations.

In response to the Reviewer’s comment, we have clarified this limitation in the Discussion and incorporated a paragraph summarizing representative numerical findings from individual studies (e.g., semi-quantitative histological scores, mineralized barrier formation, and reported proportions of mineralized foci). These examples illustrate consistent trends toward early mineralized tissue deposition and odontoblast-like differentiation within the ex vivo model, while avoiding inappropriate quantitative aggregation.

Lines 440-458

 

 

Comments 2:

2. Bringing it back to the clinic  

You’ve got great histology data, so let’s spell out what it means for the dentist holding the handpiece. A short paragraph that links the microscopic findings to everyday choices—like which cement to pick, what sort of reparative dentin we can realistically expect, and how this fits into modern vital pulp therapy—will help bridge lab and chairside worlds.

 

Response 2: We thank you for this valuable suggestion. We agree that clarifying the clinical relevance of the histological findings strengthens the manuscript.

In response, we have added a paragraph in the Discussion linking the microscopic observations to vital pulp therapy. We explain how the intact tooth ex vivo model allows anticipation of early pulpal responses following direct pulp exposure and biomaterial placement, particularly regarding mineralized tissue deposition and odontoblast-like cell differentiation. Based on these tissue-level findings, we also highlight that conventional calcium silicate–based materials without resin components showed more favorable and organized reparative responses compared with resin-modified formulations under the conditions evaluated.

 

We believe this addition helps bridge the experimental and clinical perspectives while remaining consistent with the scope of the review.

Lines 552-563

 

Comments 3:

3. A word on generalizability  

Most of the included teeth came from young donors with healthy, immature third molars. That is perfectly valid for the model, yet mature teeth or inflamed pulps might behave differently. Expanding your limitations section to touch on age, baseline inflammation, and how these factors could sway outcomes will give readers a fair sense of where the evidence does or doesn’t reach.

 

Response 3: We sincerely thank the Reviewer for this thoughtful comment regarding the generalizability of the findings.

In response, we have expanded the Limitations section of the Discussion to address this point more explicitly. We now clarify that most included studies were conducted using teeth from young donors with healthy, immature pulps, which represents a controlled and biologically favorable condition for maintaining pulp vitality in the ex vivo model. We further discuss that mature teeth, pulps with pre-existing inflammation, or different baseline biological conditions may exhibit altered reparative and inflammatory responses, and therefore the outcomes observed in this model should be interpreted within this specific experimental context.

Lines 470-493

 

Comments 4:

4. Reading the risk of bias  

You have already laid out the QUIN scores nicely. A brief comment on how a “mostly moderate” risk of bias should temper our confidence—without undermining the entire message—would round out the methods story and keep everything transparent.

 

Response 4: We appreciate this insightful comment regarding the interpretation of the risk-of-bias assessment.

Following this suggestion, we have expanded the Limitations section to clarify how the predominance of a moderate risk of bias among the included studies should be understood. We now specify that this level of risk does not invalidate the reported findings, but indicates that certain methodological aspects may not have been consistently controlled across studies. Consequently, the results should be interpreted as reflecting general biological trends rather than precise quantitative estimates of material performance.

 

We believe this clarification improves methodological transparency and provides readers with a more balanced understanding of the strength and boundaries of the available evidence.

Lines 539-545

 

Comments 5:

5. Tidying up the discussion  

A few sentences repeat the point that hydraulic calcium-silicate cements tend to come out on top. Trimming those echoes will tighten the flow while leaving your core conclusion intact.

Response 5: We thank you for this helpful observation.

We agree that certain statements concerning the favorable performance of hydraulic calcium silicate–based cements may have been reiterated. In response, we have revised the Discussion to reduce redundancy and improve the overall flow of the text, while preserving the main conclusions of the review.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

the manuscript seems pertinent to the journal and appears well conducted. It requires however several improvements to improve clarity and broadness to non expert in this topic.

Major revisions are below reported:

Introduction

Among the favorable characteristics of newly marketed materials, authors should mention premixed sealers (root canal sealers and putty materials) and their chemical and biological properties. The chemical reactions of CaSi in phosphate buffered solutions relies on the formation of hydrated silica, calcium release and amorphous calcium phosphate nucleation. Authors should implement introduction with the biological properties of new premixed sealers or experimental materials that used CaSi in its formulations. I report below some articles that can be used to clarify these crucial aspects:

 

DOI: 10.3390/nano11123439

doi.org/10.1038/s41598-023-36690-4

DOI: 10.1002/term.2769

 

The use of vascular wall–derived mesenchymal stem cells (VW-MSCs) represent a novel and compelling animal model strategy, as these perivascular multipotent cells persist in inflamed periapical tissues and are closely associated with lesion vascularization. Accumulating evidence indicates that VW-MSCs contribute to bone regeneration both directly, through participation in bone formation, and indirectly, via interactions with resident skeletal cells, supporting their relevance for studying periapical bone repair mechanisms. I recommend to implement introduction section with this paragraph.

 

Methods:

The methodology appears adequate, some clarifications in relation to the search gray literature.

Authors need to better discuss why meta analysis was not performed (eterogeneity of the studies).

 

Results:

Tables: consider to divide the results in early observation period (3-7 days) to late observation periods (e.g 14-28 days). Different markers and cytological observation are expected (for example, differentiation markers are usually observed at longer follow.up).

Add further analyses that compare materials used from the extracted data (e.g. HCSC vs calcium hydroxide vs control): for examples, X studies compared HCSC versus calcium hydroxide; X studies compared with control (cells without materials).

 

Discussion:

Regarding the mineralization nodules, authors should also include recently published studies that analysed premixed sealers, as doi: 10.1111/iej.14098.  doi: 10.1016/j.joen.2018.08.007

The current evidence extracted from this study (risk of bias from the studies) should be enhanced and more deeply reported.

Conclusions: 

please report with more detail the clinical recommendations of the present review

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

3. Point-by-point response to Comments and Suggestions for Authors Comments 1: Introduction   Among the favorable characteristics of newly marketed materials, authors should mention premixed sealers (root canal sealers and putty materials) and their chemical and biological properties. The chemical reactions of CaSi in phosphate buffered solutions relies on the formation of hydrated silica, calcium release and amorphous calcium phosphate nucleation. Authors should implement introduction with the biological properties of new premixed sealers or experimental materials that used CaSi in its formulations.   Response 1: We appreciate the Reviewer’s valuable perspective and suggestion to further develop the background on contemporary calcium silicate–based materials. In response, we have expanded the Introduction to better describe the evolution and growing diversity of calcium silicate formulations, including ready-to-use premixed putty-type materials. We now explain how these newer formulations were developed to improve handling properties and clinical applicability across different operative scenarios.   Additionally, we have incorporated discussion of the available evidence regarding the physicochemical and biological characteristics of these premixed putty formulations, linking their compositional and functional features with the histological responses reported in the reviewed studies. We believe this addition strengthens the conceptual framework of the manuscript and provides a clearer context for interpreting the biological outcomes observed in the intact tooth ex vivo culture model. Lines 387-418   Comments 2: The use of vascular wall–derived mesenchymal stem cells (VW-MSCs) represent a novel and compelling animal model strategy, as these perivascular multipotent cells persist in inflamed periapical tissues and are closely associated with lesion vascularization. Accumulating evidence indicates that VW-MSCs contribute to bone regeneration both directly, through participation in bone formation, and indirectly, via interactions with resident skeletal cells, supporting their relevance for studying periapical bone repair mechanisms. I recommend to implement introduction section with this paragraph.   Response 2: We thank you for highlighting the role of vascular wall–derived mesenchymal stem cells. In response, we have incorporated a paragraph in the Discussion addressing perivascular mesenchymal stem/stromal cell populations and their potential contribution to mineralized tissue formation. Evidence indicates that multipotent mesenchymal progenitors reside in vascular walls and microvessels, remain viable under inflammatory conditions, and can participate in mineralized tissue repair. This perspective provides a plausible cellular explanation for the proximity of mineralization nodules to pulpal microvasculature observed in the included histological studies. Lines 340-356   Comments 3: Methods:   The methodology appears adequate, some clarifications in relation to the search gray literature.   Authors need to better discuss why meta analysis was not performed (eterogeneity of the studies).

Response 3:

We thank you for these methodological observations.

Regarding the search of gray literature, we have clarified the corresponding subsection of the Methods to better describe the sources consulted and the strategy used to identify potentially relevant non-indexed studies.

Concerning the absence of a meta-analysis, we agree that this point required clearer justification. A quantitative synthesis was not considered feasible due to the marked heterogeneity among the included studies in terms of experimental design, outcome definitions, scoring systems, culture durations, and histological assessment methods. In addition, many of the included studies reported descriptive findings without providing quantitative or semi-quantitative data suitable for statistical pooling. We have now explicitly discussed these aspects in the Discussion section to clarify why a meta-analytic approach was not appropriate for this body of evidence.

We appreciate the Reviewer’s suggestion, which helped us improve the transparency of the methodological rationale.

 

Lines 440-458

 

Comments 4:

Results:

 

Tables: consider to divide the results in early observation period (3-7 days) to late observation periods (e.g 14-28 days). Different markers and cytological observation are expected (for example, differentiation markers are usually observed at longer follow.up).

 

Add further analyses that compare materials used from the extracted data (e.g. HCSC vs calcium hydroxide vs control): for examples, X studies compared HCSC versus calcium hydroxide; X studies compared with control (cells without materials).

 

 

Response 4:

We thank the Reviewer for these constructive suggestions regarding the presentation of the Results.

Regarding the proposal to divide the results according to early (3–7 days) and late (14–28 days) observation periods, we acknowledge the biological rationale behind this approach. However, the included studies exhibited substantial variability in culture durations, experimental endpoints, and reporting formats. Time points were not consistently aligned across studies, and histological evaluations often focused on structural outcomes rather than stage-specific molecular markers. As a result, grouping the findings strictly according to early versus late periods could lead to artificial categorization and potentially misleading comparisons. For this reason, we retained the current presentation, which reflects the original study designs more faithfully.

Concerning additional comparative analyses between material categories (e.g., hCSCs versus calcium hydroxide or control groups), we agree that such comparisons are conceptually relevant. However, only a limited number of studies included direct comparative arms, and outcome measures were heterogeneous in definition and reporting. Nevertheless, wherever direct comparisons were available, these have been described narratively in the Results and discussed in the Discussion section. We believe that further numerical aggregation or subgroup quantification would not be methodologically reliable given the available data.

 

Comments 5:

Discussion:

 

Regarding the mineralization nodules, authors should also include recently published studies that analysed premixed sealers, as doi: 10.1111/iej.14098.  doi: 10.1016/j.joen.2018.08.007

 

The current evidence extracted from this study (risk of bias from the studies) should be enhanced and more deeply reported.

 

Response 5: We thank you for these valuable comments aimed at strengthening the Discussion.

Regarding the suggestion to include recently published studies on premixed materials, we have incorporated the study by Bernardini et al., 2024 into the Discussion, as it provides relevant information related to calcium silicate–based biomaterials and their biological behavior. We appreciate this helpful recommendation. The other suggested reference focuses primarily on root canal sealers. As the scope of the present review is centered on reparative calcium silicate–based biomaterials evaluated using the intact tooth ex vivo culture model, we considered that studies specifically addressing sealers fall outside the primary focus of our work and therefore were not included.

Concerning the risk-of-bias assessment, detailed scoring for each study and each domain is provided in Supplementary Tables S2 and S3. These materials allow a transparent evaluation of the methodological quality of the included studies. We have ensured that the Discussion also summarizes the overall level of risk of bias and its implications for interpreting the evidence.

 

 

 

Comments 6:

Conclusions: 

 

Please report with more detail the clinical recommendations of the present review

Response 6: We appreciate the Reviewer’s suggestion to further elaborate on the clinical implications of the present review.

In response, we have expanded the Discussion section to include a clearer clinical interpretation of the findings. Specifically, we now relate the observed histological and immunohistochemical outcomes to their potential relevance for vital pulp therapy decision-making, highlighting how the biological performance of different calcium silicate–based biomaterials may inform material selection and expectations regarding reparative responses.

We thank the Reviewer for this helpful comment, which has improved the translational clarity of the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

 

Authors revised the graphical abstract in accordance with the reviewer’s comments. I have no further recommendations for this manuscript and therefore recommend ”accept”.

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

the manuscript is now suitable for pubblication