Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

++ highlighted strengths
-- major/moderate concerns
-minor concerns

Abstract
-Please see author instructions regarding abstract format
Introduction
++ The abstract is clear and informs the reader the relevance of the project
- Discussing denture stomatitis should be expanded to discuss the clinical relevance of the project including how this potential affects medically complex patients

Methods
++ the methods present sufficient details for the experiments to be repeated
++ Biofilm experiments were presented with important details.
++ Attention to correct stats was performed, notably non-parametric methods
-Table 1 requires reformatting and should include estimated percentage ranges when known (for example, there are estimated % for Denture Base OP Resin,RS-F2-DBOP-01).  
--It is unclear when the bacterial media was replenished and if QA contamination checks were routinely performed.  
Results
++ The results clearly demonstrate that manufacturing method influence S. san over S. mut.
-The results section discusses SEM to examine morphology but the contamination later found (noted in Figure 4 and in the text before SEM) wasn't discussed in terms of morphology.  The reviewer appreciates the authors mentioning the contamination issue.
Discussion
++ The discussion is directly related to the results of the study and discusses the relevance of the results, the limitations of the study, and future directions
-- The authors should discuss how the results could be different if S. mutans was exposed to sucrose
which is a more favorable condition to supplement with TSY.  

Author Response

Point by point response – Reviewer 1

We thank the reviewer for the thorough evaluation of our manuscript and the constructive comments, which helped to improve the clarity, methodological transparency, and clinical relevance of the study. All suggested revisions have been carefully addressed, and the manuscript has been revised accordingly. In addition, the labeling of the measurement days was corrected throughout the manuscript. Specifically, T4 was revised to day 7 (instead of day 6), and T5 to day 9 (instead of day 7). We hope that in the light of the revisions the manuscript can be recommended for publication.

 

Abstract - Please see author instructions regarding abstract format

OUR RESONSE

We agree upon this aspect.

OUR ACTION

We adjusted the abstract format regarding the author instructions. (P.1 L10-28)

 

Introduction - Discussing denture stomatitis should be expanded to discuss the clinical relevance of the project including how this potential affects medically complex patients

OUR RESPONSE

We agree with the reviewer on the relevance of this aspect.

OUR ACTION

We added further details to the Introduction. (P.2 L60-65)

 

Methods - Table 1 requires reformatting and should include estimated percentage ranges when known (for example, there are estimated % for Denture Base OP Resin,RS-F2-DBOP-01). 

OUR RESPONSE

We thank the reviewer for raising this aspect.

OUR ACTION

Table 1 has been reformatted, and all information has been included when known. (P.4)

 

Methods - It is unclear when the bacterial media was replenished and if QA contamination checks were routinely performed. 

OUR RESPONSE

We agree that this aspect requires additional information.

OUR ACTION

We added the corresponding information to the Material and Methods section. (P.6 L188-196)

 

Results - The results section discusses SEM to examine morphology but the contamination later found (noted in Figure 4 and in the text before SEM) wasn't discussed in terms of morphology.  The reviewer appreciates the authors mentioning the contamination issue.

OUR RESPONSE

We thank the reviewer for pointing out this aspect. The contamination observed at this time point was macroscopically visible and likely represented fungal growth. The SEM images of the specimens after 12 days of incubation revealed structures consistent with bacterial biofilms formed by the investigated species. Although contamination occurred on day 6 with S. mutans based on agar plating and macroscopic inspection, no morphological features indicating contamination were detected in the SEM images.

OUR ACTION

We added further these aspects to the manuscript in the Results section and Discussion section. (P8, L238-239; P11, L309-313)

 

Discussion - The authors should discuss how the results could be different if S. mutans was exposed to sucrose which is a more favorable condition to supplement with TSY. 

OUR RESPONSE

We thank the reviewer for this valuable suggestion. We agree that sucrose supplementation represents an important factor influencing S. mutans biofilm formation.

OUR ACTION

A corresponding paragraph discussing the influence of sucrose on biofilm formation has been added to the Discussion section. (P13, L365-376)

 

Reviewer 2 Report

Comments and Suggestions for Authors

Contemporary dentistry is undergoing a major technological shift due to the increasing use of 3D Printed and Milled polymeric dental materials. Accordingly, research on newly developed materials and the acquisition of comprehensive knowledge regarding their properties in comparison to conventional materials is essential. The present study on biofilm formation fits within this research trend.

Overall Structure
The manuscript is well-structured

Introduction

The introduction is clearly written. One limitation is the omission of Candida yeasts, which can represent an important and pathogenic component of the oral microbiota. While the focus of this study is on bacterial biofilms, mentioning Candida could provide additional context, especially in relation to denture-associated microbial communities.

Methodology
The methodology section is generally well presented; however, certain points need clarification:

1. The number of specimens per group and the total number of samples are not explicitly stated. While mentioned in the discussion, the rationale for the chosen sample size should be provided in methodolog. This is important for assessing statistical power and reproducibility.
2. From the description of specimen surface preparation, it may appear that the two sides of each sample were prepared differently. This was likely intentional (to mimic the “intaglio surface” versus external surfaces), but the manuscript should clarify which side was considered standard for observation and how this relates to real clinical conditions.
3. It should also be specified which side of the specimen faced upwards during immersion in the microbial suspension, as this could influence biofilm formation.
4. Including additional statistical analyses for each material to assess time-dependent changes would strengthen the conclusions regarding trends in biofilm accumulation.

Results
The results are adequately described. Some points could be improved for clarity:

1. The rationale for selecting 12 days for SEM analysis is unclear, and it is not explained why the final 21-day time point was omitted. While this is not a critical flaw, providing a justification would help interpret the data. Optionally, it will be fine to see SEM results also after 21 days.

1. Microphotographs should be provided at higher resolution and enlarged; in their current form, the legends and markers are largely illegible. The “Original Images” appear adequate, suggesting the issue may have occurred during figure preparation or PDF generation.

Discussion

1. The statement: “In contrast, S. sanguinis exhibited a pronounced increase in biomass, including an intermediate peak during the mid-experimental phase followed by a temporary decline and a subsequent rise toward the end of the incubation period.” - is not fully supported by the presented data, as no statistical analysis of changes over time was performed. Variability in the data is high, and the sample size is not provided, making it difficult to determine whether the observed trends are meaningful.
2. “Material-related properties likely influenced bacterial adhesion and biofilm accumulation. Although all specimens underwent standardized finishing and polishing, subtle differences in surface roughness, surface energy, internal microstructure, and degree of polymerization should be considered. Material surface properties such as roughness and surface free energy significantly impact bacterial adhesion and the resulting development of biofilms on dental materials — increased surface roughness and higher surface energy have been associated with enhanced microbial retention and biofilm formation, whereas smoother surfaces tend to resist initial bacterial attachment [18,19]” 

However, the study did not measure these properties quantitatively, and surface standardization was performed to minimize roughness differences. Therefore, while the discussion is scientifically valid, the manuscript should clarify that the influence of material chemistry or microstructure, rather than surface roughness, likely explains differences in biofilm formation.

Editorial

• The notation:

“Source: own figure” is unnecessary when reporting original data.

Author Response

Point by point response – Reviewer 2

We thank the reviewer for the thorough comments and suggestions for our manuscript, which helped to improve the clarity, methodological transparency, and clinical relevance of the study. In addition, the labeling of the measurement days was corrected throughout the manuscript. Specifically, T4 was revised to day 7 (instead of day 6), and T5 to day 9 (instead of day 7). All suggested revisions have been carefully addressed, and we hope that in the light of the revisions the manuscript can be recommended for publication.

 

Introduction - The introduction is clearly written. One limitation is the omission of Candida yeasts, which can represent an important and pathogenic component of the oral microbiota. While the focus of this study is on bacterial biofilms, mentioning Candida could provide additional context, especially in relation to denture-associated microbial communities.

OUR RESPONSE

We thank the reviewer for this helpful comment. We agree on this aspect. However, the present study was intentionally designed to focus on bacterial mono-species biofilm formation by Streptococcus mutans and Streptococcus sanguinis under standardized conditions.

OUR ACTION

We added a brief statement to the Introduction to acknowledge the relevance of Candida in denture-associated biofilms while clarifying that the present study focused on bacterial early colonizers. (P.2, L70-74)

 

Methodology - The number of specimens per group and the total number of samples are not explicitly stated. While mentioned in the discussion, the rationale for the chosen sample size should be provided in methodolog. This is important for assessing statistical power and reproducibility.

OUR RESPONSE

We thank the reviewer for this important comment. We agree on this aspect.

OUR ACTION

We revised the Materials and Methods section. (P.5, L.140-145)

 

 

Methodology - From the description of specimen surface preparation, it may appear that the two sides of each sample were prepared differently. This was likely intentional (to mimic the “intaglio surface” versus external surfaces), but the manuscript should clarify which side was considered standard for observation and how this relates to real clinical conditions.

OUR RESPONSE

We thank the reviewer for this helpful comment. The different preparation of the two specimen sides was intentional.

OUR ACTION

We revised the Materials and Methods to provide clarification. (P.5, L.131-136)

 

Methodology - It should also be specified which side of the specimen faced upwards during immersion in the microbial suspension, as this could influence biofilm formation.

OUR RESPONSE

We thank the reviewer for this important comment. We agree that the orientation of the specimens during immersion should be stated explicitly

OUR ACTION

We added further clarification to the Materials and Methods section. (P.5, L.161-163)

 

Methodology - Including additional statistical analyses for each material to assess time-dependent changes would strengthen the conclusions regarding trends in biofilm accumulation.

OUR RESPONSE

We thank the reviewer for this valuable suggestion. We agree that additional analyses across time points could further strengthen the interpretation of temporal patterns in biofilm accumulation. However, the present study was primarily designed to compare biofilm formation between denture base materials and bacterial species at defined measurement time points rather than to perform a comprehensive time-course analysis within each material.

OUR ACTION

We revised the Results section to present temporal changes more cautiously. (P.11, L.309-313)

 

Results - The rationale for selecting 12 days for SEM analysis is unclear, and it is not explained why the final 21-day time point was omitted. While this is not a critical flaw, providing a justification would help interpret the data. Optionally, it will be fine to see SEM results also after 21 days.

OUR RESPONSE

We agree with the reviewer that a justification is required – we had the SEM analysis after 12 days as a representative later-stage time point to qualitatively assess established biofilm morphology.

OUR ACTION

We added further details to the Material and Methods to justify this. (P.7, L.207-209)

 

Results - Microphotographs should be provided at higher resolution and enlarged; in their current form, the legends and markers are largely illegible. The “Original Images” appear adequate, suggesting the issue may have occurred during figure preparation or PDF generation.

OUR RESPONSE

We thank the reviewer for this helpful observation. We agree that the readability of the images should be improved. The original images are provided in sufficient quality.

OUR ACTION

We will ensure that appropriately sized and high-resolution figures are provided for the final publication version.

 

Discussion - The statement: “In contrast, S. sanguinis exhibited a pronounced increase in biomass, including an intermediate peak during the mid-experimental phase followed by a temporary decline and a subsequent rise toward the end of the incubation period.” - is not fully supported by the presented data, as no statistical analysis of changes over time was performed. Variability in the data is high, and the sample size is not provided, making it difficult to determine whether the observed trends are meaningful.

OUR RESPONSE

We thank the reviewer for this important comment.

OUR ACTION

The corresponding sentence in the Results section was rephrased more cautiously. (P.13, L.375-376)

Discussion - “Material-related properties likely influenced bacterial adhesion and biofilm accumulation. Although all specimens underwent standardized finishing and polishing, subtle differences in surface roughness, surface energy, internal microstructure, and degree of polymerization should be considered. Material surface properties such as roughness and surface free energy significantly impact bacterial adhesion and the resulting development of biofilms on dental materials — increased surface roughness and higher surface energy have been associated with enhanced microbial retention and biofilm formation, whereas smoother surfaces tend to resist initial bacterial attachment [18,19]” However, the study did not measure these properties quantitatively, and surface standardization was performed to minimize roughness differences. Therefore, while the discussion is scientifically valid, the manuscript should clarify that the influence of material chemistry or microstructure, rather than surface roughness, likely explains differences in biofilm formation.

OUR RESPONSE

We thank the reviewer for this important comment.

OUR ACTION

The corresponding paragraph in the Discussion was revised to clarify that surface roughness and surface free energy were not quantitatively measured in this study. We also specified that, due to standardized surface finishing, the observed differences in biofilm formation are more likely related to material-specific properties such as chemistry, microstructure, degree of polymerization, or other manufacturing-related characteristics. (P.13, L.386-397)

 

Editorial - The notation: “Source: own figure” is unnecessary when reporting original data.

OUR RESPONSE

We agree with the reviewer.

OUR ACTION

We corrected this.

Reviewer 3 Report

Comments and Suggestions for Authors

Minor suggestions for improvement: Methods: Measure Ra (roughness) by profilometry and surface energy (goniometry). Results: Calculate size effect (η²) and post-hoc power. Discussion: Test viability (LIVE/DEAD) or qPCR for biomass/extracellular matrix. Overall: Include C. albicans or saliva in vitro.

 

Author Response

Point by point response – Reviewer 3

We thank the reviewer for the thorough comments and suggestions for our manuscript. In addition, the labeling of the measurement days was corrected throughout the manuscript. Specifically, T4 was revised to day 7 (instead of day 6), and T5 to day 9 (instead of day 7). We hope that in the light of the revisions the manuscript can be recommended for publication.

 

Methods: Measure Ra (roughness) by profilometry and surface energy (goniometry)

OUR RESPONSE

The authors appreciate this valuable suggestion. However, the present study was designed to focus on species-specific biofilm biomass formation using a standardized crystal violet assay in order to ensure comparability between different manufacturing methods. Additional analyses such as surface roughness measurements or surface energy measurements would certainly provide further insights into the mechanisms of bacterial adhesion. However, these analyses were beyond the scope of the present study and were not part of the original experimental design. We agree that such investigations would be valuable and plan to address these aspects in future studies.

OUR ACTION

We addressed this aspect in the Discussion. (P.13, L. 389-397)

 

Results: Calculate size effect (η²) and post-hoc power.

OUR RESPONSE

We thank the reviewer for raising this aspect.

OUR ACTION

We calculated effect sizes and post-hoc power and reported these in the manuscript. (Table 3 and Table 4)

 

 

 

Discussion: Test viability (LIVE/DEAD) or qPCR for biomass/extracellular matrix.

OUR RESPONSE

The authors agree that additional analytical approaches such as LIVE/DEAD staining or qPCR could provide further insights into bacterial viability and extracellular matrix composition within the biofilms.

OUR ACTION

We addressed this in the Discussions section. (P.14, L. 445-451)

 

Overall: Include C. albicans or saliva in vitro.

OUR RESPONSE

The authors appreciate the reviewer’s suggestion to include additional biological factors such as Candida albicans or saliva in future experimental models. While the present study intentionally employed monoculture models to investigate species-specific biofilm formation under controlled conditions, more complex multispecies biofilm models including fungal species or salivary components would better reflect the clinical situation.

OUR ACTION

This aspect has been added to the limitations and future perspectives section of the Discussion. (P.15, L. 474-483)

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript has been revised by the authors to an appropriate extent. I recommend it for publication.