Mechanobiological and Molecular Alterations in the Aging Dentin–Pulp Complex

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper investigated known molecular signals related to tooth aging in relation to the mechanical changes (stiffness, fracture resistance) to human teeth with age. Importantly, the authors considered the roots and crowns separately since their function and mechanical uses differ. The candidates signals were appropriate and the discussion was thorough and interesting. I have only a few suggestions to improve the manuscript:

1) Please white balance all of your brightfield microscope images. This will improve the ability to see your immunohistochemical stains and is easy to adjust on any microscope of modern age (less than 60 years old). It would also help to have images of the tooth at lower magnifications with boxes to indicate where we are looking in the teeth. It's not apparent from the images and it's not even clear if you are imaging the same areas in each group.

2) Lines 101-103 are repetitive from earlier in the discussion - remove them or do not discuss the same point earlier.

3) Please explain why you chose a Poisson's ration of 0.3 for Young's modulus calculations (line 216). 

4) Tables 1-3 are difficult to understand. Please at least put what these percentages mean in each figure legend, but perhaps you can find a way to better clarify what you are looking at. 

5) Provide your p values in Table 4. Without this, your statements mean relatively little. I also suggest putting these numbers into graph form so we can confirm the error bars do not overlap. 

6) Please provide a cartoon or graphical depiction of your story. You did a good job in the discussion but it is so many details that it's difficult to remember what is what. A figure will help the reader integrate the study. 

Author Response

We thank the reviewers for their valuable and constructive comments, which have significantly helped improve the clarity and quality of the manuscript.

 

 

Comments and Suggestions for Authors

This paper investigated known molecular signals related to tooth aging in relation to the mechanical changes (stiffness, fracture resistance) to human teeth with age. Importantly, the authors considered the roots and crowns separately since their function and mechanical uses differ. The candidates signals were appropriate and the discussion was thorough and interesting. I have only a few suggestions to improve the manuscript:

1) Please white balance all of your brightfield microscope images. This will improve the ability to see your immunohistochemical stains and is easy to adjust on any microscope of modern age (less than 60 years old). It would also help to have images of the tooth at lower magnifications with boxes to indicate where we are looking in the teeth. It's not apparent from the images and it's not even clear if you are imaging the same areas in each group.

Thank you for this comment. We agree that white balancing and lower-magnification overview images would improve figure clarity. However, the images were obtained during the original study and cannot be re-acquired at this stage. All samples were analyzed using the same standardized protocol from comparable anatomical regions, and this has been clarified in the revised manuscript.

2) Lines 101-103 are repetitive from earlier in the discussion - remove them or do not discuss the same point earlier.

This sentence has been revised.

3) Please explain why you chose a Poisson's ration of 0.3 for Young's modulus calculations (line 216). 

Thank you for this comment. A Poisson’s ratio of 0.3 was selected because it is a commonly accepted reference value for human dentin in nanoindentation studies and has been widely used in the literature for elastic modulus calculations. Since dentin exhibits limited variation in Poisson’s ratio, this assumption provides reliable and comparable results across samples. We have clarified this rationale in the revised manuscript.

4) Tables 1-3 are difficult to understand. Please at least put what these percentages mean in each figure legend, but perhaps you can find a way to better clarify what you are looking at. 

Data are presented as percentages of cases (n) according to immunohistochemical score. Scores: 0 = negative, 1 = weak positivity (<50% of cells), 2 = strong positivity (≥50% of cells).

5) Provide your p values in Table 4. Without this, your statements mean relatively little. I also suggest putting these numbers into graph form so we can confirm the error bars do not overlap. 

Table 4. Modulus of elasticity and hardness values for Groups 1–3.

Coronal dentin
Samples	Modulus of elasticity (Mean ± SD)	Hardness  (Mean ± SD)
Group 1	21.135 ± 1.169	0.705 ± 0.071
Group 2	18.364 ± 0.440	0.726 ± 0.029
Group 3	24.446 ± 0.587	0.837 ± 0.040
Root dentin
Samples	Modulus of elasticity (Mean ± SD)	Hardness  (Mean ± SD)
Group 1	7.052 ± 0.801	0.345 ± 0.053
Group 2	18.300 ± 0.767	0.605 ± 0.049
Group 3	19.823 ± 0.946	0.753 ± 0.053

Table 4. Mean values (±SD) are presented for each group. Statistically significant differences were observed between groups (p < 0.001).

Figure 6. Bar graphs showing modulus of elasticity and hardness in coronal and root dentin across three experimental groups. Statistically significant differences between groups were observed (ANOVA/Kruskal-Wallis test, p < 0.001).

 

6) Please provide a cartoon or graphical depiction of your story. You did a good job in the discussion but it is so many details that it's difficult to remember what is what. A figure will help the reader integrate the study. 

5) and 6) We agree that a graphical depiction would improve presentation of the main findings. Therefore, we have added a Table 4 and new fig. 6 summarizing the proposed age-related molecular and mechanical changes within the dentin–pulp complex and their potential impact on pulp vitality and regenerative capacity.

Figure 7. Biological roles of examined markers and their clinical relevance.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This study presents an interesting multi-modal approach combining immunohistochemistry and nanoindentation to investigate age-related changes in the dentin–pulp complex. The relatively large sample size and the effort to integrate molecular and mechanical perspectives are notable strengths, and the manuscript is generally well organized with a clear narrative.

However, some critical issues limit the current manuscript:

1. The inclusion of different tooth types introduces significant confounding, making it difficult to attribute observed differences solely to aging.
2. The reliance on semi-quantitative immunohistochemical scoring , without complementary quantitative validation, limits the strength of the conclusions.
3. While the study proposes interactions between molecular pathways and mechanical properties, no direct experimental or statistical evidence  is provided to support these links.
4. There are inconsistencies between the statistical analyses and the interpretation of results, particularly regarding significance across variables and the use of parametric tests for ordinal data.
5. Several conclusions, especially those related to mechanistic interplay and regenerative implications, extend beyond what is directly supported by the data.

Overall, the study provides descriptive and potentially valuable observations, with appropriate revisions addressing the points raised above, the manuscript would be substantially improved and more convincing.

Author Response

 

We thank the reviewers for their valuable and constructive comments, which have significantly helped improve the clarity and quality of the manuscript.

This study presents an interesting multi-modal approach combining immunohistochemistry and nanoindentation to investigate age-related changes in the dentin–pulp complex. The relatively large sample size and the effort to integrate molecular and mechanical perspectives are notable strengths, and the manuscript is generally well organized with a clear narrative.

However, some critical issues limit the current manuscript:

1. The inclusion of different tooth types introduces significant confounding, making it difficult to attribute observed differences solely to aging.

We thank the reviewer for this important comment. Although different tooth types were necessarily included due to the clinical source of extracted specimens, all teeth met strict inclusion/exclusion criteria and were stratified primarily by the biological stage of the pulp–dentin complex; moreover, age groups were equally sized, supporting that the observed differences reflect age-related changes rather than tooth type alone.

1. The reliance on semi-quantitative immunohistochemical scoring , without complementary quantitative validation, limits the strength of the conclusions.

While immunohistochemical assessment was based on semi-quantitative scoring, all samples were processed under standardized conditions and evaluated comparatively across identical protocols, allowing reliable detection of group-related differences; nevertheless, we acknowledge that additional quantitative methods would further strengthen the findings and have noted this as a limitation of the study.

1. While the study proposes interactions between molecular pathways and mechanical properties, no direct experimental or statistical evidence  is provided to support these links.

We thank the reviewer for this important comment. The proposed relationship between molecular pathways and mechanical properties is based on the parallel age-related changes observed in immunohistochemical expression and nanoindentation measurements, which provide supportive but indirect evidence of association; however, direct statistical correlation analyses were beyond the scope of the present study and are acknowledged as a limitation.

 

1. There are inconsistencies between the statistical analyses and the interpretation of results, particularly regarding significance across variables and the use of parametric tests for ordinal data.

We have carefully reviewed and corrected the statistical analysis and the corresponding interpretation of the results, ensuring consistency in the reported significance levels and the appropriate use of statistical tests for the data structure.

1. Several conclusions, especially those related to mechanistic interplay and regenerative implications, extend beyond what is directly supported by the data.

We have revised the Conclusion section to remove overinterpretation and to ensure that all statements are strictly aligned with the directly supported data, with mechanistic and regenerative implications now presented in a more cautious and descriptive manner.

Overall, the study provides descriptive and potentially valuable observations, with appropriate revisions addressing the points raised above, the manuscript would be substantially improved and more convincing.

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

We have read with interest your manuscript addressing a relevant and timely topic, namely the age-related molecular and mechanobiological changes occurring within the dentin-pulp complex, with potential implications for regenerative dentistry. The study is original in its integrative approach, combining histological and immunohistochemical characterization with nanomechanical assessment of dentin, and the overall dataset is substantial for a human observational study. The manuscript is generally well structured, the objectives are clear, and the conclusions are, in broad terms, consistent with the results presented.

The work has several strengths. In particular, the inclusion of 90 human teeth distributed across three age groups provides a solid descriptive basis, and the combination of markers related to apoptosis, inflammation, autophagy, vascularization, and tissue mechanics gives the study a genuinely multidimensional character. The methodology is described in reasonable detail, including region selection, semi-quantitative scoring, blinded evaluation, and nanomechanical testing.

There are only few modification/clarification to add, mainly because some aspects of interpretation should be refined rather than because of any major methodological flaw:

(1) Several biological interpretations are stated somewhat too strongly in relation to the statistical support, especially where the manuscript discusses age-related “increases” or mechanistic implications despite non-significant comparisons. These statements should be softened and framed more consistently as trends or associations.

(2) The Results and Discussion would benefit from improved internal consistency, particularly regarding the interpretation of JAK1 activity and certain age-dependent patterns, so that the narrative fully matches the tabulated data.

(3) The manuscript still contains numerous typographical, grammatical, and stylistic issues, as well as a few inconsistencies in wording and referencing, which require careful editorial revision before publication.

Comments on the Quality of English Language

“mechanical properties in human teeth remains underexplored”
Correct with : “mechanical properties in human teeth remain underexplored”

“Modernhigh-resolution techniques provide a unique opportunity for in-depth study…”
(space missing)

“theeth” >> correct with "teeth"

“The age-dependent naonomichanical changes…”
Correct with: “... nanomechanical changes…”

“in the contex of preserving pulp vitality”
Correction with: “in the context ....”

“The overall period including fixation, decalsification, paraffin embedding and complete IHC analyzing of total of 90 dentin-pulp samples took 12 months…”
Suggested correction: “The overall process, including fixation, decalcification, paraffin embedding, and complete IHC analysis of all 90 dentin–pulp samples, took 12 months…”
(for both spelling, article use, and unnatural wording)

“The comparative hystological and imunohistochemical data are presented in Fig.1”
Correct with “The comparative histological ..."

“Interstengly, autophagic dynamics…” correct with “Interestingly, autophagic dynamics…”

“Figure S1 availble as a supplementary file”
Correct with : “Figure S1, available as a supplementary file”

“Images obtained from an atomically powered microscope.”
Do you mean “Images obtained by atomic force microscopy.” ???

“Aging of the pulpo-dentin complex…”
Should be written “Aging of the pulp–dentin complex…”

Author Response

We have read with interest your manuscript addressing a relevant and timely topic, namely the age-related molecular and mechanobiological changes occurring within the dentin-pulp complex, with potential implications for regenerative dentistry. The study is original in its integrative approach, combining histological and immunohistochemical characterization with nanomechanical assessment of dentin, and the overall dataset is substantial for a human observational study. The manuscript is generally well structured, the objectives are clear, and the conclusions are, in broad terms, consistent with the results presented.

The work has several strengths. In particular, the inclusion of 90 human teeth distributed across three age groups provides a solid descriptive basis, and the combination of markers related to apoptosis, inflammation, autophagy, vascularization, and tissue mechanics gives the study a genuinely multidimensional character. The methodology is described in reasonable detail, including region selection, semi-quantitative scoring, blinded evaluation, and nanomechanical testing.

There are only few modification/clarification to add, mainly because some aspects of interpretation should be refined rather than because of any major methodological flaw:

(1) Several biological interpretations are stated somewhat too strongly in relation to the statistical support, especially where the manuscript discusses age-related “increases” or mechanistic implications despite non-significant comparisons. These statements should be softened and framed more consistently as trends or associations.

We thank the reviewer for this valuable comment. We have revised the relevant sections to temper the interpretation of the findings, replacing overly strong causal language with more cautious wording (e.g., “tendencies” and “associations”) and ensuring that all statements are fully consistent with the statistical significance of the results.

(2) The Results and Discussion would benefit from improved internal consistency, particularly regarding the interpretation of JAK1 activity and certain age-dependent patterns, so that the narrative fully matches the tabulated data.

We have carefully revised the Results and Discussion sections to improve internal consistency, particularly in the interpretation of JAK1 activity and age-dependent patterns, ensuring that the narrative now fully aligns with the tabulated data. These changes have been incorporated into the revised manuscript.

(3) The manuscript still contains numerous typographical, grammatical, and stylistic issues, as well as a few inconsistencies in wording and referencing, which require careful editorial revision before publication.

The corrections have been made.

 

Comments on the Quality of English Language

Spelling errors have been corrected.

“mechanical properties in human teeth remains underexplored”
Correct with : “mechanical properties in human teeth remain underexplored”

“Modernhigh-resolution techniques provide a unique opportunity for in-depth study…”
(space missing)

“theeth” >> correct with "teeth"

“The age-dependent naonomichanical changes…”
Correct with: “... nanomechanical changes…”

“in the contex of preserving pulp vitality”
Correction with: “in the context ....”

“The overall period including fixation, decalsification, paraffin embedding and complete IHC analyzing of total of 90 dentin-pulp samples took 12 months…”
Suggested correction: “The overall process, including fixation, decalcification, paraffin embedding, and complete IHC analysis of all 90 dentin–pulp samples, took 12 months…”
(for both spelling, article use, and unnatural wording)

“The comparative hystological and imunohistochemical data are presented in Fig.1”
Correct with “The comparative histological ..."

“Interstengly, autophagic dynamics…” correct with “Interestingly, autophagic dynamics…”

“Figure S1 availble as a supplementary file”
Correct with : “Figure S1, available as a supplementary file”

“Images obtained from an atomically powered microscope.”
Do you mean “Images obtained by atomic force microscopy.” ???

“Aging of the pulpo-dentin complex…”
Should be written “Aging of the pulp–dentin complex…”

 

 

Author Response File: Author Response.docx