Review Reports - Spondin-1 Inhibits Odontoblastic Differentiation of Human Dental Pulp Stem Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors presented findings suggesting that spondin-1 inhibits odontoblastic differentiation. While the ideas and theme are novel, the following additional data are required to strengthen the study:

Experimental Groups: Additional experimental groups using methods such as SPON1 inhibitors or SPON1 receptor knockout models should be included.
Marker Analysis: The analysis of dentin-related markers was limited to DSPP. Results for additional markers, such as Runx2, Dentin Sialoprotein (DSP), and Dentin Matrix Protein 1 (DMP1), are necessary.
Characterization: Data comparing morphology after cell differentiation and immunohistochemical staining should be provided.
Pathway Analysis: Western blotting results for signaling pathways, such as the Wnt pathway, are required.

Author Response

General response

We sincerely thank the reviewer for the careful evaluation of our manuscript and for the constructive comments. We agree that additional experimental evidence and clearer presentation were necessary to strengthen the study. In the revised manuscript, we added new experimental data, including SPON1 loss-of-function analysis by siRNA-mediated knockdown, expanded odontoblast-related marker analysis, including DMP1, morphological and immunocytochemical characterization after differentiation, and Wnt/β-catenin pathway analysis by western blotting. We also revised the Introduction, Materials and Methods, Results, Discussion, Conclusions, and figure presentation to improve the overall clarity and strength of the manuscript.

Reviewer: 1

Thank you very much for your suggestion.

Major Comments:

Q1 : Experimental Groups: Additional experimental groups using methods such as SPON1 inhibitors or SPON1 receptor knockout models should be included.

Thank you very much for this important suggestion. To strengthen the experimental design, we added a loss-of-function experiment using siRNA-mediated SPON1 knockdown. SPON1 knockdown was confirmed by qRT-PCR 48 h after transfection, and the cells were then cultured under odontoblastic differentiation conditions for 7 days. The siSPON1 group showed stronger Alizarin Red S staining and significantly higher mineralized areas compared with the siControl group. In addition, odontoblast-related marker expression was increased after SPON1 knockdown. Together with the SPON1-neutralizing antibody experiment, these data provide complementary evidence that inhibition of endogenous SPON1 promotes odontoblastic differentiation of HDPSCs (p12, 13, line 441 - 469 in Results).

Q2 : Marker Analysis: The analysis of dentin-related markers was limited to DSPP. Results for additional markers, such as Runx2, Dentin Sialoprotein (DSP), and Dentin Matrix Protein 1 (DMP1), are necessary.

Thank you very much for this helpful comment. We expanded the marker analysis in the revised manuscript. In addition to DSPP, NESTIN, OCN, and OPN, we added DMP1 as an additional dentin/mineralized matrix-related marker. DMP1 expression was reduced by recombinant SPON1 treatment and increased after SPON1 neutralization and siRNA-mediated SPON1 knockdown. We also added immunocytochemical staining for DSP and NESTIN in the Supplementary Figure to further support the odontoblastic differentiation phenotype at the protein/localization level. These additional analyses strengthen the evidence that SPON1 regulates odontoblastic differentiation and mineralized matrix formation (p11, 12, line 378-440 in Results).

Q3 : Characterization: Data comparing morphology after cell differentiation and immunohistochemical staining should be provided.

Thank you very much for this suggestion. We added immunocytochemical staining of HDPSCs for odontoblast-related markers, including NESTIN and DSP, in CM, DM, and DM + SPON1 group in the Supplementary Fig. 4. These data provide additional cellular characterization and support the changes observed in mineralization and gene expression analyses.

Q4 : Pathway Analysis: Western blotting results for signaling pathways, such as the Wnt pathway, are required.

Thank you very much for this important comment. We showed western blotting analysis of the Wnt/β-catenin pathway. In Figure 7, non-phosphorylated active β-catenin was increased under odontoblastic differentiation conditions, whereas recombinant SPON1 treatment reduced active β-catenin levels. Total β-catenin levels were not markedly changed. We also included Supplementary Figure 4 to show the supporting/original western blot data and confirm the reliability of the analysis. These results suggest that the inhibitory effect of SPON1 on odontoblastic differentiation may be associated, at least in part, with suppression of canonical Wnt/β-catenin signaling. However, we revised the Discussion to state this interpretation cautiously, and further pathway-specific inhibition or rescue experiments will be needed to confirm the precise mechanism (p17, line 600-617 in Discussion).

Q5 : Evaluation items marked as “Must be improved”

The reviewer marked the following items as “Must be improved”: introduction/background/references, research design, methods, results presentation, conclusions supported by results, and figures/tables presentation.

Thank you very much for these important overall evaluations. We carefully revised the manuscript to address each of these points.

First, we revised the Introduction to provide a clearer background on reparative

dentin formation, the need for negative regulatory mechanisms to prevent excessive

mineralization, and the rationale for investigating SPON1 based on its reported role

in mineralized tissue homeostasis. We also revised and updated the relevant

references where necessary.

Second, we strengthened the research design by adding additional experimental

evidence. Specifically, we added siRNA-mediated SPON1 knockdown as a loss-of-

function approach. This complements the recombinant SPON1 gain-of-function

experiment and the SPON1-neutralizing antibody experiment, providing stronger

evidence for the inhibitory role of endogenous SPON1 in odontoblastic

differentiation.

Third, we improved the Materials and Methods by adding descriptions of the newly

included experiments, including SPON1 siRNA transfection, additional marker

analysis, morphological/immunocytochemical characterization, and western blotting

analysis of Wnt/β-catenin signaling.

Fourth, we revised the Results section to present the newly added data more clearly.

We added results showing that SPON1 knockdown enhanced Alizarin Red S-

positive mineralization and increased odontoblast-related marker expression. We also added DMP1 analysis, supplementary morphology and immunocytochemistry data, and Wnt/β-catenin western blotting data.

Fifth, we revised the Discussion and Conclusions to more clearly explain how the

conclusions are supported by the results. We now explain that the conclusion is

supported by multiple lines of evidence: the transient reduction of SPON1 during the

early stage of reparative dentin formation in vivo, the inverse association between

SPON1 expression and odontoblast-related markers during HDPSC differentiation,

the inhibitory effect of recombinant SPON1, and the enhanced differentiation

observed after SPON1 neutralization and siRNA-mediated SPON1 knockdown. We

also revised the wording to avoid overstatement and to clarify that SPON1 may

contribute to the regulation of reparative dentin formation.

Finally, we improved the figures and supplementary data by adding new figures and

supplementary figures to present the additional experiments more clearly, including t

he knockdown experiment, morphology/immunocytochemistry data, Wnt/β-catenin

western blotting data, and supporting/original western blot data.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the opportunity to review this manuscript. This is an interesting original article that describes the role of Spondin-1 as a negative regulator of odontoblastic differentiation during reparative dentin formation. The article is straightforward, well written, and concise. The methodology is well described.

The authors characterize the role of Spondin-1 in odontoblastic differentiation in human DPSC culture and in the model to evaluate the formation of reparative molar dentin. Based on the results obtained, the authors conclude that Spondin-1 functions as a negative regulator of odontoblastic differentiation in the formation of reparative dentin.

In my opinion, the article requires a discussion of the results obtained after neutralizing the Spondin-1. In Figure 5 shows that Spondin-1 neutralization causes a stronger mineralization of HDPSCs in the DM+SP group than in the DM group, while the level of the Spondin-1 gene expression (5g) in these groups is the same. There is no discussion of this result in the article.

There are no fundamental comments. The manuscript is well illustrated, definitely deserves to be published, and represents a valuable contribution to the journal ‘Biomolecules’.

The article, after a small revision, can be published in the Biomolecules.

Author Response

General response

We sincerely thank the reviewer for the positive evaluation of our manuscript and for recognizing the novelty and value of our study. We are also grateful for the helpful comment regarding the SPON1 neutralization results. We agree that this point required further clarification, and we have revised the Discussion to explain why SPON1 neutralization enhanced mineralization even though SPON1 mRNA expression was not markedly different between the DM and DM + nSPON1 groups.

Reviewer: 2

Thank you very much for your suggestion.

There are no fundamental comments. The manuscript is well illustrated, definitely deserves to be published, and represents a valuable contribution to the journal ‘Biomolecules’.

The article, after a small revision, can be published in the Biomolecules.

A. We sincerely thank the reviewer for this insightful comment. We agree that this result required further discussion. In the revised Discussion, we added an explanation that the neutralizing antibody is expected to block SPON1 protein activity rather than directly suppress SPON1 transcription. Therefore, enhanced mineralization in the DM + neuSPON1 group may occur even when SPON1 mRNA expression is not markedly different from that in the DM group. This suggests that residual extracellular or cell-associated SPON1 protein may still exert an inhibitory effect during odontoblastic differentiation, and that blocking this activity can further promote mineralization (p16, line 566 - 584 in Discussion).

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

This study investigates the inhibitory role of spondin-1 in odontoblast differentiation. By incorporating additional data, the author has improved the overall quality of the paper.

It is expected to serve as a valuable reference for future research on dentin regeneration.