Effect of Autologous Bioactive Concentrated Growth Factor and Residual Dental Pulp on Dentin–Pulp Complex Regeneration

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates the regenerative potential of autologous concentrated growth factor when combined with residual dental pulp tissue in immature dog teeth. The topic is clinically relevant and updatedy, particularly in the field of regenerative endodontics. The study is well designed, and presented data support authors conclusions.

Nevertheless, some minor issues should be addressed: 

1. The novelty of the technique should be more clearly emphasized in the introduction and discussion sections, underninging the existing gap in knowledge, the new insight that this study provides and deepening the biological effects of CGF + residual pulp. Especially about this last point has to be explained giving more information about the possible mechanism at the base of tissue regeneration.

2. Referring to group III description, the authors state that "pulp tissue is removed, yet residual pulp (1–4 mm) remains": please, describe how the method/operator to leave the same amount of residual pulp has been standardized. On the same idea, specify if the hystological analysis has been performed with blinded operator or not. 

3. The conclusion that CGF + residual pulp results in “true regeneration” should be softenened, Just as example "pulp-like tissue closely resembling normal pulp"

Author Response

 

 

Response to Reviewer 1 Comments

 We sincerely thank the reviewer for the insightful and constructive comments, which have helped us to improve the clarity, scientific depth, and rigor of the manuscript. All comments have been carefully addressed, and the manuscript has been revised accordingly. Our detailed responses are provided below.

Comment 1:

The novelty of the technique should be more clearly emphasized in the introduction and discussion sections, underlining the existing gap in knowledge, the new insight that this study provides, and deepening the biological effects of CGF + residual pulp, particularly regarding the possible mechanisms underlying tissue regeneration.

Response 1:  We appreciate your valuable comment. To address it, we have substantially revised both the Introduction and Discussion sections to more clearly emphasize the novelty of the proposed approach and its biological significance.

Specifically:

• We have highlighted the existing gap in knowledge, namely that most regenerative endodontic strategies rely solely on cell homing from apical tissues using blood clot or platelet concentrates, while the biological contribution of deliberately preserved residual pulp tissue remains insufficiently investigated.
• We clarified that the novelty of the present study lies in the intentional combination of autologous CGF with a standardized amount of residual vital pulp (1–4 mm), aiming to exploit both the scaffold-driven regenerative potential of CGF and the intrinsic biological signaling capacity of remaining pulp tissue.
• The Discussion now includes a deeper mechanistic interpretation, proposing that residual pulp acts as a native reservoir of dental pulp stem cells, neurovascular elements, and endogenous growth factors, while CGF provides a mechanically stable fibrin scaffold with sustained release of bioactive molecules (e.g., PDGF, VEGF, TGF-β). The interaction between these two components likely enhances angiogenesis, odontoblastic differentiation, collagen maturation, and organized pulp-like tissue formation.

These revisions strengthen the conceptual framework of the study and better define the new biological insight provided by our findings.

Location of revision:

• Page: 2, 9
• Paragraph: 5 in page 2, 2 in page 9
• Lines: 83-89, 282-287

Comment 2:

Regarding Group III, the authors state that pulp tissue is removed, yet residual pulp (1–4 mm) remains. Please describe how the method/operator to leave the same amount of residual pulp was standardized. Also, specify whether the histological analysis was performed by a blinded operator.

Response 2:
Thank you for highlighting this important methodological point. The Materials and Methods section has been revised to clarify both aspects.

• Standardization of residual pulp length:

The amount of residual pulp tissue (1–4 mm) was standardized by controlled mechanical removal using large K-files under radiographic guidance. Canal enlargement was performed to a predetermined working length measured from the radiographic apex, ensuring that instrumentation stopped consistently 1–4 mm short of the apex in all Group III specimens. The same experienced operator performed all procedures using identical instruments and protocols to minimize operator-related variability.

• Blinding of histological analysis:

We have clarified that the histological and histomorphometry evaluations were performed by an experienced examiner who was blinded to group allocation, thereby reducing observational bias.

These clarifications have now been explicitly stated in the revised manuscript.

Location of revision:

• Page: 4, 5
• Paragraph: 4 in page 4, 1 in page 5
• Lines: 149-156, 182-186

Comment 3:

The conclusion that CGF + residual pulp results in “true regeneration” should be softened, for example, to “pulp-like tissue closely resembling normal pulp.”

Response3:
We agree with the reviewer’s suggestion and appreciate this important point regarding scientific precision. To avoid overstatement, the language in the Conclusions section has been carefully revised.

The revised text:

In this preclinical descriptive study, the combination of CGF with 1–4 mm of residual pulp resulted in enhanced structural tissue characteristics and higher collagen density compared to the blood clot group. While these results are promising for dentin–pulp complex reconstitution within this experimental model, further clinical trials are required to determine functional effectiveness and clinical applicability.

This revised wording more appropriately reflects the histological and histomorphometric findings while acknowledging the limitations inherent to preclinical animal studies.

Location of revision:

• Page: 10
• Paragraph: 1
• Lines: 327-337

Once again, we thank the reviewer for these insightful comments, which have significantly strengthened the scientific quality and clarity of the manuscript.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

  Abstract

 

1. The abstract states that 30 teeth were included but does not clarify that these teeth were obtained from only four animals. This information is essential, as it affects data independence and interpretation. The abstract should explicitly indicate that multiple teeth were harvested from the same animals.
2. 2. The expression “most favorable outcomes” and the reference to “normal pulp-like tissue” are evaluative and imply equivalence to native pulp. Given the descriptive histological nature of the study, these phrases should be replaced with neutral, descriptive terminology.
3. The statement that the combination “demonstrated a synergistic effect” is not supported by the study design, as no factorial or interaction analysis was performed. Causal and mechanistic terminology should therefore be replaced with associative language.
4. The phrase “promising approach for dentin–pulp complex regeneration” implies clinical applicability. Given the preclinical animal model and descriptive outcomes, such statements should be explicitly restricted to the experimental context.

 

Introduction

 

1. The Introduction provides a broad overview of dentin–pulp biology and regenerative endodontics; however, the specific knowledge gap addressed by the present study should be defined more explicitly and earlier in the section to better frame the rationale of the investigation.
2. The description of concentrated growth factor (CGF) emphasizes its biological advantages; however, clearer distinction between established evidence and hypothetical benefits within the context of the present experimental model would improve the scientific balance of the Introduction.
3. Although the study aim is clearly stated, its wording would benefit from closer alignment with the descriptive histological and histomorphometric nature of the applied methodology.

 

Materials and Methods

 

1. The study includes multiple teeth obtained from the same animals; however, the statistical analysis treats each tooth as an independent observation. This clustering at the animal level should be explicitly acknowledged and its implications for data interpretation clearly stated.

 

2. Histomorphometric assessment is limited to the percentage of collagen-positive area. The methodological section should clarify the descriptive nature of this outcome and avoid implying functional or biological equivalence beyond structural tissue characteristics.

 

Results

 

1. The Results are presented at the tooth level without indicating that multiple measurements originate from the same animals. This should be explicitly acknowledged in the Results section to avoid overinterpretation of statistical independence.
2. Several expressions used in the Results section (e.g., “regenerated”, “well-organized”, “resembled normal pulp”) imply interpretative conclusions. The Results should remain strictly descriptive, with such interpretations reserved for the Discussion.

 

Discussion

 

1. The Discussion employs causal and mechanistic language (e.g., “synergistic effect”, “accounts for”, “resulting in odontoblastic differentiation”) that exceeds the level of evidence provided by descriptive histology and histomorphometric analysis. Such statements should be reformulated using associative, non-causal terminology.
2. Histological similarity and increased collagen deposition are interpreted as functional dentin–pulp regeneration. Given the absence of functional or long-term outcome measures, such interpretations should be clearly limited.
3. The Discussion would benefit from a more explicit acknowledgment of key methodological limitations, including the small number of animals, clustering of samples at the animal level, and the preclinical nature of the model, to appropriately contextualize the findings.

 

Conclusions

 The Conclusions reiterate causal and functional claims that extend beyond the level of evidence provided by this preclinical, descriptive study. The conclusions should be reformulated to strictly reflect the reported results without implying functional regeneration or clinical effectiveness.

 

Author Response

We sincerely thank the reviewer for the detailed, thoughtful, and methodologically rigorous comments. We appreciate the emphasis on precision in interpretation, statistical transparency, and appropriate framing of preclinical findings. All comments have been carefully addressed, and the manuscript has been revised accordingly. Our responses are provided point by point below.

Abstract

Comment 1:

The abstract states that 30 teeth were included but does not clarify that these teeth were obtained from only four animals. This information is essential, as it affects data independence and interpretation.

Response1:
Thank you for highlighting this point. The Abstract has been revised to explicitly state that the 30 immature teeth were obtained from four animals, and that multiple teeth were harvested from the same experimental subjects. This clarification ensures transparency regarding sample clustering and data interpretation.

• Revised Sample Description: "Thirty immature anterior and premolar teeth, harvested from four dogs, were randomly assigned to three groups...".

Location of revision:

• Page: 1
• Paragraph: 1 in the abstract
• Lines: 15-16

Comment 2

The expressions “most favorable outcomes” and “normal pulp-like tissue” are evaluative and imply equivalence to native pulp. These should be replaced with neutral, descriptive terminology.

Response2:
Thank you for highlighting this point. We have revised the Abstract to replace evaluative and comparative language with strictly descriptive terminology. Phrases implying equivalence to native pulp have been removed and replaced with neutral descriptions reflecting histological appearance only.

• Neutral Results: "The CGF-treated group exhibited newly formed tissue with morphological characteristics comparable to the negative control group after partial pulp removal".

Location of revision:

• Page: 1
• Paragraph: 1 in the abstract
• Lines: 21-23

Comment 3:

The statement that the combination “demonstrated a synergistic effect” is not supported by the study design and should be replaced with associative language.

Response3:
We fully agree. The term “synergistic effect” has been removed from the Abstract and replaced with non-causal, associative language describing the observed coexistence of CGF placement and residual pulp tissue in relation to histological findings, without implying interaction or causality.

• Associative Language: Replace "demonstrated a synergistic effect" with: "The combination of CGF with 1–4 mm of residual pulp was associated with enhanced tissue organization".

Location of revision:

• Page: 1
• Paragraph: 1 in the abstract
• Lines: 23-24

Comment 4:

The phrase “promising approach for dentin–pulp complex regeneration” implies clinical applicability and should be restricted to the experimental context.

Response4:
This comment is well taken. The Abstract has been revised to explicitly restrict conclusions to the preclinical experimental setting, avoiding any implication of direct clinical applicability.

• Associative Conclusion: The combination of CGF with 1–4 mm of residual pulp was associated with enhanced tissue organization, representing a promising approach for dentin–pulp complex regeneration within this experimental context

The phrase "promising approach" is now explicitly restricted to the "experimental context" of this preclinical model.

Location of revision:

• Page: 1
• Paragraph: 1 in the abstract
• Lines: 25-26

Introduction

Comment 1:

The specific knowledge gap addressed by the present study should be defined more explicitly and earlier in the Introduction.

Response1:
We appreciate this valuable comment. To address it, we have substantially revised both the Introduction section to more clearly emphasize the novelty of the proposed approach and its biological significance.

Specifically:

• We have highlighted the existing gap in knowledge, namely that most regenerative endodontic strategies rely solely on cell homing from apical tissues using blood clot or platelet concentrates, while the biological contribution of deliberately preserved residual pulp tissue remains insufficiently investigated.
• We clarified that the novelty of the present study lies in the intentional combination of autologous CGF with a standardized amount of residual vital pulp (1–4 mm), aiming to exploit both the scaffold-driven regenerative potential of CGF and the intrinsic biological signaling capacity of remaining pulp tissue.

These revisions strengthen the conceptual framework of the study and better define the new biological insight provided by our findings.

Location of revision:

• Page: 2
• Paragraph: 5 in page 2
• Lines: 85-89

Comment 2:

Clearer distinction between established evidence and hypothetical benefits of CGF is needed.

Response2:
We agree and have revised the CGF-related paragraphs to clearly distinguish between:

• Established evidence derived from previous experimental and clinical studies, and
• Hypothetical or proposed biological effects relevant to the present experimental model.

Speculative statements have been appropriately qualified.

• Evidence vs. Hypothesis: "CGF is established as a rich source of growth factors in surgical healing; however, its hypothetical benefits in facilitating specific odontoblastic differentiation within the pulp chamber require further preclinical validation".

Location of revision:

• Page: 2
• Paragraph: 4 in the page 2
• Lines: 80-82

Comment 3:

The study aim should be better aligned with the descriptive histological and histomorphometric methodology.

Response3:
The study aim has been rephrased to emphasize its descriptive and exploratory nature, focusing on histological and histomorphometric evaluation rather than implying functional or regenerative outcomes.

• Aim Alignment: Refine the aim: "This study aimed to characterize the histological and histomorphometric features of tissue formed after the application of autologous CGF combined with residual pulp".

Location of revision:

• Page:2
• Paragraph: 5 in page 2
• Lines: 89-91

Materials and Methods

Comment 1:

Clustering of multiple teeth within the same animals should be explicitly acknowledged, as statistical analysis treats each tooth as independent.

Response1:
We agree. The Statistical Analysis subsection has been revised to explicitly acknowledge that multiple teeth were obtained from the same animals and that analyses were performed at the tooth level. The potential implications of sample clustering and limited independence have been clearly stated to guide cautious interpretation of the findings.

Clustering Acknowledgment: "Because multiple teeth were obtained from each of the four animals, the potential for intra-animal correlation (clustering) exists. While statistical analysis treated each tooth as an independent observation (n=30), this limitation should be considered when interpreting the data's generalizability".

Location of revision:

• Page: 5
• Paragraph: 2
• Lines: 195-198

Comment 2

The histomorphometric outcome should be clarified as descriptive and structural rather than functional.

Response2:
This clarification has been incorporated. The Methods section now explicitly states that the histomorphometric assessment of collagen-positive area reflects structural tissue characteristics only and does not imply functional equivalence or biological performance beyond morphological description.

• Histomorphometric Clarification: Clarify the outcome: "Histomorphometric assessment of the percentage of collagen-positive area was performed to provide a descriptive measure of structural tissue density and should not be interpreted as a measure of functional pulp recovery".

Location of revision:

• Page: 5
• Paragraph: 1 in page 5
• Lines: 183-185

Results

Comment 1:

Results are presented at the tooth level without acknowledging clustering at the animal level.

Response1:
The Results section has been revised to explicitly state that measurements were obtained from multiple teeth within the same animals and that data are reported at the tooth level. This clarification prevents misinterpretation of statistical independence.

• Acknowledge Sample Source: Begin the results with: "Histological findings from 30 teeth across four animals revealed distinct patterns...".

Location of revision:

• Page: 5
• Paragraph: 3 in page 5
• Lines: 202

Comment 2

Interpretative expressions (e.g., “regenerated”, “resembled normal pulp”) should be avoided in the Results section.

Response2:
The Results section has been carefully edited to remove interpretative or inferential language. Descriptions are now strictly morphological and observational, with interpretative statements reserved exclusively for the Discussion.

• Descriptive Terminology:
• Replace "regenerated" with "newly formed".
• Replace "well-organized" and "resembled normal pulp" with densely packed tissue that resembles Group I in morphology, and more densely arranged similar to the collagen fibers morphology in group I.

Location of revision:

• Page:  7, 8
• Paragraph: 2, 3, 4in page 7, and 1 in page 8
• Lines: 242, 246, 253,258,259

Discussion

Comment 1:

Causal and mechanistic language exceeds the level of evidence provided by descriptive histology.

Response1:
We agree and have revised the Discussion to replace causal or mechanistic language with associative, non-causal phrasing. Statements implying direct biological mechanisms or cause–effect relationships have been reformulated to reflect observed associations only.

• Non-Causal Language: Replace "synergistic effect" and "resulting in" with associative terms: "The concomitant presence of CGF and residual pulp correlated with improved tissue structure... This may be associated with the sustained release of growth factors".

• Causal to Associative Language:
• Changed "synergistic effect" to "concomitant positive association".
• Changed "accounts for" to "is consistent with".
• Changed "resulting in odontoblastic differentiation" to promoting the morphological characteristics of odontoblastic differentiation

Location of revision:

• Page:  9
• Paragraph: 2, 3 in page 9
• Lines: 289, 295- 297.

Comment 2:

Histological similarity and collagen deposition should not be interpreted as functional regeneration.

Response2:
This important point has been fully addressed. Interpretations in the Discussion have been explicitly limited to histological resemblance and structural organization, without implying functional dentin–pulp regeneration or long-term biological performance.

• Functional Limitations: Explicitly limit interpretations: "While histological similarity and increased collagen deposition were observed, these structural findings are not proof of functional dentin-pulp regeneration, as no long-term clinical or physiological markers were assessed".

Location of revision:

• Page: 9
• Paragraph: 5 in page 9
• Lines: 314-316

Comment 3:

Key methodological limitations should be more explicitly acknowledged.

Response3:
The Discussion now includes a dedicated paragraph explicitly acknowledging major limitations, including:

• The small number of experimental animals
• Clustering of samples at the animal level
• The descriptive nature of the outcomes
• The preclinical animal model context

These limitations are clearly stated to appropriately contextualize the findings.

• Methodological Limitations: "Key limitations include the small number of animals (n=4), the clustering of samples at the animal level, and the preclinical nature of the model, which may not fully replicate human clinical healing".

Location of revision:

• Page: 9
• Paragraph: 7 in page 9
• Lines: 325-327

Conclusions

Comment1:

The Conclusions extend beyond the level of evidence and should be reformulated to avoid functional or clinical claims.

Response1:
We fully agree. The Conclusions have been carefully rewritten to strictly reflect the reported histological and histomorphometric findings. All causal, functional, and clinical implications have been removed, and conclusions are now explicitly limited to descriptive outcomes observed within this preclinical experimental model.

• Revised Statement:

In this preclinical descriptive study, the combination of CGF with 1–4 mm of residual pulp resulted in enhanced structural tissue characteristics and higher collagen density compared to the blood clot group.

Location of revision:

• Page: 10
• Paragraph: 1 in page 10
• Lines: 332-334

We sincerely thank the reviewer for these rigorous and constructive comments, which have substantially improved the scientific clarity, methodological transparency, and interpretative balance of the manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

all comments are in the file

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 3 Comments

Introduction

 Comment 1:

Add references for this paragraph: "CGF, first described by Sacco in 2006, is an autologous platelet concentrate obtained by variable-speed centrifugation of venous blood without anticoagulants. Compared with PRP and PRF, CGF yields a more stable fibrin scaffold capable of entrapping higher concentrations of bioactive molecules and progenitor cells.

Response 1:

 We thank the reviewer for this suggestion. We have incorporated the following references:

11. Sacco L Lecture. International academy of implant prosthesis and osteoconnection. Lecture. 2006; 12:4.
12. Rodella LF, Favero G, Boninsegna R, Buffoli B, Labanca M, Scarì G, et al. Growth factors, CD34 positive cells, and fibrin network analysis in concentrated growth factors fraction. Microsc Res Tech. 2011;74(8):772–7.
13. Chen L, Cheng J, Cai Y, Zhang J, Yin X, Luan Q. Efficacy of concentrated growth factor (CGF) in the surgical treatment of oral diseases: a systematic review and meta-analysis. BMC Oral Health. 2023 Oct 4;23(1):712.

 

14. Mozzati M, Gallesio G, Tumedei M, Del Fabbro M. Concentrated growth factors vs. leukocyte-and-Platelet-Rich fibrin for enhancing post extraction socket healing. a longitudinal comparative study. Applied Sciences. 2020 Nov 20;10(22):8256.
15. Bharti R, Tikku AP, Verma P, Yadav RK, Pant AB. Effect of platelet-rich fibrin and concentrated growth factor on the regenerative potential of human-induced pluripotent stem cells: A comparative analysis. Journal of Conservative Dentistry and Endodontics. 2024 Sep 1;27(9):975-82.

Location of revision:

• Page: 2
• Paragraph: 4 in page 2
• Lines: 73-76

 

Comment 2:

What is the null hypothesis

Therefore, the present study aimed to evaluate the regenerative potential of autologous CGF combined with residual pulp tissue in immature dog teeth, using histological and histomorphometric evaluation.

Response 2:

We thank the reviewer for this clarification.

Inserted text:

The null hypothesis of the present study was that the application of autologous concentrated growth factor (CGF) combined with residual pulp tissue would not result in significantly different histological or histomorphometric regenerative outcomes in immature dog teeth compared with the control treatment.

Location of revision:

• Page: 2,3
• Paragraph: 5 in page 2 and 1 in page 3
• Lines: 91-94

 

Procedure

Comment 1:

Who was conducting the clinical procedure?

Response1:
We thank the reviewer for this important comment. The manuscript has been revised to clarify that all endodontic procedures were performed by a single experienced endodontist to ensure procedural standardization and to minimize operator-related variability.

Inserted text:

All endodontic procedures were performed by a single experienced endodontist under standardized aseptic conditions to minimize operator-related variability.

Location of revision:

• Page: 4
• Paragraph: 2
• Lines: 139–140

Comment 2:

What is the size of the file used?

Response2:
We appreciate the reviewer’s request for clarification. The file sizes used for canal enlargement have now been explicitly stated in the Materials and Methods section.

Inserted text:

Canals were enlarged using large stainless-steel K-files up to sizes 100–120 to 1–4 mm short of the radiographic apex.

Location of revision:

• Page: 4
• Paragraph: 4 in page 4
• Lines: 149–151

Comment 3:

MTA requires moisture to set; usually a wet sterile cotton swab is placed and temporary material for 24 h. Please clarify.

Response 3:
We thank the reviewer for this valuable technical comment. The manuscript has been revised to clearly describe the moisture-controlled setting protocol used for MTA placement.

Inserted text:

After MTA placement, a sterile saline-moistened cotton pellet was placed over the material to provide the moisture required for proper setting, and the access cavity was temporarily sealed before final restoration.

Location of revision:

• Page: 4
• Paragraph: 5
• Lines: 159-162

 

 

Discussion

Comment 1

Discuss whether the null hypothesis was discussed or rejected.

Response 1:
We agree with the reviewer and have revised the Discussion section to explicitly state whether the null hypothesis was supported or rejected based on the study findings.

Inserted text:

Based on the statistically significant histological and histomorphometric differences observed between the CGF-treated group and the control groups, the null hypothesis was rejected. The findings indicate that the combination of CGF with residual pulp tissue positively influences dentin–pulp complex regeneration compared with blood clot regeneration or no treatment.

Location of revision:

• Page: 8,9
• Paragraph: 4 in page 8. 1 in page 9
• Lines: 277–281

Comment 3:

Add references to this paragraph:

Previous vivo studies demonstrated…………

Response 3:

We thank the reviewer for this suggestion. We have incorporated the following references:

22. Torabinejad M, Alexander A, Vahdati S A, Grandhi A, Baylink D, Shabahang S. Effect of Residual Dental Pulp Tissue on Regeneration of Dentin-pulp Complex: An In Vivo Investigation. J Endod. 2018; 44:1796-801.
23. Astudillo-Ortiz E, Babo PS, Sunde PT, Galler KM, Gomez-Florit M, Gomes ME. Endodontic tissue regeneration: a review for tissue engineers and dentists. Tissue Engineering Part B: Reviews. 2023 Oct 1;29(5):491-513.

Location of revision:

• Page: 9
• Paragraph: 4 in page 9
• Lines: 302

 

Conclusion

Comment 1:

 Add clinical Significance.

Response1:
We thank the reviewer for this constructive suggestion. A clinical significance statement has now been added to the Conclusions section.

 Inserted text:

The clinical significance of this study lies in the shift from "repair" to "functional regeneration." By preserving even minimal residual pulp (1–4 mm), clinicians can leverage native "biological signaling centers" to achieve better outcomes than traditional cell-homing techniques.

Location of revision:

• Page: 10
• Paragraph: 1 in page 10
• Lines: 328-331

Comment 2:

Could you add some more limitations of this study?

Response 2:
We appreciate the reviewer’s insightful comment. A paragraph outlining the limitations of the study has now been included.

Inserted text:

While these results are promising for dentin–pulp complex reconstitution within this experimental model, further studies with longer follow-up periods and clinical trials are required to determine functional effectiveness and clinical applicability.

Location of revision:

• Page: 10
• Paragraph: 1
• Lines: 333–336

 

Reference

Comment 7:

 Very good and up to date.

• Response 7:

We sincerely thank the reviewer for this positive and encouraging comment. We have ensured that all the references represent the most recent and relevant high-impact studies in the field of regenerative endodontics.

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have adequately addressed the main methodological and interpretative concerns. Minor language polishing is recommended to further limit causal phrasing.

Author Response

 

 

Point 1: Introduction, Background, and References

Reviewer Comment: Does the introduction provide sufficient background and include all relevant references? (Can be improved). The introduction provides a solid foundational understanding but requires more depth regarding the specific molecular signaling of CGF and the biological rationale for using residual pulp as a signaling center.

Author Response: We thank the reviewer for this constructive feedback. We have significantly expanded the Introduction to incorporate the molecular signaling pathways of Concentrated Growth Factor (CGF). Specifically, we have added details on the release kinetics of Vascular Endothelial Growth Factor (VEGF) and Transforming Growth Factor-beta 1 (TGF-β1). We have also refined the concept of "residual pulp" as a "biological signaling center" that provides the endogenous cues necessary for stem cell recruitment and differentiation.

Revised Manuscript Text:

Compared with PRP and PRF, CGF yields a more fibrin scaffold capable of entrapping higher concentrations of bioactive molecules [Vascular Endothelial Growth Factor (VEGF) and Transforming Growth Factor-beta 1 (TGF-β1)] and progenitor cells. These bioactive molecules are released more gradually, stimulating cellular proliferation and supporting predictable tissue repair [11-15]. The dense fibrin matrix of CGF allows for a sustained release of these molecules, which is critical for the recruitment of dental pulp stem cells (DPSCs) and the promotion of angiogenesis [16,17]. By preserving 1–4 mm of residual pulp, this study utilizes these tissues as 'biological signaling centers', providing the necessary neurovascular cues and native stem cell niches required to guide the differentiation of odontoblast-like cells [18].

Location of revision:

• Page: 2
• Paragraph: 4
• Lines: 77- 86

Point 2: Conclusions and Support of Results

Reviewer Comment: Are the conclusions supported by the results? (Must be improved). The current conclusions regarding "functional regeneration" must be moderated. The results demonstrate structural and histomorphometric similarity, but histological data alone cannot confirm "functional" recovery.

Author Response: We agree with the reviewer’s assessment that histological and histomorphometric data (collagen-positive area %) describe structural outcomes rather than functional ones. We have revised the manuscript to replace the term "functional regeneration" with "structural reconstitution" or "structural tissue organization." We have also included a statement in the Discussion and Conclusion acknowledging that while the morphological results are promising, physiological vitality requires further validation.

Location of revision:

• Page: 10
• Paragraph: 1
• Lines: 335-347

Revised Manuscript Text (Conclusion Section):

"In this preclinical descriptive study, the combination of autologous CGF with 1–4 mm of residual pulp was associated with enhanced structural tissue organization and a collagen density percentage (52.49% ± 4.70) that was statistically comparable to native pulp (56.48% ± 4.88). These findings suggest that CGF facilitates a favorable environment for the structural reconstitution of the dentin–pulp complex. While the presence of organized odontoblast-like cells is a promising morphological indicator, these results do not constitute proof of physiological vitality or sensory function. Future clinical trials are essential to validate the long-term functional effectiveness and clinical applicability of this approach in human subjects."

Point 3: Language Polishing and Causal Phrasing

Reviewer Comment: Minor language polishing is recommended to further limit causal phrasing. The authors should use more conservative, scholarly language to describe the relationship between the experimental intervention and the observed outcomes.

Author Response: In accordance with the reviewer’s recommendation, we have performed a comprehensive linguistic revision. Definitive causal verbs such as "achieved," "produced," or "caused" have been replaced with associative or probabilistic terms such as "was associated with," "potentially facilitated," and "suggests." Furthermore, we have ensured the consistent use of the term "odontoblast-like cells" to accurately describe the newly formed cells in the experimental group.

Revised Manuscript Text (Discussion Snippet):

The histomorphometric analysis revealed that the CGF group exhibited characteristics associated with advanced matrix maturation. The dense fibrin network potentially facilitated a stable scaffold for cell migration and collagen deposition. This suggests a synergistic effect between the sustained growth factor release from the CGF and the native signaling molecules provided by the residual pulp. While the results point toward a more predictable morphological outcome compared to blood clot induction, the lack of electrophysiological data remains a limitation for confirming total biological recovery [17,32].

Location of revision:

• Page: 9
• Paragraph: 5
• Lines: 315-322