Review Reports - Polydeoxyribonucleotide (PDRN) Selectively Promotes Osteoblast Differentiation Without Affecting Osteoclastogenesis

Round 1

Reviewer 1 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

The authors submitted a revised manuscript, which differs from the original one for the better. However, there are a number of issues and comments.

1 Lines 144-147 - The sentenses should be removed.

2 The abstract should be aligned with the amended main text of the manuscript.

3 Figures 2 and 3 are still small. The axis signatures of some graphs are unreadable.

4 The authors explain the decrease in formazan production in the MTS test by ‘proliferation–differentiation switch’. However, to confirm this assumption, it would be advisable to conduct additional experiments, for example, with direct cell counting, cell cycle investigation and others. Moreover, the authors themselves admit the discrepancy between their data and the available literature data.

5 The authors discussed that "our findings imply that PDRN triggers this switch via a specific functional interaction between adenosine signaling and osteogenic pathways." (Lines 186-194 and 212-). However, there is no experimental evidence to support this reasoning in this manuscript.

Even if other studies have shown a link between the A2AR receptor and Ranx2, there is no convincing evidence that this is the only way. Cellular machinery is a very intricate and complex network of interactions, and the same effector molecules can be under the control of different receptors. Receptor pathways can intersect and transmit a signal in a non-canonical way.

Thus, with the available experimental data, one can only assume that this pathway may be involved, and the statement "PDRN-mediated A2AR activation robustly promoted osteogenic differentiation markers (Runx2, ALP) and mineralization." (Lines 218-2019) has no evidence.

The authors state "the proposed A2AR-mediated mechanism is supported by literature, direct validation using pharmacological inhibitors was not performed in this study." (Lines 274-275). However, this statement has no reference to the article. I can only assume that the authors are referring to reference 12, which they indicated in the Introduction. This review (https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2017.00224/full) contains two links to relevant publications. However, data on the effect on the adenosine receptor were obtained only in a competitive test using other cells, and they cannot be considered convincing evidence that the adenosine receptor was also involved in PDRN effects this study.

6 The conclusions also assert the effect of PDRN on the cell proliferation, which is not confirmed in the manuscript.

Thus, it is necessary to carefully bring the conclusions and discuccions in line with the experimental data obtained and the well-known literary facts.

Author Response

The authors submitted a revised manuscript, which differs from the original one for the better. However, there are a number of issues and comments.

Response: We sincerely appreciate your insightful review and constructive suggestions to improve our manuscript. We have carefully addressed your concerns by incorporating new experimental data (direct cell counting and extended viability assays) and refining the text to ensure full consistency between the Abstract and the main text."

1 . Lines 144-147 - The sentenses should be removed.

Response: As suggested, we have removed the indicated sentences from the manuscript.

2. The abstract should be aligned with the amended main text of the manuscript.

Response: We agree that consistency throughout the manuscript is cruial. We have revised the Abstract to fully align with the amended main text. Specifically:

We moderated the conclusion regarding the molecular mechanism (changing "confirmed" to "suggested"). (Page 1 lines 24)
We updated the statement on cell proliferation to accurately reflect our new direct counting data, clarifying that PDRN induces a "metabolic shift" rather than simple toxicity. (Page 1 lines 21-23)

3. Figures 2 and 3 are still small. The axis signatures of some graphs are unreadable.

Response: We apologize for the poor visibility in the previous version. In the revised manuscript, we have:

Resizedall graphs in Figure 2 and 3 to be larger and clearer.
Significantlyincreased the font size of all axis labels, legends, and significance markers to ensure they are easily readable even when printed.

4. The authors explain the decrease in formazan production in the MTS test by ‘proliferation–differentiation switch’. However, to confirm this assumption, it would be advisable to conduct additional experiments, for example, with direct cell counting, cell cycle investigation and others. Moreover, the authors themselves admit the discrepancy between their data and the available literature data.

Response: We agree with your insightful comment that the MTS assay primarily measures metabolic activity and is insufficient to act as a sole proxy for proliferation. To address this and confirm our hypothesis, we performed the suggested additional experiments:

Direct Cell Counting (Added to Figure 1a):We performed direct nuclei and cell counting. As shown in the revised Figure 1a (middle and lower panels), the results confirmed a significant reduction in cell numbers in PDRN-treated groups at 24 hours, proving that the effect involves growth arrest.
Extended Viability Assay (Added to Figure S1):To ensure this was a regulatory "switch" and not permanent toxicity, we performed an extended time-course MTS assay. The results (Figure S1) showed that metabolic activity recovered to control levels by 48 hours, confirming that cells remained viable. These combined data (reduced number but preserved viability) strongly support the 'proliferation-differentiation switch' hypothesis. We have updated the Results(Section 2.1) and Discussion to reflect these findings accurately. (Page 3 lines 166-168, Page 6 lines348-350)

5. The authors discussed that "our findings imply that PDRN triggers this switch via a specific functional interaction between adenosine signaling and osteogenic pathways." (Lines 186-194 and 212-). However, there is no experimental evidence to support this reasoning in this manuscript.

Response: We accept the reviewer’s critical assessment regarding the mechanistic explanation. We agree that stating " activation" as a proven fact without direct pharmacological validation (e.g., inhibitor studies) in our specific model is premature.

To strictly align our claims with the experimental data, we have made the following revisions:

Refining causal interpretation:
- Original (Criticized):"In this specific cellular context, PDRN-mediated A2AR activation robustly promoted..."
- Revised (Page 8 Lines 487-488):"In this specific cellular context, PDRN treatment robustly promoted..."
- We have removed the phrase "A_2AR activation" from this sentence to describe the result purely based on the treatment administered, not the assumed receptor mechanism.
Toning Down the Discussion:
- We have rephrased the mechanistic discussion to present thepathway as a "plausible hypothesis" or "suggested model" supported by literature consistency, rather than a proven conclusion of this study.
- We explicitly acknowledged in theLimitations section (Page 9 Lines 563-569) that definitive confirmation requires future antagonist studies.

We believe these changes accurately reflect the scope of our data, distinguishing clearly between the observed anabolic phenotype and the hypothesized molecular pathway.

6. The conclusions also assert the effect of PDRN on the cell proliferation, which is not confirmed in the manuscript.

Thus, it is necessary to carefully bring the conclusions and discuccions in line with the experimental data obtained and the well-known literary facts.

Response: We sincerely appreciate this constructive comment. We agree that drawing conclusions about "cell proliferation" based solely on metabolic assays (MTS) is insufficient. To align our conclusions with the experimental facts, we have taken the following actions:

Experimental Confirmation (Direct Cell Counting):Base on the new Direct Nuclei and Cell Counting (Figure 1a), we confirmed a significant reduction in cell number at 24 hours.
Viability Check (Figure S1):To ensure this reduction was not due to cytotoxicity, we performed an extended time-course MTS assay (Figure S1). The results showed that metabolic activity recovered to control levels by 48 hours, confirming that the cells remained viable but underwent a temporary growth arrest.
Refined Conclusion: Based on thisconfirmed decrease in cell number, we have revised the Conclusion. Instead of claiming a generic "effect on proliferation," we now specifically describe the finding as "growth arrest (reduced cell number) consistent with cellular differentiation." (Page 12 Lines 725-726). We explicitly cite literature (Refs 25-27) stating that osteoblasts typically exit the cell cycle to initiate matrix synthesis, framing our result as a phenotypic alignment with this well-known biological principle rather than a proven mechanistic discovery. (Page 6 Lines 353-355).

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The authors have addressed all comments and recommendations; therefore, the manuscript can be accepted.

In line 193, change “Wnt/β-signaling” to “Wnt/β-catenin.”

Author Response

The authors have addressed all comments and recommendations; therefore, the manuscript can be accepted.

In line 193, change “Wnt/β-signaling” to “Wnt/β-catenin.”

Response: "We sincerely appreciate your positive evaluation and recommendation for acceptance. We are grateful for your time and effort in reviewing our manuscript.

Regarding your specific correction: As requested, we have corrected the terminology from 'Wnt/β-signaling' to 'Wnt/β-catenin signaling' in the revised manuscript (Page 7, Line 402)."

Reviewer 3 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

Jeon and colleagues investigated how exposure to PDRN may influence osteogenic differentiation and the viability of MC3T3-E1 cells, as well as bone-marrow-derived macrophages. Unfortunately, the study remains incomplete, and the conclusions drawn are not adequately supported by the results presented.

The authors continue to make the same mistake regarding the nomenclature of adenosine receptors; the abbreviations used are incorrect. Greater care and rigor are therefore required when referring to these metabotropic receptors, in accordance with IUPHAR recommendations.
In an attempt to better contextualize the work, the authors added new information about adenosine receptors; however, this information is very incomplete. They still fail to describe the full enzymatic cascade involved in adenosine metabolism, as well as the mechanisms of adenosine release or uptake, as was clearly highlighted in the previous submission.
The rationale for using MC3T3-E1 cells is not provided, and the authors fail to specify passage numbers beyond subclone 10 in their methods. They also did not address the earlier recommendation to use BMMs from the same animal, which would have yielded stronger and more relevant results.
The absence of additional assays to properly assess cell proliferation compromises the authors’ conclusions. The explanation provided regarding the proliferation–differentiation switch remains insufficient. With relatively little additional effort, the authors could clarify this issue. Similarly, the justification for the absence of additional time-points is unconvincing. For instance, ALP activity is a dynamic osteogenic marker and should always be monitored throughout the culture period. Once again, how can the effect of PDRN on proliferation versus differentiation be assessed with such limited data? It must be emphasized that the MTS assay does not measure cell proliferation. To support the authors’ (premature) conclusions, additional experiments are required. In this context, ALP activity would be a more robust indicator, alongside key transcription factors such as Osterix and Runx2.
In Figure 1, regarding the immunofluorescence data, it is unclear what the dots represent. Their number varies considerably between experimental groups. Are these data derived from image fluorescence intensity, individual cells, or another metric? This must be clearly explained. Once again, the criteria for image selection should be described (for example, using predefined coordinates within the well). Random image selection can introduce bias, particularly if images with stronger staining are preferentially chosen. This issue must be explicitly addressed.
An n = 3 is insufficient, regardless of variability. Please consult the bibliography from the previous submission. It is unclear where the authors derived the consensus that n = 3 is widely accepted for in vitro experiments. The experiments must therefore be repeated with a higher number (n=5, or more) of biological replicates to increase confidence in the results and conclusions, in the interest of clarity and scientific rigor.
It remains premature to conclude that A_2A receptors are involved in the effects mediated by PDRN. The authors could have easily included parallel experimental groups using selective adenosine receptor antagonists to determine whether the effects of PDRN were blocked or attenuated. It would also be important to test the effect of these antagonists alone, as these receptors may exhibit tonic activity, particularly considering that these cells may release adenosine or ATP (which is subsequently converted to adenosine by ecto-NTPDases and CD73). Additionally, different time-points should be included, since ALP activity is dynamic during culture as differentiation progresses and mineralization begins.
As previously noted, A_2A receptors have been associated with other mechanisms in osteoblasts and osteoclasts. The authors must critically discuss the existing literature and its apparent contradictions, rather than relying exclusively on data that seem to support their hypothesis.
Including staining for adenosine receptors would have been appropriate, as previously suggested. What effect does PDRN have on receptor expression? This should be investigated.

Final recommendation: The study is not suitable for publication due to missing methodological details, lack of parallel controls, and inadequate statistical analysis. Major revisions and further experiments are needed.

Author Response

Response: "We sincerely appreciate the reviewer’s rigorous and critical assessment. Your detailed critique regarding experimental design, statistical power, and mechanistic validation has been instrumental in significantly improving the quality and scientific accuracy of our manuscript. We have carefully addressed each point to meet the high standards of Marine Drugs."

1. The authors continue to make the same mistake regarding the nomenclature of adenosine receptors; the abbreviations used are incorrect. Greater care and rigor are therefore required when referring to these metabotropic receptors, in accordance with IUPHAR recommendations.

Response: We sincerely appreciate the reviewer’s keen attention to detail regarding scientific rigor. We apologize for the typographical errors and incorrect nomenclature in the previous draft. In the revised manuscript, we have:

Corrected all typos (e.g.,extracellular, diphosphohydrolase).
Strictly adhered toIUPHAR nomenclature by using the correct subscripts for receptor subtypes (e.g., adenosine receptor, A_2AR) throughout the text.

2. In an attempt to better contextualize the work, the authors added new information about adenosine receptors; however, this information is very incomplete. They still fail to describe the full enzymatic cascade involved in adenosine metabolism, as well as the mechanisms of adenosine release or uptake, as was clearly highlighted in the previous submission.
Response: We sincerely apologize for failing to fully address the reviewers’ previous concern regarding the complexity of adenosine metabolism. We agree that a comprehensive description of the full enzymatic cascade and transport mechanisms is essestial to properly contextualize the mechanism of action of PDRN.

In the revised Introduction, we have significantly expanded this section to provide the “complete picture” as requested (Page 1-2, lines 39-82). Specifically, we have added:

Mechanisms of ATP release: Explicitly mentioning Connexin 43 and Pannexin 1 channels, which are critical in osteoblast mechanotransduction.
The full enzymatic cascade: We now include Tissue-Nonspecific Alkaline Phosphates (TNAP) alongside CD39 and CD73, emphasizing its dual role in mineralization and adenosine generation.
Uptake and Intracellular Fate: We clarified the roles of ENTs/CNTs for uptake and the subsequent purine salvage pathway (via Adenosine Kinase and Deaminase)

We believe this rigorous description now provides a. solid theoretical foundation for our study.

3. The rationale for using MC3T3-E1 cells is not provided, and the authors fail to specify passage numbers beyond subclone 10 in their methods. They also did not address the earlier recommendation to use BMMs from the same animal, which would have yielded stronger and more relevant results.

Response: We appreciate the reviewer’s keen observation regarding the cell sources. We acknowledge that using an autologous system is ideal; however, our choice of models was dictated by the distinct physiological limitations and temporal requirements of each assay, rather than an arbitrary selection

Rationale for the Hybrid Model (Stability vs. Fidelity)
- For Osteoblasts (The issue of Long-term Culture): The mineralization assay requires a long-term culture period (usually up to 28 days). Primary murine osteoblasts are notoriously prone to replicative senescence and phenotypic drift (dedifferentiation) during such extended protocols, which compromises the reliability of late-stage data. Therefore, we selected MC3T3-E1 cell line, a validated model that retains stable osteogenic potential over long durations, ensuring reproducible mineralization results.
- For osteoclasts (Short-term Precision): In contrast, osteoclast differentiation is a relatively acute process (3-5 days). For this short timeframe, Primary BMMs provide superior physiological fidelity compared to leukemic cells lines without the concerns of long-term instability.
Genetic Consistency: Furthermore, it is important to note that MC3T3-E1 cells are derived from C57BL/6 mice, which is the same genetic background as the mice used for our BMM isolation. Thus, although the cells were not harvested from the same individual anima, they share a concordant genetic background, minimizing potential discrepancies due to strain differences.

We have clarified this “fit-for-purpose” rationale in the revised Materials and Methods section (4.2. cell culuture, Page 10, lines 617-655).

4. The absence of additional assays to properly assess cell proliferation compromises the authors’ conclusions. The explanation provided regarding the proliferation–differentiation switch remains insufficient. With relatively little additional effort, the authors could clarify this issue. Similarly, the justification for the absence of additional time-points is unconvincing. For instance, ALP activity is a dynamic osteogenic marker and should always be monitored throughout the culture period. Once again, how can the effect of PDRN on proliferation versus differentiation be assessed with such limited data? It must be emphasized that the MTS assay does not measure cell proliferation. To support the authors’ (premature) conclusions, additional experiments are required. In this context, ALP activity would be a more robust indicator, alongside key transcription factors such as Osterix and Runx2.

Response: We sincerely appreciate the reviewers’ insistence on rigorous data interpretation. We fully agree that the MTS assay primarily measures metabolic activity and is insufficient to conclude on proliferation.

Clarification on Proliferation (Direct Counting): Addressing exactly this concern, we did not rely sole on MTS. As shown in the middle and lower panels of Figure 1a, we performed “Direct Nuclei and Cell Counting”. The results clearly show a statistically significant decrease in the absolute number of cells in PDRN-treated groups. Thus, we have direct evidence of suppressed proliferation.
Validation of cell viability (Figure S1 added): To further rule out the possibility that the reduced cell number was due to cytotoxicity, we performed an extended time-course MTS assay (Figure S1). The results confirmed that while metabolic activity was transiently reduced at 24 h, it significantly recovered in all groups by 48 h. This proves that the cells remained viable and capable of growth, supporting our interpretation that the initial arrest is a regulatory event rather than toxicity.
Title Modification (Fact-based): We strongly agree with the reviewer that asserting a direct causal “switch” mechanism without cell cycle analysis might be premature. Accordingly, we have revised the title of Section 2.1 to be purely descriptive: “ PDRN upregulates early osteogenic markers concomitant with reduced cell proliferation.”. This accurately reflects our observed data-specifically the simultaneous reduction in cell number and increase in Runx2-without implying a definitive mechanistic trigger.
Refined Interpretation: In the Discussion, we have tempered our conclusion. Instead of claiming definitive proof, we now state that these findings are “consistent with the phenotypic pattern” of the proliferation-differentiation switch reported in the literature. (Page 6 lines 354-356)

5. In Figure 1, regarding the immunofluorescence data, it is unclear what the dots represent. Their number varies considerably between experimental groups. Are these data derived from image fluorescence intensity, individual cells, or another metric? This must be clearly explained. Once again, the criteria for image selection should be described (for example, using predefined coordinates within the well). Random image selection can introduce bias, particularly if images with stronger staining are preferentially chosen. This issue must be explicitly addressed.

Response: We sincerely appreciate the reviewer’s insistence on data transparency and rigorous image acquisition standards. We apologize that the representation of data points and the selection criteria were not explicitly defined in the previous version.

We have addressed these issues in the revised manuscript as follows:

Clarification of Data Points (The "Dots"):
- We have clarified in theFigure 1 Legend that each dot represents the mean fluorescence intensity of one independent biological replicate (N = 5), not individual cells or technical replicates.
- To calculate this single value, we averaged the intensity from four distinct fields within that replicate. This ensures that the variance shown reflects true biological variation, not intra-sample heterogeneity.
Systematic Image Selection (Anti-bias Strategy):
- We fully agree that random selection can introduce bias. To eliminate the possibility of preferentially selecting areas with stronger staining ("cherry-picking"), we employed aSystematic Sampling Strategy.
- Predefined Coordinates:Images were acquired from four fixed positions (four cardinal points: 3, 6, 9, and 12 o’clock positions) within each well, regardless of the staining appearance.
- Blinding Method:Furthermore, focusing was performed using the DAPI channel (nuclei). This ensured that the investigator was blinded to the target protein's fluorescence intensity during image acquisition.
- This methodology has been explicitly added to theMaterials and Methods section (Section 4.4, Page 11 lines 685-687) and the Figure 1 Legend (Page 4 lines 236-238).

6. An n = 3 is insufficient, regardless of variability. Please consult the bibliography from the previous submission. It is unclear where the authors derived the consensus that n = 3 is widely accepted for in vitro experiments. The experiments must therefore be repeated with a higher number (n=5, or more) of biological replicates to increase confidence in the results and conclusions, in the interest of clarity and scientific rigor.

Response: We treat the reviewer’s concern regarding sample size and statistical rigor with the utmost seriousness. We respectfully submit that the core quantitative conclusions of this study are already supported by high-replicate data (N ≥ 5).

Core Mechanistic Data: Fully Compliant (N = 5): addressing your main concern, the primary quantitative assays driving our conclusions were performed with high biological replicates
- mRNA Expression (Figure 2c): As clearly shown in the revised Figure 2c, qRT-PCR for Osteocalcin, Runx2, and Osterix was performed with five independent biological replicates (N = 5). This unequivocally confirms that PDRN activates the osteogenic genetic program with high statical power.
- Early differentiation (Figure 1): Immunofluorescence and cell counting were also analyzed with N = 5.
Rationale for Phenotypic Assays (N = 3-4): For the functional differentiation assays, we maintained the current sample sizes based on technical feasibility
- ALP activity (Figure 2a): Analyzed with N =4 independent biological replicates
- Mineralization (ARS, Figure 2b): Analyzed with N = 3.
- Justification: The ARS assay requires 28-day long term culture. Due to the technical challenges of maintaining monolayer integrity over this extended period (e.g., risk of cell sheet detachment). We applied stric quality control, resulting in N = 3. However, the results (p < 0.01) are perfectly consistent with the robust mRNA data (N = 5). Since the phenotypic outcomes aligns with the high-power genetic data, we believe the current dataset is scientifically valid without further repetition.
Action on Western Blot (Figure 2d): To strictly adhere to your standards of rigor, we have removed the densitometry bar graphs for Western blotting. We agree that seim-quantitative analysis from representative image ( N = 1 for the plot) could be misleading. We not present images solely as qualitative biochemical confirmation.

7. It remains premature to conclude that A_2A receptors are involved in the effects mediated by PDRN. The authors could have easily included parallel experimental groups using selective adenosine receptor antagonists to determine whether the effects of PDRN were blocked or attenuated. It would also be important to test the effect of these antagonists alone, as these receptors may exhibit tonic activity, particularly considering that these cells may release adenosine or ATP (which is subsequently converted to adenosine by ecto-NTPDases and CD73). Additionally, different time-points should be included, since ALP activity is dynamic during culture as differentiation progresses and mineralization begins.

Response: We appreciate the reviewer’s insightful critique regarding the mechanistic validation and experimental design.

Regarding the A_2AR Antagonist Study:We fully agree with the reviewer that pharmacological blockade using selective antagonists is the gold standard to definitively prove receptor involvement. We acknowledge that without these inhibitor studies, identifying the A_2A receptor (A_2AR)as the sole mediator remains a hypothesis rather than a confirmed conclusion.
However, the primary objective of this study was to elucidate the phenotypic effects of PDRN—specifically its anabolic impact on osteoblasts versus its neutral effect on osteoclasts—to support its clinical potential. Given the complexity of PDRN as a macromolecular mixture (deoxyribonucleotides) rather than a single synthetic ligand, we aimed first to establish its direct cellular efficacy.

To address your valid concern without overstepping our data:

We havetoned down our mechanistic claims throughout the manuscript. We now refer to the pathway as a "plausible mechanistic model" supported by literature, rather than a definitive conclusion of this study. (Page 12, lines 731)
We have explicitly added aLimitations section in the Discussion stating: "Direct validation using pharmacological inhibitors was not performed... Therefore, our conclusion regarding the mechanism relies on the consistency between our observed phenotypes and the well-documented signaling cascades... rather than direct pharmacological blockade." (Page 9 lines 562-568)

Regarding ALP Time-points:We appreciate the suggestion to monitor ALP dynamics. We selectedDay 7 based on the well-established differentiation timeline of MC3T3-E1 cells.

Biological Rationale:In this cell line, ALP activity typically follows a bell-shaped curve, peaking at the early-to-mid stage (Day 7) of differentiation (matrix maturation phase) and subsequently declining as cells transition into the mineralization phase (Day 14-21).
Experimental Design:To capture the full spectrum of differentiation, we employed a complementary approach: measuring ALP at its peak (Day 7) to assess early commitment, and Alizarin Red S at the terminal stage (Day 28) to verify functional mineralization.
Since our Day 7 data showed a robust, dose-dependent increase in enzymatic activity (p < 0.01), and this was successfully translated into terminal mineralization at Day 28, we believe these two pivotal time-points effectively demonstrate the continuous osteogenic progression driven by PDRN.

8. As previously noted, A_2Areceptors have been associated with other mechanisms in osteoblasts and osteoclasts. The authors must critically discuss the existing literature and its apparent contradictions, rather than relying exclusively on data that seem to support their hypothesis.

Response: We appreciate the reviewer’s guidance to provide a balanced and critical view of the existing literature. We agree that the role of adenosine signaling in bone homeostasis is complex and often appears contradictory depending on the experimental model.

To address this, we have ensured that the Discussion section explicitly juxtaposes our findings with conflicting reports and offers a mechanistic explanation for these discrepancies, rather than ignoring them.

Contradictions in Osteoblasts (Proliferation vs. Differentiation):We have explicitly citedCosta et al. (2011), who reported that activation delayed osteogenic differentiation in human bone marrow mesenchymal stem cells (hBM-MSCs).

Our Critical Discussion (Page 7-8 Lines 421-495) : We argue that this contradiction arises from the"differentiation stage." While signaling may favor the maintenance/proliferation of the stem cell pool (as seen in Costa et al.), our data using committed MC3T3-E1 pre-osteoblasts suggests that in lineage-committed cells, the same signal drives terminal differentiation. We propose a "biphasic role" of adenosine signaling to resolve this apparent conflict.

Contradictions in Osteoclasts (Inhibition vs. No Effect):We have also addressed the conflict withMediero et al. (2012), who demonstrated that synthetic agonists strongly inhibit osteoclastogenesis.

Our Critical Discussion (Page 8-9 Lines 522-551:We attribute this difference to "pharmacological kinetics."Unlike potent synthetic agonists (e.g., CGS21680) that forcibly saturate receptors to trigger inhibition, PDRN acts as a pro-drug, releasing physiological levels of adenosine. Our discussion emphasizes that PDRN’s mode of action—providing a sustained, physiological ligand supply—distinctly differs from the supraphysiological blockade observed with synthetic agonists.

We believe this comparative analysis provides the fair and critical discussion requested by the reviewer.

9. Including staining for adenosine receptors would have been appropriate, as previously suggested. What effect does PDRN have on receptor expression? This should be investigated.

Response: We appreciate the reviewer’s suggestion to investigate the expression dynamics of adenosine receptors. We agree that understanding whether PDRN upregulates or downregulates its target receptor is an interesting pharmacological question.

However, we respectfully submit that quantifying receptor expression levels (e.g., via staining) may not directly correlate with signaling efficacy in this specific context, due to the complex kinetics of G-protein coupled receptors (GPCRs).

Receptor Desensitization vs. Signaling:As noted in the literature, prolonged exposure to agonists often leads to internalization or desensitization. Therefore, a snapshot of receptor density might not accurately reflect the magnitude of the intracellular signal.
Functional Validation (Downstream Targets):Instead of monitoring the receptor capability at the membrane level, we focused on the functional outcome of the signaling pathway. The robust upregulation of Runx2 (the downstream target of the /cAMP/PKA/-catenin axis) observed in our study provides definitive evidence that the receptor-mediated signaling was successfully transmitted to the nucleus .
Revised Discussion:We have addressed this limitation in the Discussion section (Page 8, Lines 496-500). We explicitly state that while receptor internalization is a possibility, the sustained osteogenic markers indicate that PDRN-induced signaling remains sufficiently active to drive differentiation.

We hope this explanation clarifies why we prioritized downstream functional markers over static receptor staining in this therapeutic efficacy study.

Round 2

Reviewer 1 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

The corrected version has become suitable for publication.

I am grateful to the authors for their constructive interaction and that they recognized the correctness of my arguments.

Author Response

Comment: The corrected version has become suitable for publication. I am grateful to the authors for their constructive interaction and that they recognized the correctness of my arguments.

Response:

We sincerely thank the reviewer for the highly positive evaluation and for recommending our manuscript for publication. We deeply appreciate the time, effort, and constructive feedback you have provided throughout the review process. Your insightful arguments were absolutely instrumental in improving the scientific rigor, clarity, and overall quality of our study. We are truly grateful for your valuable guidance and are very pleased that the revised manuscript now meets your expectations.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Jeon and colleagues investigated how exposure to PDRN may influence osteogenic differentiation and the viability of MC3T3-E1 cells, as well as bone-marrow-derived macrophages. Although the topic is of interest, the study is incomplete, and the conclusions drawn are not adequately supported by the results obtained.

Overall assessment:

A) Introduction:
Although well written, the authors should exercise additional care regarding nomenclature, particularly in the designation of adenosine receptors. They should therefore refer to IUPHAR (this must be corrected throughout all sections). To better contextualize the work, it would be relevant to include one or two paragraphs on adenosine receptors, the origin of the nucleoside, how it is formed, and how it is released/uptaken by cells, including relevant literature.

B) Methodology:

In the first section, when referring to MC3T3-E1 cells, the authors should briefly justify their choice. Given the use of BMMs, why weren’t stromal mesenchymal osteoprogenitor cells isolated for osteogenic induction? This would have allowed a more rigorous and comparable assessment of the effects of PDRN on osteoclastogenesis versus osteogenesis, using the same animals.
How many passages were used for these cultures? More concerningly than in MC3T3-E1 cells, it is well known and widely discussed in the literature that bone marrow stem cells are particularly sensitive to passaging; passages can significantly affect their proliferative and differentiation capacity, and therefore the authors must clearly state between which passages the in vitro experiments were performed. Additional citations should be included in this part of the methodology, similar to those mentioned in the subsequent paragraphs of the Methods section.
Why were males used? Would differences be expected in females? Would the results be comparable? It is also necessary to provide justification for this issue.
The MTS assay does not, in fact, indicate cell proliferation, but cellular viability. Which other controls were performed to assess cell proliferation? There are several straightforward methods available that the authors could have used to confirm the findings obtained with this assay.
Statistics: It is not clear, anywhere in the manuscript — neither in the Methods nor in the figure legends — how many independent experiments were performed for each assay. Omitting this information is problematic and incompatible with the level of scientific rigor expected for an international publication. Ideally, unless constrained by relevant limitations (e.g., when using human biological material of limited availability), the number of experimental replicates should not be fewer than five ( https://doi.org/10.1111/bph.15868) . Which statistical test was used to assess the distribution of the values per group for each assay? Indeed, the tests used assume a normal distribution. Moreover, for the sake of rigor, the authors must specify the statistical tests used in all figures.
A section listing all reagents should be included in the Methods.

C) Results and Conclusions:

When evaluating proliferation and differentiation of MC3T3-E1 cells, the authors chose a 24-h exposure. Why was only a single time-point assessed? Evaluating the impact of PDRN over extended time periods would be more informative and not technically demanding.
It should be noted that the effect of PDRN on cell viability is minimal, although statistically significant, at 10 and 20 μg/ml. What was the reason for selecting these concentrations (rather than doses) for the subsequent assays? This is not clearly justified.
Why didn’t the authors quantify immunoreactivity in Figure 1b? As presented, the data are not conclusive. In addition to quantifying these images, the authors must: (1) explain how the images were selected within each well; how many images were analysed per well; (2) specify which antibodies were used and at what dilutions. This information must be clearly detailed in this section; again, the absence of this information is unacceptable and raises concerns regarding scientific transparency.
Quantifying ALP activity in these cells would have been more informative than staining. Once more, how were the representative images per well selected? Why did the authors not include statistical comparisons among the PDRN groups? Where is the quantification of the Western blot results, alongside the presented gel, with appropriate statistics?
The authors suggest that A_2A receptors mediate the effect; however, this conclusion appears to be premature. The authors could have simply added parallel experimental groups using selective adenosine receptor antagonists to determine if the effects of PDRN were blocked or reduced. It would also be relevant to test the effect of the antagonists alone, since these receptors may exhibit tonic activity (bearing in mind that these cells may release the nucleoside or ATP, which is then converted to adenosine by ecto-NTPDases and CD73). Here, it would also be relevant to include different time-points, given that ALP activity is dynamic in culture, as differentiation progresses and mineralization begins.
It should be noted that A_2A receptors have been associated with other mechanisms in osteoblasts and osteoclasts; studies such as those by Costa and colleagues, for example, using human BM-MSCs, suggested that A_2A receptors may in fact be associated with cell proliferation, inhibiting osteogenic differentiation (J. Cell. Physiol. 226: 1353–1366, 2011). The authors must discuss the literature and its apparent contradictions, rather than relying solely on data that appear to align with their results.
Including staining for adenosine receptors would have been suitable. What impact does PDRN have on their expression?
Another important point to include is a discussion of PDRN stability in these cultures — relevant literature should be cited.
What characterization was performed on the BMMs? How can the authors confirm, alongside the markers presented, that they obtained a culture enriched in BMMs? Phenotypic characterization of these cultures is essential.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript marinedrugs-4001399, entitled “Polydeoxyribonucleotide (PDRN) Selectively Promotes ………” addresses the cellular effects of PDRN on osteogenic differentiation and mineralization, with potential therapeutic implications for bone‑related disorders. The topic is relevant and of interest to the researchers. However, several critical issues must be resolved before the manuscript can be considered for publication.

Major Comments:
The authors should reconcile the discrepancy between their findings (PDRN inhibits osteoblast proliferation) and the results reported in references 4 and 12, which indicate that PDRN promotes proliferation. Current explanations are insufficient. A more detailed discussion is required, specifically addressing methodological differences, concentration ranges, and any different experimental conditions etc.
The statement that “A2AR activation preferentially triggers a proliferation‑differentiation switch” needs elaboration with precise mechanistic detail, with any related available citations.
The claim that “other studies show A2AR engagement enhances differentiation via pathways like Wnt/β‑catenin” in Lines 164–165 must be supported with stronger evidence (or specific references) and clarify how these findings align with your current data.
The comparison in lines 148–151 between PDRN and agents such as simvastatin and intermittent PTH is not convincing due to their differences in chemical structures as ligands. Unless there is experimental evidence showing similar anti‑proliferative effects on a common biological target, this section should be revised properly or removed.
The conclusion “…….our data suggest that its anti-resorptive properties are context-dependent rather than intrinsic” mentioned in Lines 176–177 is currently vague and rhetorical. It should be rewritten to provide a precise, evidence‑based scientific statement that reflects the data presented.

Figures and References:

Figure 1 should be revised to Figure 3 (line 119).
Insert the reference to Figure 3b at the appropriate place on page 4.
Add publication years after cited authors’ names (e.g., lines 156, 171, and throughout the manuscript).

Formatting, Terminology, and others:

Ensure the following words/phrases are written in plain font (not italicized or underlined): “proliferation‑differentiation switch” - words in lines 207–210.
In the Materials and Methods section, please provide full sourcing details for all major reagents (company name, city/state, country).

Reviewer 3 Report

Comments and Suggestions for Authors

First of all, I would like to point out that all Figures are very small and difficult to read. This makes it difficult to understand the presented results.

Secondly, the results of the MTT/MTS test are incorrectly interpreted in the manuscript. The metabolic activity of cells is called "proliferation", although no evidence has been provided for the effect or non-effect of the PDRN on osteoblast proliferation.

Obviously, any effect on proliferation can only be determined by the results of a cell cycle study, an EdU/BrdU or tritium-labeled thymidine incorporation test, cell counting, CFDA flow cytometry test, and the like. In this regard, the authors should clarify what exactly they studied and bring their conclusions in line with the results obtained.

Reviewer 4 Report

Comments and Suggestions for Authors

Comments to the Authors

In the Manuscript titled “Polydeoxyribonucleotide (PDRN) Selectively Promotes Osteoblast Differentiation Without Affecting Osteoclastogenesis”, the effect of salmon sperm-derived polydeoxyribonucleotide (PDRN, source/preparation unknown) on osteogenic (MC3T3-E1 murine cell line, supplier unknown) and osteoclastic precursors (bone marrow macrophages, BMM) is preliminarily investigated as follows: a) for MC3T3-E1 the osteoblast protocol uses MTS assay to demonstrate reduction of progenitor cells (Fig. 1A, other possible causes of cell death not investigated, was osteoblast differentiation induced – if not why not?) and confocal microscopy to show increased expression of Runx2 but not Osterix (Fig. 1B, quantitative analysis missing), followed by ALP staining and Alizarin Red S staining to demonstrate promotion of bone matrix formation, and protein blots for Runx2/Osterx (ImageJ or equivalent quantitative data not presented), and b) MTS assay of BMM cells to demonstrate no viability loss (cells referred as “osteoclasts” even though differentiation and extraction protocols as well as expression of osteoclasts markers are absent) in combination with RT-PCR results that osteoclast differentiation markers Cathepsin K (CTSK), NFATc1 (primers do not amplify target), and TRAP are unchanged after PDRN.

General comments: Exploration of the marine environment as source material for new biomaterials is a field of active interest and PDRN could be a promising new material for bone regeneration. The Manuscript presents interesting preliminary information, but the data are too limited and insufficient to warrant publication at this stage.

Major issues: This is an early-stage project that does not provide sufficient data to warrant publication.

Minor issues: Please see attached reviewed Manuscript and address all 23 comments.

Comments for author File: Comments.pdf