Preclinical Evaluation of Human Donor-Derived Micronized Bone Marrow Stroma/Parenchyma Versus Bone Marrow Aspirate Concentrate in a Rat Model of Post-Traumatic Knee Osteoarthritis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This preclinical study compares ECM-retaining micronized bone marrow product BMAX with conventional BMAC in a rat DMM model of PTOA. BMAX showed stronger effects on pain behavior, swelling, cartilage preservation, and possibly subchondral bone protection. The concept is relevant to orthobiologics and extends the authors’ prior work.

Major comments

Was a power calculation performed? With 10–12 rats per group and n=5–6 for some endpoints, are the study arms sufficiently powered to support claims of BMAX superiority?

How do the authors justify strong efficacy conclusions when several key comparisons, especially micro-CT outcomes, show only trends or borderline significance?

Since all human material came from a single donor, how generalizable are the findings given known donor-to-donor variability in MSC content, secretome, and ECM composition?

Was the lack of surgeon blinding during treatment administration considered as a potential source of bias, and can this limitation be stated more clearly?

Can the authors correct the figure formatting, labeling, and consistency issues, and ensure that all stated p-values are fully supported by the presented data?

Suggestions 

Please report a post-hoc power analysis.

If available, provide quantitative information on injected cell number, ECM content, or other product composition metrics.

Please address donor variability more directly, either by including a second donor or clearly stating replication plans.

The abstract and figure legends should be tightened to avoid overstating superiority where data show trends.

The proposed mechanisms, including IRAP/IL-6-related effects from prior work, should be discussed more cautiously unless supported by direct data in this study.

Author Response

1. Was a power calculation performed? With 10–12 rats per group and n=5–6 for some endpoints, are the study arms sufficiently powered to support claims of BMAX superiority?

Response:
We thank the Reviewer for this important comment. An a priori power calculation was not performed because this study was initiated as an extension of an ongoing animal experiment, and the number of animals was determined based on logistical and budgetary considerations while adhering to the principles of animal welfare and reduction of animal use. The selected sample size was also informed by our previous rat OA study, in which eight animals per group were used to evaluate five treatment groups across two time points. Given that the present study included only three treatment groups, we considered a smaller group size to be appropriate.

As recommended, we performed a post-hoc power analysis using G*Power based on the 12-week knee bend score, which represented the primary functional outcome demonstrating the greatest difference between the BMAC and BMAX groups. The analysis yielded an achieved power of 0.940 (Cohen's d = 2.40) using a two-tailed independent-samples t-test (α = 0.05), indicating that the comparison between the BMAC and BMAX groups was adequately powered. The corresponding description has been added to the Materials and Methods section, and the results of the post-hoc power analysis have been included in the Results section of the revised manuscript.

 

2. How do the authors justify strong efficacy conclusions when several key comparisons, especially micro-CT outcomes, show only trends or borderline significance?

Response:
We thank the Reviewer for this thoughtful comment. We agree that the original Conclusion may have overstated the overall efficacy of ECM-retaining micronized bone marrow. Although ECM-retaining micronized bone marrow demonstrated superior functional improvement and significantly greater cartilage regeneration than BMAC in the femoral condyle, the micro-CT findings showed comparable effects between the two treatment groups, with only modest regeneration of subchondral bone architecture relative to PBS controls. In addition, we have revised the Results and Discussion to report pain outcomes by assay and timepoint rather than in aggregate: BMAX™ was statistically superior to BMAC only for movement-evoked (knee-bend) pain at the final timepoint (P=0.0090), while the von Frey nociception measure showed no significant BMAX–BMAC difference; borderline comparisons (e.g., P=0.0686 and P=0.0517 versus PBS) are now described as non-significant trends rather than as significant effects.

To better reflect these findings, we have revised the Conclusion to more accurately distinguish the observed cartilage-protective/regenerative and functional benefits from the more limited effects on subchondral bone structure. The revised text emphasizes that preservation of the native extracellular matrix may enhance cartilage-protective/regenerative effects, while acknowledging that the micro-CT outcomes did not demonstrate superiority over conventional BMAC.

 

3. Since all human material came from a single donor, how generalizable are the findings given known donor-to-donor variability in MSC content, secretome, and ECM composition?

Response:
We thank the Reviewer for this important observation and agree that donor-to-donor variability is an important consideration for the interpretation and translation of our findings. Because this study was designed as a preclinical proof-of-concept investigation, BMAC and ECM-retaining micronized bone marrow preparations were generated from a single donor to minimize experimental variability and enable a controlled comparison between processing methods. We acknowledge that MSC abundance, secretory profile, and extracellular matrix composition may vary among donors and could influence therapeutic efficacy.

We have revised the Limitations section of the Discussion to explicitly acknowledge this limitation and to emphasize that future studies should validate our findings using samples obtained from multiple donors with diverse demographic and clinical backgrounds, including different ages and sexes, to assess the reproducibility and generalizability of the observed effects.

 

4. Was the lack of surgeon blinding during treatment administration considered as a potential source of bias, and can this limitation be stated more clearly?

Response:
We thank the Reviewer for this thoughtful comment. We would like to clarify that the DMM surgery was completed before treatment allocation. Specifically, all animals underwent the same surgical procedure, and randomization to the BMAX, BMAC, or PBS groups was performed four weeks later, immediately before intra-articular treatment administration. Therefore, surgeon blinding during the DMM procedure was not applicable because treatment allocation had not yet occurred.

We acknowledge that the investigator administering the intra-articular injections was not blinded to treatment allocation due to the nature of the intervention. However, all behavioral assessments, histological evaluations, and micro-computed tomography analyses were performed by investigators who were blinded to treatment allocation, thereby minimizing the potential for assessment bias. Furthermore, the investigator administering the treatments has no competing interests. To improve clarity, we have revised the Animal Study Design section of the Materials and Methods to explicitly describe the timing of randomization, treatment administration, and blinding procedures.

 

5. Can the authors correct the figure formatting, labeling, and consistency issues, and ensure that all stated p-values are fully supported by the presented data?

Response:
We thank you for these suggestions.  All P values are exact P values generated from Graphpad Prism 10.  P values between group bars are the Tukey’s post-hoc multiple comparison generated by the software. Each dot in each bar represents a sample. The bar heights indicated mean.

 

Suggestions

1. Please report a post-hoc power analysis.

Response:
Thank you for the comment. As recommended, we performed a post-hoc power analysis using G*Power based on the 12-week knee bend score, which represented the primary functional outcome demonstrating the greatest difference between the BMAC and BMAX groups. The analysis yielded an achieved power of 0.940 (Cohen's d = 2.40) using a two-tailed independent-samples t-test (α = 0.05), indicating that the comparison between the BMAC and BMAX groups was adequately powered. The corresponding description has been added to the Materials and Methods section, and the results of the post-hoc power analysis have been included in the Results section of the revised manuscript.

 

2. If available, provide quantitative information on injected cell number, ECM content, or other product composition metrics.

Response:

We thank the Reviewer for this suggestion. Absolute composition of the fresh injectate – nucleated cell number and MSC number per mL – was not enumerated, and treatments were not dose-matched by cell number; instead, all groups received an equal, fixed injection volume (100 µL). The MSC content of the donor-matched BMAC and BMAX™ preparations was characterized functionally and phenotypically on culture-expanded cells (CFU-F clonogenicity and ISCT surface phenotype; Figure 2). The differential ECM retention and secretome of BMAX™ relative to donor-matched BMAC were previously quantified [9]. A statement to this effect has been added to the Materials and Methods.

3. Please address donor variability more directly, either by including a second donor or clearly stating replication plans.

Response:
We thank the Reviewer for this important observation and agree that donor-to-donor variability is an important consideration for the interpretation and translation of our findings. Because this study was designed as a preclinical proof-of-concept investigation, BMAC and ECM-retaining micronized bone marrow preparations were generated from a single donor to minimize experimental variability and enable a controlled comparison between processing methods. We acknowledge that MSC abundance, secretory profile, and extracellular matrix composition may vary among donors and could influence therapeutic efficacy.

We have revised the Limitations section of the Discussion to explicitly acknowledge this limitation and to emphasize that future studies should validate our findings using samples obtained from multiple donors with diverse demographic and clinical backgrounds, including different ages and sexes, to assess the reproducibility and generalizability of the observed effects.

 

4. The abstract and figure legends should be tightened to avoid overstating superiority where data show trends.

Responses:
We thank the reviewer for this valuable suggestion. We substantially revised the Abstract by using more precise language, avoiding general statements, and removing any descriptions of non-significant trends as meaningful findings. We also further refined the Results and Discussion so that the superiority of BMAX™ over BMAC is stated only where statistically supported, specifically for movement-evoked pain at the final time point (P = 0.0090) and reduced swelling at 4 weeks (P = 0.0039).
The original figure legends are descriptive in nature and simply explain the contents of each figure without overstating the findings. Therefore, we have left the figure legends unchanged.

5. The proposed mechanisms, including IRAP/IL-6-related effects from prior work, should be discussed more cautiously unless supported by direct data in this study.

Response:
We thank the Reviewer for this valuable comment and agree that the mechanistic discussion should not overinterpret findings that were not directly examined in the present study. The discussion of IL-6 and IRAP was based on our previously published in vitro work and was intended to provide a potential biological context for the observed therapeutic effects rather than to imply that these mechanisms were demonstrated here.

To address the Reviewer's concern, we have revised the Discussion to explicitly state that inflammatory mediators, including IL-6 and IRAP, were not directly assessed in the current study and that any mechanistic contribution of these pathways remains speculative. We also emphasize that future studies incorporating cytokine profiling and molecular analyses will be required to determine whether these mechanisms contribute to the reduced joint inflammation and improved functional outcomes observed following BMAX™ treatment.

Reviewer 2 Report

Comments and Suggestions for Authors

The scientific paper Enhanced outcomes using micronized bone marrow stroma/parenchyma compared to BMAC using post-traumatic knee osteoarthritis model rats aimed to evaluate the therapeutic effects of BMAX™ in a rat destabilization of the medial meniscus (DMM) model of post-traumatic osteoarthritis and compared its efficacy with conventional BMAC.After careful reading, I can make the following recommendations:

1) Clearly specify in the title that the study is preclinical (using rats) and that the products are derived from a human donor.

2) In the Abstract, include the "n" (group size per experiment) and key p-values, and indicate that the BMAC/BMAX samples came from a single donor.

3) In the introduction, I recommend explicitly stating the specific gaps in the literature that this study aims to fill (e.g., BMAC limitations due to blood dilution and the absence of ECM).

4) In Materials and Methods, clarify and justify the use of a single human donor (age, sex, clinical condition) and discuss the limitations of this choice.

5) Also describe the exact volume collected for BMAC and BMAX and final concentrations (TNC, MSC estimated per mL) with units and standard deviations.

6) Provide details of the composition of the anticoagulant used and times from collection to processing.

7) Describe negative/positive controls for cytometry and provide a gating strategy (perhaps attach a supplementary figure/file with cytometry plots).

8) Specify exact inclusion/exclusion criteria (e.g., surgical complications, mortality) and confirm that no animals were excluded.

9) Please explain the reason for single injection (versus multiple injections) and discuss the implications.

10) Provide complete numerical values ​​(means ± SD, N per time) in figures/tables; currently only bars with p-values ​​are available.

11) In Micro-CT, describe segmentation criteria and ROI in more detail (anatomical-metric limits), and report reproducibility measures.

12) In the results section, include tables with N per group, means ± SD/SEM, and exact p-values ​​for all primary and secondary outcomes.

13) Explicitly discuss limitations: single donor, relatively small animal N, absence of multiple donors/replications, and potential bias due to involvement of authors related to the company.

14) Include clear recommendations for future studies: multiple donors, dose-response calculation, repeated injections, hematocrit monitoring, and long-term immunological safety assessment.

 

 

Author Response

1) Clearly specify in the title that the study is preclinical (using rats) and that the products are derived from a human donor.

Response:
We thank the Reviewer for this helpful suggestion. We agree that the original title did not clearly indicate the preclinical nature of the study or the origin of the bone marrow products. To improve clarity and accurately reflect the study design, we have revised the title as follows:

Revised title:

Preclinical Evaluation of Human Donor-Derived Micronized Bone Marrow Stroma/Parenchyma versus Bone Marrow Aspirate Concentrate in a Rat Model of Post-Traumatic Knee Osteoarthritis

2) In the Abstract, include the "n" (group size per experiment) and key p-values, and indicate that the BMAC/BMAX samples came from a single donor.

Response:
We thank the Reviewer for this helpful suggestion. We have revised the Abstract to improve the transparency and completeness of the study design and results. Specifically, we have clarified that both BMAC and BMAX™ were prepared from bone marrow obtained from a single human donor, included the number of animals used for each experimental endpoint, and added the key P-values for the principal outcome measures. We have also revised the concluding statement to more accurately reflect the findings of this preclinical study.

3) In the introduction, I recommend explicitly stating the specific gaps in the literature that this study aims to fill (e.g., BMAC limitations due to blood dilution and the absence of ECM).

Response:
We thank the Reviewer for this helpful suggestion. We have revised the Introduction to state the specific gap addressed by this study. We now make explicit the fundamental distinction between the two products: conventional BMAC is prepared from aspirated marrow fluid, which is diluted by peripheral blood, whereas BMAX™ is harvested as intact marrow tissue (subcortical bone cores) that preserves the native stromal extracellular matrix and stem-cell niche. We further state that no prior in vivo study had tested whether this tissue-preserving, ECM-retaining preparation improves outcomes over conventional BMAC in post-traumatic osteoarthritis – the gap this study addresses. In the Discussion we further contextualize this with recent single-cell and spatial mapping of human marrow, which shows that aspiration is biased toward adipocytic MSCs and largely fails to capture the bone-adherent, ISCT-defined (CD73/CD90/CD105) MSCs of highest clonogenic capacity [12], providing a mechanistic rationale for the tissue-based BMAX™ approach.

4) In Materials and Methods, clarify and justify the use of a single human donor (age, sex, clinical condition) and discuss the limitations of this choice.

Response:
We thank the Reviewer for this helpful suggestion. We have expanded the Materials and Methods to justify and describe the single donor. A single donor was used by design so that BMAX™ and BMAC could be prepared from the same marrow, enabling a controlled, donor-matched comparison of the two processing methods free of inter-donor variability. The donor was a 50-year-old female (height 5 ft 6 in, weight 121 lb, BMI 19.5) with Ehlers–Danlos syndrome, selected from among several prospective donors specifically because she carried among the highest burden of senescent cells on pre-procedural screening – a deliberately conservative, worst-case regenerative profile and the focus of one of the studies of which this study is an extension; the demonstration of therapeutic benefit even with aged, high-senescence cells strengthens rather than weakens the finding. The Limitations acknowledge that single-donor material precludes assessment of inter-donor variability and that validation across multiple donors of differing age and sex is required.

5) Also describe the exact volume collected for BMAC and BMAX and final concentrations (TNC, MSC estimated per mL) with units and standard deviations.

Response:
We thank the Reviewer for this comment. We have added the collection and preparation details to the Materials and Methods. BMAC was prepared from 50 mL of iliac-crest bone marrow aspirate by two-step centrifugation. For BMAX™, subcortical bone cores were harvested with an 11-gauge Jamshidi needle and pressed into the BMAX™ device (washed with heparin and containing 1 mL of saline); after BMAC processing, approximately 2 mL of bone marrow-derived plasma was added for a total volume of 3 mL, the device underwent two automated processing cycles, and the product was withdrawn into a 3 mL syringe. The final intra-articular injectate volume was 100 µL for every group. Absolute total nucleated cell (TNC) and MSC counts per mL of the fresh injectate were not enumerated; because both products derived from a single donor prepared in single preparations, a mean ± standard deviation across preparations cannot be computed. MSC content was instead characterized functionally (CFU-F) and phenotypically (ISCT flow cytometry) on culture-expanded cells (Figure 2). We would also note that BMAC and BMAX™ are not directly comparable on a per-mL cellular basis: BMAC is a liquid concentrate whose cellularity is expressed per unit volume, whereas BMAX™ is a micronized tissue construct whose cellularity was necessarily expressed per unit mass of processed tissue in our prior characterization [9]. Consistent with the tissue-based approach, BMAX™ is lower in overall cellularity and leukocyte content than BMAC [9].

6) Provide details of the composition of the anticoagulant used and times from collection to processing.

Response:
We thank the Reviewer for this helpful suggestion. The Materials and Methods now specify that the aspirate was anticoagulated with preservative-free heparin at a final concentration of 10 U/mL (the BMAX™ bone cores were not treated with heparin). Bone marrow was harvested from the iliac crest on two occasions; on each occasion the harvest was bilateral, with one side used for the donor’s clinical treatment and the contralateral side used for this study. BMAC and BMAX™ were processed immediately after harvest, without interim storage, and were then transported to the animal facility located approximately 30 minutes away for intra-articular administration.

7) Describe negative/positive controls for cytometry and provide a gating strategy (perhaps attach a supplementary figure/file with cytometry plots).

Responses:
We thank the Reviewer for this helpful suggestion. We have revised the Flow Cytometry section in the Materials and Methods to provide a more detailed description of the gating strategy and the negative/positive controls used for flow cytometric analysis.
Specifically, we have clarified that the cultured cell population was initially identified using FSC/SSC plots, and antibody isotype controls were used for each fluorescent channel to establish negative fluorescence and define positive/negative gating thresholds. For the ISCT MSC characterization panel, sequential back-gating was performed beginning with the WBC-negative population (CD34^−, CD45^−, CD11b^−, CD19^−, HLA-DR^−), followed by CD73^+, CD105^+, and CD90^+ cells. For the CD146 analysis, antibody isotype controls were similarly used to establish gating, and the CD146^+ population was back-gated to the CD45^− population to determine the percentage of CD45^−/CD146^+ cells.
In addition, we have included Supplementary Figure S1, which illustrates the gating strategy using representative histogram- and dot plot-based gating examples. We believe these additions improve the transparency and reproducibility of our flow cytometric analysis.

8) Specify exact inclusion/exclusion criteria (e.g., surgical complications, mortality) and confirm that no animals were excluded.

Response:
We thank the Reviewer for this helpful suggestion. We have revised the Animal Study Design section of the Materials and Methods to explicitly describe the inclusion and prespecified exclusion criteria. Animals were eligible for inclusion if they successfully underwent the DMM procedure and survived to the designated experimental endpoint. Prespecified exclusion criteria included major surgical complications, such as postoperative infection or perioperative mortality, that would preclude completion of the study protocol. No animals met these exclusion criteria, and no animals or data points were excluded from the final analyses.

9) Please explain the reason for single injection (versus multiple injections) and discuss the implications.

Response:
We thank the Reviewer for this important comment. A single intra-articular injection was selected to evaluate the therapeutic potential of BMAX™ under a standardized proof-of-concept experimental design while minimizing variability associated with repeated administrations. In addition, because the study used bone marrow products prepared from a single human donor, the available material was limited, and repeated injections were not feasible. We agree that repeated administration may better reflect certain clinical orthobiologic treatment protocols. Therefore, we have added this point to the Limitations section and now emphasize that future studies should investigate the efficacy of repeated BMAX™ administration and optimize treatment timing and dosing regimens.

10) Provide complete numerical values (means ± SD, N per time) in figures/tables; currently only bars with p-values are available.

Response:  
Thank you for these suggestions. The bars actually showed the mean±SD, each dot on the bar represents one sample. According to your suggestions, we have added 5 tables to match quantification graphs in Figure 4,5,6.  But they appeared redundant with the quantification Figures. Since microCT only has medial subchondral bone Tb.Sp showed significance, we did not make table for this quantification because it will not enhance the clarity of the paper.

11) In Micro-CT, describe segmentation criteria and ROI in more detail (anatomical-metric limits), and report reproducibility measures.

Response:
We thank you for these suggestions.  We have added a new panel in Figure 7 to show the view of interest.

12) In the results section, include tables with N per group, means ± SD/SEM, and exact p-values for all primary and secondary outcomes.

Response:
We thank the Reviewer for this helpful suggestion. We have added summary tables for the primary and secondary outcome measures, including the sample size (n) for each group and the corresponding mean ± SD values. Exact P-values are provided in the corresponding figures, where all statistical comparisons are presented. To avoid unnecessary duplication and maintain the readability of the tables, we did not repeat the same P-values within the tables.

13) Explicitly discuss limitations: single donor, relatively small animal N, absence of multiple donors/replications, and potential bias due to involvement of authors related to the company.

Response:
We thank the Reviewer for this important suggestion. We have expanded the Limitations section to acknowledge that several co-authors have disclosed professional relationships with the company developing BMAX™, as described in the Conflict of Interest statement. We also emphasize that behavioral, histological, and micro-computed tomography assessments were performed by investigators blinded to treatment allocation to minimize potential assessment bias, and that independent external validation will be important to further confirm the reproducibility of our findings.

14) Include clear recommendations for future studies: multiple donors, dose-response calculation, repeated injections, hematocrit monitoring, and long-term immunological safety assessment.

Response:
We thank the Reviewer for this valuable suggestion. We have expanded the Future Directions in the Discussion to provide more specific recommendations for subsequent studies. In addition to validating our findings using multiple human donors, we now emphasize the need to investigate dose–response relationships, repeated injection regimens, hematocrit and other product characteristics that may influence therapeutic efficacy, and the long-term immunological safety and durability of treatment effects. These additions better define the next steps required for the preclinical and translational development of ECM-retaining micronized bone marrow therapy.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This study examines outcomes in micronized bone marrow stromal/parenchymal vs bone marrow aspirate concentrate using rat model.

Main concerns and comments:

1. The title has a little bit confused and needs to rewrite it.
2. Also using full name of BMAC in title will be better
3. Since bone marrow is from a 50-year-old woman. Please provide the patient's info. Including race, lab data, history of diseases, etc.
4. Will gender of the bone marrow donor affect the results? Please discuss.
5. Figure 7: Please explain why some groups have n=5, n=6, or n=4? Would it be supposed to be the same sample size?
6. The list of reference is appropriate.

Author Response

1. The title has a little bit confused and needs to rewrite it.

 

Response:

We thank the Reviewer for this helpful suggestion. We agree that the original title did not clearly indicate the preclinical nature of the study or the origin of the bone marrow products. To improve clarity and accurately reflect the study design, we have revised the title as follows:

 

Revised title:

Preclinical Evaluation of Human Donor-Derived Micronized Bone Marrow Stroma/Parenchyma versus Bone Marrow Aspirate Concentrate in a Rat Model of Post-Traumatic Knee Osteoarthritis

 

2. Also using full name of BMAC in title will be better
   
   

Response:

We thank the Reviewer for this helpful comment. The title has been revised accordingly.

3. Since bone marrow is from a 50-year-old woman. Please provide the patient's info. Including race, lab data, history of diseases, etc.

 

We thank the Reviewer for this suggestion. The donor was a 50-year-old female (height 5 ft 6 in, weight 121 lb, BMI 19.5) with a diagnosis of Ehlers–Danlos syndrome, selected from among several prospective donors specifically because pre-procedural screening showed she carried among the highest burden of senescent cells (C12FDG+/CD87+) (See Table below), representing a conservative, worst-case regenerative profile. These details have been added to the Materials and Methods. Additional identifying clinical information is limited by the donor’s consent and privacy protections under the governing IRB approval (WCG-IRB# 20230413).

               Table: Senescence profile of different donors

Sample	C12FDG+	CD87+	C12FDG+/CD87+
1(Gradient)	8.82%	8.05%	8.57%
1A	22.39%	17.46%	18.93%
1B	39.88%	33.11%	35.81%
2A	5.10%	6.54%	4.42%
3A	4.57%	8.94%	3.82%
4A	19.61%	12.49%	14.91%
4B	19.22%	9.42%	11.59%

 

We tested samples from four different patients, and the sample from Patient #1 showed the highest senescent burden.

 

4. Will gender of the bone marrow donor affect the results? Please discuss.

 

Response：

We thank the Reviewer for this thoughtful question. Donor sex can influence MSC yield, secretome, and regenerative capacity, and we cannot exclude a contribution of donor sex to the observed effects, as all material derived from a single female donor. Importantly, this donor represented a conservative, worst-case biological profile (advanced age relative to typical preclinical donors, high senescent-cell burden, and a connective-tissue disorder), such that the observed benefit likely underestimates rather than overstates the effect achievable with more favorable donors. Donor sex is included among the factors identified in the Limitations as requiring validation across multiple donors in future studies.

 

5. Figure 7: Please explain why some groups have n=5, n=6, or n=4? Would it be supposed to be the same sample size?

 

Response:

We thank the Reviewer for this careful observation. The intended sample sizes for the histological and micro-computed tomography analyses were the same. However, minor differences occurred because of technical issues during sample processing.

For the micro-CT analysis, one specimen from the BMAX™ group was not included in the imaging dataset. This discrepancy was identified only after the specimens had already been transferred for histological processing and decalcification, making retrospective micro-CT acquisition impossible.

For the histological analysis, although all specimens were processed, only three PBS samples yielded histological sections of sufficient quality for quantitative evaluation because of technical difficulties encountered during tissue sectioning. These differences were unrelated to the experimental outcomes, and no specimens were selectively excluded based on the study results. We have clarified the sample sizes for each analysis in the revised manuscript and the corresponding figure legends.

 

6. The list of reference is appropriate.

Response:

Thank you so much for the comment.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I thank the authors for their efforts to revise and improve the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

No more comments