Natural Nacre-Derived Biomimetic Materials for In Vivo Bone Regeneration

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The present study assesses the capacity of natural nacre-derived proteins to enhance bone regeneration in a critical-size defect model in rats, either alone or in combination with bioactive porous silicon (pSi) microparticles. Natural proteins were extracted from nacre powder, while the microparticles were produced from oxidized pSi. Although the topic is of interest, several points of concern should be addressed.

First, it is unclear whether any characterization of the extracted proteins and the pSi microparticles was performed prior to the in vivo experiments. Were the physicochemical and mechanical properties of these materials evaluated? In particular, information regarding the resorption rate of oxidized pSi, as well as its potential cytotoxicity and genotoxicity, is missing and should be provided.

Lines 109–111 state: “Shells were then subjected to a decalcification–recalcification protocol adapted from previously published nacre extraction procedures.” The previously published procedures should be clearly and correctly cited.

In line 116, the term “ball-mill grinder” is used. Please provide the full name of the equipment, including the manufacturer and country of origin.

The third dimension of the bone defect is not specified. Please clearly state the complete defect dimensions and provide a schematic illustration of the defect model.

Animal welfare during the 60-day experimental period should be described in detail. Additionally, were any other parameters monitored during this period, such as biochemical blood markers or radiographic imaging (X-ray and micro-CT at intermediate time points) to follow bone regeneration over time?

Section 3.2. “Histological Evidence of Mineralized Tissue Formation” does not correspond to the described methodology, which includes collagen and Alizarin Red S staining. Please present results that directly reflect the applied methodology. Furthermore, the histological figures should be of adequate resolution and quality.

Figure 2. is confusing: panels A–C are described as representing regeneration with pSi microparticles, while panels B–D correspond to regeneration with nacre proteins. Please clarify the figure layout and provide clearly labeled, representative images for all experimental groups.

Line 234 states: “A comparison of all groups (micro-CT and histology) is shown in Figure 3.” This statement is inaccurate, as Figure 3 presents only micro-CT results, as indicated in the caption “Histogram presents BMD values as measured on μCT slices”. Please revise the text accordingly.

The absence of an inflammatory response should be adequately demonstrated. Based on the presented figures, this conclusion cannot be reliably drawn. Additional histological or immunohistochemical evidence is required to support this claim.

Finally, the Discussion section is written in an unusual manner. Rather than integrating the authors’ own findings with previously published studies, it reads more like a mini-review of the field. The Discussion should be restructured to focus on interpreting the current results in the context of relevant literature.

Author Response

Comment 1:

The present study assesses the capacity of natural nacre-derived proteins to enhance bone regeneration in a critical-size defect model in rats, either alone or in combination with bioactive porous silicon (pSi) microparticles. Natural proteins were extracted from nacre powder, while the microparticles were produced from oxidized pSi. Although the topic is of interest, several points of concern should be addressed.

First, it is unclear whether any characterization of the extracted proteins and the pSi microparticles was performed prior to the in vivo experiments. Were the physicochemical and mechanical properties of these materials evaluated? In particular, information regarding the resorption rate of oxidized pSi, as well as its potential cytotoxicity and genotoxicity, is missing and should be provided.

Response 1:

We thank the reviewer for this important comment. The objective of the present study was not to introduce a newly developed material, but rather to evaluate the in vivo proof-of-concept of combining a well-documented bioresorbable porous silicon platform with a naturally derived nacre-based organic/mineral fraction.

Porous silicon microparticles used in this study were produced following previously published protocols, and their physicochemical properties, oxidation state, biodegradation behavior, and biocompatibility have been extensively characterized and validated in prior works. We have now clarified this point in the Materials and Methods section with addition of appropriate references, and in the Introduction section, with a re-written paragraph detailing pSi resorption kinetics, surface chemistry, and cytocompatibility, including long-term in vivo biocompatibility and absence of cytotoxic effects.

Regarding nacre-derived material, the organic fraction was used in its native mineral context, following established nacre processing protocols reported in the literature. The study was not designed to provide exhaustive molecular characterization, which has been reported elsewhere, but to investigate the biological response elicited by this biomimetic composite in vivo. This limitation and the rationale for this choice are now explicitly stated in the revised manuscript, in both Introduction section and Limitations paragraph.

Comment 2:

Lines 109–111 state: “Shells were then subjected to a decalcification–recalcification protocol adapted from previously published nacre extraction procedures.” The previously published procedures should be clearly and correctly cited.

Response 2:

We agree and thank the reviewer for pointing this out. The corresponding section has been revised to include explicit citations to the original nacre extraction and processing protocols on which our procedure was based. These references are now clearly indicated in the Materials and Methods section.

Comment 3:

In line 116, the term “ball-mill grinder” is used. Please provide the full name of the equipment, including the manufacturer and country of origin.

Response 3:

This information has now been added. The full name of the equipment, manufacturer, and country of origin are specified in the revised Materials and Methods section to ensure reproducibility.

Comment 4:

The third dimension of the bone defect is not specified. Please clearly state the complete defect dimensions and provide a schematic illustration of the defect model.

Response 4:

We thank the reviewer for this remark. The full three-dimensional dimensions of the defect have now been clearly specified in the Materials and Methods section (in red in the MS), and a figure (Figure 1) was added to illustrate the defect model.

Comment 5:

Animal welfare during the 60-day experimental period should be described in detail. Additionally, were any other parameters monitored during this period, such as biochemical blood markers or radiographic imaging (X-ray and micro-CT at intermediate time points) to follow bone regeneration over time?

Response 5:

Animal welfare monitoring has now been described in more detail, including daily clinical observation, body weight monitoring, and postoperative care in accordance with institutional and national ethical guidelines.

No intermediate imaging or biochemical analyses were performed, as the study design focused on end-point evaluation at 60 days to assess final defect bridging and mineralized tissue formation. This has now been clearly stated in the revised manuscript (in red in the MS).

Comment 6:

Section 3.2. “Histological Evidence of Mineralized Tissue Formation” does not correspond to the described methodology, which includes collagen and Alizarin Red S staining. Please present results that directly reflect the applied methodology. Furthermore, the histological figures should be of adequate resolution and quality.

Response 6:

We thank the reviewer for this accurate comment. Indeed, in the original experimental plan, calcium deposits were supposed to be stained with Alizarin Red. However, the use of undecalcified samples is somehow difficult to handle. Therefore, we only used Masson Trichrome staining on undecalcified sections. To further investigate collagen maturation, we have decalcified some sections, and stained them with Sirius Red. The Materials & Methods section and the Results section have been revised to better align the description with the applied histological methodologies. The text has been clarified to explicitly refer to collagen staining outcomes. Figure legends have been revised for clarity, and representative images have been reorganized to improve readability and consistency with the described methods, with the addition of images stained with Sirius Red.

Comment 7:

Figure 2. is confusing: panels A–C are described as representing regeneration with pSi microparticles, while panels B–D correspond to regeneration with nacre proteins. Please clarify the figure layout and provide clearly labeled, representative images for all experimental groups.

Response 7:

We agree and apologize for the confusion. Figure 2 (renamed Figure 3) has been reorganized, and the panel labeling and legend have been corrected to clearly identify each experimental group, with addition of Red Sirius staining. The revised figure now presents representative images in a consistent and unambiguous layout.

Comment 8:

Line 234 states: “A comparison of all groups (micro-CT and histology) is shown in Figure 3.” This statement is inaccurate, as Figure 3 presents only micro-CT results.

Response 8:

We thank the reviewer for noticing this inconsistency. The text has been corrected to accurately reflect that Figure 3 (renamed Figure 4) presents micro-CT–derived quantitative data only.

Comment 9:

The absence of an inflammatory response should be adequately demonstrated. Based on the presented figures, this conclusion cannot be reliably drawn. Additional histological or immunohistochemical evidence is required to support this claim.

Response 9:

We agree with the reviewer. The manuscript has been revised to avoid overinterpretation regarding the absence of inflammation. The conclusions have been softened, and the text now states that no overt inflammatory reaction was observed in standard histological sections, without claiming definitive absence of inflammation. This limitation is now explicitly acknowledged in the Discussion, with the addition of a specific paragraph “4.4 Biocompatibility and host response”. The Conclusions section has been re-written.

Comment 10:

Finally, the Discussion section is written in an unusual manner. Rather than integrating the authors’ own findings with previously published studies, it reads more like a mini review of the field.

Response 10:

We thank the reviewer for this constructive suggestion. The Discussion has been substantially restructured to focus more directly on interpreting the present findings in light of relevant literature, rather than providing a general overview of the field. Redundant review-style content has been reduced, and greater emphasis has been placed on the implications and limitations of the current study. Paragraph 4.1 and 4.2 have been entirely re-written.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript investigates how natural nacre proteins and their combination with porous silicon microparticles (pSi-MP) support bone regeneration in critical-size defects in rats. The study demonstrates a synergistic effect arising from the bioactive properties of nacre proteins and the structural and biodegradation characteristics of pSi-MP, offering a meaningful contribution to the literature. The writing is clear, the methodology is well described, and the results are convincing. However, several analytical limitations and interpretational considerations should be addressed.

1. The manuscript does not report the purification degree, protein composition, or molecular profile (e.g., SDS-PAGE) of the natural nacre proteins used. Providing basic characterization data would clarify the nature and reproducibility of the biomacromolecule and enhance the scientific reliability of the study.

2. The study lacks a positive control group, which is important for establishing a robust comparative standard in bone regeneration research. At minimum, this limitation should be explicitly acknowledged in the Discussion to properly contextualize the findings.

3. A broader discussion on the relationship between material structure and bioactivity would strengthen the manuscript. Relevant literature shows that naturally derived bioactive glass-based microarchitectures can achieve both osteogenic and controlled-release functionalities through structural design (e.g., https://doi.org/10.1016/j.cej.2020.126667). Highlighting structural and functional parallels between your nacre–pSi composite and these bioactive glass microstructures—such as multifunctionality and biomimetic design—may provide valuable context.

4. The manuscript states that nacre proteins were mixed directly with nacre powder; however, it remains unclear how much of the protein fraction actually dissolved, what types of proteins were present, and whether any filtration or separation steps were performed after extraction. This ambiguity makes the term “natural nacre proteins” insufficiently defined.

5. The histological findings indicate that the regenerated tissue remained at the woven bone stage. The implications of this—such as long-term mechanical stability and the expected timeline for maturation into lamellar bone—would benefit from further discussion.

6. Although BMD values are reported, the manuscript does not specify which statistical tests were used, the sample size for each group (n), or whether assumptions such as variance homogeneity were verified. Clarifying these points would improve the transparency and rigor of the quantitative analysis.

Author Response

Comment 1:

The manuscript does not report the purification degree, protein composition, or molecular profile (e.g., SDS-PAGE) of the natural nacre proteins used. Providing basic characterization data would clarify the nature and reproducibility of the biomacromolecule and enhance the scientific reliability of the study.

Response 1:

We thank the reviewer for this important comment. In this study, the nacre-derived material was intentionally used without extensive purification, in order to preserve the native organic/mineral context of nacre, which is considered central to its biomimetic bioactivity. The material therefore does not correspond to a purified protein fraction, but rather to a naturally derived organic fraction embedded within residual mineral phases.

We fully agree that molecular characterization such as SDS-PAGE would be required for studies focusing on isolated protein mechanisms. However, the objective of the present work was to evaluate the in vivo biological response to a nacre-derived biomimetic material, rather than to characterize individual macromolecular components. This distinction is now more clearly stated in the Materials and Methods section, and the terminology has been clarified throughout the manuscript to avoid ambiguity regarding the nature of the nacre-derived fraction (in red in the MS). The title has also been modified accordingly.

Comment 2:

The study lacks a positive control group, which is important for establishing a robust comparative standard in bone regeneration research. At minimum, this limitation should be explicitly acknowledged in the Discussion to properly contextualize the findings.

Response 2:

We agree with the reviewer and thank them for highlighting this point. The absence of a positive control group (e.g., autograft or clinically approved substitute) is now explicitly acknowledged as a limitation of the study in the Discussion section.

The present work was designed as an exploratory proof-of-concept study aimed at comparing the relative effects of nacre-derived material, porous silicon microparticles, and their combination within the same experimental model. The lack of a positive control limits direct clinical comparison, and this is now clearly stated in the revised manuscript, in the fully re-written paragraph “Limitations and future directions”

Comment 3:

A broader discussion on the relationship between material structure and bioactivity would strengthen the manuscript. Relevant literature shows that naturally derived bioactive glass-based microarchitectures can achieve both osteogenic and controlled-release functionalities through structural design.

Response 3:

We thank the reviewer for this valuable suggestion. The Discussion section has been expanded to better address the relationship between material structure and bioactivity, with particular emphasis on how the nacre–pSi composite combines biochemical cues from the nacre-derived organic fraction with the structural, degradable, and carrier properties of porous silicon.

Relevant literature on bioactive glass microarchitectures has now been cited and discussed to highlight conceptual parallels in multifunctionality, controlled degradation, and biomimetic design, while clearly distinguishing the distinct material chemistries involved.

Comment 4:

The manuscript states that nacre proteins were mixed directly with nacre powder; however, it remains unclear how much of the protein fraction actually dissolved, what types of proteins were present, and whether any filtration or separation steps were performed after extraction. This ambiguity makes the term “natural nacre proteins” insufficiently defined.

Response 4:

We fully agree with the reviewer. To address this ambiguity, the manuscript has been revised to clarify that the nacre-derived material corresponds to a whole nacre-derived organic fraction associated with mineral components, rather than a purified or selectively extracted protein solution.

No post-extraction filtration or molecular separation was performed, and the term “natural nacre proteins” has been revised throughout the manuscript to more accurately reflect the nature of the material used. This clarification improves transparency and reproducibility and aligns the terminology with the actual experimental approach. The title of the manuscript has also been corrected accordingly.

Comment 5:

The histological findings indicate that the regenerated tissue remained at the woven bone stage. The implications of this—such as long-term mechanical stability and the expected timeline for maturation into lamellar bone—would benefit from further discussion.

Response 5:

We thank the reviewer for this insightful comment. The Discussion has been expanded to address the observation that regenerated tissue predominantly corresponds to woven bone at the 60-day time point. We now discuss this finding in the context of the non-load bearing vertebral model, the expected temporal progression of bone remodeling, and the need for longer-term studies to assess maturation toward lamellar bone and mechanical competence. These considerations are now clearly presented as perspectives for future work.

Comment 6:

Although BMD values are reported, the manuscript does not specify which statistical tests were used, the sample size for each group (n), or whether assumptions such as variance homogeneity were verified.

Response 6:

We thank the reviewer for pointing this out. A specific “Statistical analysis” paragraph has been added, in Materials & Methods section, to clearly specify the statistical tests used, the sample size per group, and the criteria applied for statistical comparison. The exploratory nature of the quantitative analysis is now explicitly stated to ensure appropriate interpretation of the results.

Reviewer 3 Report

Comments and Suggestions for Authors

Please see the attached File

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Please see the attached File

Author Response

Comment 1:

In introduction section, there is limited information about the latest research in the field of proposed research, indicating the need to revise the introduction section according to the latest research.

Response 1:

We thank the reviewer for this constructive comment. The Introduction has been revised to better reflect recent advances in the field, including updated literature on biomimetic bone regeneration strategies, naturally derived bioactive materials, and composite systems combining organic and inorganic cues. This revision strengthens the contextual framework of the study and highlights its relevance within current research trends. Added and corrected paragraphs are in red in the MS (added references are also in red in the References section)

Comment 2:

In last section of introduction, author should clearly mention objectives of this study along with novelty statement. Comparative analysis of the study with reference to recent research related to present research should be highlighted.

Response 2:

We agree with the reviewer. The final paragraph of the Introduction has been rewritten to clearly state the objectives of the study and to explicitly identify its novelty. The revised text (in red in the MS) now emphasizes the originality of evaluating a nacre-derived biomimetic organic/mineral material, alone and in combination with porous silicon microparticles, in an in vivo critical-size defect model.

Comment 3:

Materials and Methods Section: It is recommended to add information about all materials, their purity along with their source at the start of the Section.

Response 3:

We thank the reviewer for this suggestion. The Materials and Methods section has been revised to include a clearer description of all materials used, their source, and relevant specifications. Especially, processing details are now indicated for nacre-derived materials to improve transparency and reproducibility.

Comment 4:

Characterization: Characterization section is incomplete, please provide more detailed physicochemical characterization of nacre proteins and pSi-MP (composition, molecular weight distribution, porosity, surface chemistry, and degradation behavior, etc.). Either provide this information or provide a suitable reason for not adding this important information.

Response 4:

We acknowledge the reviewer’s concern. As clarified in the revised manuscript, the study was not designed to introduce or optimize new materials, but rather to assess the in vivo biological response to a biomimetic nacre-derived organic/mineral fraction combined with a well-established porous silicon platform.

Extensive physicochemical characterization of similar nacre-derived materials and porous silicon microparticles has been reported previously and is now appropriately cited, and is also included in the discussion. The rationale for not performing additional characterization within the present study has been explicitly stated, and this limitation is acknowledged in the Discussion.

Comment 5:

In materials section, author should clearly mention all materials, manufacturing details and strategies to synthesize. Also add the information of standard test methods that are used in this study.

Response 5:

We agree and thank the reviewer for this comment. The Materials section has been expanded to clearly describe material preparation steps, processing strategies, and experimental procedures. Standard methodologies used for in vivoimplantation, histology, and µCT analysis are now explicitly referenced or described to improve methodological clarity. A specific paragraph “Statistical analysis” has been added.

Comment 6:

After materials section, author should put characterizations details in a paragraph along with standards, if authors perform any physiochemical or any other characterization.

Response 6:

We thank the reviewer for this suggestion. As explained in Response 4, the study was not designed to introduce or optimize new materials, but clarifications have been added in the Discussion section, to indicate the properties that were supported by previously published data. We hope that this structure improves readability and avoids ambiguity regarding the experimental scope.

Comment 7:

Article lacks the information about the dose and ratio optimization; please justify how did concentration and nacre–pSi ratios were selected.

Response 7:

We appreciate this comment. The selection of nacre-derived material and pSi microparticle ratios was based on prior in vitro and in vivo feasibility studies reported in the literature, as well as preliminary handling considerations to ensure defect filling and material stability. The study was not designed as a dose-optimization investigation, and this point is now clearly stated as a limitation in the Discussion, with dose optimization identified as an important perspective for future work.

Comment 8:

Comparison: did the authors carry out comparative study with clinically relevant bone graft materials (e.g., autograft or commercial substitutes)?

Response 8:

We thank the reviewer for this relevant question. No comparative study with clinically approved bone graft materials was performed in the present work, but we had investigated this in a previously published work (Renaud et al., 2023). This limitation is now explicitly acknowledged in the Discussion. The study was designed as an exploratory comparison between nacre-derived material, porous silicon, and their combination within a controlled experimental framework, rather than as a direct clinical benchmarking study.

Comment 9:

Reference: Most of the references are not latest; addition of latest research references (2021–2025) would enhance the significance.

Response 9:

We agree with the reviewer. The reference list has been updated to include additional recent publications (2021–2025), with 5 new references relevant to biomimetic bone regeneration, composite biomaterials, and nacre-inspired strategies.

Comment 10:

There are minor grammatical mistakes that needs a revision.

Response 10:

We thank the reviewer for this remark. The manuscript has undergone careful language revision to correct grammatical issues and improve clarity and readability.

Reviewer 4 Report

Comments and Suggestions for Authors

Authors evaluated the bone regenerative capacity of natural nacre proteins alone and combined with oxidized porous silicon microparticles (pSi-MP) in rat caudal vertebrae defects. The nacre-pSi composite showed superior bone regeneration compared to individual components, demonstrating synergistic effects. Micro-CT and histological analyses revealed enhanced bone mineral density and integration. Comments,

1. What specific mechanisms explain the observed synergy between nacre proteins and pSi?
2. How does the osteogenic potential of natural nacre proteins compare to synthetic BMPs?
3. What are the long-term degradation profiles of the composite material?
4. How would mechanical loading affect the regeneration outcomes?
5. Can you elaborate on the potential immunogenicity of the materials used?
6. How might the findings apply to different bone defect types or locations?
7. What are the limitations of using a non-load-bearing model for this study?
8. How might the results differ in older or osteoporotic animal models?

Author Response

Comment 1:

What specific mechanisms explain the observed synergy between nacre proteins and pSi?

Response 1:

We thank the reviewer for this insightful question. While the present study was not designed to elucidate molecular mechanisms, the observed synergy can be interpreted as the result of complementary biological and structural functions. The nacre-derived organic fraction provides bioactive cues that may promote osteogenic cell recruitment and differentiation, whereas porous silicon microparticles act as a resorbable scaffold offering high surface area, spatial confinement, and progressive degradation. Their combination likely enhances local retention and presentation of bioactive components within the defect site. This mechanistic interpretation has now been more clearly articulated in the Discussion as a working hypothesis, with addition of a specific paragraph (in red in the MS).

Comment 2:

How does the osteogenic potential of natural nacre proteins compare to synthetic BMPs?

Response 2:

We thank the reviewer for this relevant comparison. The manuscript has been revised to clarify that nacre-derived materials are not intended to replicate the potency or specificity of synthetic BMPs. Rather, they represent a biomimetic, multi-component bioactive system that may support bone regeneration through moderate, physiologically inspired signaling rather than strong single-factor induction. This distinction, along with the respective advantages and limitations of nacre-derived materials versus BMP-based approaches, is now discussed in the revised Discussion, with a extended paragraph added in the discussion “4.5. Relevance to the broader nacre literature and comparison with BMP-based strategies”

Comment 3:

What are the long-term degradation profiles of the composite material?

Response 3:

We appreciate this comment. Long-term degradation profiles were not directly assessed in the present study. However, porous silicon biodegradation kinetics and biocompatibility have been extensively reported in the literature, and these references have now been included. The degradation behavior of the nacre-derived mineral and organic components is also discussed based on existing studies. The lack of long-term degradation assessment is now explicitly acknowledged as a limitation and identified as a priority for future investigations.

Comment 4:

How would mechanical loading affect the regeneration outcomes?

Response 4:

We thank the reviewer for raising this important point. The present study was conducted in a non-load-bearing vertebral defect model, which was selected to evaluate biological compatibility and regenerative potential in a controlled environment. Mechanical loading is expected to significantly influence remodeling, maturation, and alignment of newly formed bone. This limitation is now clearly stated, and the potential impact of mechanical loading is discussed as a key perspective for future studies using load-bearing models.

Comment 5:

Can you elaborate on the potential immunogenicity of the materials used?

Response 5:

We thank the reviewer for this question. The manuscript has been revised to clarify that no overt inflammatory response was observed in standard histological sections, while avoiding definitive claims regarding immunogenicity. In addition, the Discussion now includes references to published studies reporting good biocompatibility and low immunogenicity of both nacre-derived materials and porous silicon. The absence of dedicated immunological analyses is acknowledged as a limitation.

Comment 6:

How might the findings apply to different bone defect types or locations?

Response 6:

We appreciate this comment. The Discussion has been expanded to address the potential applicability of the findings to other bone defect types and anatomical locations. While the current results are specific to a non-load-bearing vertebral defect, the biomimetic principles underlying the composite material may be relevant to other contexts, with appropriate adaptation of material formulation, defect size, and mechanical environment.

Comment 7:

What are the limitations of using a non-load-bearing model for this study?

Response 7:

The limitations associated with the non-load-bearing model, including the inability to assess mechanical competence and long-term functional performance, are now explicitly discussed. This clarification helps contextualize the scope and translational relevance of the study.

Comment 8:

How might the results differ in older or osteoporotic animal models?

Response 8:

We thank the reviewer for this forward-looking question. The Discussion now includes a paragraph addressing how age-related or osteoporotic conditions could alter the regenerative response, potentially affecting cellular recruitment, remodeling kinetics, and material–tissue interactions. These aspects are presented as important directions for future research.

Reviewer 5 Report

Comments and Suggestions for Authors

In this manuscript, the authors investigate the effect of “nacre proteins” alone and in combination with oxidized porous silicon microparticles (pSi-MP) on bone regeneration in a rat caudal vertebra critical-size defect model, using µCT and non-decalcified histology to evaluate healing after 60 days. The composite group (nacre + pSi-MP) appears to show more complete bridging and higher BMD than nacre or pSi-MP alone, with good local biocompatibility and no apparent inflammation. Overall, the manuscript is clearly written and well-structured. However, there are some concerns before the publication.

1. Section 2.1 describes preparing whole nacre powder (1–5 µm) and states that this “whole nacre powder—containing the natural water-soluble protein fraction—was used for all in vivo experiments.” That is not “purified nacre proteins” or even a defined “protein extract”; it is mineral (CaCO₃) + organics. This is a big issue because the mineral phase itself can act as an osteoconductive filler, and also strongly affects µCT attenuation.
2. In Fig. 3 the initial µCT images show a bright radiopaque mass in the nacre and pSi–nacre groups. At 2 months, part of the measured density may still come from residual nacre CaCO₃ and/or pSi rather than new bone. This is the central quantitative problem: you are interpreting higher BMD as “more bone”, but the materials themselves contribute to attenuation.
3. The study uses 6 adult male Wistar rats, with 4 defects per tail, and four treatment conditions (control, nacre, pSi-MP, nacre + pSi-MP). It is implied that each rat received one defect for each condition, which is a sensible way to reduce inter-animal variability. However, this design makes the animal, not the individual defect, as the true experimental unit.

The statistical analysis is not described at all (type of test, whether you averaged per animal, treatment of repeated measures). In Fig. 3 you show a BMD histogram with “(*) p<0.05” but no information on n, dispersion (SD/SEM), or what comparison was tested.

If all four conditions are present in each rat, then the analysis should use either per-animal averages (n=6 per group) or a mixed-effects model accounting for within-animal correlation. Treating each of the 24 defects as independent would artificially inflate n and risk pseudoreplication.

4. Fig. 2 shows histology for pSi and nacre, but I did not see representative histology for control and especially the composite group in comparable format/low magnification overview. If composite is the key result, it must be shown clearly.
5. For pSi: particle size distribution, porosity/surface area, oxidation confirmation (FTIR/XPS), and sterilization approach are not described. For nacre powder: how was protein content measured (10% w/w seems high compared with commonly cited nacre organic fraction), and how did you sterilize the powder? Also, you must report the mass or volume loaded per defect for each condition; “filled with nacre” is not reproducible.
6. The µCT section is quite brief. For reproducibility, more detail is needed: acquisition parameters, Reconstruction and segmentation and quantitative outputs.

Author Response

Comment 1:

Section 2.1 describes preparing whole nacre powder (1–5 µm) and states that this “whole nacre powder—containing the natural water-soluble protein fraction—was used for all in vivo experiments.” That is not “purified nacre proteins” or even a defined “protein extract”; it is mineral (CaCO₃) + organics. This is a big issue because the mineral phase itself can act as an osteoconductive filler, and also strongly affects µCT attenuation.

Response 1:

We thank the reviewer for this critical and well-founded comment. We fully agree that the material used in this study does not correspond to purified nacre proteins or to a defined protein extract. Rather, it consists of a nacre-derived organic fraction embedded within its native mineral matrix, intentionally preserved to maintain biomimetic integrity.

To address this concern, the manuscript has been carefully revised to clarify the nature of the nacre-derived material throughout the text. The terminology “natural nacre proteins” has been replaced where appropriate with more accurate descriptions, and the contribution of the mineral phase is now explicitly acknowledged. We agree that the CaCO₃ component may contribute to osteoconduction and radiopacity, and this aspect is now discussed as an integral part of the biomimetic design rather than a confounding factor.

A complete paragraph has been added in the Materials & Methods section, and it has also been explicated in the Discussion section (“In the present model, the mineral phase of the nacre-based material was considered an integral component of the biomimetic design, contributing to osteoconduction and radiodensity rather than representing a confounding factor.”) (in red in the MS). The title has also been modified accordingly.

Comment 2:

In Fig. 3 the initial µCT images show a bright radiopaque mass in the nacre and pSi–nacre groups. At 2 months, part of the measured density may still come from residual nacre CaCO₃ and/or pSi rather than new bone. This is the central quantitative problem: you are interpreting higher BMD as “more bone”, but the materials themselves contribute to attenuation.

Response 2:

We thank the reviewer for highlighting this very important limitation. We fully agree that µCT-derived BMD values in this study represent apparent mineral density, and that part of the attenuation signal may originate from residual mineral phases of nacre and/or porous silicon, particularly at early and intermediate stages of healing.

The manuscript has been revised to explicitly acknowledge this limitation. The interpretation of µCT data has been softened accordingly, and BMD is now discussed in conjunction with qualitative parameters such as defect bridging, spatial distribution of mineralized tissue, and histological observations. This clarification ensures a more cautious and accurate interpretation of the quantitative data. A full paragraph has been added at the beginning of the Discussion section to address this issue (“4.1. Interpretation of bone mineral density (BMD) in the presence of mineral biomaterials”)

Comment 3:

The study uses 6 adults male Wistar rats, with 4 defects per tail, and four treatment conditions. This design makes the animal, not the individual defect, the true experimental unit. The statistical analysis is not described at all and may suffer from pseudo replication.

Response 3:

We thank the reviewer for this insightful methodological observation. The experimental design indeed involved multiple defects within the same animal, with each treatment condition represented per animal to minimize inter-animal variability. To address this point, a specific “Statistical Analysis” paragraph has been added to clarify the analytical approach, and the exploratory nature of the quantitative comparisons is now explicitly stated.

Comment 4:

Fig. 2 shows histology for pSi and nacre, but I did not see representative histology for control and especially the composite group in comparable format.

Response 4:

We thank the reviewer for this important comment regarding histological presentation. In the present study, histological analysis was primarily intended as a qualitative and illustrative approach to visualize material–tissue interactions, rather than as a comprehensive comparative assessment across all experimental groups. In this context, representative sections from the pSi-alone and nacre-alone groups were selected to illustrate the distinct degradation behaviors of these materials and their respective integration within newly forming bone tissue. We chose to focus on the two individual material conditions to clearly illustrate how pSi and nacre-based materials interact with the surrounding tissue during degradation and bone formation.

To further support interpretation of tissue organization around degrading particles, we have added Sirius Red staining images, allowing improved visualization of collagen deposition and organization in the vicinity of the materials. The key conclusions regarding the composite group are therefore drawn primarily from µCT analyses and overall defect bridging, with histology serving as a complementary qualitative illustration of material–tissue interactions rather than as a direct quantitative comparison between all groups.

Comment 5:

For pSi: particle size distribution, porosity/surface area, oxidation confirmation, and sterilization approach are not described. For nacre powder: how was protein content measured, and how did you sterilize the powder? Also, you must report the mass or volume loaded per defect.

Response 5:

We thank the reviewer for this detailed comment. The Materials and Methods section has been expanded to provide additional details regarding pSi microparticle preparation, including particle size range, porosity, pores diameter, and sterilization procedure (in red in the MS). References to prior detailed characterizations of similar pSi platforms have been added.

For the nacre-derived material, the sterilization method is now specified, and an extended paragraph has been added to describe the methodology of nacre recovery and characterization. Finally, the volume of material implanted per defect for each experimental condition is now clearly reported to improve reproducibility (in red in the MS; 20µL per defect, corresponding to the drilled defect of approximately 19mm³).

Comment 6:

The µCT section is quite brief. For reproducibility, more detail is needed: acquisition parameters, reconstruction, segmentation, and quantitative outputs.

Response 6:

We have revised the µCT methodology section to include detailed acquisition parameters, reconstruction settings, segmentation thresholds, and the quantitative outputs used for analysis. These additions improve methodological transparency and reproducibility (in red in the MS).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revised manuscript could be accepted for publication. The only concern is the 29% similarity reported by iThenticate. This should be addressed and corrected prior to publication.

All the best in the New Year!

Author Response

Comment 1: The revised manuscript could be accepted for publication. The only concern is the 29% similarity reported by iThenticate. This should be addressed and corrected prior to publication.

Response 1: We thank the reviewer for their precious time spent to review our manuscript.

We have checked the manuscript with antiplagiat software (Compilatio Magister). For the main text (excluding references and affiliation), we obtained 1% similarity.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is acceptable for publication.

Author Response

Comment 1: The manuscript is acceptable for publication.

Response 1: We thank the reviewer for their precious time spent to review our manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

Authors have addressed the comments to my satisfaction. No further corrections required.

Author Response

Comment 1: Authors have addressed the comments to my satisfaction. No further corrections required.

Response 1: We thank the reviewer for the time used to review our manuscript

Reviewer 5 Report

Comments and Suggestions for Authors

The authors have effectively addressed all previous comments. The manuscript is now improved in terms of clarity, accuracy, and completeness. Thus, I recommend acceptance of this revised manuscript.

Author Response

Comment 1: The authors have effectively addressed all previous comments. The manuscript is now improved in terms of clarity, accuracy, and completeness. Thus, I recommend acceptance of this revised manuscript.

Response 1: We thank the reviewer for the time spent to review our manuscript