Advances in Decellularization of Fish Wastes for Extracellular Matrix Extraction in Sustainable Tissue Engineering and Regenerative Medicine

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review addresses an increasingly relevant topic: valorizing fish-processing by-products as sources of decellularized extracellular matrix (dECM) for tissue engineering, and it attempts to cover decellularization approaches, downstream processing, functionalization, and representative biomedical applications.

However, in its current form the manuscript reads as “more descriptive than analytical”, and it has some issues in structure, terminology consistency, and English presentation. Several claims and sections would benefit from tighter logic, clearer sourcing, and more fish-specific synthesis.

1. The manuscript states it provides a “comprehensive examination” and even cites an estimated share of fish-focused studies (e.g., “~7% … from 2022 to 2024”), but there is no transparent description of how literature was identified/selected (databases, keywords, inclusion/exclusion, timeframe, screening). Please add a brief “Methods” paragraph (even for a narrative review) or a PRISMA-style summary if the intent is systematic.
2. Please more explicitly define what is meant by “fish wastes” in the scope (skin, scales, bones, viscera, swim bladder, etc.) and whether the review targets waste-derived tissues only or fish tissues broadly.
3. A core message is that protocols are not standardized. That is true, but the review would be much more valuable if it proposes a **standard reporting checklist** for fish dECM studies (e.g., tissue preprocessing, decellularization parameters, washing strategy, residual detergent quantification, DNA metrics, histology, ECM composition retention, mechanical testing, cytocompatibility, and sterilization validation).
4. Tables are a strength (protocol summaries, pros/cons). Consider adding one “high-impact” summary table that compares fish tissue type → best-performing protocol families → key parameters → typical pitfalls → recommended characterization endpoints.
5. The manuscript transitions from Section “3.2 Physical Disinfection/Sterilization” and then later introduces “4. Sterilization and Disinfection…” but the content that follows is actually about “preservation” (freeze-drying/cryopreservation/immersion). Please correct section titles/numbering so the outline matches the content. Also, please standardize capitalization/terminology in headings (e.g., “DECM” vs “dECM”).
6. The “clinical translation” discussion is currently high-level. Please add concrete considerations relevant to fish-derived biomaterials: batch variability (species/season/processing), endotoxin/bioburden control, residual detergent risk (especially after SDS use), sterility assurance level considerations, and regulatory expectations for implantable biomaterials. The sterilization section mentions SAL 10^-6—this is good; expand on how studies should validate sterility without compromising ECM bioactivity.
7. Some comparative statements need clearer framing. For example, the text implies fish dECM reduces risk of mammalian pathogens (BSE, swine influenza, PERV), which is reasonable as a “comparison”, but the sentence is written in a way that could be misread as fish transmitting those pathogens. Please rephrase.
8. Multiple figures appear adapted from a single review source (Gadre et al., 2024). Consider replacing at least some with original schematics created by the authors that better reflect “fish-waste-specific” workflows and decision points (e.g., tissue type → decell method → wash → sterilize → preserve → formulate). Ensure permissions/attribution comply with journal requirements.

 

Minor comments

* in Section 2: “The methods used include chemical, enzymatic, and physical approaches…” appears twice consecutively. Please remove one occurrence.

* Typo/terminology: “Freeze-Frying” should be **Freeze-drying**. Also spacing/punctuation: “[96].But …” needs a space.

* Typo in applications heading: “Bone Regeneration Cartillage Repair” → **Cartilage**; also the sentence “Bone and cartilage regeneration … due to the lack of vascularity and limited cell activity” is logically incorrect as written (these are reasons repair is difficult). Please revise.

* Consider revising ambiguous/contradictory phrasing: “lessen inflammatory responses” but “encourage pro-inflammatory cytokines” in the same sentence.

* Several sentences would benefit from grammar cleanup (examples include subject–verb agreement and missing articles), e.g., “Gamma Irradiation uses Cobalt-60 radioisotopes which a radioactive materials …”.

* Standardize formatting of species names (italicize Latin binomials consistently) and unify abbreviations (TX-100 vs Triton X-100; ScCO₂ vs ScCO2).

Author Response

Comments 1: The manuscript states it provides a “comprehensive examination” and even cites an estimated share of fish-focused studies (e.g., “~7% … from 2022 to 2024”), but there is no transparent description of how literature was identified/selected (databases, keywords, inclusion/exclusion, timeframe, screening). Please add a brief “Methods” paragraph (even for a narrative review) or a PRISMA-style summary if the intent is systematic.

Response 1: We thank the reviewer for emphasizing the importance of transparency in the literature identification and selection process. To address this concern, we have added a dedicated “Literature Search and Selection” paragraph at the end of Section 1 (line 121), outlining the databases consulted, keyword strategy, inclusion and exclusion criteria, screening approach, and the timeframe covered by the review.

Comments 2: Please more explicitly define what is meant by “fish wastes” in the scope (skin, scales, bones, viscera, swim bladder, etc.) and whether the review targets waste-derived tissues only or fish tissues broadly.

Response 2: The comment regarding scope clarification is well taken. In the revised manuscript, “fish wastes” are explicitly defined as commonly discarded by-products of fish processing, including skin, scales, bones, swim bladder, and other collagen-rich membranes. The review now clarifies that while emphasis is placed on waste-derived fish tissues because of their sustainability and circular-economy relevance, studies involving fish tissues more broadly are also included when they provide transferable insights into decellularization strategies and extracellular matrix preservation. This clarification has been incorporated into the Introduction (line 52) to clearly delineate the scope of the review.

Comments 3: A core message is that protocols are not standardized. That is true, but the review would be much more valuable if it proposes a **standard reporting checklist** for fish dECM studies (e.g., tissue preprocessing, decellularization parameters, washing strategy, residual detergent quantification, DNA metrics, histology, ECM composition retention, mechanical testing, cytocompatibility, and sterilization validation).

Response 3: The reviewer raises an important point regarding the need to move beyond identifying variability toward providing practical guidance for the field. In response, we have added a new Section 4: Proposed Framework for Standardized Reporting of Fish Tissue Decellularization (line 541), which introduces a minimum reporting checklist for fish-derived dECM studies. This framework, summarized in Table 6 (line 549), outlines essential parameters spanning tissue sourcing and preprocessing, decellularization and washing conditions, residual detergent and DNA assessment, extracellular matrix composition retention, mechanical characterization, cytocompatibility, and sterilization validation.

Comments 4: Tables are a strength (protocol summaries, pros/cons). Consider adding one “high-impact” summary table that compares fish tissue type → best-performing protocol families → key parameters → typical pitfalls → recommended characterization endpoints.

Response 4: The suggestion to include a single, high-impact synthesis table is appreciated. In response, we have added a new subsection, Section 2.4: Strategic Selection and Optimization Framework (line 347), which introduces Table 5 (line 358) as an integrative decision-making guide. This table maps major fish tissue types to their best-performing decellularization protocol families, key processing parameters, commonly reported pitfalls (e.g., detergent-induced collagen loss), and recommended characterization endpoints. By consolidating these elements, the table translates the reviewed literature into a practical framework that supports protocol selection, optimization, and cross-study comparability.

Comments 5: The manuscript transitions from Section “3.2 Physical Disinfection/Sterilization” and then later introduces “4. Sterilization and Disinfection…” but the content that follows is actually about “preservation” (freeze-drying/cryopreservation/immersion). Please correct section titles/numbering so the outline matches the content. Also, please standardize capitalization/terminology in headings (e.g., “DECM” vs “dECM”).

Response 5: We have revised the section titles and numbering to ensure that they accurately reflect the content discussed, with a clear distinction between sterilization/disinfection and preservation strategies such as freeze-drying, cryopreservation, and immersion. Specifically, both topics are now consolidated under Section 3, “Sterilization and Preservation of Fish-Derived dECM (line 368)” which is further organized into Section 3.1, “Sterilization Methods of Fish-Derived dECM (385)” and Section 3.2, “Preservation Methods of Fish-Derived dECM (line 453)”. In addition, capitalization and terminology have been standardized throughout the manuscript (e.g., consistent use of “dECM” in headings and text).

Comments 6: The “clinical translation” discussion is currently high-level. Please add concrete considerations relevant to fish-derived biomaterials: batch variability (species/season/processing), endotoxin/bioburden control, residual detergent risk (especially after SDS use), sterility assurance level considerations, and regulatory expectations for implantable biomaterials. The sterilization section mentions SAL 10^-6—this is good; expand on how studies should validate sterility without compromising ECM bioactivity.

Response 6: This comment prompted a substantial revision of the manuscript’s discussion on clinical translation. In response, the former Sections 3 and 4 were merged and fully restructured into a single, revised section (Section 3: Sterilization and Preservation of Fish-Derived dECM), now focused exclusively on fish-derived dECM and explicitly framed within the context of tissue engineering and regenerative medicine.

The revised section incorporates concrete clinical translation considerations specific to fish-derived biomaterials, including batch variability arising from species, seasonal, and processing differences; endotoxin and bioburden control; risks associated with residual detergents, particularly following SDS-based decellularization; sterility assurance level (SAL) requirements; and regulatory expectations for implantable biomaterials. In addition, a new subsection (Section 3.3: Clinical Translation Considerations for Fish-Derived dECM - line 489) was added to synthesize these issues, expand the discussion on sterility validation strategies that preserve ECM bioactivity, and highlight remaining challenges relevant to translational and regulatory pathways.

Comments 7: Some comparative statements need clearer framing. For example, the text implies fish dECM reduces risk of mammalian pathogens (BSE, swine influenza, PERV), which is reasonable as a “comparison”, but the sentence is written in a way that could be misread as fish transmitting those pathogens. Please rephrase.

Response 7: This comment identified an important issue regarding clarity in comparative risk statements. In response, we have carefully rephrased the relevant passages to explicitly frame pathogen-related comparisons as relative risk reductions compared with mammalian-derived biomaterials, rather than implying transmission risks associated with fish-derived dECM. The revised text now clearly distinguishes fish-derived sources from mammalian pathogens such as those associated with bovine or porcine tissues and avoids ambiguous wording that could lead to misinterpretation. These clarifications were applied consistently throughout the manuscript to improve precision and reader understanding.

Comments 8: Multiple figures appear adapted from a single review source (Gadre et al., 2024). Consider replacing at least some with original schematics created by the authors that better reflect “fish-waste-specific” workflows and decision points (e.g., tissue type → decell method → wash → sterilize → preserve → formulate). Ensure permissions/attribution comply with journal requirements.

Response 8: This comment is well taken. For Figure 1, the source acknowledgment has been clarified and expanded by compiling all relevant references from which the figure and its conceptual elements were adapted, including Gadre et al. (2024), Poo et al. (2021), and Dzobo et al. (2023), in full compliance with journal permission and citation requirements. In addition, Figure 2 has been replaced with an original schematic developed by the authors. This figure specifically illustrates a fish-waste-derived decellularized extracellular matrix (dECM) workflow, highlighting key decision points from tissue selection and decellularization methods through washing, sterilization, preservation, and formulation toward final biomedical applications.

Comments 9: in Section 2: “The methods used include chemical, enzymatic, and physical approaches…” appears twice consecutively. Please remove one occurrence.

Response 9: The duplicated sentence in Section 2 has been removed, and the text has been revised accordingly to eliminate redundancy at line 141.

Comments 10: Typo/terminology: “Freeze-Frying” should be **Freeze-drying**. Also spacing/punctuation: “[96].But …” needs a space.

Response 10: These typographical and formatting errors have been corrected in the revised manuscript, including replacing “Freeze-Frying” with “Freeze-drying” (line 459) and correcting spacing and punctuation.

Comments 11: Typo in applications heading: “Bone Regeneration Cartillage Repair” → **Cartilage**; also the sentence “Bone and cartilage regeneration … due to the lack of vascularity and limited cell activity” is logically incorrect as written (these are reasons repair is difficult). Please revise.

Response 11: The typographical error in the subsection heading has been corrected (“Cartillage” revised to “Cartilage”) (line 608). In addition, the sentence describing bone and cartilage regeneration was revised to clarify that the research interest in these tissues arises from their limited intrinsic healing capacity, which is attributed to poor vascularity and restricted cellular activity. The revised wording now accurately reflects the intended cause–effect relationship and improves clarity.

Comments 12: Consider revising ambiguous/contradictory phrasing: “lessen inflammatory responses” but “encourage pro-inflammatory cytokines” in the same sentence.

Response 12: This comment identified an instance of ambiguous and potentially contradictory phrasing. In response, the sentence was revised (line 573) to clarify the biological mechanism by replacing “encourage pro-inflammatory cytokines” with wording that reflects the immunomodulatory role of omega-3 polyunsaturated fatty acids in regulating inflammatory signaling. The revised text now avoids implying pro-inflammatory effects and more accurately represents the reported anti-inflammatory and pro-healing roles described in the cited literature.

[Revised sentence: These bioactive components are known to modulate inflammatory responses, regulate pro-inflammatory cytokine activity [16], enhance angiogenesis, and support granulation tissue formation [80,81].]

Comments 13: Several sentences would benefit from grammar cleanup (examples include subject–verb agreement and missing articles), e.g., “Gamma Irradiation uses Cobalt-60 radioisotopes which a radioactive materials …”.

Response 13: The entire manuscript was carefully edited to correct the subject–verb agreement, missing articles, and sentence structure, including the example noted for the description of gamma irradiation.

Comments 14: Standardize formatting of species names (italicize Latin binomials consistently) and unify abbreviations (TX-100 vs Triton X-100; ScCO₂ vs ScCO2).

Response 14: Formatting and terminology have been standardized throughout the manuscript. Latin binomial species names are now consistently italicized, and abbreviations have been unified to ensure consistency.

General Assessment: 

This review addresses an increasingly relevant topic: valorizing fish-processing by-products as sources of decellularized extracellular matrix (dECM) for tissue engineering, and it attempts to cover decellularization approaches, downstream processing, functionalization, and representative biomedical applications.

However, in its current form the manuscript reads as “more descriptive than analytical”, and it has some issues in structure, terminology consistency, and English presentation. Several claims and sections would benefit from tighter logic, clearer sourcing, and more fish-specific synthesis.

Response: 

We sincerely thank the reviewer for the thoughtful and constructive assessment of our manuscript and for recognizing the relevance of valorizing fish-processing by-products as sources of decellularized extracellular matrix (dECM) for tissue engineering.

We acknowledge the reviewer’s observation that the original manuscript was more descriptive than analytical and required improvements in structure, terminology consistency, English presentation, and fish-specific synthesis. Guided by these comments, we have undertaken substantial revisions to enhance analytical depth, strengthen logical organization, improve clarity of presentation, and provide more integrated, fish-focused synthesis across sections. We now believe that the revised manuscript more clearly reflects an analytical and application-driven review of fish-derived dECM.

Reviewer 2 Report

Comments and Suggestions for Authors

The review deals with an interesting approach to use decellularized fish wastes for extracellular matrix in tissue engineering. The review is well written and easy to read. 

There are several minor issues need to be addressed:

1. in the section to deal with decellularize fish wastes, parameters (conditions) used in the decellularization process should be included in the review. Although some of the conditions are listed in tables, not for all the methods.
2. Line 625 "microfluidic" should be removed from the text. 
3. Lines 713 to 719 should be removed. 
4. Overall, Section 7 should be shortened by at least 50%. 
5. Table 1 is only for one example. There must be a better way to show this one example.  

Comments on the Quality of English Language

some minor changes are still needed. The authors should go through the text to make the necessary changes. 

Author Response

Comments 1: In the section to deal with decellularized fish wastes, parameters (conditions) used in the decellularization process should be included in the review. Although some of the conditions are listed in tables, not for all the methods.

Response 1: We thank the reviewer for this critique. In response, Section 2 and the associated tables were revised and expanded to ensure that all decellularization approaches include key experimental conditions, such as reagent type and concentration, exposure time, temperature, and relevant processing steps.

Comments 2: Line 625 "microfluidic" should be removed from the text.

Response 2: The term “microfluidic” has been removed from the indicated line in the revised manuscript (now in line 671).

Comments 3: Lines 713 to 719 should be removed.

Response 3: Lines 713–719 have been removed from the revised manuscript as suggested.

Comments 4: Overall, Section 7 should be shortened by at least 50%.

Response 4: Section 7 has been substantially revised and condensed to improve focus and conciseness. Redundant descriptions were removed, and overlapping content was streamlined, resulting in a reduction of more than 50% while preserving the key conclusions and forward-looking perspectives.

Comments 5: Table 1 is only for one example. There must be a better way to show this one example. 

Response 5: Table 1 has been removed, and the relevant information has been incorporated directly into the main text of Section 2.1 (Physical Treatment) to improve clarity and flow.

General Assessment: The review deals with an interesting approach to use decellularized fish wastes for extracellular matrix in tissue engineering. The review is well written and easy to read.

Response: We appreciate the reviewer’s positive assessment of the manuscript and their recognition of the relevance of using decellularized fish wastes as sources of extracellular matrix for tissue engineering. We are encouraged that the review was found to be well written and easy to read, and we have further refined the manuscript to maintain clarity while strengthening analytical depth and synthesis in response to the specific comments provided below.

Reviewer 3 Report

Comments and Suggestions for Authors

Please see the file attached.

Comments for author File: Comments.pdf

Author Response

Comments 1: 

Significant Discrepancy Between the Abstract and the Main Text
The most critical issue is the pronounced inconsistency between the abstract and the body of the manuscript. The manuscript is structured into seven sections (1. Introduction; 2. Fish Tissue Decellularization Techniques; 3. Sterilization and Disinfection of Fish-Derived dECM; 4. Sterilization and Disinfection of Fish-Derived dECM [duplicate heading]; 5. Biomedical Applications of Fish-Derived dECM; 6. Advances in Post-Processing of FishDerived dECM; 7. Conclusion and Future Perspectives).

In the abstract, the authors state that a major goal of this review is “to maximize the potential of fish-derived dECM in regenerative medicine and tissue engineering,” which is likely to be the main point of interest for readers. However, this central theme is scarcely addressed in the main text. In particular, Sections 3 and 4 (which appear to concern long-term preservation and sterilization of dECM) have little relevance to regenerative medicine or tissue engineering applications, despite the strong emphasis placed on these fields in the abstract.

Response 1: 

In response to this insightful comment, the manuscript was substantially revised and reorganized to ensure that the central objective stated in the abstract; maximizing the potential of fish-derived dECM in regenerative medicine and tissue engineering, is clearly and consistently reflected throughout the main text.

To address this, the former Sections 3 and 4 were merged and fully rewritten as Section 3: Sterilization and Preservation of Fish-Derived dECM (line 368), eliminating the duplicate heading and re-framing the discussion away from procedural description toward regenerative performance, scaffold functionality, and translational relevance. The revised section now focuses exclusively on fish-derived dECM and explicitly discusses how sterilization and preservation strategies influence ECM integrity, biocompatibility, bioactivity retention, and downstream tissue engineering outcomes. In addition, a new subsection (Section 3.3: Clinical Translation Considerations for Fish-Derived dECM - line 489) was added to connect these processing steps directly to regenerative medicine applications and translational challenges.

Comments 2: 

Insufficient Depth and Imbalanced Coverage of Topics

Sections 5 and 6, which should form the core of the review, lack both quantitative and qualitative depth. The number of subheadings is limited, and the discussion within each subheading is superficial. Several important aspects that are critical for evaluating the biomedical potential of fish derived dECM are either underdeveloped or entirely omitted. Within Section 5, Subsections 5.1 (Wound Healing and Skin Regeneration) and 5.2 (Bone Regeneration and Cartilage Repair) are appropriate and potentially engaging. In contrast, Subsection 5.3 (3D Printing) introduces a completely different perspective and appears abruptly, without sufficient conceptual linkage to the preceding subsections. This topic would be more appropriate as an independent section rather than being placed alongside biological application-focused subsections.

Response 2: 

We sincerely thank the reviewer for this constructive and insightful comment, which helped us substantially strengthen the analytical depth, structure, and coherence of Sections 5 and 6.

In response, Sections 5 and 6 were extensively revised and expanded to improve both quantitative and qualitative depth and to establish clearer conceptual continuity across subsections. Specifically, we introduced comparative tables, integrative figures, and synthesis subsections that collectively strengthen the evaluation of the biomedical potential of fish-derived dECM and address the reviewer’s concerns regarding superficial discussion and imbalanced coverage.

Within Section 5, quantitative and qualitative depth was enhanced by the inclusion of Table 7 (line 562), which systematically summarizes reported quantitative outcomes, biological advantages, and key limitations of fish-derived dECM across major biomedical applications (Section 5, opening paragraph). In addition, Figure 6 provides a qualitative, application-driven comparison of bioactivity, mechanical demand, structural stability, and translational readiness across wound healing, skin grafting, bone regeneration, cartilage repair, and biofabrication. These additions are synthesized and critically interpreted in the newly added Section 5.4 (Comparative Performance and Application-Driven Insights), which explicitly evaluates where fish-derived dECM performs effectively, where it remains constrained, and how application-specific requirements govern its suitability.

Regarding Subsections 5.1 and 5.2, the discussions on wound healing, skin regeneration, bone regeneration, and cartilage repair were expanded to include clearer mechanistic explanations, quantitative performance indicators, and explicit identification of biological–mechanical mismatches, thereby addressing the reviewer’s concern regarding insufficient depth (Sections 5.1 and 5.2).

With respect to Subsection 5.3 (3D Printing), we acknowledge the reviewer’s concern regarding its abrupt introduction and conceptual divergence from biologically focused subsections. To address this, Subsection 5.3 was substantially revised to explicitly position 3D printing and biofabrication as enabling technologies rather than independent biomedical applications. The revised text clarifies the role of biofabrication in overcoming the intrinsic mechanical and processing limitations of fish-derived dECM, thereby providing a clear conceptual bridge between biological performance (Sections 5.1–5.2) and the post-processing strategies discussed in Section 6. This linkage is explicitly articulated at the end of Subsection 5.3 and further reinforced in Section 5.4.

In Section 6, depth and balance were similarly strengthened through the introduction of Table 8 (line 752), which provides a comparative, quantitative overview of post-processing strategies, targeted limitations, reported performance improvements, and associated trade-offs. In addition, Figure 7 presents a conceptual engineering trade-off framework that qualitatively illustrates how crosslinking, polymer–dECM composites, and advanced biofabrication strategies balance mechanical reinforcement, bioactivity preservation, and translational complexity. These elements are integrated and critically discussed in the newly added Section 6.3 (Engineering Trade-Offs and Design Considerations).

Comments 3:

Lack of Synthesis Across Studies
Throughout the manuscript, particularly in Sections 5 and 6, the discussion largely consists of a sequential listing of individual studies (e.g., “Author A reported…,” “Author B demonstrated…”). There is little attempt to synthesize findings across studies, identify overarching trends, reconcile conflicting results, or highlight unresolved questions in the field. Consequently, readers are left without a clear understanding of what conclusions can be drawn from the accumulated literature.

Response 3: 

We agree that meaningful synthesis across studies is essential for a review article to provide clear conclusions and forward-looking insights, rather than a purely descriptive summary of the literature. In response, we have substantially revised Sections 5 and 6 to move beyond sequential reporting of individual studies and to explicitly integrate findings, identify overarching trends, reconcile limitations, and highlight unresolved challenges in the field.

In Section 5, synthesis across studies was strengthened through the introduction of Table 7, which consolidates quantitative outcomes, biological advantages, and major limitations of fish-derived dECM across multiple biomedical applications. This table enables direct cross-comparison of reported performance metrics and facilitates identification of recurring trends and constraints that are not apparent when studies are discussed individually. Furthermore, the newly added Section 5.4 (Comparative Performance and Application-Driven Insights) explicitly synthesizes evidence across wound healing, skin grafting, bone regeneration, cartilage repair, and biofabrication applications. Rather than reiterating individual reports, this subsection evaluates collective trends, such as the strong alignment of fish-derived dECM with soft tissue repair and the recurring mechanical and compositional limitations observed in load-bearing applications.

Qualitative synthesis was further enhanced by Figure 6, which visually integrates multiple performance dimensions—bioactivity, mechanical demand, structural stability, and translational readiness—across applications. This figure, together with the accompanying discussion in Section 5.4, provides readers with a clear, integrative framework for understanding where fish-derived dECM performs consistently well, where outcomes are constrained, and why these differences arise across tissue types.

In Section 6, synthesis across post-processing studies was similarly reinforced. Table 8 was added to summarize post-processing strategies, targeted limitations, reported quantitative improvements, and associated trade-offs across multiple studies, allowing readers to identify common patterns rather than isolated results. In addition, Figure 7 presents a conceptual engineering trade-off framework that integrates findings across crosslinking, polymer–dECM composites, and advanced biofabrication approaches. This figure highlights the overarching trend that no single post-processing strategy simultaneously optimizes mechanical reinforcement, bioactivity preservation, and translational feasibility.

To further address the reviewer’s concern, a new Section 6.3 (Engineering Trade-Offs and Design Considerations) was added to explicitly synthesize outcomes across studies, reconcile contrasting results, and emphasize unresolved challenges such as balancing crosslinking density with cell infiltration, polymer reinforcement with ECM cue preservation, and fabrication complexity with clinical translatability.

Comments 4:

Conceptual Inconsistencies in Section 6
In Section 6, the relationship between the section title (Advances in PostProcessing of Fish-Derived dECM) and its subsections is unclear. Subsection 6.1 (Crosslinking for Surface Modification) and Subsection 6.2 (Polymer–dECM Composites Fabrication) do not align well with the stated scope of the section. In particular, Subsection 6.2 discusses content that is largely unrelated to fish-derived dECM, further diluting the focus of the review.

Response 4: 

We agree and appreciate the reviewer for this constructive comment, which highlighted the need for clearer conceptual alignment between the title of Section 6 and its subsections. In response, Section 6 was comprehensively revised to explicitly frame all subsections within the scope of post-processing strategies applied to fish-derived dECM, thereby improving coherence and focus.

First, the opening paragraph of Section 6 was rewritten to clearly define post-processing as encompassing surface crosslinking, chemical stabilization, and polymer hybridization strategies specifically employed to restore or enhance the functional performance of fish-derived dECM following decellularization. This revised introduction explicitly introduces Table 8, which summarizes post-processing approaches, targeted limitations, quantitative performance improvements, and trade-offs, thereby establishing a clear conceptual framework for the subsections that follow (Section 6, opening paragraph).

Subsection 6.1 (Crosslinking for Surface Modification) was refined to emphasize crosslinking as a post-decellularization intervention directly applied to fish-derived dECM to improve mechanical stability, regulate degradation, and mitigate immunogenicity. The discussion was streamlined to focus on crosslinking strategies reported for fish-derived ECM systems, with particular emphasis on EDC/NHS-based approaches and their documented effects on mechanical performance, porosity, and biocompatibility (Section 6.1).

Regarding Subsection 6.2 (Polymer–dECM Composites Fabrication), we acknowledge the reviewer’s concern that the previous version lacked sufficient focus on fish-derived dECM. To address this, the subsection was substantially revised to ensure that all examples and discussions explicitly involve fish-derived dECM or fish-derived collagen-based systems integrated with polymeric matrices. The revised text now frames polymer–dECM composites as a post-processing strategy aimed at overcoming the inherent mechanical weakness, rapid degradation, and limited processability of fish-derived dECM. Non-relevant or overly general polymer hydrogel content was removed or reframed to maintain a consistent focus on fish-derived matrices (Section 6.2).

Furthermore, to strengthen conceptual continuity and avoid dilution of scope, a new Section 6.3 (Engineering Trade-Offs and Design Considerations) was added. This subsection synthesizes findings across post-processing strategies and, together with Figure 7, explicitly illustrates how crosslinking, polymer–dECM composites, and advanced fabrication approaches represent distinct but complementary post-processing pathways for optimizing fish-derived dECM performance. This integrative framework reinforces the internal consistency of Section 6 and aligns all subsections with the section’s stated theme.

Comments 5:

Weak Conclusion and Limited Forward-Looking Perspective
The concluding section primarily reiterates points already described in the main text and fails to provide a meaningful synthesis or critical outlook. Despite the statement that “fish-derived ECM has demonstrated significant potential as a biomaterial in various biomedical applications,” this claim is not adequately substantiated or critically evaluated in the conclusion. Moreover, the manuscript does not clearly articulate future challenges, unresolved issues, or specific directions for future research.

Response 5:

The reviewer’s observation regarding the limited synthesis and forward-looking perspective of the original conclusion is well taken. In response, the Conclusion and Future Perspectives section has been substantially revised to move beyond reiteration of earlier content and instead provide a clear integrative synthesis of the review’s key findings.

The revised conclusion now explicitly articulates future challenges, unresolved issues, and priority research directions relevant to the biomedical translation of fish-derived dECM. These include the need for standardized decellularization and characterization protocols, improved control of mechanical stability and degradation behavior, optimization of sterilization strategies that preserve ECM bioactivity, and the development of application-specific post-processing and composite design frameworks. The conclusion also highlights sustainability-driven research opportunities and regulatory considerations that remain underexplored in current literature.

General assessment:

This narrative review manuscript aims to comprehensively summarize recent progress in the decellularization of fish-derived tissues and wastes, with a particular focus on decellularization strategies, extracellular matrix preservation, and emerging applications of fish-derived dECM in regenerative medicine and tissue engineering. However, there is a substantial mismatch between the title, the stated aims, and the actual content of the manuscript. The overall scope is unclear and excessively broad, making it difficult to identify the central question or objective of the review. As a result, the manuscript lacks a coherent narrative framework and does not meet the standards expected of a high-quality narrative review. For these reasons, I regret recommending rejection.

Response: 

We appreciate the reviewer’s candid and detailed assessment of the manuscript. We acknowledge that the original submission exhibited weaknesses in clearly defining its scope, aligning the title and stated aims with the core content, and maintaining a coherent narrative framework. In particular, the reviewer’s comments underscored the need to move beyond a broad, descriptive compilation toward a more focused and integrative narrative review.

In response, the manuscript has undergone substantial restructuring and revision. The scope has been clarified and refined to emphasize fish-derived dECM from processing by-products within a tissue engineering and regenerative medicine context. Key sections were reorganized to strengthen conceptual continuity, analytical synthesis across studies was added, and application-driven and design-oriented perspectives were explicitly incorporated. Comparative tables, conceptual figures, and synthesis subsections were introduced to improve coherence and to articulate clear conclusions and translational insights rather than isolated summaries.

We believe that these comprehensive revisions have significantly improved the alignment between the title, objectives, and content of the manuscript, and that the revised version now presents a clearer, more focused, and analytically driven narrative consistent with the standards of a high-quality review article for Bioengineering.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

the authors have addressed my concerns.

Author Response

Comments 1: the authors have addressed my concerns.

Response 1: We sincerely thank the reviewer for the valuable comments, which have significantly improved the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Please see the attachment

Comments for author File: Comments.pdf

Author Response

Comments 1: Section 4: Table 6 is shown as a Checklist for standardization for fish-derived dECM scaffolds, which is unusual and should be justified. Thus, this section is not necessary.
Response 1: Thank you for the comment. We agree that presenting Table 6 as a checklist is unconventional in this context. Therefore, we have removed Table 6 and Section 4 accordingly to avoid redundancy and improve the overall coherence of the manuscript.

Comments 2: Section 5: Table 7 does not provide sufficient information. The authors have attempted to summarize the changes in biological effects resulting from various decellularization treatments in a table using symbols (e.g., ↑, ↓). While visual summaries are generally helpful, the current presentation is problematic for the following reasons:

1. Limited Evidence and Overgeneralization: The trends indicated in the table are supported by only a few references (1–2 papers). Relying on such a limited number of sources to define "universal" trends is scientifically risky and lacks the rigor expected of a narrative review.
2. Lack of Specificity to Fish-derived dECM: Many of the described changes appear to be general phenomena observed in various mammalian tissues rather than characteristics unique to fish-derived dECM. As a review focused on fish wastes, it is essential to highlight how these specific biological sources respond differently—or similarly—to treatments compared to conventional sources.
3. Oversimplification of Data: Using only arrows (↑/↓) to represent complex material behaviors obscures crucial quantitative details and potential contradictions in existing literature. A narrative review should critically synthesize conflicting data rather than merely categorizing it into binary trends.
4. The authors should conduct a more extensive literature search to broaden the evidence base for this table. Furthermore, please provide a more nuanced discussion in the text regarding how treatment parameters influence these properties.

Response 2: 

1. We thank the reviewer for this important observation and agree that the original presentation risked implying generalized trends that were not sufficiently supported by the available evidence. In the revised manuscript, we have substantially revised Table 7 and the associated text in Section 5 to address this concern. Specifically, the arrow-based symbols (e.g., ↑/↓), which could be interpreted as defining universal or consensus trends, have been removed. Table 7 has been reconceptualized from a trend-defining summary into a comparative evidence map that reports context-dependent observations drawn from individual studies rather than generalized outcomes. Language implying uniform or universal biological responses has been explicitly eliminated. In addition, the description following Table 7, as well as the narrative in Section 5.4, now explicitly emphasizes that the reported outcomes are derived from limited and heterogeneous datasets and should be interpreted as context-dependent observations rather than universal trends. A synthesis paragraph has been added in Section 5.4 (Comparative Performance and Application-Driven Insights) to explicitly discuss why the current evidence base does not justify universal conclusions and to frame Table 7 as a qualitative reference rather than a prescriptive guideline. 
2. We agree with the reviewer that a clear distinction between fish-derived dECM behavior and general mammalian dECM phenomena is essential for a review focused on fish wastes. In the revised manuscript, both Table 7 and the surrounding narrative in Section 5 have been explicitly revised to ensure that all reported observations are clearly anchored to fish-derived tissues and matrices, rather than inferred from mammalian systems. 

   Specifically, Table 7 now explicitly identifies the fish tissue source and matrix type (e.g., fish skin ECM, fish-derived DBM, fish cartilage dECM) associated with each reported biological outcome. This ensures that the summarized observations reflect responses reported for marine-derived matrices, including fish skin, scales, bone, and cartilage, rather than generalized dECM behavior.

   In the text immediately following Table 7 and in the synthesis presented in Section 5.4, we now explicitly distinguish between outcomes that are reported for fish-derived dECM and those that are mechanistically similar to trends described in mammalian systems, clarifying that such similarities are discussed for comparative context rather than as fish-unique characteristics. Where biological responses appear to depend on factors such as glycosaminoglycan retention, lipid preservation, mineral integrity, or decellularization severity, these dependencies are described as reported sensitivities specific to the fish-derived matrices studied, rather than universal mechanisms.

   Where fish-specific evidence remains limited or restricted to a small number of studies, this is now explicitly acknowledged as a knowledge gap, consistent with the scope and intent of a narrative review.

3. We agree with the reviewer that the original arrow-based representation risked oversimplifying complex and context-dependent material behaviors. In response, we have removed the use of directional symbols (↑/↓) from Table 7 and revised both the table and the associated narrative to better reflect the heterogeneity and, where applicable, the conflicting nature of reported findings.

   In the revised Table 7, binary indicators have been replaced with descriptive summaries of reported observations, explicitly stating the biological outcome assessed, the experimental context (e.g., in vitro vs. in vivo), and the processing sensitivities associated with each observation. This revision avoids collapsing diverse outcomes into categorical trends and instead presents the reported behaviors as study-specific observations.

   In addition, the text immediately following Table 7 and the synthesis in Section 5.4 have been revised to explicitly discuss parameter-dependent variability and reported contradictions, such as differences arising from decellularization chemistry, exposure severity, and tissue-specific susceptibility. Rather than resolving these differences into simplified trends, the revised narrative critically interprets them as evidence of trade-offs and context-dependent performance, which is consistent with the goals of a narrative review.
4. We agree with the reviewer that both broader literature coverage and a more nuanced discussion of treatment parameters are essential for accurately interpreting the reported biological outcomes. In the revised manuscript, we have addressed this comment through targeted expansion and refinement rather than by implying exhaustive coverage beyond what the current fish-derived dECM literature supports.

   Specifically, additional fish-derived and marine-specific studies were incorporated into Table 7 and the surrounding narrative where available. Where the evidence base remains limited to a small number of studies, this limitation is now explicitly acknowledged rather than implicitly generalized. This approach ensures that the table reflects the current state of the literature without overstating consensus.

   In parallel, the narrative discussion in Section 5, including the text immediately following Table 7 and the synthesis in Section 5.4, has been expanded to provide a more detailed, parameter-level interpretation of reported outcomes. The revised text now explicitly discusses how detergent type, concentration, exposure duration, tissue thickness, and matrix composition influence biological responses such as cell adhesion, angiogenesis, inflammatory modulation, and mechanical integrity. Rather than treating these effects as uniform trends, the discussion emphasizes protocol-dependent trade-offs and variability across studies.

   Together, these revisions broaden the evidentiary basis where possible while providing a more critical and nuanced interpretation of how decellularization parameters govern the observed performance of fish-derived dECM.

Comments 3: Section 5: categorizing subsections. The classification logic in the application sections needs refinement.

1. '3D printing' is a fabrication technology and should not be listed parallel to biomedical applications like 'Wound healing'. This reviewer recommends moving it to a separate section on fabrication or integrating it as a sub-section within each tissue type.
2. 'Bone' and 'Cartilage' regeneration/repair should be discussed in separate sections, as the biological requirements and material properties (e.g., vascularization, mechanical stiffness) for these tissues are fundamentally different" as authors stated.

Response 3: 

1. We agree with the reviewer that three-dimensional (3D) printing is a fabrication strategy rather than a biomedical application and should not be positioned in parallel with tissue-specific application sections. In the revised manuscript, the classification logic of Section 5 has been refined accordingly.

   Specifically, content previously associated with 3D printing has been removed from the application-level categorization in Section 5 and repositioned within Section 6, where fabrication and post-processing strategies are discussed. In this revised structure, 3D printing and related biofabrication approaches are explicitly framed as enabling technologies that support the implementation of fish-derived dECM across different tissue targets, rather than as standalone biomedical applications.

   The revised narrative now clearly distinguishes between biomedical application domains (e.g., wound healing, bone regeneration, cartilage repair) discussed in Section 5 and fabrication and engineering strategies addressed in Section 6. This restructuring improves conceptual clarity and aligns the manuscript organization with the functional role of biofabrication technologies.
2. We agree with the reviewer that bone and cartilage regeneration involve fundamentally different biological requirements and material constraints and should therefore be discussed in separate sections. In the revised manuscript, the structure of Section 5 has been revised to explicitly reflect this distinction.

   Specifically, the previously combined discussion has been separated into Section 5.2 (Bone Regeneration) and Section 5.3 (Cartilage Repair). Each section now independently addresses tissue-specific biological requirements, dominant failure modes, and material performance constraints. The revised bone regeneration section emphasizes vascularization, mineralized matrix formation, and early-stage mechanical stability, whereas the cartilage repair section focuses on avascularity, glycosaminoglycan retention, compressive performance, and the risk of chondrocyte hypertrophy.

   The comparative synthesis has been correspondingly consolidated in Section 5.4 (Comparative Performance and Application-Driven Insights) to maintain coherence across application domains.

Comments 4: Section 6: Figures 6 and 7 are not sound, "In Figure 6 and Figure 7, the criteria for the 'High' labels and the directional arrows are not explicitly defined. Without a clear methodology or comparative framework for these assessments, these figures lack scientific objectivity. For a review to be informative for the field, evaluations of material performance must be supported by a standardized comparison of the cited literature. I recommend defining these parameters clearly in the figure legends or replacing them with more descriptive, evidence-based labels."

Response 4: 

We highly appreciate the reviewer’s constructive comment regarding the objectivity and interpretability of Figures 6 and 7. We agree that the use of qualitative labels (e.g., “High”) and directional arrows without explicitly defined criteria may introduce ambiguity and limit scientific rigor, particularly in the context of a narrative review.

In response, Figures 6 and 7 have been removed from the revised manuscript, as redefining categorical labels or directional arrows would still risk subjective interpretation and the appearance of unsupported performance ranking given the heterogeneity and limited comparability of available datasets. Instead, the comparative insights originally conveyed by these figures have been fully integrated into an evidence-based narrative synthesis, primarily within Section 5.4 (Comparative Performance and Application-Driven Insights) and Section 6.4 (Engineering Trade-Offs and Design Considerations). These discussions are further supported by Tables 7 and 8, where all evaluations are explicitly linked to and traceable from the cited literature.

This revision ensures that material performance comparisons are presented transparently and critically, allowing parameter-dependent variability and conflicting findings to be discussed explicitly without reliance on implicit scoring schemes or undefined visual criteria.

Comments 5: Regarding the relationship between Subsection 5.3 (3D printing) and Subsection 6.2 (Polymer dECM composited fabrication), the current structure feels somewhat redundant. Subsection 5.3 appears to function primarily as a precursor to justify the discussion in 6.2, which creates a biased flow. I recommend either integrating these two subsections for a more cohesive narrative or clearly separating their scopes to avoid repetitive or leading descriptions. Furthermore, the content in Subsection 6.2 is not exclusively specific to fish-derived dECM, but rather discusses broader principles of dECM composites. The authors should refine the text to either focus strictly on marine-derived materials or acknowledge the general nature of these findings to ensure scientific accuracy.

Response 5: 

We appreciate the reviewer’s careful observation and agree that the original structure risked redundancy and a leading narrative flow. In the revised manuscript, the organization and scope of these sections have been explicitly refined to address this concern.

Specifically, all discussion of three-dimensional (3D) printing has been removed from the biomedical application sections and repositioned within Section 6 as a fabrication-focused discussion. In this revised structure, 3D printing is explicitly framed as an enabling biofabrication strategy, rather than as a precursor or justification for polymer–dECM composite fabrication. This change clearly separates application-level analysis (Section 5) from processing and engineering strategies (Section 6).

In parallel, Section 6.2 (Polymer–dECM Composite Fabrication) has been revised to clarify scope and scientific specificity. Where composite design principles are broadly applicable across dECM systems, this generality is now explicitly acknowledged. Fish-derived dECM examples are highlighted where available, and limitations in fish-specific evidence are clearly stated when broader principles are discussed for comparative or illustrative purposes.

We now believe that these revisions remove redundancy, eliminate biased narrative progression, and ensure a clear distinction between application-driven discussion and fabrication or materials-engineering strategies, thereby improving conceptual clarity and scientific accuracy.

Comments 6: Please use full name when appears at the first time, line67, 270.

Response 6: We have revised the manuscript to use the full names at their first appearance. Specifically, we used the full names of biochemical oxygen demand and chemical oxygen demand at line 67 and removed the abbreviations since they are not mentioned again in the manuscript. In addition, we introduced sodium dodecyl sulfate in full at line 208 and used its abbreviation (SDS) in the subsequent text, including at line 270.

Comments 7: Please use abbreviation when appears at the second time, line 130, 136.

Response 7: We have revised the manuscript to use the abbreviation ECM after its first full mention, including at lines 130 and 136, as suggested.

Comments 8: In Figure 1, what does the red box on the fish image indicate?

Response 8: We understand the confusion brought by this element. The red box in the original fish image was not intended to indicate a specific anatomical region, but rather to serve as a visual cue linking the fish to the extracellular matrix (ECM) schematic. To avoid any potential misinterpretation, the figure has been revised by removing the boxed region and replacing it with a generalized fish tissue icon and a directional arrow. This revision clarifies that Figure 1 refers to the extracellular matrix derived from fish tissue in general, rather than a region-specific anatomy.

Comments 9: Figure 3 describes too details, can be deleted

Response 9: We appreciate the reviewer’s suggestion. Figure 3 has been deleted. The corresponding discussion has been retained to ensure clarity and conciseness.

Comments 10: Is the copyright fine with Figure 4? Nonetheless, it shows too specific and does not contain any legends. This figure is inappropriate in a narrative review.

Response 10: We acknowledge this point. We have confirmed that the copyright status of Figure 4 permits its use. However, we agree that the figure is overly specific, lacks sufficient legend information, and is not well suited for a narrative review format. For the sake of consistency and uniformity of the manuscript, Figure 4 has therefore been removed, and the corresponding text has been revised accordingly.

Comments 11: Figure 5 does not provide any information, and should be deleted.

Response 11: Thank you for the comment. Figure 5 has been deleted from the manuscript. We also verified its copyright status and found that permission is required for reuse; therefore, it has been removed accordingly.

 

 

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for addressing this reviewer's comments and restructured the manuscript.

Although the quality of this work improved a lot, there are still several issues to be addressed for publication.

(1) Please delete the row of "Biofabrication" in Table 6 because it is independent on biomedical application.

(2) Most of the reference paper in the section 4 should be included in Table 6, such as Ref # 13, 76, (Line 541), 99 (Line 579), and etc.

(3) In Table 7, in the column of Reported Qualitative Effect, Please start a new line at the directional symbols, like ↓ degradation rate.  

(4) Most of the reference paper in the secion 5 should be included in Table 7, such as Ref #139, 140, 141 (Line 743, 745), Ref # 153, 154 (Line 821, 824, and etc.

(5) Authors should include the 3D Printing under the "Nanocomposite" in the Table 7.

Author Response

Comments 1: Please delete the row of "Biofabrication" in Table 6 because it is independent on biomedical application.
Response 1: Thank you for this suggestion. The "Biofabrication" row has been removed from Table 6 to maintain focus on biomedical applications.

Comments 2: Most of the reference paper in the section 4 should be included in Table 6, such as Ref # 13, 76, (Line 541), 99 (Line 579), and etc.
Response 2: We appreciate this feedback. Table 6 has been updated to include the relevant references discussed throughout Section 4.

Comments 3: In Table 7, in the column of Reported Qualitative Effect, Please start a new line at the directional symbols, like ↓ degradation rate.  
Response 3: Thank you for this helpful formatting note. The formatting in the "Reported Qualitative Effect" column of Table 7 has been adjusted. Entries now start on a new line at each directional symbol to improve readability.

Comments 4: Most of the reference paper in the secion 5 should be included in Table 7, such as Ref #139, 140, 141 (Line 743, 745), Ref # 153, 154 (Line 821, 824, and etc.
Response 4: We have updated Table 7 to include the relevant references cited in Section 5. This ensures that the table and the corresponding text are now fully aligned.

Comments 5: Authors should include the 3D Printing under the "Nanocomposite" in the Table 7.
Response 5:  We appreciate this specific point. 3D printing has been added under the "Nanocomposite" category in Table 7 including its relevant references found in Section 5.