Relevant Properties and Potential Applications of Sericin in Bone Regeneration

Round 1

Reviewer 1 Report

Dear authors,

I read your paper entitled "Sericin as bone grafts" which is interesting for the potential consideration of silk proteins in bone tissue engineering. The paper reviews the main characteristics of silk sericin and its potential use in bone grafts. Although the topic is challenging with real practical potential, the authors should explore more literature and discuss in detail the use of sericin for BTE. There are various papers in literature on the use of silk proteins in medical domain and the novelty and added value must be considered here. Some comments:

1. I found your preprint version of the paper on the internet (https://www.preprints.org/manuscript/202307.1738/v1). I think it is better first to follow a review and then to have it on the internet.

2. I think you should add more examples and discuss in more details about sericin characteristics for bone tissue engineering. The paper is a review and it should explore more details about the selected topic.

3. Please add the added value of the paper with respect to similar papers in literature. The novelty and added value must be specified as there are similar papers on this topic in the literature ( Das, G., Shin, HS., Campos, E.V.R. et al. Sericin based nanoformulations: a comprehensive review on molecular mechanisms of interaction with organisms to biological applications. J Nanobiotechnol 19, 30 (2021). https://doi.org/10.1186/s12951-021-00774-y,  Seo SJ, Das G, Shin HS, Patra JK. Silk Sericin Protein Materials: Characteristics and Applications in Food-Sector Industries. Int J Mol Sci. 2023 Mar 3;24(5):4951. doi: 10.3390/ijms24054951. PMID: 36902381; PMCID: PMC10003638, Veiga Anabela, Castro Filipa, Rocha Fernando, Oliveira Ana L., Recent Advances in Silk Sericin/Calcium Phosphate Biomaterials, Frontiers in Materials, VOLUME=7, 2020 DOI=10.3389/fmats.2020.00024 )

4. The conclusion section should be extended and show the main advantages of using sericin in bone grafts.

5. The references are appropriate, but they could be improved with recent papers on the use of silk sericin in the medical field.

6. Section 4: Comparative analysis: should be extended with more examples and characteristics of the sercin scaffolds to support bone grafting.

7. You can also add some examples of 3D printing methods for the fabrication of silk sericin supports for medical use (biofabrication for example).

Author Response

I read your paper entitled "Sericin as bone grafts" which is interesting for the potential consideration of silk proteins in bone tissue engineering. The paper reviews the main characteristics of silk sericin and its potential use in bone grafts. Although the topic is challenging with real practical potential, the authors should explore more literature and discuss in detail the use of sericin for BTE. There are various papers in literature on the use of silk proteins in medical domain and the novelty and added value must be considered here. Some comments:

1. I found your preprint version of the paper on the internet (https://www.preprints.org/manuscript/202307.1738/v1). I think it is better first to follow a review and then to have it on the internet.

[Answer] We appreciate your thoughtful suggestion. The preprint version was shared online upon the request of the publisher. We have received the following correspondence from the editorial office: "Dear Professor Kim, We are delighted to inform you that we have established a direct connection with “Preprints.org”, a free preprint platform (https://www.preprints.org). Your article has been recommended to the Preprints.org platform based on their initial screening policy and may be accepted directly."

2. I think you should add more examples and discuss in more details about sericin characteristics for bone tissue engineering. The paper is a review and it should explore more details about the selected topic.

[Answer] We appreciate your insightful suggestion. We have incorporated additional examples and detailed characteristics of sericin scaffolds for bone grafting. Moreover, we've included a section on 3D printing using sericin.

3. Please add the added value of the paper with respect to similar papers in literature. The novelty and added value must be specified as there are similar papers on this topic in the literature ( Das, G., Shin, HS., Campos, E.V.R. et al. Sericin based nanoformulations: a comprehensive review on molecular mechanisms of interaction with organisms to biological applications. J Nanobiotechnol 19, 30 (2021). https://doi.org/10.1186/s12951-021-00774-y, Seo SJ, Das G, Shin HS, Patra JK. Silk Sericin Protein Materials: Characteristics and Applications in Food-Sector Industries. Int J Mol Sci. 2023 Mar 3;24(5):4951. doi: 10.3390/ijms24054951. PMID: 36902381; PMCID: PMC10003638, Veiga Anabela, Castro Filipa, Rocha Fernando, Oliveira Ana L., Recent Advances in Silk Sericin/Calcium Phosphate Biomaterials, Frontiers in Materials, VOLUME=7, 2020 DOI=10.3389/fmats.2020.00024

[Answer] We are thankful for your valuable input. We had already referenced Seo et al. (2023) and Anabela et al. (2020) in our original draft (ref. 16 and 27). However, based on your suggestion, we have included an additional reference (Das, G. et al. 2021).

4. The conclusion section should be extended and show the main advantages of using sericin in bone grafts.

[Answer] We sincerely appreciate your constructive suggestion. We have amended our conclusion in line with your advice.

5. The references are appropriate, but they could be improved with recent papers on the use of silk sericin in the medical field.

[Answer] We are grateful for your helpful suggestion. Following your advice, we have incorporated an additional 21 references.

6. Section 4: Comparative analysis: should be extended with more examples and characteristics of the sercin scaffolds to support bone grafting.

[Answer] We value your suggestion and have added further examples and characteristics of sericin scaffolds for bone grafting.

7. You can also add some examples of 3D

[Answer] We appreciate your suggestion and have included some examples of 3D as requested.

Reviewer 2 Report

Figure 1. Provide sericin image after cocoon, not this fig shows the biomedical application of cocoon, not sericin. 

Figure 2.: Scheme in this fig is wrong. Osteoblasts do not come from macrophages, but osteoclasts do. 

Figure 2: (c). It is hypothesized that a protein: This review focuses on sericin not general protein (both Fibroin and sericin are silk protein), so this Figure is irrelevant to the paper. Be specific with sericin protein. Now its confusing which protein this fig is describing.

Figure 3. Foreign proteins often elicit immune responses: Why the authors discussed about signaling pathways of Foreign proteins here? Though you clarified the statement earlier (Sericin, being an insect-derived protein, is considered a foreign protein for mammals), mentioning foreign protein is not specifically describing sericin. The authors should describe the typical molecular interaction of sericine and TLRs recognition (Not general protein) and elucidate how the sericine triggers paracrine and juxtacrine signaling pathways through receptor interaction. Now, the current image is deviating from the major focus of this paper. Revise it. 

The major concept is missing in this review: THe authors should describe the role of sericine in osteoblasts differentiation from precursors and osteoclasts formation from macrophages, their receptor's interaction with precursors cells for paracrine signals, role in mineralization and hydroxyapatite/ca-P deposition and their different forms (hydrogels, scaffolds, 3D matrix..) in bone grafts.

Need to check spelling and grammar throughout.

Author Response

Figure 1. Provide sericin image after cocoon, not this fig shows the biomedical application of cocoon, not sericin.

[Answer] We appreciate the reviewer's comment, however, we would like to clarify that sericin is intricately intertwined with fibroin, forming the primary components of the cocoon. We believe the cocoon's image is apt as it vividly portrays both sericin and fibroin. Additionally, the presence of sericin is highlighted on the right side of the figure.

Figure 2.: Scheme in this fig is wrong. Osteoblasts do not come from macrophages, but osteoclasts do.

[Answer] We would like to assure the reviewer that their interpretation is not in alignment with the illustration's intent. If we were to accept the reviewer's argument, it would imply that the silk mat is derived from macrophages, which is not the case. 

Figure 2: (c). It is hypothesized that a protein: This review focuses on sericin not general protein (both Fibroin and sericin are silk protein), so this Figure is irrelevant to the paper. Be specific with sericin protein. Now its confusing which protein this fig is describing.

[Answer] We appreciate the reviewer's observation, however, we would like to highlight that the main text clearly identifies the protein in question as sericin. It's important to note that fibroin cannot be the protein in solution as it hasn't been identified as such.

Figure 3. Foreign proteins often elicit immune responses: Why the authors discussed about signaling pathways of Foreign proteins here? Though you clarified the statement earlier (Sericin, being an insect-derived protein, is considered a foreign protein for mammals), mentioning foreign protein is not specifically describing sericin. The authors should describe the typical molecular interaction of sericine and TLRs recognition (Not general protein) and elucidate how the sericine triggers paracrine and juxtacrine signaling pathways through receptor interaction. Now, the current image is deviating from the major focus of this paper. Revise it.

[Answer] We kindly disagree with the need for revision here. The TLR response is a crucial mechanism in sericin-mediated bone formation, and while two figures were omitted due to their unpublished status, an additional figure (Figure 5) has been included in the revised version for better comprehension.

The major concept is missing in this review: THe authors should describe the role of sericine in osteoblasts differentiation from precursors and osteoclasts formation from macrophages, their receptor's interaction with precursors cells for paracrine signals, role in mineralization and hydroxyapatite/ca-P deposition and their different forms (hydrogels, scaffolds, 3D matrix..) in bone grafts.

[Answer] We appreciate the insightful comment. However, we would like to clarify that the main focus of this review is sericin's role in immunomodulation. While the subject of osteoblast and osteoclast differentiation is indeed intriguing, it does not align with the primary theme of this review. It is important to note that sericin stimulates tissue resident macrophages, leading to osteoblast activation. This mechanism, specifically the role of BMP-2 in osteoblast activation, has been adequately detailed in prior publications.

Reviewer 3 Report

The scientific paper "Sericin as Bone Grafts" aimed to review and explore the properties of sericin, a silkworm-derived protein, and explore its applications in bone grafting.

The review was well conducted and is clear and easy to read for researchers interested in the area.

It can be considered that:

1)      Correct the word "biodegradability" in figure 1;

2)      On line 189, the authors write that "has been extensively investigated in several studies [16]" but only reference 1 study. Add more studies on the same subject;

3)      On line 42, where it says "Allografts and xenografts, obtained from human donors and animal species respectively, bypass the issue of limited material availability [7,8]", I suggest adding a current reference in the subject: https://doi .org/10.3390/polym14102075

4)      The title can be made more attractive by adding information such as: “Relevant properties and potential applications of sericin in bone regeneration”

5)      Part b of Figure 3 is difficult to visualize. Please improve.

6)      The call to figure 4 in the text must be before the image. Adjust your position in the manuscript.

Moderate editing of English language required

Author Response

The scientific paper "Sericin as Bone Grafts" aimed to review and explore the properties of sericin, a silkworm-derived protein, and explore its applications in bone grafting.

The review was well conducted and is clear and easy to read for researchers interested in the area.

It can be considered that:

1)      Correct the word "biodegradability" in figure 1;

[Answer] We are grateful for your observant advice. We have rectified the typographical error in Figure 1.

2)      On line 189, the authors write that "has been extensively investigated in several studies [16]" but only reference 1 study. Add more studies on the same subject;

[Answer] We thank you for your attentive advice. We have corrected the citation error to reference multiple studies [14-16].

3)      On line 42, where it says "Allografts and xenografts, obtained from human donors and animal species respectively, bypass the issue of limited material availability [7,8]", I suggest adding a current reference in the subject: https://doi .org/10.3390/polym14102075

[Answer] We appreciate your thoughtful suggestion. We have added the following reference: Reis, C.H.; Buchaim, R.L.; Pomini, K.T.; Hamzé, A.L.; Zattiti, I.V.; Duarte, M.A.; Alcalde, M.P.; Barraviera, B.; Ferreira Júnior, R.S.; Pontes, F.M.; Grandini, C.R. Effects of a biocomplex formed by two scaffold biomaterials, hydroxyapatite/tricalcium phosphate ceramic and fibrin biopolymer, with photobiomodulation, on bone repair. Polymers 2022, 14(10), 2075.

4)      The title can be made more attractive by adding information such as: “Relevant properties and potential applications of sericin in bone regeneration”

[Answer] We value your insightful suggestion. We have revised the title as per your recommendation.

5)      Part b of Figure 3 is difficult to visualize. Please improve.

[Answer] We thank you for your careful observation. We have improved the visibility of Figure 3 by increasing its size.

6)      The call to figure 4 in the text must be before the image. Adjust your position in the manuscript.

[Answer] We appreciate your attention to detail. We have accordingly repositioned Figure 4 in the manuscript.

Round 2

Reviewer 1 Report

Dear authors,

Your paper has been improved after the revision. I think the title is more representative now, and the comments have been taken into consideration.

New references have been added, and more examples and discussion on sericin applications in bone tissue engineering are shown. 3D printing with silk sericin bioinks are shown in the future directions sub-chapter.

I am ready to give the final positive feedback for this manuscript.

Author Response

Thank you for your insightful feedback. We deeply appreciate all of your comments on our paper. With the benefit of your review, we have been able to successfully revise our manuscript. Once again, thank you for your guidance.

Reviewer 2 Report

The authors literally refused all my comments and their justification is not at all convinced. Their responses and arguments are not scientifically right. Therefore,  I am forced to REJECT this paper for publication.

[Author Answer] Additionally, the presence of sericin is highlighted on the right side of the figure.

Reviewer Comment: The authors wanted to focus this review on sericin (not cocoon or proteins from cocoon), now the image is more confusing, on right side displayed sericin and fibroin for biocompatibility and metabolic regulation on the other hand, the left side displayed cocoon for biodegradability and antioxidant. There are plenty of studies already described this information even with more details. Try to focus on the potential applications of sericin as mentioned in the title, if not change the title, because this image is contradictory to the title of this MS. 

 [Author Answer] it would imply that the silk mat is derived from macrophages, which is not the case. 

Reviewer comment: The authors did not understand my previous comment. My previous comment talks about macrophages and osteoblasts, not silk mat, Fig 2d shows that silk mat releases proteins (again not specific, it can be fibroin and sericin, but this review only focuses on sericin not fibroin, then why do you talk about general proteins) to macrophages and osteoblasts derived from macrophages, which is wrong. The osteoblasts come from Mesenchymal stem cells and osteoclasts are derived from macrophages. Need proper clarification. 

 [Author Answer] It's important to note that fibroin cannot be the protein in solution as it hasn't been identified as such.

Reviewer Comments: Why is the fibroin protein not present in the solution and sericin present? clarify

[Authour Answer] The TLR response is a crucial mechanism in sericin-mediated bone formation.

Reviewer Comments: If you want to talk about bone formation, then why did the authors state immune cells in this Fig.? [Fig.3. Foreign proteins often elicit immune responses through the recognition of toll-like receptors (TLRs) by immune cells],  What was the link? Here the authors should try to explain How sericin triggers and recognize TLRs receptor (Pattern of TLRs and sericin attachment) and mediate bone formation?

[Author Answer] We appreciate the insightful comment. However, we would like to clarify that the main focus of this review is sericin's role in immunomodulation.

Reviewer Comment: In this case, why did the authors keep the title as "Relevant Properties and Potential Applications of Sericin in 2 Bone Regeneration", So, the major focus of this review is Bone regeneration not immunomodulation.  The authors' response is contradictory to their title and seems they don't even know the major theme of their review. If your review talks about bone regeneration then "the role of sericine in osteoblasts differentiation from precursors and osteoclasts formation from macrophages" is highly relevant to your review. In this case, no novelty in this review.

Spell-check and grammar throughout MS.

Author Response

[Author Response] To begin with, the reviewer's comment contained several grammatical errors, which made comprehension challenging. For instance, the initial sentence was fragmented and unclear. Phrases such as "on right side displayed sericin..." were not smoothly articulated. Moreover, "There are plenty of studies already described this information even with more details." was not properly constructed. These inconsistencies persisted throughout the comments. Despite these obstacles, we have done our utmost to address your queries.

Reviewer Comment: The authors wanted to focus this review on sericin (not cocoon or proteins from cocoon), now the image is more confusing, on right side displayed sericin and fibroin for biocompatibility and metabolic regulation on the other hand, the left side displayed cocoon for biodegradability and antioxidant. There are plenty of studies already described this information even with more details. Try to focus on the potential applications of sericin as mentioned in the title, if not change the title, because this image is contradictory to the title of this MS.

[Author Answer] The image was designed to provide a comprehensive view, contrasting sericin with related proteins. The emphasis remains on sericin. We will not adjust the image. The title stands as it accurately reflects the main theme of the paper.

Reviewer comment: The authors did not understand my previous comment. My previous comment talks about macrophages and osteoblasts, not silk mat, Fig 2d shows that silk mat releases proteins (again not specific, it can be fibroin and sericin, but this review only focuses on sericin not fibroin, then why do you talk about general proteins) to macrophages and osteoblasts derived from macrophages, which is wrong. The osteoblasts come from Mesenchymal stem cells and osteoclasts are derived from macrophages. Need proper clarification.

[Author Answer] We stand by our interpretation. The role of bone resident macrophages in bone regeneration through immunomodulation is central to our argument. We're clear on the fact that osteoblasts come from Mesenchymal stem cells. Our focus is on the interaction of sericin with macrophages. We recommend a thorough review of the references provided to understand this crucial mechanism.

1: MIRON, Richard J.; BOSSHARDT, Dieter D. OsteoMacs: Key players around bone biomaterials. Biomaterials, 2016, 82: 1-19.

2: Weivoda MM, Bradley EW. Macrophages and Bone Remodeling. J Bone Miner Res. 2023 Mar;38(3):359-369.

3: Schlundt C, Fischer H, Bucher CH, Rendenbach C, Duda GN, Schmidt-Bleek K. The multifaceted roles of macrophages in bone regeneration: A story of polarization, activation and time. Acta Biomater. 2021 Oct 1;133:46-57.

4: Raggatt LJ, Alexander KA, Kaur S, Wu AC, MacDonald KP, Pettit AR. Absence of B cells does not compromise intramembranous bone formation during healing in a tibial injury model. Am J Pathol. 2013 May;182(5):1501-8.

5: Miyamoto T. Role of osteoclasts in regulating hematopoietic stem and progenitor cells. World J Orthop. 2013 Oct 18;4(4):198-206.

6: Batoon L, Millard SM, Raggatt LJ, Wu AC, Kaur S, Sun LWH, Williams K, Sandrock C, Ng PY, Irvine KM, Bartnikowski M, Glatt V, Pavlos NJ, Pettit AR. Osteal macrophages support osteoclast-mediated resorption and contribute to bone pathology in a postmenopausal osteoporosis mouse model. J Bone Miner Res. 2021 Nov;36(11):2214-2228.

7: Winkler IG, Sims NA, Pettit AR, Barbier V, Nowlan B, Helwani F, Poulton IJ, van Rooijen N, Alexander KA, Raggatt LJ, Lévesque JP. Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs. Blood. 2010 Dec 2;116(23):4815-28.

8: Bozec A, Soulat D. Latest perspectives on macrophages in bone homeostasis. Pflugers Arch. 2017 Apr;469(3-4):517-525. doi: 10.1007/s00424-017-1952-8.

9: Alexander KA, Raggatt LJ, Millard S, Batoon L, Chiu-Ku Wu A, Chang MK, Hume DA, Pettit AR. Resting and injury-induced inflamed periosteum contain multiple macrophage subsets that are located at sites of bone growth and regeneration. Immunol Cell Biol. 2017 Jan;95(1):7-16.

10: Horibe K, Hara M, Nakamura H. M2-like macrophage infiltration and transforming growth factor-β secretion during socket healing process in mice. Arch Oral Biol. 2021 Mar;123:105042.

11: Mohamad SF, Gunawan A, Blosser R, Childress P, Aguilar-Perez A, Ghosh J, Hong JM, Liu J, Kanagasabapathy D, Kacena MA, Srour EF, Bruzzaniti A. Neonatal Osteomacs and Bone Marrow Macrophages Differ in Phenotypic Marker Expression and Function. J Bone Miner Res. 2021 Aug;36(8):1580-1593.

Reviewer Comments: Why is the fibroin protein not present in the solution and sericin present? clarify

[Author Answer] Fibroin was not detected in the solution, as clearly demonstrated by the study: Jo et al. Int J Biol Macromol. 2021;190:607-617. This is not a matter of interpretation; it's a fact.

Reviewer Comments: If you want to talk about bone formation, then why did the authors state immune cells in this Fig.? [Fig.3. Foreign proteins often elicit immune responses through the recognition of toll-like receptors (TLRs) by immune cells],  What was the link? Here the authors should try to explain How sericin triggers and recognize TLRs receptor (Pattern of TLRs and sericin attachment) and mediate bone formation?

[Author Answer] The connection is clearly elaborated in Figure 5. It illustrates the interaction of sericin with TLRs and its subsequent role in bone regeneration. If there's any ambiguity, the figure should resolve it.

Reviewer Comment: In this case, why did the authors keep the title as "Relevant Properties and Potential Applications of Sericin in 2 Bone Regeneration", So, the major focus of this review is Bone regeneration not immunomodulation.  The authors' response is contradictory to their title and seems they don't even know the major theme of their review. If your review talks about bone regeneration then "the role of sericine in osteoblasts differentiation from precursors and osteoclasts formation from macrophages" is highly relevant to your review. In this case, no novelty in this review.

[Author Answer] Our title is apt. The manuscript emphasizes the importance of sericin in bone regeneration, where immunomodulation plays a key role. While we respect your viewpoint, we believe that a holistic understanding of how sericin impacts both the immune response and bone cell differentiation is essential. We strongly suggest revisiting the references provided to grasp this connection.

Reviewer 3 Report

No comments 

Minor editing of English language required

Author Response

Thank you for your insightful feedback. We deeply appreciate all of your comments on our paper. With the benefit of your review, we have been able to successfully revise our manuscript. Once again, thank you for your guidance.