Embroidered Silk Fibroin Scaffolds for ACL Tissue Engineering

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents relevant results. However, several aspects of data presentation and text organization require revision to improve clarity, consistency, and scientific rigor.

1. Although it contains the necessary information to contextualize the study, the Introduction section should be revised to improve the English and ensure a more fluent and cohesive flow of ideas.
2. Section 2.1.3; Figure 5 - Capital letters were used to indicate statistical differences, which might cause confusion since the figure is already divided into panels A and B. It is recommended to use lowercase letters or another symbol system to avoid misinterpretation.
3. Section 2.2 (line 219) - The sentence “cell vitality was significantly higher in all the scaffold groups compared to the cells treated with 10% DMSO (Fig. 6)”, please clarify whether 10% DMSO represents the positive (K⁺) control, as typically this control should present very low viability (close to 0–10%). Probably 70% ethanol would have been a more appropriate positive control. Also compare with non-treated cells (negative control).
4. Section 2.2; Figure 6:
   • This figure needs significant improvement for better readability and interpretation. For example, ensure uniformity in graph dimensions and consider reorganizing the data into two separate figures to enhance clarity.
   • I suggest replacing the abbreviation GM with K– or NC (negative control) for standardization.
   • Clarify why the Live/Dead assay was performed only for LCL fibroblasts and not for L929 cells.
5. Some subsection titles in Section Results read more like methodological descriptions than results. Consider rephrasing them to highlight the findings of the study.
6. Section Results, Figure 12 - It would be clearer to present gene expression results for days 7 and 14 in the same graph, facilitating comparison and identification of significant differences.
   • Also, the statement “The comparison of day 7 and 14 revealed an overall increase in all scaffold groups on day 14.” (line 350) lacks statistical support, as no p-values or test types are indicated in either the text or the figure caption. Consequently, the sentence on Discussion “Gene expression analysis showed on day 14 of culture that connexin 43 was higher expressed in p-silk scaffolds without or with P(LA-CL) fibers.” (line 468) is not fully supported by the presented data and should be revised accordingly.
7. The Discussion section is too extensive and somewhat repetitive. It could be made more concise by focusing on key findings and reducing redundancy. Study limitations are appropriately mentioned.
8. Minor points: Revise text alignment in Lines 109–124; Figure 2: The image quality should be improved; scale bars are currently unclear and difficult to perceive.

Author Response

The manuscript presents relevant results. However, several aspects of data presentation and text organization require revision to improve clarity, consistency, and scientific rigor.

Response: The authors would like to thank the reviewer for his/her time and careful review of the manuscript. The authors have attempted to address all comments and make all necessary improvements (the changes are marked in red in the manuscript).

1. Although it contains the necessary information to contextualize the study, the Introduction section should be revised to improve the English and ensure a more fluent and cohesive flow of ideas. Response: The authors have thoroughly improved the introduction section, thereby hopefully increasing its readability.

 

1. Section 2.1.3; Figure 5 - Capital letters were used to indicate statistical differences, which might cause confusion since the figure is already divided into panels A and B. It is recommended to use lowercase letters or another symbol system to avoid misinterpretation.

Response: The authors agree and have changed the symbols in the Figure accordingly.

 

1. Section 2.2 (line 219) - The sentence “cell vitality was significantly higher in all the scaffold groups compared to the cells treated with 10% DMSO (Fig. 6)”, please clarify whether 10% DMSO represents the positive (K⁺) control, as typically this control should present very low viability (close to 0–10%). Probably 70% ethanol would have been a more appropriate positive control. Also compare with non-treated cells (negative control).
   Response: Yes, in our view, the sentence is correct, because the authors interpret the cytotoxicity assay as follows: based on its name, the authors understand that anything that has a toxic effect (DMSO) on cells should be labeled “positive.” Whereas medium should have a no effect on toxicity and therefore, also represents the negative control. We perform the cytotoxicity assay with DMSO because we want to adhere closely to the DIN standard (DIN EN ISO 10993-5 2009-10 norm) in order to obtain certification for future biomaterials. This is also important for our further planned experiments in animal models. We hope we have been able to convince the reviewer that the designation already used is correct. It also goes conform with our published previous studies (Gögele et al., 2021 and 2020 PMID: 34702508; PMID: 32046263).

 

1. Section 2.2; Figure 6:

• This figure needs significant improvement for better readability and interpretation. For example, ensure uniformity in graph dimensions and consider reorganizing the data into two separate figures to enhance clarity.
• Response: The authors thank the reviewer for his/her comment and have rearranged and revised Fig. 6 in two figures (now Figures 6 and 7).
• I suggest replacing the abbreviation GM with K– or NC (negative control) for standardization.
• Response: The authors have changed it in “negative control.” (NC)
• Clarify why the Live/Dead assay was performed only for LCL fibroblasts and not for L929 cells.
  
  

Response: The live/dead assay was only performed with LCL fibroblasts and not with L929, because L929 are a genetically modified cell line from mice that are subcutaneous tissue fibroblasts. However, the aim of our work is to reconstruct the cruciate ligament using a newly developed 3D silk construct. For this reason, the authors wanted to conduct in vitro experiments that were as realistic as possible, which is why we decided to use primary cruciate ligament fibroblasts from rabbits. These cells are primary cells that are not only capable of proliferating but, as our study has shown, also of expressing tissue-specific extracellular matrix. This would only be possible to a limited extent with a cell line. Furthermore, we decided to use cruciate ligament fibroblasts from rabbits in order to build on previous work (Gögele et al., 2020 PMID: 32046263, Hahn et al., 2019 PMID31546928), to be able to compare the study with other studies we have conducted, and to generate preliminary work for future in vivo implantations in the rabbit model. Overall, it is important for the authors to gain a better understanding of the characterization of primary cruciate ligament fibroblasts on 3D tissue constructs.

1. Some subsection titles in Section Results read more like methodological descriptions than results. Consider rephrasing them to highlight the findings of the study.
   Response: The authors would like to thank the reviewer for his/her constructive criticism and have attempted to make the headings of the results more concise and meaningful pointing out results. The improvements are highlighted in red in the manuscript.

 

1. Section Results, Figure 12 - It would be clearer to present gene expression results for days 7 and 14 in the same graph, facilitating comparison and identification of significant differences.
   Response: The authors would like to thank the reviewer for his/her suggestion, which the authors have implemented and now summarized in Fig. 13 for days 7 and 14.

• Also, the statement “The comparison of day 7 and 14 revealed an overall increase in all scaffold groups on day 14.” (line 350) lacks statistical support, as no p-values or test types are indicated in either the text or the figure caption. Consequently, the sentence on Discussion “Gene expression analysis showed on day 14 of culture that connexin 43 was higher expressed in p-silk scaffolds without or with P(LA-CL) fibers.” (line 468) is not fully supported by the presented data and should be revised accordingly.
  
  

Response: Unfortunately, there were no significant differences in gene expression neither between the groups nor within the two selected time points. The new presentation makes our statement even clearer. We hope that this will convince the reviewer.

 

1. The Discussion section is too extensive and somewhat repetitive. It could be made more concise by focusing on key findings and reducing redundancy. Study limitations are appropriately mentioned.
   
   

Response: The authors would like to thank the reviewer for his/her suggestions for improvement. Redundancies have been removed, key findings have been presented more clearly, and the text has been shortened overall.

 

1. Minor points: Revise text alignment in Lines 109–124; Figure 2: The image quality should be improved; scale bars are currently unclear and difficult to perceive.

           

            Response: Thank you for the suggestion. We have revised Fig. 2 and hope that the quality is now          better.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigates embroidered silk fibroin scaffolds for anterior cruciate ligament (ACL) tissue engineering. The findings demonstrate these embroidered scaffolds, with tunable porosity and mechanical properties, are promising candidates for ACL tissue engineering and future graft development. Research is valuable, but there are some issues that need to be addressed before its publication. Below are my opinions：

1. The images of moths and silkworm eggs in Figure 1 are redundant, serve no practical purpose, and even deviate from the theme. It is recommended to remove them.
2. The unit of tensile strength should be MPa or GPa, not N. N is the unit of force, not tensile strength. The statement "tensile strength was highest in purified silk scaffolds (~684 N)" in the abstract is incorrect, and revisions should be made throughout the entire manuscript.
3. Only rabbit LCL fibroblasts were used in the study; neither human-derived cells nor primary ACL cells were employed, and no in vivo animal experiments were conducted. There may be differences between rabbit LCL and human ACL in terms of cell phenotype, mechanical environment, and healing mechanism.
4. The longest culture period was only 14 days, and long-term stability (such as degradation, fibrosis, and calcification) was not evaluated. Why not consider extending the culture period to 4-8 weeks and introducing mechanical stimulation (e.g., dynamic stretching) to simulate the in vivo environment?
5. There are obvious errors in the figure legend of Figure 1: the legend for Figure 1E cannot be found, and the legend labeled for Figure 1C seems to belong to Figure 1D. The authors are requested to check and correct this.
6. The arrangement and figure legend of Figure 6 are too disorganized. It is recommended not to include B1-B8 and D1-D6; only A, B, C, and D should be retained. Additionally, D8 follows D6 directly—where is D7? The images also need to be rearranged, as they are very irregular in their current layout.
7. There are many obvious errors in the references, such as missing page numbers and the failure to italicize species names. The authors are requested to revise these.
8. The expression of inflammatory factors (e.g., IL-6, TNF-α) was not investigated in Figure 12, which is necessary to rule out the risk of immune reactions induced by silk fibroin scaffolds.

 

Author Response

This study investigates embroidered silk fibroin scaffolds for anterior cruciate ligament (ACL) tissue engineering. The findings demonstrate these embroidered scaffolds, with tunable porosity and mechanical properties, are promising candidates for ACL tissue engineering and future graft development. Research is valuable, but there are some issues that need to be addressed before its publication. Below are my opinions:

Response: The authors would also like to express their sincere thanks to Reviewer2 for his/her time and careful review of the manuscript.

1. The images of moths and silkworm eggs in Figure 1 are redundant, serve no practical purpose, and even deviate from the theme. It is recommended to remove them.
   
   

Response: In Fig. 1, the authors must disagree with the reviewer, because in our view, the depiction of the moth and the silkworm cocon makes perfect sense, as it is this that makes the “common thread” running through the entire manuscript fully comprehensible. Furthermore, the moth is necessary for the caterpillar to develop and thus produce silk. The entire illustration, as it currently stands, once again clearly illustrates that this is a natural product and also highlights the complex manufacturing process involved.

 

1. The unit of tensile strength should be MPa or GPa, not N. N is the unit of force, not tensile strength. The statement "tensile strength was highest in purified silk scaffolds (~684 N)" in the abstract is incorrect, and revisions should be made throughout the entire manuscript.
   
   

Response: The authors would like to thank the reviewer for his comments. The authors have revised this issue in the entire manuscript and marked the corrected sections in red.

 

1. Only rabbit LCL fibroblasts were used in the study; neither human-derived cells nor primary ACL cells were employed, and no in vivo animal experiments were conducted. There may be differences between rabbit LCL and human ACL in terms of cell phenotype, mechanical environment, and healing mechanism.
   
   

Response: The authors thank the reviewer for his/her comments and agree that there may be differences between             rabbit and human ACL fibroblasts. However, the authors would like to build on previous experiments             in order to be able to compare results. Furthermore, rabbit cruciate ligament fibroblasts were used             for future in vivo experiments in the rabbit animal model. The approach of using human ACL             fibroblasts is certainly exciting and can be pursued further in the future.

 

1. The longest culture period was only 14 days, and long-term stability (such as degradation, fibrosis, and calcification) was not evaluated. Why not consider extending the culture period to 4-8 weeks and introducing mechanical stimulation (e.g., dynamic stretching) to simulate the in vivo environment?
   
   

Response: The authors would like to thank the reviewer for his/her constructive suggestions. Unfortunately, due to         time constraints (10 days reevision time!), the authors are currently unable to add further long-term cultures to this manuscript,        which is why we were only able to add one sentence to the limitations section stating that this should be carried out in the future.

 

1. There are obvious errors in the figure legend of Figure 1: the legend for Figure 1E cannot be found, and the legend labeled for Figure 1C seems to belong to Figure 1D. The authors are requested to check and correct this.
   Response: The authors thank the reviewer for his comment and have corrected the errors in Fig. 1.

 

1. The arrangement and figure legend of Figure 6 are too disorganized. It is recommended not to include B1-B8 and D1-D6; only A, B, C, and D should be retained. Additionally, D8 follows D6 directly—where is D7? The images also need to be rearranged, as they are very irregular in their current layout.
   Response: Fig. 6 has been completely revised and redesigned by the authors. It is now figure 6 and 7.
2. There are many obvious errors in the references, such as missing page numbers and the failure to italicize species names. The authors are requested to revise these.
   Response: We appreciate your comment and have changed the reference style according to journal´s guidelines.   
3. The expression of inflammatory factors (e.g., IL-6, TNF-α) was not investigated in Figure 12, which is necessary to rule out the risk of immune reactions induced by silk fibroin scaffolds.

Response: The authors agree with the reviewer that inflammatory and immune reactions may also occur after in vivo experiments and that IL-6 and TNF should therefore, be investigated. Unfortunately, we had not enough RNA to conduct a broader spectrum of analyses.

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The author has addressed part of the issues, and the manuscript has undergone certain revisions. It is recommended to accept the manuscript.