Small-Molecule Loaded Biomimetic Biphasic Scaffold for Osteochondral Regeneration: An In Vitro and In Vivo Study

Round 1

Reviewer 1 Report

This paper by Chih-Hsiang Fang et al. reported a study on kartogenin (KGN) and metformin (MET) loaded biomimetic biphasic scaffold for osteochondral regeneration.

This article has innovative points, meaningful results, and discussion, but the experimental design has unreasonable points. Therefore, due to the following reasons, I recommend acceptance of this manuscript for publication after the major revision.

1.      In the abstract, the authors mention the effect of biphasic scaffold on cell homing and recruitment of endogenous stem cells, however, no corresponding data is proving this in the results.

2.      Fig 1, Fig 2, Fig 3, Fig 4, Fig 5, and Fig 6, please indicate the order of the pictures with consecutive letters and describe them accordingly in the figure notes.

3.      Please make a statistical analysis of the stents in SEM images in Fig 1.

4.      Fig 2A, B illustration, and picture location are inconsistent.

5.      Please supplement the control group data with statistics in Fig 5.

6.      The units and quantities of reagents used in the pretreatment of 96-well plates are incorrect in experimental section 2.7.

7.      Please supplement the in vitro experiments on scaffold drug release and degradation in experimental section 3.1.

8.      Please explain the design reason for not having a control scaffold set in vitro study.

9.      Please explain the reason that BMSCs were not selected in the experimental section 3.2.1.

10.  Please provide an explanation of the rationale for choosing a concentration gradient for the Live/dead staining in the experimental section 3.2.1.

11.  Please count the results of the live-dead experiment in the experimental section 3.2.1.

12.  Relative osteogenesis gene expresses in Fig 3 and the experimental section 3.2.2(qPCR), “ALP” writing does not correspond.

13.  Please provide an explanation for the division of biphasic scaffold for GHSM and NGFK to validate osteogenic and cartilaginous expression separately, rather than validating them simultaneously as a whole in the experimental section 3.2.2(qPCR and WB), 3.2.3(ELISA) and 3.2.4(Immunofluorescence).

14.  Please supplement ALP and ToluidineBlueO staining to verify osteogenesis and chondrogenesis in the experimental section 3.2.

15.  Please supplement the modeling flow chart in experiment section 3.3.

16.  Please provide medical imaging evidence to support the results of in vivo experiments in experiment section 3.3.

17.  Please supplement with more immunofluorescence or immunohistochemistry data to verify the in vivo results and correspond to the in vitro results in experiment section 3.3.

The manuscript requires further English Language correction. Authors may consult a professional language editing service to avoid language errors including use of acronyms and prepositions.

Author Response

To Reviewer #: 1

Ref: Manuscript ID: bioengineering-2415695

Title: Small-molecule loaded biomimetic biphasic scaffold for oste-ochondral regeneration: An in vitro and in vivo study

Authors: Chih-Hsiang Fang, Yi-Wen Lin, Chung-Kai Sun and Jui-Sheng Sun.

Thank you very much for your kind review and comments on our paper. We have learned much from your comments. After looking over your remarks, we feel confident that the revised manuscript addresses your concerns in detail and bring the paper into accordance with the standards expressed in your review. We are resubmitting the revised manuscript and would like to request a further review. We have responded to your original comments on a point-by-point basis below. Additional commentary on the revised manuscript would be highly appreciated.

Comments and Suggestions for Authors

1. In the abstract, the authors mention the effect of biphasic scaffold on cell homing and recruitment of endogenous stem cells, however, no corresponding data is proving this in the results.

Ans.:

Thank you for your comments. This statement had been revised in the revised manuscript as the following:

… (GHSM and NGFK layers). The biphasic scaffolds upregulated both osteogenic and chondrogenic gene expression, sulfated glycosaminoglycan (sGAG), osteo- and chondrogenic biomarker, and relative mRNA gene expression. ..

2. Fig 1, Fig 2, Fig 3, Fig 4, Fig 5, and Fig 6, please indicate the order of the pictures with consecutive letters and describe them accordingly in the figure notes.

Ans.:

Thank you for your comments. The order of the pictures with consecutive letters had been added in the revised manuscript and description of them had also been added in the figure notes. Please refer to the revised manuscript for the details.

3. Please make a statistical analysis of the stents in SEM images in Fig 1.

Ans.:

Thank you for your comments. In Figure 1, we tried to describe the characteristics of biphasic scaffold (BSP) by representative SEM images; we did not don many samples for this kind of study, we are sorry that we can not provide statistical analysis about SEM images.

4. Fig 2A, B illustration, and picture location are inconsistent.

Ans.:

Thank you for the comment. We had changed the sequence of pictures of Fig. 2A and 2B in the revised manuscript.

5. Please supplement the control group data with statistics in Fig 5.

Ans.:

Thank you for the comment. In the original figure, the control data was presented as blue bars, while the experimental data was described as the orange cure.  Since this may confuse the readers, we had changed both data and presented in blue and orange bars in the revised manuscript.

6. The units and quantities of reagents used in the pretreatment of 96-well plates are incorrect in experimental section 2.7.

Ans.:

Thank you for the comment. The mistake in experimental section 2.7 had been corrected as the following:

… For the WST-1 assay, each well was pretreated with 10 µL reagent for 4 h. …

7. Please supplement the in vitro experiments on scaffold drug release and degradation in experimental section 3.1.

Ans.:

Thank you for the comment. The in vitro experiments on scaffold drug release and degradation were not done in this study, we choose the selected concentration for different components according to previous reports of our instiytutes as the following:

Chih-Hsiang Fang, Yi-Wen Lin, Feng-Huei Lin, Jui-Sheng Sun, Yuan-Hung Chao, Hung-Ying Lin, and Zwei-Chieng Chang. Biomimetic Synthesis of Nanocrystalline Hydroxyapatite Composites: Therapeutic Potential and Effects on Bone Regeneration. Int J Mol Sci. 2019 Dec; 20(23): 6002.

Ching-Yun Chen, Chunching Li, Cherng-Jyh Ke, Jui-Sheng Sun, Feng-Huei Lin. Kartogenin Enhances Chondrogenic Differentiation of MSCs in 3D Tri-Copolymer Scaffolds and the Self-Designed Bioreactor System. Biomolecules . 2021 Jan 16;11(1):115. doi: 10.3390/biom11010115.

But, we must admit this is a shortcoming of this study, we mentioned this fact in the last 2 sentence of “Discussion” in the revised manuscript as the following:

Lane 459-462: …. promoted the complete regeneration of osteochondral defects in rats. This study effectively enabled the recruitment of endogenous bone marrow MSCs for osteogenesis and chondrogenesis, facilitating osteochondral regeneration. However, the in vitro drug release and degradation profile was not evaluated in this study, the relative time-related osteogenic and chondrogenic gene expression effects were not analyzed; the possible time-related benefit from different component is overlooked. ….

8. Please explain the design reason for not having a control scaffold set in vitro study.

Ans.:

Thank you for the comment. In this study, for the sake of animal safety, only one leg per animal was used for experiment and two femoral condyles can be used per animal; one of these two condyles was used as the sham (non-Implanted control). We apologize that we can not provide data of implanted with control scaffold. This had been described in the revised manuscript as the following:

2.15. Generation of osteochondral defect

To generate an osteochondral defect, a transverse medial parapatellar incision was made, and the patella was laterally dislocated. A circular hole (1 x 1 mm) was drilled in both the medial and lateral femoral condyle till bleeding from the subchondral bone was observed. Then, the scaffolds were implanted into one of the defective sites. After three months of implantation, the joints were harvested for histological evaluation. Buffered 4% paraformaldehyde was used to fix the joints, the specimens were demineralized, dehydrated, defatted, cleared with xylene, and then embedded in wax.

Lane 466- 472:

…. However, the in vitro drug release and degradation profile was not evaluated in this study, the relative time-related osteogenic and chondrogenic gene expression effects were not analyzed; the possible time-related benefit from different components maybe overlooked. In this study, we use empty osteochondral defect (without control scaffold) as control, another sham control with control scaffold can be used to validate the role of small-molecule in the osteochondral regeneration.  

9. Please explain the reason that BMSCs were not selected in the experimental section 3.2.1.

Ans.:

Thank you for the comment. As described in “ 2.7. Cell viability”; following the ISO 10993-5 standard, we use L929 cells (instead of BMSC) to perform and to evaluate the cell viability.

10. Please provide an explanation of the rationale for choosing a concentration gradient for the Live/dead staining in the experimental section 3.2.1.

Ans.:

Thank you for the comment. As described at 2.7. Cell viability, the rationale for choosing concentration gradient is the same following the ISO 10993-5 standard.

11. Please count the results of the live-dead experiment in the experimental section 3.2.1.

Ans.:

Thank you for the comment. As shown in Figure 2, excellent cell viability (WST-1 assay) and minimal cytotoxicity (LDH assay) at different concentrations of biphasic scaffold (BPS) were observed when compared with control. Only scanty dead cells were observed at different concentrations of biphasic scaffold (BPS) extract, so did not count the live/dead staining in the experimental section 3.2.1.

12. Relative osteogenesis gene expresses in Fig 3 and the experimental section 3.2.2(qPCR), “ALP” writing does not correspond.

Ans.:

Thank you for your comment. We had corrected this mistake of Fig. 3 in the revised manuscript.

13. Please provide an explanation for the division of biphasic scaffold for GHSM and NGFK to validate osteogenic and cartilaginous expression separately, rather than validating them simultaneously as a whole in the experimental section 3.2.2(qPCR and WB), 3.2.3(ELISA) and 3.2.4(Immunofluorescence).

Ans.:

Thank you for your comment. We had revised section 2.3 and 2.4 to validate the division of biphasic scaffold for GHSM and NGFK for their osteogenic and cartilaginous expressions as the following in the revised manuscript.

2.3 Preparation of metformin embedded gelatin/hydroxyapatite scaffold (GHSM)

….. Metformin (MET) was added to achieve a final concentration of 50 μM. Finally, 0.1% microbial transglutaminase (mTGase; Activa, Ajinomoto, Japan) was used as a cross-linking agent to crosslink the GHSM sponge. The specimens were moved to 4 °C for 24 h, frozen at −20 °C for 24 h, −80 °C 24 h, and then lyophilized for 72 h.

2.4. Synthesis of biphasic scaffold and study of morphology

The biphasic scaffold used for osteochondral regeneration was synthesized by coupling the GHSM sponge (lower layer) and NGFK scaffold (upper layer) with 0.1% microbial transglutaminase. The samples were ….

14. Please supplement ALP and ToluidineBlueO staining to verif

y osteogenesis and chondrogenesis in the experimental section 3.2.

Ans.:

Thank you for your comment. The osteogenesis and chondrogenesis had been verified by immunofluorescence staining of osteo- (A,B) and chondro- (C,D) specific biomarkers (Figure 6) dy osteocalcin, aggrecan and the different types of collagen.

15. Please supplement the modeling flow chart in experiment section 3.3.

Ans.:

Thank you for your comment. The modeling flow chart of experiment had been provided as Supplementary Figure 1: Flow chart of experimental design in the revised manuscript.

16. Please provide medical imaging evidence to support the results of in vivo experiments in experiment section 3.3.

Ans.:

Thank you for your comment. As the osteochondral defect is quite small and it is quite hard to visualize by image (or radiograph image), we can not provide the medical images of this study, we really apologize for this.

17. Please supplement with more immunofluorescence or immunohistochemistry data to verify the in vivo results and correspond to the in vitro results in experiment section 3.3.

Ans.:

Thank you for your comment. In this study, we hematoxylin and eosin (H&E) staining of subchondral bone. and Alcian blue and Periodic acid–Schiff (AB/PAS) double staining of cartilage regeneration, we are not able to provide any more immunofluorescence or immunohistochemistry data at present time, we really apologize for this.

Thank you very much for your kind review and comments on our paper.

Sincerely yours,

Jui-Sheng Sun, MD, Ph.D.

Department of Orthopedic Surgery, College of Medicine, China Medical University,

No. 2, Yu-Der Rd, Taichung City 40447, Taiwan. Tel: +886-4-22062121

Department of Orthopedic Surgery, National Taiwan University Hospital,

No.7, Chung-Shan South Rd., Taipei 10035, Taiwan, Tel: +886-2-23123456

Email: [email protected]

Author Response File: Author Response.pdf

Reviewer 2 Report

This paper described a cell-free, small-molecule loaded biphasic biomimetic scaffold that supports human mesenchymal stem cells adhesion, proliferation and differentiate to either the osteogenic and chondrogenic pathways. In in vivo rat models, this scaffold showed regeneration of osteochondral defects. However, this paper didn’t state what controls were used for each experiments making it difficult to judge the outcome of the results. Some methods are lacking details also make it hard to interpret the results. The figures could be improved by having lettered numbering. The conclusion that can be drawn from in vivo experiment could be strengthen by having at least one other methods to assess recovery such as immunohistological staining of collagen type II, or behavior assessment of the rat to indicate recovery. Discussion section could be improved by focusing on discussing the results and providing meanings to the results.

Specifically:

Line29, Reviewer couldn’t find data that indicates “upregulate double-stranded DNA”.

Methods,

1.       The author described the synthesis of NGFK and GHSM, but it is not clear how the two scaffolds are put together as a biphasic scaffold. It might be helpful to include a figure to illustrate the scaffold structure as well.

2.       What are the culture conditions?

3.       Line 195. “incubating cells with the scaffold” can authors be more specific on how the cells were incubated with the scaffold? Did you seed the cells on-top of the scaffold, or you had a cell layer on plastic and had the scaffold floating in the well?

4.       If cells were seeded on the scaffold, it is not clear how the cells were harvested for RNA and Western blot analysis.

Figure 1, author might want to label XRD and FTIR graphs as D and E instead of refer to them as Lower/right etc.

Figure 2,

1.       A,B,C are not labeled in the figures but mentioned in the caption.

2.       The order of the LDH and WST-1 graphs in the figure is different to the order they are mentioned in the caption.

3.       What is used for control, negative control, and positive control?

4.       Scale bar is missing

Line 342-343, author mentioned DMMB results for both GHSM and NGFK in Fig3, but only data for NGFK is shown in Fig 3.

Figure 3,

1.       Number the different graphs would make referring to them more clear.

2.       What are the control conditions?

3.       For the Western blots, each lane should be labeled

4.       Not sure how the “relative quantification of protein” is calculated? Is this showing the fold change in densitometry between control vs scaffold?

Figure 5.

1.       No information is given on how this experiment was perform in the figure caption nor in methods section. Which scaffold GHSM or NGFK was used or was this the jointed biphasic scaffold?

2.       How were the hMSCs seeded, what were the culture conditions? What controls were included?

Figure 6.

1.       What was used in the control group?

2.       Scale bars are illegible

Discussion,

1.       Line 395-423 These information are more relevant for introduction

2.       Line 446 authors didn’t show any gene expression from in vivo study

3.       Line 447 Was human serum used for this study?

4.       Line 472 No data indicate modulation of stem cells in vivo

It would be good to have this paper proofread by an editor

Author Response

To Reviewer #: 2

Ref: Manuscript ID: bioengineering-2415695

Title: Small-molecule loaded biomimetic biphasic scaffold for oste-ochondral regeneration: An in vitro and in vivo study.

Authors: Chih-Hsiang Fang, Yi-Wen Lin, Chung-Kai Sun and Jui-Sheng Sun.

Thank you very much for your kind review and comments on our paper. We have learned much from your comments. After looking over your remarks, we feel confident that the revised manuscript addresses your concerns in detail and bring the paper into accordance with the standards expressed in your review. We are resubmitting the revised manuscript and would like to request a further review. We have responded to your original comments on a point-by-point basis below. Additional commentary on the revised manuscript would be highly appreciated.

Comments and Suggestions for Authors

Line 29, Reviewer couldn’t find data that indicates “upregulate double-stranded DNA”.

Thank you for your comments. This statement had been revised in the revised manuscript as the following:

… (GHSM and NGFK layers). The biphasic scaffolds upregulated both osteogenic and chondrogenic gene expression, sulfated glycosaminoglycan (sGAG), osteo- and chondrogenic biomarker, and relative mRNA gene expression. ..

Methods,

1. The author described the synthesis of NGFK and GHSM, but it is not clear how the two scaffolds are put together as a biphasic scaffold. It might be helpful to include a figure to illustrate the scaffold structure as well.

Ans.:

Thank you for your comment. We had revised section 2.3 and 2.4 to explain how the two scaffolds are put together as a biphasic scaffold in the revised manuscript as the following:

2.3 Preparation of metformin embedded gelatin/hydroxyapatite scaffold (GHSM)

….. Metformin (MET) was added to achieve a final concentration of 50 μM. Finally, 0.1% microbial transglutaminase (mTGase; Activa, Ajinomoto, Japan) was used as a cross-linking agent to crosslink the GHSM sponge. The specimens were moved to 4 °C for 24 h, frozen at −20 °C for 24 h, −80 °C 24 h, and then lyophilized for 72 h.

2.4. Synthesis of biphasic scaffold and study of morphology

The biphasic scaffold used for osteochondral regeneration was synthesized by coupling the GHSM sponge (lower layer) and NGFK scaffold (upper layer) with 0.1% microbial transglutaminase. The samples were ….

While extra details was provided in the revised Figure 1.Please refer to the revised manuscript for the details.

2. What are the culture conditions?

Ans.:

Thank you for the comment. As described in “ 2.7. Cell viability”; the culture conditions is following the ISO 10993-5 standard, we use L929 cells (instead of BMSC) to perform and to evaluate the cell viability.

3. Line 195. “incubating cells with the scaffold” can authors be more specific on how the cells were incubated with the scaffold? Did you seed the cells on-top of the scaffold, or you had a cell layer on plastic and had the scaffold floating in the well?

Ans.:

Thank you for the comment. The words “incubating cells with the scaffold” means we put the biphasic scaffold on the bottom of culture disk and then put the cells and culture media on the top of the scaffold. Thank you for the reminds.

4. If cells were seeded on the scaffold, it is not clear how the cells were harvested for RNA and Western blot analysis.

Thank you for the comment. As the cells were put the biphasic scaffold on the bottom of culture disk and then put the cells and culture media on the top of the scaffold, harvests for RNA and Western blot analysis is just similar to routine cell culture. Thank you for the reminds.

Figure 1, author might want to label XRD and FTIR graphs as D and E instead of refer to them as Lower/right etc.

Ans.:

Thank you for your comments. The order of the pictures with consecutive letters had been added in the revised manuscript and description of them had also been added in the figure notes. Please refer to the revised manuscript for the details.

Figure 2,

1. A,B,C are not labeled in the figures but mentioned in the caption.
2. The order of the LDH and WST-1 graphs in the figure is different to the order they are mentioned in the caption.

Ans.:

Thank you for the comment. We had changed the sequence of pictures of Fig. 2A and 2B in the revised manuscript. The order of the pictures with consecutive letters had been added in the revised manuscript and description of them had also been added in the figure notes. Please refer to the revised manuscript for the details.

3. What is used for control, negative control, and positive control?

Ans.:

Thank you for the comment. In this study, we just use the sham-operated osteochondral defect control (but without control scaffold), we had clarified this drawback in the revised manuscript as the following:

Lane 466- 472:

…. However, the in vitro drug release and degradation profile was not evaluated in this study, the relative time-related osteogenic and chondrogenic gene expression effects were not analyzed; the possible time-related benefit from different components maybe overlooked. In this study, we use empty osteochondral defect (without control scaffold) as control, another sham control with control scaffold can be used to validate the role of small-molecule in the osteochondral regeneration. 

4. Scale bar is missing

Thank you for the comment. The scale bars had been added in the revised Figure 7, please refer to the revised manuscript for the details.

Line 342-343, author mentioned DMMB results for both GHSM and NGFK in Fig3, but only data for NGFK is shown in Fig 3.

Thank you for the comment. The data shown in Figure 3 present the change for biphasic scaffold (not only for NGFK or GHSM only). Thank you for your reminds.

Figure 3,

1. Number the different graphs would make referring to them more clear.

Ans.:

Thank you for your comments. The order of the pictures with consecutive letters had been added in the revised manuscript and description of them had also been added in the figure notes. Please refer to the revised manuscript for the details.

2. What are the control conditions?

Ans.:

Thank you for the comment. In this study, we just use the sham-operated osteochondral defect control (but without control scaffold), we had clarified this drawback in the revised manuscript as the following:

Lane 466- 472:

…. However, the in vitro drug release and degradation profile was not evaluated in this study, the relative time-related osteogenic and chondrogenic gene expression effects were not analyzed; the possible time-related benefit from different components maybe overlooked. In this study, we use empty osteochondral defect (without control scaffold) as control, another sham control with control scaffold can be used to validate the role of small-molecule in the osteochondral regeneration. 

3. For the Western blots, each lane should be labeled

Ans.:

Thank you for the comment. We had labeled each lane of the Western blots.

4. Not sure how the “relative quantification of protein” is calculated? Is this showing the fold change in densitometry between control vs scaffold?

Ans.:

Thank you for the comment. As mentioned in “2.11. Western blotting”: …The membranes were then incubated at 37°C with secondary antibodies for 2 h. The proteins were visualized with enhanced chemiluminescence (ECL), and their relative intensity was normalized to the internal control and control group using ImageJ software (National Institutes of Health, Bethesda, MD, USA)… , this showing the fold change in densitometry between control vs scaffold.

Figure 5.

1. No information is given on how this experiment was perform in the figure caption nor in methods section. Which scaffold GHSM or NGFK was used or was this the jointed biphasic scaffold?

Ans.:

Thank you for your comment. We had revised section 2.3 and 2.4 to explain how the two scaffolds are put together as a biphasic scaffold in the revised manuscript as the following:

2.3 Preparation of metformin embedded gelatin/hydroxyapatite scaffold (GHSM)

….. Metformin (MET) was added to achieve a final concentration of 50 μM. Finally, 0.1% microbial transglutaminase (mTGase; Activa, Ajinomoto, Japan) was used as a cross-linking agent to crosslink the GHSM sponge. The specimens were moved to 4 °C for 24 h, frozen at −20 °C for 24 h, −80 °C 24 h, and then lyophilized for 72 h.

2.4. Synthesis of biphasic scaffold and study of morphology

The biphasic scaffold used for osteochondral regeneration was synthesized by coupling the GHSM sponge (lower layer) and NGFK scaffold (upper layer) with 0.1% microbial transglutaminase. The samples were ….

While extra details were provided in the revised Figure 1. Please refer to the revised manuscript for the details.

2. How were the hMSCs seeded, what were the culture conditions? What controls were included?

Ans.:

Thank you for your comment. The culture condition was described at 2.7. Cell viability following the ISO 10993-5 standard, except the L929 cells were changed to hMSC in the other in vitro study,

Figure 6.

1. What was used in the control group?

Ans.:

Thank you for the comment. In this study, for the sake of animal safety, only one leg per animal was used for experiment and two femoral condyles can be used per animal; one of these two condyles was used as the sham (non-Implanted control). We apologize that we can not provide data of implanted with control scaffold. This had been described in the revised manuscript as the following:

2.15. Generation of osteochondral defect

To generate an osteochondral defect, a transverse medial parapatellar incision was made, and the patella was laterally dislocated. A circular hole (1 x 1 mm) was drilled in both the medial and lateral femoral condyle till bleeding from the subchondral bone was observed. Then, the scaffolds were implanted into one of the defective sites. After three months of implantation, the joints were harvested for histological evaluation. Buffered 4% paraformaldehyde was used to fix the joints, the specimens were demineralized, dehydrated, defatted, cleared with xylene, and then embedded in wax.

Lane 466- 472:

…. However, the in vitro drug release and degradation profile was not evaluated in this study, the relative time-related osteogenic and chondrogenic gene expression effects were not analyzed; the possible time-related benefit from different components maybe overlooked. In this study, we use empty osteochondral defect (without control scaffold) as control, another sham control with control scaffold can be used to validate the role of small-molecule in the osteochondral regeneration.  

2. Scale bars are illegible

Ans.:

Thank you for the comment. The scale bars had been added in the revised Figure 7, please refer to the revised manuscript for the details.

Discussion,

1. Line 395-423 These information are more relevant for introduction

Ans.:

Thank you for the comment. Indeed, the information on Line 395-423 are more relevant for introduction, but we mentioned here to reiterate what we mentioned in the introduction and also to quote the following about the following statement on discussion as: “Johnson's first report showed that kartogenin (KGN) could induce in vitro differentiation of human mesenchymal stem cells (hMSCs) into…..”

2. Line 446 authors didn’t show any gene expression from in vivo study

Ans.:

Thank you for the comment. We deleted the word “in vivo” in the revised manuscript. Thank you for the remind.

3. Line 447 Was human serum used for this study?

Ans.:

Thank you for the comment. We did not use human serum in this study.

4. Line 472 No data indicate modulation of stem cells in vivo

Ans.:

Thank you for the comment. From Figure 7, we do observed cartilage regeneration as shown by  Alcian blue and Periodic acid–Schiff (AB/PAS) double staining. This indicate modulation of stem cells in vivo. Thank you four remind.

Thank you very much for your kind review and comments on our paper.

Sincerely yours,

Jui-Sheng Sun, MD, Ph.D.

Department of Orthopedic Surgery, College of Medicine, China Medical University,

No. 2, Yu-Der Rd, Taichung City 40447, Taiwan. Tel: +886-4-22062121

Department of Orthopedic Surgery, National Taiwan University Hospital,

No.7, Chung-Shan South Rd., Taipei 10035, Taiwan, Tel: +886-2-23123456

Email: [email protected]

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The work of this paper is clear and logical. However, I have to reject it because of the following problem:

1.      In response to question 8, the authors answered the reason for the lack of blank scaffolds in the in vivo experiments, which is inconsistent with the lack of blank scaffold groups in the in vitro experiments in the question.

2.      In response to question 9, I don't think L929 cells are a substitute for hBMMSCs, and L929 cells are not the same source as the rats used in animal experiments.

3.      In response to questions 9 and 10, The author's answer does not match the question.

4.      In response to question 13, the authors added a cross-linking step for the material but still did not explain the use of a single layer of GHSM or NGFK rather than the entire composite to describe the data in the subsequent content.

5.      In response to question 16, the authors' response makes experiments lack macroscopic evidence for the extent of chondrogenesis.

6.      In response to question 17, the author could not provide more data. Regretfully, in vivo, experimental data are too scarce to better validate the function of the scaffold

Reviewer 2 Report

The authors have address some of the concerns from the first review comments. The main concern is still the lack of information on what were used for controls for each in vitro experiments. Figure numbers are mismatch between text and figures. Maybe author can revisit your Fang et. al. 2019 IJMS paper for guidance on how to write the figure captions and methods.

Specific comments:

·         please correct all figure references, they are off by 1, and please refer to them with alphabets as well

·         remove all excess “biphasic scaffold (BPS)”, you only need to spell it out once, and can use abbreviation afterwards.

·         It is confusing what scaffolds are used in each experiments, whether it is the GHSM or NGFK separately or the cross-linked BPS.

·         Line 290 should be (BPS)?

·         For figure 3, scale bar is missing, and what were used for control, negative control, and positive control?

·         Figure 4, what was used as control for each experiment?

·         Figure 4D, For Western blots, there are two lanes for each protein, I assume one is control and one is your scaffold? Each lane should be labeled.

·         Figure 5, what was used as control?

·         Figure 6, what scaffold or scaffolds were used for this experiments?