Controlled release of basic fibroblast growth factor from a peptide biomaterial for bone regeneration


 Self-assembled peptide scaffolds based on D-RADA16 are an important matrix for controlled drug release and three-dimensional cell culture. In this work, D-RADA16 peptide hydrogels were coated on artificial bone composed of nano-hydroxyapatite/polyamide 66 (nHA/PA66) to obtain a porous drug-releasing structure for treating bone defects. The developed materials were characterized via transmission electron microscopy and scanning electron microscopy. The proliferation and adhesion of bone mesenchymal stem cells (BMSCs) were examined by confocal laser microscopy and CCK-8 experiments. The osteogenic ability of the porous materials towards bone BMSCs was examined
 in vitro
 by staining with Alizarin Red S and alkaline phosphatase, and bioactivity was evaluated
 in vivo
 . The results revealed that nHA/PA66/D-RADA16/bFGF reduces the degradation rate of D-RADA16 hydrogels and prolongs sustained release of bFGF, which would promote BMSCs proliferation, adhesion and osteogenesis
 in vitro
 and bone repair
 in vivo
 . Thus, it deserves more attention and is worthy of further research.



Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
The work entitled 'Controlled release of basic fibroblast growth factor from a peptide biomaterial for bone regeneration' is well conducted and needs some minor revisions, as commented below: 1. Pls analyze other cell morphological factors (other than cell roundness) that are related with cell properties. 2. Reason how more proliferative cells are better in osteogenic differentiation. 3. It is better to analyze ECMs of in vivo tissue samples to see the effect of bFGF release more clearly. 4. Other approaches of novel scaffolds and stem cell therapies (apart from delivery approach) should also be cited to feedback the recent research trend in bone regeneration (some representative review papers helpful for this are shown below) -de Grado, Gabriel Fernandez; Keller, Laetitia; Idoux-Gillet, Ysia. Bone substitutes: a review of their characteristics, clinical use, and perspectives for large bone defects management. J. Tissue

Review form: Reviewer 2
Is the manuscript scientifically sound in its present form? No

Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
The manuscript contains interesting analysis and data about the combination of commonly used materials (NHA/PA66) with self-assembling peptides, in the field of bone defects regeneration.
The developed experiments and the obtained results are promising at all levels: material characterization, in vitro cell analysis and animal models. However, the text is not easy to follow and in general it does not explain properly the realized experiments and the obtained results. Moreover, the discussion and conclusions do not properly encompass the results of the experiments.
Specific considerations: 1-Material and methods -Need to be better described to allow the reader following the experiment or even repeat it. In its current form it is not possible.
-It is stated that during the electron microscopy assay, only the surface of the composites are analysed. It is not what it can be seen in the results. -I miss the supplier of the reagents -Control and samples groups need to be clearly defined for each experiment -Cell roundness analysis needs to be explained. Why is it measured using DAPI staining? It is not cell roundess, it is nuclei roundess, -I miss the passage of the BMSC used for experimentation and the time from cell seeding until the begining of the experiment. -Why is cell proliferation analyzed in well plate? The control group should be the composite without RAD16 and/or bFGF. -I miss an explanation of statistical analysis for each experiment (N,n and statistical analysis).

2-Results
-Material Characterization --> needs to be better described in the results -Cellular deformation? Do you mean cell phenotype? - Figure 2: control group needs a better picture. Which is the control group in this case: 2D well plate or NHA/PA66? -Cell proliferation, alzarin red and ALP activity needs to be normalized and expressed as % compared to control groups -Consider rewriting figure captions.
-I miss an explanation of bFGF interaction with the composite. Is it embedded? -In vitro osteogenesis results could be better described.
3-Discussion -Proliferation is not explained.
-The release of bFGF can be adjusted by varying RAD16 concentration, but never varying the amount of bFGF.
-The stability of RAD16 is improved due to it is protected by the macroporus of the NHA/PA66. -Consider revising avoiding subsections and describing the work as a whole.
-I miss an explanation of the impact of in vivo results. 4-General considerations -It is important to maintain the nomenclature during all the mansucript. -As a recommendation, I would recommend to revise the paragraph describing the self assembling peptide RAD16 in the introduction.
-A brief description of the mechanical properties of the used materials should be added since the final application of the composite is bone regeneration. -A description of the abbreviations is needed. -To follow the text easily, I recommend to maintain the titles of the subsections of materials and methods and the ones in the results. -Mechanical assay results should be included in the discussion and compared with previous bibliography.
Decision letter (RSOS-191830.R0) 18-Dec-2019 Dear Dr zhao, The Subject Editor assigned to your paper ("Controlled release of basic fibroblast growth factor from a peptide biomaterial for bone regeneration") has now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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Kind regards, Andrew Dunn Royal Society Open Science Editorial Office Royal Society Open Science openscience@royalsociety.org on behalf of Professor Guy Genin (Associate Editor) and Malcolm White (Subject Editor) openscience@royalsociety.org Associate Editor Comments to Author (Professor Guy Genin): Associate Editor Comments to the Author: The paper is much improved since the original submission. However, several aspects of the manuscript still need to be brought into a more standard form. Both reviewers note that many required aspects of the manuscript are missing (for example, the suppliers for reagents), and both note as well that extra care must be made to remove any conclusions that are not specifically supported by the results. Finally, the manuscript could benefit from a thorough rewriting that streamlines the authors' arguments and places these in the context of the current literature.
In addition to strengthening these aspects of the write-up, which in the associate editor's opinion is a requirement for publication in RSOS, Reviewer #1 has some very nice ideas for further analyzing your data. The associate editor recommends considering these carefully, but notes as well that following these suggestions for additional plots is not a requirement for publication.
Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) The work entitled 'Controlled release of basic fibroblast growth factor from a peptide biomaterial for bone regeneration' is well conducted and needs some minor revisions, as commented below: 1. Pls analyze other cell morphological factors (other than cell roundness) that are related with cell properties. 2. Reason how more proliferative cells are better in osteogenic differentiation. 3. It is better to analyze ECMs of in vivo tissue samples to see the effect of bFGF release more clearly. 4. Other approaches of novel scaffolds and stem cell therapies (apart from delivery approach) should also be cited to feedback the recent research trend in bone regeneration (some representative review papers helpful for this are shown below) Comments to the Author(s) The manuscript contains interesting analysis and data about the combination of commonly used materials (NHA/PA66) with self-assembling peptides, in the field of bone defects regeneration. The developed experiments and the obtained results are promising at all levels: material characterization, in vitro cell analysis and animal models. However, the text is not easy to follow and in general it does not explain properly the realized experiments and the obtained results. Moreover, the discussion and conclusions do not properly encompass the results of the experiments.
Specific considerations: 1-Material and methods -Need to be better described to allow the reader following the experiment or even repeat it. In its current form it is not possible.
-It is stated that during the electron microscopy assay, only the surface of the composites are analysed. It is not what it can be seen in the results.
-I miss the supplier of the reagents -Control and samples groups need to be clearly defined for each experiment -Cell roundness analysis needs to be explained. Why is it measured using DAPI staining? It is not cell roundess, it is nuclei roundess, -I miss the passage of the BMSC used for experimentation and the time from cell seeding until the begining of the experiment. -Why is cell proliferation analyzed in well plate? The control group should be the composite without RAD16 and/or bFGF.
-I miss an explanation of statistical analysis for each experiment (N,n and statistical analysis).

2-Results
-Material Characterization --> needs to be better described in the results -Cellular deformation? Do you mean cell phenotype? - Figure 2: control group needs a better picture. Which is the control group in this case: 2D well plate or NHA/PA66? -Cell proliferation, alzarin red and ALP activity needs to be normalized and expressed as % compared to control groups -Consider rewriting figure captions.
-I miss an explanation of bFGF interaction with the composite. Is it embedded? -In vitro osteogenesis results could be better described.

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Comments to the Author(s)
The authors have answer all the questions presented in previous revision and have include the recommendations in the text. I consider that the the text can be published in the current form.

12-Feb-2020
Dear Dr Zhao, It is a pleasure to accept your manuscript entitled "Controlled release of basic fibroblast growth factor from a peptide biomaterial for bone regeneration" in its current form for publication in Royal Society Open Science. The comments of the reviewers who reviewed your manuscript are included at the foot of this letter.
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Please see the Royal Society Publishing guidance on how you may share your accepted author manuscript at https://royalsociety.org/journals/ethics-policies/media-embargo/. The authors have answer all the questions presented in previous revision and have include the recommendations in the text. I consider that the the text can be published in the current form.
Follow Royal Society Publishing on Twitter: @RSocPublishing Follow Royal Society Publishing on Facebook: https://www.facebook.com/RoyalSocietyPublishing.FanPage/ Read Royal Society Publishing's blog: https://blogs.royalsociety.org/publishing/ Editor comments to the Author: The authors try an interesting new approach to bone defect healing. However, the study seems to be lacking a negative control, and the write-up has discussion mixed in with the results. I would welcome a reworked version of the manuscript that shows negative controls (that is, compares results of the treatment cases to what happens when the animal is left alone; these data almost certainly exist somewhere for this model, and the authors likely need not perform new experiments). In this reworked manuscript, please be careful to ensure that every statement in the results is an actual result rather than an interpretation, as this journal is very strict about ensuring that every statement in the results section is backed up by experiment. More speculative interpretations could be included in a discussion section. Minor notes: please be careful about the number of "significant digits" in your results, and please check the language in the figure captions carefully.

Respond:
Dear editor： Thanks for your comments. A negative control (black control) was added in the manuscript speculative interpretations have been removed from "Results" best, Weikang Appendix A Reviewer: 1 1. Pls analyze other cell morphological factors (other than cell roundness) that are related with cell properties.
Answer: I have added other cell morphological factors in the discussion.
Cell morphology was observed by immunofluorescence staining, we can conclude that bFGF affects early cell adhesion. After being cocultured with different groups for 12 h, the morphology of BMSCs in these groups with bFGF was flat and the area large, indicating that the cell spreading is good. Many pseudopodium were observed in the cells, indicating that the cell extending is good, while the cells in the groups without bFGF were smaller with fewer pseudopods.
2. Reason how more proliferative cells are better in osteogenic differentiation.
Answer: No direct results from this experiment suggest that proliferation can promote BMSCs osteogenic differentiation. bFGF promotes cell adhesion, proliferation, and osteogenic differentiation have been reported. But bFGF's rapid inactivation restricts its bioactivity. The main purpose of this experiment is to verify the slow-release ability of this slow-release material in the future, so that the working time of bFGF will be extended. The long-term retention of bFGF may be the reason that the cells of the nHA / PA66 / D-RADA16 / bFGF group have better proliferation and osteogenesis 3. It is better to analyze ECMs of in vivo tissue samples to see the effect of bFGF release more clearly.
Answer: In this study, the u-CT results showed that the bone density of the bFGF controlled-Appendix B release group was significantly increased compared with the other two groups. Since the formation of new bone is closely related to EMC, this result can indirectly reflect the correlation between bFGF and EMC in osteogenesis. ECM is a very broad concept that plays a major role in bone formation. ECM contains bone morphogenetic proteins, fibronectin, laminin, vitronectin, etc. The detection of EMC secretion can indeed make the research deeper explore the relationship between ECM and bFGF, and we will conduct further research in subsequent experiments.

Other approaches of novel scaffolds and stem cell therapies (apart from delivery approach)
should also be cited to feedback the recent research trend in bone regeneration (some representative review papers helpful for this are shown below) Answer: Reviewers recommend very helpful articles, and I will add all of them in subsequent revisions.
Reviewer: 2 1-Material and methods -Need to be better described to allow the reader following the experiment or even repeat it. In its current form it is not possible.

Answer: Materials and methods have been Revised
-It is stated that during the electron microscopy assay, only the surface of the composites are analyzed. It is not what it can be seen in the results.
Image-pro plus was employed to measure the macropore size of nHA/PA66 and the length as well as width of nanofibers (n=12). The porosity of porous nHA/PA66 was measure as followsa: 1.Prepare the same volume of nHA / PA66 porous materials and non-porous solid nHA / PA66 material.
2.Measure the mass of each material on the analytical balance to calculate the density of the porous material and non-porous material for the next step of material pores Calculation of rate.
3.The calculation formula of the material porosity is as follows: Porosity of porous bone filling material = (1-

Porous bone filling material density
Non−porous solid material density ) × 100% -I miss the supplier of the reagents Answer: I will add them in subsequent revisions.
-Cell roundness analysis needs to be explained. Why is it measured using DAPI staining? It is not cell roundess, it is nuclei roundess, Cellular deformation? Do you mean cell phenotype?
Answer: I apologize for the inaccuracy description. Since DAPI stains the nucleus, phalloidin stains the cytoplasm. A live cell requires both signals. Phalloidin stains were used for the cell roundness test Cell roundness is employed to measure the cell shape. There are reports that after the cells adhere, the cells will spread slowly to increase the attach strength. This experiment measured cell roundness and analyzed of cytoskeleton morphology to indirectly reflect cell adhesion in the early stage of co-culture. Answer: I apologize for the inaccuracy. All experiments were co-cultured with different materials in well plates. In this experiment, CCK8 and Edu were used to evaluate the cell proliferation ability.
CCK8 uses a method of measuring absorbance to indirectly detect cell proliferation ability. Edu staining statistics rely on the method of staining proliferating cells to reflect cell proliferation ability.
The control group used a glass circle. We have previously verified that there are no significant differences between the results of porous nHA/PA66 and control groups. The main purpose of this experiment is to test the sustained release ability of nHA/PA66/D-RADA16/bFGF materials. nHA/PA66/D-RADA16 was used to compare the effect with or without bFGF and nHA/PA66/bFGF was used to compare the control release effect.
-Cell proliferation, Alizarin red and ALP activity needs to be normalized and expressed as % compared to control groups Answer: Thanks for your suggestion. These results have been normalized to control groups and put in revised manuscript -Consider rewriting figure captions.
Answer: It has been revised -I miss an explanation of bFGF interaction with the composite. Is it embedded?
Answer: A better detailed description has been revised in manuscript.
Lyophilized peptide powder was dissolved in deionized water to obtain a 10 mg/ml (w/v) peptide stock solution at 25 °C. Peptide hydrogels in each group were mixed well at 25 °C. In group one, combining 10 mg/ml (w/v) d-RADA16 peptide solution with PBS (pH 7.4) by the volume ratio of 1:1, and with the stimulation of ionic solution PBS, 5 mg/ml d-RADA16 peptide solution would undergo self-assembly for 24 h in order to produce d-RADA16 peptide hydrogel. While in the bFGF group, 50 μg/ml (w/v) bFGF solution was prepared by dissolving bFGF powder into PBS (pH 7.4). And then 10 mg/ml (w/v) d-RADA16 peptide solution was combined well with 50 μg/ml (w/v) bFGF by the volume ratio of 1:1. As a result, bFGF was embedded in D-RADA16 nanofibers.
-In vitro osteogenesis results could be better described.

It has been revised in results
Includes description of 3D images and histological pictures, and quantitative analysis of uCT 3-Discussion -Proliferation is not explained.
Answer: Add more explanation of proliferation The CCK-8 results indicated that there were no significant differences between each group at 6 h.
Specifically, the cell proliferation in nHA/PA66/bFGF and nHA/PA66/D-RADA16/bFGF groups were significantly higher than groups (control and nHA/PA66/D-RADA16) without bFGF at 12 h and 24 h. This effect was more persistent in the nHA/PA66/D-RADA16/bFGF group than in the nHA/PA66/bFGF group, and cell proliferation remained significantly increased after 72 h in the former case. We can conclude that bFGF-containing materials can significantly promote cell proliferation, while slow-release bFGF materials can extend the effect of cell proliferation to a minimum of 72h.
-The release of bFGF can be adjusted by varying RAD16 concentration, but never varying the amount of bFGF.
Answer: Thanks for this very good suggestion, I will add this in my discussing.
In this experiment, we mainly studied the adequacy of this slow-release system and have not thoroughly explored the effects of different concentrations of sustained-release bFGF on cell proliferation, adhesion, and differentiation at various time points. The role of bFGF is very important, including the effects on blood vessels, immune system, etc. We will study in the next experiment.
-The stability of RAD16 is improved due to it is protected by the macroporus of the NHA/PA66.
Answer: Yes, compared with exposed peptides, D-RADA16 nanofibers agglomerated in the gap can have a longer degradation time I will add more about this in discussing.
-Consider revising avoiding subsections and describing the work as a whole.
Answer: Thanks for the suggestions, I have revised it in the paper.
-I miss an explanation of the impact of in vivo results.
Answer: Thanks for the suggestions, I have revised it in the paper.

4-General considerations
-It is important to maintain the nomenclature during all the manuscript.
Answer: Thanks for the suggestions, I will revise it in the paper -As a recommendation, I would recommend revising the paragraph describing the self-assembling peptide RAD16 in the introduction.
Answer: Thanks for the suggestions, I will revise it in the paper -A brief description of the mechanical properties of the used materials should be added since the final application of the composite is bone regeneration.
A better detailed description has been revised in manuscript.
In the field of bone tissue replacement and repair, biomaterials composed of bioactive inorganic compounds (such as nHA) and macromolecular compounds (such as PA66) can provide better mechanical properties and ductility. Specifically, nHA is the main mineral component of bones.
Therefore, HA is extremely biocompatible and does not promote inflammatory responses.
Furthermore, PA66 mimics the collagen component in bones, making the material more ductile.
-A description of the abbreviations is needed.
Answer: I have added the description of the abbreviations in supplement.
-To follow the text easily, I recommend maintaining the titles of the subsections of materials and methods and the ones in the results.
Answer: Thanks for the suggestions, I will modify it in the paper -Mechanical assay results should be included in the discussion and compared with previous bibliography.
Answer: Thanks for the suggestions, I will modify it in the paper Sever previous bibliographies have been compared in discussion