Degradation Behavior, Transport Mechanism and Osteogenic Activity of Mg–Zn–RE Alloy Membranes in Critical-Sized Rat Calvarial Defects

Round 1

Reviewer 1

Thanks a lot for your reviews. I 'd like to answer your concerns. We think our study content may be consistent with journal requirements, because this Mg-Zn-RE alloy was prepared as the membrane implant, and we focused on the interaction between the surface of the membranes and the surrounding tissues, discussed the degradation of the membranes, deposition of products, subsequent transport mechanism, and osteogenic properties of the membrane, which led us to believe that the article may be within the scope of journal contents.

We look forward to hearing from you regarding our submission. We would be glad to respond to any further questions and comments that you may have.

Thanks a lot for your reviews. I 'd like to answer your concerns. We think our study content may be consistent with journal requirements, because this Mg-Zn-RE alloy was prepared as the membrane implant, and we focused on the interaction between the surface of the membranes and the surrounding tissues, discussed the degradation of the membranes, deposition of products, subsequent transport mechanism, and osteogenic properties of the membrane, which led us to believe that the article may be within the scope of journal contents. We look forward to hearing from you regarding our submission. We would be glad to respond to any further questions and comments that you may have.

Reviewer 2 Report

 Dear Authors,

The manuscript is very interesting and it could be published after a minor revision.

I mentioned in text some observations/corrections/suggestions as follows:

1. Lines 78-79: The authors must specify whether they have prepared the “Mg-6Zn-2.7RE alloy” themselves and describe how to prepare it, and if not, the authors must specify the name of the supplier of the Mg-6Zn-2.7RE alloy.

Thank you for your advice. The relevant description was provided in our revised manuscript.

2. Line 82: Authors must specify the name of the UV source and the wavelength used in their research experiments.

We have provided the description in our revised manuscript.

3. Lines 110-111: Please specify if these instruments were sterilized and how.

The relevant description was provided in our revised manuscript.

4. Lines 238, 243, 371, 457, 464, 472, 492, 499, 501, 506, 531, 537, 553, 556, 562: Latin names must be written in italics.

Thanks for the corrections, it has been corrected.

5. At Lines 291-293, the sentences:

“Authors should discuss the results and how they can be interpreted in perspective of previous studies and of the working hypotheses. The findings and their implications should be discussed in the broadest context possible. Future research directions may also be highlighted.”

need to be reformulated and replaced with others. The use of the verb "should" must be avoided. For example, paragraph could begin as follows:

"Furthermore, our results and their interpretations will be presented in the broadest possible context. Future research directions will also be highlighted."

Thanks a lot for your careful reading, this paragraph is the content of the template. We apologize for forgetting to delete it. In the revised manuscript, we have deleted it. Sorry again for this mistake.

6. At Line 304, the authors must specify the geometric parameters of the Mg-6Zn-2.7RE membrane (from microscopy images). What is its thickness?

We have corrected the description in Line 86-87, and also provide the description in Line 304.

7. Line 307: It must be written “α-Mg”, not α Mg.

The format problems have been corrected.

8. Line 324: Please replace “transition into“ with a verb. For example, “converted into...”.

Thank you for your correction. We have taken your specific suggestions.

9. Lines 353-354: Authors must delete 2 rows (lines).

Thanks a lot. We have corrected it in our revised manuscript.

10. Lines 365, 379, 380, 485: Insert a space in the yellow highlighted regions!

Thank you. We have corrected them in our revised manuscript.

11. Lines 404-406: The authors must point out the originality and novelty of their research work, as compared to other studies in the scientific literature.

We have emphasized the innovation of our work in the Lines 404-406 of the revised manuscript.

12. Lines 417-718: Figure 5 does not show the change in bone porosity, but only the change in bone volume.

Sorry for the mistake. We think the results could be seen in Figure 6. So we have corrected it in the revised manuscript.

13. Line 436: Delete space in “node ,”

We have corrected it in our revised manuscript.

14. The section References must be corrected according to the Journal format, and updated with recent studies (2019, 2020). Moreover, authors must check the references: 11; 14; 30; 32; 35; 43.

Thank you for your patience. We have corrected the References format and updated with some studies (2019-2020) in our revised manuscript (reference 4,7,12,45,46).

Reviewer 3 Report

1. First of all, the sentence should be expressed in the third person (Line 291 and 344).

Thanks a lot for your careful reading. The paragraph in Line 291 is the content of the template. We apologize for forgetting to delete it. In the revised manuscript, we have deleted it. Sorry again for this mistake. As for Line 344: We have changed the expression as you suggest. (Line 345 for the revised manuscript).

2. Please indicate what does RE stand for.

RE stands for rare element. ‘RE’ is used to represent rare earth elements when writing Mg-based alloy names. The Line 75-76 may also indicate the issue.

RE stands for rare element. ‘RE’ is used to represent rare earth elements when writing Mg-based alloy names. The Line 75-76 may also indicate the issue.

3. The authors selected a Mg-Zn alloy containing several rare-earth elements as a candidate for biomedical appreciation. What is the reason to examine that specific alloy?

Why a specific composition, 2.7 % rare-earth elements? 

Why the elements such as Y, Gd, La,and Ce were chosen ?

Thanks for your questions. This Mg-based alloy have not been used for the biomedical application before, and when rare earth elements were added for alloying, a mischmetal of various compositions was often used. Y, Gd , La and Ce were often used to modify the mechanical and corrosion properties of Mg-based alloys, so we choose them for
alloying(Line 49-50,57-58). As for 2.7wt% REEs, this specific content is finally determined by the material provider. We think this content is just suitable for studying the whole process of material degradation in the short term.

4. The authors heat-treated the alloy specimens at 175 degree centigrade for 10 hours. Based on what this condition was chosen? In addition, need to indicate the company name where authors get the alloys.

Through preliminary experiments, we believe that the alloy after this heat treatment has good mechanical properties and its degradation behaviour is suitable for shortterm research. In addition, we have provided the company name which supplies the Mgbased alloy in Line 84 of our revised manuscript.

5. Toxicity of rare-earth elements is well known. What is the rational of using alloys containing these elements?

Thanks a lot for your consideration. In the past few years, many REEscontaining Mg-based alloys have been successfully used in in vivo applications. As far as the toxicity of these Mg-based alloys is concerned, there are many differences in reports. Therefore, the REEs’ potential effects and their clearance from the body in vivo are still need
to be studied. In addition, the toxicity and transport mechanism of rare-earth elements may be various because of different intended use of these alloys, and there is no report about the transport of Mg-RE alloy used in craniomaxillofacial bone defect. So we use REEscontaining Mg-based alloys to evaluate the above items.

6. The authors claimed that the alloy tested showed an excellent biocompatibility. Based on what one can says this statement.

We think the 3.2.1Clinical examination and 3.2.3Histological evaluation (Figure 6) could show an excellent biocompatibility, because (1) The movement and daily behaviour of all the tested animals were not affected and no animals died during the observation.(2) There are few inflammatory cells in the samples, except for one sample with
moderate inflammation.

7. Titanium alloys have been well accepted for various biomedical appreciations. Are there any merits of examining Mg alloy?

Compared with Titanium alloys, the density of magnesium alloy is closer to that of human natural bone, and the elastic modulus is more similar to that of natural bone, thus avoiding "stress shielding" effect caused by mismatch of elastic modulus between Ti-alloy implants and human bone tissue, promoting the growth and healing of bone tissue, and preventing the occurrence of secondary fracture. In addition, Mg-based alloys is degradable and their degradation rate are controllable, which is required in some clinical implantation applications. In some biostable metal (such as Titanium alloys) applications, implants usually require secondary surgery to remove them. Mg-based alloy avoids these and reduces many risks. We have provided relevant supplements in the Line 37-43 of the revised manuscript.

8. The authors indicate the background of this study using many references. However, they did just list the references No. Please add the details of each reference as needed. For example, how is the excellent biomechanical properties of the Mg-based alloys compared other alloys used as the biomedical applications.

Thank you for your advices. We have taken your specific suggestions. The corrections were green highlighted in Line 37-43 of our revised manuscript.

Reviewer 4 Report

In general terms the paper is well written and organized. Nevertheless, I have some recommendations for the authors:

1. The EDS spectra tables would be better as separate tables sections.

Thank you for your advices. We have taken your specific suggestions. The corrections were green highlighted in Line 37-43 of our revised manuscript.

2. Figure 10 is hard to read and compare, please make it bigger and the indicate the n of the animals used for this biodistribution.

Thanks for your advice, we have made Figure 10 bigger. In addition, we have provided the number of the animals used for biodistribution tests in Line 292 of our revised manuscript.

3. Discussion: Typo: the first paragraph is the journal instructions, please remove it.

Thanks a lot for your careful reading. I feel so sorry for forgetting to delete it. In the revised manuscript, we have deleted it. Sorry again for this mistake.

4. Histological evaluation

Please discuss the possible effect that the osteoclasts had in the remodeling process as high pH is expected in the area and the osteoclasts are unable to form in this conditions. These cells are essential for bone remodeling. The most problematic issue with Mg based materials is due to the rapid degradation, did the authors consider performing some kind of coating? Have these kinds of materials evaluated in vitro as extracts? Please add some information to the discussion. Which quantity of REE would the cells be able to uptake? as you mention the action of the macrophages.

Thanks for your advices. (1) We have added some discussions about the effect of the pH value on the osteoclasts in Line 408-410 of our revised manuscript. (2) We appreciate that your proposal is the key direction of future research. While our study focused on the degradation and transport mechanism of this Mg alloy, we still need to improve its
corrosion resistance. In our opinion, some kinds of coatings or other surface treatments may be done in the future study. We added the discussion in Lines 426-427. (3) We did not conduct the cell test, since we think the in vitro evaluation may have a certain gap from practical application. While still necessary, we would do that in the future study. We added the discussion in Lines 427-429. (4) Your focus is great, while we think this problem still need further research with flow cytometry, it is very difficult to get a clear conclusion now. However, the result of tissue pathology in present study shows that there are few macrophages cell can be found, the uptake of macrophage maybe not the main transport channel of REEs. This is another direction for our further study.

Round 2

Reviewer 1 Report

1. As I've mentioned previously, this paper has many merits. It is well written, methodologies are logical, the data presentation and discussion is structured. I commend for an excellent work.

I just have some minor suggestions, as will be seen in the attached file.

However, I did have reservations on its relevance to 'çoatings' as a theme. You have pointed out that the implant is a membrane, and therefore is more 'surface' than a 'bulk' material. I can agree on this point.

Going along this theme, this is just a personal query. I understand that your use of a membrane is intentional since it will enhance degradation and will allow you to elicit and study the necessary cellular response within a reasonable period. The question is: Do you think that your proposed mechanisms will change if the specimen is thicker?  Or if the implant is inserted in other sites such as within a bone, away from a lymph node? 

Otherwise, you may have to include a short discussion on the perceived limitations/applicability of the proposed mechanisms/pathways in the paper.

Thank you very much for your time and careful reviews. According with your advices, we have amended the relevant part and the corrections were red highlighted in the revised manuscript.

2. Lines 80-81: Is this thickness? If cutting to a thickness of 130 µm, the cutting technique should be indicated to give an idea of possible damage to membrane.

Thank you for your advice. We have provided the cutting technique-mechanical wire cutting in our revised manuscript (Line 86).

3. Lines 163, 245: Rewrite the expressions.

Thanks for your correction, we have rewritten the sentences/words as you corrected in Lines 170-171 ,250, 252 of our revised manuscript.

4. Line 186: Correct ‘maintains’ to ‘maitained’. 

Thanks a lot for your careful reading. We have corrected it in our revised manuscript (Line 194).

5. Lines 291-293: Delete the portion.

Thanks a lot for your careful reading. I feel so sorry for forgetting to delete it. In the revised manuscript, we have deleted it. Sorry again for this mistake.

6.  Lines 430-432: Alter the sentence.

Thanks for your advice. We have altered the first paragraph of the Conclusions part.

7.  Lines 433,434: Correct the sentence/words.

Thanks a lot. We have corrected them in Lines 437,439 in the revised manuscript.

8. However, I did have reservations on its relevance to 'çoatings' as a theme. You have pointed out that the implant is a membrane, and therefore is more 'surface' than a 'bulk' material. I can agree on this point.

Thanks for your concern. Just as you say, I think our study may be still within the Aims and Scope of Coatings, so we determine to continue. We appreciate your consideration again and thanks for giving it a chance to proceed further.

9.  Going along this theme, this is just a personal query. I understand that your use of a membrane is intentional since it will enhance degradation and will allow you to elicit and study the necessary cellular response within a reasonable period. The question is: Do you think that your proposed mechanisms will change if the specimen is thicker? Or if the implant is inserted in other sites such as within a bone, away from a lymph node? Otherwise, you may have to include a short discussion on the perceived limitations/applicability of the proposed mechanisms/pathways in the paper.

Thank you for your good question. The Mg-alloy can be made to the stent, screw and fixed plate according to various intended use in clinic. Furthermore, the Mg-alloy can also be made to the membrane for Guiding bone regeneration (GBR). There may be different degradation mechanism and tissue response of Mg-alloys in various shapes and
various physiological environments. The present study is to investigate the performance of Mg-alloy membranes in cranio-maxillofacial bone defects, and imitate the clinic usage situation for guiding bone regeneration. We have provided the discussion related in the manuscript (Lines 394-398)

Reviewer 3 Report

I have reviewed "Degradation behavior, transport mechanism and osteogenic activity of Mg-Zn-RE alloy membranes in critical-sized rat calvarial defects".

I think the authors made changes following reviewer suggestions.

This manuscript will be suitable for your journal.

Thank you so much for giving a chance of reviewing this paper to me.

We are grateful to your careful reviews. Many thanks for your interesting and approval for our manuscript.