In Vitro Degradation Behavior and Biocompatibility of Bioresorbable Molybdenum

Round 1

Reviewer 1 Report

Mo as a biodegradation metallic materials is a potential selection due to its relatively superior mechanical property and biocompatibility. However, its degradation velocity is still need to be improved compared than Mg, Zn and Fe alloy. It is contradictory to control the relation between ion release and biosafety. The test of biocompatibility in this work used pure Mo ions solution, I think that author should use the immersion solution ion after different immersion time to carry out related experiment. In addition, in vivo experiment is still need to further evaluate its actual properties.

Author Response

Dear Sir or Madam,

thank you very much for the review of our article. Concerning you comment, I will try to make our methodical approach clearer:

First, the dissolution of molybdenum in aqueous media follows a reaction cascade outlined in the article and described in detail in our first article on the subject [1]. The last step in the cascade is the dissolution of molybdenum trioxide MoO₃ to molybdate ions MoO₄^2-. I did not write this in the text and have changed the passaged accordingly now (line 333 f.).

 

I hope this explanation and the mentioned additions to the article are satisfactorily. Also, we checked grammar and orthography again and hope to have enhanced the comprehensibility and readability of the manuscript.

 

With best regards,

Christian Redlich

 

References

1. Redlich, C.; Quadbeck, P.; Thieme, M.; Kieback, B. Molybdenum - A biodegradable implant material for structural applications? Acta Biomater. 2020, 104, 241–251, doi:10.1016/j.actbio.2019.12.031.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Authors,

 

The paper is for sure worth for publication. As a Reviewer, I would like to express my positive opinion. Below I have few minor comments:

1. 2.4 – please add ASTM standard to bibliography
2. Please add more comments in sec. Results – Tafel constants
3. Please check English language.

Author Response

Dear Sir or Madam,

thank you very much for the review of our article. In the following, I will answer to your individual comments:

1. 2.4 – please add ASTM standard to bibliography

I have added the norm to the bibliography under number [35].

2. Please add more comments in sec. Results – Tafel constants

I have added a table (Table 4) comprising the electrochemical data for all performed tests and added the corrosion current densities as well as the Tafel constants. I adapted the corresponding text accordingly.

3. Please check English language.

I hope the mentioned additions to the article are satisfactorily. Also, we checked grammar and orthography again and hope to have enhanced the comprehensibility and readability of the manuscript.

 

With best regards,

Christian Redlich

Reviewer 3 Report

Highly assessing the research and obtained results, I have several comments.

1. There is no composition of neither P/M Mo nor commercial Mo. It always should be done, in the first case by EDX (EDS), in the second – even following the certificate. It is important for further discussion of observed effects.
2. Line 255: please give the number and title of a standard(s).
3. The key point is a discussion about the presence of oxides and their role in corrosion. It is stated that „The microstructure shows signs of localized corrosion attacks in the

vicinity of the incorporated oxides, e.g., at the measurement point 2. The degradation product are most likely solid molybdenum oxides since they consist mostly of molybdenum

and oxygen” (lines 335-338).

At first, please show such oxides in photos. Secondly, please identify them with XRD. Third, please explain how such oxides may appear in CA Mo. Or are they absent?

4. Lines 362-364: „From the data published so far, it appears highly probable that the same mechanism that results in dense, uniform and slowly dissolving products is also valid for in-vivo environment”. Please give the appropriate references.
5. Lines 378-380: „Like the electrochemical corrosion rates, the dissolution rate of PM Mo is

slightly higher than for CA Mo. Most probably this is due to the incorporated oxides that

dissolve slightly faster than the Mo matrix”. This statement is highly controversial as to my best knowledge, ceramic oxides are highly resistant to dissolution, and technical metals dissolve quickly. Please either explain it in detail and testify to this conclusion by results, e.g., EDX tests before and after corrosion, detailed analysis of surfaces, and cross-sections focused on quantities of elements and presence of oxides. I do not believe in this speculative explanation, on the other side. If it would be true, why the effect of Mo form is almost negligible, and is it certainly due to a rougher surface for the PM sample?

6. Lines 382-383: „However, the influence of oxides on the degradation of Mo remains a field for future investigations”. It is speculation, what oxides, in both metals? I suggest deleting this sentence.
7. Lines 384-387: „Therefore, future studies on PM Mo should focus on intergranular oxygen reduction through the addition of small amounts of carbon [37] or reducing hydrogen atmosphere during the sintering process”. Please, repeat, to identify the oxides on cross-sections of native materials. It is also a mistakemin the text: oxygen is either interstitial (sometimes intergranular, sometimes not) solution or appears in phase form (oxides). I see no such results to discuss both two forms.
8. Lines 391-392: „in both cases Ca and P were incorporated into the oxide layer”. How do you know? In my opinion, they simply form phosphate films. To obtain such a layer as you suggest, it is necessary to make MAO in a phosphate-containing solution.
9. 2. Biocompatibility. This subchapter says nothing about biocompatibility, and the title should be changed, e.g. for „Modelling of degradation of Mo stent in a human body” or so.

Summarizing, I suggest avoiding these numerous and controversial statements assuming that small differences in corrosion potentials and rates are due to the different toughness of the surface, and not discuss any doubtful role of oxide. In CA Mo corrosion, oxygen plays a minor role even in an aerated solution, and the presence of oxides in PM Mo material, which is very likely but not here justified, can affect rather a strength property.

Some grammar errors have been indicated by color in the attached manuscript.

Comments for author File: Comments.pdf

Author Response

Dear Sir or Madam,

thank you very much for the review of our article. In the following, I will answer to your individual comments:

1.     There is no composition of neither P/M Mo nor commercial Mo. It always should be done, in the first case by EDX (EDS), in the second – even following the certificate. It is important for further discussion of observed effects.

I have added a table comprising the composition/impurities for all purchased materials as specified by the manufacturers (Table 1) and referenced it in the text (lines 128f.). Also I pointed out the EDX analysis for PM Mo in Figure 2f, data points 2-4, in the text in lines 360 ff.

2.    Line 255: please give the number and title of a standard(s).

For unknown reasons, the list of primers for qRT-PCR was deleted in the submitted manuscript. I have added it again as Table 2.

3.     The key point is a discussion about the presence of oxides and their role in corrosion. It is stated that „The microstructure shows signs of localized corrosion attacks in the vicinity of the incorporated oxides, e.g., at the measurement point 2. The degradation product are most likely solid molybdenum oxides since they consist mostly of molybdenum and oxygen” (lines 335-338). At first, please show such oxides in photos. Secondly, please identify them with XRD. Third, please explain how such oxides may appear in CA Mo. Or are they absent?

I extended the paragraphs on degradation and dissolution of the tested materials to hopefully achieve a better comprehensibility for the reader. First, I clarified the correlation between the described color change and the formation of surface oxides (lines 349 ff.).
Furthermore, I specified the existence of oxides in the PM Mo with EDX data shown in Figure 2f (lines 360 ff.). I then pointed out the differing composition of the surface oxide analyzed in data point 1 in Figure 2f (lines 373 f.). We were not able to conduct an XRD measurement in short time, however, because the XRD equipment is under construction at the moment.
The commercially manufactured material does not contain such oxides. However, an oxide layer with incorporated Ca and P, which slightly differs in composition from the PM Mo oxide layer, was also found here (data point 5). I clarified this in the text.

4.    Lines 362-364: „From the data published so far, it appears highly probable that the same mechanism that results in dense, uniform and slowly dissolving products is also valid for in-vivo environment”. Please give the appropriate references.

In all publications on the anodic dissolution of molybdenum in near neutral media, the findings agree with ours findings. I have added the references concerning the overall corrosion mechanism of molybdenum (Johnson et al., Petrova et al.) [1,2] and supporting experimental results (Redlich et al., Yin et al.) [3,4] (line 405).

5.    Lines 378-380: „Like the electrochemical corrosion rates, the dissolution rate of PM Mo is slightly higher than for CA Mo. Most probably this is due to the incorporated oxides that dissolve slightly faster than the Mo matrix”. This statement is highly controversial as to my best knowledge, ceramic oxides are highly resistant to dissolution, and technical metals dissolve quickly. Please either explain it in detail and testify to this conclusion by results, e.g., EDX tests before and after corrosion, detailed analysis of surfaces, and cross-sections focused on quantities of elements and presence of oxides. I do not believe in this speculative explanation, on the other side. If it would be true, why the effect of Mo form is almost negligible, and is it certainly due to a rougher surface for the PM sample?

The formed oxides in the microstructure (see Figure 2d and 2f in data points 2 and 3) are most likely oxides that are part of the dissolution reaction cascade:

Mo à MoO₂ à Mo₂O₅ à MoO₃ à MoO₄^2- (Johnson et al. [1] and Petrova et al. [2])

Thus, they form on native Mo and ultimately dissolve into molybdate anions. MoO₂, which is formed first, was shown to be slightly soluble in physiological media by Mörsdorf et al. [5]. However, since we cannot differentiate the Mo oxide species at the moment and do not have data on their individual dissolution rates, I changed the sentence (lines 421 ff.). It is most likely due to the different microstructure, in which the oxides play a (unclear) role for PM Mo. The surface roughness should be approx. the same, since we treated all samples similarly.

6.     Lines 382-383: „However, the influence of oxides on the degradation of Mo remains a field for future investigations”. It is speculation, what oxides, in both metals? I suggest deleting this sentence.

I have deleted the sentence since the content of microstructural oxygen and oxides must be as low as possible for any practical application of Mo-based implants, anyway.

7.    Lines 384-387: „Therefore, future studies on PM Mo should focus on intergranular oxygen reduction through the addition of small amounts of carbon [37] or reducing hydrogen atmosphere during the sintering process”. Please, repeat, to identify the oxides on cross-sections of native materials. It is also a mistakemin the text: oxygen is either interstitial (sometimes intergranular, sometimes not) solution or appears in phase form (oxides). I see no such results to discuss both two forms.

I have deleted the sentence, too, since the content of microstructural oxygen and oxides must be as low as possible for any practical application of Mo-based implants, anyway.

8.    Lines 391-392: „in both cases Ca and P were incorporated into the oxide layer”. How do you know? In my opinion, they simply form phosphate films. To obtain such a layer as you suggest, it is necessary to make MAO in a phosphate-containing solution.

I changed the wording to “Ca and P are incorporated in the degradation product layer” (line 435) since I cannot prove in which form. However, we already know that Ca and P accumulate in the degradation product layer over time whilst Mo amount decreases, independent of the position in the layer.

9.    2. Biocompatibility. This subchapter says nothing about biocompatibility, and the title should be changed, e.g. for „Modelling of degradation of Mo stent in a human body” or so.

I added a subheading “3.2.1 Model correlation for a hypothetical Mo stent”.

Summarizing, I suggest avoiding these numerous and controversial statements assuming that small differences in corrosion potentials and rates are due to the different toughness of the surface, and not discuss any doubtful role of oxide. In CA Mo corrosion, oxygen plays a minor role even in an aerated solution, and the presence of oxides in PM Mo material, which is very likely but not here justified, can affect rather a strength property.

Some grammar errors have been indicated by color in the attached manuscript.

Thank you very much. I hope the mentioned additions to the article are satisfactorily. Also, we checked grammar and orthography again and hope to have enhanced the comprehensibility and readability of the manuscript.

 

With best regards,

Christian Redlich

 

References

1. Johnson, J.W.; Chi, C.H.; Chen, C.K.; James, W.J. The Anodic Dissolution of Molybdenum. Corrosion 1970, 26, 338–342, doi:10.5006/0010-9312-26.8.338.
2. Petrova, M.; Bojinov, M.; Zanna, S.; Marcus, P. Mechanism of anodic oxidation of molybdenum in nearly-neutral electrolytes studied by electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy. Electrochimica Acta 2011, 56, 7899–7906, doi:10.1016/j.electacta.2010.12.022.
3. Redlich, C.; Quadbeck, P.; Thieme, M.; Kieback, B. Molybdenum - A biodegradable implant material for structural applications? Acta Biomater. 2020, 104, 241–251, doi:10.1016/j.actbio.2019.12.031.
4. Yin, L.; Cheng, H.; Mao, S.; Haasch, R.; Liu, Y.; Xie, X.; Hwang, S.-W.; Jain, H.; Kang, S.-K.; Su, Y.; et al. Dissolvable Metals for Transient Electronics. Adv. Funct. Mater. 2014, 24, 645–658, doi:10.1002/adfm.201301847.
5. Mörsdorf, A.; Odnevall Wallinder, I.; Hedberg, Y. Bioaccessibility of micron-sized powder particles of molybdenum metal, iron metal, molybdenum oxides and ferromolybdenum--Importance of surface oxides. Regul. Toxicol. Pharmacol. 2015, 72, 447–457, doi:10.1016/j.yrtph.2015.05.027.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

I am fully satisfied with answers to my remarks and changes made in the revised manuscript. I have not other comments.