Comparison of Corrosion Resistance and Cytocompatibility of MgO and ZrO₂ Coatings on AZ31 Magnesium Alloy Formed via Plasma Electrolytic Oxidation

Round 1

Reviewer 1 Report

Summary: The authors claimed the coating of the medical grade Mg alloy AZ31 with MgO and ZrO₂ layers by using one-step plasma electrolytic oxidation (PEO) procedure in order to increase the alloy corrosion and cytocompatibility. The results obtained conducted the authors to the conclusion that AZ31 Mg alloy coated with ZrO₂ presents the highest corrosion resistance and cytocompatibility in indirect contact in vitro assays.

The authors should consider the following points:

1. “Introduction” provides the context for the approached topic but there is the penultimate phrase However, although AZ31 magnesium is a medical-grade magnesium alloy, the long-term corrosion resistance of ZrO₂-coated AZ31 magnesium alloy in simulated body fluid, and the cytocompatibility of ZrO2-coated AZ31 magnesium alloy are seldom reported” that requires to include those references that are seldom reported. Please also change "Stefan et al." to "Shultzemosgan et al." and improve the English language within the whole manuscript.

2. “Materials and methods”

a) Please provide methodology of the hydrogen evolution measurement.

b) The authors are advised to revise the Section 2.4. For instance they do not mention whether the cell culture medium contained serum which is an important aspect.

c) Likewise, it is written „Cells were cultured in 96-well plates for 24 h with a cell density of 2.5 × 10³). It is not about cell density here because this a measure of the number of cells plated/squared cm but it seems to show the number of cells seeded in a well. Please revise.

d) Why the authors chose an extraction ratio of 0.5 square cm/ml culture medium?

3. “Results and discussion”

a) Although I am not a specialist it seems to me that the authors do not show EDS spectra of the coated Mg alloy as it is mentioned within the text and the figure legend. Please revise. Also, the pictures are not labeled a) or b).

b) The authors wrote „However, the viabilities of L-929 cells cultured with the extract media of ZrO₂-coated AZ31 for 4 and 7 days are much higher than those of MgO-coated AZ31 and uncoated AZ31, with the difference between ZrO₂-coated AZ31 and MgO-coated AZ31 being statistically significant”. Please correct since statistically significant differences seem to be also between ZrO₂-coated AZ31 and uncoated AZ31 alloy at these two time points.

c) In my opinion the paragraph „The relationship between biological materials and cell adhesion properties is affected by many parameters, such as the roughness of the material surface, interface energy, surface element composition metallographic structure, and others [35]. Also, the snowflake cluster particles deposited on the surface of the ZrO₂ coating improved the roughness of the surface, thereby facilitating the adhesion of cells [31] has no sense in the context of the indirect contact assays such those performed in this study.

d) The authors wrote „Figure 11b shows the morphologies of the L-929 cells cultured in extraction medium from (a) AZ31, (b) MgO-coated AZ31, (c) ZrO₂-coated AZ31, and (d) negative control for 7 days”. It is about Figure 11 and it seems that the sequence of the pictures is“(a) negative control, (b) AZ31, (c) MgO-coated AZ31, and (d) ZrO₂-coated AZ31” instead of “(a) AZ31, (b) MgO-coated AZ31, (c) ZrO₂-coated AZ31, and (d) negative control”.

4. “References”

Please carefully check this section and correct it by presenting it in accordance with the Coating journa’s styling.

Author Response

Point 1:” Introduction” provides the context for the approached topic but there is the penultimate phrase” However, although AZ31 magnesium is a medical-grade magnesium alloy, the long-term corrosion resistance of ZrO₂-coated AZ31 magnesium alloy in simulated body fluid, and the cytocompatibility of ZrO₂-coated AZ31 magnesium alloy are seldom reported” that requires to include those references that are seldom reported. Please also change” Stefan et al” to” Shultzemosgau et al” and improve the English language within the whole manuscript.

Thank you for your advice. We are grateful for the suggestions. We have added the related references into the revised manuscript according to the comment. Also, we are sorry for our carelessness, the author’s name was modified according to the comment (Line 77, page 2).

Point 2:” Materials and methods”

a) Please provide methodology of the hydrogen evolution measurement.

a) We are grateful for your kind suggestions. Thank you for underlying this deficiency. We have added the methodology of the hydrogen evolution according to the comment (Line 268, page 11).

b) The authors are advised to revise the Section 2.4. For instance, they do not mention whether the cell culture medium contained serum which is an important aspect.

b) Thank you for underlining this deficiency. The cell culture medium contained 10% (v/v) fetal bovine serum. And the description of this section has been modified according to the comment (Line 131, page 3).

c) Likewise, it is written “Cells were cultured in 96-well plates for 24h with a cell density of 2.5 × 10³”. It is not about cell density here because this a measure of the number of cells plated/squared cm but it seems to show the number of cells seeded in a well. Please revise.

c) We are grateful for your advice. This sentence has been changed into “Cells were cultured (2.5 × 10³ cell/mL) in 96-well plates for 24h”.

d) Why the authors chose an extraction ratio of 0.5 square cm/ml culture medium?

d) Thank you for your suggestions. According to IOS standard, we need to immerse the sample completely into the medium. Due to material size limitations, we made a slight change to this ratio. So we ended up using this ratio.

Point 3. ” Results and discussion”

a) Although I am not specialist it seems to me that the authors do not show EDS spectra of the coated Mg alloy as it is mentioned within the text and the figure legend. Please revise. Also, the pictures are not labeled a) or b).

a) Thank you for your kind suggestions. I am sorry that I did not provide complete information. We have added the EDS spectra of the coated Mg alloy in the article. Also, we have modified the pictures according to the comments. (Line 190, page 6).

b) The authors wrote “However, the viabilities of L-929 cells cultured with the extract media of ZrO₂-coated AZ31 for 4 and 7 days are much higher than those of MgO-coated AZ31 and uncoated AZ31, with the difference between ZrO₂-coated AZ31 and MgO-coated AZ31 being statistically significant”. Please correct since statistically significant differences seem to be also between ZrO₂-coated AZ31 and uncoated AZ31 alloy at these two time points.

b) Thank you for your kind suggestions. In this part, we emphasized the difference in cytotoxicity between ZrO₂-coated AZ31 and MgO-coated AZ31 instead of describing all the differences. Now we have added all the description into the revised manuscript. (Line 292, page 13).

c) In my opinion the paragraph” The relationship between biological materials and cell adhesion properties is affected by many parameters, such as the roughness of the material surface, interface energy, surface element composition metallographic structure, and others [35]. Also, the snowflake cluster particles deposited on the surface of the ZrO₂ coating improved the roughness of the surface, thereby facilitating the adhesion of cells [31],” has no sense in the context of the indirect contact assays such those performed in this study.

c) Thank you for your kind suggestions. And we are sorry for our carelessness. We have already modified this part according to the comments.

d) The authors wrote “Figure 11b shows the morphologies of the L-929 cells cultured in extraction medium from (a) AZ31, (b) MgO-coated AZ31, (c) ZrO₂-coated AZ31, and (d) negative control for 7 days”. It is about Figure 11 and it seems that the sequence of the pictures is “(a) negative control, (b) AZ31, (c) MgO-coated AZ31, and (d) ZrO₂-coated AZ31” instead of “(a) AZ31, (b) MgO-coated AZ31, (c) ZrO₂-coated AZ31, (d) negative control”.

d) Thank you for your suggestions. We are sorry for our carelessness. We have modified the part according to the comments (Line 298, page 13).

Point 4. ” References”

Please carefully check this section and correct it by presenting it in accordance with the Coating journal’s styling.

We are grateful to your suggestions. Thank you for underlying this deficiency. We have already modified those references’ styling according to the comments.

Reviewer 2 Report

Line 77: put the correct name of the author of the reference [25].

Line 87: SiC instead of Sic.

Table 1: Na₃PO₄ instead of (NaPO₃)₆. Add the pH values of the electrolytes.

Line 124. Describe “hydrogen evolution test” in details.

Figures 2–4: The symbols a), b), c), d) are not seen at the SEM pictures. In addition, the order of appearance of the images is not everywhere the same. Figure 2b appears on the right upper side, while Figure 3b is on left down side.

Tables 2–4. What is a precision of your EDX measurement? What are the deviations of the EDX measurements? How many measurements were performed to obtain these values? It is known that the composition of the PEO coatings is not constant laterally. In this case, one cannot justify such a precision of the EDX measurements. It is not clear if the composition of the ZrO₂ coating is stable inside and outside the micropores.

Line 165: It is not clear why the pH values are fluctuating between alkaline and acidic.

3.3.1. Electrochemical measurements.

The precision of the measurements is incredibly high. The deviations are missing.

Line 229: “Bode” instead of “bode”.

Figures 7 are blurring. Please, improve their quality.

The cytotoxicity tests should be conducted during 21 days in order to observe a long-term cell response. It is very possible that during first 7 days the cell response is positive due to an absence of corrosion, then it turns to negative because the corrosion starts.

If it is not possible to repeat these tests and to conduct them during 21 days, you should at least write in the conclusions that the cell response is positive during first 7 days. The long-term cell response is unknown.

Line 325: “In-vitro” instead of “the vitro”.

Author Response

Point 1: Line 77. Put the correct name of the author of the reference [25].

Thank you for underlining this deficiency. This mistake has already modified according to the comment (Line 77, page 2).

Point 2: Line 87. SiC instead of Sic.

Thank you for underlining this deficiency. We are sorry for our incorrect writing. We have changed it according to the comment (Line 89, page 2).

Point 3: Table 1. Na₃PO₄ instead of (NaPO₃)₆. Add the pH values of the electrolytes.

Thank you for your kind suggestions. In this experiment, we use (NaPO₃)₆ as the concentration of electrolyte. And we are sorry that the pH value of the electrolyte is not written clearly. We have already added the PH value to the Table 1 (Line 99, page 3).

Point 4: Line 124. Describe” hydrogen evolution test” in details.

Thank you for your suggestions. We have already added the detailed process into the revised manuscript (Line 268, page 11).

Point 5: Figures 2–4. The symbols a), b), c), d) are not seen at the SEM pictures. In addition, the order of appearance of the images is not everywhere the same.Figure 2b appears on the right upper side, while Figure 3b is on left down side.

We are grateful to your suggestions. The order of these symbols has been modified according to the comment.

Point 6: Tables 2–4. What is a precision of your EDX measurement? What are the deviations of the EDX measurements? How many measurements were performed to obtain these values? It is known that the composition of the PEO coatings is not constant laterally. In this case, one cannot justify such a precision of the EDX measurements. It is not clear if the composition of the ZrO₂ coating is stable inside and outside the micropores.

Thank you for underlining this deficiency. The precision of this EDX measurement is two numbers behind the decimal point. This EDX measurement was repeated three times, all the data were directly from the instrument and we chose the most stable one. As a result of your suggestion, we found out the deficiencies in our work, we will think more carefully in this section in the future.

Point 7: Line 165. It is not clear why the pH values are fluctuating between alkaline and acidic.

Thank you for your suggestion. The pH value of the electrolyte does not fluctuate. This sentence means, snowflake cluster particles can be formed when the PH value of the electrolyte is alkaline or too acidic. In this experiment, the PH of the electrolyte was acidic, so snowflake cluster particles were formed on the surface of the coating.

Point 8: 3.3.1 Electrochemical measurements. The precision of the measurements is incredibly high. The deviations are missing.

Thank you for underlining this deficiency. We have added the deviations in the Table 2 (Line 211, page 8).

Point 9: Line 229. ” Bode” instead of “bode”.

Thank you for underlining this deficiency. We are sorry for our carelessness. We have modified this place according to comment (Line 228, page 9).

Point 10: Figures 7 are blurring. Please, improve their quality.

Thank you for underlining this deficiency. We have improved the quality of this picture according to the comment (Line 247, page 10). In addition, the quality of this picture was limited by the size of A4 paper, so we recommend the editor that the picture will be clearer when zoom in it.

Point 11: The cytotoxicity tests should be conducted during 21 days in order to observe a long-term cell response. It is very possible that during first 7days the cell response is positive due to an absence of corrosion, then it turns to negative because the corrosion starts. If it is not possible to repeat these tests and to conduct them during 21 days, you should at least write in the conclusions that the cell response is positive during first 7 days. The long-term cell response is unknown.

Thank you for your suggestions. As the cells were raised about 7 days, contact inhibition occurred and it influenced the experiment result. So we cut the experiment down to 7 days. In addition, we added expression in the conclusion part (Line 325, page 15).

Point 12: Line 325.” In-vitro” instead of “the vitro”.

Thank you for underlining this deficiency. This phrase was modified according to the comment (Line 324, page 15).

Round 2

Reviewer 1 Report

The authors considered my comments. However, I would suggest to make some minor changes in the final form of the manuscript:

1) “Chen [22] et al.” (lines 73, 81) and “Depprich [26] et al.” (line 82) changed to “Chen et al. [22]” and “Depprich et al. [26]”, respectively; 2) “Cells were cultured (2.5 × 10³ cell/mL) in 96-well plates for 24 h. The L-929 cells were cultured in RMPI-1640 as negative group. Then, the culture medium was replaced by the extraction medium” changed to “Cells were seeded at a concentration of 2.5 × 10³ cells/mL in 96-well plates and cultured for 24 h. Then, the culture medium was replaced by the extraction medium excepting the cytotoxicity negative group which was represented by L-929 cells cultured in RMPI-1640”; 3) “corrosion densities (I_corr)” changed to “corrosion current densities (I_corr)”.

Author Response

The authors considered my comments. However, I would suggest to make some minor changes in the final form of the manuscript:

1) “Chen [22] et al.” (lines 73, 81) and “Depprich [26] et al.” (line 82) changed to “Chen et al. [22]” and “Depprich et al. [26]”, respectively; 2) “Cells were cultured (2.5 × 10³ cell/mL) in 96-well plates for 24 h. The L-929 cells were cultured in RMPI-1640 as negative group. Then, the culture medium was replaced by the extraction medium” changed to “Cells were seeded at a concentration of 2.5 × 10³ cells/mL in 96-well plates and cultured for 24 h. Then, the culture medium was replaced by the extraction medium excepting the cytotoxicity negative group which was represented by L-929 cells cultured in RMPI-1640”; 3) “corrosion densities (I_corr)” changed to “corrosion current densities (I_corr)”.

Thanks for your comments. We have revised the text in the paper according to your comments.

Reviewer 2 Report

Good improvement!