Effect of Phase Distribution on the Antibacterial Property and Cytotoxicity of Ti-5Al-2.5Cu Alloy after Heat Treatment at Various Temperatures

Round 1

Reviewer 1 Report

You did an interesting work but the presentation of it does not satisfy. My remarks are as follows:

In the Abstract, abbreviation (SEM, EDS, TEM) should not be given.

P2 L46: Many authors questioned Al as a biomaterial regarding the allergic. whether the authors knew it and if so, why they did not take it into account in this investigation?

P2 L71: Please explain why did you decide to research the alloy of this composition (Ti-5Al-2.5Cu).

P2 L80: what was the mas of the ingot? whether the ingot was so small that remelting 2 times was enough?

P2 L81: what were the initial dimensions and shape of the casting ingot?

P2 L82: on what basis the authors chose temperatures for heat treatments (720-1040 C)?

P2 L83: please specify the total number of the specimens and also the number of specimens for each heat treatment.

P2 L86: please state the conditions of etching (time and temperature of etching solution)

P2 L93: how many specimens were selected for the cytotoxicity tests?

P3 L111 and L127: what is the origin of the formula (1) and (2) and why the authors chose them? Formula in line 127 should be designated as (2).

P3 L143: How could you state that there was no β phase observed from the SEM micrographs? By which analysis was that established? By SEM??

P3 Figure 2: It cannot be clearly seen magnification scale. Are the all micrographs taken at the same magnification?

P5 L172/173: Figure 6 should be stated as Figure 5.

P6 Figure 5: Distribution of titanium should be given also (EDS map).

P6 Table 1. Are the EDS results average values? If they are, how many measurements were provided and what are the deviations? If the average values are measured, the mean square deviation is needed. If the single values are measured, how to ensure the reliability of the values?

P7 L187: I suppose that Figure 7 should be Figure 6.

P7 194: authors stated: In addition, the antibacterial property decreased when the annealing temperature was increased to 920 °C and 1040 °C. Please provide an explanation.

Author Response

25/06/2020

Prof. Hugo F. Lopez

Editor-in-Chief

Metals

Dear Prof. Hugo F. Lopez:

We would like to resubmit our revised manuscript, titled " Effect of phase distribution on the antibacterial property and cytotoxicity of Ti-5Al-2.5Cu alloy after heat treatment at various temperatures " (Manuscript ID: metals-846648), for consideration for publication in Metals.

   The editor and reviewers have recommended major revisions. We have carefully reviewed these suggestions and revised our text accordingly. Please note that those words within manuscript, tables and figures are written with red words for correction. Our responses to each of the reviewers’ comments are detailed below. The details are provided in the manuscript and mark with red for correction.

Comments And Suggestions for Authors:

# Reviewer 1.

You did an interesting work but the presentation of it does not satisfy. My remarks are as follows: In the Abstract, abbreviation (SEM, EDS, TEM) should not be given.

Answer : Thank you for your suggestions. The abbreviation (XRD, SEM, EDS, TEM) has been replaced at line 17-20 Page 1 of revised manuscript and marked with red.

P2 L46: Many authors questioned Al as a biomaterial regarding the allergic. whether the authors knew it and if so, why they did not take it into account in this investigation?

Answer : Thanks for the question. Al does have a risk of affecting biocompatibility. So, the V and Al were both mentioned in the introduction at line 50-52 Page 2 of revised manuscript and marked with red. Also, we highlight the biocompatibility test for the present alloy was also conducted in the last paragraph of introduction at line 81-82 Page 2 of revised manuscript and marked with red.

P2 L71: Please explain why did you decide to research the alloy of this composition (Ti-5Al-2.5Cu).

Answer : The maximum addition of Al in titanium alloys is about 9 wt%, because it will appear embrittlement with higher Al content, so usually 6 wt% Al is the upper limit. But in bata-Ti, it is 3~4 wt% Al, such as BT14 titanium. Our design is to consider the effect of solid solution strengthening, so the Al content is about 5 wt%. 1~5 wt% Cu is a common additive component used in antibacterial titanium alloy, so our choice falls in the middle.

P2 L80: what was the mas of the ingot ? whether the ingot was so small that remelting 2 times was enough?

Answer : Thank you for your question. The ingot was 5 kg. It was 1 time of melting while casting and 2 times of re-melting was conducted. Overall, it has melted three times.

P2 L81: what were the initial dimensions and shape of the casting ingot?

Answer : Thanks. The ingot was cylindrical in a diameter=10 cm and thickness=15 cm. This information has been supplemented in methods at line 89-90 Page 2 of revised manuscript and marked with red.

P2 L82: on what basis the authors chose temperatures for heat treatments (720-1040 C)?

Answer : Thank you for your pointing out. Based on the Ti-Al and Ti-Cu binary phase diagram, there are two important temperature. One is 790℃ which is the eutectoid temperature for Ti-2.5Cu alloy. The other one is 910℃ which is the transformation temperature for Ti-5Al alloy. To further verification of phase transformation, it is reasonable to conduct the heat treatment between 740-1040℃ which could cover these two temperature.

P2 L83: please specify the total number of the specimens and also the number of specimens for each heat treatment.

Answer : Thank you for your deep concern. In this study, a total of 270 samples were produced and divided into each heat treatment group (n=15) and randomly assigned to subsequent experiments. This information has been supplemented in methods from line 92-94 Page 2 to line 95 Page 3 of revised manuscript and marked with red.

P2 L86: please state the conditions of etching (time and temperature of etching solution)

Answer : Thank you for your kind suggestion. For SEM observation, the specimens were etched for 3 min at room temperature (approximately 25℃) at line 100-102, 105-107 Page 3 of revised manuscript and marked with red.

P2 L93: how many specimens were selected for the cytotoxicity tests?

Answer : Thanks. The sample size for cytotoxicity tests is n=3 and each specimens repeat three times at line 134 Page 3 of revised manuscript and marked with red.

P3 L111 and L127: what is the origin of the formula (1) and (2) and why the authors chose them? Formula in line 127 should be designated as (2).

Answer: Thank you for your suggestions. The formula (1) and (2) have cited appropriate references [10] at line 127 Page 3 and line 144 Page 4 of revised manuscript and marked with red. Also, the formula in line 127 has been designated as (2) at line 144 Page 4 of revised manuscript and marked with red.

P3 L143: How could you state that there was no β phase observed from the SEM micrographs? By which analysis was that established? By SEM??

Answer : Thank you for your deep concern. The phase identification was conducted by XRD and TEM. Based on both of the XRD and TEM examination, it is demonstrated that there are no β phase could be observed. So, the description “there was no β phase observed” was moved to another section at line 198-199 Page 6 of revised manuscript and marked with red because this statement was based on both of the XRD and TEM examination.

P3 Figure 2: It cannot be clearly seen magnification scale. Are the all micrographs taken at the same magnification?

Answer : Thank you for your kind suggestion. The magnification scale was enlarged and all micrographs were taken at the same magnification (x3,000).

P5 L172/173: Figure 6 should be stated as Figure 5.

Answer : We appreciate this helpful comment from the reviewer. The figure order has been corrected at line 204 Page 7 of revised manuscript and marked with red.

P6 Figure 5: Distribution of titanium should be given also (EDS map).

Answer : Thanks for the suggestion of reviewer. The distribution of titanium has been given at line 214 Page 7 of revised manuscript.

P6 Table 1. Are the EDS results average values? If they are, how many measurements were provided and what are the deviations? If the average values are measured, the mean square deviation is needed. If the single values are measured, how to ensure the reliability of the values?

Answer : Thank you for your pointing out. The chemical composition from EDS was average value and it was measured by five different positions to obtain the average and standard deviation. This information has been supplemented at Table 1 Page 8 of revised manuscript and marked with red.

P7 L187: I suppose that Figure 7 should be Figure 6.

Answer : Thank you for your correction. The figure order has been corrected.

P7 194: authors stated: In addition, the antibacterial property decreased when the annealing temperature was increased to 920 °C and 1040 °C. Please provide an explanation.

Answer : Thank you for your suggestions. The explanation for the different antibacterial property after heat treatment at 920 °C and 1040 °C has been discussed from line 342-343 Page 11 to line 344-350 Page 12 of revised manuscript. It has been pointed out that the antibacterial property was affected by the phase distribution. So, the specimen processed 920 °C and 1040 °C heat treatment did not express good antibacterial property due to low volume fraction of Ti₂Cu+Cu-rich phases.

Thank you for your consideration. We hope our manuscript is suitable for publication in your journal.

Sincerely,

Je-Kang Du

Associate professor, School of Dentistry, Kaohsiung Medical University

100 Shih-Chuan 1st Road, San-Ming District, Kaohsiung, Taiwan 807

Tel.: + 886-7-3121101 ext. 7006, Fax: + 886-7-3121510

E-mail: [email protected]

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript presets the results of study of growth of Cu-rich precipitates in TiCuAl alloys and their antibacterial activity. I don't feel competent to asses the biology art of the work, but the physics/material-science part must be improved as for the following items:

1. The authors determined the lattice parameters of the Ti2Cu precipitates, however their numerical values are given without any information on their accuracy (error bars?) The authors probably used x-ray diffraction but the measurement is not commented at all.
2. The topotactical relation between alpha-Ti and Ti2Cu is mentioned in the text but not demonstrated in experimental data
3. Figure 3c is completely unclear - does it refer to direct or reciprocal space and what is the meaning of black and yellow dots?
4. Table 1 is not clear as well. The caption says that the table contains "chemical composition of the Ti2Cu precipitates". This statement seems to me misleading; an intermetallic Ti2Cu precipitate has a given stoichiometry, thus it always contains 33 %at Cu.
5. The crystal structure of the precipitates must be given; in my opinion Ti2Cu is usually tetragonal (space group 139), however from the text it seems to me that the authors found hexagonal Ti2Cu
6. Formal point: the affiliations of the authors are not given (only e-mails). I think it is not possible to publish anything without proper affiliation

Author Response

25/06/2020

Prof. Hugo F. Lopez

Editor-in-Chief

Metals

Dear Prof. Hugo F. Lopez:

We would like to resubmit our revised manuscript, titled " Effect of phase distribution on the antibacterial property and cytotoxicity of Ti-5Al-2.5Cu alloy after heat treatment at various temperatures " (Manuscript ID: metals-846648), for consideration for publication in Metals.

   The editor and reviewers have recommended major revisions. We have carefully reviewed these suggestions and revised our text accordingly. Please note that those words within manuscript, tables and figures are written with red words for correction. Our responses to each of the reviewers’ comments are detailed below. The details are provided in the manuscript and mark with red for correction.

Comments And Suggestions for Authors:

# Reviewer 2.

The manuscript presets the results of study of growth of Cu-rich precipitates in TiCuAl alloys and their antibacterial activity. I don't feel competent to asses the biology art of the work, but the physics/material-science part must be improved as for the following items:

1. The authors determined the lattice parameters of the Ti2Cu precipitates, however their numerical values are given without any information on their accuracy (error bars?) The authors probably used x-ray diffraction but the measurement is not commented at all.

Answer : Thanks for the question. Lattice parameters of the Ti₂Cu precipitates were calculated from both XRD (not shown in the previous manuscript) and TEM-SADPs at line 174-175 Page 4 of revised manuscript and marked with red. For the accuracy of lattice parameters, it was calculated from six different diffraction spots of SADP and the standard deviation of each Lattice parameters was presented. Moreover, the XRD data was added and analyzed in the present version.

2. The topotactical relation between alpha-Ti and Ti2Cu is mentioned in the text but not demonstrated in experimental data

Answer : Thank you for your question. Two more SADPs were added in Fig. 3 to detail the relation between alpha-Ti and Ti₂Cu at line 176-178 Page 4 of revised manuscript and marked with red.

3. Figure 3c is completely unclear - does it refer to direct or reciprocal space and what is the meaning of black and yellow dots?

Answer : Thank you for your kind suggestion. The schematic diagram was a reciprocal space and used to enlarge the SADP to show the diffraction spots more clearly. Different color of dots was used to display different phases in Figure 4 at line 187-192 Page 6 of revised manuscript and marked with red.

4. Table 1 is not clear as well. The caption says that the table contains "chemical composition of the Ti2Cu precipitates". This statement seems to me misleading; an intermetallic Ti2Cu precipitate has a given stoichiometry, thus it always contains 33 %at Cu.

Answer : We appreciate this helpful comment from the reviewer. Alternative words have been used to indicate the precipitates (precipitation-1~3) of different specimens at Table 1 Page 8 of revised manuscript and marked with red. In addition, supplementary explanation: EDS component analysis may detect the nearby matrix, so the components obtained are relative to each other.

5. The crystal structure of the precipitates must be given; in my opinion Ti2Cu is usually tetragonal (space group 139), however from the text it seems to me that the authors found

hexagonal Ti2Cu

Answer : Thank you for your correction. Ti₂Cu is indeed a BCT structure, which has been demonstrated at line 175-178 Page 4 of revised manuscript and marked with red.

6. Formal point: the affiliations of the authors are not given (only e-mails). I think it is not possible to publish anything without proper affiliation.

Answer : Thank you for your pointing out. The affiliations of the authors have been given at line 6-12 Page 1 of revised manuscript and marked with red.

Thank you for your consideration. We hope our manuscript is suitable for publication in your journal.

Sincerely,

Je-Kang Du

Associate professor, School of Dentistry, Kaohsiung Medical University

100 Shih-Chuan 1st Road, San-Ming District, Kaohsiung, Taiwan 807

Tel.: + 886-7-3121101 ext. 7006, Fax: + 886-7-3121510

E-mail: [email protected]

Author Response File: Author Response.pdf

Reviewer 3 Report

1. The authors highlight that the antibacterial properties of Ti-5Al-2.5Cu were 'enhanced' in the abstract but do not say against which sample/how the Ti2Cu / Cu rich phase was increased 
2.  It would be helpful to add details of the different heat treatment temperatures and times used to section 2.1
3. I found the images in Figure 2 rather small and really struggled to view the scale bars. The captions for SEM images should also state the size of scale bars
4. It would have been interesting to include standard Ti alloys (e.g. Ti64) as a control for the bacterial and cell culture assays 
5. It is know that surface roughness and wetability may have influence on bacterial adhesion - Can the authors comment on whether these properties are likely to vary between samples and play a part in the differences observed? 
6. I find the grey colours used for the different groups in Figure 7 very similar and therefore it makes interpretation more difficult
7. An R2 value should be added to figure 8 to confirm suitability of linear regression
8. There are a few spelling / grammatical errors - the authors are encouraged to check the whole manuscript before re-submission
9. The authors should justify the selection of E.coli as the species for antibacterial testing. If these alloys are intended for use as medical devices in orthopaedics then the most common infecting species is typically S.Aureus. It would also be interesting to test both gram positive and negative.
10. Furthermore, bacteria are most commonly observed in a biofilm state when associated with medical device infections - perhaps the authors could include some thoughts on further testing that could be completed and make their findings more clinically relevant?  

Author Response

25/06/2020

Prof. Hugo F. Lopez

Editor-in-Chief

Metals

Dear Prof. Hugo F. Lopez:

We would like to resubmit our revised manuscript, titled " Effect of phase distribution on the antibacterial property and cytotoxicity of Ti-5Al-2.5Cu alloy after heat treatment at various temperatures " (Manuscript ID: metals-846648), for consideration for publication in Metals.

   The editor and reviewers have recommended major revisions. We have carefully reviewed these suggestions and revised our text accordingly. Please note that those words within manuscript, tables and figures are written with red words for correction. Our responses to each of the reviewers’ comments are detailed below. The details are provided in the manuscript and mark with red for correction.

Comments And Suggestions for Authors:

# Reviewer 3.

1.The authors highlight that the antibacterial properties of Ti-5Al-2.5Cu were 'enhanced' in the abstract but do not say against which sample/how the Ti2Cu / Cu rich phase was increased

Answer : Thank you for your suggestions. The description has been re-written at line 24-29 Page 1 of revised manuscript and marked with red.

2. It would be helpful to add details of the different heat treatment temperatures and times used to section 2.1

Answer : We appreciate this helpful comment from the reviewer. The heat treatment temperatures and times were described in section 2.1 at line 92-94 Page 2 of revised manuscript and marked with red.

3.I found the images in Figure 2 rather small and really struggled to view the scale bars. The captions for SEM images should also state the size of scale bars

Answer : Thank you for your kind suggestion. The magnification scale was enlarged and all micrographs were taken at the same magnification (x3,000) in Figure 2 Page 5.

4. It would have been interesting to include standard Ti alloys (e.g. Ti64) as a control for the bacterial and cell culture assays

Answer : Thank you for your deep concern. We’ve done the antibacterial test and cytotoxicity test of both CP-Ti and Ti-64 before, but we didn’t show. Because we wanted to simply compare the antibacterial ability or biocompatibility of Ti-5Al-2.5Cu under different microstructures in the previous version. The comparison of these two alloys seems to be more meaningful for comparing the newly developed alloys. So, we have showed the antibacterial properties and biocompatibility of CP-Ti and Ti-64 in Figure 7 Page 8 of revised manuscript.

5. It is know that surface roughness and wetability may have influence on bacterial adhesion - Can the authors comment on whether these properties are likely to vary between samples and play a part in the differences observed?

Answer : Thank you for your question. Reviewer has mentioned an important issue for bacterial behavior (such as: adhesion). In our current research, the relationship and mechanism between roughness, wettability and bacterial adhesion was processing. A pilot results showed that the lower roughness and higher contact angle express lower bacterial adhesion and biofilm formation. So, we have also considered this point of view, all the specimens used in antibacterial measurement are ground by #100-1500 grit SiC sandpaper to obtain a consistent roughness values (about 0.096 μm) which did not affect the antibacterial behavior. Thus, the difference of microstructure become the only one factor to affect the antibacterial behavior. This information has been supplemented in 2.1. Sample preparation at line 95-97 Page 3 of revised manuscript and marked with red.

6. I find the grey colours used for the different groups in Figure 7 very similar and therefore it makes interpretation more difficult.

Answer ; Thank you for your kind suggestion. The figure has been applied with different patterns for different specimens in Figure 8 Page 9 of revised manuscript.

7. An R2 value should be added to figure 8 to confirm suitability of linear regression

Answer : Thank you for your suggestions. R2 value has been added in Figure 9 Page 11 of revised manuscript.

8. There are a few spelling / grammatical errors - the authors are encouraged to check the whole manuscript before resubmission

Answer : Thank you for your suggestion. The manuscript has been submitted to MDPI English editing service for English editing (English editing ID: English-19898). Every revision had been clearly highlighted with red.

9. The authors should justify the selection of E.coli as the species for antibacterial testing. If these alloys are intended for use as medical devices in orthopaedics then the most common infecting species is typically S.Aureus. It would also be interesting to test both gram positive and negative.

Answer : Thanks for the suggestion of reviewer. The main reason why we chose E coli. as the test object for the present alloy is because once the Ti-Al-Cu alloy can produce antibacterial to E. coli, so it can already initially show that this alloy has antibacterial properties. Moniri Javadhesari et.al indicated that the antibacterial rate of Ti-Cu alloy against E. coli and S. aureus was 96.45 % and 97.99 %, respectively. They believed that Ti-Cu alloy could express same antibacterial property due to the same antibacterial mechanism (Cu ion release and contact sterilization). As the reviewer mentioned, we also believed that exploring a variety of bacteria is meaningful for evaluating antibacterial biomedical materials. However, this study was focus on the E coli first. We will investigate the antibacterial test of both gram positive and negative in next manuscript.

(Moniri Javadhesari, S.; Alipour, S.; Akbarpour, M.R. Biocompatibility, osseointegration, antibacterial and mechanical properties of nanocrystalline Ti-Cu alloy as a new orthopedic material. Colloids and Surfaces B: Biointerfaces 2020, 189, 110889.)

10. Furthermore, bacteria are most commonly observed in a biofilm state when associated with medical device infections perhaps the authors could include some thoughts on further testing that could be completed and make their findings more clinically relevant?

Answer : We appreciate this helpful comment from the reviewer. S. aureus has been used by our team as a strain to explore the mechanism of biofilm growth. And the anti-biofilm ability of different alloys (such as Ti-Ta-Nb, Ti-Zr and Ti-Nb-Ag) were conducting by different surface treatment. We also try to find out the relationship between surface roughness, surface energy and biofilm to avoid infection during the surgery. We sincerely hope that the further article could be published again in Metals. Based on the present study, Ti-5Al-2.5Cu is a potential antibacterial material for using as a dental implant material.

Thank you for your consideration. We hope our manuscript is suitable for publication in your journal.

Sincerely,

Je-Kang Du

Associate professor, School of Dentistry, Kaohsiung Medical University

100 Shih-Chuan 1st Road, San-Ming District, Kaohsiung, Taiwan 807

Tel.: + 886-7-3121101 ext. 7006, Fax: + 886-7-3121510

E-mail: [email protected]

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors answered satisfactorily all comments and criticism in my previous report and modified the manuscript accordingly. The manuscript is now ready for publication