Bioactivity and Mechanical Properties of Hydroxyapatite on Ti6Al4V and Si(100) Surfaces by Pulsed Laser Deposition

Round 1

Reviewer 1 Report

The authors investigated the effects of substrate temperature and ablation wavelength on the mechanical, structural, adhesive, and bioactive properties of HAP coatings. The deposition conditions can stably control the HAP coating. But above 700 °C, its mechanical, bioactivity, and structural properties decline. The data gives optimism that crystallinity is a vital factor in the functionality of the coatings. While I suggest making a few minor revisions before its consideration of publication. There are some comments as follows: 

1. Some punctuation marks in the manuscript are inappropriate, such as "(byXRD?)" in line 303.

2. The legend in Figure 7 of the manuscript covers part of the data, and the data has no error bars.

3. The resolution of some graphs in the manuscript may not be enough, such as figure 8, and the error bars of the box plots are also deviated.

4. In the part of verifying the biocompatibility of materials, only cell proliferation is verified by means of cell counting, and it is also necessary to design experiments to verify the toxicity of materials to cells.

5. The author can refer to more relevant references in the introduction and discussion sections, such as “ Biomaterials translational 2021,2(3):248-256.; Journal of Orthopaedic Translation,2021,30: 82-92.; Biomaterials Translational, 2021, 2(3): 188-196.; Journal of Orthopaedic Translation,2023,40: 27-36.; Journal of Orthopaedic Translation,2022,33: 107-119. ”

Some grammar and words can be considered again.

Author Response

The authors investigated the effects of substrate temperature and ablation wavelength on the mechanical, structural, adhesive, and bioactive properties of HAP coatings. The deposition conditions can stably control the HAP coating. But above 700 °C, its mechanical, bioactivity, and structural properties decline. The data gives optimism that crystallinity is a vital factor in the functionality of the coatings. While I suggest making a few minor revisions before its consideration of publication. There are some comments as follows: 

1. Some punctuation marks in the manuscript are inappropriate, such as "(by XRD?)" in line 303.

Response (R): We thank the Reviewer for the careful analysis of the text and would like to assure her/him that all punctuation issues were fixed.

2. The legend in Figure 7 of the manuscript covers part of the data, and the data has no error bars.

R: This issue is now fixed in the revised version of the manuscript.

3. The resolution of some graphs in the manuscript may not be enough, such as figure 8, and the error bars of the box plots are also deviated.

R: We have fixed this issue.

4. In the part of verifying the biocompatibility of materials, only cell proliferation is verified by means of cell counting, and it is also necessary to design experiments to verify the toxicity of materials to cells.

R: Following the Reviewer’s suggestion, additional MTT assays were performed. No differences in colorimetric results were observed, thus it could be concluded that the coated surfaces did not exhibit any toxicity. Furthermore, Trypan Blue was used to check viability of cells during cell counting and no difference in cell viability among different coated surfaces was found.

5. The author can refer to more relevant references in the introduction and discussion sections, such as “ Biomaterials translational 2021,2(3):248-256.; Journal of Orthopaedic Translation,2021,30: 82-92.; Biomaterials Translational, 2021, 2(3): 188-196.; Journal of Orthopaedic Translation,2023,40: 27-36.; Journal of Orthopaedic Translation,2022,33: 107-119. ”

R: We have added those references in addition to new references. Our introduction is now much stronger. We would like to thank the reviewer sincerely for her/his crucial comments.

The authors would like to thank the reviewers again. We believe that all reviewer concerns have been addressed in the revised version of our manuscript. The authors believe that the manuscript is now significantly improved in terms of concept, technical content, and clarity of exposition.  We look forward to hearing from you.

Sincerely,

Ali Oguz Er

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript reports a detailed study on the coating of bioinert substrates with  hydroxyapatite thin films by nanosecond PLD. Authors compare the effect of laser wavelength and substrate temperature on the coatings physic-chemical and biological properties.  In my opinion some issue have to be considered to improve the manuscript soundness.

1)      The effect of laser wavelength on the physic-chemical properties has been discussed. Can the authors suppose a relationship between the different properties of films obtained by 355nm,532nm and 1064nm and the biological properties (in terms of protein absorption and cells growth)?

2)      can the author report the ablation rate obtained for the three different laser wavelengths that were considered in the manuscript? Moreover, the respective fluencies and the target to substrate distance should be reported

3)      considering figure 4: can the authors discuss the shift of the position of the signals of the titanium substrate in the XRD spectra of the coated substrates?

4)      can the authors evaluate the amorphous amount of the coatings by XRD measurements?

5)      The chemical composition of coatings is missing EDS or XPS measurements should be considered.

6)      There are several typing and editing errors (i.e. line 304 figure 1 instead of figure 6, line 71, line 303), please, check whole the manuscript.

Author Response

The manuscript reports a detailed study on the coating of bioinert substrates with  hydroxyapatite thin films by nanosecond PLD. Authors compare the effect of laser wavelength and substrate temperature on the coatings physic-chemical and biological properties.  In my opinion some issue have to be considered to improve the manuscript soundness.

1)      The effect of laser wavelength on the physic-chemical properties has been discussed. Can the authors suppose a relationship between the different properties of films obtained by 355nm,532nm and 1064nm and the biological properties (in terms of protein absorption and cells growth)?

This discussion is added to the lines between 344-347.  We thank reviewer for pointing out this important point.

2)      can the author report the ablation rate obtained for the three different laser wavelengths that were considered in the manuscript? Moreover, the respective fluencies and the target to substrate distance should be reported. à Wrote a paragraph about the requested analysis.

This is now added to line 114.

3)      considering figure 4: can the authors discuss the shift of the position of the signals of the titanium substrate in the XRD spectra of the coated substrates?

The apparent shift in the titanium substrate could be due to residual, thermal, and epitaxial strain. In addition, thermal strain may play a role due to variations in the cell parameters because of substrate and film having different thermal expansion coefficients.  In addition, some residual strain induced the laser-ablated high-energy particle impingement may cause intrinsic residual strain which may result a slight change of XRD peaks of substrate.  This is added into line 253.

4)      can the authors evaluate the amorphous amount of the coatings by XRD measurements amorphous amount of the coatings by XRD measurements?

We have added additional measurements and comments in line 291 and line 325. We hope that this issue is now resolved.

5)      The chemical composition of coatings is missing EDS or XPS measurements should be considered.

EDS measurement are added into manuscript and we also provided XPS measurement in the supplementary file.

6)      There are several typing and editing errors (i.e. line 304 figure 1 instead of figure 6, line 71, line 303), please, check whole the manuscript.

Those issues are fixed.

Sincerely,

Ali Oguz Er

Reviewer 3 Report

It's a quality job well done. The results presented do not raise doubts and questions. I recommend the work for publication and a little clarification to the authors:
1. Vickers microhardness. Item 2.4. The authors specified one of the methods of mechanical analysis as Vickers microhardness. The results of such a study are not presented. Remove this paragraph from the title, or provide the results of the study, as microhardness measurement would provide a more complete picture of the effect of deposition parameters on the mechanical properties of hydroxyapatite coatings. Supplementing the article with microhardness data would improve its informative value for readers.2. For practical applications, it would be important to investigate the long-term stability of HAP coatings in model biofluids.

3. I recommend that the authors familiarize themselves with the additive manufacturing approach to bioimplants with the example of article 10.3390/jfb14050259. It may be worth expanding the literature review to include a list of advantages and disadvantages of the chosen synthesis method. 

Author Response

Please find attached the revised manuscript entitled “Bioactivity and mechanical properties of hydroxyapatite on Ti6Al4V and Si(100) surfaces by pulsed laser deposition” in accordance with the referees' suggestions. We are grateful for their constructive criticism and suggestions which have helped us in our understanding of this problem and have led to a clearer and stronger manuscript.

This letter includes our revisions and our responses to the reviewers. The authors would like to thank the reviewers for their time and their valuable comments which have helped us improve its scientific level. We have revised our manuscript and have incorporated major new changes in accordance with the reviewers' suggestions. Please find below a detailed response to each of the comments and criticisms

It's a quality job well done. The results presented do not raise doubts and questions. I recommend the work for publication and a little clarification to the authors:

1. Vickers microhardness. Item 2.4. The authors specified one of the methods of mechanical analysis as Vickers microhardness. The results of such a study are not presented. Remove this paragraph from the title, or provide the results of the study, as microhardness measurement would provide a more complete picture of the effect of deposition parameters on the mechanical properties of hydroxyapatite coatings. Supplementing the article with microhardness data would improve its informative value for readers.2. For practical applications, it would be important to investigate the long-term stability of HAP coatings in model biofluids.

We apologize for the mistake. We intended to have microhardness data but due to scheduling conflict we were not able to do that. We remove the part referring to microhardness. We sincerely appreciate reviewer’s comment on this issue.

3. I recommend that the authors familiarize themselves with the additive manufacturing approach to bioimplants with the example of article 10.3390/jfb14050259. It may be worth expanding the literature review to include a list of advantages and disadvantages of the chosen synthesis method.

We have added additional references in to the manuscript. We also added the disadvantages of PLD, which are mainly the formation of particulate formation. We also discussed the possible ways to reduce the particles.  

The authors would like to thank the reviewers again. We believe that all reviewer concerns have been addressed in the revised version of our manuscript. The authors believe that the manuscript is now significantly improved in terms of concept, technical content, and clarity of exposition.  We look forward to hearing from you.

Sincerely,

Ali Oguz Er

Round 2

Reviewer 2 Report

The authors fully addressed all my comments