Characterization, Bioactivity and Antibacterial Properties of Copper-Based TiO2 Bioceramic Coatings Fabricated on Titanium
Round 1
Reviewer 1 Report
Review Manuscript Coatings 380815
Manuscript title: Characterization, Bioactivity and Antibacterial 2 Properties of Copper-Based Tio Bioceramic Coatings Fabricated on Titanium
Authors: Salih Durdu
In this paper the authors report the preparation bioactive and anti-bacterial Cu-based bioceramic (Cu/TiO2) coatings on cp-Ti (Grade 2) by two-steps combining micro-arc oxidation (MAO) and physical vapor deposition – thermal evaporation (PVD-TE) techniques for dental implant applications. In their study, the authors have investigated phase structures, morphologies, elemental amounts, thicknesses, roughnesses and wettabilities of the MAO and Cu-based MAO coating surfaces by XRD (powder- and TF-XRD), SEM, EDS, eddy 16 current device, profilometer and contact angle goniometer, respectively.
In my opinion this paper should be considered for publication with the following major changes:
· The authors should consider adding some biocompatibility and cytotoxicity assays of the coatings.
· Moreover, Figure 7 caption does not explain the figure.
· In order to have good comparative discussions of SEM images, the SEM images should be presented at the same magnification. Otherwise, no sound conclusion could be drawn.
· Also, for the antimicrobial assay a Ti disc should be used as control in order to have the certainty that the antimicrobial activity of the coatings is due to the layer and not to the substrate.
Author Response
Response to Reviewer 1 Comments
In this paper the authors report the preparation bioactive and anti-bacterial Cu-based bioceramic (Cu/TiO2) coatings on cp-Ti (Grade 2) by two-steps combining micro-arc oxidation (MAO) and physical vapor deposition–thermal evaporation (PVD-TE) techniques for dental implant applications. In their study, the authors have investigated phase structures, morphologies, elemental amounts, thicknesses, roughnesses and wettabilities of the MAO and Cu-based MAO coating surfaces by XRD (powder- and TF-XRD), SEM, EDS, eddy 16 current device, profilometer and contact angle goniometer, respectively.
In my opinion this paper should be considered for publication with the following major changes:
Point 1: The authors should consider adding some biocompatibility and cytotoxicity assays of the coatings.
Response 1: Thank you for your useful comment. The formation, characterization and investigation of in vitro bioactive and antimicrobial of the Cu-based MAO coatings were focused respect to the MAO coatings in this study. However, after this work, in vitro (biocompatibility and cytotoxic) and in vivo experimental tests of these types coatings produced at various parameters will be systematically carried out at the future research.
Point 2: Moreover, Figure 7 caption does not explain the figure.
Response 2: Thank you for your useful comment. Figure 7 caption was added in the revised manuscript.
Point 3: In order to have good comparative discussions of SEM images, the SEM images should be presented at the same magnification. Otherwise, no sound conclusion could be drawn.
Response 3: Thank you for your useful comment. The SEM images such as Figures 3a,b and 4a,b have the same magnification (500×) at pre- and post-immersion process in SBF. However, in order to prove the formation of apatite structure on both surfaces, SEM images with high magnification such as Figure 4c,d (20000×) were added to the manuscript at post-immersion in SBF.
Point 4: Also, for the antimicrobial assay a Ti disc should be used as control in order to have the certainty that the antimicrobial activity of the coatings is due to the layer and not to the substrate.
Response 4: Thank you for your useful comment. In this study, the MAO and Cu-based MAO coatings were compared in terms of antimicrobial activity. However, antimicrobial test results of Ti substrate were added into the revised manuscript.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The paper is very similar to the papers published previously and has a lack of novelty (especially with the paper entitled “The dual function of Cu-doped TiO2 coatings on titanium for application in percutaneous implants” published in journal of materials chemistry B). I cannot agree with the publication of this paper.
Author Response
Response to Reviewer 2 Comments
Point 1: The paper is very similar to the papers published previously and has a lack of novelty (especially with the paper entitled “The dual function of Cu-doped TiO2 coatings on titanium for application in percutaneous implants” published in journal of materials chemistry B). I cannot agree with the publication of this paper.
Response 1: Thank you for your comment. However, this study completely differs from aforementioned published work.
Zhang et al. directly fabricated Cu-doped TiO2 coatings in alkaline electrolyte containing β-glycerophosphate disodium, calcium acetate and various amounts of copper acetate on Ti via single step MAO process [Ref. 55]. And then, they investigated in vitro biocompatibility and antibacterial activity for gram positive S. aureus [Ref. 55]. Moreover, in vitro bioactivity of the Cu-MAO coatings were not investigated although in vitro biocompatibility investigations were carried out in above aforementioned published study.
In this work, unlike the literature, a convenient two-steps MAO and PVD-TE techniques were devoted to synthesis uniform, bioactive, biocompatible and anti-bacterial novel Cu-based TiO2 bioceramic composite coatings on cp-Ti substrate. A bioactive and biocompatible anatase and rutile-based bioceramic structures on cp-Ti substrate were coated by MAO technique in an alkaline electrolyte, consisting of Na3PO4 and KOH in de-ionized water at the first step. And then, a copper (Cu) nano-layer with 5 nm thickness was accumulated on the MAO coatings by PVD-TE technique at the second step. The phase structures, morphologies, elemental amounts, functional groups, thicknesses, roughnesses and wettabilities of the MAO and Cu-based MAO coating surfaces were characterized by XRD (powder- and TF-XRD), SEM, EDS, FTIR, eddy current device, profilometer and CAG in detail, respectively. In vitro bioactivity of all coatings was evaluated by immersion tests in SBF at body temperature (36.5 °C) during 28 days. And then, for gram-positive bacteria (Staphylococcus aureus) and gram-negative bacteria (Escherichia coli), in vitro antibacterial properties of all coatings were investigated in detail.
Author Response File: Author Response.pdf
Reviewer 3 Report
Characterization, Bioactivity and Antibacterial Properties of Copper-Based TiO2 Bioceramic Coatings Fabricated on Titanium by S. Durdu
The paper describes the bioactivity and antibacterial properties of Cu-based TiO2 bioceramics. The results presented in the paper are a full complete characterization of this material.
Before publication some small details have to be corrected and revised carefully:
1.- In the title the chemical formula of TiO2 has to be corrected and also other chemical formula in section 2.5. In addition, the TiO2 (no tio2) and other chemical compounds should be corrected in many references: For instance references 12, 13, 14, 15, 16, 18, 20, 21, 22, 28, 35, 36, 37, 39, 40, 42 and many others.
2.- Line 260: Units in the roughness measurements are needed.
3.- I consider that Figure 4 is not needed. With the images of Figure 3 and the data of the roughness are enough. Figure 4 does not provide additional information.
4.- Line 263: I consider that title of section 3.3 is not adequate. I suggest to change “Elemental amounts of the coatings” by “Elemental chemical analysis of the coatings” or something similar.
5.- Figure 5 is also not needed, taking into account the compositions given in Table 3.
6.- Figure 6 is also not needed and the data provide in Table 4 will provide a more clear information in a Figure.
7.- Table 4: Indicate that the contacting time is in seconds.
8.- In the discussion of Figure 7 it is important to indicate if the distribution of the fine particles that are deposited is similar in both samples because taking into account the images presented in Figure 7, the coating seem to be thinner in the Cu coated sample.
9.- In the analysis of Figure 8 and Table 5 it should be important to indicate what happens with peaks that have not been considered in the quantification, for instance, peaks between 1 and 2 eV. It is important to consider that the EDS is not of these particles because due to the penetration of the electrons, an important contribution od the substrate is measured. This should be pointed in the discussion presented in lines 343–348.
10.- Author has described in the paper the behaviour of these novel Cu-based TiO2 coatings. It should be desirable to include in the conclusions section a final paragraph comparing the performance of these coatings with the different alternatives that have been mentioned in the paper.
Author Response
Response to Reviewer 3 Comments
The paper describes the bioactivity and antibacterial properties of Cu-based TiO2 bioceramics. The results presented in the paper are a full complete characterization of this material.
Before publication some small details have to be corrected and revised carefully:
Point 1: In the title the chemical formula of TiO2 has to be corrected and also other chemical formula in section 2.5. In addition, the TiO2 (no tio2) and other chemical compounds should be corrected in many references: For instance references 12, 13, 14, 15, 16, 18, 20, 21, 22, 28, 35, 36, 37, 39, 40, 42 and many others.
Response 1: Thank you for your useful comment. The chemical formula of TiO2 and other compounds are written in original word file according to your useful comments. All chemical formulas (TiO2 and other chemical compounds) were checked throughout the whole text and corrected in the revised manuscript.
Point 2: Line 260: Units in the roughness measurements are needed.
Response 2: Thank you for your useful comment. A unit of the roughness was added as µm in the revised manuscript.
Point 3: I consider that Figure 4 is not needed. With the images of Figure 3 and the data of the roughness are enough. Figure 4 does not provide additional information.
Response 3: Thank you for your useful comment. Figure 4 was removed from the text in the revised manuscript.
Point 4: Line 263: I consider that title of Section 3.3 is not adequate. I suggest to change “Elemental amounts of the coatings” by “Elemental chemical analysis of the coatings” or something similar.
Response 4: Thank you for your useful comment. Title of Section 3.3 was corrected as “Elemental chemical analysis of the coatings” in the revised manuscript.
Point 5: Figure 5 is also not needed, taking into account the compositions given in Table 3.
Response 5: Thank you for your useful comment. Figure 5 was removed from the text in the revised manuscript.
Point 6: Figure 6 is also not needed and the data provide in Table 4 will provide a more clear information in a Figure.
Response 6: Thank you for your useful comment. Figure 6 was removed from the text in the revised manuscript.
Point 7: Table 4: Indicate that the contacting time is in seconds.
Response 7: Thank you for your useful comment. The contacting time was indicated as second in Table 4 in the revised manuscript.
Point 8: In the discussion of Figure 7 it is important to indicate if the distribution of the fine particles that are deposited is similar in both samples because taking into account the images presented in Figure 7, the coating seem to be thinner in the Cu coated sample.
Response 8: Thank you for your useful comment. This section was modified in the direction of your useful comments in the revised manuscript.
Point 9: In the analysis of Figure 8 and Table 5 it should be important to indicate what happens with peaks that have not been considered in the quantification, for instance, peaks between 1 and 2 eV. It is important to consider that the EDS is not of these particles because due to the penetration of the electrons, an important contribution od the substrate is measured. This should be pointed in the discussion presented in lines 343–348.
Response 9: Thank you for your useful comment. You are right. There are some undefined and missing peaks between 1 and 2 eV. These peaks could not be defined by EDS. Also, as removed from Figure 5 in the direction of your useful comments, Figure 8 was removed from this section due to the existence of Table 5 in the manuscript. So, this was pointed in the revised manuscript. This section was modified in the direction of your useful comments in the revised manuscript.
Point 10: Author has described in the paper the behaviour of these novel Cu-based TiO2 coatings. It should be desirable to include in the conclusions section a final paragraph comparing the performance of these coatings with the different alternatives that have been mentioned in the paper.
Response 10: Thank you for your useful comment. This section was modified in the direction of your useful comments in the revised manuscript.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
In my opinion the manuscript could be considered for publication after performing a spell check of the entire manuscript.
Also, for future studies I strongly recommend the authors to revise the XRD experimental set up for the investigation of the coatings because the XRD patterns presented for the investigated coatings are of poor quality.
Author Response
Response to Reviewer 1 Comments
Point 1: <span style="; ;Times New Roman", serif;">In my opinion the manuscript could be considered for publication after performing a spell check of the entire manuscript.
Response 1: Thank you for your useful comment. All manuscript was checked. The spell checked process was carried out through whole text in the revised manuscript.
Point 2: Also, for future studies I strongly recommend the authors to revise the XRD experimental set up for the investigation of the coatings because the XRD patterns presented for the investigated coatings are of poor quality.
Response 2: Thank you for your useful comment. I will keep in mind your useful comments on this for future studies.
Author Response File: Author Response.pdf
Reviewer 2 Report
Although the author explain the difference current study with the mentioned reference, but the work is very similar, and changing the method and performing the in vitro study do not mean a scientific contribution to the field and still I believe that there is a lack of novelty. Novelty is the quality of being new, original, or unusual however non of these aspects can be seen in the present work. I can not agree with the publication of this paper.
Author Response
Response to Reviewer 2 Comments
Point 1: Although the author explain the difference current study with the mentioned reference, but the work is very similar, and changing the method and performing the in vitro study do not mean a scientific contribution to the field and still I believe that there is a lack of novelty. Novelty is the quality of being new, original, or unusual however non of these aspects can be seen in the present work. I can not agree with the publication of this paper.
Response 1: Thank you for your comment. I respect your comment on this subject. However, I disagree with you on this. This study completely differs from aforementioned published work as mentioned my previous response. Moreover, I supported this with other literature studies in the revised manuscript. Also, the novelty of this work was clearly emphasized as below:
Zhang et al. directly fabricated Cu-doped TiO2 coatings in alkaline electrolyte containing β-glycerophosphate disodium, calcium acetate and various amounts of copper acetate on Ti via single step MAO process [Ref. 55]. And then, they investigated in vitro biocompatibility and antibacterial activity for gram positive S. aureus [Ref. 55]. Moreover, in vitro bioactivity of the Cu-MAO coatings were not investigated although in vitro biocompatibility investigations were carried out in above aforementioned published study.
Zhang et al. claimed: “TiO2 coatings doped with Cu2+ were directly prepared via MAO by adding various amounts of copper acetate into electrolytes. Fibroblasts (L-929) were seeded onto the TiO2 surfaces to investigate the effect of Cu2+ on cytocompatibility and skin regeneration potential. Previous evidence has shown that pocket formation can provide a moist and warm environment for the enhancement of bacterial colonization and enhance epithelial downgrowth, leading to the implant failure. In this process, S. aureus usually plays a major part.12 So, the antibacterial activities of the Cu-doped TiO2 coatings against S. aureus were also examined.”
In this work, unlike the literature, a convenient two-steps MAO and PVD-TE techniques were devoted to synthesis uniform, bioactive, biocompatible and anti-bacterial novel Cu-based TiO2 bioceramic composite coatings on cp-Ti substrate. A bioactive and biocompatible anatase and rutile-based bioceramic structures on cp-Ti substrate were coated by MAO technique in an alkaline electrolyte, consisting of Na3PO4 and KOH in de-ionized water at the first step. And then, a copper (Cu) nano-layer with 5 nm thickness was accumulated on the MAO coatings by PVD-TE technique at the second step. The phase structures, morphologies, elemental amounts, functional groups, thicknesses, roughnesses and wettabilities of the MAO and Cu-based MAO coating surfaces were characterized by XRD (powder- and TF-XRD), SEM, EDS, FTIR, eddy current device, profilometer and CAG in detail, respectively. In vitro bioactivity of all coatings was evaluated by immersion tests in SBF at body temperature (36.5 °C) during 28 days. And then, for gram-positive bacteria (Staphylococcus aureus) and gram-negative bacteria (Escherichia coli), in vitro antibacterial properties of all coatings were investigated in detail.
Author Response File: Author Response.pdf
Reviewer 3 Report
In Table 2 author has included two values of “Pressure of vacuum evaporation system” without explaining the difference. This information is in the previous paragraphs but if Table is included (is it really needed?) it should be autoconsitent. In addition, these values do not coincide with the values presented in the text: lines 133 and 135.
Please consider revising equation in line 190. Considering dimensions is not correct. Maybe it should be [(CI-CF)/CI] x 100 or [1-CF/CI] × 100. It is important to specify that CF is the number of colonies in the surface that is being compared with a reference surface CF.
Line 222: Correct “positively charge cationic” by “positively charged cationic”
Correct sentence “Furthermore, another reason of hydrophilicity of the TiO2-based MAO surfaces have rough surface as reported [75]” in line 308.
Figure caption of Figure 4 is not correct. MAO surface corresponds to images a and c and Cu-based MAO surface to images b and d.
Also, some errors in chemical formula remain after revision: References 4 (niti), 21 (tio2), 33 (mg), 49 (cu), 56 (cu-tiO2)
Author Response
Response to Reviewer 3 Comments
Point 1: In Table 2 author has included two values of “Pressure of vacuum evaporation system” without explaining the difference. This information is in the previous paragraphs but if Table is included (is it really needed?) it should be autoconsitent. In addition, these values do not coincide with the values presented in the text: lines 133 and 135.
Response 1: Thank you for your useful comment. I am really sorry for this mistake. This section was corrected and Table 2 was removed from the revised manuscript.
Point 2: Please consider revising equation in line 190. Considering dimensions is not correct. Maybe it should be [(CI-CF)/CI] × 100 or [1-CF/CI] × 100. It is important to specify that CF is the number of colonies in the surface that is being compared with a reference surface CF.
Response 2: Thank you for your useful comment. This section was modified in the revised manuscript.
Point 3: Line 222: Correct “positively charge cationic” by “positively charged cationic”.
Response 3: Thank you for your useful comment. Line 222: “positively charge cationic” was corrected as “positively charged cationic” in the revised manuscript.
Point 4: Correct sentence “Furthermore, another reason of hydrophilicity of the TiO2-based MAO surfaces have rough surface as reported [75]” in line 308.
Response 4: Thank you for your useful comment. This sentence was corrected in the revised manuscript.
Point 5: Figure caption of Figure 4 is not correct. MAO surface corresponds to images a and c and Cu-based MAO surface to images b and d.
Response 5: Thank you for your useful comment. Figure caption of Figure 4 was corrected in the revised manuscript.
Point 6: Also, some errors in chemical formula remain after revision: References 4 (niti), 21 (tio2), 33 (mg), 49 (cu), 56 (cu-tiO2).
Response 6: Thank you for your useful comment. All errors in chemical formula were checked through whole text and they were corrected in the revised manuscript.
Author Response File: Author Response.pdf
Round 3
Reviewer 3 Report
Suggested corrections have been made.
