Investigation of Ion Release and Antibacterial Properties of TiN-Cu-Nanocoated Nitinol Archwires

Round 1

Reviewer 1 Report

1- The English language used in the manuscript should be improved to enhance clarity and comprehensibility. Some sentences need to be rephrased for better clarity.

2- All abbreviations should be written in their full form when mentioned for the first time.

3- In line 17, the sentence should be revised to state that the antibacterial activity was tested, not analyzed.

4- In the Materials and Methods section, all techniques and methods should be properly referenced.

5- In Table 2, there seems to be an inconsistency with the standard deviation (SD) values, which appear to be higher or equal to the result values. For example, if the result value is 5000 cells, the SD should be around ±5, not 4000 cells. Please double-check the SD values as this seems incorrect.

6- It is important to mention the source of the materials and chemicals used in the study. Please include this information in the Materials and Methods section.

7- How was the metabolic activity tested? It would be more accurate to describe it as cell viability rate or cytotoxicity activity instead of metabolic activity.

1- The English language used in the manuscript should be improved to enhance clarity and comprehensibility. Some sentences need to be rephrased for better clarity.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding corrections highlighted in the re-submitted file.

Comment 1: The English language used in the manuscript should be improved to enhance clarity and comprehensibility. Some sentences need to be rephrased for better clarity.

Response 1: The paper has been reviewed by the person with the English proficiency, and the corrections have been made in order to improve its quality.

Comment 2: All abbreviations should be written in their full form when mentioned for the first time.

Response 2: We have checked the abbreviations and made the corrections so their full name is written when mentioned for the first time.

Comment 3:  In line 17, the sentence should be revised to state that the antibacterial activity was tested, not analyzed.

Response 3: The verb in the sentence was corrected.

Comment 4:  In the Materials and Methods section, all techniques and methods should be properly referenced.

Response 4: We have added the references regarding the MTT test and antibacterial test.

Comment 5:  In Table 2, there seems to be an inconsistency with the standard deviation (SD) values, which appear to be higher or equal to the result values. For example, if the result value is 5000 cells, the SD should be around ±5, not 4000 cells. Please double-check the SD values as this seems incorrect.

Comment 6:  It is important to mention the source of the materials and chemicals used in the study. Please include this information in the Materials and Methods section.

Response 6: We have provided additional information regarding the composition of the tested archwires: Investigated archwires (0.018 × 0.025-inch; Superelastic NiTi, OC Orthodontics- containing 55% nickel and 45% of titanium; Stainless Steel- American Orthodontics- containing 18–20% of chromium and 8–10% of nickel; and chemicals used for MTT test (e.g. 5 mg/mL in a phosphate buffer; Thermo Fisher Scientific, Inc., sodium dodecylsulfate in 0.01 mol/L HCl (Serva, Heidelberg, Germany).

Comment 7: How was the metabolic activity tested? It would be more accurate to describe it as cell viability rate or cytotoxicity activity instead of metabolic activity.

Response 7: Biocompatibility was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT). This test is commonly used colorimetric test for biocompatibility investigation. The MTT reagent passes through the cell membrane as well as the mitochondrial inner membrane of viable cells where it is reduced to formazan by metabolically active cells, so it is more precise to use metabolic activity, because we cannot claim if the cell proliferation occurred.

 Ghasemi M, Turnbull T, Sebastian S, Kempson I. The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis. Int J Mol Sci. 2021 Nov 26;22(23):12827. doi: 10.3390/ijms222312827. PMID: 34884632; PMCID: PMC8657538.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript contains valuable information. However, it needs some modifications as follows:

1. The hypothesis for the synthesis is lacking in the introduction.

2. Please provide the state-of-art. On the other hand, why such nanoparticles are useful and selected? What is the knowledge gap and novelty of the work?

3. In the section of Discussion, the authors should also mention the factors that may have an influence on the biological activity of inorganic nanoparticles. These factors include size distribution, morphology, surface charge, surface chemistry, capping agents, etc. Follow and cite the articles below to support the above explanations.

https://doi.org/10.1016/j.talanta.2022.123374

doi: 10.22037/ijpr.2020.113820.14504

4. Statistical analysis is suggested to be included in MTT analysis to compare the significance among groups.

5. More information should be included in the section of Discussion.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding corrections highlighted in the re-submitted file.

Comment 1: The hypothesis for the synthesis is lacking in the introduction.

Response 1: We have further explained the reasons why combination of two methods have been used and defined new hypothesis: Based on the introductory information, the aim of this study was to coat NiTi archwire with TiN-Cu by applying combined technologies of CAE and DC-MS and further to investigate ion release and antibacterial properties in comparison to the NiTi and Stainless Steel (SS) archwires.

Comment 2: Please provide the state-of-art. On the other hand, why such nanoparticles are useful and selected? What is the knowledge gap and novelty of the work?

Response 2: We have provided more state-of-art findings that explain why we have selected nano Cu as coating metal.

• In order to introduce antibacterial properties to NiTi archwires, most commonly used inorganic nanoparticles are silver (Ag) and titanium dioxide (TiO₂) [17]. Arising issues of nano Ag safety and increased friction respecting TiO₂ nanocoatings [18], address different strategy must be implemented. Copper (Cu) nanoparticles possess the antibacterial effects against different bacterial strains [19]. Moreover, Cu is one of essential elements in cell metabolism, cell proliferation and differentiation, as well as in fight against cancer [20]. It is estimated that the Cu content of the maximum 2% wt in the coatings is biocompatible, making it suitable for the NiTi archwires surface modification [21].

Comment 3: In the section of Discussion, the authors should also mention the factors that may have an influence on the biological activity of inorganic nanoparticles. These factors include size distribution, morphology, surface charge, surface chemistry, capping agents, etc. Follow and cite the articles below to support the above explanations.

https://doi.org/10.1016/j.talanta.2022.123374

doi: 10.22037/ijpr.2020.113820.14504

Response 3: We thank the reviewer for this very valuable comment because we think it improved the manuscript significantly. The influence of particle size and shape has been discussed, as well as its influence on biocompatibility. However, we have found references that in our opinion better reflect the nature of used inorganic metal- copper.

- combination of spherical and elongated morphologies, and their agglomerations. Such topography promotes corrosion resistance and consequently increases biocompatibility, due to decreased surface roughness [18]. In addition, favorable mechanical properties, especially lower friction may be accomplished, which is very crucial during OT [18].

- Recent studies show that antibacterial effects of nano Cu not only depend on size, but also on particles morphology and bacterial strain nature [38,39]. The different results acquired regarding the nano Cu particle size may be contributed to the usage of different bacterial strains and methodologies applied. Spherical shaped copper nanoparticles showed more antibacterial property on gram positive bacteria than on gram negative bacteria [38,39]. These findings support our result because both investigated bacterial strains are gram positive.

Comment 4: Statistical analysis is suggested to be included in MTT analysis to compare the significance among groups.

Response 4: We have included the information regarding the significance among groups (marked in yellow in the paragraph MTT Analysis.

Comment 5: More information should be included in the section of Discussion.

Response 5: More information regarding the SS archwires in terms of chromium ion release has been discussed: The release of Cr²⁺ increased with time in acidic environment, comparably to the study of Laird et al. [34]. Furthermore, the lowest metabolic activity observed in case of SS archwires may be contributed to the highest Cr²⁺ release. Besides NiTi archwires, most commonly used during OT are SS archwires. Therefore, it was the main reason we have included SS archwires in the present study.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript describes the investigation of the enhanced corrosion resistance and antibacterial properties to NiTi archwire by coating them with TiN-Cu.

The scope is adequate; the methods are comprehensive for achieving significant results to achieve the objectives.  

The reproducibility is compromised by the lack of some experimental details and clarity, such as full product info of the procured samples, sample size immersed in Petri dishes. Result presentation needs to be improved. Statistical analysis results as well as EDS have not been presented.

The title should be modified to reflect the corrosion resistance.

Thus a major revision is recommended before further consideration.

Some suggestions for consideration:

Paragraph Line 85 needs clarity.

Please double check equation in Line 147 and give what each term means.

Fig. 1 a, b, the scale bars are not visible. Please mark the surface characteristics and the particle size. What is EDS result?

Which SS did you use? Does it contain Ti? What was the purpose of measuring SS wires? SS results have not been discussed though.

The general introduction of nitinol needs references, please consider citing the recent doi10.3390/met12030406

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding corrections highlighted in the re-submitted files.

The manuscript describes the investigation of the enhanced corrosion resistance and antibacterial properties to NiTi archwire by coating them with TiN-Cu.

The scope is adequate; the methods are comprehensive for achieving significant results to achieve the objectives.  

Comment 1: The reproducibility is compromised by the lack of some experimental details and clarity, such as full product info of the procured samples, sample size immersed in Petri dishes. Result presentation needs to be improved. Statistical analysis results as well as EDS have not been presented.

Response 1: The full product names have been added with more detailed information regarding their chemical composition and the sample number and the sample size immersed in Petri dishes- Investigated archwires (Superelastic NiTi, OC Orthodontics, Oregon, USA- containing 55% nickel and 45% of titanium; Stainless Steel- American Orthodontics, Washington, USA- containing 18–20% of chromium and 8–10% of nickel; and TiN-Cu nanocoated archwires) were sterilized using UV and then immersed in Petri dishes (n= 6, per archwire0.018 × 0.025-inch) containing DMEM at 37°C in for 7 days, 21 days and 28 days (W/V ratio was 0.1 mg/mL).

The results regarding FESEM analysis were better explained and the Figure 1 has been changed. We have made the separate paragraph for the EDS results, so they can be more visible and easier to follow. We have changed the presentation of Table 2., so it would be easier to follow.

Comment 2: The title should be modified to reflect the corrosion resistance.

Response 2: We have changed the title of the paper into „Investigation of ion release and antibacterial properties of TiN-Cu nanocoated nitinol archwires”

Comment 3: Paragraph Line 85 needs clarity.

Response 3: We have rephrased the sentence so it would be more clear to read: Prior to deposition, the substrate was ultrasonically cleaned in trichlorethylene, rinsed with alcohol, and then dried with N gas.

Comment 4: Please double check equation in Line 147 and give what each term means.

Response 4: We have check equation and define the abbreviation OD as optical density.

Comment 5: Fig. 1 a, b, the scale bars are not visible. Please mark the surface characteristics and the particle size. What is EDS result?

Response 5: We would like to thank the Referee for these comments. According to the Referee’s comment, we provide the more visible bar lines in Figure 1 a,b. Also, in Figure 1b, we have marked the surface characteristics, as well as the size of the different particles. In line with these changes, additional text was added in the manuscript (Results section), which was highlighted in yellow. We also revised the text related to FESEM analysis. In addition to the EDS results, i.e. the spectrum indicating the peaks of the elements contained in the sample, we have now included a table with the percentage composition in the inset of Figure 1c.

Comment 6: Which SS did you use? Does it contain Ti? What was the purpose of measuring SS wires? SS results have not been discussed though.

Response 6: We have used Stainless Steel archwires- American Orthodontics containing 18–20% of chromium and 8–10% of nickel which does not contain Ti. We have investigated SS archwires because they also contain Ni, and besides NiTi, these archwires are most commonly used during orthodontic treatment. More information regarding the release of chromium have been presented. Furthermore, the results obtained for SS archwires have been discussed.  

Comment 7: The general introduction of nitinol needs references, please consider citing the recent doi10.3390/met12030406

Response 7: We have provided more information regarding NiTi archwires in the Introduction part and implemented the reference you have recommended. During the 1960s a new alloy made of nickel and titanium- named nitinol was developed by W. F. Buehler at the Naval Ordnance Laboratory. The use of NiTi archwires in orthodontic applications has expanded significantly due to unique mechanical properties such as shape memory effect (SME) and superelasticity (SE).

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors should check on all abbreviations

That's ok

Author Response

The paper has been checked by the person with the English proficiency, and small corrections have been made. We have checked the abbreviations once again and made the corrections.

Author Response File: Author Response.docx

Reviewer 3 Report

All the issues have been addressed.

Author Response

There are no comments to be answered.

Author Response File: Author Response.docx