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Article
Peer-Review Record

Effect of Plasma Treatment of Titanium Surface on Biocompatibility

Appl. Sci. 2019, 9(11), 2257; https://doi.org/10.3390/app9112257
by Daiga Ujino 1, Hiroshi Nishizaki 2, Shizuo Higuchi 2, Satoshi Komasa 3,* and Joji Okazaki 3
Reviewer 1: Anonymous
Reviewer 2: Anonymous
Reviewer 3: Anonymous
Appl. Sci. 2019, 9(11), 2257; https://doi.org/10.3390/app9112257
Submission received: 5 May 2019 / Revised: 23 May 2019 / Accepted: 24 May 2019 / Published: 31 May 2019
(This article belongs to the Special Issue Nanoscale Materials for Drug Delivery and Tissue Engineering)

Round 1

Reviewer 1 Report

Ujino et al investigated the effects of cold atmospheric plasma treatment with a piezo brush on the surface properties of titanium discs and the subsequent effects on cell adhesion and osteogenic differentiation of rat bone marrow cells.

The study is clearly described, results are comprehensibly presented and the manuscript is well-written. However similar approaches have been taken by other researchers. In my opinion, some more discussions of already published studies on the use of cold plasma for surface modification/biocompatibility should be included, especially ones pertaining to dentistry (e.g. Coelho, Argon-based atmospheric pressure plasma enhances early bone response to rough titanium surfaces.)

Please find some more detailed comments blow:

Introduction:

- “However, the devices performing plasma processing are very large and may be impractical for clinical application. In contrast, the plasma apparatus used in the present study was comparatively small and easy to use.”

Other small (hand-held) plasma devices have been studied for their use in surface modification for clinical applications such as plasma jets. Please discuss some more of these studies in your introduction.

Materials and Methods

- Line83/84: “It was used as the active gas in atmospheric-pressure, low-temperature plasma treatment under irradiation at 0.2 MPa for 30 s at 10 mm.” What do the authors refer to by ‘it’? Was plasma generated in air? Please clarify.

- Line 101: “The rate of albumin adsorption was calculated as the % albumin adsorbed onto the samples relative to the total amount of albumin in the solution.” This does not seem to agree with Figure 5 where BSA adsorption is shown in μg/dL. Please correct/clarify.

- Line 125-127: this section is not clear and does not seem to correspond to the CEllTiter Blue protocol: Did the authors use fluorescence measurements at 560/590 or absorbance measurements, in which case the measurements should have been taken at 570 and 600nm.

- Line 127: “Absorbances of the remaining solution were measured at OD560/590.” Does this refer to the remaining solution after removal of 100μl or was the absorbance of the 100μl that were transferred to a 96-well plate measured. In that case: Were 3 x 100μl (triplicates) of the solution transferred to a 96-well pate and measured? Please clarify this section.

- Line 127/128:  “The difference between optical densities was defined as the proliferation value.” Please explain this. What is the proliferation value? Figure 6 does not show proliferation but cell adhesion. How were the units in figure 6 determined? μg/dL seems a strange unit for cell adhesion? I would expect cells/cm2 or similar.

Results

- Figure legends: Please expand the figure legends to describe briefly what is shown in the figures especially in figures consisting of multiple panels.

- Fig. 2: why are the scales on the images different? The authors state that there was no difference in roughness – how did they determine this? Visually the images seem different. Please explain.

- Fig. 3: Please include the values in the pie charts.

- Figure 4: quantification of contact angles? Without quantitative data, you can not really refer to “significant difference”. Please include quantitative data (statistically significant) or rephrase to say ‘clear difference’ or similar.

- Fig 5 and 6: y-axis titles are missing (e.g. conc. BSA): unit needs to be corrected in all panels of figure 5 and panels 2-4 in figure 6: μg dL-1 (-1 is missing). Also I think it would be better to calculate the adsorption/adhesion per surface area (e.g. μg/cm2) rather than per volume…

- Figure 7: Please include the size on the scale bar in these images

- 3.4 Fig. 8: Why was the gene expression of these genes analysed on different days? Please explain the rationale e.g. expression depending on early differentiation/late differentiation stages.

- Figure 9: What is the unit of the y-axis (ALP activity/DNA)? I would also recommend using the same scale (e.g. 0-1) for both graphs as it will make it easier to see the increase in ALP between day 7 and 14.

- Fig. 10: it would be preferable to express the calcium deposition per surface area rather than per volume.

- Line 252: “Conventional plasma devices require vacuum and processing is both constrained and costly.” The authors seem to be considering predominantly large plasma processing devices employed for surface modification. However, there are many atmospheric pressure, cold plasma devices under investigation in ‘plasma medicine’, including plasma jets, DBD systems, micro-barrier discharge etc, which are small, inexpensive to operate and can be used for chairside/bedside applications. Please consider these in your discussions.

- Line 276: “To the best of our knowledge, the present study is the first to compare RBM cell proliferation on plasma-treated titanium surfaces with that on unprocessed controls.”

While it may be correct that no previous studies used rat bone marrow cells, cell proliferation e.g. of bone marrow stromal cells on plasma-treated titanium surfaces has been reported and should be referenced/discussed here. (e.g. Karaman, Synergistic Effect of Cold Plasma Treatment and RGD Peptide Coating on Cell Proliferation over Titanium Surfaces)

- Line 278: “Plasma-treated materials have larger surface areas than untreated controls.” Please explain this statement or reference other studies. Your own analysis found no difference between the surface of treated and untreated titanium.

- Line 297: “The piezobrush plasma device used in the present study is invaluable to clinicians because it is smaller and easier to operate than conventional plasma devices.” Please refer to other handheld plasma devices as well (e.g. plasma jets) as the one described here is not the only one.

- Line 298: “Moreover, the findings of the in vivo efficacy assays performed in the present study suggest that this novel technology could be highly useful in dental practice.”

The authors do not show any in vivo data (e.g. animal study) but only in vitro data. Please correct this statement.


Overall I recommend minor revisions of the manuscript prior to acceptance.

 


Author Response

Thank you very much for circulating our manuscript entitled “Effect of plasma treatment of titanium surface on biocompatibility” by Satoshi Komasa et al. among the members of the editorial board of the Applied Science and forwarding two reviewers’ suggestions to me. I have enclosed our responses to the reviewers’ comments. Attached are ten files associated with our manuscript.

 

Reviewer #1:

 

Thank you very much for your comments. We have revised our manuscript in accordance with your suggestions as follows:

 

1. The study is clearly described, results are comprehensibly presented and the manuscript is well-written. However similar approaches have been taken by other researchers. In my opinion, some more discussions of already published studies on the use of cold plasma for surface modification/biocompatibility should be included, especially ones pertaining to dentistry (e.g. Coelho, Argon-based atmospheric pressure plasma enhances early bone response to rough titanium surfaces.)

 

We agree with your suggestion and have corrected our manuscript as follows:

We unified representation with “alkali-modified” and “unmodified”.

 

2. Introduction:

- “However, the devices performing plasma processing are very large and may be impractical for clinical application. In contrast, the plasma apparatus used in the present study was comparatively small and easy to use.”

Other small (hand-held) plasma devices have been studied for their use in surface modification for clinical applications such as plasma jets. Please discuss some more of these studies in your introduction.

 

We agree with your suggestion and have corrected our manuscript as follows:

Lines 70-78

 For past decades, several techniques have been used for generating plasmas at atmospheric pressure and with a temperature close to ambient such as Radio-frequency plasmas (RF) [23], Dielectric barrier discharges (DBD) plasmas [24], Corona discharge plasmas [24-26], Gliding arc discharge plasmas [27]. The general advantage of these techniques is that the formation of a large number of reactive species could be obtained which was used for the treatment of surface, gases and aqueous solutions. Meanwhile, the miniaturization of the generator employed to create plasma has always been an important subject of research. The aim of these technologies, such as Piezoelectric direct discharge plasmas [28, 29], is to generate plasmas as “thin and small” as possible in terms of clinical application.

             

3. Materials and Methods

- Line83/84: “It was used as the active gas in atmospheric-pressure, low-temperature plasma treatment under irradiation at 0.2 MPa for 30 s at 10 mm.” What do the authors refer to by ‘it’? Was plasma generated in air? Please clarify.

 

We agree with your suggestion and have corrected our manuscript as follows:

Page 2, lines 82-84

 

A Piezobrush PZ2 (Relyon Plazma GmbH, Regensburg, Germany) was used to coat the disks by using active gas in atmospheric-pressure, low-temperature plasma treatment under irradiation at 0.2 MPa for 30 s at 10 mm.

Additionally, we used air to generate plasma.

 

4. - Line 101: “The rate of albumin adsorption was calculated as the % albumin adsorbed onto the samples relative to the total amount of albumin in the solution.” This does not seem to agree with Figure 5 where BSA adsorption is shown in μg/dL. Please correct/clarify.

 

We agree with your suggestion and have corrected our manuscript as follows:

We deleted “The rate of albumin adsorption was calculated as the % albumin adsorbed onto the samples relative to the total amount of albumin in the solution.”

 

5. - Line 125-127: this section is not clear and does not seem to correspond to the CEllTiter Blue protocol: Did the authors use fluorescence measurements at 560/590 or absorbance measurements, in which case the measurements should have been taken at 570 and 600nm

 

According to following references, we used celltiterblue protocol in order to clear cell adhesion on titanium surface.

Xing H, Komasa S, Taguchi Y, Sekino T, Okazaki J. Osteogenic activity of titanium surfaces with nanonetwork structures. Int J Nanomed. 2014; 9: 1741-55.

Su Y, Komasa S, Sekino T, Nishizaki H, Okazaki J. Characterization and Bone Differentiation of Nanoporous Structure Fabricated on Ti6Al4V Alloy. J Nanomater. 2015; http://dx.doi.org/10.1155/2015/358951

Komasa S, Kusumoto T, Taguchi Y, Nishizaki H, Sekino T, Umeda M, Okazaki J. Effect of Nanosheet Surface Structure of Titanium Alloys on Cell Differenciation. J Nanomater. 2014; http://dx.doi.org/10.1155:642527

 

6. - Line 127: “Absorbances of the remaining solution were measured at OD560/590.” Does this refer to the remaining solution after removal of 100μl or was the absorbance of the 100μl that were transferred to a 96-well plate measured. In that case: Were 3 x 100μl (triplicates) of the solution transferred to a 96-well pate and measured? Please clarify this section.

 

We agree with your suggestion and have corrected our manuscript as follows:

Lines 125-126

After incubation at 37 °C for 1 h, the solution was removed and 3 x 100μl (triplicates) of it was transferred to a new Falcon 96-well tissue culture plate

 

7. - - Line 127/128:  “The difference between optical densities was defined as the proliferation value.” Please explain this. What is the proliferation value? Figure 6 does not show proliferation but cell adhesion. How were the units in figure 6 determined? μg/dL seems a strange unit for cell adhesion? I would expect cells/cm2 or similar.

 

We agree with your suggestion and have deleted this sentence.

Additionaly, we have corrected Figure 6.

 

8. - -Results

- Figure legends: Please expand the figure legends to describe briefly what is shown in the figures especially in figures consisting of multiple panels.

 

We agree with your suggestion and have expand the figure legends.

 

9. -Fig. 2: why are the scales on the images different? The authors state that there was no difference in roughness – how did they determine this? Visually the images seem different. Please explain.

 

We agree with your suggestion and have reshoot SPM. 

 

10. - Fig. 3: Please include the values in the pie charts.

 

We agree with your suggestion and have corrected Fig. 3.

 

11. - - Figure 4: quantification of contact angles? Without quantitative data, you can not really refer to “significant difference”. Please include quantitative data (statistically significant) or rephrase to say ‘clear difference’ or similar.

 

We agree with your suggestion and have corrected our manuscript as follows:

Lines 191-192

 There was clear difference between the contact angles of the plasma-treated titanium disks (titanium; 32°, plasma-modified titanium; 0°) and those of the untreated disks (Fig. 4).

 

12. - Fig 5 and 6: y-axis titles are missing (e.g. conc. BSA): unit needs to be corrected in all panels of figure 5 and panels 2-4 in figure 6: μg dL-1 (-1 is missing). Also I think it would be better to calculate the adsorption/adhesion per surface area (e.g. μg/cm2) rather than per volume…

 

We agree with your suggestion and have corrected Figure 5 and 6.

 

13. - Figure 7: Please include the size on the scale bar in these images

 

We agree with your suggestion and have included the size on the scale bar in Figure 7.

 

14. - 3.4 Fig. 8: Why was the gene expression of these genes analysed on different days? Please explain the rationale e.g. expression depending on early differentiation/late differentiation stages.

 

Runx2 mRNA and ALP mRNA were expressed on early differentiation, and BMP-2 mRNA and Bglap mRNA (as well as OCN mRNA) were expressed on late differentiation. According to following references, we used same analysis in order to clear PCR analysis on titanium surface.

 

Nishizaki, M; Komasa, S; Taguchi, Y; Nishizaki, H; Okazaki, J. Bioactivity of NANOZR Induced by Alkali Treatment. Int J Mol Sci 2017

Terada C, Komasa S, Kusumoto T, Kawazoe T, Okazaki J. Effect of Amelogenin Coating of a Nano-Modified Titanium Surface on Bioactivity. Int J Mol Sci 2018;19:1274.

 

15. Figure 9: What is the unit of the y-axis (ALP activity/DNA)? I would also recommend using the same scale (e.g. 0-1) for both graphs as it will make it easier to see the increase in ALP between day 7 and 14.

 

We agree with your suggestion and have corrected Figure 9.

 

16. Fig. 10: it would be preferable to express the calcium deposition per surface area rather than per volume.

 

According to following references, we used this protocol in order to clear calcium deposition on titanium surface.

Xing H, Komasa S, Taguchi Y, Sekino T, Okazaki J. Osteogenic activity of titanium surfaces with nanonetwork structures. Int J Nanomed. 2014; 9: 1741-55.

Su Y, Komasa S, Sekino T, Nishizaki H, Okazaki J. Characterization and Bone Differentiation of Nanoporous Structure Fabricated on Ti6Al4V Alloy. J Nanomater. 2015; http://dx.doi.org/10.1155/2015/358951

Komasa S, Kusumoto T, Taguchi Y, Nishizaki H, Sekino T, Umeda M, Okazaki J. Effect of Nanosheet Surface Structure of Titanium Alloys on Cell Differenciation. J Nanomater. 2014; http://dx.doi.org/10.1155:642527

Nishizaki, M; Komasa, S; Taguchi, Y; Nishizaki, H; Okazaki, J. Bioactivity of NANOZR Induced by Alkali Treatment. Int J Mol Sci 2017

Terada C, Komasa S, Kusumoto T, Kawazoe T, Okazaki J. Effect of Amelogenin Coating of a Nano-Modified Titanium Surface on Bioactivity. Int J Mol Sci 2018;19:1274.

 

17. Line 252: “Conventional plasma devices require vacuum and processing is both constrained and costly.” The authors seem to be considering predominantly large plasma processing devices employed for surface modification. However, there are many atmospheric pressure, cold plasma devices under investigation in ‘plasma medicine’, including plasma jets, DBD systems, micro-barrier discharge etc, which are small, inexpensive to operate and can be used for chairside/bedside applications. Please consider these in your discussions.

 

We agree with your suggestion and have corrected our manuscript as follows:

Lines 70-78

 For past decades, several techniques have been used for generating plasmas at atmospheric pressure and with a temperature close to ambient such as Radio-frequency plasmas (RF) [23], Dielectric barrier discharges (DBD) plasmas [24], Corona discharge plasmas [24-26], Gliding arc discharge plasmas [27]. The general advantage of these techniques is that the formation of a large number of reactive species could be obtained which was used for the treatment of surface, gases and aqueous solutions. Meanwhile, the miniaturization of the generator employed to create plasma has always been an important subject of research. The aim of these technologies, such as Piezoelectric direct discharge plasmas [28, 29], is to generate plasmas as “thin and small” as possible in terms of clinical application.

             

18. - Line 276: “To the best of our knowledge, the present study is the first to compare RBM cell proliferation on plasma-treated titanium surfaces with that on unprocessed controls.”

While it may be correct that no previous studies used rat bone marrow cells, cell proliferation e.g. of bone marrow stromal cells on plasma-treated titanium surfaces has been reported and should be referenced/discussed here. (e.g. Karaman, Synergistic Effect of Cold Plasma Treatment and RGD Peptide Coating on Cell Proliferation over Titanium Surfaces)

 

We agree with your suggestion and have corrected our manuscript as follows:

Lines 289-291

To the best of our knowledge, the present study is to compare RBM cell proliferation on plasma-treated titanium surfaces with that on unprocessed controls.

 

19. Line 278: “Plasma-treated materials have larger surface areas than untreated controls.” Please explain this statement or reference other studies. Your own analysis found no difference between the surface of treated and untreated titanium.

 

We agree with your suggestion and have corrected our manuscript as follows:

Lines 291

This sentence have deleted, Plasma treated titanium surface enhances the adhesion of cells such as osteoblasts and fibroblasts.

 

20. Line 297: “The piezobrush plasma device used in the present study is invaluable to clinicians because it is smaller and easier to operate than conventional plasma devices.” Please refer to other handheld plasma devices as well (e.g. plasma jets) as the one described here is not the only one.

 

We agree with your suggestion and have corrected our manuscript as follows:

Lines 70-78

 For past decades, several techniques have been used for generating plasmas at atmospheric pressure and with a temperature close to ambient such as Radio-frequency plasmas (RF) [23], Dielectric barrier discharges (DBD) plasmas [24], Corona discharge plasmas [24-26], Gliding arc discharge plasmas [27]. The general advantage of these techniques is that the formation of a large number of reactive species could be obtained which was used for the treatment of surface, gases and aqueous solutions. Meanwhile, the miniaturization of the generator employed to create plasma has always been an important subject of research. The aim of these technologies, such as Piezoelectric direct discharge plasmas [28, 29], is to generate plasmas as “thin and small” as possible in terms of clinical application.

             

 

21. Line 298: “Moreover, the findings of the in vivo efficacy assays performed in the present study suggest that this novel technology could be highly useful in dental practice.”

The authors do not show any in vivo data (e.g. animal study) but only in vitro data. Please correct this statement.

 

We agree with your suggestion and have corrected our manuscript as follows:

Lines 311

 Moreover, the findings of the in vitro efficacy assays performed in the present study suggest that this novel technology could be highly useful in dental practice.

Reviewer 2 Report

Please see the attached file 

Author Response

Thank you very much for your comments. We have revised our manuscript in accordance with your suggestions as follows:

 

line 26, abstract: authors mention the reduction of C-1s peak. It was confusing to think where the carbon is coming from. Please have one sentence before mentioning the carbon contamination of titanium surface.

 

We agree with your suggestion. XPS results shows the reduction of C1s peak between plasma-modified titanium and unmodified titanium disk by the effect of plasma treatment. C1s peak of control titanium disk are originating from air contamination (almost) and TiO2.

 

2.  Results session: Authors did not explain the data in detail. One or two sentence for each figure was too short and the flow was missing. Although the figures explain the message, the trends and its magnitude should explain in detail.

 

We agree with your suggestion and have expand the figure legends.:

 

3.  Figure 3. XPS data shows presence of oxygen. Is it from the air-oxidation or associated with carbon? Please explain.

We agree with your suggestion. O1s peak of unmodified titanium disk and plasma-treated titanium disk are originating from air-oxidation.

 

4.  Figure 7. Please add the scale bar dimension.

 

We agree with your suggestion and have included the size on the scale bar in Figure 7.

Reviewer 3 Report

-

Author Response

Thank you very much for your comments.

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