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Article
Peer-Review Record

Low-Pressure Plasma Sterilization for Test Specimens to be Worn on Splints in the Oral Cavity

by Ella A. Naumova 1,*, Alexander-Simon Engel 1, Hagen Tizian Kranz 1, Marvin Schneider 1, Jan Tietze 1, Thomas Dittmar 2, Marcel Fiebrandt 3, Katharina Stapelmann 3,4, Andree Piwowarczyk 5, Thorsten Kuczius 6 and Wolfgang H. Arnold 7
Reviewer 1: Anonymous
Reviewer 2: Anonymous
Reviewer 3: Anonymous
Submission received: 15 January 2019 / Revised: 1 February 2019 / Accepted: 3 February 2019 / Published: 6 February 2019

Round  1

Reviewer 1 Report

The authors investigated the possibility of plasma sterilization for test specimens to be worn on splints in oral cavity. In general, the study is well made and the manuscript is well written. However, a few concerns need to be addressed by the authors.

Please clarify the purpose of your study in the title and the abstract (plasma sterilization for test specimens to be worn on splints in oral cavity)

Please exchange the term “probe” by specimen thoroughly.

How were the enamel disks sterilized before applying to oral cavity?

suggest deleting storage conditions from Figure 1 and 2. This is confusing in part. Please write in the text for how long at the latest the samples were stored at the respective temperatures.

Figure 2: Please specify that you isolated single bacterial strains from the microbiological culture and thereafter you cultured them on new enamel discs in vitro.

Microbiology: I suggest separating the microbiological analysis of biofilm samples obtained from test specimens and checking for sterility after plasma sterilization. (both methods and results).

You isolated single bacterial strains and processed them further to plasma sterilization (according to Materials and methods). Those are obviously presented in Table 1?  Please clarify and add a comment into the text.

Did you made also SEM photographs immediately before (after placing specimens into 0.9% w/v NaCl) and after plasmasterilization? It might be of interest if plasmasterilzation can really destroy biofilms (matrix).

Is there any data about influence of autoclaving on surface roughness and chemical composition of enamel? Please discuss.   

Author Response

First of all we would like to thank the reviewer for the ontructive and helpful comments.

 

Response to reviewer 1

 

Please clarify the purpose of your study in the title and the abstract (plasma sterilization for test specimens to be worn on splints in oral cavity)

 

The title has been changed.

 

Please exchange the term “probe” by specimen thoroughly.

Has been done.

 

How were the enamel disks sterilized before applying to oral cavity?

 

Thank for mentioning this point. They were sterilized by plasma sterilization before they were placed into the oral cavity.

 

I suggest deleting storage conditions from Figure 1 and 2. This is confusing in part. Please write in the text for how long at the latest the samples were stored at the respective temperatures.

 

Has been done

 

Figure 2: Please specify that you isolated single bacterial strains from the microbiological culture and thereafter you cultured them on new enamel discs in vitro.

 

We changed Figure 2 accordingly.

 

 

Microbiology: I suggest separating the microbiological analysis of biofilm samples obtained from test specimens and checking for sterility after plasma sterilization. (both methods and results).

 

The reviewer is right. The microorganisms were obtained from untreated disks and analyzed by biotyping. Streptococci and Staphylococci growing aerobically were seen on plates at increased counts. As plaques grew under identical conditions we considered very similar bacterial growth on all disks. In contrast to untreated disks with high numbers of microorganisms no bacteria were identified after the sterilization process demonstrating high efficiency of the plasma sterilization.  

The text has been changed accordingly

 

You isolated single bacterial strains and processed them further to plasma sterilization (according to Materials and methods). Those are obviously presented in Table 1?  Please clarify and add a comment into the text.

 

Considering identical plaque contamination of the samples, one sample was plasma sterilized, the other one was untreated. Bacterial growth was identified only on untreated disks and as well  in increased bacterial counts indicating high contamination and plaque formation with natural microorganisms. The identified isolates are listed in the table. The strains were not processed to a further sterilization process, because these isolates were not embedded additionally in a biofilm. To show this we should regrow bacteria in a biofilm under identical conditions which is technically not possible because the biofilm matrix would show another composition as the original biofilm on the disks and the microbiological composition is another one as on these disks, so that upcoming results would never be comparable with the original results. It is important to note that we only detected the microorganisms which were able to use the media for growth, however most of natural bacteria are not able to be cultivated. We showed that no growth was observed after plasma sterilization indicating a high efficient sterilization process. We only could demonstrate this with natural but cultivable isolates. In contrast, high bacterial counts were determined with untreated samples which indicated high biofilm formation.

 

Did you made also SEM photographs immediately before (after placing specimens into 0.9% w/v NaCl) and after plasmasterilization? It might be of interest if plasmasterilzation can really destroy biofilms (matrix).

No we did not, because placing the specimens in 0.9% NaCl would not fix the biofilm. Organic matrix must be fixed for microscopy either in glutaraldehyde or formalin.

 

Is there any data about influence of autoclaving on surface roughness and chemical composition of enamel? Please discuss.

 

Thank you very much. An important point. Wee added it to the discussion section. 

 

 

Reviewer 2 Report

In the current manuscript entitled “Characterization and Low-Pressure Plasma Sterilization of Oral Biofilms” the authors investigated the effect of plasma sterilization on the biofilm formation on enamel surfaces by means of scanning electron microscopy, confocal laser microscopy and microbiological analysis. The experiments are carried out properly; the system of interest is of great importance in dental restorations. Presenting and discussing the findings are weakly addressed. On the other side, the manuscript is well-written. Therefore, I believe that the manuscript deserve to be published in Coatings after addressing the following points.

1) In designing the study as shown in Figure 2, the authors split the samples after plaque growth and then subject them for investigations with various methods without clear purpose. The whole work actually can be divided into two packages (as indicated in the title); i) biofilm characterization mainly with all methods which are mentioned in the manuscript and ii) the impact of plasma sterilization on the biofilm formation. Unfortunately the latter is not characterized with SEM or CLSM. It will be extremely important, very convincing for the readers and to support the main conclusion of the study to see difference in the SEM or CLSM before and after plasma treatment.

2) The authors mentioned in the introduction that the commonly used chemical disinfectants include hydrogen peroxide (l.59) which might be the motivation to use plasma treatment from hydrogen and oxygen gas. But this does not mean the resulted plasma is that of  hydrogen peroxide and thus the term “H2O2 plasma sterilization “ is confusing and must be changed all over the manuscript with proper shortcut i.e  H2 / O2 (e.g., Figure 3, 8 captions, l.138, l.170).

3) The figure captions are very short. I suggest adding more information to indicate the experimental conditions.

4) Figure 7 is presented and it is not clear for me why and what we can learn from it. Is it just to say we have live and dead bacteria? I think it is more crucial to confirm the impact of plasma treatment by CLSM. I suggest the authors to add more sentences in the main text to explain more about the image or modify the image with /without plasma treatment to indicate the effect LPP sterilization.

5) In Figure 8, the authors mention that there is no significant difference in the median values. However, I see a clear difference in the standard deviation or the distribution width of the roughness values. This is clearly supports the formation of biofilms in the control experiments which is likely to happen while after sterilization with plasma the biofilm is unlikely to be formed or undergo perfect growths. Do the authors agree?

6) In Figure 3, the plasma treatment is clearly increases the enamel surface roughness in the micro/mesoscopic length scales. Does the increase in surface roughness prompts or prohibit the bacterial growth?

Author Response

First of all we would like to thank the reciewer for the constructive and helpful comments which we hope improved the manuscrpit.

 

Response to reviewer 2

 

1) In designing the study as shown in Figure 2, the authors split the samples after plaque growth and then subject them for investigations with various methods without clear purpose. The whole work actually can be divided into two packages (as indicated in the title); i) biofilm characterization mainly with all methods which are mentioned in the manuscript and ii) the impact of plasma sterilization on the biofilm formation. Unfortunately the latter is not characterized with SEM or CLSM. It will be extremely important, very convincing for the readers and to support the main conclusion of the study to see difference in the SEM or CLSM before and after plasma treatment.

Both figures have been redesigned.

ii) Basically, this is correct. However, as there were no bacteria detectable after plasma sterilization we refrained from another SEM and CLSM investigation.

2) The authors mentioned in the introduction that the commonly used chemical disinfectants include hydrogen peroxide (l.59) which might be the motivation to use plasma treatment from hydrogen and oxygen gas. But this does not mean the resulted plasma is that of  hydrogen peroxide and thus the term “H2O2 plasma sterilization “ is confusing and must be changed all over the manuscript with proper shortcut i.e  H2 / O2 (e.g., Figure 3, 8 captions, l.138, l.170).

Correct. Thank you for mentioning this point. We changed it.

3) The figure captions are very short. I suggest adding more information to indicate the experimental conditions.

The figure captions have been changed.

4) Figure 7 is presented and it is not clear for me why and what we can learn from it. Is it just to say we have live and dead bacteria? I think it is more crucial to confirm the impact of plasma treatment by CLSM. I suggest the authors to add more sentences in the main text to explain more about the image or modify the image with /without plasma treatment to indicate the effect LPP sterilization.

Principally the reviewer is right. However, after plasma sterilization of the biofilm no bacteria could be detected. Therefore, an additional CLSM picture would show nothing, but background noise.

5) In Figure 8, the authors mention that there is no significant difference in the median values. However, I see a clear difference in the standard deviation or the distribution width of the roughness values. This is clearly supports the formation of biofilms in the control experiments which is likely to happen while after sterilization with plasma the biofilm is unlikely to be formed or undergo perfect growths. Do the authors agree?

Not really. The purpose of the roughness measurements was to find out if the surface roughness changes after LPP sterilization. An increased surface roughness would promote biofilm growth. This was not the case here. We changed the description of the boxplot.

A boxplot graph does not show standard deviation, but the data distribution.

6) In Figure 3, the plasma treatment is clearly increases the enamel surface roughness in the micro/mesoscopic length scales. Does the increase in surface roughness prompts or prohibit the bacterial growth?

Sorry, but we choose the wrong picture out of so many. We changed Figure 3 and added a better example.

Reviewer 3 Report

Introduction

If bacterial biofilms are essential for oral health why are you trying to develop materials that inhibit growth? Can you clarify a “good” biofilm as opposed to the “bad” biofilm that causes cavities? You also may want to comment on this in either the discussion or the conclusion. This could be basic information for researchers in this specific area of work but adding a sentence in a discussion can extend the readership for this article.

Could you clarify which of the methods you listed changes the surface of the dental implant as opposed to which are not efficient in destroying the biofilm.

Could you make it clear if you are looking for a rough or smooth surface and the advantages and disadvantages of biofilm growth on each.

Discussion

Could you please clarify this sentence “Oral biofilms are complex systems of bacteria adhering to the oral surfaces [26]. They are the important biological characteristics of the surfaces of the oral cavity and have protective and destroying functions.”

Authors need to discuss in more detail the significance of their findings and how these findings are useful to this field of study.

I believe the effectiveness of the explanation would be enhanced if the discussion and results were discussed together.

This type of information would be more effective in the introduction. The discussion should only be for the discussion of the experimental results and their significance to the field.

“Medical devices may be categorized into three different groups. The first are the critical devices which must be sterile before application. The second are the semicritical devices which are in contact with mucous membranes such as the oral cavity and the third are the non-critical devices which are in contact with skin [9]. Dental materials belong to the second or third group and have to be decontaminated or sterilized before application. It is a prerequisite for dental materials that their surface properties are not altered after disinfection or sterilization. The influence of the biofilm development on material surfaces and their destructive effect can be investigated experimentally. For this often material probes are mounted on dental devices and worn by subjects for a certain time. As control material usually human enamel is used. In this study dental enamel has been used as experimental material to test the effects of plasma sterilization on the enamel surface using either oxygen or a mixture of hydrogen and oxygen as the process gas.”

Conclusion

Weak conclusion. Authors say that they successfully showed that the plasma sterilization is a nondestructive method but did not explain how they reached that conclusion.

Need to give an overview of the major results and then discuss the conclusion based on those results.

Author Response

First of all we would like to thank the reciewer for the constructive and helpful comments which we hope improved the manuscrpit.

Response to reviewer 3:

If bacterial biofilms are essential for oral health why are you trying to develop materials that inhibit growth? Can you clarify a “good” biofilm as opposed to the “bad” biofilm that causes cavities? You also may want to comment on this in either the discussion or the conclusion. This could be basic information for researchers in this specific area of work but adding a sentence in a discussion can extend the readership for this article.

This point has been discussed in the discussion section lines We added one more sentence to clarify this.

Could you clarify which of the methods you listed changes the surface of the dental implant as opposed to which are not efficient in destroying the biofilm.

Sorry, but we did not investigate dental implants. This was not the topic of this study.

Could you make it clear if you are looking for a rough or smooth surface and the advantages and disadvantages of biofilm growth on each.

Has been done. See discussion lines 225-226.

Discussion

Could you please clarify this sentence “Oral biofilms are complex systems of bacteria adhering to the oral surfaces [26]. They are the important biological characteristics of the surfaces of the oral cavity and have protective and destroying functions.”

The whole oral cavity is covered by biofilm which are different in different areas. They are dependent from the surface properties. We added explanatory details in the discussion section including literature.

Authors need to discuss in more detail the significance of their findings and how these findings are useful to this field of study.

 

I believe the effectiveness of the explanation would be enhanced if the discussion and results were discussed together.

 

We dot not agree to this. Most journals still require a clearly structured manuscript.

This type of information would be more effective in the introduction. The discussion should only be for the discussion of the experimental results and their significance to the field.

“Medical devices may be categorized into three different groups. The first are the critical devices which must be sterile before application. The second are the semicritical devices which are in contact with mucous membranes such as the oral cavity and the third are the non-critical devices which are in contact with skin [9]. Dental materials belong to the second or third group and have to be decontaminated or sterilized before application. It is a prerequisite for dental materials that their surface properties are not altered after disinfection or sterilization. The influence of the biofilm development on material surfaces and their destructive effect can be investigated experimentally. For this often material probes are mounted on dental devices and worn by subjects for a certain time. As control material usually human enamel is used. In this study dental enamel has been used as experimental material to test the effects of plasma sterilization on the enamel surface using either oxygen or a mixture of hydrogen and oxygen as the process gas.”

We disagree with this, because it discusses why there is a need for this investigation.

Conclusion

Weak conclusion. Authors say that they successfully showed that the plasma sterilization is a nondestructive method but did not explain how they reached that conclusion.

We strengthened the conclusion.

Need to give an overview of the major results and then discuss the conclusion based on those results.

 

Usually a conclusion is nit discussed. This has been done in the discussion section.

 

Round  2

Reviewer 2 Report

In the revised version of the article submitted by Naumova et al. and entitled “Low-Pressure Plasma Sterilization for Test Specimens to be Worn on Splints in the Oral cavity”, the authors responded to and addressed satisfactorily my comments. I think the quality of the current manuscript has improved and reached the level to be published in Coatings.


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