The Invasion of Bacterial Biofilms into the Dentinal Tubules of Extracted Teeth Retrofilled with Fluorescently Labeled Retrograde Filling Materials

Round 1

Reviewer 1 Report

This is an ex vivo study aimed at comparatively evaluating the viability and depth E. faecalis penetration into tubuli of extracted teeth retrofilled using MTA, IRM and Biodentine. 

Few minor misspellings and misusage can be found through the article. Please, check lines 33-34, 80, 150-151, 170-172, 178, 200.

Materials and methods related to figure 1b SEM images are missing. 

The statistical evaluation paragraph should be rewritten in clearer fashion; specifically, the comparison undertaken should be better explained.  Was the statistical comparison undertaken between the different groups (MTA, IRM and Biodentine) or just within each group at buccal, lingual, mesial and distal areas? 

Please, add pvalue to table 1 and spell out the superscripts (a, b, c, d). Also, add the statistical test to figures 3 and 4.

In the results section, lines 173-179, it should be added which filling material showed the very variable penetration depths. These results should also be discussed. 

The first half of the discussion is focused on discussing the different models used to investigate bacterial penetration and invasion into dentinal tubuli of retrofitted extracted human teeth.  It should be shortened and focused on the comparison with other studies using the CLSM model. Moreover, differences and improvements of the present model should be commented on.  

To this reviewer opinion, acceptance can be considered pending the revisions suggested above. 

Author Response

Thanks you for your comments, please see our response:

• Few minor misspellings and misusage can be found through the article. Please, check lines 33-34, 80, 150-151, 170-172, 178, 200

Response: these misspellings were corrected accordingly.

• Materials and methods related to figure 1b SEM images are missing

Response: M&M for the SEM were added as follows: 

"In order to validate the model, one slice from each tooth was scanned in Environmental SEM (ESEM). The ESEM slices were viewd in environmental “wet” mode with a Philips XL30 ESEM-Feg (FEI/Philpips Electron Otpics, Eindhoven, The Netherlands) (working conditions: 5°C, 2.9-5.9 torr gas pressure, 80% relative humidity, 6-9 kV). 5 interesting spots on each sample were selected"

 

• The statistical evaluation paragraph should be rewritten in clearer fashion; specifically, the comparison undertaken should be better explained.  Was the statistical comparison undertaken between the different groups (MTA, IRM and Biodentine) or just within each group at buccal, lingual, mesial and distal areas? 

Response: The statistics paragraph was edited as follows:

"The results were statistically analyzed using SPSS software (SPSS version 22; SPSS Inc, Chicago, IL). One-way ANOVA tests were done to assess the fluorescence at the buccal, lingual, mesial and distal areas, and to evaluate the level of fluorescence staining within each area (in the same group and between the different groups), the depth of bacterial penetration into the dentinal tubules and the depth of the filling penetration into the tubules. Pearson’s Chi-squared test was performed to check for dependency between the bacterial viability and the filling material used. Chi-squared tests with Yates' continuity correction were performed to evaluate the bacterial viability effect difference between the materials. P<0.05 was considered as statistically significant"

• Please, add pvalue to table 1 and spell out the superscripts (a, b, c, d). Also, add the statistical test to figures 3 and 4.

Response: p-values were added to table 1, the superscripts (a, b, c,) were spelled out, and the statistical test was added to figures 3 and 4 as follows:

"Table 1: Presents the penetration depths (in μm) of the retrograde filling materials and bacteria within the tubuli. The minimal and maximal bacterial penetration depths into the dentinal tubules were 1 and 1480 μm, respectively, with a mean of 167 μm. The minimal and maximal filling penetration depths into the dentinal tubules were 0 and 957 μm, respectively, with a mean of 130 μm. In the Biodentine group, in comparison to the other materials (MTA, IRM), Bacteria penetrated deeper into the dentin (b: One way Anova, p=0.0021), while the filling depth was lower (a: One way Anova, p=0.04). In the control group bacteria penetrated deeper into the dentin, in comparison to other groups (c: One way Anova, p=0.00018)"

"Figure 3. Average percentage of detected dead bacteria (red) and live bacteria (green). There were significantly more dead bacteria than live bacteria in all the experimental groups (Chi-squared, p=0.003)"

"Figure 4. Fluorescence Penetration amount for the different groups (presents as the number of pixel X 9800). (One way Anova)".

• In the results section, lines 173-179, it should be added which filling material showed the very variable penetration depths. These results should also be discussed.

Response: The results include now the following: "Looking on all groups, the filling materials and the bacterial penetration depths within the  tubuli were variable, with a minimal and maximal filling penetration depths of 11 and 957 μm, respectively (mean of 130 ±158 μm), and a minimal and maximal bacterial penetration depth of 9 and 1480 μm, respectively (mean of 167±317 μm) (Table1)." 

Table 1 in the results presents the min max and standard deviation of these variable penetration depths.

• The first half of the discussion is focused on discussing the different models used to investigate bacterial penetration and invasion into dentinal tubuli of retrofitted extracted human teeth.  It should be shortened and focused on the comparison with other studies using the CLSM model. Moreover, differences and improvements of the present model should be commented on.

Response: The first half of the discussion was shorten and revised to ensure it is   focused on the comparison with other studies using the CLSM model, and that   differences and improvements of the present model are commented on.

 

Reviewer 2 Report

1) The abstract and introduction say very little about relevance. This is a very important point in scientific articles. Write it down better.

2) It is not correct to use the concept of optimal filling. Because there was no mathematical model and the optimization method was not used. In your case, we are talking about the rational or best option for filling.

3) There is no quantitative information in the conclusions (specify specific percentages of parameter improvement).

4) There is no perspective in the annotation and conclusions. New scientific knowledge always raises new questions. Therefore, you need to indicate the direction of further research.

5) There is a lot of textual information in the discussion chapter. It would be better if you added drawings, diagrams, or tables.

6) The subject of the article should reflect the novelty.

Author Response

Thank you for your comments. Please see our point-by-point response:

1) The abstract and introduction say very little about relevance. This is a very important point in scientific articles. Write it down better.

Response: the following was added to the abstract, introduction and conclusions, in order to emphasise the relevance and significance of the study:

"this study enable better understanding of the microbiological-pathological course after endodontic surgical procedures"

2) It is not correct to use the concept of optimal filling. Because there was no mathematical model and the optimization method was not used. In your case, we are talking about the rational or best option for filling.

Response: we edited to use the term "rational"

3) There is no quantitative information in the conclusions (specify specific percentages of parameter improvement).

Response: quantitative information was added to the conclusions

4) There is no perspective in the annotation and conclusions. New scientific knowledge always raises new questions. Therefore, you need to indicate the direction of further research.

Response: the conclusions were edited to indicate the direction of further research, as follows:

"Additional clinical studies are indicated to elucidate the clinical implications of the bacterial invasion into the retrofilled root-end following endodontic surgery. In addition, the clinical and experimental assessment of retrograde filling materials should take into consideration not only the direct antimicrobial effect of the filling material but also its chemical and physical properties that affect its penetration capacity deep into the tubuli".

5) There is a lot of textual information in the discussion chapter. It would be better if you added drawings, diagrams, or tables.

Response: There are 5 figures and tables in the manuscript.

  

 

Reviewer 3 Report

Dear Authors,

According to my review this manuscript may have the merit to be accepted for publication.

Abstract and manuscript text is well written and clearly describe the purpose, methods, theoretical support and limitations of your in vitro study.

The only main limitations found in my review were:

• although the seventy single rooted extracted human teeth included in your study no sample size calculation was provided or was explained by authors.
• Implications for future research should also have been discussed.

 

 

Author Response

Thank you for your comments. Please see our response: 

• although the seventy single rooted extracted human teeth included in your study no sample size calculation was provided or was explained by authors.

Response: We selected these numbers since the current experiment is based on the methods we used in our previously published article, in which we used this experimental model. We explained it in the methods as follows:

"Based on a previously established experimental model [2] seventy single rooted freshly extracted human teeth were kept in 0.05% sodium hypochlorite liquid, and were selected for the experiment"

• Implications for future research should also have been discussed.

Response: the conclusions were edited as follows:

"Additional clinical studies are indicated to elucidate the clinical implications of the bacterial invasion into the retrofilled root-end following endodontic surgery. In addition, the clinical and experimental assessment of retrograde filling materials should take into consideration not only the direct antimicrobial effect of the filling material but also its chemical and physical properties that affect its penetration capacity deep into the tubuli"

Reviewer 4 Report

In this study the authors investigated a self-designed ex vivo teeth model for invasion of bacterial biofilms into dentinal tubules. However there are some points and drawbacks, which do not justify publication at the moment.

critics:

1.What is the novelty of this study to already known publcations about artifical root canal infection system? This is not clear. What is the novelty here?

2. Figure 1: Where are bacteria visible? I do not see any bacteria in figure 2B.

What is describe by the green-dotted line? Unclear. 

3. Furthermore, please see  e.g. Hecker et al. 2012 (Establishment of an optimized ex vivo system for artificial root canal infection evaluated by use ofsodium hypochlorite and the photodynamic therapy) published in Interantional Endotonic Journal. In this paper you can clearly see bacteria which are located on the surface and inside dentinal tubules.

4.Figure 2: magnification scale is not ready for publication. It's not readable without special software. Which filling material is shown? Not given.

5.Figure 3: How many samples did you have measured and evaluated? Here in figure 3 only bars are shown in percentage without any deviation? Is it a single measurement? Unclear.

6.Figure 4: Again, how many samples did you have measured and evaluated? Here only bars are shown without any further information.

7. figure 5: What means low and high magnification? Unclear.

8. General: All figure legends are not self-explanatory and relevant information is missing.

9 .Line 17-18: “The roots were filled with Enterococcus-faecalis bacteria from their coronal part for 21-days”. How was this done exactly? The schematic drawing of figure 1 must be revised for a better understanding.

10. table 1: "The penetration depths (in um) of the retrograde": What is meant by "um"? Small letters (a, b, c) are not explained????

 

Author Response

Thank you for your comments. Please see our response:

1.What is the novelty of this study to already known publcations about artifical root canal infection system? This is not clear. What is the novelty here?

Response: The introduction was edited to better explain the novelty as follows:

"In a recent study confocal laser scanning microscopy (CLSM) was used to assess the invasion of bacteria into the root-apices following retrograde filling [2]. It was demonstrated that even in the presence of retrograde fillings viable bacteria penetrated deep into the dentinal tubules [2]. However, in that study only the bacteria were stained and evaluated using CLSM, and the filling material penetration into the tubuli and its effect on the proliferating bacteria were not evaluated, and remains unknown.

Recently, in another study CLSM was used to evaluate fluorescently labeled filling materials penetration into the dentinal tubules [6]. Thus, it seems beneficial to use CLSM to simultaneously evaluate both the retrograde filling material interface with the dentinal walls and its penetration into the tubuli, and its effects on the invasion of bacteria at the apically prepared and filled root canal and dentinal tubules [2,6–8]. This study enable better understanding of the microbiological-pathological course after endodontic surgical procedures"

2. Figure 1: Where are bacteria visible? I do not see any bacteria in figure 2B.

Response: Figure 1b was replaced to show the bacteria within the dental tubuli.

What is describe by the green-dotted line? Unclear. 

Response: figure was edited

3. Furthermore, please see  e.g. Hecker et al. 2012 (Establishment of an optimized ex vivo system for artificial root canal infection evaluated by use ofsodium hypochlorite and the photodynamic therapy) published in Interantional Endotonic Journal. In this paper you can clearly see bacteria which are located on the surface and inside dentinal tubules.

Response:The figure now corresponds to the Hecker et al. 2012 figure

4.Figure 2: magnification scale is not ready for publication. It's not readable without special software. Which filling material is shown? Not given.

Response: Magnifaction scale was also added in legend, and stated that this is the control group (no filling).

5.Figure 3: How many samples did you have measured and evaluated? Here in figure 3 only bars are shown in percentage without any deviation? Is it a single measurement? Unclear.

Response: All samples were measured and evaluated

6.Figure 4: Again, how many samples did you have measured and evaluated? Here only bars are shown without any further information.

Response: All samples were measured and evaluated

7. figure 5: What means low and high magnification? Unclear.

Response: Figure 5 was edited to include the details of the magnification.

8. General: All figure legends are not self-explanatory and relevant information is missing.

Response: The figure legends were edited so they are now self-explanatory and include all relevant information

9 .Line 17-18: “The roots were filled with Enterococcus-faecalis bacteria from their coronal part for 21-days”. How was this done exactly? The schematic drawing of figure 1 must be revised for a better understanding.

Response: The bacteria were filled using a pipette, this was added to illustration for a better understanding, and also in the text as follows:

"The roots were then coronally filled with E.Faecalis bacterial suspension using a pipette..."

10. table 1: "The penetration depths (in um) of the retrograde": What is meant by "um"? Small letters (a, b, c) are not explained????

Response: um was corrected to μm, and the small letters (a, b, c) were explained.

Reviewer 5 Report

In general, the study is well done.
This study evaluated the invasion of bacteria to dentinal tubules of retrofilled extracted human teeth, and the influence of different fluorescently labeled retrograde filling materials on the bacterial invasion and viability.
Abstract - the conclusion in the abstract it is a little bit confusing. It should be rewritten.
Introduction - the last paragraph of the introduction should be rewritten without include the words aim 1 and aim 2.
Material and methods- figure 1 b could have a better resolution to see the bacteria penetration inside the dentinal tubules.

 

Author Response

Thank you for your comments. Please see our response:

Abstract - the conclusion in the abstract it is a little bit confusing. It should be rewritten.

Response: the conclusions in the abstract were edited as follows:

"In conclusion, the current study enables better understanding of the microbiological-pathological course after endodontic surgical procedures. It was found that even with retrograde fillings bacteria invade deep into the dental tubules, where deeper filling penetration prevents deeper penetration of the bacteria and adversely affects the viability of the bacteria"

Introduction - the last paragraph of the introduction should be rewritten without include the words aim 1 and aim 2.

Response: the last paragraph of the introduction was edited without include the words aim 1 and aim 2

Material and methods- figure 1 b could have a better resolution to see the bacteria penetration inside the dentinal tubules. 

Response: Figure 1b was replaced with a better one.

Round 2

Reviewer 2 Report

There are no comments.

Author Response

Thank you for your review.

 

Reviewer 4 Report

In general I'm wondering why the revised version isn't marked by the authors to show all the changes? I couldn't found any revised version of the manuscript.

So far I downloaded a version of the authors which I now reviewed again:

1)2. Figure 1: Where are bacteria visible? I do not see any bacteria in figure 2B.: Q: No scale bar is inserted. 

2)What is describe by the green-dotted line? Unclear. 

Response: figure was edited

Query: I do not see any explanation within the revised version.

3)3. Furthermore, please see  e.g. Hecker et al. 2012 (Establishment of an optimized ex vivo system for artificial root canal infection evaluated by use ofsodium hypochlorite and the photodynamic therapy) published in Interantional Endotonic Journal. In this paper you can clearly see bacteria which are located on the surface and inside dentinal tubules.

Response:The figure now corresponds to the Hecker et al. 2012 figure

Q: Which figure do you mean? 

4)Figure 2: magnification scale is not ready for publication. It's not readable without special software. Which filling material is shown? Not given.

Response: Magnification scale was also added in legend, and stated that this is the control group (no filling).

Q: Not done??? I couldn't see it.

5)Figure 3: How many samples did you have measured and evaluated? Here in figure 3 only bars are shown in percentage without any deviation? Is it a single measurement? Unclear.

Response: All samples were measured and evaluated

Q: Not done so far. Where is it shown? 

The manuscript is not ready for publication because a careful re-review is not possible due to the point that my comments and recommendations were not answered sufficiently by the authors.

Author Response

Thank you for your comments. please see our response: 

1)2. Figure 1: Where are bacteria visible? I do not see any bacteria in figure 2B.:

Q: No scale bar is inserted. 

Response: Image was revised. a scale bar was added.

2)What is describe by the green-dotted line? Unclear. 

Response: figure was edited

Query: I do not see any explanation within the revised version.

Response: The green dotted line was removed.

3)3. Furthermore, please see  e.g. Hecker et al. 2012 (Establishment of an optimized ex vivo system for artificial root canal infection evaluated by use ofsodium hypochlorite and the photodynamic therapy) published in Interantional Endotonic Journal. In this paper you can clearly see bacteria which are located on the surface and inside dentinal tubules.

Response:The figure now corresponds to the Hecker et al. 2012 figure

Q: Which figure do you mean? 

Response: Figure 1b

4)Figure 2: magnification scale is not ready for publication. It's not readable without special software. Which filling material is shown? Not given.

Response: Magnification scale was also added in legend, and stated that this is the control group (no filling).

Q: Not done??? I couldn't see it.

Response: Image was revised. New and clearer scale bar was added.

5)Figure 3: How many samples did you have measured and evaluated? Here in figure 3 only bars are shown in percentage without any deviation? Is it a single measurement? Unclear.

Response: All samples were measured and evaluated

Q: Not done so far. Where is it shown? 

Response: Each slice was measured using the LAS AF software in 4 mesial, distal, buccal and lingual direction, but all the confocal staining was measured (not only one point). Calculation of the result is shown in percentage in order to ease the understanding of the reader,  whereas presenting the original number of pixels measured would have confused the reader.

 

Round 3

Reviewer 4 Report

The authors revised the manuscript and marked the changes within the revised version. However there are some points which need a revision:

I do not understand clear what is shown in figure 3. No information is given within the figure legend, if this is a representative result or from n= x ? independent experiments. I'm wondering about the given information.

However the authors stated that "each slice was measured using the LAS AF software in 4 mesial, distal, buccal and lingual direction, but all the confocal staining was measured (not only one point)". How often did the authors do independently? 

This must be revised. 

Table 1: However the authors explained (a: One way Anova, p=0.04), but I miss the information about which values were compared. The given value in combination with the letter "a" is significant compared to which other values? To all values? I don't now. 

This must be revised.

Author Response

Thank you for your comments, see our response: 

Regarding figure 3 - as presented in the figure and stated in the figure legends, figure 3 presents the "average percentage of detected dead bacteria (red) and live bacteria (green) for each group. There were significantly more dead bacteria than live bacteria in all the experimental groups (Chi-squared, p=0.003)"

 

Regarding "each slice was measured using the LAS AF software in 4 mesial, distal, buccal and lingual direction" - this is based on our original methods that were previously published in another manuscript (ref 2): "The CLSM images of the bacterial biofilm were acquired at a resolution of 1024X1024 pixels and analyzed by the LAS AF software (version 2.6.0.7266; Leica Microsystems CMS GmbH). The specimens were observed using a X4 lens. The mesial, distal, buccal, and lingual areas of the specimens were evaluated by the software". Based on these methods in the current study "The images were then evaluated by a dedicated software (LAS AF, version 2.6.0.7266; Leica Microsystems CMS, Wetzlar, Germany). The slices were assessed by a ×4 lens, and the extent of fluoresce staining within the buccal, lingual, mesial and distal areas of the slices was assessed [2,6]. The following measurements were performed to assess the invasion of the bacteria into the dental tubuli (penetration depth and viability), and to assess the influence of the retrograde filling on the bacterial invasion (Figure 2):

1. The depths of bacterial invasion and filling penetrations within the tubuli were measured at the buccal, lingual, mesial and distal areas of the root dentin axial slices defining the root canal wall as the beginning point (Figure 2f).
2. The bacterial viability was calculated as the proportion between live and dead bacteria.

    3. The correlation between the filling material type and penetration depth, and the

        bacterial invasion depth and viability.

 

"Table 1: However the authors explained (a: One way Anova, p=0.04), but I miss the information about which values were compared. The given value in combination with the letter "a" is significant compared to which other values? To all values? I don't now"

Please note that each letter (A,b,c) makes a statistically significant value compared to the other groups. This is specified and explained in the manuscript as following:

In the Biodentine group, in comparison to the other materials (MTA, IRM), Bacteria penetrated deeper into the dentin (b: One way Anova, p=0.0021), while the filling depth was lower (a: One way Anova, p=0.04). In the control group bacteria penetrated deeper into the dentin, in comparison to other groups (c: One way Anova, p=0.00018).