Analysis of Coating Loss from Coated Stainless Steel Orthodontic Wire

Round 1

Reviewer 1 Report

This study examined coating loss associated with low pH exposure, bending, and brushing of various types of coated orthodontic SS wire. Its main contribution is in the field of orthodontics, to make known to the dental community if the loss of coating of orthodontic wires can cause a release of metal ions and thus cause inconveniences in patients with allergies to metals.

The manuscript is clear, relevant and presented in a well-structured manner. Furthermore, it is scientifically sound and presents an appropriate experimental design to test the hypothesis. About 50% of the cited references are from the last 5 years. The results are reproducible based on the details given in the methods. The figures, tables and images are appropriate, correctly display the data and are easy to interpret and understand. The data is interpreted appropriately and consistently throughout the manuscript. A correct statistical analysis is performed and the conclusions are consistent with the evidence and arguments presented.

Author Response

Reviewer 1

 

Thank you very much for kindly comments.

Reviewer 2 Report

This is a well performed study on an interesting topic, and well written. However, some issues regarding the study design/statistical analysis are of concern and should be corrected or at least clarified.

-The objective is not clearly stated, as authors aparently want to evaluate the effect of different immersion conditions and angulation of bending on different materials of wire on the concentration of different metal ions on the acid solution (ion elution), however as they do not compare between materials, it seems that the material type is not an independent variable (study factor of interest). However, it is the first impression according to the statement of the problem and objective stated.

-The hypothesis or hypotheses of the study is not clearly stated.

-Important basic data as "n" (sample size) for each experiment are not clearly stated.

-Regarding statistical analysis: First of all, data should be analyzed to see if they follow a parametric or non-parametric behavior. So a normality and homoscedasticity test should have been applied first to see which type of statistical test is the right one in this case, and to know if data should be presented in means, or in medians, between other details. All these information should be clarify in the methods and results sections.

-Second issue regarding the statistical analysis. The confusion regarding the actual objective of the study, is also present in the statistical evaluation design (regarding numerical dependent variables: ion elution and roughness), as authors only applied a multiple comparison test (Tukey post-hoc test). No hypothesis test was applied, which is mandatory on a factorial design as this, and is needed before applying a multiple comparison test such as Tukey (authors can see McHugh ML. Multiple comparison analysis testing in ANOVA. Biochemia Medica 2011;21 (3):203-9). The proper statistical analysis should have been at least a one-way ANOVA, applied to each dependent variable (concentration of each metal ions measured, and roughness). The study factor in this case (metal ions concentration) would be the "wire condition" (not-immersed, uncoated and TiN-coated). So for the first experiment, the one-way ANOVA should have been applied to each metal ion for each wire material (assuming that- as it seems-, comparing between materials was not an objective of this work). If comparing between materials is also of interest, then a two-way ANOVA should have been applied. And for the second experiment (of ion concentration), the study factor is the bending angle. So again, there are more than 2 levels (30°, 60°, 90° and 120°), so a one-way ANOVA should have been applied here. After performing a hypothesis test to see first if the study factor was statistically significant, then a multiple comparison (post-hoc test) can be applied, such as Tukey, to compare between groups (levels of the factor). Similarly to roughness variable, however in this experiment the study-factor is indeed the type of material (according to the presented results on table 1.

-EDS analysis, is only descriptive. A quantitative analysis would be more interesting, to associate with the obtained outcomes from the other variables (mainly metal ions elution). This is a simple analysis, performed by obtaining rates from the element of interest and one reference. Then as a numerical variable, also a hypothesis and multiple comparisons statistical tests should be applied to analyze if statistical differences are present between different brushing conditions, or even both: brushing conditions and material acting together (2 factors: 2-way ANOVA).

If authors correct or clarify these issues, and derive results and conclusions from a proper statistical analysis, then this work will be very interesting and strong.

 

Author Response

Reviewer 2
1
-The hypothesis or hypotheses of the study is not clearly stated.
Response:
The hypotheses of the study have been added to the text (line 69 –71). In addition, the results of the hypotheses (H01-3) have been added to the text (line 239-241, 273-274, 296-297)

Revision:
・To our knowledge, there have been no previous investigations of corrosion re-sistance of orthodontic wires with multiple coating materials. The null hypotheses to be researched in this study were that exposure to low pH conditions(H01), bending(H02), and brushing(H03) of several types of coated orthodontic SS wire would not cause coating loss.
・On the other hand, Rh-coated wire showed more corrosion than Au-coated wire, even though Rh has a higher standard electrode potential than hydrogen. This increased corrosion may have been due to the greater Ra of Rh-coated wire compared to the Au-colated wire, as shown in Table 1 [24]. The null hypothesis (H01) was rejected, as exposure to low pH conditions of TiN-coated and Rh-coated wires caused coating loss.
・Furthermore, the results of the present study suggest that PEN has sufficient durability for the range of bends used in orthodontic treatment. The null hypothesis (H02) was rejected, because bending of TiN-coated, Au-coated and Rh-coated wires caused coating loss.
・Taken together, these results suggest that brushing does not result in coating loss from TiN-coated and Au-coated wires, but that it may cause loss of the coating from Rh-coated and PEN-coated wires. Therefore, the null hypothesis (H03) was rejected.

2
-Important basic data as "n" (sample size) for each experiment are not clearly stated.
Response:
Sample size information is already provided on line 113-114 and 125-126. 
Revision:
No correction needed.

3
-Regarding statistical analysis: First of all, data should be analyzed to see if they follow a parametric or non-parametric behavior. So a normality and homoscedasticity test should have been applied first to see which type of statistical test is the right one in this case, and to know if data should be presented in means, or in medians, between other details. All these information should be clarify in the methods and results sections.
Response:
The all data showed standard normal distribution, so we present the values as means. I have added 
the information on the statistical analyses in the revised manuscript (line140, 167-168, 203-204, 191).
Revision:
・The results of analyses of metal ions release from coated wires immersed in HCl are shown in Figure 2. Statistical analysis showed that all data follow a parametric behavior. All of the coatings examined showed suppression of metal ion release from the substrate metal in comparison to uncoated SS wire.
・On the other hand, the PEN-coated wire showed no surface deterioration on bending. Figure 5 showed metal ions derived from substrate metal, SS, released from four types of coated wire with various bend angles from 30° to 120° immersed in HCl. Statistical analysis showed that all data follow a parametric behavior. Examination of metal ion release showed that the amount of metal ion release increased with increasing wire bend angle, exception for PEN-coated wire (Figure 5).
・Table 1 showed Surface roughness of coated wire. Statistical analysis showed that all data follow a parametric behavior. There were significant differences in Ra of each type of coated wire (P < 0.01) except between TiN-coated and Rh-coated wire, with a maximum Ra of 0.532 ± 0.014 μm for PEN-coated wire and minimum Ra of 0.028 ± 0.004 μm for Au-coated wire (Table 1).

4
-Second issue regarding the statistical analysis. The confusion regarding the actual objective of the study, is also present in the statistical evaluation design (regarding numerical dependent variables: ion elution and roughness), as authors only applied a multiple comparison test (Tukey post-hoc test). No hypothesis test was applied, which is mandatory on a factorial design as this, and is needed before applying a multiple comparison test such as Tukey (authors can see McHugh ML. Multiple comparison analysis testing in ANOVA. Biochemia Medica 2011;21 (3):203-9). The proper statistical analysis should have been at least a one-way ANOVA, applied to each dependent variable (concentration of each metal ions measured, and roughness). The study factor in this case (metal ions concentration) would be the "wire condition" (not-immersed, uncoated and TiN-coated). So for the first experiment, the one-way ANOVA should have been applied to each metal ion for each wire material (assuming that- as it seems-, comparing between materials was not an objective of this work). If comparing between materials is also of interest, then a two-way ANOVA should have been applied. And for the second experiment (of ion concentration), the study factor is the bending angle. So again, there are more than 2 levels (30°, 60°, 90° and 120°), so a one-way ANOVA should have been applied here. After performing a hypothesis test to see first if the study factor was statistically significant, then a multiple comparison (post-hoc test) can be applied, such as Tukey, to compare between groups (levels of the factor). Similarly to roughness variable, however in this experiment the study-factor is indeed the type of material (according to the presented results on table 1.

Response:
I have revised the statistics section in materials and methods (line128-136).
Revision:
JMP® Pro software (version 16.0.0; SAS Institute Inc., Cary, NC, USA) was used for statistical analyses. Descriptive analyses of the ICP-MS and Ra data were carried out and all data were presented as mean ± standard deviation. In a first step, the Anderson-Darling test was applied on each data set to determine its normality. O’Brien’s test was then used to check significant difference between the variances of the data sets. One-way analysis of variance (one-way ANOVA) was also used to evaluate statistical differences between the data sets. For post-hoc analysis, we used Tukey–Kramer honestly significant difference (HSD) was used to determine which groups had statistically significant differences. In all analyses, P < 0.05 was taken to indicate statistical significance.

5
-EDS analysis, is only descriptive. A quantitative analysis would be more interesting, to associate with the obtained outcomes from the other variables (mainly metal ions elution). This is a simple analysis, performed by obtaining rates from the element of interest and one reference. Then as a numerical variable, also a hypothesis and multiple comparisons statistical tests should be applied to analyze if statistical differences are present between different brushing conditions, or even both: brushing conditions and material acting together (2 factors: 2-way ANOVA).
Response:
The purpose of the EDS analysis in this study was to determine whether SS, the base metal, was exposed or not. Therefore, the degree of exposure and quantification were not an issue in this study. However, because the coating loss of Rh-coated wires is obvious, but the coating loss of PEN-coated wires is difficult to determine from the EDS analysis in this study. So, I have revised text (line279-281, 289-293, 295-296,309,311-315). In addition, we think that future studies examining coating loss under different brushing conditions are necessary, so we have added that information to line 311-315 of the text. In that case, we believe that quantitative and statistical analysis is necessary to clarify the differences between different brushing conditions.
Revision:
・As it was not possible to determine whether brushing affected coating loss on coated wires by stereomicroscopy, we next examined the surface properties of the wires by SEM. On SEM analysis, each type of wire showed black areas after brushing. EDS analysis was performed to determine whether there was coating loss and exposure of the substrate metal at these areas. TiN-coated, Au-coated, and PEN-coated wires do not appear to show significant changes in graph before and after brushing. On the other hand, a change was observed in Rh-coated wires after brushing. In the Rh-coated wire, a peak of Rh was observed before brushing, which decreased after brushing and a new peak of Al was observed. It is possible that Al used in the pretreatment was exposed due to detachment of the Rh coating. However, although the manufacturer of the wire stated that they pretreat the Rh-coated wire to improve its coloration, precise data on the materials used were not available due to their policy of keeping this information confidential. In addition, Ti peaks were detected in the PEN coating both before and after brushing, which were due to the use of Ti oxide directly beneath the coating. As the PEN coating was as thin as 50um, the Ti peak may have been detected even before brushing. Because of the thin thickness of the PEN coat and the substrate metal is detected both before and after brushing, it may be difficult to discuss the coating loss of the PEN coated wire by EDS analysis in this study. However, a new trace peaks of Fe were detected after brushing, suggesting that coating loss may have occurred in the PEN-coated wire. Taken together, these results suggest that brushing does not result in coating loss from TiN-coated and Au-coated wires, but that it may cause loss of the coating from Rh-coated and PEN-coated wires. In addition, coating loss may have occurred on the PEN coated wire, but further analysis is needed. Therefore, the null hypothesis (H03) was rejected. 
・In this study, brushing using an electric toothbrush corresponding in hardness to that likely to be encountered in clinical situations may have caused detachment of the PEN coating from the wire. Therefore, it would be advisable to select a toothbrush with softer filaments in patients treated with PEN-coated wire.
・Therefore, it would be advisable to select a toothbrush with softer filaments in patients treated with PEN-coated wire. To examine this issue in more depth, it is necessary to compare the degree of coating loss between different brushing conditions. Brushing conditions could include changing such as the filament hardness and number of brushings, and statistical analysis is needed when comparing the amount of metal exposure between conditions.

Reviewer 3 Report

In this manuscript (applied sciences-1918263) titled "Analysis of coating loss from coated stainless steel orthodontic wire," the authors demonstrated the resilience and durability of different types of orthodontic wire coatings. 

Generally, this is a well-designed work providing the structural analysis of the coatings in response to the change in pH and mechanical stress.

Orthodontic braces (wires) have been widely used to align and straighten teeth and help position them. Overall, this work is likely to receive some interest in the dental field.

The levels of insight and depth of experimental analysis are sufficiently strong; therefore, it is recommended this manuscript be considered for publication.

Please find the following (minor) comments which could improve the quality of the manuscript further:

1.      Authors used strong acid (35% HCl) treatment to examine the ion release from the orthodontic wires, which is much more acidic than the oral pH. The authors should explain the relevance of this experimental setup to oral (pH) conditions.

2.      Figure 8: It is very hard to interpret the relative composition of each specimen from EDXS graphs. I suggest proving each ion's cumulative abundance (composition) in a separate table to make it easier to read.

Overall, the manuscript is of good quality and novel and suggested publishing with minor revision. 

Author Response

Reviewer 3

1. Authors used strong acid (35% HCl) treatment to examine the ion release from the orthodontic wires, which is much more acidic than the oral pH. The authors should explain the relevance of this experimental setup to oral (pH) conditions.
Response: The method used in this study was based on previous studies [Ito et al Applied Sciences 2021]. This has been added in the revised manuscript (lines 106). 
Revision:
2.5. Metal Ion Release Tests
As described previously [10], Orthodontic wire samples were separately immersed in 35-mm plastic dishes (Iwaki, Osaka, Japan) containing 3 mL of 35% HCl (pH -1.1) (Wako Pure Chemical Industries, Ltd., Osaka, Japan), placed at 25°C for 30 min. Of the total length of the wire samples, 4 mm were immersed in hydrochloric acid. The concentrations of Cr, manganese (Mn), iron (Fe), and Ni ions released from the coated wires into the solutions were measured by triple quadrupole inductively coupled plasma mass spectrometry (ICP-MS) (ICP-QQQ-Agilent 8800; Agilent Technologies, Santa Clara, CA, USA). The concentrations of metal ions are shown as the mean ± SD of four replicates for each sample.

2. Figure 8: It is very hard to interpret the relative composition of each specimen from EDXS graphs. I suggest proving each ion's cumulative abundance (composition) in a separate table to make it easier to read. 
Response:
The purpose of the EDS analysis in this study was to determine whether SS, the base metal, was exposed or not. Therefore, the degree of exposure and quantification were not an issue in this study. However, because the coating loss of Rh-coated wires is obvious, but the coating loss of PEN-coated wires is difficult to determine from the EDS analysis in this study. So, I have revised text (line279-281, 289-293, 295-296,309, 311-315). In addition, we think that future studies examining coating loss under different brushing conditions are necessary, so we have added that information to line 311-315 of the text. In that case, we believe that quantitative and statistical analysis is necessary to clarify the differences between different brushing conditions.
Revision:
・As it was not possible to determine whether brushing affected coating loss on coated wires by stereomicroscopy, we next examined the surface properties of the wires by SEM. On SEM analysis, each type of wire showed black areas after brushing. EDS analysis was performed to determine whether there was coating loss and exposure of the substrate metal at these areas. TiN-coated, Au-coated, and PEN-coated wires do not appear to show significant changes in graph before and after brushing. On the other hand, a change was observed in Rh-coated wires after brushing. In the Rh-coated wire, a peak of Rh was observed before brushing, which decreased after brushing and a new peak of Al was observed. It is possible that Al used in the pretreatment was exposed due to detachment of the Rh coating. However, although the manufacturer of the wire stated that they pretreat the Rh-coated wire to improve its coloration, precise data on the materials used were not available due to their policy of keeping this information confidential. In addition, Ti peaks were detected in the PEN coating both before and after brushing, which were due to the use of Ti oxide directly beneath the coating. As the PEN coating was as thin as 50um, the Ti peak may have been detected even before brushing. Because of the thin thickness of the PEN coat and the substrate metal is detected both before and after brushing, it may be difficult to discuss the coating loss of the PEN coated wire by EDS analysis in this study. However, a new trace peaks of Fe were detected after brushing, suggesting that coating loss may have occurred in the PEN-coated wire. Taken together, these results suggest that brushing does not result in coating loss from TiN-coated and Au-coated wires, but that it may cause loss of the coating from Rh-coated and PEN-coated wires. In addition, coating loss may have occurred on the PEN coated wire, but further analysis is needed. Therefore, the null hypothesis (H03) was rejected. 
・In this study, brushing using an electric toothbrush corresponding in hardness to that likely to be encountered in clinical situations may have caused detachment of the PEN coating from the wire. Therefore, it would be advisable to select a toothbrush with softer filaments in patients treated with PEN-coated wire.
・Therefore, it would be advisable to select a toothbrush with softer filaments in patients treated with PEN-coated wire. To examine this issue in more depth, it is necessary to compare the degree of coating loss between different brushing conditions. Brushing conditions could include changing such as the filament hardness and number of brushings, and statistical analysis is needed when comparing the amount of metal exposure between conditions.

 

Round 2

Reviewer 2 Report

Thank you for addressing all pointed issues. Actually, the paper has been improved. Only be carefull with typos and minor writing issues, mainly in newly added sentences and avoid writing in first person, for example: "For post-hoc analysis, we used Tukey–Kramer honestly significant difference (HSD) was used to determine which groups had statistically significant differences."

Author Response

Reviewer 2

 

Only be carefull with typos and minor writing issues, mainly in newly added sentences and avoid writing in first person, for example: "For post-hoc analysis, we used Tukey–Kramer honestly significant difference (HSD) was used to determine which groups had statistically significant differences."

 

Response:

Thank you for your comment and suggestion.

Our manuscript has been checked by English editing company. Changed parts are marked in yellow.