Tooth Discoloration Induced by Endodontic Phenothiazine Dyes in Photodynamic Therapy

Abstract

Objective: This study sought to assess if discoloration of tooth structures occurs after photodynamic therapy (PDT) and to determine the efficacy of a protocol to remove the photosensitizers. Background data: PDT has been used in root canal treatment to enhance cleaning and disinfection of the root canal system. PDT uses a low power laser in association with a dye as a photosensitizer. Photosensitizers can induce staining of the dental structures, resulting in an unaesthetic appearance. Methods: Forty teeth were randomly divided into four groups according to the photosensitizer used and pre-irradiation time: 0.01% methylene blue for 5 min (MB5); 0.01% methylene blue for 10 min (MB10); 0.01% toluidine blue for 5 min (TB5); and 0.01% toluidine blue for 10 min (TB10). Specimens were irradiated with a 660 nm diode laser with a 300 μm diameter optical fiber, at 40 mW power setting for 3 min. Immediately after, the photosensitizers were removed with Endo-PTC cream+2.5% sodium hypochlorite (NaOCl). The shade was measured by a Vita Easyshade spectrophotometer based on the CIELAB color system (L*a*b* values) at three different experimental times: before PDT (T0), immediately after PDT (T1), and after removal of the photosensitizer (T2). Results: The results showed a decrease in the averages of the L*a*b* coordinate values after PDT (T1) in all the groups, when compared with the number at T0, with a significant statistical difference in group MB10. After photosensitizer removal (T2), all the values of the coordinates increased with significant statistical differences (p<0.05) between T1 and T2 in L* and a*. Conclusions: It can be concluded that both methylene blue and toluidine blue dyes cause tooth discoloration, and that Endo-PTC cream associated with 2.5% NaOCl effectively remove these dyes, regardless of the pre-irradiation time used for PDT.

Introduction

The goal of root canal treatment is the prevention or elimination of the root canal system as a locus for bacteria, which serve as a source of inflammation to the periodontium.^1,2 Infected root canals have complex microbial flora and some microorganisms, such as Enterococcus faecalis, can survive cleaning and shaping procedures because of internal mechanisms such as a proton pump.^3
–5 The anatomic complexity of the root canal system with its nooks, crannies, anastomoses, re-entrances, and dentin tubles provides an environment where microorganisms can hide and not be reached by irrigants and medicaments.^6
–8 New techniques and resources to enhance cleaning and disinfection of the root canal system have been sought, including low power lasers associated with a photosensitizer.^9,10 This treatment modality, named “photodynamic therapy,” (PDT) began to be used in the 1990s, showing effective results in decreasing the bacterial load in root canal systems.^6

–13

In PDT, a photosensitizer with an appropriate spectrum of absorption for the wavelength produced by the low power laser is delivered into the root canal system. The photosensitizer will bind the microorganisms, and when the low power laser is activated, a photochemical reaction will take place, with generation of free radicals and singlet oxygen.¹⁴ This reaction will cause the rupture of the bacterial cell wall, leading to microbial cell death.^11,15 This treatment is very specific, as it affects only the bacterial cells that are impregnated by the photosensitizer, which makes it a safe a procedure for the periapical tissues.¹⁶

The time elapsed between the delivery of the photosensitizer into the root canal system and the actual photoactivation is called the “pre-irradiation time.” The pre-irradiation time is a key factor in PDT, as it permits the photosensitizer to penetrate through the dentin. Research is controversial regarding the pre-irradiation time, and available data show pre-irradiation times of 5–15 min.^7,9,17

Phenothiazine dyes such as toluidine blue (TB) and methylene blue (MB) have been the most frequently used photosensitizers in PDT, with favorable results. ^{8,10
–12,18,19} At low concentrations, these dyes are biocompatible, because they are not cytotoxic when in contact with keratinocytes and fibroblasts.⁶

An adverse effect of using these dyes in PDT is that they can stain the tooth structures, resulting in an unaesthetic appearance. Very few studies have addressed this problem using either visual or digital image methodologies.^8,20

The aim of this study was to assess if discoloration of tooth structures occurs after PDT when MB and TB are used as photosensitizers, and if improvement of discoloration occurs after removal of the photosensitizers with the suggested protocol. The null hypotheses are that both photosensitizers will discolor the tooth structure and that discoloration will be improved after the photosensitizers are removed from the tooth with suggested protocol.

Material and Methods

Sample selection and preparation

This study was approved by the Research Ethics Board of the University of Taubate (CEP/UNITAU 031/11). Forty human teeth (single-rooted, freshly extracted as part of an orthodontic treatment plan) were selected. Teeth were cleaned of gross debris with a brush and a curette and autoclaved. All teeth were then stored in single bottles with saline.

After access opening, cleaning and shaping were performed with hand instruments (crown down technique, Dentsply/Maillefer S.A., Ballaigues, Switzerland) and 2.5% sodium hypochlorite (NaOCl). The working length was visually established at 1 mm short of the apical foramen. The canals were irrigated with 2.5% mL of NaOCl after each file, and the master apical file was #40 for all the teeth. After completion of cleaning and shaping, the canals were filled with 17% ethylenediaminetetracetic acid (EDTA) (Biodinâmica Química e Farmacêutica, Ibiporã, PR, Brasil) for 3 min for smear layer removal. The canals were then rinsed with 5 mL of saline for removal of residues of the previous irrigants, and dried with suction and paper points (Dentsply/Maillefer).

Shade recording

The samples were individually positioned in a plaster apparatus, and a silicone impression was made over the teeth with perforations of equal diameter in the center of the buccal surface of the teeth to standardize the procedure for shade recording (Fig. 1a.). Shade recording of the crown was done with a Vita Easyshade Compact spectrophotometer (Vita Zahnfabrik Rauter GmbH & Co. KG, Bad Säckingen, Germany). This equipment quantifies the shade through a system named CIELAB-CIE1976 (L*a*b*) (Comission Internationale de L'Eclairage, Vienna, Austria)²¹. The system is composed of three coordinates L*, a*, and b*. The L* value indicates brightness and varies between 0 for black and 100 for white, a* determines the amount of red (positive values) or green (negative values), and b* exhibits the amount of yellow (positive values) or blue (negative values).

FIG. 1.

(a) Silicone jig positioned on the buccal surface of the specimens. (b) Openings for positioning of the tip of the spectrophotometer.

One examiner was calibrated for shade analysis in the same room under the same light in three different time intervals: before PDT (T0), immediately after PDT (T1), and after the procedure for dye removal (T2). During each time interval, three measurements of L*, a*, and b* were taken, and ΔL, Δa, and Δb were then calculated by subtracting the final data from the initial data from each time interval. The shade stability was then calculated by using the following formula: ΔE=[(ΔL)²+(Δa)²+(Δb)²]^1/2 (Friele-Max Adam-Chickering - Comission Internacionale de L'Eclairage, 1978).

Experimental groups

Specimens were randomly assigned to a group (n=10), according to the photosensitizer and the pre-irradiation time, as following: MB5, methylene blue and pre-irradiation time of 5 min; MB10, methylene blue and pre-irradiation time of 10 min; TB5, toluidine blue and pre-irradiation time of 5 min, and TB10, toluidine blue and pre-irradiation time of 10 min. Both dyes were at 0.01% concentration (Farmácia de Manipulação Fórmula & Ação, São Paulo, SP, Brazil).

PDT

The photosensitizers were delivered into the canals with a 30 gauge needle (ProRinse, Dentsply-Tulsa Dental, Tulsa, OK) up to the cavosurface bevel and remained in the canal according to the pre-irradiation time. The canals were then irradiated with low power diode laser (Thera Lase DMC equipamentos ltda., São Carlos, SP, Brazil) with a 660 nm red wavelength, through a 300 μm diameter optic fiber and at 40 mW output power setting. The optic fiber was inserted 1 mm short of the working length and withdrawn with spiral movements so that the optic fiber could touch as much of the canal walls as possible. The irradiation time was 3 min. According to Soukos et al.,⁶ the root canal surface area is ∼0.316 cm²; therefore, the output power used in the present study corresponded with an energy fluency of ∼30.4 J/cm² and power density of 126.6 mW/cm².

Immediately after PDT (T1), the shade was again recorded as follows: the silicone jig was placed over the tooth, and the tip of the spectrophotometer was fitted inside the perforations created in the silicone jig, in the center of the buccal surface.

Procedures for photosensitizer removal

The Endo-PTC cream (10% urea peroxide, 15% Tween 80 15%, and 75% carbowax - Farmácia de Manipulação Fórmula & Ação) was placed in a plastic syringe (5 mL), and ∼0.05 mg of the cream was delivered through a plastic tip into the pulp chamber. Then, irrigation with 15 mL of 2.5% NaOCl was performed for 3 min. Immediately after, the third shade record was done (T2).

Statistical analysis

The data were analyzed at 5% significance. T and Wilcoxon tests were performed to analyze intragroup data, to detect changes within the same group in the different experimental time periods. Kruskal–Wallis and Dunn's test were used to analyze intergroup data, to detect the differences among the experimental groups in the same time period.

Results

Assessment of discoloration After PDT in the time periods of T0 and T1

In the intragroup analysis, all groups exhibited negative values of ΔL after PDT, indicating discoloration of the tooth structure (TB10>MB10>TB5>MB5) (Table 1). Statistical difference was found only in the group TB10 between the time periods of T0 and T1.

Table 1.

Average Values of L, a, and b Before (T0) and Immediately After Photodynamic Therapy (PDT) (T1)

	LT0	LT1	ΔL	aT0	aT1	Δa	bT0	bT1	Δb	ΔE
MB5	81.40^a	81.23^a	−0.17^A	−2.00^a	−2.42^a	−0.42^A	25.91^a	25.86^a	−0.05^A	2.57±1.96^A
MB10	82.89^a	82.55^a	−0.34^AB	−1.67^a	−2.56^b	−0.89^A	28.89^a	28.14^a	−0.75^A	2.76±.1.41^A
TB5	82.59^a	82.37^a	−0.22^AB	−1.69^a	−1.94^a	−0.25^A	27.36^a	27.14^a	−0.22^A	2.33±1.75^A
TB10	84.49^A	82.68^b	−1.81^B	0.67^a	0.23^b	−0.43^A	38.22^a	37.15^b	−1.07^A	2.62±1.67^A

□ Intragroup statistical analysis. Statistical differences are indicated by different lower case letters in columns (t test and T Wilcoxon test, p<0.05).

Intergroup statistical analysis. Statistical differences are indicated by different capital letters in rows (Kruskal–Wallis test, p<0.05).

Average values of ΔL (LT1-LT0), Δa (aT1-aT0), Δb (bT1-bT0), and ΔE, with standard deviation. Results of statistical analysis.

Inter-group analysis indicated a statistical difference of ΔL only between groups MB5 and TB10. The average values of ΔE varied between 2.33 and 2.76 (MB10>TB10>MB5>TB5), with no statistical difference among the groups.

Assessment of efficacy of procedures for photosensitizer removal in the time periods of T1 and T2

In the intra-group analysis, the ΔL value was positive in all groups, as there was improvement of discoloration after photosensitizer removal procedure, with statistical difference in all groups between the time periods of T1 and T2 (Table 2).

Table 2.

Average Values of L, a, and b, Immediately After Photodynamic Therapy (PDT) (T1) and After Photosensitizer Removal with Endo-PTC Cream and 2.5% NaOCl (T2)

	LT1	LT2	ΔL	aT1	aT2	Δa	bT1	bT2	Δb	ΔE
MB5	81.23^a	82.38^b	1.15^A	−2.42^a	−1.87^b	0.56^A	25.86^a	26.19^a	0.32^A	2.18±1.51^A
MB10	82.55^a	84.60^b	2.05^A	−2.56^a	−1.89^b	0.67^A	28.14^a	29.43^b	1.29^A	2.92±2.26^A
TB5	82.37^a	84.48^b	2.11^A	−1.94^a	−1.39^b	0.55^A	27.14 ^a	27.93^a	0.79^A	3.01±1.47^A
TB10	82.68^a	84.52^b	1.84^A	0.23^a	0.67^b	0.44^A	37.15^a	38.08^b	0.93^A	2.26±1.35^A

□ Intragroup statistical analysis. Statistical differences are indicated by different lower case letters in columns (t test and T Wilcoxon test, p<0.05).

Intergroup statistical analysis. Statistical differences are indicated by different capital letters in rows (Kruskal–Wallis test, p<0.05).

Average values of ΔL (LT2-LT1), Δa (aT2-aT1), Δb (bT2-bT1), and ΔE, with standard deviation. Results of statistical analysis.

In the intergroup analysis, no statistical differences were shown among the groups. The average values of ΔE varied between 2.18 and 3.02 (TB5>MB10>TB10>MB5).

Assessment of total discoloration in the time periods of T0 and T2

In the intragroup analysis, all groups exhibited positive values of ΔL, that is, the values of the coordinate L* obtained after the procedures for photosensitizer removal (T2) were closer to white than the values of L* at the beginning of the experiment (T0), with statistical difference in the groups MB10 and TB5 between the time periods of T0 and T2 (Table 3).

Table 3.

Average Values of L, a, and b, Before Photodynamic Therapy (PDT) (T0) and After Procedures for Photosensitizer Removal (T2)

	LT0	LT2	ΔL	aT0	aT2	Δa	bT0	bT2	Δb	ΔE
MB5	81.40^a	82.38^a	0.98^A	−2.00^a	−1.87^a	0.13^A	25.91^a	26.19^a	0.28^A	2.06±0.94^A
MB10	82.89^a	84.60^b	1.71^A	−1.67^a	−1.89^a	−0.22^A	28.89^a	29.43^a	0.54^A	2.50±1.40^A
TB5	82.59^a	84.48^b	1.89^A	−1.69^a	−1.39^b	0.41^A	27.36^a	27.93^b	0.57^A	2.76±1.10^A
TB10	84.49^a	84.52^a	0.03^A	0.67^a	0.67^a	0.03^A	38.22^a	38.08^a	−0.14^A	1.86±1.51^A

□ Intragroup statistical analysis. Statistical difference are indicated by different lower case letters in columns (t test and T Wilcoxon test, p<0.05).

Intergroup statistical analysis. Statistical differences are indicated by different capital letters in rows (Kruskal–Wallis test, p<0.05).

Average values of ΔL (LT2-LT0), Δa (aT2-aT0), Δb (bT2-bT0), and ΔE, with standard deviation. Results of statistical analysis.

The intergroup statistical analysis did not show statistical differences among the groups.

Discussion

There is growing interest in PDT in endodontics, as there is a constant search to eliminate microorganisms from the root canal system, in particular, in persistent intracanal infections.¹

Both null hypotheses were accepted. The results show that tooth discoloration occurred at both pre-irradiation times (5 and 10 min) when both photosensitizers (MB and TB) were used. Overall, the pre-irradiation time of 10 min yielded to more severe discoloration of the tooth structure when compared with the pre-irradiation time of 5 min. The longer time likely allowed the photosensitizers to penetrate deeper into the dentin and closer to the dentin–enamel interface, making the discoloration more noticeable.

It is important to understand that, according to the CIE L*a*b* system, the shade changes (ΔE) with values between 0.0 and 0.5 are considered vestiges, values between 1.5 and 3.0 are considered clinically perceptible, and values >3.3 are considered clinically unacceptable.^22,23 Concerning the final discoloration (ΔE), the average values between T0 and T1 ranged from 2.33 and 2.76. That being said, the shade changes caused by both photosensitizers can be classified as clinically perceptible. When the experiment was being conducted, the discoloration could be easily visually appreciated in most specimens. In all the groups, there were 1–3 specimens (MB5=1, MB10=2, TB5=3, and TB10=2) with values of ΔE>3.3 (clinically unacceptable). Such differences may be attributed to individual characteristics of each tooth, such as tooth age and dentin permeability, which may interfere with the photosensitizer penetration.

Specimens in groups TB5 and TB10 suffered more overall discoloration when compared with groups MB5 and MB10. TB has a lower molecular weight (107.17 g/moL) compared with MB (373.91 g/moL), which may have allowed deeper penetration into the dentin tubules, yielding to a more noticeable discoloration. Studies on discoloration in PDT are rare, and we could only compare the results of the present study with the results of a previous study in which the use of MB resulted in tooth discoloration.²⁰

After PDT was concluded and discoloration occurred, Endo-PTC cream associated with 2.5% NaOCl was used to remove the photosensitizers.²⁰ The values of ΔL (difference in brightness) were all positive in all groups in T2, which meant that the differences between T1 and T2 were significant, and that improvement of discoloration occurred. NaOCl is broken down into chlorine and oxygen that will promote bleaching of the dentin.²⁴ Endo-PTC cream is a mixture of Tween 80 (detergent), urea peroxide, and carbowax, and when associated with NaOCl, will likely increase and prolong the oxygen release, facilitating bleaching and improving the aesthetic results.^20,25

Considering that the average values of ΔE varied between 2.18 and 3.02, the bleaching that occurred after the photosensitizers were removed could be classified as clinically perceptible, which was visually confirmed in all samples.

There are a few methods to measure tooth shade and assess shade variation. The advantage of the spectrophotometer used in this study is that the method that it uses to verify shade changes is based on the CIE L*a*b* system. The advantage of this system is that the shade differences are expressed numerically, which can be related to the visual perception and to the clinical significance.¹⁵ The system uses three different coordinates (L, a and b). The L values represent the brightness, and it seems to be the most significant parameter, whereas the a* and the b* values represent saturation and are less significant in this type of analysis. However, all the three values were collected, as they were needed in the formula used to calculate the ΔE, which represents the final discoloration.

It is interesting to note that ΔL values were all positive in T2 when compared with the values in T0, showing that the teeth were lighter in T2 than in T0, in the beginning of the experiment. Statistically significant differences were found in groups MB10 and TB5. The average values of ΔE varied between 1.86 and 2.76 (TB5>MB10>MB5>TB10), which can be considered clinically perceptible. This shade change could not be visually noticed in all specimens, as demonstrated between T0 and T1.

Based on this study, it is clear that the photosensitizers used for PDT will discolor the tooth, causing an unaesthetic appearance. However, this discoloration is not permanent and can be improved by simply using Endo-PTC cream associated with 2.5% NaOCl rinse.

Conclusions

Based on the results and on the limitations of this study, it can be concluded that both MB and TB dyes cause tooth discoloration and that Endo-PTC cream associated with 2.5% NaOCl effectively removes these dyes, improving discoloration, regardless of the pre-irradiation time used for PDT.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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