Evaluation of the effectiveness of different treatment approaches in preventing coronal discoloration caused by regenerative endodontic treatment

This study aimed to evaluate the effectiveness of Teethmate desensitizer, a dentin bonding agent (DBA), Nd:YAG laser, and Er:YAG laser, which provides dentin tubule occlusion in the pulp chamber with different mechanisms, in preventing tooth discoloration due to regenerative endodontic treatment. One hundred five extracted maxillary human incisors with single roots and single canals were included in the study. The apical third of each tooth was resected below the enamel-cementum junction (CEJ) to obtain a standard root length as 10 ± 1 mm. Root canal preparation was performed using the ProTaper Next files up to X5. Root canals were prepared with Gates Glidden (# 2–4) burs to simulate the immature root apex and an apical diameter of 1.1 ± 0.1 mm was obtained. The teeth were randomly divided into 7 groups (n = 15): DBA, Teethmate, Nd:YAG, Er:YAG, Biodentine, Blood, and Negative Control. Relevant dentin tubule occlusion methods were applied to DBA, Teethmate, Nd:YAG, and Er:YAG groups. Following dentin tubule occlusion procedures, Biodentine was placed on the blood clot after filling the root canals with blood up to 4 mm below the CEJ. No dentin tubule occlusion procedure was applied for Blood and Biodentine groups. Color measurement was performed with the spectrophotometer Vita Easyshade Advance before treatment, immediately after treatment, and at days 7, 30, and 90. Data were converted to L*a*b color values of Commission International de I'Eclairage (CIE L*a*b) and ΔE values were calculated. Two-way ANOVA and post hoc Tukey test (p = 0.05) were performed for statistical analysis. A clinically detectable color change was observed in all groups except for the negative control (ΔE ≥ 3,3). It was observed that Biodentine used alone has a potential for discoloration. It was determined that as the contact time with blood increased, tooth discoloration increased. However, no significant difference was found between dentin tubule occlusion methods in preventing color change (p > 0.05). It was determined that no dentin tubule occlusion method could 100% prevent discoloration caused by RET. DBA and Teethmate, which do not have a significant difference in terms of preventing color change, are considered to be suitable for dentin tubule occlusion due to their ease of application and low cost compared to Nd:YAG laser and Er:YAG laser.


Introduction
Regenerative endodontic treatment (RET), which aims to regenerate damaged pulp and other teeth tissues, including dentin and other root structures, has become a treatment approach with increasing popularity and applied frequency [1]. In RET, a scaffold is needed for the regeneration of tissues. For this purpose, the use of many tissue scaffolds, including platelet-rich plasma (PRP), platelet-rich fibrin (PRF), and blood clot, has been reported [2]. Today, blood clot is one of the most preferred tissue scaffolds due to their autologous provision, low cost, and ease of clinical application. In addition, it has an important role in the differentiation, maturation, and regeneration of cells such as cementoblasts, odontoblasts, and fibroblasts, as it is a rich source of growth factors needed for blood clot regeneration [3]. However, despite all these advantages, when a blood clot comes into contact with the coronal tooth structures, erythrocytes that penetrate through the dentinal tubules are hemolyzed and hemoglobin and hematin molecules released after this reaction accumulate in the dentinal tubules, resulting in tooth discoloration [4].
Blood products are not the only cause of tooth discoloration due to RET. It has been reported that various calcium silicatebased cements (CSCs) also cause tooth discoloration by different mechanisms [5]. It has been suggested that the entry of mineral trioxide aggregate (MTA) into the dentinal tubules in the pulp chamber or its passage through the enamel dentin structure causes tooth discoloration [6]. In addition, it has been reported that the iron and bismuth oxide which provides radioopacity, contained in MTA cause tooth discoloration [7]. It was reported that possible other mechanisms of undesirable tooth discoloration associated with MTA that the oxidation of the iron content of the powder to the calcium aluminoferrite phase, the oxidation of bismuth oxide to produce bismuth carbonate, and the interaction of bismuth oxide with dentin collagen [8]. Biodentine is currently widely used in RET for color stability, as it has better color preservation compared to MTA and contains zirconium oxide as a radioopacifier instead of bismuth oxide, which is an important component in tooth discoloration [5]. In addition to these advantages, the use of Biodentine in RET is increasing due to its short setting time and ease of use [9]. However, despite all these advantages, it has been reported that even in RET using Biodentine, there is a certain degree of tooth discoloration [10]. Therefore, today, with the increase in dental aesthetic concerns, and to prevent tooth discoloration that may be caused by RET, the search for the ideal treatment approach continues [4].
It has been reported in various studies that by applying DBA to the pulp chamber before starting RET procedures, occlusion of the dentinal tubules can be achieved and tooth discoloration caused by blood clot and MTA can be reduced to a certain extent [10,11]. Similarly, when Teethmate desensitizer is applied to dentin, it turns into hydroxyapatite with tetracalcium phosphate and dicalcium phosphate in its content and clogs the dentinal tubules so that tooth discoloration can be reduced [12]. Neodymium-doped yttrium aluminum garnet (Nd:YAG) laser, which is offered as an alternative treatment for dentinal tubule occlusion, has been shown to occlude dentinal tubules through a procedure called "melting and resolidification" [13]. Erbium-doped yttrium aluminum garnet (Er:YAG) laser provides occlusion of dentinal tubules by crystallization or melting of dentin [14].
The comprehensive literature review found a study in which the preventive efficacy of Teethmate desensitizer, DBA, and Nd:YAG laser was evaluated against tooth discoloration caused by triple antibiotic paste (TAP) [12]. However, there was no study comparing the effectiveness of DBA, Teethmate desensitizer, Nd:YAG laser, Er:YAG laser in preventing discoloration due to blood clot and Biodentine, which is a cause of tooth discoloration in RET. Therefore, in the current in vitro study, the effectiveness of DBA, Teethmate desensitizer, Nd:YAG laser, Er:YAG laser, which provides occlusion of the dentinal tubule by different mechanisms, in preventing the color change caused by Biodentine in contact with blood in RET was investigated. The null hypothesis of the study; There will be no difference between DBA, Teethmate desensitizer, Nd:YAG laser, and Er:YAG laser in terms of preventing tooth discoloration.

Sample selection and preparation
The study design was approved by the XX University Clinical Research Ethics Committee (Ethics No: 2020/165). The sample size was calculated using G*Power software 3.1.2 (Universitat, Düsseldorf, Germany) concerning a recent study's power analysis [15] in the literature with a similar design, 0.05 probability of alpha error and 80% power (effect size = 0.25). It was determined that at least 15 teeth in each group were statistically necessary, and a total of 105 teeth for 7 groups were included in the study.
One hundred five permanent maxillary incisors with single root and single canal, extracted for periodontal, orthodontic, and prosthetic reasons, were included in the present study. The initial apical diameter of all included teeth was equal to #15 K-files. The teeth were stored in 0.9% saline solution at 25 °C room temperature until used in the study.

Root canal preparation
Conventional endodontic access cavities were prepared using a high-speed handpiece and diamond bur (G&Z Instrumente, Lustenau, Austria) under water cooling. The apical patency of the teeth was checked with a #10 K-file (Dentsply Maillefer, Ballaigues, Switzerland). In order to simulate teeth with immature apex, the apical parts of all teeth were removed under low speed water cooling using diamond disk (Sunshine, Langenhagen, Germany) as previously described in the literature [12], and the roots were standardized to be 10 ± 1 mm from CEJ. The root canals were prepared with the ProTaper Next (Dentsply Maillefer, Ballaigues, Switzerland) X1, X2, X3, X4, and X5 files respectively. All preparations were performed with a VDW Gold endomotor (VDW, Munich, Germany) using the torque and rpm values specified in the manufacturer's instructions. Root canals were irrigated with 2 ml of 1.5% NaOCl (Wizard, Guide Chemistry, Istanbul, Turkey) during each file change. After root canal preparation, root canals were prepared with gates glidden burs (Proud, London, UK) #2 to #4, respectively, to obtain an immature tooth model with a standard canal diameter of 1.1 ± 0.1 mm, similar to the methodology performed by Chen et al. [10].

Irrigation procedures
After the preparation procedures are completed, each root canal was irrigated with 20 ml 1.5% NaOCl (Wizard) for 5 min and then was irrigated with 20 ml 17% EDTA (Nazar Chemistry Ltd, Istanbul, Turkey) based on the RET protocol of American Association of Endodontists (AAE) [9]. A 30 gauge needle with a side-vented was used for all irrigation of root canals (NaviTip; Ultradent, South Jordan, UT, USA). Root canals were dried with paper points (Dentsply Tulsa Dental, Tulsa, USA) after irrigation procedures were completed. The root ends were sealed with composite resin (Clearfill Majesty Esthetic, Okayama, Japan) using the incremental technique.

Preparation for color measurement
To make color measurements on all dental crowns from the same point each time, an acrylic model obtained by copying the tip of the spectrophotometer was placed on the buccal surface of the teeth above CEJ, and a fixed frame made of flowable composite (Filtek Z250, 3 M ESPE, Germany) was prepared around it.
The teeth selected for use in our study were numbered from 1 to 105. Randomization was accomplished by using computer sequence generation (www. random. org), which provided by a table for 7 groups with randomized tooth numbers (n = 15) in each group. Afterward, the teeth were placed in Eppendorf tubes filled with distilled water.

Negative control
No procedure was applied to the pulp chamber of the teeth in this group.

Blood
In this group, no procedure was performed to occlude the dentinal tubules. Only blood was applied to all teeth in this group to evaluate blood-induced discoloration. Whole fresh blood was drawn from a healthy volunteer (M.O.A) in K2-EDTA tubes (BD Vacutainer®, Plymouth, UK) by a trained person. Root canals were filled with blood taken with a syringe, 4 mm below the CEJ, without any waiting time [16]. A blood clot was allowed to form for 15 min. Then a collagen matrix spongostan (Cutanplast, Milano, Italy) was placed on the blood clot. Blood and spongostan (Cutanplast) were placed in the root canals of the samples in all groups except the negative control and Biodentine groups, as described above, after dentin tubule occlusion procedures.

Biodentine
In this group, no procedure was performed for the occlusion of the dentin tubules. Only Biodentine (Septodont, Saint Maur-des-Fosses, France) was applied to all teeth in this group to determine the discoloration caused by Biodentine (Septodont) alone. By the manufacturer's instructions, the capsule containing the powder was opened in a single dose container, and 5 drops of liquid containing water-soluble polymer and calcium chloride were dropped into the powder, and then the capsule was closed. It was mixed with an amalgamator (Linea Tac, Montegrosso, Italy) for 30 s. Biodentine (Septodont) with a thickness of 3 mm thick was condensed with hand plugger No. 2 (Buchanan, Kerr, USA) to be positioned in the CEJ. All processes were carried out considering the setting time of 12 min.

DBA
Nova Compo B Plus (IMICRYL, Konya, Turkey), a 7th generation all-in-one dentin bonding agent, was applied to the inner surfaces of the pulp chamber of the teeth in this group for 20 s with the help of a micro-applicator. The teeth were gently air-dried for 5 s. For polymerization, it was cured with a light-cure device (Monitex BlueLex GT) with a light power of 1200 mW/cm 2 for 20 s [17]. After the procedures of blood placement in the root canal, 3 mm thick Biodentine (Septodont) was placed in the root canal to be positioned in the CEJ. A setting time of 12 min was expected.

Teethmate
After applying Teethmate desensitizer (Kuraray Noritake Dental, Nigata, Japan) prepared by the manufacturer's instructions to the dentin walls of the pulp chamber of the teeth in this group, with the help of a micro applicator, a 60-s rubbing motion was made. After the procedures of blood placement in the root canal, 3-mm thick Biodentine (Septodont) was placed in the root canal to be positioned in the CEJ. A setting time of 12 min was expected.

Nd:YAG laser
Laser device (Fotona AT, Fidelis III, Ljubljana, Slovenia) is set to Nd:YAG dentin hypersensitivity prevention mode. With the help of a 300-μm optical fiber tip, a laser was applied to the dentin walls of the pulp chamber of the teeth in this group, from a distance of 1 mm, with a vertical sweeping motion at 10 Hz, 1 W parameters for 60 s without using air/water [18,19]. After the procedures of blood placement in the root canal, 3 mm thick Biodentine (Septodont) was placed in the root canal to be positioned in the CEJ. A setting time of 12 min was expected.

Er:YAG laser
Er:YAG mode was selected by attaching the non-contact head (R02) to the laser device (Fotona AT, Fidelis III). Laser was applied at the parameters of 80 mJ, 2 Hz, 0.15 W for 60 s without using air/water spray, perpendicular to the dentin walls in the pulp chamber of the teeth in this group, from a distance of 1 mm [19,20]. After the procedures of blood placement in the root canal, 3 mm thick Biodentine (Septodont) was placed in the root canal to be positioned in the CEJ. A setting time of 12 min was expected.
Samples of all groups were restored with composite (Clearfill Majesty Esthetic) using the incremental technique. The samples were stored at 37 °C and 100% humidity during the entire experiment.

Tooth color assessment
The color change of the teeth over time was measured with Vita Easyshade Advance (VITA Zahnfabrik, Bad Sackingen, Germany). Vita Easyshade Advance is the spectrophotometer of choice for reliable color measurement, which has standards of the international CIE L*a*b* color system (Commission Internationale de L'Eclairage, Vienna, Austria) [12].
Before each color assessment procedure, the device was calibrated using the calibration table by the manufacturer's instructions. During the assessments, the head of the spectrophotometer was placed in the composite frame and the measurement was made. Color measurements were performed three times from the same spot under white light by placing the tip of the spectrophotometer in the frame on the labial surfaces of the teeth. L*, a*, and b* values were obtained for each measurement and the average of these measurements was calculated. Color measurement was performed and evaluated by a single researcher. L* values indicate color change ranging from black (0) to white (100), a * values from red (+ 80a *), green (− 80a *), and b * values from yellow (+ 80b *) to blue (− 80b *)) denotes varying color variations [10].
Color measurements were evaluated at the following 5-time points; T0: before the procedure T1: immediately after the procedure T7: 1 week after the procedure T30: 1 month after the procedure T90: 3 months after the procedure The mean color change (ΔE) value was calculated using the following formula: If the ΔE value was 3.3 ≥ , it was accepted that there was a visible color change [5,10,21].

Statistical analysis
Shapiro-Wilk test was applied to confirm the normality of the obtained data. Since the data showed normal distribution, ΔE values were analyzed using two-way ANOVA and post hoc Tukey tests. All statistical analyzes were performed using SPSS software version 17 (IBM, Armonk, NY, USA). Statistically significant level was determined as 5%.

Results
Clinically detectable color change was observed in the samples from all groups except the negative control (ΔE ≥ 3.3) (Fig. 1). In the T0-T90 time interval, ΔE values for all groups except the negative control group were in the range of 4. 26-19.44. There was no significant difference in the increase in coloration at all time intervals in the negative control, DBA, and Teethmate (p > 0.05). Compared to T0-T1 in the Nd:YAG group, in the T0-T7 time interval; in the Er:YAG group, at T0-T7 and T0-T30 time intervals compared to T0-T1; in all other time intervals compared to T0-T1 in the Biodentine group; In the blood group, the increase in coloration was statistically higher in the T0-T90 time interval compared to T0-T1 (p < 0.05) (Fig. 2).
In the T0-T1 time interval, tooth discoloration was statistically significantly higher in the Teethmate, Nd:YAG, Er:YAG, Biodentine, and blood-treated compared to the negative control group (p < 0.05). There was no statistical difference between the DBA and the negative control (p > 0.05).
Discoloration was statistically significantly higher in the Teethmate, Nd:YAG, Er:YAG, and blood-treated compared to the negative control in the T0-T90 time interval (p < 0.05). Discoloration was statistically significantly higher in the blood-treated compared to the DBA, Nd:YAG, and Biodentine (p < 0.05).

Discussion
The effectiveness of different treatment approaches to prevent tooth discoloration after various RET techniques have been investigated in the literature. However, no treatment protocol provides definite success in preventing discoloration. In this study, the potential of four different treatment approaches; Teethmate desensitizer, a DBA, and Nd:YAG, Er:YAG which occlude dentinal tubules by different mechanisms, to prevent tooth discoloration due to RET using blood clot and Biodentine was investigated.
In the current literature, tissue scaffolds such as blood, PRF, PRP are used for cell growth, differentiation, and organization in RET procedures [22]. Although PRP and PRF scaffolds have a high potential in terms of growth factors, they have disadvantages such as the risk of pediatric patients not adapting to the blood collection process, additional equipment and reagents required to process PRP in the clinic, variability of composition, rapid growth factor reduction, insufficient mechanical strength, and difficulty in application [23,24]. In addition, studies are reporting that PRP and PRF do not show superior efficacy to blood clot in promoting root development [2]. Blood clot, on the other hand, is one of the most preferred tissue scaffolds due to their autologous delivery, low cost, and ease of clinical application [25,26]. It has been reported that the use of intracanal bleeding method in RET provides an optimal mixture of growth factors obtained from the patient's blood for pulp regeneration, as well as growth factors released from dentin in terms of density and quality [27]. Considering all these factors, RET with intracanal bleeding method was applied in our study. Despite all these advantages, it has been reported in many studies in the literature that Fig. 1 Photograps of a randomly selected tooth from all groups at T0, T1, T7, T30, and T90 time intervals Fig. 2 Comparison of the mean ΔE values of the groups for each treatment step with the graph tooth discoloration occurs in teeth that have been applied intracanal bleeding method as tissue scaffold [8,28].
It has been reported in various case reports and ex vivo studies that certain rates of discoloration of teeth occur with various CSCs used as a coronal barrier in RET procedures [29][30][31]. It is thought that the iron content of MTA, which is one of the CSCs widely used in RET, passes into the calcium aluminoferrite phase by oxidation and causes color change due to its penetration into the dentinal tubules [32]. Marciano et al. [33] reported that amino acids in dentin collagen interact with bismuth oxide in MTA, causing black discoloration in the tooth. However, Biodentine containing zirconium oxide as a radiopacifier has been shown to have better color stability. It has been reported that Biodentine does not show any color change under light and anaerobic conditions [30]. Biodentine's color change with blood contamination is minimal [34]. In addition, Biodentine has the advantage of completing a permanent restoration in a single session, as it has a shorter setting time compared to other CSCs [10]. Considering all these factors, Biodentine was preferred as the coronal barrier material in the current study.
To ensure the size standardization of the teeth, the apical third of all teeth were removed so that they had equal root length from the CEJ. To simulate the immature apex and standardize the apical diameter, the procedure indicated by Chen et al. [10] was applied.
No clinically detectable color change (ΔE < 3.3) occurred in any of the samples belonging to the negative control group at all time intervals measured in the current study. Chen et al. [10], evaluating the discoloration of CSCs in the presence of blood with RET, supported our findings and reported that ΔE was not at a clinically detectable level in the negative control group on the 180th day (ΔE = 1.71 ± 0.34, ΔE < 3.3). Fundaoglu et al. [15] also reported that the color change of different antibiotic pastes was measured by spectrophotometric analysis, and it occurred at a level lower than the detectability level in the control group (ΔE = 1.79). For this reason, we think that there is no additional discoloration of the teeth due to storage conditions. The group with the highest color change was in blood application. The discoloration of the blood-treated group gradually increased at all time intervals and the maximum color change was observed at day 90. Similar to the findings of our study, Yoldaş et al. [31] reported that the highest color change values were measured at the end of 1 year compared to the baseline of the blood-treated group in their study in which they evaluated the discoloration caused by various CSCs and blood. However, similar to our findings, it was observed that as the contact time of tooth tissue with blood increased, tooth discoloration increased. Madani et al. [35] also reported that the group with the most discoloration at the end of 6 months was the teeth in which only blood was applied, in a study in which they measured the color change of various CSCs in the presence of blood and without blood, and that the increase in coloration gradually increased in parallel with the findings in our study. One of the hypotheses explaining the highest color change values in the bloodtreated group can be explained by the use of a CSC that prevents the inflow of blood components [31]. A general trend observed for all teeth to which the materials were applied, except the blood-treated group and the negative control group, was similar to the study by Beatty et al. [36] in which they compared the color change of CSCs, with maximum color change immediately after treatment followed by a rebound on day 1. the effect was observed. Despite this rebound effect, clinically detectable discoloration was also observed in teeth treated with Biodentine at all time intervals in the current study.
Zızka et al. [4] reported that when CSCs are used for the coronal barrier, priority should be given to bismuth-free CSCs with lower coronal discoloration potential. However, he reported that CSCs without bismuth oxide also caused minimal color change when in contact with blood. This situation is explained by the theory that Biodentine may have blocked blood components more rapidly by setting them faster than other CSCs [10]. Oliveira et al. [37] also reported that coronal barrier materials with blood caused tooth discoloration. Shokouhinejad et al. [8] reported that in the presence of blood, ProRoot MTA, OrthoMTA, Biodentine, and Endosequence RRM occurred at a clinically detectable level (ΔE ≥ 3.3) in ΔE after 6 months. Similarly, Adl et al. (8) reported that clinically detectable color change was observed in the blood-treated group together with Biodentine. Although the exact mechanism of the color change potential of CSCs in the presence of blood is not yet fully understood, it is thought that the CSCs may be related to blood contact during the setting period [31].
In the literature, it has been reported in various studies that DBAs should be applied to occlude the dentinal tubules of the pulp chamber to prevent possible discoloration caused by RET [5]. Poly et al. [38] and Bezgin et al. [39] showed that DBA is a good option for reducing crown discoloration due to minocycline antibiotic paste in RET. Akbari et al. [11] suggested occlusion of dentinal tubules with DBA to prevent tooth discoloration due to MTA use. Kim et al. [40] and Shokouhinejad et al. [5] reported that DBAs reduced the coloration due to antibiotic paste, but could not completely prevent it. However, there are concerns about the toxic effect of DBAs on stem cells, especially in unpolymerized forms [41]. Therefore, the findings of our study could not be directly compared with the results of other studies in the literature.
It has been reported in the literature that the use of Nd:YAG and Er:YAG lasers at high power and exposure time can cause damage to both the hard tissues of the teeth and the surrounding tissues [18,42,43]. However, it has been reported in many studies that the laser parameters used for hypersensitivity do not damage the dental hard tissues and surrounding tissues during application [18,44]. Oclusion of dentinal tubules is a treatment approach that can be applied not only for hypersensitivity but also to prevent discoloration [45]. Considering this information in the present study, both Nd:YAG and Er:YAG lasers were used in hypersensitivity mode [19].
Fundaoglu et al. [12] evaluated the preventive effect of three different dentin tubule occlusion methods against discoloration caused by TAP, and reported that there was no significant difference in preventing discoloration between Teethmate, DBA, and Nd:YAG laser after 21 days. Clinically perceptible color change was observed in all three groups, supporting our study. In this study and our current study, the reason for the clinically perceptible discoloration in the teeth treated with Nd:YAG and Teethmate can be explained by the fact that they cannot occlude the dentinal tubules 100% and the plug they form can deteriorate over time.
Despite standardized requirements, the current in vitro study model has limitations in fully mimicking the clinical situation. In case of leakage at the restoration margins in clinical conditions, the interaction of the endodontic coronal barrier material with salivary components and bacteria may occur. In this case, it may lead to different coloration mechanisms in vivo [29]. A possible limitation of using human teeth is the expected natural discoloration of the teeth used. Only the hypersensitivity mode of the laser systems tested in this study was used. However, we think that the effectiveness of laser systems to prevent tooth discolorations needs to be investigated using different parameters.
To evaluate the effect of dentin tubule occlusive techniques in preventing discoloration, there is a need for more randomized controlled clinical studies with longer follow-ups, when different results may be obtained by examining the long-term efficacy.

Conclusions
In the current study, the clinically detectable color change was observed at all time intervals in groups except for the negative control. This showed that none of the dentin tubule occlusion methods used in our study could 100% prevent discoloration due to RET procedures. DBA and Teethmate, which do not have a significant difference in preventing color change, are considered to be suitable for primary use as dentinal tubule occlusive due to their ease of application and low cost compared to Nd:YAG and Er:YAG lasers. It was observed that the coloration increased as the contact time of tooth tissue with blood increased. It was determined that Biodentine used alone has a potential for discoloration.
Author contribution MOA and ZUA have made substantial contributions to conception and design; MOA and ZUA acquisition of data, analysis, and interpretation of data; MOA and ZUA have been involved in drafting the manuscript and revising it critically for important intellectual content; MOA and ZUA have given final approval of the version to be published.
Funding The costs of this study were covered by the researchers.

Data availability
The study materials and data can be requested in writing from the corresponding author.

Declarations
Ethical approval All procedures performed in studies were in accordance with the ethical standards of the institutional and/or national research committee. The ethics committee of the XXX University approved the study (Ethics No: 2020/165).

Conflict of interest
The authors declare no competing interests.