Sample Selection and Preparation
The study design was approved by the XX University Clinical Research Ethics Committee (Ethics No: XX). 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 and five permanent maxillary incisors with single root and single canal, extracted for periodontal, orthodontic, and prosthetic reasons, were included in the present study. 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 were 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 minutes 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, 3M ESPE, Germany) was prepared around it.
Division Of Samples Into Experimental Groups And Application Of The Dentin Tubule Occlusion Procedures
The samples were numbered and placed in Eppendorf tubes filled with distilled water. All samples were then randomly divided into 7 experimental groups (n = 15):
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 minutes. 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) 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 seconds. 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 minutes.
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 seconds with the help of a micro applicator. The teeth were gently air-dried for 5 seconds. For polymerization, it was cured with a light-cure device (Monitex BlueLex GT) with a light power of 1200 mW/cm² for 20 seconds [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 minutes was expected.
Teethmate
After applying Teethmate desensitizer (Kuraray) 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-second 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 minutes 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 seconds without using air/water [18]. 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 minutes 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 seconds 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]. 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 minutes 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. 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:
ΔE*= ((ΔL*)²+(Δa*)²+(Δb*)²)½
ΔL*=L1*-L0*; Δa*=a1*-a0*; Δb*=b1*-b0*
If the ΔE value was 3.3 ≥, it was accepted that there was a visible color change [5].
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%.