2.1.

Sample Preparation

All crowns were removed using a diamond wheel under water cooling and the length of the roots was standardized between 13 and 15 mm. A size 15 K file (Dentsply/Maillefer, Baillaigues, Switzerland) was inserted into the canal until visible at the apical foramen. The working length of each root canal was then established 1 mm short of the apical foramen. All samples were prepared using the ProTaper Universal rotary system (Dentsply/Maillefer) up to a F4 file together with 5 ml 2.5% sodium hypochlorite with each change of file. Next, the root canals were rinsed with 10 ml of EDTA-T (17% ethylenediaminetetraacetic acid in combination with 1.25% sodium lauryl ether sulfate solution) to remove the smear layer and debris, followed by a final rinse with 10 ml sterile water. All irrigating solutions were delivered with 30-gauge NaviTip needles (Ultradent Products Inc., South Jordan, Utah) and 3 ultrasonic activations of 20 s. The roots were kept moistened with sterile water in gauge during instrumentation.

2.2.

Experimental Groups

The samples were randomly divided into four groups of 42 teeth each ($n=42$), according to the treatment employed before obturation. The specimens in Group GØ received no laser treatment and served as control teeth. Teeth in Group Neodymium had the root canals irradiated with a 1064-nm Nd:YAG laser (Pulse Master 1000; American Dental Technologies, Southfield, Michigan) operated at 1.5 W, 100 mJ, 15 Hz, and $\mathrm{124,34}\mathrm{J}/{\mathrm{cm}}^2$, in pulsed mode.^9,11,16 Group diode was irradiated with an 808-nm diode laser (Zap Softlase; Zap Lasers Ltd., Pleasant Hill, California) with a setting of 2.5 W in continuous mode.^5,6,19,20 In the fourth group (Erbium), the root canals were irradiated with a 2940 nm Er:YAG laser (KavoKey laser 2; KaVo Co., Biberach, Germany) operated at 1 W, 100 mJ, 10 Hz, and $\mathrm{38,03}\mathrm{J}/{\mathrm{cm}}^2$ in pulsed mode.^12,13,18 All laser treatments were carried out according to the manufacturer’s instructions. The roots treated with a laser contained sterile water and were irradiated four times with circular movements, from apex to crown at a speed of $2\mathrm{mm}/\mathrm{s}$ with a 20 s interval between applications. For that, a $300\text{-}\mu \mathrm{m}$ fiber optic tip was inserted into the root canal until it reached the working length. At this moment, the laser was activated, and the irradiation was performed as described. All power settings were checked using a power meter (model 841P, Newport Corp., Irvine, California) before and after each irradiation.

2.3.

Scanning Electron Microscope Observation of Surface Morphology

Two samples of each group were randomly selected to analyze the surface micromorphology after the previous procedures. The roots were split in a bucco-lingual direction and the sections flushed with 20 ml of sterile water. They were then fixed in 2.5% glutaraldehyde, dehydrated in a series of graded ethanol solutions (30%, 50%, 70%, 80%, 90%, and 100%), and then coated with a gold 15 nm layer using a sputter coater (MED 020; Bal-Tec AG, Liechtenstein). The dentin surface of the apical third, 4 mm from the apex and in a central region was examined with a scanning electron microscope (SEM) at $2500\times$ magnification operating at 15 kV (Quanta 600 FEG; FEI Company, Eindhoven, The Netherlands).

2.4.

Root Canal Obturation

The remaining teeth ($n=40/\text{group}$) were randomly divided into four subgroups ($n=10$), according to the root canal sealer used for obturation. Prior to obturation, all teeth were externally coated with two layers of nail varnish, except for the last 1 mm at the apical third. One operator (CMN) filled the root canals of all groups using a single cone technique. A standard-size taper. 04 cone was fitted to the working length. In the AH Plus (Dentsply/deTrey) subgroups, a gutta-percha cone (Dentsply/deTrey) was used. The EndoREZ (Ultradent Products Inc.) subgroups used EndoREZ points (Ultradent Products Inc.), a gutta-percha point covered with a thin layer (approximately $15\mu \mathrm{m}$) of UDMA resin. The Epiphany and Epiphany SE (SybronEndo Corp., Orange, California) subgroups used Resilon cones (SybronEndo Corp.), a synthetic polymer-based resin (polycaprolactone). The sealers were mixed according to the manufacturer’s instructions and placed in a Skini syringe (Ultradent Products Inc.) attached to a 27G needle to fill the canal. Because Epiphany is not a self etch sealer, it was necessary to apply a layer of Epiphany primer in the root canal walls before the resin filling. For that, a ProTaper F4 sterile paper point moistened with the primer was used, brushing all root canal surfaces.

Before obturation, the teeth from the AH Plus subgroups were completely dried with suction using 30G NaviTip needle (attached to a air-vacuum suction device) and ProTaper F4 sterile paper points, while the roots from the other groups were lightly dried using suction with 30 G NaviTip needle only, to maintain a moist dentin condition. The canals were filled with the sealers and the master cone gently seated to the working length. Excess material was removed using a hot plugger followed by cold vertical condensation. After obturation, the apical foramen was covered with a thin plastic film to avoid oxygen contact and all the samples were stored in an incubator for 72 h at 37°C and 100% humidity.

2.5.

Silver Nitrate Penetration Assay

Upon completion of the root canal filling procedures, the teeth were immersed into 50% ammoniacal silver nitrate solution (pH 9.5) for 24 h, according to a previously published methodology.^6,21,22 This assay aims to indirectly measure the quality of the dentin tubule sealing after obturation of the root canal. The samples were washed in sterile water and embedded individually in acrylic blocks. The roots were cut in a longitudinal direction using a 0.3-mm-thick diamond blade (Isomet 1000 Precision Saw, Buehler Ltd., Illinois), operated at 200 rpm and 400 g of load, under running water. The cut root sections were then polished with 600 and 1200 grit abrasive papers (3 M St. Paul, Minnesota) using an Ecomet 3 polisher (Buehler) and ultrasonicated with sterile water for 1 min. The blocks were immersed in photo developing solution (Kodak Bras. Ind. Com., São Paulo, Brazil) for 8 h under fluorescent light to reduce silver ions into metallic grains within voids along the interface root surface/filling material. The samples were then scanned with a high-optical resolution scanner (24,000 dpi–CX7300, Epson, São Paulo, Brazil), and the penetration on each side of the sample was blindly measured (mean value) by one operator, with Image J Software (NIH, Bethesda).

2.6.

Environmental Scanning Electron Microscopy Analysis

The root sections were prepared for environmental scanning electron microscopy (ESEM) analysis in order to evaluate the adaptation of the filling material to the dentin walls as well as sealer penetration into the dentinal tubules. In order to visualize the morphology, the sectioned surfaces were conditioned with 37% phosphoric acid for 30 s and then rinsed with 10 ml of distilled water. Subsequently, the roots were immersed in 2.5% NaOCl solution for 10 min followed by a 10 ml rinse of distilled water according to Tay et al.²² The sample was kept hydrated and attached to a specific platform for ESEM analysis using a Quanta 600 FEG microscope (FEI Company, Eindhoven, The Netherlands). To standardize the analysis of the surface, the ESEM images ($1500\times$ magnification) were acquired in the same region as previously mentioned, 4 mm coronal to the apex examining the interface between filling material and dentin on both mesial and distal sides. Twenty ESEM images were acquired for each group. Three calibrated examiners did a blind evaluation of the adaptation of the filling material to the dentin wall and sealer penetration into the dentinal tubules (tag formation) using a four-grade score table for each parameter (Table 1). The criteria have been reported in previous studies.^23,24

Table 1

Adaptation to dentin walls and sealer penetration criteria.

2.7.

Statistical Analysis

For adaptation and sealer penetration, all data of each examiner were tabulated and a Kappa test was used to confirm inter-examiner agreement. Then, the scores were compared using the Kruskal-Wallis nonparametric test complemented by the Dunn’s test ($p<0.05$). For the silver nitrate penetration assay, the normality of the data was verified using the Kolmogorov-Smirnov test. After that, the groups were compared using an analysis of variance (ANOVA), complemented by the Tukey’s test ($p<0.05$).

3.1.

Scanning Electron Microscope Observation of Surface Morphology

SEM analysis of the control group [Fig. 1(a)] showed a dentin surface, free of smear layer with open dentin tubules. In Neodymium [Fig. 1(b)], an irregular surface with fusion of dentin and resolidification was observed and the absence of a smear layer and debris. There were few open dentin tubules while others were partially or completely occluded. The surfaces irradiated with the diode laser [diode group-Fig. 1(c)] also showed aspects of fusion and resolidification of the dentin wall with more open and partially occluded dentin tubules than in neodymium group.

Fig. 1

Composite scanning electron microscope (SEM) images of dentin. Control group with no surface changes (a); neodymium group (Nd:YAG laser) showing melting and resolidification of dentin surface (b); diode group (diode laser) also showing melting and resolidification but in a different pattern (c); erbium group (Er:YAG) showing ablation of the dentin surface (d).

In group erbium [Fig. 1(d)], the dentin surface appeared clean with some localized fusion of dentin and mostly opened dentinal tubules that demonstrated ablated intertubular dentin.

3.2.

Environmental Scanning Electron Microscopy Analysis

The scores for sealer adaptation to the dentin walls were mostly 3 and 4 (more than 50% of adaptation) irrespective of the laser irradiation or sealer used. The Kappa analysis showed a strong agreement between examiners ($K>0.7$). The Kruskal-Wallis’ test did not reveal statistically significant differences between groups ($p>0.05$).

The results of the sealer penetration into dentin tubules are presented in Table 2. Despite the surface changes caused by laser irradiation, all sealers tested penetrated into the dentinal tubules. The Kappa analysis showed a strong inter-examiner agreement ($K>0.75$). The Kruskal-Wallis’ test revealed significant differences between groups ($p<0.05$). The Dunn post-hoc test showed differences with regard to the sealers used. Table 2 shows the comparison between sealers in the same group and between groups in the same sealer. ESEM images in Fig. 2 illustrate the pattern of adaptation and tag formation of each sealer.

Table 2

Scores of sealer tag formation into dentinal tubules.

Fig. 2

Environmental scanning electron microscope (ESEM) images of resin tags from sealers. AH Plus (a) and EndoREZ (b) showing considerable tag formation; Epiphany (c) with more discrete tags; Epiphany SE (d) showing few tags.

3.3.

Silver Nitrate Penetration Assay

The penetration of tracer solution was observed in all groups. The means and standard deviations of penetration measurements are presented in Table 3. The ANOVA test demonstrated that the factors laser irradiation ($p=0.003$), sealer ($p<0.001$), and the interaction between them ($p<0.001$) significantly affected the silver nitrate penetration.

Table 3

Means and standard deviations (mm) of silver nitrate penetration.

Independently of the dentin treatment, the silver nitrate penetration results were similar in the teeth filled with Epiphany sealer. For all other sealers, the laser irradiation affected the outcome of the silver nitrate penetration. The use of the Nd:YAG and diode lasers had a positive effect on the AH Plus sealer penetration leading to a significant lower index of tracer penetration, when compared with the nonlased group. However, the irradiation with the Nd:YAG laser significantly increased the tracer penetration index in the roots filled with EndoREZ and Epiphany SE. The tracer penetration in the Er:YAG laser group in the teeth filled with Epiphany SE was significantly higher when compared with those filled with the other sealers. The surface irradiation with Er:YAG laser did not interfere in the outcome of the root canal fillings, independently of the sealer used when compared with the control group ($p>0.05$).