The effect of smear layer removal on E. faecalis leakage and bond strength of four resin-based root canal sealers

Two methacrylate resin-based sealers Endo Rez (ER, Ultradent Products Inc., South Jordan, UT) and Epiphany/ Resilon (RS, SybronEndo, Orange, CA) and two epoxy-based sealers AH Plus (AH, Dentsply/ DeTrey, Konstanz, Germany) and MTA Fill Apex (MTAF) (Angelus Soluções Odontológicas, Londrina, PR, Brazil) were used in this study.

A total of 230 human maxillary central incisors extracted for reason not related to this study were used. Periapical radiographs (Bel-Ray II AC, Belmont Equipment, Somerset, NJ, USA) were taken in mesio-distal and bucco-lingual planes to exclude severe root canal calcification, apical curvatures, or any resorptive alteration of the canal lumen. All the teeth were subject to surface disinfection by immersion in 0.5% Chloramine T, followed by removal of all adhering soft tissues and debris by scaling, washed under running tap water, placed in distilled water, and refrigerated at 4C° for 24 hours before use.

The crowns of 130 teeth were removed with a diamond bur in a high-speed hand piece under water-cooling, leaving 10mm of the root segment. All roots were inspected for the presence of cracks with a stereomicroscope under x40 magnification. ProTaper Universal NiTi rotary files (Dentsply/Maillefer, Ballaigues, Switzerland) were used to prepare each root canal to size #50 and stainless steel K-files (Dentsply/Maillefer) to finish preparation up to size #60. The root canals were irrigated with 2.5% 3ml NaOCl after the use of each file. All the roots were randomly divided into two groups. Half of instrumented roots were rinsed with 5ml of 17% EDTA for 5 min to remove the smear layer [12, 13]. Distilled water was finally used to rinse the roots thoroughly. All the roots were autoclaved at 121+/- 2°C for 20 min. After sterilization, the root canals were dried with sterile paper points (Dentsply/Maillefer). All the roots with and without the smear layer were assigned to eight experimental subgroups (n=15) and two control groups (n=5) as shown in Table 1. All the tested sealers were mixed according to manufacturer’s instructions and applied into the root canals on the master gutta-percha (GP) cone size #60 (Dentsply/Maillefer) or Resilon cones (SybronEndo), respectively. The canals were obturated with the lateral condensation method. Additional GP cones size A (Dentsply/Maillefer) were placed after lateral compression with same size spreader until the cervical part of the root was filled. Eventually excess GP from the coronal part was removed with a heated hand plugger and condensed vertically. Aseptic techniques were employed throughout the procedure. The specimens were kept in sealed tubes with sterile water at 37°C for 7 days to allow the sealers to set. The positive controls prepared and rinsed with EDTA as described above were obturated without a sealer (core material only), simulating poor obturation.

The two-chamber microleakage device [8, 14] was used with minor modifications. The specimens were inserted through a cut tip of 15ml polyethylene tubes (upper chambers), leaving 3mm of the cervical part inside the tube and the remaining part hanging out of it. Melted sticky wax was first applied on the outer surface of the 3mm cervical part, leaving the surface with the canal orifice exposed in the experimental and positive control groups, but fully covering it in the negative control group. Thereafter the wax was applied on the hanging part of the root and the tube interface, leaving 3mm of the apical part uncovered similarly in all the groups. These mounts were then tightly sealed with sticky wax to sterile 50 ml polyethylene tubes (lower chambers) containing 8 ml of sterile Trypticase Soy Broth (TSB; Oxoid Ltd, Basingstoke, UK). The apices extruding from the upper chambers were hanging vertically 2mm in the broth.

On observation of turbidity, the seal was broken and the bacterial cultures were then streaked on Trypticase Soy Agar (TSA; EMD Millipore Corporation, Billerica, MA, USA) plates for colony morphology observation and PCR identification. The bacteria growing on 25 plates, randomly chosen from the eight experimental subgroups, were identified by PCR assay with species-specific primers targeting E. faecalis 16S rRNA [15]. The date of leakage was recorded for each leaking sample.

The method previously described by Jessop [16] was used with minor modifications for the preparation and testing of the samples.

One hundred teeth were decoronated at the cement-enamel junction using a slow speed diamond-watering blade (Ernst Leitz GmbH, Wetzlar, Germany), split longitudinally in the bucco-lingual direction and inspected for presence of cracks with a stereomicroscope under x40 magnification, grounded on a water-irrigated grinding wheel (Struers LaboPol-21) until smooth and flat surface, using 2000-grit (FEPA) silicon carbide paper and cut into three parts: cervical, middle, and apical. Each specimen was fixed in the acrylic resin (Heraeus Kulzer Dental GmbH, Laboratory Products Division, Hanau, Germany). All the roots were divided into two groups according to the final exposed dentin surface treatment. In group A, the smear layer was removed by rinsing each specimen with 3ml 2.5%NaOCl solution for 1min, followed by 3ml 17%EDTA for 1min. Group B specimens were irrigated for 1min only with 3ml 2.5% NaOCl.

Each group was divided into five subgroups: i) AH was injected into the plastic mould, with 2.4 mm diameter and 2mm cylindrical button height (Ultradent Products Inc. South Jordan, UT, USA) on the root dentin bonding surface, ii) RS was similarly applied and light-cured for 40 sec according to manufacturer’s instructions, iii) MTAF and iv) ER were applied as AH, v) composite specimens were prepared according to manufacturer’s instructions by applying 3M ESPE Scotchbond Etchant, Adhesive and Primer (3M Dental Products, St. Paul, MN, USA) on the dentin specimens. Finally, the composite 3M ESPE Filtec Supreme XP was packed through the mould on the bonding surface and light-cured for 20 sec. All the samples were left for 8 hours in a water bath at the room temperature and the jig was removed. Afterwards they were placed in an incubator at 37°C and 100% humidity for 7 days. For comparison, every third crown was used for testing the dentin bonding to simulate a restorative procedure in a class III standard cavity (1.5 mm deep).

The specimens were tested using a notched-edge shear test fixture (Crosshead, Ultradent Products Inc.) on a universal testing machine (Lloyd LRX; Lloyd Instruments, Fareham Hants, UK) and the results expressed in MPa by diving the force needed to break the bond (N) by the surface area in mm². Failure modes obtained by the shear-bond testing were reported and a mean and standard deviation calculated.

In the leakage assay, the Kaplan Meier test for survival analysis was used. The median time of leakage was calculated and pairwise comparisons of groups were performed by using the log-rank test. Bond strength between the groups was analysed using the Two-way ANOVA with Tukey’s post hoc test. Data were entered and analysed by the statistical program package IBM SPSS statistics 21.0 (IBM, Somers, New York, NY, USA).

All the cell colonies on TSA plates appeared as small, smooth, cream or white colonies with entire edges and were identical to those of E. faecalis ATCC 29212. The PCR assay revealed 23 samples showing one band at the same size as 137 bp of positive control of E. faecalis ATCC 29212 and two samples showing one band a bit lower than the positive control (Fig. 1). Further DNA sequencing confirmed that these two strains were also E. faecalis ATCC 29212 having 99-100% sequence identity.

All the samples leaked within 41 days (range 6-41 days), except the negative controls (N=5) which remained without leakage throughout the 50 days test period. All the positive controls (N=5) samples leaked on the second day (Table 2).

The Kaplan-Meir survival curves showed that the AH-ns group significantly differed from all the other groups (p<0.001; Mantel-Cox Chi-square analysis) (Fig. 2).

All the tested sealer materials showed an increase in bond strength when going from the apical region towards the crown. Bonding to the crown dentin after the removal of the smear layer with composite resin, used as a reference material, showed the highest strength values, 13.7±1.5 MPa (Table 3). The strength values of the sealers bonded to the root canal wall dentine ranged from 0.2± 0.1 to 3.5± 0.7 MPa, depending on the material, location or removal of the smear layer with EDTA (Table 3). The removal of the smear layer increased the bond strength of all the sealers in all thirds of the root, except in the AH group in the cervical and middle third of the root and in the MTAF group in the cervical third, but none of the differences were statistically significant when tested by Tukey’s pairwise comparisons (P>0.05) (Table 3). In all the tested root dentin sites, the composite resin used for the reference showed significantly higher (P<0.001) bonding values (range from 3.1±0.5 to 10.8±1.9 MPa) compared to any of the sealers, irrespective of the site of the tooth or the smear layer removal (Table 3). In the apical third, AH showed highest bond strength from all the tested sealers, and differed significantly from all other sealers groups (AH-ns vs. RS-ns p=0.001, Tukey’s pairwise comparisons), regardless of the smear layer removal (Table 3).

There was an obvious overall trend that higher bond strength values resulted in less bacterial leakage (Fig. 3). In the apical third, the removal of the smear layer increased bond strength within all the materials tested, but had a favourable effect on bacterial sealability with two materials only, AH and MTAF.