This investigation was conducted using forty extracted human teeth, caries-free, monoradicular, single-canaled with fully formed roots; teeth with caries were excluded to minimize the possibility of preoperative bacterial contamination of the root canal. No data were collected on age, sex, or reason for extraction.
The teeth were divided into two groups, an experimental group of thirty teeth (15 for each retrograde filling material to be evaluated: MTA and EndoSequence BC RRM-fast set putty) and a control group of ten teeth (5 for positive control and five for negative control).
The extracted teeth were stored in PBS (phosphate-buffered solution) to avoid dehydration. Remains of bone, calculus, or soft tissue were gently removed from the root surface of each tooth with a sterile curette. Subsequently, they were immersed in 5% sodium hypochlorite (NaClO) for approximately 15 minutes to remove organic debris from the root surface. Preoperative radiographs were taken of each tooth and were evaluated using an operating microscope (D.F. Vasconcellos, Brazil) at 20X magnification to ensure that there were no root caries or vertical fractures. The teeth were cut with a water-cooled diamond disk (D&N, Darmstadt, Germany) at the coronary level of the root, at an approximate root length of 14 mm, to standardize the working length of the samples.
The samples were randomly divided into two groups of fifteen teeth each and two control groups of five teeth each. For the root canal preparation, the working length was first determined using #15 files, K-Flexofile (Dentsply Maillefer SA, Ballaigues, Switzerland), which were placed inside the canal until the apex was slightly exceeded and 1 mm was subtracted from the previous measurement. Instrumentation was performed using the crown-down technique. The canal preparation in the coronal and middle third of the root was completed with Gates Glidden drills (Dentsply Maillefer SA, Ballaigues, Switzerland), sizes 3, 2, 1. The WaveOne Gold reciprocating, rotating system, Primary 025/0.06 (Dentsply Maillefer SA, Ballaigues, Switzerland), was used to continue the canal shaping process.
Instrumentation was performed under copious irrigation with 2.5% NaClO using a sterile syringe with a 27-gauge Endo-Eze irrigation needle (Ultradent products, INC, Spain). All canals received final irrigation with 3 ml of 17% EDTA followed by 3 ml of 2.5% NaClO to remove the smear layer and dried with Hygenic sterile absorbent paper points (Coltene/Whaledent inc., Germany).
Under continuous water irrigation, the apical 3 mm of each root was cut at 90 degrees to the long axis of the tooth, with a multiblade bur (Dentsply Maillefer SA, Ballaigues, Switzerland) on a high-speed handpiece. At the root-end, cavities were prepared with retrograde diamond-coated tips to a depth of 3 mm using a DTE ultrasound unit at a low power setting and water coolant. The teeth were steam sterilized for 30 min at 121°C. Before using the retrofilling material, the root-end was fitted with a red Machtou condenser (Dentsply Maillefer SA, Ballaigues, Switzerland) 3 mm anterior to the sectioned root-end, which was verified with a radiograph. At this point, the condenser provided an intercanal matrix against which the filling materials were compacted.
Experimental group
It comprised two subgroups corresponding to the retrofilling material to be used. All materials were prepared according to the manufacturer's instructions.
Subgroup #1: the cavities were retrofilled with MTA-Angelus (Angelus, Londrina, PR, Brazil), which was mixed in a 3:1 powder-to-water ratio, using sterile water, and incrementally placed in the root preparations using an MTA carrier. The material was then compacted using a Machtou straight condenser (Dentsply Maillefer SA, Ballaigues, Switzerland). The root surface was cleaned with moistened sterile cotton swabs and then coated with two layers of Valmy® nail varnish (Drocosca CA, Caracas, Venezuela) except for the 3mm in the apical portion containing the obturation material.
Subgroup #2
the cavities were retrofilled with EndoSequence BC RRM-fast set putty (Brasseler, Savannah, GA, USA), which comes in paste or putty form previously mixed in an injectable syringe. It was applied to the cavities using the B&L Jetip microsurgical instrument tip (JM2, B&L Biotech, Fairfax, VA, USA) and condensed with a Machtou straight condenser (Dentsply Maillefer SA, Ballaigues, Switzerland). For surface finishing, a microbrush (FGM, Joinville, SC, Brazil) was used on the BC RRM-fast set putty as recommended by the manufacturer. The root surface was coated with two layers of Valmy® nail varnish (Drocosca CA, Caracas, Venezuela), except for the 3 mm of the apical portion containing the obturation material.
Control group
The control group, in turn, comprised two subgroups of five teeth described below.
Positive control
the root-end cavities were left without retrofilling material.
Negative control
utility wax (Asfer, Caetano do Sul, SP, Brazil) was inserted to obturate the root-end cavity, and the entire root surface was coated with two layers of Valmy® nail varnish (Drocosca CA, Caracas, Venezuela), including the apical area containing utility wax.
After retrofilling the roots, the barrier placed inside the canal was removed, and radiographs were taken to verify the density and depth of the retrofilling material. The samples were stored in 100% humidity at 37°C for one week.
Sample preparation in the leakage system
The coronary part of each tooth was connected to the end of a 2.5 ml polyethylene tube (Nelaton Catheter; MD Products, Venezuela), using cyanoacrylate and sticky wax to prevent leakage from the connection. The root surface and the tubes were coated with two layers of Valmy® nail varnish (Drocosca CA, Caracas, Venezuela), except for the apical 2 mm of the roots. The five teeth used as negative controls were completely covered with two coats of nail varnish. All samples were sterilized using ethylene oxide gas for 12 hours. The polyethylene tube was attached to the screw cap of the sterile 30 ml glass bottle, in which 10 ml of brain heart infusion broth (lower chamber) was placed and maintained at 37°C in an oven for 48 hours, for quality control before placing the bacterial inoculum in the upper chamber. With a sterile Pasteur pipette, 2 ml of brain heart infusion medium with E. faecalis, at 0.5 McFarland turbidity (108 bacteria/ml), were pipetted. Root-ends were immersed in brain heart infusion broth containing one drop of 0.04% phenol red indicator solution. The complete set was maintained at 37°C, during the 30-day observation period. (Figure 1)
Bacterial leakage control
The leakage system used in the present study was that proposed by Barthel et al., [14] Chailertvanitkul et al., [15], and Torabinejad et al., [16]. The lower chamber was observed daily to determine bacterial leakage, which was evidenced when the culture medium changed from red (alkaline) to yellow (acidic) because of acid production due to bacterial growth, or by the turbidity of the medium; the time for this to occur indicated contamination of the entire root canal and then it was evaluated whether it was the same bacteria. For this purpose, a sample was taken and sown on Columbia blood agar base plates and incubated at 37°C for 48 hours for macroscopic evaluation by colony morphology; Gram staining was performed for microscopic evaluation to confirm the presence of E. faecalis. The bacterial count of each leaked sample was performed using the serial dilution technique. Replacement of the inoculum in the upper chamber was performed aseptically with Pasteur pipettes every seven days to ensure the viability of the bacteria at a density of 0.5 McFarland.
Protocol for the processing, quantification, and identification of microorganisms by serial dilutions
The brain heart infusion broth with the leaked culture was homogenized with agitation, and then serial dilutions (10−2 to 10−6) were performed in a sterile saline solution (0.9%). 10µl of the culture, from which the dilutions were made, were taken and plated on blood agar. The plates were incubated at 37 ºC for 24 hours.
For the quantification of microorganisms, plate colonies corresponding to the dilutions that presented between 30 and 300 colonies were counted, and the results were expressed in Colony- Forming Units per milliliter of sample (CFU/ml), according to the following formula:
Bacterial titer: No. of colonies x dilution factor/ml of culture plate
Data collection and processing technique
A data collection sheet was used; it included the sample number, the retrofilling material, date of inoculum placement, date of leakage, time in which the turbidity of the medium occurred, and verification of E. faecalis in the lower chamber. This information was obtained through daily observation of the specimens. The change in color or turbidity of the culture medium in the lower chamber, and the verification of E. faecalis through its morphological identification under the optical microscope and in the culture medium, showed microleakage of E. faecalis.
The tabulation of all sample information was collected in a sheet prepared for this purpose that included the day of leakage, the specimens that presented leakage, and the number of colony-forming units per milliliter of sample (CFU/ml) for each one.
Once gathered the information in the data collection instrument, a matrix was designed in Excel, in which it was primarily transcribed.
Data analysis technique
The information was entered into a database using SPSS statistical software version 21.0. The variables were processed and presented in tables.
Statistical analysis
Considering the number and type of variable, level of measurement in which the values of the variables are expressed, manner of choice or occurrence of the subjects or elements of the study, and the sample size, non-parametric tests were used. Fisher's Exact Test was used to verify the association between the presence or absence of leakage of the samples by type of bioceramic material used and the Mann-Whitney U test to verify the existence of a difference between the average of Colony-Forming Units by type of bioceramic material used.
To evaluate the statistical significance of the hypotheses of association and differences formulated, we used as Significance Level (α = 0.05) and a 95% Confidence Level and, as a decision rule for rejecting the null hypothesis (H0) formulated, when the p value associated with the contrast statistic resulted lower than the Significance Level set (α = 0.05), that is, when p < 0.05.