This in vitro, experimental study evaluated 180 single-rooted human teeth that had been extracted within the past 6 months due to hopeless periodontal prognosis or as part of orthodontic treatment. The study was approved by the ethics committee of AJA University of Medical Sciences (ethical approval code: 97000668). The study was performed in line with the principles of the Declaration of Helsinki. All methods were performed in accordance with the relevant guidelines and regulations of AJA university of medical sciences.
The inclusion criteria were single-rooted, single-canal human teeth extracted within the past 6 months with no internal or external root resorption, dilaceration, severe root curvature, root caries, or previous endodontic treatment. The teeth were selected using convenience sampling.
Sample size was calculated to be 180 teeth assuming the effect size of 0.3, 80% study power, and alpha = 0.05.
Periapical radiographs were obtained from the teeth in buccolingual and mesiodistal directions to ensure absence of dilaceration, severe root canal curvature, internal or external root resorption, caries, and previous endodontic treatment, and also to make sure that the teeth had one single canal. The teeth were immersed in 0.2% thymol solution for disinfection, and were then transferred to saline. The teeth were then immersed in 5.25% sodium hypochlorite (Chloraxid, Cerkamed, Poland) for 15 min to eliminate organic debris. The residual tissues were removed by a curette.
Access cavity preparation:
Standard access cavity was prepared in all teeth. A reference point was selected on the tooth crown, and a #10 K-file (Mani, Nakanishi Inc., Tokyo, Japan) was introduced into the root canal such that the file tip was visible at the apex at x10 magnification. Next, 16 mm was subtracted from this length, and the teeth were decoronated at the marked level by a diamond bur (Teeskavan, Iran) and high-speed handpiece under water spray. By doing so, the root canal length was standardized in all teeth (16 mm). One millimeter was subtracted from the 16 mm length to have 15 mm working length in all teeth.
Root canal instrumentation:
The root canals were instrumented by hand files (Mani, Nakanishi Inc., Tokyo, Japan) and ProTaper rotary files (Dentsply Maillefer, Ballaigues, Switzerland) with an electronic endodontic motor (Endo Mate DT, NSK, Japan) operating at 250 rpm and 3 Ncm torque. First, the root canals were instrumented with hand files up to #25 K-file to the working length, and then SX ProTaper rotary file was used for coronal root canal flaring followed by S1, S2, F1, F2 and F3 files to the working length. After using each file, a #10 K-file was passed through the apex to ensure patency. Each rotary file was used for preparation of 3 teeth. Passive root canal irrigation between files was performed with 10 mL of 5.25% sodium hypochlorite solution (Chloraxid, Cerkamed, Poland) using a plastic syringe and 27-gauge needle. After completion of root canal cleaning, the root canals were rinsed with 17% EDTA (Asia Chemi Teb Mfg, Tehran, Iran) followed by a final rinse with 2 mL of distilled water.
Root canal obturation:
After cleaning and shaping, the root canals were dried with paper points ((Dentsply Maillefer, Ballaigues, Switzerland). Next, the teeth were randomly divided into three groups for obturation using three different sealers (n = 54). Endoseal MTA was used in group A, nano-MTA was used in group B, and GuttaFlow Bioseal was used in group C. Nine teeth were considered as the positive control, and 9 other teeth were considered as the negative control group.
In Endoseal MTA and GuttaFlow Bioseal groups, the sealer was passively injected into the canal by a syringe, and then a size 30 Lentulo spiral was used to spread the sealer to the working length. In nano-MTA group, nano-MTA was prepared in paste-like consistency on a glass slab and delivered into the canal by a Lentulo spiral. The largest gutta-percha with 6% taper that reached the working length and had tug-back was used for single-cone obturation of root canals. The gutta-percha was dipped in sealer and inserted into the canal to the working length. It was then cut 1 mm below the orifice using a heat carrier and condensed by a plugger. In the positive control group, cleaning and shaping were performed as explained earlier, but the root canals were not filled. In the negative control group, cleaning and shaping and obturation of root canals (3 teeth from each sealer group) were performed as explained earlier, and then the access cavity and apical foramen were sealed with sticky wax.
After root canal obturation, the teeth were stored in an incubator for 1 week. Next, the teeth in each group were randomly divided into three subgroups (n = 18) and subjected to ambient atmospheric pressure (1 atm), 2 atm pressure (to simulate underwater diving), and 0.5 atm pressure (to simulate aviation). For this purpose, the teeth were subjected to the abovementioned pressures for 45 min within every 24 h, and were then placed back in ambient atmospheric pressure. This cycle was repeated for 1 month for each group.
Simulation of underwater diving and aviation conditions:
For this purpose, a chamber with a monometer was experimentally designed. A compressor was used to increase the pressure in the chamber and a vacuum pump was used to decrease the pressure. The speed of pressure change was 1 atm/min.
Prior to placement of the teeth in the chambers, they were wrapped in a moist gauze and were then placed in the chamber. Increasing the pressure to 2 atm simulated 10 m depth underwater, and 0.5 atm pressure simulated the pressure at 5.5 km height from the sea level. The control teeth, and other teeth (when not in the chamber) were stored in an incubator (Memment INC10, Germany) at 37 °C and 97% relative humidity during this time period.
Sealing of root surfaces:
The entire root surface was sealed with two layers of nail varnish except for the apical 2 mm and apical foramen, which remained exposed. This was done to prevent bacterial leakage through the accessory canals. In the positive control group, root surface was sealed as in the experimental groups while in the negative control group, the entire root surface and the apical foramen were coated with 2 layers of nail varnish.
Bacterial leakage model:
A bacterial leakage model consisting of an upper and a lower chamber was used for assessment of bacterial microleakage using Streptococcus mutans (S. mutans). The upper chamber included a 15-mm polycarbonate centrifuge tube with a small hole at one end. S. mutans in Wilkins-Chalgren culture medium was placed in this chamber and in contact with the coronal part of the roots. The teeth were placed in the tube from the apical end, and gently pressed until half of the root was inside the tube. It was then sealed with sticky wax; 4 mm of the root was placed in the upper chamber.
The lower chamber included a 15-mL tube, and the apical part of the root was placed in it. This chamber contained culture medium and phenol red indicator with 1% sucrose.
Sterilization of specimens:
The culture media were autoclave-sterilized at 121 °C for 20 min. After placement of the teeth in the chambers, they were sterilized with ethylene oxide for 12 h (Andersen sterilizer, Haw River, North California, 27258 − 9564. USA). To ensure sterility, all specimens were incubated at 37 °C for 3 days. During this time period, none of the samples showed any evidence of bacterial contamination.
Standard strain S. mutans (ATCC 35668) was obtained in lyophilized form from the Iranian Research Organization for Science and Technology, and cultured in 10 cc of Wilkins-Chalgren broth for 24 h. Every 24 h, 0.2 mL of balanced bacterial suspension was added to the upper chamber. Every 2 days, 9 cc of Wilkins-Chalgren broth was removed from the upper chamber and replaced with fresh broth. The valve of the centrifuge tube was used to prevent evaporation and contamination of medium.
The teeth were incubated at 37 °C for 30 days, and the change in color of pH indicator was evaluated every 5 days. Color change from red to yellow was considered as a positive result (acid production). To confirm the presence of bacterial colonies, blood agar culture medium was used. The purity of proliferated bacteria was evaluated by Gram-staining (Gram-positive chains of cocci) and the ability to proliferate on selective S. mutans culture medium i.e. mitis salivarius agar.
The mean and standard deviation of descriptive data were reported, and comparisons were performed using the Chi-square test via SPSS version 16 at 0.05 level of significance.