Introduction
Proper endodontics treatment with knowledge of the morphology of the root canal includes cleaning and shaping and finally correct filling using a suitable sealer (1).The main purpose of filling the root canal is to create a complete flood to prevent contamination and re-infection due to leakage of liquids and microorganisms into the root canal (2).The most common root canal filling material is gutta-percha, which is used in different ways, including lateral compression, vertical compression, and thermoplastic(3).
The use of sealer is very important for long-term sealing of the root canal, because it adheres to the gutta-percha and the dentin of the root canal and fills the irregularities and spaces between the gutta-percha and the walls of the root canal (4). Root canal sealers are effective in completely filling the root canal by reducing apical and coronal microleakage (5). In all root canal filling methods, the dentist must use a type of sealer to improve the filling quality, which depends on the sealing ability of the used sealer(6).
Currently, there are various types of sealers, including zinc oxide, eugenol, calcium hydroxide, glass ionomer, silicone, resin, and bioceramic sealers (7).Recently, bioceramic materials have become one of the most famous biological materials used in endodontics after the clinical success of MTA. Bioceramic sealers have various advantages such as low cytotoxicity, excellent antimicrobial activity due to high pH, ​​stimulation of hard tissue formation and formation of hydroxyapatite layer (8).Endoseal MTA is a type of bioceramic sealer that has been introduced recently and contains a pre-mixed material that is kept in a syringe under vacuum conditions and allows its direct use inside the root canals (7). This sealer consists of calcium silicate, calcium aluminate, calcium aluminoferrite and calcium sulfate(9). During setting, the moisture is absorbed by the sealer and it sets slowly without any mixing(10).
Among some desirable mechanical and biological properties of Endoseal MTA, we can mention biocompatibility, greater resistance to washing, and bioactivity(11). On the other hand, this sealer has disadvantages such as color change due to the release of iron ions, long stinging, short working time and hard work. Also, there is no known solvent for MTA; Therefore, it is difficult to remove it from the root canal (7).
On the other hand, epoxy resin-based sealers are used in the root of the tooth as a standard gold, and with changes to the original formula, they are widely used to fill the root canal (3). Resin sealers have a greater penetration depth than ordinary sealers (12). Due to their low layer thickness, these sealers have deeper penetration and good fluidity. Also, resin sealers have high bond strength to dentin (13).
As one of the resin sealers, AH26 has good sealing power and adhesive properties and antibacterial properties (14). It has been confirmed that AH26 releases formaldehyde when it sets. These sealers do not contain formaldehyde in their chemical composition, but during the chemical reactions that occur when they are set, formaldehyde is produced and released, which is an effective substance against bacteria (15). It has been reported that AH26 sealer can maintain its antibacterial property from 24 hours to 7 days (16).
Due to the fact that Endoseal MTA is one of the newer sealers and sufficient studies were not found in the field of comparing the performance of this sealer with standard gold sealers, this study considered the bacterial microleakage of two sealers, AH26 and Endoseal MTA, with two methods of single cone filling and lateral compression.

Methods
In order to carry out this laboratory study (in vitro), according to the formula for determining the sample size, taking into account the α error of 0.05 and the β error of 0.20 and the results of previous similar studies, the minimum number of samples in each group was determined to be 14.56 extracted single-rooted human teeth, which were matched in terms of tooth type, were collected from clinics and dental offices in Mazandaran.Single-root and single-canal teeth with developed apex, without external analysis, without cracks on the root surface, without caries and without additional canal were included in the study. This project was implemented with the code of ethics IR.MUBABOL.REC.1400.177.In order to remove mass and soft tissue on the root surface, all teeth were mechanically cleaned with periodontal cort and placed in 5.25% sodium hypochlorite solution for one hour and placed in distilled water until use.
In order to homogenize the samples, the crown of the teeth was cut using a turbine and a diamond bur, so that 12 mm of each tooth remained. In order to remove the variable related to the operator, all preparation steps were done by one person.
Preparation of teeth
k-file (Mani inc, Tochigi, Japan) number 15 was used to determine the working length. In this way, the file was placed in the canal until the tip of the file was seen in the apical foramen. Then the working length was determined to be 1 mm less than this length. To prepare the root canal of the teeth, Sx to F3 nickel titanium (Ni-Ti) rotary files from the Protaper gold system (Dentsply Maillefer, Ballaigues, Switzerland) were used. To maintain patency in the filing intervals between each step and the next step, the canal was washed using 1 ml of 25.5% sodium hypochlorite and 5 ml of sterile physiological serum 0.9%, and then a manual file No. 10 was inserted along the length of the canal to remove debris. come out from the end of the apex of the root.
After preparing the canals, the teeth were sterilized by UV rays (4) and after the sterilization process in all four experimental groups of root canals, they were dried using a paper cone (Ariadent, Iran). Then the teeth were randomly divided into four groups:
1. Group one: 14 teeth filled with gutta-percha and AH26 sealer by single-cone method.
2. Group two: 14 teeth filled with gutta percha and AH26 sealer by lateral compression method.
3. Group three: 14 teeth that were filled with gutta percha and Endoseal MTA sealer by single-cone method.
4. Group four: 14 teeth that were filled with gutta percha and Endoseal MTA sealer by lateral compression method.
In each group, one tooth was placed as a positive control and one tooth as a negative control. Positive and negative control were considered as follows:
- Negative control: teeth that were prepared and filled with AH26 sealer by lateral compression method, but were not exposed to bacteria.
- Positive control: a tooth that was prepared but not filled.
AH26 sealer was stirred on a glass slab until a homogeneous consistency was obtained and it was applied to the canal wall using Maillefer (Dentsply, Switzerland) spreader No. In the single-cone method, an F3 (Meta biomed, Korea) along with the pumping movement was placed inside the canal, in such a way that the excess sealer was removed from the canal opening.
Then the additions of the sealer were removed and the gutta was cut and compacted. Also, in the lateral compression method, the original gutta-percha with a tipper of 2% (Meta biomed, Korea) was selected as MAC according to the last apical file used, that is, gutta-percha No. 30 or larger than the one that had a tug back function during a brief operation. The largest finger spreader that reached 1 mm length in the presence of MAC
was selected and secondary gutta-percha (Ariadent, Iran) was used for filling by lateral compression method. Gutta-percha sub-cones were used until the spreader penetrated more than 2 mm beyond the canal entrance. After the filling was completed, a heat carrier was used to cut the gutta-percha and remove the excess gutta-percha. Then the gutta-percha was compacted with Dentsply, Maillefer plugger (Switzerland). After completing the filling, radiographs were prepared from the samples to evaluate the filling quality. Then, all the teeth in the experimental groups were kept in 100% humidity and 37°C temperature for 1 week until the sealer set.
Preparation of samples to check microleakage
The outer surface of all the teeth of the experimental groups except for 1 mm of the apical end was covered with two layers of nail polish (Arcancil, France). In the negative control teeth, the entire external surface of the root, including the apical foramen and the canal entrance, was covered with two layers of nail polish. In the positive control teeth, the entire surface of the tooth was varnished except for the apical and coronal 1 mm. The separate chamber model was used to investigate bacterial leakage using bacterial suspension; Thus, the upper chamber containing the coronal third of the root was in contact with the bacterial suspension and the lower chamber containing the apical two thirds of the root was placed in contact with the TSB culture medium. Micropipettes with a volume of 0.5 cc with an inner diameter of 7 mm and a length of 30 mm were used to create the upper chamber in the coronal part of each tooth.
The ends of the micropipettes were cut and the coronal third of the root was placed inside the cut end of the micropipette. The junction between the micropipette and the root was sealed with cyanoacrylate glue. A test vial was used to create the lower chamber in the apical part of the root. The dimensions of these vials were 50 mm (height) x 10 mm (diameter) and had a volume capacity of 2 cc and had a plastic lid. First, the lid of these vials was removed, and using an electric drill and a drill, a hole with a diameter of 7 mm (the size of a micropipette) was created in the middle of the vial lid. Then the micropipette was passed through the created hole and the joint of the micropipette and the cap of the test vial was completely sealed with glue. Finally, the cap containing the micropipette and the tooth root were placed on the vial. The samples were sterilized by UV rays (4).(Figure 1).
After sterilization, the samples were removed from the cover and after opening the cap of the vial, sterile liquid culture medium Tryptic Soy Broth (TSB) (Merk, Germany) was poured into the lower chamber using a sterile 22-gauge syringe and needle. Be in contact with the apical 2 mm of the root that was not covered with nail polish. To prepare liquid TSB culture medium, according to the instructions of the manufacturer, 30 grams of its powder was dissolved in 1000 cc of distilled water, then poured into a sterile Erlen (Razi, Iran) and autoclaved (temperature 121 15 minutes) was sterilized.
After performing this step, all the samples were kept in an incubator at 37°C for 72 hours to ensure the sterility of the set; In this way, the absence of turbidity in the culture medium indicated the absence of contamination of the samples.
Bacterial leakage
Enterococcus faecalis (ATCC29212) was used to obtain the bacterial suspension. A suspension containing 109 bacteria was prepared from Enterococcus faecalis in TSB culture medium and 0.3 ml of the above suspension was injected into the upper chamber using a sterile syringe and needle. Then, these collections were incubated at 37°C for a maximum of 90 days and checked every day for the presence of turbidity in the TSB culture medium (lower compartment).  The turbidity of the culture medium indicated microbial leakage along the root canal into the underlying culture medium. In each sample, the time it took (in days) for turbidity to appear in the bottom culture medium was recorded. In addition, the bacterial solution was changed every 3 days to ensure the presence of live and active microorganisms. After observing the turbidity of the culture medium, one sample was randomly selected from among the samples with cloudy culture medium in each group. Then, observing the sterile conditions and in the vicinity of the flame under the hood, the vials were opened and samples were taken by a sterile analyzer (Razi, Iran).

Figure 1. How to design compartments

The removed sample was cultured in blood agar solid culture medium (Difco, Germany) to check the presence of microorganisms and possible contamination. The blood agar medium was incubated at 37°C for 48 hours. Then the grown colonies were sampled and warm staining was done. The stained samples were examined under the microscope with 100x magnification (Figure 2).

 Figure 2. Examining stained bacteria under a microscope with 100x magnification

Finally, the data was entered into SPSS software version 22 and Kruskal-Wallis, chi-square and Kaplan-Meier statistical analysis by log-rank test (mantel-cox) were used to analyze the data. P-value < 0.05 was considered statistically significant.

In the present study, positive and negative control samples were used in each group to check the correct design of the sample model for testing. The negative control sample using cyanoacrylate varnish and glue showed that the method of sealing the gaps between different compartments was effective and there was no possibility of bacterial penetration. On the other hand, the positive control sample showed that the penetration of bacteria from the upper compartment through the root canal to the culture medium of the lower compartment was possible.