This ex-vivo, study evaluated 72 human single-rooted single-canal teeth that had been extracted due to poor periodontal prognosis or as part of orthodontic treatment. The ethics committee of AJA University of Medical Sciences approved the study protocol (IR.AJAUMS.REC.1398.255). The inclusion criteria were single-canal, single-rooted teeth with closed apices, canal curvature < 25°, absence of calcification, internal resorption, and root caries. In addition, Mesiodistal and buccolingual periapical radiographs were obtained to ensure that the teeth met the inclusion criteria. The sample size was calculated assuming alpha=0.05, beta=0.2, and study power of 80%, and the teeth were selected using convenience sampling. To standardize the root length, all teeth were decoronated by a high-speed diamond disc such that the remaining root length was 15 mm in all teeth. Next, a #10 K-file was introduced into the canal until the file tip was visible at the apex to determine the working length. The file length was then measured, and 1 mm was subtracted from this length to determine the working length. Teeth in which a #10 K-file could not easily reach the apical foramen, those with an apical size larger than a #20 K-file, and those without a round cross-section were excluded and replaced. The root canals were then instrumented with ProTaper rotary files (Dentsply Maillefer, Ballaigues, Switzerland) up to F2 at a speed of 300 rpm and 1.5 Ncm torque. After instrumentation, the root canals were irrigated with 5.25% sodium hypochlorite using a 5 ml syringe with a 23-gauge needle. After root canal preparation, each tooth was packed and autoclave-sterilized at 121°C for 20 min. Next, 5 teeth were randomly selected for negative control and incubated in brain heart infusion (BHI) broth (Conda Laboratories, Madrid, Spain) at 37°C for 48 h.

1. faecalis (ATCC29212) was obtained from the Iranian Pasteur Institute in lyophilized form. BHI broth was added to it such that it completely covered the surface of the solid powder. It was then incubated at 37°C for 15 min to create a microbial suspension. The prepared microbial suspension was transferred to a solid culture medium by a sterile syringe. After bacterial culture on BHI agar plate (Conda Laboratories, Madrid, Spain) and 24 h of incubation, safranin staining was performed and the culture was inspected under a light microscope at x100 magnification to ensure the correct type of microorganism, and no contamination of culture medium. For bacterial culture in the liquid medium, one independently grown colony was removed from the plate by a sterile loop under aseptic conditions and transferred into a test tube containing 3 mL of BHI broth. The test tube was then incubated at 37°C for 24 h. In order to standardize the microbial suspension used for inoculation, sterile BHI broth was transferred to the test tube containing bacteria and the turbidity was adjusted at 0.5 McFarland standard concentration. The obtained standard microbial suspension was transferred into 72 test tubes under aseptic conditions such that each test tube contained 1 mL of the suspension. Next, each root was placed in one test tube with a sterile hemostat. The test tubes were incubated at 37°C for 2 days. Afterward, 1 mL of sterile BHI broth was added to each test tube by a sterile syringe under aseptic conditions. This process was repeated for one week. To ensure no contamination with other microorganisms during this period, a few drops of the contents of two randomly selected test tubes were collected by a sterile loop and Gram-stained.

The apical 3 mm of a ProTaper F2 rotary file (Dentsply Maillefer, Ballaigues, Switzerland) was cut by a diamond bur  and high-speed hand-piece. Next, a small notch was created at 4 mm from the tip of the remaining file segment to weaken the file using a knife-edge bur. The files were then autoclave-sterilized at 121°C for 20 min. After 1 week of incubation, each tooth was removed from the test tube with a sterile hemostat under aseptic conditions. The sterilized file was introduced into the root canal and rotated until the apical 4 mm segment broke at the notch. To further aggravate the canal obstruction by the fractured file, a small amount of sterile dentin chips (collected by using a #4 Gates-Glidden drill in the canal of a sterile tooth) was added to each root canal. A radiograph was obtained from each tooth to ensure instrument fracture at the desired location (apical 3 mm). Afterward, the teeth were randomly divided into 6 groups (n=12) according to the type of intervention to be performed:

G1: 2% CHX + electrophoresis

G2: Cupral paste + electrophoresis

G3: Electrophoresis alone

G4: 2% CHX alone

G5: Cupral paste alone

G6: Positive control group

In electrophoresis groups (G1, G2 and G3), alginate (Chromogel, Iran) was used as a periodontal tissue substitute. For this purpose, alginate powder was mixed with water and transferred into 5 ml Eppendorf plastic microtubes. The roots were mounted in the tubes such that their coronal 2 mm was out of the alginate.

In G1, 2% CHX (Cerkamed, Gluco) was injected into the canal through the orifice by a sterile syringe such that it did not overflow after insertion of the cathode end of the depot phoresis (Humanchemie GmbH, Germany) in the root canal filled with CHX. Next, the tooth was mounted in a microtube containing alginate. The anode end of the device was placed in the alginate next to the root without contacting it. The electric current was then started (0.9-1.2 mA) and it was automatically discontinued when it reached 7.5 mA min. and incubated at 37°C under humid conditions.

In G2, a small amount of distilled water was added to Cupral paste (Humanchemie GmbH, Germany) to reach a creamy consistency. The paste was then delivered into the canal by a #20 hand file with counter-clockwise motion up to the orifice. The roots were then mounted in microtubes containing alginate. The rest of the procedure was the same as that in G1. In G3, 20 µL of sterile saline was used in each root canal to enhance electric conduction. The rest of the procedure was the same as that in G1. In G4, 2% CHX was used alone. In G5, Cupral paste was used alone as in G2. In G6, no intervention was performed, and this group served as the positive control. The roots in all 6 groups were then removed from the microtubes and incubated at 37°C under humid conditions for 1 week. After 1 week of incubation, the interventions were repeated and the roots were incubated again at 37°C under humid conditions for 1 more week.

Finally, the antimicrobial efficacy of the interventions was evaluated by counting the colony-forming units (CFUs). For this purpose, the apical 3 mm of the roots was cut under aseptic conditions, and the root canal of the separated segment was filled with sterile BHI broth. A sterile #20 paper point was placed inside the canal with a sterile hemostat. After 1 min, the upper part of the paper point which was out of the canal was cut, and the remaining segment was transferred into a test tube containing 1 mL of sterile BHI broth with a sterile hemostat. The test tube was shaken (in order to separate the bacteria from the paper point), and it was then incubated at 37°C for 2 h. Next, 20 µL of the contents of each tube was transferred onto a culture plate containing BHI agar with a disposable insulin syringe under aseptic conditions. The plates were incubated at 37°C for 24 h. The number of CFUs was counted after incubation.

Data were analyzed using SPSS version 25 (SPSS Inc., IL, USA). The mean and standard deviation of colony count were reported for each group. One-way ANOVA was used for the general comparison of the groups, while the Tukey’s test was applied for pairwise comparisons. A P<0.05 was considered statistically significant.