This Experimental in vitro study evaluated 81 single-canal human teeth that had been extracted as part of periodontal or orthodontic treatment. The study was approved by the ethics committee of Tehran University of Medical Sciences (IR.TUMS.DENTISTRY.REC.1398.118). The medicaments evaluated in this study included CH (Golchadent, Tehran, Iran), Cupral (HUMANCHEMIE, Alfeld, Germany), and CH plus 5% AgNPs (nano-sized metal particles with 20 nm dimensions, US Research nanomaterials, Huston, USA).
Inclusion and exclusion criteria:
Single-rooted single-canal teeth with straight canals, mature apices, no internal or external root resorption, no cracks on the root surface, and no caries on the root surface were selected .The teeth with oval-shaped canals and those with a length shorter than 20 mm were excluded and replaced.
To eliminate the soft tissue from the root surface, the teeth were immersed in 5.25% sodium hypochlorite for 30 min, and stored in saline until the experiment. Next, for the purpose of standardization of root canals, the incisal edge of the teeth were reduced by a diamond fissure bur and high-speed handpiece such that all teeth had 15 mm length. Next, the access cavity was prepared by high-speed handpiece under air and water spray. To determine the working length, a #15 K-file was introduced into the canal until its tip was visible at the apex. Working length was determined to be 1 mm shorter than this length. All root canals were prepared by the same technique to minimize the confounding factors. All root canals were prepared by the single-length technique using Endo-Mate DT rotary motor (NSK, Japan) and S1 to F3 ProTaper Gold rotary files (Dentsply Maillefer, Ballaigues, Switzerland) to the working length. Next, #1 and 2 (#70 and 90) peeso reamers were used to widen the canals to 3 mm shorter than working length. Accordingly, all root canals had the same diameter in 3–11 mm of the working length. During the instrumentation period, 1 ml of 2.5% sodium hypochlorite (Morvabon, Tehran, Iran) was used for irrigation after using each file. After completion of root canal preparation, 2 ml of 2.5% sodium hypochlorite was used for 10 minutes. Subsequently, canals were flushed with 10 ml of sterile normal saline. Then 2 ml of 17% ethylenediaminetetra-aceticacid (Morvabon, Tehran, Iran) was used for 1 minute to eliminate the smear layer. A final rinse with 10 ml of saline was then performed to eliminate the effect of disinfecting agents (20). The teeth were then packed and autoclave-sterilized at 121°C for 30 min (21). After sterilization, two teeth were randomly selected and incubated in brain heart infusion broth (Merck, Darmstadt, Germany) at 37°C for 48 h to ensure complete sterilization of the teeth. Next, inoculation of canals with E faecalis was performed in all teeth, except for the negative control group.
The process of microbial inoculation of root canals was as follows:
For E faecalis biofilm formation, the methodology by Luther et al, (22)with slight modifications was followed. In brief, the 24-h culture of E faecalis in the stationary phase was diluted with 1% ratio (vol/vol) with tryptic soy broth containing 1% glucose, and 50 mg/L calcium chloride. This suspension contained 108 colony forming units (CFUs) per milliliter, and 500 µL of this suspension was inoculated into a tube containing the teeth under sterile conditions. The suspension was refreshed every 48 h. According to the literature (22–24)biofilm forms within 24 h. The teeth in the tubes were incubated at 37°C for 14 days to allow biofilm formation and its penetration into dentinal tubules. Formation of biofilm was confirmed by random culture of some samples. According to the results of the Fuss study(19), using the One Way ANOVA Power Analysis option of the PASS II software, considering alpha equal to 0.05 and beta equal to 2. The standard deviation of the logarithm of the average colony number is 1.65 and the effect size is 0.46. The minimum sample size required in each of the 9 studied groups is 9 (total 81 teeth).
The teeth were then randomly divided into 9 groups (n = 9):
Group I: CH without electrophoresis
Group II: CH plus copper (Cupral) without electrophoresis
Group III: CH plus 5% AgNPs without electrophoresis
Group IV: CH with electrophoresis
Group V: CH plus copper (Cupral) with electrophoresis
Group VI: CH plus 5% AgNPs with electrophoresis
Group VII: Electrophoresis alone
Group VIII: Positive control group to ensure presence of E faecalis after culture
Group IX: Negative control group to ensure sterility of the teeth
Preparation of medicaments was as follows:
A total of 1000 mg of CH [Ca (OH) 2; Golchadnet, Tehran, Iran] was mixed with 1 mL of sterile saline (Darou Pakhsh Pharmaceutical Mfg. Co, Tehran, Iran).
To prepare CH paste plus 5% AgNPs, 950 mg CH was mixed with 50 mg AgNPs and 1 mL of sterile saline. Cupral paste was readily available. The medicaments were delivered into the canals by a #30 Lentulo spiral (Dentsply, Sirona, USA). For electrophoresis, the samples were placed in alginate (25)(for electric current transfer). Depotphorese®-Gerät Original II (Humanchemie, Germany) was used to transfer electric current and perform electrophoresis. The positive electrode (anode) was placed outside the canal and in the alginate, while the negative electrode (cathode) was placed in the canal filled with medicament at 3–4 mm depth (Fig. 1). The electric current transfer in electrophoresis groups was such that 7.5 mA was applied during 2.5 min. The teeth were then incubated for 2 weeks. After 2 weeks, 7.5 mA current was applied. In total, 15 mA was applied within 5 min. Next, the samples were incubated for 2 weeks. In groups without electrophoresis, only the medicaments were applied into the canals with #30 Lentulo spiral, and they were then incubated for 4 weeks. Finally, they underwent microbial analysis.
Microbial tests:
After incubation of the samples, the 3 mm of apical third of the roots were cut by a diamond disc (thickness: 0.2 mm) (D + Z, Germany) and a micromotor (NSK, Japan). Also, the coronal part of the roots were cut by a diamond disc to create an 8 mm cylinder (Fig. 2). Next, the canals were rinsed with saline. To assess the efficacy of different techniques for inactivation of microbial biofilm and elimination of residual bacteria from the dentinal tubules, dentin chips were collected from the root canal walls by using sterile peeso reamers; #3 (#110) peeso reamer was used for depth 1, #4 (#130) was used for depth 2, and #5 (#150) was used for depth 3. Next, the dentin chips obtained at each phase were weighed, and 0.0005 mg of each was transferred into sterile 0.2 ml Eppendorf tubes containing sterile saline. They were then incubated at 37°C for 24 h to increase the number of viable bacteria; 100 µL of the obtained bacterial suspension was placed in brain heart infusion agar plates and also in thioglycolate liquid culture medium and were incubated at 37°C for 24 h. Next, the number of CFUs/mL was counted and proliferation of microorganisms was evaluated in thioglycolate liquid culture medium. The data were analyzed using PASS 11 software.
Diagram 1 explain the methods of article.