This study was approved by the Research Ethical Committee of the University of Passo Fundo (protocol number 5.783.928).
Antimicrobial evaluation
Sixty single-rooted extracted human teeth were used in the present evaluation. All teeth were obtained from the Biobank of the School of Dentistry of the University of Passo Fundo (Passo Fundo, RS, Brazil). Dental crowns were sectioned with diamond disc so that all roots remained with 15 mm in length. All roots were prepared to remove pulp tissues and to standardize the canal diameter. Working length (WL) was established by introducing #10 K-file (Dentsply-Sirona, York, PA, USA) into the root canal until its tip was visualized at the apical foramen. From this measure, 1 mm was subtracted from WL. Roots were enlarged using manual K-files (Dentsply-Sirona) and serial instrumentation, up to a #35 file. Distilled water (DW) (Natupharma, Passo Fundo, RS, Brazil) was used as an irrigant solution and renewed at each instrument change. Subsequently, root canals were filled with 17% EDTA (Natupharma, Passo Fundo, RS, Brazil) for 1 minute to remove the smear layer, followed by irrigation with 5 mL of DW and drying with absorbent paper (Tanari, Manacapuru, AM, Brazil).
Roots were sterilized at 120oC in an autoclave (Kavo, Joinville, RS, Brazil) for 30 minutes. Five samples were randomly selected for sterilization control. The sterile paper point was placed in contact with the canal walls of each sample for 15 seconds and transported to a microtube containing 1 mL of 0.9% saline solution (Basso, Caxias do Sul, RS, Brazil). The material was homogenized and a 100 μL aliquot was cultivated on blood agar after five minutes. Samples were incubated at 37oC for 48 hours and showed no sign of bacterial growth (16).
The reference strain was Enterococcus faecalis (E.faecalis) (ATCC 19433), which was cultivated in brain-heart infusion (BHI) broth (Acumedia–Neogen, Lansing, MI, USA) for 24 hours at 37oC in bacteriological incubator (Kavo, Joinville, SC, Brazil). The turbidity degree was adjusted to the McFarland's 1.0 scale, corresponding to 3.0 x 108 CFU/ml and optical density from 0.25 to 550 nm. A 100-μL culture aliquot was inoculated into the root canal of each sample, until extravasation to the root canal entrance. The culture was maintained for 14 days for biofilm formation, and the remaining volume was replaced every 48 hours with sterile BHI. Once a week, one BHI aliquot was collected from a randomly selected specimen of each group and submitted to Gram staining and cultured on blood agar, followed by catalase and esculin tests, to verify the absence of other microorganisms.
After contamination, the 60 samples were irrigated with 5 mL of DW and randomly distributed into six groups (n=10), according to test decontamination protocols: G1: DW (control); G2: 1% NaOCl; G3: 2.5% NaOCl; G4: 5,25% NaOCl; G5: 250 ppm HClO; G6: 500 ppm HClO. This number of samples was based on previous study that focused on antimicrobial strategies in endodontics (16).
DW and NaOCl solutions were obtained by manufacturing pharmacy (Natupharma, Passo Fundo, RS, Brazil). The 250 ppm and 500 ppm HClO solutions were obtained by specific Dentaqua device (Dentaqua, Conmel, Ireland) for each concentration. This device consists of a compartment on the top, a connector for the smaller bottle on the back, and a connector for the larger bottle on the front. Figure 1 illustrates the Dentaqua device. The electrochemical activation technology involves the generation of electrochemically activated solutions by passing a dilute NaCl solution through an electric field in a Flow-through Electrolytic Module (FEM), segregating the ions formed and producing two oppositely charged solutions with altered physical and chemical properties. The positively charged solution (anolyte) consists of a mixture of unstable mixed oxidants, such as HClO, in a physically excited state, which is capable of penetrating biofilms and is highly microbicidal. The negatively charged antioxidant solution (catholyte) predominantly consists of sodium hydroxide in an excited state.
The upper compartment is filled with sterile DW. The smaller bottle on the back is filled with brine solution, which is composed of sodium chloride. After filling, the smaller bottle was connected to the back of the device, while the larger bottle was connected to the front of the device. The Water Bottle button was pressed, performing a washing cycle of the device with DW for 3 minutes, discarding this solution in the larger bottle. The DW was then discarded, and the larger bottle was again connected to the device to produce HClO. The Ecasol button was pressed to produce HClO for a period of 10 minutes, which was obtained by the electrolysis of saline solution mixed with DW in the device, dispensing the HClO solution at 250 ppm and 500 ppm in the larger bottle, respectively, being ready to use.
In all groups, the root canals were completely filled with the tested solution, by using 5-mL syringe with 19-G needle, until extravasation to the root canal entrance. The tested solution remained in contact with root canal walls for 5 minutes. Subsequently, irrigation with 5 mL of tested solution was performed and the tested solution was renewed. Six 5-minute decontamination cycles were performed, totaling 30 minutes of the tested solution into root canals. Finally, irrigation with 5 mL of DW was performed and the root canals of all groups were dried using aspiration cannula and absorbent paper points (Tanari).
Microbiological analysis was performed in two stages: after contamination (S1) and after irrigation procedures (S2). Root canals were filled with sterile saline solution and #35 K-file promoted contact with root canal walls for 30 seconds, and #35 sterile absorbent paper points promoted the same intentional contact with root canal walls for 30 seconds. It was transferred to a tube containing 450 μL of 0.85% sterile saline solution, being subsequently homogenized and diluted to 10-3. Aliquots of 100 μL of each dilution were cultivated on blood agar in duplicate, being incubated for 24 h at 37oC. Subsequently, the number of CFUs was counted on plates. The antimicrobial activity was analyzed by the reduction percentage in S1 and S2 CFUs counts.
Cytotoxicity evaluation
In this evaluation, L929 gingival fibroblastic cells were used, which were provided by the Laboratory of Applied Virology of the Federal University of Santa Catarina (UFSC, Florianópolis, SC, Brazil). For cell maintenance, Minimum Essential Media (Sigma-Aldrich, Rio de Janeiro, RJ, Brazil) supplemented with 10% fetal bovine serum medium (Invitrogen, São Paulo, SP, Brazil) were used, being kept in 75 cm2 culture flasks under humidity at 37°C and 5% CO2.
The assay was performed with 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide or MTT (Sigma-Aldrich), corresponding to a colorimetric test to evaluate cell viability. L929 cells were trypsinized, counted and distributed in 96-well plates at concentration of 1 x 105 cells per well. The Dulbecco's Modified Eagle Medium (DMEM) (Sigma-Aldrich, Rio de Janeiro, RJ, Brazil) was used, enriched with 5% fetal bovine serum. After incubation for 24 hours at 37°C in oven with 5% CO2, cell confluence was observed under inverted microscope. Then, the DMEM medium was removed by aspiration and 100 µL of test chemical substances were added, being incubated for 3 minutes.
Wells were washed with 200 µL of sterile PBS and 50 mL of MTT solution (1 mg/mL in DMEM) were added for incubation for 4 hours. MTT was carefully removed avoiding damage to cells and 100 mL of dimethylsulfoxide (DMSO) were added to solubilize formazan crystals. DMSO was also added to empty wells for white colorimetric calculation. The plate was placed on mixer for 10 minutes and the absorbance was measured on a 490 nm filter. All experiments were performed in triplicate and cell viability percentages were calculated in relation to control group, according to the following formula: viability (%) = (sample absorbance – mean blank absorbance) x100 / control absorbance – mean blank absorbance.
Cleaning ability and Erosion Potential Analysis (Scanning Electron Microscopy – SEM)
Fifty-six single-rooted bovine lower incisors were selected after extraction with levers and forceps. They were stored in distilled water and 2.5% bleach (in a 50/50 ratio) until used. The criteria for the selected teeth to be included in the study were: predominantly straight canals that had similar and ideal diameters for carrying out the work and complete formation of the root apex. Exclusion criteria were deciduous teeth, teeth with incomplete development, any type of fracture or apical lacerations.
Tooth Cleaning and Sterilization: After selecting the samples, the external surface of the teeth was cleaned by root scaling. The remaining periodontal ligament attached to the roots was removed with a Gracey type nº 1/2, 3/4 and 5/6 periodontal curette (Neumar Instrumentos Cirúrgicos Ltda - Brazil) and a nº 12 scalpel blade. Then, samples were washed in running water for one minute and dried at room temperature.
The crown of the teeth was removed below the cementoenamel junction perpendicular to the long axis of the tooth with the aid of a diamond disc (KG Sorensen Indústria e Comércio Ltda., Barueri, São Paulo, Brazil) to standardize root length of 16mm (Figure 2 a). Grooves were made in the long axis of the buccal and lingual surfaces of the samples, with sufficient depth so that at the end of the experiment they could be cleaved more easily into two hemisections, but taking care to ensure that the interior of the root canal was not reached (Figure 2 b).
For root canal gauging, 21mm type K manual files (Dentsply Sirona Maillefer, Catanduva, São Paulo - SP) calibrated at 16mm were used. For larger-caliber canals, 20-gauge K-type files were selected, while for smaller-caliber canals, #10 K-type files were used.
After exploring the root canals, a small amount of New Max U utility wax (Technew Comércio e Indústria Ltda, Quintino Bocaiuva, RJ, Brazil) was fixed in the region corresponding to the apex, providing an apical seal to allow tooth irrigation protocol to be carried out (Figure 2 c). The samples were attached to a bench vise so that the grooved faces were perpendicularly attached to the vise, and below a container was placed to collect waste (water, solutions, organic waste).
All teeth received an adapted irrigation protocol for root canal disinfection: protocol by Grundling et al. [21]. Briefly, it consisted of an initial irrigation with distilled water (Asfer, São Caetano do Sul, São Paulo, São Paulo, Brazil) and activation for a time of three seconds. Afterwards, irrigation was performed with 2.5% sodium hypochlorite (Soda Chlorinated Asfer 2.5%, Dental Marc, Caxias do Sul, Rio Grande do Sul, Brazil) and activated again for three seconds. This was followed by using 17% trisodium EDTA (Iodontec Indústria e Comércio de Produtos Odontológicas Ltda, Porto Alegre, Rio Grande do Sul, Brazil) and activation for three seconds. The protocol was repeated three times. For the final wash, distilled water was used. Teeth were dried at room temperature. Helse E1 Irrisonic ultrasonic inserts (Helse, Santa Rosa de Viterbo, SP, Brazil) were used to clean the samples. The insert was positioned 3mm from the working length (16mm) and activated with power corresponding to one third of the equipment (Mini-Endo, SybronEndo, São Paulo, Brazil).
The decoronated teeth were positioned in 1.5ml microtubes (Kasvi, São José dos Pinhais, Paraná, Brazil) with the cervical portion upwards. A small hole was made in the center of the microtube lids with the help of an instrument heated over a flame. In this hole, the teeth were positioned and, subsequently, strips of Nexcare Micropore Tape (3M, Sumaré, São Paulo, Brazil) were used around the cap to adequately fix the tooth and reduce the chances of possible contamination. Next to the central hole, two smaller lateral holes were made with a disposable irrigation needle (Injex 21G, Ourinhos, São Paulo, Brazil); these holes were used to change the culture medium (Figure 2 d).
The microtube/tooth assembly was positioned in polypropylene boxes (Heathrow Scientific, Vernon Hills, Illinois, USA), which were individually packaged and sterilized in an autoclave (Figure 2 e). Sterilization time occurred in a 15-minute cycle at a temperature of 120°C.
Before inoculating the samples, they underwent sterility test using sterile paper cones (Absorbent paper tips 15-40 and 45-80, Diadent/TDK, Dental Web, Porto Alegre, Rio Grande do Sul, Brazil). These tips were introduced into the root canal and, shortly after, were transferred to a tube containing 3ml of BHI. These tubes were incubated at a temperature of 37°C for 24 hours. Sterility testing was performed on all samples. No type of bacterial growth was detected in the broth of the tubes after the sterility test, so the samples passed to the bacteria inoculation stage.
Culture Preparation: Enterococcus faecalis (ATCC 29212) was taken from -20 stock, cultivated in an aliquot of 200ul in 3ml of BHI broth and incubated for a period of 24 hours in the Immunology and Microbiology Laboratory of the School of Sciences of the Pontifical Catholic University of Rio Grande do Sul. Then, a 200 μL aliquot was removed and cultured again in a new 3ml BHI broth and incubated again for 24 hours.
After preparing the inoculum, its initial count was carried out by diluting it to -9. In the dilution box, 450 μL of saline solution was added to each of the 9 wells (each well corresponded to a dilution). In the first well, 50 μL of the initial inoculum was added. Then, serial dilution was carried out: 50 μL from the first well was passed to the second well, and so on, homogenizing the broth. After the serial dilution was completed, 10 μL of each dilution was inoculated using the drop technique, in triplicate.
E. faecalis strains were cultivated in a brain heart infusion (BHI) broth for 24 hours positioned in a shaker. Subsequently, a Gram stain test was performed to check whether there was contamination. After confirming the negative contamination test, the culture was kept in an oven at 37°C until use. E. faecalis were cultivated in BHI medium containing 1% trypticase (BBL, Cockeysville, MD), 1% proteosis peptone (Difco, Detroit, MI), 0.5% yeast extract (Difco, Detroit, MI), 0.5% sodium chloride, 5 mg/mg hemin, and 0.5% mg/ml vitamin K supplemented with 0.2% glucose at 37°C for 24 hours.
Inoculation of Samples: In the microtubes, the Enterococcus faecalis culture was added equally into the root canal of each sample (amount corresponding to 100 μL of the initial inoculum) through the central hole, for biofilm development. Furthermore, BHI broth was added through the side holes until the entire length of the tooth was submerged. In the negative control group, only BHI broth was introduced without the presence of E. faecalis. The boxes with the microtubes inside were wrapped with plastic film and incubated in an oven at 37°C until the medium was changed again.
Every 48 hours, the culture medium was changed with the aid of disposable irrigation needles and 5ml luer lock disposable syringes (Injex, Ourinhos, São Paulo, Brazil). The largest amount of BHI broth possible (on average 2/3) was removed through the side holes made in the lids of the microtubes and discarded. A new culture medium was inserted at the same location. The samples were then taken to the oven at the same temperature of 37°C for another 48 hours, until the next exchange for a period of 7 days. The renewal of the medium was always carried out three times a week.
During each medium change, a drop of material from each group was collected from the inside of the microtube and placed on a blood agar plate. The 7 plates were placed in an oven at 37°C for a period of 48 hours, after which a visual assessment, collection and Gram analysis were carried out under a microscope (100x objective). This analysis aimed to verify whether there was staphylococcus-type bacteria contamination.
Classification of Treatment Groups
The samples were numbered from 1 to 56 and randomly divided into 7 groups, totaling 8 samples per group, as follows:
G1: NEGATIVE CONTROL - to prove the absence of erosive areas, changes in dentinal tubules, and bacterial growth, the samples were not inoculated and did not receive any type of treatment;
G2: E. faecalis + BHI + 1% NaOCl
G3: E. faecalis + BHI + 2.5% NaOCl
G4: E. faecalis + BHI + 5.25% NaOCl
G5: E. faecalis + BHI + 250 ppm HClO;
G6: E. faecalis + BHI + 500 ppm HClO;
G7: POSITIVE CONTROL - E. faecalis + BHI (no treatment but with bacterial growth)
All groups were, after carrying out the procedures, analyzed using Scanning Electron Microscopy (SEM). After the 7-day cultivation period, the samples were removed from the microtubes. Again, utility wax was fixed to the apex of each root (apical seal) and they were attached to the bench vice.
The irrigation protocol was carried out in the same way in samples from Dentaqua and Sodium Hypochlorite groups: 3 irrigation cycles with the solution belonging to each group; each cycle lasted 3 seconds. The irrigating needle was inserted 3mm short of the working length. During irrigation, a back-and-forth movement was carried out.
The 250 ppm and 500 ppm HClO solutions were obtained by specific Dentaqua device (Dentaqua, Conmel, Ireland) for each concentration, using the previously mentioned protocol.
Scanning Electron Microscopy (SEM): After teeth cleavage, samples remained for 7 days in a 2.5% glutaraldehyde solution (Glicolabor – Ribeirão Preto, São Paulo, Brazil). They were dehydrated and dried using a paper tip (Absorbent paper tips 15-40 and 45-80, Diadent/TDK, Burnaby, Canada), on a surface with the root canal portion facing upwards.
Samples were covered with 30nm gold/palladium alloy (Emitech K650 Sputter Coater, London, England), in a BAL-TEC SCD 005 Sputter Coater machine (BAL-TEC AG, Liechtenstein, Germany), at a vacuum level of approximately 5 x 10-2 mbar.
Observations using Scanning Electron Microscopy (XL 30; Philips, Eindhoven, Netherlands) were carried out at 5000x magnifications. Areas were standardized: between the cervical and middle third (cervical) and the middle and apical third (apical).
The most representative images of these regions were saved and analyzed on a ranking system, for two criteria (cleaning ability and erosion). Rank 1 was given for the image with the cleanest look, and the higher the rank, the worse was the cleaning ability. In the erosion analysis, rank 1 was given to the least eroded, and the higher the score, the greater the erosion profile. The positive control group could not be assessed because of the number of bacteria did not allow visualization of the dentine. For each area (cervical and apical) averages of each group were obtained. The objective was to verify the changes in the dentin structure after the applied treatment, making it possible to compare the cleaning ability, and the erosion magnitude in each group tested.
Statistical analysis
Data were analyzed using SPSS 11.0 software (SPSS, Chicago, IL, United States). Normal data distribution was confirmed by the Shapiro-Wilk test in antimicrobial and cytotoxicity evaluations. Data of antimicrobial and cytotoxicity evaluations were analyzed by one-way ANOVA followed by post-hoc Tukey’s test (p<0.05). SEM analyses of cleaning ability and Erosion were performed using the average with min-max, followed by Kruskal-Wallis and Mann-Whitney tests, with values adjusted for multiple comparisons by Benjamini-Hochberg procedure (p<0.05).