The manuscript of this laboratory study has been written according to the Preferred Reporting Items for Laboratory Studies in Endodontology (PRILE) 2021 Guidelines (25).
This study was approved by the Clinical Research Ethics Committee of Gazi University, Faculty of Dentistry (No: 21071282-050.99-). Freshly extracted human premolar teeth with a single root and one root canal were selected. Teeth with caries, cracks, fractures, calcified root canals, immature apices, root resorption, and previous endodontic treatment were excluded. The presence of a single canal was confirmed with periapical radiographs. One hundred and fifteen teeth meeting these criteria were stored in sterile water at 4℃ before the experiment.
The residual tissues on the root surfaces of the teeth were cleaned using periodontal scalers. The teeth were separated from their crowns under water cooling, remaining with a root length of 13 mm. Each root was sectioned horizontally at the middle third to obtain one slice with a thickness of 3 mm using a precision saw (Isomet, Buehler, NY, USA). The thickness of each slice was confirmed using a digital caliper.
The root canal space of the dentine slices was enlarged to a size of 1 mm using a #2 peeso reamer (Mani, Tochigi, Japan). The dentine discs were irrigated using 17% EDTA solution (Wizard, Rehber Chemistry, Turkey) for 1 minute to remove the smear layer. They were then washed with 2.25% sodium hypochlorite (NaOCl) (Wizard). The action of NaOCl was neutralized with 5% sodium thiosulfate. The specimens were sterilized using ethylene oxide. Enterococcus faecalis ATCC #29212 standard strain was produced by incubation at 37℃ for 24 hours in petri dishes containing tryptic soy agar (Merck, Darmstadt, Germany) (TSA). Purity controls were performed on the produced colonies. The produced cell suspension was then adjusted in 100x15 mm sterile glass tubes containing 10 ml tryptic soy broth (TSB) (Merck) to match the turbidity of 1.5x108 colony forming unit (CFU/mL) equivalent to 0.5 McFarland standards. The determined number of sterile dentine discs in each group were placed on sterile polystyrene 24-well cell culture plates for biofilm development. Sterile pipettes transferred one hundred ml of the E. faecalis suspension into the dentine discs. Then, 2 mL of TSB growth culture was added to cover the whole surface of the prepared dentine disc. One hundred mL of 5% sucrose concentration was added to the dentine discs. The dentine specimens were incubated at 37°C in an aerobic atmosphere for seven days. One day later, the samples were checked for bacterial growth. The specimens were replenished with E. faecalis biofilm every 48 hours to supply nutrients to the bacteria to ensure their survival.
Treatment of the infected dentine discs with different irrigation protocols
One hundred fifteen infected dentine samples were randomly divided into 11 experimental (n = 10) and one control group (n = 5) according to the irrigation regimen:
Group 1 [6 ml 2.5% sodium hypochlorite (NaOCl)]
2.5% NaOCl was obtained by mixing 5.25% NaOCl (Wizard, Rehber Chemistry, Istanbul, Turkey) with water in equal proportion. The solution was applied manually using a disposable dental syringe (Ultradent Products, South Jordan. UT, USA) and 27-G needle (NaviTip) for 2 minutes without any activation.
Group 2 [6 ml 17% ethylene diamine tetra acetic acid (EDTA)]
17% EDTA (Wizard, Rehber Chemistry, Istanbul, Turkey) was purchased from a commercial source at the indicated concentrations. The solution was applied manually for 2 minutes as described above.
Group 3 [6 ml 1% peracetic acid (PAA)]
A 36–40% peracetic acid solution (Code: 433241) supplied from a company (Sigma-Aldrich, St Louis, MO) was diluted with non-ionized water to obtain a solution with a weight/volume ratio of 1%. This solution was stored in the refrigerator at + 40C and warmed to room temperature before use. The solution was applied manually for 2 minutes as described above.
Group 4 [6 ml 2% peracetic acid (PAA)]
A 36–40% peracetic acid solution (Code: 433241) (Sigma-Aldrich, St Louis, MO) was diluted with non-ionized water to obtain a solution with a weight/volume ratio of 2%. This solution was stored in the refrigerator at + 4 0C and warmed to room temperature before use. The solution was applied manually for 2 minutes as described above.
Group 5 [6 ml 9% etidronic acid (HEBP)]
Aqueous 60% HEBP solution (Code: H6773) was obtained from a commercial market (Sigma-Aldrich, St Louis, MO). It was mixed with ultrapure water with a weight/volume ratio of 9% and stored at room temperature in a glass bottle until use. The solution was applied manually for 2 minutes as described above.
Group 6 [6 ml 18% etidronic acid (HEBP)]
Aqueous 60% HEBP solution (Code: H6773) was obtained from a commercial market (Sigma-Aldrich, St Louis, MO). It was mixed with ultrapure water with a weight/volume ratio of 18% and stored at room temperature in a glass bottle until use. The solution was applied manually for 2 minutes as described above.
Group 7 [3 ml 2.5% NaOCl- 3 ml 17% EDTA]
3 ml 2.5% NaOCl solution was applied manually using a disposable dental syringe (Ultradent Products, South Jordan. UT, USA) and 27-G needle (NaviTip) for 1 minute without any activation. Then, 3 ml 17% EDTA was applied manually as stated above for 1 minute.
Group 8 [3 ml 2.5% NaOCl- 3 ml 1% PAA]
3 ml 2.5% NaOCl solution was applied manually for 1 minute as stated above. Then, 3 ml 1% PAA was applied manually for 1 minute.
Group 9 [3 ml 2.5% NaOCl- 3 ml 2% PAA]
3 ml 2.5% NaOCl solution was applied manually for 1 minute as stated above. Then, 3 ml 2% PAA was applied for 1 minute.
Group 10 [6 ml 2.5% NaOCl + 9% HEBP]
A 6 ml 2.5% NaOCl + 9% HEBP single irrigation solution was obtained by mixing 3 ml 5.25% NaOCl solution and 3 ml 18% HEBP solution. This solution was applied manually for 2 minutes as described above.
Group 11 [6 ml 2.5% NaOCl + 18% HEBP]
A 6 ml 2.5% NaOCl + 18% HEBP single irrigation solution was obtained by mixing 3 ml 5.25% NaOCl solution and 3 ml 36% HEBP solution. This solution was applied manually for 2 minutes as described above.
Group 12 [Saline (contol)]
A 6 ml saline solution was applied for 2 minutes as described above.
All the solutions were delivered with the application rate of 1min/3ml. After the application period, 6 ml of water was added to neutralize the solution.
Investigation of bacterial biofilms with confocal laser scanning microscope (CLSM)
After the irrigation regimen, all the experimentally infected dentin discs were stained with Live/Dead BacLightTM Bacterial Molecular Kit L7012 (Molecular Probes, Invitrogen, Carlsbad, CA, USA), which includes Syto-9 and propidium iodide (PI). Styo-9 is a green fluorescent stain and targets the intact membranes of the live bacteria. Propidium iodide is a red fluorescent strain that marks damaged membranes of dead bacteria. According to the manufacturer's guidelines, 9 ml dye was added to 3 ml distilled water. The mixture was vortexed for 2 minutes to obtain homogeneity. Following staining the samples using a 200 ml dye mixture for 15 minutes, each sample was washed with saline solution. A specialist operator examined each sample under a confocal laser scanning microscope (LSM 510 META, Zeiss, Jena, Germany) at 20x magnification. The recorded 2D images were converted to 3D images using Imaris 9.2.1 software (Bitplane, Oxford Instrument Company, Concord, MA, USA). The biological volumes of the dead and live bacteria were calculated and recorded. Then, the percentage of the dead bacterial biovolume was calculated using the data.