1- The antimicrobial test:
▪ Sample preparation:
1. Teeth will be cleaned from any hard debris using ultrasonic scalers 3 and then immersed in 5.25% NaOCl4 for 30 minutes to facilitate removal of soft periodontal tissues and debris. The teeth will be kept in a saline solution until their use to keep natural teeth hydrated.
2. Crowns of all teeth will be cut off at cemento-enamel junction using a high-speed hand piece and the root lengths will be standardized to a 16 mm length.
3. Patency will be established using a #10 5 hand k file, the working length (WL) will be established 1 mm shorter than the apical foramen.
4. All the root canals will be prepared with ProTaper Next 6 rotary system till files up to X4 (0.40 tip size and 6% taper) according to the manufacturer’s specifications.
3 (ultrasonic scalers , Woodpecker, China).
4 (Sodium Hypochlorit, JK Dental vision, Mansoura; Egypt).
5 (M-Access K-Files, Dentsply Sirona, USA).
6 (Protaper Next rotary system, Dentsply Sirona, USA).
5. After each instrument change, canals will be passively irrigated with 5 ml of 2.5% NaOCl solution using 30 Gauge side vented needle.
6. After complete root canal instrumentation, the canals will be irrigated with 5 ml saline, followed by 5 ml of 17% Ethylenediaminetetraacetic Acid (EDTA) for 1 min, then a final flush with saline.
Culture and inoculum preparation:
1. Standard strain of E. faecalis (ATCC 29212) will be used and cultured aerobically on Brain heart agar at 37°C for 24 h. Inoculum will be prepared in sterile brain heart infusion broth (BHI) and turbidity will be adjusted to 0.5 McFarland corresponding to approximately1.5 × 108 CFU (CFU/mL).
2. All root canals will be filled with 30μ E. faecalis suspension by using a micropipette.
3. The sterilized teeth will be inoculated with this solution, then placed inside a sterile box and incubated aerobically for one week at 37 C with refreshment by sterile BHI media every 48 hours.
The specimens will be randomly divided into five groups according to intracanal medication as follow:
- Group one: Medication with CPCP inserted into the pulp chamber on a cotton pellet.
- Group two: Intracanal medication with CPCP using paper point.
- Group three: Intracanal medication with Ca (OH)2.
- Group four: Intracanal medication with a combination of Ca (OH)2 and CPCP.
- Group five: Root canals will be kept empty (control).
Intervention:
- For G1: Intracanal BHI broth will be dried using sterile paper points, then the tooth will be medicated with CPCP using a cotton pellet placed into the pulp chamber to evaluate the antibacterial efficacy.
- For G2: Intracanal BHI broth will be dried using sterile paper points, then the canal will be medicated with CPCP using paper point till the apical 1/3 of the canal to evaluate the antibacterial efficacy.
-For G3: Intracanal BHI broth will be dried using sterile paper points, then the canal will be medicated with Ca (OH)2 till the apical 1/3 of the canal to evaluate the antibacterial efficacy.
-For G4: Intracanal BHI broth will be dried using sterile paper points, then the canal will be medicated with a combination of Ca (OH)2 and CPCP till the apical 1/3 of the canal to evaluate the antibacterial efficacy.
-For G5: Intracanal BHI broth will be dried using sterile paper points, but the canals will be kept empty without any medications (control).
Outcome assessment for antibacterial efficacy:
It will be determined using culture technique in the microbiological department, Cairo University.
After a period of one week at 37 C, the first samples will be treated as follow:
A first sample (S1) will be taken from each canal before implementing the tested protocols. The canal will be filled with a sterile saline solution, H file will loosen biofilm bacteria by scraping the root canal walls for 15 seconds, and dentinal shavings from each canal will be collected using sterile paper points (40size). The paper point from each tooth will be placed in a test tube containing (1 ml of BHI broth).
Samples in the transport medium (1 ml of BHI broth) will be dispersed with vortex in the mixer for 60 seconds by the laboratory technician. Serial 10-fold dilution will be prepared of 1/10, 1/100, and 1/1000 dilutions to assess the microbial load of E. faecalis in each root canal.50 μl of these diluted samples will be transferred and spread over BHI agar plates and cultured under aseptic conditions, followed by incubation at 37o C for 24 hours (Gajan et al., 2009).
Eventually, the resultant growth will be visually quantified by counting the number of colony forming units per milliliter of each dilution (CFUs) on the agar medium using unaided eye (Molander et al., 1998; Martinho et al., 2014). Only plates with 30-300 colonies will be counted. The number of CFUs/ml will be then transformed into actual counts based on the previously determined dilution factors.
After a period of 24 hours, the second samples will be treated as follow:
A second sample (S2) will be taken from each canal after implementing the investigated intracanal medication. The number of CFUs will be determined as described for S1.
2- The cytotoxicity test:
Experimental groups:
• CPCP Group : Cells grown in medium conditioned by CPCP.
• Ca (OH)2 Group: Cells grown in medium conditioned by Ca (OH)2.
• Combination Group : Cells grown in medium conditioned by a combination of Ca (OH)2 and CPCP.
• Control Group (CG): Cells grown in a fresh medium.
Cell culture:
Normal fibroblast cell line BHK will be obtained from the National Cancer Institute of Egypt in a medium containing 10% fetal bovine serum (FBS), penicillin, amphotericin B and streptomycin. To adapt to the new environment, the cells in the afore-mentioned medium will be incubated at 37°C for 48 hours under 95% humidity and 5% CO2. To obtain higher number of cells, the cells will be cultured again in a culture medium containing 15% FBS and this process will be repeated 5 to 8 times to obtain more cells. This cell line will be cultured in a culture medium containing 10% bovine serum in a sterile flask. The medium will be refreshed every 2‒3 days and the cells will be passaged after one week.
Conditioned medium:
Intracanal medicaments will be prepared according to the manufacturer’s instructions. The culture media conditioned by the intracanal medicaments will be obtained .
Outcome assessment for biocompatibility
Cytotoxicity test:
Cells will be seeded (2 x103 cells/well) into 24-well culture plates and maintained in humid chamber for 24 hours at 37 C in 5% CO2. Then, the culture medium will be replaced by the conditioned medium according to experimental groups. The control group will receive fresh medium. Cytotoxicity will be evaluated in 1, 3, 5 and 7 days, analyzing the mitochondrial activity measured by the MTT reduction assay. Half of the medium in each well will be replaced every other day by 150μL of fresh medium.
A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide dye solution MTT will be prepared as 5 mg Ml in PBS at 37C just before use. A total of 20 lL MTT dye will be added to each well and incubated at 37C, in air containing 5% CO2 and at 95% relative humidity for 4 hours in the dark. After incubation, the MTT will be aspirated and the formazan product was solubilized in 0.1 mL in HCl (0.04 mol L) in isopropanol. The plates will be shaken before the optical densities are measured at 570 nm, using a Multiskan EX spectrophotometer.