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Abstract The purpose of this study was to: - evaluate cytotoxicity of different concentrations of three intracanal medications Neem oil, double antibiotic paste (DAP) and calcium hydroxide (Ca(OH)2) using MTT and Apoptosis/necrosis assays. In addition, to compare subcutaneous tissue reaction of the three intracanal medicaments in rats at two different time intervals (7 and 21 days). Materials and Methods: The research was approved by the Ethics Committee of Faculty of Dentistry, Suez Canal University (number 17/2017). Cell culture:- Immortalized human gingival fibroblast cells were retrieved from the cell bank of Egyptian Company for Production of Vaccines (Vacsera). Cells were cultured in plastic tissue culture dishes in complete growth medium: Dulbecco’s Modified Eagle Medium (DMEM), and then were maintained in an incubator at 37°C in humidified 95% air and 5% CO2. Cells were detached with 0.5% (w/v) trypsin–EDTA for subcultures. Part I (cytotoxicity assays / in vitro):- A. MTT dye reduction assay:- Three intracanal medicaments, calcium hydroxide paste Ca(OH)2, Double antibiotic paste (DAP) and Neem oil were diluted using Dulbecco’s Summary and Conclusions 92 Modified Eagle medium to achieve five descending concentrations (0.5, 0.25, 0.125, 0.0625 and 0.00781 mg/mL). Growing fibroblasts were seeded into 96-well plates, and then incubated with 100 μl/well of serial dilutions of the used intracanal medicaments for 24 h. To determine cell viability, a total of 10 μl MTT dye was added to each well and the plate was incubated at 37°C in air containing 5% CO2 and at 95% relative humidity for 2–12 h to allow mitochondrial succinate dehydrogenases in viable cells to reduce intracellular soluble yellow tetrazolium dye MTT to insoluble violet formazan dye. Optical densities of each plate were read with microplate reader at 550–600 nm. The percentage of cell viability was calculated and the cytotoxicity of the medicaments was categorized as severe (30%), moderate (30-60%), mild (60-90%) and non-toxic (>90%). The experiments were done in triplicates. B. Apoptosis/necrosis assay:- Fibroblasts were incubated in binding buffer containing 5 μL of FITC‐coupled annexin V and 5 μL of propidium iodide (PI), and the samples were incubated for 15 min in the dark at room temperature according to the manufacturer’s instructions. The samples were analyzed on a fluorescenceactivated cell sorter (FACS) flow cytometer. This software only allows assessing specific populations, individualized by gates according to size (FSC), granularity (SSC), and fluorescence (FL) parameters. Early apoptotic cells stained positively with annexin V, whereas late apoptotic cells stained positively with annexin V and PI. Percentages of viable cells, apoptotic cells, and necrotic cell populations were determined as described by Vermes et al., (96) . The experiments were done in duplicate.Summary and Conclusions 93 Part II (subcutaneous tissue reaction/ ex vivo):- A total of forty-eight male Albino rats were distributed into four groups according to type of intracanal medicament (n = 12): DAP, Neem oil, Ca(OH)2 and control group. Each group was then subdivided into two subgroups according to the time interval (7 and 21 days). Animals were anesthetized with 10% Ketamine Chloride (75 mg/kg) along with Xylazine (10 mg/kg) by intramuscular injection. Single (1 cm) incision was made on the back of each rat. Then, sterile polyethylene tubes filled with one of the medicaments were implanted in the dorsal subcutaneous tissues of the rats while empty tubes served as controls and the skin borders were sutured. After 7 and 21 days, rats were sacrificed by intravenous injection of an overdose of pentobarbital sodium and perfused with 10% buffered formalin. The implants with the surrounding tissue were removed. The tissues were fixed and embedded in paraffin wax and stained with hematoxylin and eosin. Qualitative and quantitative analysis were carried out for the stained histological sections. Results of our study were then gathered, tabulated and statistically analyzed. Results: MTT dye reduction assay:- The intergroup analysis showed statistically significant difference between the tested groups Ca(OH)2 and Neem oil at concentrations (0.5 and 0.25 mg/mL) with obvious toxicity to the fibroblasts. For DAP, four concentrations (0.5, 0.25, 0.125 and 0.0625 mg/mL) were significant slight cytotoxic to fibroblasts. However, at concentration 0.00781 mg/mL there Summary and Conclusions 94 was no statistically significant difference between the three intracanal medications, all three materials were biocompatible and were considered non cytotoxic with viable cells concentration > 90%. Apoptosis/necrosis assay:- Regarding the healthy apoptotic cells, DAP showed the statistically significant highest mean value followed by Ca(OH)2 then Neem oil. On the other hand, the early and late apoptotic cells, Neem oil exhibited the statistically significant highest apoptotic rate and necrosis, followed by Ca(OH)2. Meanwhile DAP exhibited the statistically significant lowest apoptotic rate. Subcutaneous tissue reaction in rats:- At 7 days: there was a statistically significant difference between all tested groups (P-value <0.001). Ca(OH)2 showed severe inflammatory reaction followed by Neem oil which showed moderate inflammatory reaction. While there was no statistically significant difference between DAP and control groups; both showed mild degree of inflammation. At 21 days, Ca(OH)2 showed statistically significant difference than Neem oil and DAP. Ca(OH)2 represented a moderate degree of inflammation, while there was no significant difference between Neem oil and DAP; both showed slight degree of inflammation.Summary and Conclusion |