الفهرس 
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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