This in vitro experimental study was conducted on sound primary central incisors extracted within the past month due to either severe mobility or over-retention.
The sample size was calculated to be 9 in each group, according to a pilot study considering α = 0.10, β = 0.25, and minimum effect size between each two groups to be 1.81. Thus, a total of 90 teeth were enrolled. Teeth with carious lesions, fractures, and enamel structural defects (such as hypoplasia) were excluded. The teeth were stored in saline after extraction, and the saline was refreshed every 48 h.
S. mutans (ATCC35668 and PTCC1683) was purchased in lyophilized form from the Iranian Microbial Culture Collection. The frozen vial was rinsed under lukewarm water to defrost. The bacteria were then transferred to blood agar (Liofilchem, Roseto degli Abruzzi, Italy) and incubated in the presence of CO2 for 18–24 h. Next, the bacteria were transferred to brain heart infusion broth (Merck, Darmstadt, Germany) under a hood.
For preparation of the solution containing 25 mg iron in 3 cc saline, 228.19 mg of ferrous fumarate, 353.21 mg of ferrous ammonium citrate, 373.2 mg of ferrous sulfate, and 647.6 mg of ferrous gluconate (based on the molecular weight of iron salts and atomic number of iron) were required for each tube (Chimi®, Alvand, Iran).
The collected teeth were cleaned with pumice paste and a low-speed handpiece. Next, the root and crown were separated at the cementoenamel junction, and the pulp chamber was sealed with composite resin.
To prepare the artificial cariogenic solution for ACC, 3.7 g of brain heart infusion broth, 0.5 g of extracted yeast (Merck, Germany), 2 g of sucrose (Sigma-Aldrich, Burlington, MA, USA), and 1 g of glucose (Sigma) were dissolved in 100 mL of distilled water; 100 µL of freshly cultured standard-stain (ATCC35668) S. mutans (18–24 h) was added to the cariogenic medium, while the pH remained at 4. One test tube was allocated to each specimen.
The sample size was calculated to be 9 in each group, according to a pilot study considering α = 0.10, β = 0.25, and minimum effect size between each two groups to be 1.81. Thus, a total of 90 teeth were enrolled. Teeth with carious lesions, fractures, and enamel structural defects (such as hypoplasia) were excluded. The teeth were stored in saline after extraction, and the saline was refreshed every 48 h.
S. mutans (ATCC35668 and PTCC1683) was purchased in lyophilized form from the Iranian Microbial Culture Collection. The frozen vial was rinsed under lukewarm water to defrost. The bacteria were then transferred to blood agar (Liofilchem, Roseto degli Abruzzi, Italy) and incubated in the presence of CO2 for 18–24 h. Next, the bacteria were transferred to brain heart infusion broth (Merck, Darmstadt, Germany) under a hood.
For preparation of the solution containing 25 mg iron in 3 cc saline, 228.19 mg of ferrous fumarate, 353.21 mg of ferrous ammonium citrate, 373.2 mg of ferrous sulfate, and 647.6 mg of ferrous gluconate (based on the molecular weight of iron salts and atomic number of iron) were required for each tube (Chimi®, Alvand, Iran).
The collected teeth were cleaned with pumice paste and a low-speed handpiece. Next, the root and crown were separated at the cementoenamel junction, and the pulp chamber was sealed with composite resin.
To prepare the artificial cariogenic solution for ACC, 3.7 g of brain heart infusion broth, 0.5 g of extracted yeast (Merck, Germany), 2 g of sucrose (Sigma-Aldrich, Burlington, MA, USA), and 1 g of glucose (Sigma) were dissolved in 100 mL of distilled water; 100 µL of freshly cultured standard-stain (ATCC35668) S. mutans (18–24 h) was added to the cariogenic medium, while the pH remained at 4. One test tube was allocated to each specimen.
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