Caries free, permanent premolars were extracted for periodontological purposes after the approval of the Ethics Committee. Teeth with fully developed root apices were included in the present study. After calculus and soft tissue removal by curettes, 12 nonsterile teeth were immediately put into one of the following storage solutions: De-ionized water (pH ~6.5), 0.2% glutaraldehyde (pH ~4.8), HBSS (HBSS, with phenol red, with calcium and magnesium, pH ~7.5, Sigma-Aldrich Corporation, St. Louis, MO, USA), 0.1% NaOCl (pH ~10), and 0.1% thymol (pH ~9.1). Only thymol solution, 0.1% was prepared in our laboratory.
Solutions were not replenished during the 12 months storage, which limits the dissolution of the inorganic fraction of teeth.[11 12 (link)] Six teeth from each group were taken out of the storage solution at the 2nd month of the storage, and the other six teeth were taken out at the 12th month. Teeth with the vestibular surfaces facing up were horizontally embedded in polyester resin. To prepare a flat surface which exposes enamel and dentin together, teeth crowns were ground 2 mm on their vestibular surface using 180, 220, 320, 500, 800, 1000, 1200 grit silicon carbide papers under copious water-cooling, followed by polishing with 0.05 μm alumina slurry using a low-speed metallurgical polisher.
After transferring teeth to the Vickers Hardness Testing Machine (Buehler, Lake Bluff, IL, USA) six indentations on enamel and six indentations on dentin were done by a pyramid diamond indenter tip with 100 g load for 15 s. On enamel and dentin, the indentations were started 200 μm away from dentino-enamel junction and made toward the coronal surface on enamel and towards apical end on dentin, with keeping approximately 200 μm distance between the indentations.
Means of the hardness values (HV) for enamel and dentin were separately calculated per sample, and data was processed using SPSS 15.0 (SPSS, Inc., Chicago, IL, USA). First, normalization of the data was evaluated, and statistically homogenous data distribution was verified using one-sample Kolmogorov-Smirnov test, and two-way ANOVA was used.
Solutions were not replenished during the 12 months storage, which limits the dissolution of the inorganic fraction of teeth.[11 12 (link)] Six teeth from each group were taken out of the storage solution at the 2nd month of the storage, and the other six teeth were taken out at the 12th month. Teeth with the vestibular surfaces facing up were horizontally embedded in polyester resin. To prepare a flat surface which exposes enamel and dentin together, teeth crowns were ground 2 mm on their vestibular surface using 180, 220, 320, 500, 800, 1000, 1200 grit silicon carbide papers under copious water-cooling, followed by polishing with 0.05 μm alumina slurry using a low-speed metallurgical polisher.
After transferring teeth to the Vickers Hardness Testing Machine (Buehler, Lake Bluff, IL, USA) six indentations on enamel and six indentations on dentin were done by a pyramid diamond indenter tip with 100 g load for 15 s. On enamel and dentin, the indentations were started 200 μm away from dentino-enamel junction and made toward the coronal surface on enamel and towards apical end on dentin, with keeping approximately 200 μm distance between the indentations.
Means of the hardness values (HV) for enamel and dentin were separately calculated per sample, and data was processed using SPSS 15.0 (SPSS, Inc., Chicago, IL, USA). First, normalization of the data was evaluated, and statistically homogenous data distribution was verified using one-sample Kolmogorov-Smirnov test, and two-way ANOVA was used.