2.1

Characterization of materials

Both un-hydrated cement powders and the set materials were characterized by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD) analysis.

2.1.1

Microscopy and elemental analysis

The powders were prepared by mixing the powder with epoxy resin (Epoxyfix, Struers GmbH, Ballerup, Denmark). For the hydrated materials cylindrical specimens 10 mm in diameter and 2 mm high were prepared and stored in Hank’s Balanced Salt Solution (HBSS, H6648, Sigma–Aldrich) for 28 days at 37 °C. At the end of the storage period the specimens were removed form the solution and dried in a vacuum desiccator. They were then embedded in resin (Epoxyfix, Struers GmbH, Ballerup, Denmark). Both embedded powders and set materials were ground and polished using progressively finer diamond discs and pastes using an automatic polishing machine (Tegramin 20, Struers GmbH, Ballerup, Denmark). The polished specimens were attached to aluminum stubs, carbon coated and viewed under the scanning electron microscope (SEM; Zeiss MERLIN Field Emission SEM, Carl Zeiss NTS GmbH, Oberkochen, Germany). Scanning electron micrographs of the different material microstructural components at different magnifications in back-scatter electron mode were captured and energy dispersive spectroscopy (EDS) was carried out.

2.1.2

X-ray diffraction (XRD) analysis

Phase analysis was carried out using X-ray diffraction. After storage in HBSS for 28 days the set materials were dried in a vacuum desiccator and crushed to a very fine powder using an agate mortar and pestle. The diffractometer (Rigaku, Tokyo, Japan) used Cu Kα radiation at 40 mA and 45 kV and the detector was set to rotate between 15° and 45°, with a sampling width of 0.05° and scan speed of 1°/min at 15 revs/min using the Bragg Brentano method. Phase identification was accomplished using a search-match software utilizing ICDD database (International Centre for Diffraction Data, Newtown Square, PA, USA).

2.2

pH and calcium ion release in solution

Eighty acrylic teeth ( n = 10) made of resin with a cavity of 3-mm depth were filled with the different materials and immersed individually in 10 mL of deionized water and stored at 37 °C. To avoid any interference with the results, all glass flasks were pre-treated with nitric acid. After 3, 24, 72 and 168 h, the teeth were placed in new flasks containing an equal volume of new deionized water. The pH of the water in which the teeth had been kept was measured with a pH meter (model 371; Micronal, São Paulo, SP, Brazil), previously calibrated using buffer solutions of pH 4, 7 and 14. After the removal of the specimens, the container was placed in a shaker (model 251; Farmem, São Paulo, SP, Brazil) for 5 s before measuring. The temperature of the room during the reading was 25 °C.

For determination of calcium ion release, an atomic absorption spectrophotometer (AA6800; Schimadzu, Tokyo, Japan) equipped with a calcium- specific hollow cathode lamp was used. The water in which calcium ion release was measured was the same used in the pH measurements. To prevent potential interferences from alkaline metals, a solution of lanthanum was prepared by diluting 9.8 g of lanthanum nitrate in 250 mL of acid solution. A stock solution of calcium was prepared by diluting 2.4972 g of calcium carbonate in 50 mL of ultrapure water. A total of 10 mL of concentrated hydrochloric acid was added to this solution. Subsequently, it was diluted with 1000 mL of ultrapure water. After the preparation, 1 mL of this solution corresponded to 1 mg of calcium. With this solution, standard solutions containing 10, 20, 40 and 80 mg L ⁻¹ of calcium were prepared. Then, 2 mL of lanthanum nitrate solution was added to 6 mL of the standard calcium or test solutions. For the preparation of the blank solution, the same amount of lanthanum nitrate solution was added to 6 mL of ultrapure water. The calcium standards, the blank and the test solutions were read in the atomic absorption spectrophotometer. To bring the device to zero absorbance, a solution of nitric acid was used. The readings of the calcium ion release were compared with a standard curve obtained from readings of the standard solutions. This reading was performed in the same periods used for the pH level measurement.

2.3

Radiopacity

Three cylindrical samples of each sealer with 10 mm in diameter and 1 mm in thickness were prepared. The thickness was checked with a digital caliper (Mitutoyo Corp, Tokyo, Japan) and the samples radiographed on occlusal films (D-speed; Kodak Comp, Rochester, NY) with an aluminum step-wedge graduated from 2 to 16 mm. A radiographic unit (Gnatus XR 6010; Gnatus, Ribeirão Preto, SP, Brazil) was used with exposures set at 60 kVp, 10 mA, 0.3 s and a focus-film distance of 30 cm. The radiographs were digitized and analyzed using Digora 1.51 software (Soredex, Helsinki, Finland). The radiopacity was determined as previously described .

2.4

Flowability

A volume of 0.5 mL of sealer was placed on a glass plate according to ANSI/ADA’s specification No. 57/2000 . Three minutes after the start of mixing, another plate with a mass of 20 ± 2 g and a load of 100 g plus was applied centrally on top of the plate. Ten minutes after the start of mixing, the load was removed, and the average of the major and minor diameters of the compressed sealer was measured using a digital caliper (Mitutoyo MTI Corporation, Tokyo, Japan). Three measurements were performed for each sealer.

2.5

Film thickness

Two 5-mm-thick flat glass plates were joined and their thickness measured. A total of 0.5 mL of sealer was placed on the center of one plate and the second was positioned centrally on top of the sealer, according to ANSI/ADA No. 57/2000 . Three minutes after the start of mixing, a load of 150 N was applied vertically on top of the glass plate. Ten minutes after the start of mixing, the thickness of the two glass plates and the interposed sealer was measured with a digital caliper (Mitutoyo MTI Corporation). The difference of three measurements for each sealer in thickness of the two glass plates, with and without sealer, was taken as the film thickness of the material.

2.6

Solubility

Three 1.5-mm-thick cylindrical polytetrafluoroethylene molds with an inner diameter of 20 mm were filled with freshly mixed sealer. The mold was supported by a larger glass plate and covered with a cellophane sheet. A nylon thread was placed inside the material, and another glass plate, also covered with cellophane film in such a way that the plates touched the entire mold in a uniform manner. The assembly was placed in an incubator (37 °C, 95% relative humidity) for a period corresponding to three times the setting time. The sealers were removed from the mold and weighed three times each with an accuracy of 0.0001 g (UMark 210, Bel Engineering, Monza, Italy). The samples were suspended by nylon thread and placed two by two inside a plastic vessel containing 50 mL of deionized distilled water. The containers were stored for 24 h in an incubator (37 °C, 95% relative humidity). Then, the samples were rinsed with deionized distilled water, blotted dry with absorbent paper, placed in desiccators for 24 h, and then reweighed. The experiment was repeated three times for each sealer. The percentage of weight loss of each sample (initial mass minus final mass) was considered the solubility of the sealer, according to ANSI/ADA specification No. 57/2000 .

2.7

Assessment of material porosity

The porosity of the set sealers was assessed after 28-day hydration in Hank’s Balanced Salt Solution (HBSS; H6648, Sigma Aldrich, St. Louis, MO, USA). The method of mercury intrusion porosimetry was used to measure the average pore diameter and percentage of porosity. Three cube specimens 7 mm × 7 mm × 7 mm were prepared for each material, allowed to set for 24 h at 37 °C and subsequently immersed in at 37 °C for 1 or 28 days. The materials were taken out of solution and dried for 4 days in an incubator at 60 °C. The porosity was measured in a two-stage process using a mercury intrusion porosimeter (PoreMaster, Quantachrome Instruments, New York, NY, USA). Calibrated mercury displacement pycnometry followed by low and high-pressure mercury intrusion porosimetry were performed. The volume of mercury intruded in the pores of the specimen was measured. Porosimetry data was processed using the software supplied with the machine (Poremaster, Quantachrome Instruments, New York, NY, USA).

A gas pycnometer (Quantachrome Instruments, New York, NY, USA) with helium was used to determine the average absolute density (g/cm ³ ). The pycnometer operates on Archimedes principle of gas displacement to determine the volume. Density of the sample can be calculated from the sample weight and volume.

2.8

Statistical analysis

The statistical analysis for porosity was performed using Kruskal–Wallis and Dunn’s tests and for physical tests, pH and calcium ion release, one-way ANOVA and Tukey’s tests as a result of normal distribution confirmed by Kolmogorov–Smirnov test ( p < 0.05). The data were evaluated using SPSS (Statistical Package for the Social Sciences) software (PASW Statistics 18; SPSS Inc., Chicago, IL, USA).

2.1

Characterization of materials

Both un-hydrated cement powders and the set materials were characterized by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD) analysis.

2.1.1

Microscopy and elemental analysis

The powders were prepared by mixing the powder with epoxy resin (Epoxyfix, Struers GmbH, Ballerup, Denmark). For the hydrated materials cylindrical specimens 10 mm in diameter and 2 mm high were prepared and stored in Hank’s Balanced Salt Solution (HBSS, H6648, Sigma–Aldrich) for 28 days at 37 °C. At the end of the storage period the specimens were removed form the solution and dried in a vacuum desiccator. They were then embedded in resin (Epoxyfix, Struers GmbH, Ballerup, Denmark). Both embedded powders and set materials were ground and polished using progressively finer diamond discs and pastes using an automatic polishing machine (Tegramin 20, Struers GmbH, Ballerup, Denmark). The polished specimens were attached to aluminum stubs, carbon coated and viewed under the scanning electron microscope (SEM; Zeiss MERLIN Field Emission SEM, Carl Zeiss NTS GmbH, Oberkochen, Germany). Scanning electron micrographs of the different material microstructural components at different magnifications in back-scatter electron mode were captured and energy dispersive spectroscopy (EDS) was carried out.

2.1.2

X-ray diffraction (XRD) analysis

Phase analysis was carried out using X-ray diffraction. After storage in HBSS for 28 days the set materials were dried in a vacuum desiccator and crushed to a very fine powder using an agate mortar and pestle. The diffractometer (Rigaku, Tokyo, Japan) used Cu Kα radiation at 40 mA and 45 kV and the detector was set to rotate between 15° and 45°, with a sampling width of 0.05° and scan speed of 1°/min at 15 revs/min using the Bragg Brentano method. Phase identification was accomplished using a search-match software utilizing ICDD database (International Centre for Diffraction Data, Newtown Square, PA, USA).

2.2

pH and calcium ion release in solution

Eighty acrylic teeth ( n = 10) made of resin with a cavity of 3-mm depth were filled with the different materials and immersed individually in 10 mL of deionized water and stored at 37 °C. To avoid any interference with the results, all glass flasks were pre-treated with nitric acid. After 3, 24, 72 and 168 h, the teeth were placed in new flasks containing an equal volume of new deionized water. The pH of the water in which the teeth had been kept was measured with a pH meter (model 371; Micronal, São Paulo, SP, Brazil), previously calibrated using buffer solutions of pH 4, 7 and 14. After the removal of the specimens, the container was placed in a shaker (model 251; Farmem, São Paulo, SP, Brazil) for 5 s before measuring. The temperature of the room during the reading was 25 °C.

For determination of calcium ion release, an atomic absorption spectrophotometer (AA6800; Schimadzu, Tokyo, Japan) equipped with a calcium- specific hollow cathode lamp was used. The water in which calcium ion release was measured was the same used in the pH measurements. To prevent potential interferences from alkaline metals, a solution of lanthanum was prepared by diluting 9.8 g of lanthanum nitrate in 250 mL of acid solution. A stock solution of calcium was prepared by diluting 2.4972 g of calcium carbonate in 50 mL of ultrapure water. A total of 10 mL of concentrated hydrochloric acid was added to this solution. Subsequently, it was diluted with 1000 mL of ultrapure water. After the preparation, 1 mL of this solution corresponded to 1 mg of calcium. With this solution, standard solutions containing 10, 20, 40 and 80 mg L ⁻¹ of calcium were prepared. Then, 2 mL of lanthanum nitrate solution was added to 6 mL of the standard calcium or test solutions. For the preparation of the blank solution, the same amount of lanthanum nitrate solution was added to 6 mL of ultrapure water. The calcium standards, the blank and the test solutions were read in the atomic absorption spectrophotometer. To bring the device to zero absorbance, a solution of nitric acid was used. The readings of the calcium ion release were compared with a standard curve obtained from readings of the standard solutions. This reading was performed in the same periods used for the pH level measurement.

2.3

Radiopacity

Three cylindrical samples of each sealer with 10 mm in diameter and 1 mm in thickness were prepared. The thickness was checked with a digital caliper (Mitutoyo Corp, Tokyo, Japan) and the samples radiographed on occlusal films (D-speed; Kodak Comp, Rochester, NY) with an aluminum step-wedge graduated from 2 to 16 mm. A radiographic unit (Gnatus XR 6010; Gnatus, Ribeirão Preto, SP, Brazil) was used with exposures set at 60 kVp, 10 mA, 0.3 s and a focus-film distance of 30 cm. The radiographs were digitized and analyzed using Digora 1.51 software (Soredex, Helsinki, Finland). The radiopacity was determined as previously described .

2.4

Flowability

A volume of 0.5 mL of sealer was placed on a glass plate according to ANSI/ADA’s specification No. 57/2000 . Three minutes after the start of mixing, another plate with a mass of 20 ± 2 g and a load of 100 g plus was applied centrally on top of the plate. Ten minutes after the start of mixing, the load was removed, and the average of the major and minor diameters of the compressed sealer was measured using a digital caliper (Mitutoyo MTI Corporation, Tokyo, Japan). Three measurements were performed for each sealer.

2.5

Film thickness

Two 5-mm-thick flat glass plates were joined and their thickness measured. A total of 0.5 mL of sealer was placed on the center of one plate and the second was positioned centrally on top of the sealer, according to ANSI/ADA No. 57/2000 . Three minutes after the start of mixing, a load of 150 N was applied vertically on top of the glass plate. Ten minutes after the start of mixing, the thickness of the two glass plates and the interposed sealer was measured with a digital caliper (Mitutoyo MTI Corporation). The difference of three measurements for each sealer in thickness of the two glass plates, with and without sealer, was taken as the film thickness of the material.

2.6

Solubility

Three 1.5-mm-thick cylindrical polytetrafluoroethylene molds with an inner diameter of 20 mm were filled with freshly mixed sealer. The mold was supported by a larger glass plate and covered with a cellophane sheet. A nylon thread was placed inside the material, and another glass plate, also covered with cellophane film in such a way that the plates touched the entire mold in a uniform manner. The assembly was placed in an incubator (37 °C, 95% relative humidity) for a period corresponding to three times the setting time. The sealers were removed from the mold and weighed three times each with an accuracy of 0.0001 g (UMark 210, Bel Engineering, Monza, Italy). The samples were suspended by nylon thread and placed two by two inside a plastic vessel containing 50 mL of deionized distilled water. The containers were stored for 24 h in an incubator (37 °C, 95% relative humidity). Then, the samples were rinsed with deionized distilled water, blotted dry with absorbent paper, placed in desiccators for 24 h, and then reweighed. The experiment was repeated three times for each sealer. The percentage of weight loss of each sample (initial mass minus final mass) was considered the solubility of the sealer, according to ANSI/ADA specification No. 57/2000 .

2.7

Assessment of material porosity

The porosity of the set sealers was assessed after 28-day hydration in Hank’s Balanced Salt Solution (HBSS; H6648, Sigma Aldrich, St. Louis, MO, USA). The method of mercury intrusion porosimetry was used to measure the average pore diameter and percentage of porosity. Three cube specimens 7 mm × 7 mm × 7 mm were prepared for each material, allowed to set for 24 h at 37 °C and subsequently immersed in at 37 °C for 1 or 28 days. The materials were taken out of solution and dried for 4 days in an incubator at 60 °C. The porosity was measured in a two-stage process using a mercury intrusion porosimeter (PoreMaster, Quantachrome Instruments, New York, NY, USA). Calibrated mercury displacement pycnometry followed by low and high-pressure mercury intrusion porosimetry were performed. The volume of mercury intruded in the pores of the specimen was measured. Porosimetry data was processed using the software supplied with the machine (Poremaster, Quantachrome Instruments, New York, NY, USA).

A gas pycnometer (Quantachrome Instruments, New York, NY, USA) with helium was used to determine the average absolute density (g/cm ³ ). The pycnometer operates on Archimedes principle of gas displacement to determine the volume. Density of the sample can be calculated from the sample weight and volume.

2.8

Statistical analysis

The statistical analysis for porosity was performed using Kruskal–Wallis and Dunn’s tests and for physical tests, pH and calcium ion release, one-way ANOVA and Tukey’s tests as a result of normal distribution confirmed by Kolmogorov–Smirnov test ( p < 0.05). The data were evaluated using SPSS (Statistical Package for the Social Sciences) software (PASW Statistics 18; SPSS Inc., Chicago, IL, USA).