2.1

Physical properties

Specimens of GIC control and CPP–ACP modified cement (lot # 0609270) were made, and physical/mechanical properties were tested following the International Organization for Standardization (ISO) Standard 9917:2003 for dental water-based cements as outlined in the following sections.

2.2

Setting time

Five discs 12 mm diameter × 5 mm thick were made to determine the net setting time for each concentration of CPP–ACP. The tests were performed in an incubator maintained at 37 ± 1 °C with a relative humidity (RH) of at least 90% as specified in the ISO standard. After the end of mixing, an indenter needle of mass 400 ± 5 g and flat end of diameter 1.0 ± 0.1 mm was lowered vertically onto the surface of the cement for 5 s.

The net setting time was recorded as the time elapsed between the end of mixing and the time when the needle failed to make a complete indentation when viewed at 2× magnification.

2.3

Compressive strength

Cylindrical specimens 4 mm diameter ( D ) × 6 mm long ( t ) were made using brass molds. One hour after the end of mixing, the ends or each sample were ground flat using wet 600 grit silicon carbide paper, then removed from the molds and stored in 100% RH for 24 h prior to testing. The specimens were tested using an Instron universal testing machine at a crosshead speed of 1 mm/min. Each specimen was placed with the flat ends between the platens of the testing machine, and compressive load was applied along the long axis of the specimen. The compressive strength ( C ), measured in megapascals (MPa), was calculated using the following formula: C = 4 P / πD ² (where P is the maximum force applied in Newtons (N)).

2.4

Diametral tensile strength

Five cylindrical specimens were made using the same dimensions as for the compressive strength test and stored under the same conditions. After 24 h, a compressive load was applied across the diameter of each specimen, producing a tensile stress perpendicular to the axis of the loading. Load was applied at a crosshead speed of 1 mm/min, and recorded until the specimen fractured. The tensile stress ( T ) measured in MPa was calculated by the following formula: T = 2 P / πDt .

2.5

Flow test

The test was carried out whereby a sufficient amount of the cement was dispensed onto a glass plate, to fill a ring with a 3 mm internal diameter. This was followed by the placement of another glass plate on top of the setting GIC, then application of a 2.5 kg load onto the top plate. After 10 min, the load was removed and the major and minor diameters of the discs formed by the dispensed material were measured, and the mean value was recorded as the flow. The flow of each material should produce a disc with an average diameter >20 mm, and the difference between the measured diameters of the discs should not be more than 1 mm to pass the test.

2.6

Ion release measurement

Five discs 6 mm in diameter × 1.5 mm in thickness were made for each experimental material tested (GIC 0, 3 and 5% CPP–ACP lot # 0609270. GC Corporation, Tokyo, Japan). Six groups were formed which varied depending on the amount of CPP–ACP incorporated and storage medium. Fuji VII with 0% CPP–ACP served as the control group. The cement was mixed, dispensed into stainless steel molds, then compressed between two glass plates and allowed to set for 1 h in an incubator at 37 ± 1 °C and relative humidity of at least 90%. The discs were removed from the molds and stored in separate plastic tubes containing 2 mL solution of either de-ionized water (pH 6.9) (Milli-Q Water, Millipore Corporation. Melbourne, Victoria, Australia), or 50 mmol/L lactic acid buffer solution (pH 4.8). The tubes were stored in an incubator maintained at 37 ± 1 °C for the duration of the study. Each storage solution was changed every 24 h for the first 3 days, and then on days 7, 14, 21, and 28. The experiment was carried out for a further 4 month period for the GICs stored in water, and the storage solution was changed every 28 days in order to measure prolonged fluoride release.

The release of fluoride was measured using an ion-selective electrode (Radiometer Analytical, ISE C301F, France) connected to an ion analyzer (Radiometer Analytical S.A., Ion check 45, France). Standard solutions were prepared at concentrations ranging from 1 to 10 ppm for water storage solutions, and from 2 to 20 ppm for the analysis of acid storage solutions. Adequate dilution of the sample solutions and acid hydrolysis using HCl were performed following a previously described method in order to minimize interactions of ions that form complexes with fluoride which may interfere with accurate detection .

The release of calcium ions from control and CPP–ACP modified GIC was measured by atomic absorption spectrometry (Varian AA240 AAS, Varian Australia Pty. Ltd.) . Finally, the release of inorganic phosphate was determined colorimetrically using a spectrophotometer (UV–vis spectrophotometer, Varian Australia Pty. Ltd.) .

The degree of saturation with respect to calcium fluoride (DS CaF ₂ ) in solution was also calculated using a computer program, which takes into account fluoride, calcium, inorganic phosphate, sodium and chloride concentrations and pH in the calculations . Values of DS greater than one, equal to one, and less than one, represent states of supersaturation, saturation, and under saturation respectively with respect to CaF ₂ .

2.7

Acid demineralization test on enamel

Eighteen extracted human molars without caries or obvious defects were selected, stored in 0.5% chloramine-T and used-within 3–6 months following extraction. The use of extracted human teeth in this study was approved by the University of Melbourne Human Research Ethics Committee. The roots of the teeth were removed and the crowns were stored in distilled water at 4 °C.

Box-shaped cavities, 8 mm long × 2 mm wide × 2 mm deep were prepared close to the cemento-enamel junction of the lingual surface of extracted human molars. One cavity was prepared on each tooth using a flat fissure tungsten carbide bur 1 mm in diameter in a high-speed hand piece with air-water coolant. The cavity margins were finished with a slow speed steel flat fissure burs 0.9 mm in diameter to create a cavo-surface angle of 90°.

Following cavity preparation, 11 teeth were sectioned bucco-lingually to obtain two cavities per tooth. Each pair of cavities obtained from the same tooth was filled with two of the following materials:

• •

  Fuji VII control (0% CPP–ACP)–Fuji VII with 3% CPP–ACP;

• •

  Fuji VII control–Fuji VII with 5% CPP–ACP; or

• •

  Fuji VII with 3% CPP–ACP–Fuji VII with 5% CPP–ACP.

using 3, 4, 4 pairs of teeth respectively. GIC was allowed to set in a controlled environment maintained at 37 °C and 100% relative humidity (RH) for 24 h. This was allowed for setting and maturation of the GICs. All restorations were finished and polished using Soflex discs (Soflex coarse, medium, fine and superfine grit, 3M ESPE Dental Products, St. Paul, MN, USA), and the integrity of the margins of each restoration was verified under a light microscope at 25× magnification.

The cavities in the remaining seven teeth, without sectioning, were restored by bulk placement with a non-fluoride releasing hybrid resin composite (Point 4 hybrid resin composite lot # 448807; Ken Co., Orange, CA, USA) which was light cured for 40 s. All materials were mixed and placed according to manufacturers’ instructions. Two coats of acid-resistant nail varnish were applied to the tooth surface, leaving a window not less than approximately 1 mm wide surrounding the cavity margin on the occlusal side. Each tooth segment was stored in individual plastic containers containing 40 mL of acid buffer solution consisting of 50 mmol/L lactic acid (Analytical Reagent. Chem-Supply Pty. Ltd., Australia) adjusted to pH 4.8. The teeth were stored in the demineralizing solution for 4 days at 37 °C. The solution was replaced once after 48 h from the beginning of the experiment.

The teeth were removed from the lactate solution after 4 days and rinsed with deionized water. Each segment was embedded in epoxy resin (Polymer Daystar Pty. Ltd. VIC, Australia) and allowed to set for 24 h prior to sectioning to maintain the integrity of each section. Longitudinal sections approximately 500 μm thick were obtained using a water-cooled diamond saw (Diamond cut-off wheel, Struers, Denmark). The sections were wet ground and polished to a thickness of 80–100 μm using 1000, 2400, and 4000-grit silicon carbide abrasive papers (Waterproof sanding sheets, Norton Abrasive Pty. Ltd. NSW, Australia). The resulting sections were observed at the enamel margin of each cavity at 5× magnification under polarized light microscopy (Leica Microsystem Wetzlar GmbH. Ernst-Leitz-Strasse, Germany) to observe the degree of demineralization, and then photographed. A first order red wavelength plate was inserted between the crossed polarizers so as to highlight changes in the demineralized enamel. The area of each lesion was measured to a point arbitrarily set at 600 μm from the cavity-restoration margin, using a computer program (UTHSCSA Image tool for Windows, version 3.0; San Antonio, TX). A graticule with 10 μm line spacing was photographed and used to calculate lesion areas in μm ² .

2.8

Statistical analysis

Data were analyzed with one-way analysis of variance using Tukey’s method for multiple comparisons at the 5% level of confidence, in addition to the Student’s t -test. Nonparametric Kruskal–Wallis test was used to analyze the data when the assumptions of normality and constant variance could not be satisfied.

2.1

Physical properties

Specimens of GIC control and CPP–ACP modified cement (lot # 0609270) were made, and physical/mechanical properties were tested following the International Organization for Standardization (ISO) Standard 9917:2003 for dental water-based cements as outlined in the following sections.

2.2

Setting time

Five discs 12 mm diameter × 5 mm thick were made to determine the net setting time for each concentration of CPP–ACP. The tests were performed in an incubator maintained at 37 ± 1 °C with a relative humidity (RH) of at least 90% as specified in the ISO standard. After the end of mixing, an indenter needle of mass 400 ± 5 g and flat end of diameter 1.0 ± 0.1 mm was lowered vertically onto the surface of the cement for 5 s.

The net setting time was recorded as the time elapsed between the end of mixing and the time when the needle failed to make a complete indentation when viewed at 2× magnification.

2.3

Compressive strength

Cylindrical specimens 4 mm diameter ( D ) × 6 mm long ( t ) were made using brass molds. One hour after the end of mixing, the ends or each sample were ground flat using wet 600 grit silicon carbide paper, then removed from the molds and stored in 100% RH for 24 h prior to testing. The specimens were tested using an Instron universal testing machine at a crosshead speed of 1 mm/min. Each specimen was placed with the flat ends between the platens of the testing machine, and compressive load was applied along the long axis of the specimen. The compressive strength ( C ), measured in megapascals (MPa), was calculated using the following formula: C = 4 P / πD ² (where P is the maximum force applied in Newtons (N)).

2.4

Diametral tensile strength

Five cylindrical specimens were made using the same dimensions as for the compressive strength test and stored under the same conditions. After 24 h, a compressive load was applied across the diameter of each specimen, producing a tensile stress perpendicular to the axis of the loading. Load was applied at a crosshead speed of 1 mm/min, and recorded until the specimen fractured. The tensile stress ( T ) measured in MPa was calculated by the following formula: T = 2 P / πDt .

2.5

Flow test

The test was carried out whereby a sufficient amount of the cement was dispensed onto a glass plate, to fill a ring with a 3 mm internal diameter. This was followed by the placement of another glass plate on top of the setting GIC, then application of a 2.5 kg load onto the top plate. After 10 min, the load was removed and the major and minor diameters of the discs formed by the dispensed material were measured, and the mean value was recorded as the flow. The flow of each material should produce a disc with an average diameter >20 mm, and the difference between the measured diameters of the discs should not be more than 1 mm to pass the test.

2.6

Ion release measurement

Five discs 6 mm in diameter × 1.5 mm in thickness were made for each experimental material tested (GIC 0, 3 and 5% CPP–ACP lot # 0609270. GC Corporation, Tokyo, Japan). Six groups were formed which varied depending on the amount of CPP–ACP incorporated and storage medium. Fuji VII with 0% CPP–ACP served as the control group. The cement was mixed, dispensed into stainless steel molds, then compressed between two glass plates and allowed to set for 1 h in an incubator at 37 ± 1 °C and relative humidity of at least 90%. The discs were removed from the molds and stored in separate plastic tubes containing 2 mL solution of either de-ionized water (pH 6.9) (Milli-Q Water, Millipore Corporation. Melbourne, Victoria, Australia), or 50 mmol/L lactic acid buffer solution (pH 4.8). The tubes were stored in an incubator maintained at 37 ± 1 °C for the duration of the study. Each storage solution was changed every 24 h for the first 3 days, and then on days 7, 14, 21, and 28. The experiment was carried out for a further 4 month period for the GICs stored in water, and the storage solution was changed every 28 days in order to measure prolonged fluoride release.

The release of fluoride was measured using an ion-selective electrode (Radiometer Analytical, ISE C301F, France) connected to an ion analyzer (Radiometer Analytical S.A., Ion check 45, France). Standard solutions were prepared at concentrations ranging from 1 to 10 ppm for water storage solutions, and from 2 to 20 ppm for the analysis of acid storage solutions. Adequate dilution of the sample solutions and acid hydrolysis using HCl were performed following a previously described method in order to minimize interactions of ions that form complexes with fluoride which may interfere with accurate detection .

The release of calcium ions from control and CPP–ACP modified GIC was measured by atomic absorption spectrometry (Varian AA240 AAS, Varian Australia Pty. Ltd.) . Finally, the release of inorganic phosphate was determined colorimetrically using a spectrophotometer (UV–vis spectrophotometer, Varian Australia Pty. Ltd.) .

The degree of saturation with respect to calcium fluoride (DS CaF ₂ ) in solution was also calculated using a computer program, which takes into account fluoride, calcium, inorganic phosphate, sodium and chloride concentrations and pH in the calculations . Values of DS greater than one, equal to one, and less than one, represent states of supersaturation, saturation, and under saturation respectively with respect to CaF ₂ .

2.7

Acid demineralization test on enamel

Eighteen extracted human molars without caries or obvious defects were selected, stored in 0.5% chloramine-T and used-within 3–6 months following extraction. The use of extracted human teeth in this study was approved by the University of Melbourne Human Research Ethics Committee. The roots of the teeth were removed and the crowns were stored in distilled water at 4 °C.

Box-shaped cavities, 8 mm long × 2 mm wide × 2 mm deep were prepared close to the cemento-enamel junction of the lingual surface of extracted human molars. One cavity was prepared on each tooth using a flat fissure tungsten carbide bur 1 mm in diameter in a high-speed hand piece with air-water coolant. The cavity margins were finished with a slow speed steel flat fissure burs 0.9 mm in diameter to create a cavo-surface angle of 90°.

Following cavity preparation, 11 teeth were sectioned bucco-lingually to obtain two cavities per tooth. Each pair of cavities obtained from the same tooth was filled with two of the following materials:

• •

  Fuji VII control (0% CPP–ACP)–Fuji VII with 3% CPP–ACP;

• •

  Fuji VII control–Fuji VII with 5% CPP–ACP; or

• •

  Fuji VII with 3% CPP–ACP–Fuji VII with 5% CPP–ACP.

using 3, 4, 4 pairs of teeth respectively. GIC was allowed to set in a controlled environment maintained at 37 °C and 100% relative humidity (RH) for 24 h. This was allowed for setting and maturation of the GICs. All restorations were finished and polished using Soflex discs (Soflex coarse, medium, fine and superfine grit, 3M ESPE Dental Products, St. Paul, MN, USA), and the integrity of the margins of each restoration was verified under a light microscope at 25× magnification.

The cavities in the remaining seven teeth, without sectioning, were restored by bulk placement with a non-fluoride releasing hybrid resin composite (Point 4 hybrid resin composite lot # 448807; Ken Co., Orange, CA, USA) which was light cured for 40 s. All materials were mixed and placed according to manufacturers’ instructions. Two coats of acid-resistant nail varnish were applied to the tooth surface, leaving a window not less than approximately 1 mm wide surrounding the cavity margin on the occlusal side. Each tooth segment was stored in individual plastic containers containing 40 mL of acid buffer solution consisting of 50 mmol/L lactic acid (Analytical Reagent. Chem-Supply Pty. Ltd., Australia) adjusted to pH 4.8. The teeth were stored in the demineralizing solution for 4 days at 37 °C. The solution was replaced once after 48 h from the beginning of the experiment.

The teeth were removed from the lactate solution after 4 days and rinsed with deionized water. Each segment was embedded in epoxy resin (Polymer Daystar Pty. Ltd. VIC, Australia) and allowed to set for 24 h prior to sectioning to maintain the integrity of each section. Longitudinal sections approximately 500 μm thick were obtained using a water-cooled diamond saw (Diamond cut-off wheel, Struers, Denmark). The sections were wet ground and polished to a thickness of 80–100 μm using 1000, 2400, and 4000-grit silicon carbide abrasive papers (Waterproof sanding sheets, Norton Abrasive Pty. Ltd. NSW, Australia). The resulting sections were observed at the enamel margin of each cavity at 5× magnification under polarized light microscopy (Leica Microsystem Wetzlar GmbH. Ernst-Leitz-Strasse, Germany) to observe the degree of demineralization, and then photographed. A first order red wavelength plate was inserted between the crossed polarizers so as to highlight changes in the demineralized enamel. The area of each lesion was measured to a point arbitrarily set at 600 μm from the cavity-restoration margin, using a computer program (UTHSCSA Image tool for Windows, version 3.0; San Antonio, TX). A graticule with 10 μm line spacing was photographed and used to calculate lesion areas in μm ² .

2.8

Statistical analysis

Data were analyzed with one-way analysis of variance using Tukey’s method for multiple comparisons at the 5% level of confidence, in addition to the Student’s t -test. Nonparametric Kruskal–Wallis test was used to analyze the data when the assumptions of normality and constant variance could not be satisfied.