2.1

Experimental design

The factor under study was the incorporation of different concentrations of TiO ₂ -nt (w/w) into self-adhesive resin cement RelyX U200™. For degree of conversion (DC), the study factors were as follows: (1) concentration of TiO ₂ -nt (0.3, 0.6 and 0.9 wt.%), (2) curing mode dual- and self-curing and (3) time (3, 6, 9, 12 and 15 min). For water sorption (WS) and solubility (SL), flexural strength ( σf ), modulus of elasticity (E) and hardness (H), the study factors were: (1) concentration of TiO ₂ -nt (0.3, 0.6 and 0.9 wt%) and (2) curing mode (dual- and self-curing). For cell viability study, the factors were as follows: (1) concentration of TiO ₂ -nt (0.3, 0.6 and 0.9 wt.%) and (2) time (24, 48 and 72 h). Two polymerization conditions were used: self- and dual-cured polymerization. For the self-curing condition, the cement handling and the specimens’ preparation were performed in an environment with room light. Thirty minutes after cement handling, specimens were removed from the custom devices used in each test and stored in distilled water at 37 °C. For the dual-curing condition, the specimens were handled in an environment with room light, embedded in custom devices used in each test of the study. Nine minutes after handling of self-adhesive resin cement, the light source 780 mW/cm ³ DB LED 686 (Dabi Atlante, Ribeirão Preto, Brazil) was applied for 20 s in different areas of the specimens so that the whole specimen was reached by light. The 9 min time was used once delay in light activation had shown to improve properties of resin-based cements .

The specimens of the self-adhesive resin cement were randomly assigned into 8 groups: SCT = self-adhesive resin cement in self-cure condition, which has no TiO ₂ -nt; S03 = self-adhesive resin cement in self-cure condition with the addition of 0.3 wt.% of TiO ₂ -nt; S06 = self-adhesive resin cement in self-cure condition with the addition of 0.6 wt.% of TiO ₂ -nt; S09 = self-adhesive resin cement in self-cure condition with the addition of 0.9 wt.% of TiO ₂ -nt; DCT = self-adhesive resin cement in dual-cure condition, which has no TiO ₂ -nt; D03 = self-adhesive resin cement in dual-cure condition with the addition of 0.3 wt.% of TiO ₂ -nt, dual cure; D06 = self-adhesive resin cement in dual-cure condition with the addition of 0.6 wt.% of TiO ₂ -nt; D09 = self-adhesive resin cement in dual-cure condition with the addition of 0.9 wt.% of TiO ₂ -nt.

2.2

Specimen preparation

TiO ₂ -nt were obtained from a commercial mixture of anatase TiO ₂ powder (Sigma–Aldrich, St. Louis, USA) mixed with an alkaline solution of sodium hydroxide (NaOH; 10 M), which remained for 24 h at 120 °C in an atmospheric pressure environment Teflon container. Then, the mixture was washed with deionized water and hydrochloric acid (HCl; 0.1 M) sequentially and repeatedly until reaching a pH of 4. After that, the mixture were placed in a furnace at 200 °C for 24 h to eliminate the liquid phase and to obtain the final powdered material . The nanotubes were measured with open source Image J software and had an average diameter of 10 nm and were 200 nm in length, formed by a single sheet of spiral-wound TiO _2. The image was obtained by Transmission Electron Microscopy (TEM) (CM 200, Phillps, Netherlands) with electrons acceleration of 200 kV . The resin cement that had the same portion of base and catalyst paste dispensed by packing clicker was dispensed on a mixing pad and weighed on a precision scale of 0.0001 g (Denver Instrument, São Paulo, Brazil). Then, the nanotubes were weighed with the value corresponding to the resin cement weigh. TiO ₂ -nt was manually added to the base paste and handled for 10 s. Then, the base paste and TiO ₂ -nt were mixed with the catalyst paste for another 10 s.

2.3

Degree of conversion

The DC was evaluated by Fourier transformed infrared spectroscopy (IRPrestige-21, Shimadzu, Tokyo, Japan). The analysis was performed in reflectance mode using the attenuated total reflectance (ATR) accessory (MIRacle, Pike Technologies, Madison, USA). Cement specimens (n = 3) were analyzed at 0, 3, 6, 9, 12 and 15 min after mixing base and catalyst pastes under self- and dual-cure polymerization conditions. After mixing the pastes and adding or not the nanotubes, small portions were placed covering the ATR crystal and the initial FT-IR spectra was obtained. For the self-cured groups, the resin cements were kept undisturbed and protected from light. For dual-cured groups, the light source 780 mW/cm ³ was applied for 20 s on self-adhesive resin cement 9 min after the application on ATR crystal. The infrared spectrum of the specimen was obtained in the range of 1560–1760 cm ⁻¹ with 10 scans and consecutive resolution of 4 cm ⁻¹ . Spectral bands were obtained, and the peak wavelength of aliphatic 1638 cm ⁻¹ (functional groups C C) and aromatic 1608 cm ⁻¹ was identified for each time studied ( Fig. 1 ). The DC was calculated using the equation:

where R is the ratio of the absorbance at 1638 cm ⁻¹ and absorbance 1608 cm ⁻¹ within the time determined for each record of the analysis (represented by T in the equation).

Peaks at 1608 and 1638 cm ⁻¹ identified in the spectral bands used for calculating the degree of conversion before polymerization (0 min) and after mixing base and catalyst pastes (3, 6, 9, 12 and 15 min).

2.4

Sorption (WS) and solubility (SL)

For WS and SL, specimens were prepared (n = 8) for each of the 8 groups using a metallic mold with a cylindrical shape of 10 mm in diameter and 1-mm-thick. The method used was based on the ISO 4049 . The specimens were kept in dry and dark storage for 24 h, and then transferred to a desiccator at 37 ± 1 °C. After 22 h in the first desiccator, the specimens were removed, stored in a second desiccator at 25 ± 1 °C for 2 h, and then weighed, using a 0.0001 precision analytical balance (Denver Instrument, Sao Paulo, Brazil) until a constant weight for each specimen (m1) was obtained (with no more than a ±0.0001 g variation). The specimens were then immersed in deionized water at 37 °C. At fixed intervals, the specimens were removed from the water, blot-dried (in sequence using two absorbent papers) and weighed until a constant mass (m2) was obtained. The specimens were then stored in a desiccator in the presence of silica at 37 °C and weighed until a constant weight (m3) was obtained, following the cycle described above for m1. The values for WS and SL (in μg/mm ³ ) were calculated using the following equations:

where m ₁ is the specimen weight before immersion, m ₂ is the specimen weight after immersion, m ₃ is the specimen weight after immersion and desiccation, and V is the volume of the specimen.

2.5

Mechanical properties

2.5.1

Flexural strength ( σf ) and elastic modulus (E)

Resin cement bars (n = 10) were made for each group according to the polymerization conditions and addition of TiO ₂ -nt. After mixing, the resin cement was inserted in a stainless steel mold for standardizing specimens dimensions (2 × 2 × 25 mm), as per ISO 9917-2 . The specimens remained in distilled water at 37 °C for 24 h before testing. The σf at three points was determined using a universal testing machine (Instron, Barueri, Brazil) with load-cell connected 50 N and a crosshead speed of 0.5 mm/min. The dimensions of the specimens were previously measured with a digital caliper (Starrett, Itu, São Paulo, Brazil). To perform the 3-point σf , the specimens were placed on the device with a distance of 12 mm between the lower cylindrical support and the load applied to the center of the upper metal rod. Flexural-strength values ​​were determined according to the equation:

where P is fracture load, (N) L is the distance between the supports (12 mm), w is the specimen width (mm) and b is the thickness (mm).

2.5.2

Surface microhardness

Microhardness measurements were obtained from 48 disc-shaped specimens (10 × 2 mm; n = 6) on the surface using a microhardness instrument (Buehler, Lake Bluff, USA) with a Knoop diamond under a 50 g load for 10 s. Before the measurements, the surface of specimens was polished with 600, 800 and 1200-grit SiC (Extec CORP., Enfield, CT, USA, #1060-524) papers for 2 min each, respectively. The measurement of the indentation was performed immediately after the period of 10 s. Five indentations spaced 0.5 mm from each other were made in the central area of each specimen, and the arithmetical average was used for subsequent statistical analysis.

2.6

Biological analyses

2.6.1

Preparation of specimens

Four disks (10 mm ø × 2 mm) of resin cement were prepared for each concentration-studied TiO ₂ -nt. After light curing, the specimens were autoclaved separately. To evaluate cell viability (n = 8), an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed using only the dual-curing condition. The studied groups were DCT, D03, D06, D09, positive control (CP) and negative control (CN).

2.6.2

Cell culture

For cell culture, mouse fibroblasts were used in the NIH3T3 line P13 (ATCC ^® , Manassas, USA). The cells were cultured in DMEM-Dulbecco’s modified Eagle (Aldrich ^® Sigma, St. Louis, USA), supplemented with 1% antibiotics (penicillin/streptomycin from Gibco ^® , Waltham, USA) and 10% FBS (fetal bovine serum from Gibco ^® ) and remained incubated at 37 °C with 5% carbon dioxide (CO ₂ ). After reaching a subconfluence stage, a subculture was formed using the trypsin enzyme responsible for dissociating the culture bottle cells (0.25% trypsin, 1 mM EDTA from Sigma–Aldrich ^® ). After 5 min in an oven at 37 °C with 5% (CO ₂ ), the trypsin was inactivated with DMEM culture medium with 10% FCS. The cells were transferred to a 50-ml falcon tube (Corning, NY, USA) and centrifuged at 1200 rpm for 5 min at 20 °C. After centrifugation, the supernatant was discarded, and the cells were suspended again in a fresh DMEM medium with 10% FCS. After that, the cell count was performed using a trypan blue protocol optical microscope (Olympus, Japan). This dye is responsible for staining the viable cells as it penetrates the dead cells because their membranes can no longer eliminate it. Subsequently, the cells were plated in 96-well microplates (TPP ^® , Trasadingen, Switzerland) for a colorimetric assay, 8-well microplates for each group. The resin-cement specimens were placed in contact with the culture medium following the recommendations of ISO 10993-5 (1 cm ² /ml). To this end, the total area of the disks was calculated (28.26 cm ² ) to evaluate the amount of conditioned media (28.26 ml). The specimens remained in the oven at 37 °C for 24 h. After this, the pH at which the disks were immersed was measured. For the viability assays, cells were plated 2 × 103/well in 96-well plates (TPP ^® ). After the enrollment period of 24 h, the culture medium was replaced by DMEM 10% FCS conditioned with the cement specimens. Each plate was examined at an experimental time of 24, 48 and 72 h after the addition of conditioned medium. After each experimental period, the culture medium was removed, the cells were washed with phosphate buffered saline solution (PBS) and then an MTT reduction test.

2.6.3

Analysis of cell metabolism MTT assay

During each experimental period (24, 48 and 72 h), the cells were washed with PBS and then incubated in 0.5 mg/ml solution (MTT/DMEM) for an MTT assay. This solution was prepared at the time of use and filtered through a Millipore filter (0.22 mM) before being added to the plates. After this procedure, the plates remained in the greenhouse for 4 h at 37 °C and 5% CO ₂ ; then, the solution was removed, and the insoluble pigment reduced intracellularly was taken up in dimethylsulfoxide and left at room temperature for 30 min. The absorbance was measured at 562 nm wavelength reader (Synergy H1 monochromator-based, Biotek, Winooski, USA).

2.7

Statistical analyses

Data were analyzed using the Shapiro–Wilk normality test. The DC data were subjected to ANOVA with repeated-measures followed by Tukey’s HSD ( α = 0.05). WS, SL and each mechanical and biological property were submitted to two-way ANOVA and Tukey’s HSD ( α = 0.05).

2.1

Experimental design

The factor under study was the incorporation of different concentrations of TiO ₂ -nt (w/w) into self-adhesive resin cement RelyX U200™. For degree of conversion (DC), the study factors were as follows: (1) concentration of TiO ₂ -nt (0.3, 0.6 and 0.9 wt.%), (2) curing mode dual- and self-curing and (3) time (3, 6, 9, 12 and 15 min). For water sorption (WS) and solubility (SL), flexural strength ( σf ), modulus of elasticity (E) and hardness (H), the study factors were: (1) concentration of TiO ₂ -nt (0.3, 0.6 and 0.9 wt%) and (2) curing mode (dual- and self-curing). For cell viability study, the factors were as follows: (1) concentration of TiO ₂ -nt (0.3, 0.6 and 0.9 wt.%) and (2) time (24, 48 and 72 h). Two polymerization conditions were used: self- and dual-cured polymerization. For the self-curing condition, the cement handling and the specimens’ preparation were performed in an environment with room light. Thirty minutes after cement handling, specimens were removed from the custom devices used in each test and stored in distilled water at 37 °C. For the dual-curing condition, the specimens were handled in an environment with room light, embedded in custom devices used in each test of the study. Nine minutes after handling of self-adhesive resin cement, the light source 780 mW/cm ³ DB LED 686 (Dabi Atlante, Ribeirão Preto, Brazil) was applied for 20 s in different areas of the specimens so that the whole specimen was reached by light. The 9 min time was used once delay in light activation had shown to improve properties of resin-based cements .

The specimens of the self-adhesive resin cement were randomly assigned into 8 groups: SCT = self-adhesive resin cement in self-cure condition, which has no TiO ₂ -nt; S03 = self-adhesive resin cement in self-cure condition with the addition of 0.3 wt.% of TiO ₂ -nt; S06 = self-adhesive resin cement in self-cure condition with the addition of 0.6 wt.% of TiO ₂ -nt; S09 = self-adhesive resin cement in self-cure condition with the addition of 0.9 wt.% of TiO ₂ -nt; DCT = self-adhesive resin cement in dual-cure condition, which has no TiO ₂ -nt; D03 = self-adhesive resin cement in dual-cure condition with the addition of 0.3 wt.% of TiO ₂ -nt, dual cure; D06 = self-adhesive resin cement in dual-cure condition with the addition of 0.6 wt.% of TiO ₂ -nt; D09 = self-adhesive resin cement in dual-cure condition with the addition of 0.9 wt.% of TiO ₂ -nt.

2.2

Specimen preparation

TiO ₂ -nt were obtained from a commercial mixture of anatase TiO ₂ powder (Sigma–Aldrich, St. Louis, USA) mixed with an alkaline solution of sodium hydroxide (NaOH; 10 M), which remained for 24 h at 120 °C in an atmospheric pressure environment Teflon container. Then, the mixture was washed with deionized water and hydrochloric acid (HCl; 0.1 M) sequentially and repeatedly until reaching a pH of 4. After that, the mixture were placed in a furnace at 200 °C for 24 h to eliminate the liquid phase and to obtain the final powdered material . The nanotubes were measured with open source Image J software and had an average diameter of 10 nm and were 200 nm in length, formed by a single sheet of spiral-wound TiO _2. The image was obtained by Transmission Electron Microscopy (TEM) (CM 200, Phillps, Netherlands) with electrons acceleration of 200 kV . The resin cement that had the same portion of base and catalyst paste dispensed by packing clicker was dispensed on a mixing pad and weighed on a precision scale of 0.0001 g (Denver Instrument, São Paulo, Brazil). Then, the nanotubes were weighed with the value corresponding to the resin cement weigh. TiO ₂ -nt was manually added to the base paste and handled for 10 s. Then, the base paste and TiO ₂ -nt were mixed with the catalyst paste for another 10 s.

2.3

Degree of conversion

The DC was evaluated by Fourier transformed infrared spectroscopy (IRPrestige-21, Shimadzu, Tokyo, Japan). The analysis was performed in reflectance mode using the attenuated total reflectance (ATR) accessory (MIRacle, Pike Technologies, Madison, USA). Cement specimens (n = 3) were analyzed at 0, 3, 6, 9, 12 and 15 min after mixing base and catalyst pastes under self- and dual-cure polymerization conditions. After mixing the pastes and adding or not the nanotubes, small portions were placed covering the ATR crystal and the initial FT-IR spectra was obtained. For the self-cured groups, the resin cements were kept undisturbed and protected from light. For dual-cured groups, the light source 780 mW/cm ³ was applied for 20 s on self-adhesive resin cement 9 min after the application on ATR crystal. The infrared spectrum of the specimen was obtained in the range of 1560–1760 cm ⁻¹ with 10 scans and consecutive resolution of 4 cm ⁻¹ . Spectral bands were obtained, and the peak wavelength of aliphatic 1638 cm ⁻¹ (functional groups C C) and aromatic 1608 cm ⁻¹ was identified for each time studied ( Fig. 1 ). The DC was calculated using the equation:

where R is the ratio of the absorbance at 1638 cm ⁻¹ and absorbance 1608 cm ⁻¹ within the time determined for each record of the analysis (represented by T in the equation).

Peaks at 1608 and 1638 cm ⁻¹ identified in the spectral bands used for calculating the degree of conversion before polymerization (0 min) and after mixing base and catalyst pastes (3, 6, 9, 12 and 15 min).

2.4

Sorption (WS) and solubility (SL)

For WS and SL, specimens were prepared (n = 8) for each of the 8 groups using a metallic mold with a cylindrical shape of 10 mm in diameter and 1-mm-thick. The method used was based on the ISO 4049 . The specimens were kept in dry and dark storage for 24 h, and then transferred to a desiccator at 37 ± 1 °C. After 22 h in the first desiccator, the specimens were removed, stored in a second desiccator at 25 ± 1 °C for 2 h, and then weighed, using a 0.0001 precision analytical balance (Denver Instrument, Sao Paulo, Brazil) until a constant weight for each specimen (m1) was obtained (with no more than a ±0.0001 g variation). The specimens were then immersed in deionized water at 37 °C. At fixed intervals, the specimens were removed from the water, blot-dried (in sequence using two absorbent papers) and weighed until a constant mass (m2) was obtained. The specimens were then stored in a desiccator in the presence of silica at 37 °C and weighed until a constant weight (m3) was obtained, following the cycle described above for m1. The values for WS and SL (in μg/mm ³ ) were calculated using the following equations:

where m ₁ is the specimen weight before immersion, m ₂ is the specimen weight after immersion, m ₃ is the specimen weight after immersion and desiccation, and V is the volume of the specimen.

2.5

Mechanical properties

2.5.1

Flexural strength ( σf ) and elastic modulus (E)

Resin cement bars (n = 10) were made for each group according to the polymerization conditions and addition of TiO ₂ -nt. After mixing, the resin cement was inserted in a stainless steel mold for standardizing specimens dimensions (2 × 2 × 25 mm), as per ISO 9917-2 . The specimens remained in distilled water at 37 °C for 24 h before testing. The σf at three points was determined using a universal testing machine (Instron, Barueri, Brazil) with load-cell connected 50 N and a crosshead speed of 0.5 mm/min. The dimensions of the specimens were previously measured with a digital caliper (Starrett, Itu, São Paulo, Brazil). To perform the 3-point σf , the specimens were placed on the device with a distance of 12 mm between the lower cylindrical support and the load applied to the center of the upper metal rod. Flexural-strength values ​​were determined according to the equation:

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