Material and methods

The denture base acrylic resins selected for this study are presented in Table 1 . Specimens (n=5) were made of 20 mm in diameter and 3.0 ±0.1 mm in thickness. Metal molds in these dimensions were made by using laboratory silicone (Zetalabor-Zhermack; Labordental) between two glass plates. The mold was embedded in metal (OGP) or plastic (Vipi Ltda) flasks with Type III stone (Herodent; Vigodent Coltène SA Ind. e Com.). The materials were handled according to the manufacturer instructions ( Table 1 ) and inserted into the mold inside the flask, which was kept under a load of 12.3 kN for 30 minutes.

The specimens were then submitted to conventional (SL-155/22; Solab) or microwave (MEF 41; Electrolux) polymerization cycles: water bath polymerization; MP cycles (both as recommended by the manufacturer); microwave processing for 3 minutes at 550 W (M550/3); and microwave processing for 5 minutes at 650 W (M650/5). The flasks were bench-cooled for 30 minutes and subsequently cooled in running water for 15 minutes. The specimens were removed from the molds and subjected to a metallographic finishing and polishing procedure (Aropol E; Arotec) using silicon carbide abrasive papers (#240, #400, #600, and #1200; 3 M) for 20 seconds on both sides.

The specimens were then stored in distilled water in an oven (Nova Instruments) at 37°C for 48 hours. Each specimen was suspended with dental floss to avoid contact with the container or other specimens. This initial storage time in distilled water was considered T ₀ .

Two beverages, instant coffee and red wine, were selected based on their potential for staining and their frequency of intake by the population. Distilled water was used as a nonstainable control. The coffee solution was prepared at a ratio of 3.6-g instant coffee (Nestlé Brasil Ltda) for each 300 mL of boiling distilled water. The specimens were immersed in this solution only after the beverage had cooled to room temperature. For the wine solution, a dry red wine brand (Campo Largo) was used.

The experimental design is illustrated in Figure 1 . After T ₀ , surface roughness and color analyses were made and considered as the initial values. Subsequently, the specimens were subjected to a cycle of immersion in stainable solutions, followed by simulated brushing ( Fig. 1 ). In that cycle, the specimens were immersed in 150 mL according to each experimental group, suspended by dental floss, and stored in an oven for 6 days at 37 °C. After that period, they were removed from the solutions, rinsed in running water, and subjected to 9000 cycles of simulated brushing (T _A ). This first cycle corresponded to 6 months of denture clinical use.

Flowchart representing experimental design.

For the simulated brushing, the toothbrushes were cut at the neck and attached by screws placed on the sides and top of the support for the brush in the equipment. The correct adjustment of these screws allowed the brush to be leveled. The nylon bristles were of uniform length and flexible with rounded ends (Tek 30; Johnson & Johnson do Brasil) and were replaced when wear was observed. The dentifrice (Colgate Total 12 Clean Mint; Colgate-Palmolive) was mixed with distilled water at a ratio of 1:1 (g/mL). This suspension was homogenized in a magnetic stirrer (model 752; Fisatom) for 1 minute at room temperature. The simulated brushing machine (MSEt; Marcelo Nucci ME) allowed 10 specimens to be simultaneously brushed with a linear back and forth movement. The equipment was set to brush at a rate of 60 reciprocal strokes per minute with a 1.96-N vertical load on each specimen. Scheduled cycles were performed on both sides of each specimen at 37°C, and 2 mL of the suspension with dentifrice were injected onto the specimens every 2 minutes.

The T _A cycle was repeated once again (T _B ) to simulate 6 more months of denture use, and specimens were then stored in the stainable solutions for 12 days before they were subjected to 18 000 cycles of simulated brushing (T _C ), corresponding to 1 year of denture use. Twelve-day storage followed by 18 000 cycles of simulated brushing was repeated (T _D ), so the total immersion storage period was 36 days. The time was based on the assumption that 24-hour immersion in coffee would simulate the stainability after 1 month of consumption. Thus, 36 days would simulate 3 years of denture use. After this period, roughness and color were reevaluated and considered the final values (T ₁ ). The solutions were replaced every 3 days, when changes in the pH (DMPH-2; Digimed), visual signs of fungal contamination, and/or precipitation of the particles were observed.

The simulated brushing was an in vitro method that has been considered suitable for the quantification of abrasivity of acrylic resins according to ISO/TR 14569_1:2007. The simulated brushing protocol used in the present study was determined based on a pilot study with three protocols. To choose the protocol that represented the most relevant daily-life routine of patients, specimens (n=3) of the lightest and most translucent commercially available Vipi Cril (CA) resin were prepared and immersed in the solution with the greatest stainability potential, red wine, for 36 days. After 18 000 cycles, corresponding to 1-year regular toothbrushing period of an adult, these specimens were brushed on both sides under three different conditions. Condition 1 was 1500 cycles per day for 36 days (equivalent to 3 years). Condition 2 comprised T _A, T _B , T _C , and T _D cycles as described previously. Condition 3 was from the 12th day, 18 000 cycles, equivalent to 1 year (T _E ) and from the 36th day, 36 000 cycles, equivalent to 2 years (T _F ). After the specimens had been submitted to each simulated brushing condition, surface roughness and stainability analyses were made, and the results were analyzed with 2-way repeated measures ANOVA (statistical power of 80%; α=.05) by using a statistical software program (IBM SPSS Statistics, v19; IBM Corp). No significant difference was found among the experimental conditions for either property ( P >.05). The ΔE results were 2.1 ±1.4 for condition 1; 3.3 ±0.9 for condition 2; and 2.6 ±1.0 for condition 3. Condition 2 was selected because it resulted in specimens with a surface roughness pattern similar to that of condition 1 ( Fig. 2 ), where brushing was performed daily.

Mean and standard deviation of surface roughness (Ra, μm) of Vipi Cril (CA) specimens in previous analysis of simulated brushing.

The specimens were removed from the distilled water (T ₀ ) or from the brushing machine (T ₁ ), rinsed in running water, dried using the absorbent paper, and evaluated for surface roughness (Ra, μm). The surface roughness was analyzed using a surface roughness profilometer (Surfcorder SE 1700; Kozaka Industry) featuring a diamond stylus (tip diameter 2 μm) with the diamond perpendicular to the long axis of the specimen. After validity (coefficient of variation of 0.20%) and reliability ( P =.801) of the profilometer were checked (Wilcoxon signed rank test; α=.05), 3 measurements were made in the central area of each specimen at intervals of 0.8 mm, and the mean reading was obtained. The resolution was 0.01 μm, the interval (cutoff length) was 0.8 mm, and the transverse length was 2.4 mm at a speed of 0.5 mm/second.

After the surface roughness analyses were performed, the specimens were evaluated for color (T ₀ and T ₁ periods). Validity ( P values of L=.999, a=.996, and b=.990) and reliability ( P values of L=.998, a=.999, and b=.998) of the spectrophotometer were checked (Student t test; α=.05). The spectrophotometric reflectance technique was applied by using the Varian Cary 100 equipment, illuminant D-65, aperture size of 18 mm, wavelength ranging from 830 to 360 nm, color space CIELab, and an observer angle of 10 degrees.

Stainability was expressed by the formula <SPAN role=presentation tabIndex=0 id=MathJax-Element-1-Frame class=MathJax style="POSITION: relative" data-mathml='Δ=(ΔL∗)2+(Δa∗)2+(Δb∗)2′>?=(??∗)2+(??∗)2+(??∗)2‾‾‾‾‾‾‾‾‾‾‾‾‾‾‾‾‾‾‾‾‾‾‾‾√Δ=(ΔL∗)2+(Δa∗)2+(Δb∗)2
Δ = ( Δ L ∗ ) 2 + ( Δ a ∗ ) 2 + ( Δ b ∗ ) 2
, where ΔL*, Δa*, and Δb* represent the coordinates of the differences among 3 readings (axis y, x, and z, respectively) before and after immersion in the stainable solutions followed by brushing. The L* parameter represents lightness ranging from 0 (black) to 100 (white), and the parameters a* and b* reflect the chromaticity from red (+a*) to green (−a*) and yellow (+b*) to blue (−b*). For this analysis, ΔE greater than 3.3 was considered clinically significant staining. In addition, stainability was quantified based on the National Bureau of Standards (NBS), as NBS units provide more clinically relevant information than stainability data (ΔE). According to NBS, critical remarks of color differences were classified as “trace” (0.0 to 0.5), “slight” (0.5 to 1.5), “noticeable” (1.5 to 3.0), “appreciable” (3.0 to 6.0), “much” (6.0 to 12.0), and “very much” (≥12.0). NBS units were calculated using the following formula:

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