The effect of cooling procedures on monomer elution from heat-cured polymethyl methacrylate denture base materials

Abstract Objective To evaluate the amount of methyl methacrylate (MMA) released in water from heat-cured polymethyl methacrylate (PMMA) denture base materials subjected to different cooling procedures. Methodology Disk-shaped specimens (Ø:17 mm, h:2 mm) were fabricated from Paladon 65 (PA), ProBase Hot (PB), Stellon QC-20 (QC) and Vertex Rapid Simplified (VE) denture materials using five different cooling procedures (n=3/procedure): A) Bench-cooling for 10 min and then under running water for 15 min; B) Cooling in water-bath until room temperature; C) Cooling under running water for 15 min; D) Bench-cooling, and E) Bench-cooling for 30 min and under running water for 15 min. A, B, D, E procedures were proposed by the manufacturers, while the C was selected as the fastest one. Control specimens (n=3/material) were fabricated using a long polymerization cycle and bench-cooling. After deflasking, the specimens were ground, polished and stored in individual containers with 10 ml of distilled water for seven days (37oC). The amount of water-eluted MMA was measured per container using isocratic ultra-fast liquid chromatography (UFLC). Data were analyzed using Student’s and Welch’s t-test (α=0.05). Results MMA values below the lower quantification limit (LoQ=5.9 ppm) were registered in B, C, E (PA); E (PB) and B, D, E (QC) procedures, whereas values below the detection limit (LoD=1.96 ppm) were registered in A, D (PA); A, B, C, D (PB); C, D, E (VE) and in all specimens of the control group. A, B (VE) and A, C (QC) procedures yielded values ranging from 6.4 to 13.2 ppm with insignificant differences in material and procedure factors (p>0.05). Conclusions The cooling procedures may affect the monomer elution from denture base materials. The Ε procedure may be considered a universal cooling procedure compared to the ones proposed by the manufacturers, with the lowest residual monomer elution in water.

Many studies have assessed the levels of residual MMA monomer regarding the MMA/PMMA ratio, the curing initiation method, the curing conditions, and the post-polymerization treatments. It has been found that an increased MMA/PMMA ratio leads to an increased amount of residual MMA in the set material, 10 with heat-cured materials possessing less residual monomer than the self-cured. 16,21 Furthermore,studies show that many procedures can reduce the MMA concentration, such as choosing a curing temperature of 100°C, 5,11,29 extending the polymerization time, [29][30][31] implementing a post-polymerization regime at 55°C for 60 min by exposure to microwave irradiation 24 or by smearing acrylic resin with a light-cured coating. 32 Recently, it has been documented that the cooling procedures of the processing flasks affect some mechanical properties of heat-cured denture base PMMA materials, 33 which may be assigned to post-curing reactions. However, the literature lacks information on the effect of these procedures on the MMA release levels, which may be implicated with ealy biocompatibility issues.
The aim of the study was to evaluate the effects of different cooling procedures instructed by the specific manufacturers on the residual MMA elution of representative heat-cured denture base resin materials. The null hypothesis was that there are statistically insignificant differences in the amount of MMA monomer eluted, despite the cooling procedures used. Figure 1 shows the composition, powder/liquid ratios and polymerization methods of the heat-cured denture base materials included in the study. From each material, 15 specimens (17 mm in diameter and 2 mm in thickness) were prepared according to manufacturers' instructions using a conventional flasking and pressure-pack technique. The discs were divided into five subgroups (n=3, each) depending on the cooling procedures applied ( Figure 2). From these procedures, four (A, B, D, E) are recommended by material manufacturers, whereas the fifth one (C: 15 min in cold water) was introduced by the authors as the shortest cooling procedure used. 33 An additional group (n=3 per material) was fabricated using a generally accepted procedure (polymerization cycle: 74°C for 1.5 h + 1 h at 100°C; cooling procedure: removal from water bath and bench-cooling until room temperature, ≈ 5 h), which served as control. 34 After deflasking, the PMMA discs were ground in a dry environment using 600 grit SiC papers to remove residual material, conventionally polished with wetted pumice and polishing paste, rinsed with water, air-dried and stored under dark conditions for 24 h (23 o C/50% RH). Each specimen was then placed in a sealed container with 10 mL distilled water and stored in dark conditions at 37 o C for seven days. At the end of the storage period, the specimens were removed from the containers, the MMA eluent was subjected to three extractions with 0.6 mL of n-hexane and the final volume of the extract was adjusted to 2.0 mL with n-hexane. The amount of the MMA released was measured using isocratic ultra-fast liquid chromatography (Prominence UFLC system, with  Figure 3 shows the representative chromatograms of the MMA reference and of a water-eluent specimen.

Results
The calibration curve fitted to the linear equation y=1350x-1965.9 (r 2 =0.9997). Table 1 shows the results of the MMA concentration in the water-eluents tested.
Treatment outcomes below the limit of detection Fully quantitative data were registered only in A, B (VE) and A, C (QC). Student's t-test showed no statistically significant differences between A and C procedures within the QC group (p=0.652) and for A procedure between QC and VE materials (p=0.827).
Welch's t-test was used for comparison within the VE group (since equal variance test failed-p<0.05), which showed insignificant differences between both cooling procedures (p=0.652).    different cooling procedures could affect the amount of leachable MMA monomer and, hence, modify the biocompatibility of the set material. Therefore, the amount of labile MMA eluted in water was estimated the amount of labile MMA eluted in water, rather than the total amount of residual MMA in the set products.
In the present study the istructed polymerization cycle for each material as a default curing process to address Studies have shown that the HPLC analysis is a suitable method to estimate the residual MMA content in denture base materials 35 and it has been applied to analyze water-eluted MMA fraction by denture base polymers. 21,26,32,36,37 In the current study an UFLC unit was used an UFLC unit, which offers advantages, such as higher peak resolution, higher signal to noise ratio, faster analysis, increased sensitivity and less consumption of the mobile phase. 38 The specimen dimensions were similar to previous studies 37 for comparison purposes. The specimens used were subjected to the standard polishing procedures performed by the dental technicians to better simulate the clinical scenario. 2) The cooling procedure instructed for one material showed higher MMA release than all other manufacturers' proposed procedures applied to the same material.
3) The combination of a long polymerization cycle and bench-cooling to room temperature, such as in the control group, showed MMA values below the limit of detection in all materials tested.

Conflict of interest
The authors declare no conflict of interest. The effect of cooling procedures on monomer elution from heat-cured polymethyl methacrylate denture base materials