Long-Term Sorption and Solubility of Zirconia-Impregnated PMMA Nanocomposite in Water and Artificial Saliva

Exposure of denture base acrylic resins to the oral environment and storage media for extended periods of time results in sorption of saliva or water, leading to a reduction in physical properties and thus clinical service life. The purpose of this in vitro study was to assess the sorption and solubility of high-impact heat-polymerised denture base acrylic resin (HI PMMA) impregnated with zirconia nanoparticles after being stored for 180 days in distilled water (DW) and artificial saliva (AS). The specimens were divided into six groups for each storage medium, according to the concentration of zirconia nanoparticles (0, 1.5, 3.0, 5.0, 7.0, and 10.0 wt.%). Data were statistically analysed for sorption and solubility using one-way and two-way ANOVA statistical tests. Sorption in DW and AS for all groups containing zirconia showed sorption values lower than the control group at 90 days, though not significantly different (p > 0.05) compared to the control group. For both the DW and AS groups, the lowest solubility value was measured in the group containing 3 wt.% zirconia, however, there was no significant difference compared to the control group except when observing 10 wt.% zirconia in AS, which showed a significantly higher solubility (p < 0.05). High-impact PMMA, impregnated with low concentrations of ZrO2, showed the lowest sorption and solubility in both media, but was not significantly different compared to pure HI PMMA.

The silane-treated zirconia nanoparticles were added to the acrylic resin monomer and mixed by hand using a stainless-steel spatula to ensure all the powder was uniformly distributed within the resin monomer and to avoid any aggregation. The HI acrylic resin powder was then added to the liquid, and mixing continued until a consistent mixture was obtained, according to the manufacturer's instructions. When the mixture reached a dough-like stage that was suitable for handling, it was packed into a mould by hand. The mould was then closed and placed in a hydraulic press (Sirio P400/13045) under a pressure of 15 MPa and then excess mixture was removed from the periphery of the mould. The mould was immersed in a curing bath for 6 h of polymerisation at temperature 95 • C, and then the mould was removed from the curing bath and cooled slowly for 30 min at room temperature. The mould was then opened, and the specimens were removed. The specimens were then trimmed using a tungsten carbide bur, ground with an emery paper and polished with pumice powder in a polishing machine (Tavom, Wigan, UK).

Sorption and Solubility Test Procedures
For water sorption and solubility experiments, a brass mould was used to prepare specimens with dimensions (50 ± 1.0) mm in diameter and a thickness of (0.5 ± 0.1) mm. Seventy-two acrylic resin specimens were prepared according to EN ISO 20795-1:2008 [12]. Specimens stored in a desiccator containing fresh silica gel were placed in an incubator at (37 ± 1) • C for (23 ± 1) hr and transferred to a second desiccator maintained at room temperature (23 ± 2) • C for 1 h. The specimens were then weighed to an accuracy of 0.0002 g using a calibrated electronic analytical balance (Ohaus Analytical plus, Ohaus Corporation, Parsippany, NJ, USA). The weighing was repeated until the mass loss of each specimen was not more than 0.2 mg in any 24 h period to achieve constant mass in the samples. The final mass in gm was recorded as m1. Each specimen diameter was measured thrice using an electronic digital calliper (Draper, Eastleigh, Hants, UK) to calculate the mean values. The mean of five thicknesses measured at four equally spaced points around the circumference was calculated. The volume (V) of each sample was calculated in cm 3 by using the mean dimeter and thickness.
For each group, six specimens were immersed in DW and six specimens were immersed in AS at (37 ± 1) • C and stored in an incubator for a period of 180 days. The AS solution was prepared according to a composition shown in Table 1 by dissolving it in 1000 mL of distilled water with a pH value of 5.52 and mixing it using a magnetic stirrer until the components were completely dissolved [17]. After each period (1,2,3,4,5,6,7,14,21,30,60,90, 120, 150 and 180 days), the specimens were removed from the storage medium, wiped with a clean dry hand-towel until free from visible moisture, and mass in gm was recorded as m2. After 180 days, specimens were reconditioned in desiccators until a constant mass was reached. This desorption process was carried out over a period of 90 days. The reconditioned mass in gm was recorded as m3. The values of water sorption (w sp ) in g/cm 3 were calculated by Equation (1) [12].
The values of water solubility (w sl ) were obtained in g/cm 3 and were calculated by Equation (2): where m1 is the mass of the sample before immersing in DW or AS, m2 is the mass of the sample after immersing in DW or AS, m3 is the reconditioned mass of the sample after immersing in DW or AS and after a desorption process for 90 days and v is the volume of the specimen in cm 3 [12]. After completing the calculations, the values for water sorption and solubility were converted to µg/mm 3 . The percentage mass gain due to sorption and the percentage mass loss due to solubility, which represent the total mass loss of components, were calculated by Equations (3) and (4) [8].

Statistical Analysis
The recorded data of sorption and solubility were calculated and statistically analysed using SPSS statistics version 23 (IBM, New York, NY, USA). Non-significant Shapiro-Wilk test showed that the data of sorption and solubility tests were normally distributed and there was homogeneity of variances. The percentage of mass change for sorption and solubility data were analysed with repeated measurements (one-way ANOVA) to evaluate the effect of storage time. The mean values of sorption to solubility among different groups were compared using two-way ANOVA followed by Tukey post-hoc analysis for multiple comparisons at a pre-set alpha value level of 0.05.

Results
All experimental groups showed that mass changed with time upon storage in DW and AS. For DW, the mass steadily increased in the first 21 days of immersion for all groups. This was then followed by a reduction in the rate of increase until 60 days, and then a slow or even decrease in mass change until equilibrium was reached after 90 days. This was then followed by a slow (except for the control group) increase for the next 60 days, and finally mass was decreased at 180 days ( Figure 1). The highest rate of mass increase was with the group containing 3 wt.% of zirconia, but it was not significantly different to other groups. In AS, all the groups demonstrated a gradual mass increase within the first 7 days except the group containing 10 wt.% of zirconia whose mass was not significantly increased until day 14, as well as showing a significant difference compared to the control group. Mass change for all the groups then decreased until day 21 and then started to increase until day 90. After that, there was no significant mass change (with the course ending on day 180), which was an indication of reaching equilibrium state ( Figure 2).   In AS, all the groups demonstrated a gradual mass increase within the first 7 days except the group containing 10 wt.% of zirconia whose mass was not significantly increased until day 14, as well as showing a significant difference compared to the control group. Mass change for all the groups then decreased until day 21 and then started to increase until day 90. After that, there was no significant mass change (with the course ending on day 180), which was an indication of reaching equilibrium state ( Figure 2). In AS, all the groups demonstrated a gradual mass increase within the first 7 days except the group containing 10 wt.% of zirconia whose mass was not significantly increased until day 14, as well as showing a significant difference compared to the control group. Mass change for all the groups then decreased until day 21 and then started to increase until day 90. After that, there was no significant mass change (with the course ending on day 180), which was an indication of reaching equilibrium state ( Figure 2).
Mass change (%)  The percentage increase in mass change after 180 days for all the experimental groups in AS was significantly higher than that in DW. However, it should be noted that after 90 days there was no significant change in mass throughout the final 90 days for both solutions. Therefore, sorption values were only presented here for 90 days. The mean and standard deviation values for sorption and solubility in DW and sorption and solubility in AS are presented in Tables 2 and 3, respectively. The results showed that at 90 days, sorption by the experimental groups containing zirconia was lower than that by the control group for both solutions, but not significant (p > 0.05) (Figure 3).
At 180 days in DW, all groups of nanocomposites showed lower solubility compared to the control group, however, the difference was not significant. The lowest solubility was recorded for the group with 3 wt.% of ZrO 2 in both solutions. In general, the specimens stored in AS showed significantly lower solubility than specimens stored in DW, as shown in Figure 4. Specimens immersed in DW exhibited a greater colour change compared to the specimens immersed in AS after 180 days and specimens in the dry condition, as shown in Figure 5. However, no visible degradation of the surface after immersing in DW and AS for 180 days was noticed.

Discussion
In this study, water sorption and solubility characteristics of HI PMMA nanocomposites with different concentrations of ZrO2 were measured after being stored for up to 180 days in DW and AS. DW and AS were used as storage media in this study because they simulate oral fluids and react with the denture base inside and outside (stored overnight in water) the mouth. The experimental groups containing zirconia showed reduced sorption in both media compared to the control group, especially with the group containing 3 wt.% zirconia. As the decrease was not statistically significant (p > 0.05), the first hypothesis was accepted. The lowest solubility was found for the group containing 3 wt.% of ZrO2 in both storage media, after being stored for 180 days but not significantly different from the control group. However, the group containing 10 wt.% of ZrO2 was increased significantly different from the control group, and thus the second hypothesis was rejected.
Previous studies reported values of water sorption ranging from 11.64 μg/mm 3 to 38.31 μg/mm 3 for different types of denture base acrylic resins after 7 days of immersion in water [5,9,10,14].

Discussion
In this study, water sorption and solubility characteristics of HI PMMA nanocomposites with different concentrations of ZrO 2 were measured after being stored for up to 180 days in DW and AS. DW and AS were used as storage media in this study because they simulate oral fluids and react with the denture base inside and outside (stored overnight in water) the mouth. The experimental groups containing zirconia showed reduced sorption in both media compared to the control group, especially with the group containing 3 wt.% zirconia. As the decrease was not statistically significant (p > 0.05), the first hypothesis was accepted. The lowest solubility was found for the group containing 3 wt.% of ZrO 2 in both storage media, after being stored for 180 days but not significantly different from the control group. However, the group containing 10 wt.% of ZrO 2 was increased significantly different from the control group, and thus the second hypothesis was rejected.
Previous studies reported values of water sorption ranging from 11.64 µg/mm 3 to 38.31 µg/mm 3 for different types of denture base acrylic resins after 7 days of immersion in water [5,9,10,14]. The results of this study indicated that HI PMMA nanocomposite experimental groups complied with the British Standard (EN ISO) specification No. 20795 (not exceeding 32 µg/mm 3 for heat-cured acrylic resin) as mean values ranged from 24.80 µg/mm 3 to 25.90 µg/mm 3 compared to the control group (27.05 µg/mm 3 ) in DW storage. The mean values of specimen sorption stored in AS ranged from 23.61 µg/mm 3 to 25.41 µg/mm 3 after 90 days, compared to the control group (25.17 µg/mm 3 ). Therefore, the amount of sorption in both DW and AS is below the standard and lower than the control group. These results indicated that the addition of nanoparticles in HI PMMA did not affect its sorption property negatively, possibly due to a favourable interaction between the particles and the matrix and it is expected that the degradation would be minimal. Furthermore, the British Standard does not specify the kind of degradation and any measurement or estimation of degradation after sorption or solubility.
The findings of this study on water sorption are in agreement with those of Asar et al. who evaluated the effect of reinforcing conventional heat-cured acrylic resin with various metal oxide microparticles (TiO 2 , ZrO 2 and Al 2 O 3 using 1% and 2% by volume) on mechanical and physical properties such as, water sorption and solubility [7]. The results showed that incorporation of metal oxides significantly decreased water sorption and solubility, particularly with 2% of ZrO 2 , which showed the lowest solubility compared to the conventional acrylic resins.
In the present study, the lower water sorption could be related to the use of long curing polymerisation (cycling for 6 h in a water bath) to avoid any voids and obtain full polymerisation. Garcia et al, reported that high-temperature curing of acrylic resin increased the degree of conversion that led to an increase in crosslinking, resulting in reduced water sorption values [18]. Dogan et al. studied the effect of the curing time of heat-cured resins on water sorption and concluded that longer curing times led to a reduction in residual monomer content and water sorption [19]. Wong et al. reported that the initial water content in the specimens of denture acrylic resin that were wet heat-cured was slightly lower than that of dry heat-cured (without water bath) [20] due to an increased degree of conversion or a reduced residual monomer content.
When the control group was compared with the groups containing ZrO 2 , it was noted that reinforcement with zirconia nanoparticles led to a decrease in sorption values in DW and AS. The group containing 3 wt.% ZrO 2 showed the lowest value of sorption after storage in DW compared to the control group, even though the effect was not significantly different. However, the group with 10 wt.% of ZrO 2 stored in AS demonstrated significantly lower sorption values, followed by the groups with 5 wt.% and 3 wt.% of ZrO 2 when compared to the control group. Inorganic metal oxide fillers usually demonstrate high surface energy because of their hydrophilic nature. However, due to the difference in surface energies, the hydrophobic polymer does not wet or interact with the filler. It is therefore necessary to alter the surface of the fillers to improve wetting and adhesion at the filler-matrix interface [7], as well as to ensure even dispersion of the fillers throughout the polymer matrix [21]. The decrease in sorption by the nanocomposites in this study may be related to the silane couple agent used for treating the zirconia surface to enhance the interfacial bonding between the zirconia and PMMA matrix. However, the higher surface energy of zirconia could encourage bacterial adhesion, which can be minimised by biofunctionalization [22]. This could be a topic of further research. In the clinical situation, silica coating is also applied to reinforcing particles to increase the hydrophobicity and to enhance bond strength to resin [23]. Furthermore, it was demonstrated that dispersion stability of ZrO 2 particles can be enhanced by applying a continuous and uniform coating with silica [24]. In the future, the effect of particle coating with silica can be explored. In our previous study [25], it was also observed that the zirconia nanoparticles were fairly distributed within the PMMA matrix without observable particle clustering and the nanoparticles would presumably fill the empty spaces, which indicated a good interaction between the particles and the matrix for improved strength characteristics. Polat et al. mentioned that denture base polymers reinforced with short glass fibres exhibited decreased water sorption and solubility. The reason for this decrease could be attributed to good bonding between glass fibres conditioned by silane coupling agent and the polymer matrix [6].
Other factors that could potentially influence the sorption and solubility of resin composites include storage media, immersion period [8], or filler loading [26]. Water sorption into a polymer is a process when the water is being absorbed and at the same time the polymer material is diffused [27]. Water molecules can spread across polymer matrix due to the small size of the water molecule-the diameter of the water molecule is less than 0.28 nm-which is smaller than the polymer chain distance in the polymer matrix [6].
In this study, according to mass change as shown in Figures 1 and 2, a general trend of gradual increase in mass change over time was found and reached an equilibrium state after 90 days. However, it is interesting to note that for all specimens stored in AS, the mean mass values dropped during 7 days and 14 days, whereas for DW no such trend was observed. The reason for a decrease in mass change values in AS during the first two weeks in our study could be related to AS, a stronger solvent, which contains a mix of various salts and other additives compared to the DW and reacts with the specimen [28]. Therefore, more monomers can come out from the specimens which leads to a decrease in mass and a decrease in sorption in AS. Jagger et al. found a linear relationship between residual monomer and water sorption, which means, as the residual monomer content in the specimens increased, the water sorption also increased [29]. Dogan et al. have hypothesised that the residual monomer near the surface of the resin leaches into water, causing a decrease in polymer mass. If the residual monomer is not close to the surface of the specimen, but rather entrapped in the inner layers of resin specimen, it cannot diffuse into water after a short time [19].
The highest mass change was observed for the control group and the lowest for the group containing 10 wt.% zirconia for all of the test durations. This could be related to the fact that the addition of zirconia fills the empty spaces in the PMMA resin and reduces sorption of saliva or mass change compared to the control group. Alsharif et al., who studied the effect of different ratios of filler loading on saliva sorption and solubility in resin composite, found that samples with higher filler loadings showed the lowest change in mass when compared to the unfilled resin. They explained this as a result of filler loading [30]. In the current study, the changing of mass during long-term storage may be related to the release of residual monomer which is, at the same time, replaced with storage solution, leading to an increased sorption, particularly with the control group. However, the inclusion of ZrO 2 might reduce the amount of residual monomer in the nanocomposite composition, evidenced by less sorption in comparison to the control group.
The current study found that sorption in DW was higher than that in AS. When the water diffuses into the material it is pulled to the hydrophilic/water dissolvable sites and solution droplets are formed. The contrast in osmotic pressure between the inside solution droplet and the outside solution will result in an osmotic force to increase the sorption [28].
Ergun et al. investigated the effect of reinforcing conventional heat-cured PMMA with concentration of (5 wt.%, 10 wt.% and 20 wt.%) zirconia nanoparticles on water sorption and solubility after 28 days [31]. The findings showed that an increase in ZrO 2 concentration increased water sorption. This was explained by the weak polymer chains allowing water to permeate into the matrix and the increase in the nanoparticle filler concentration resulting in more nanoparticles at the filler-matrix interface [31]. Kundie et al. evaluated the effect of different ratios of nano-and micro-alumina fillers on water sorption and solubility in denture base acrylic resin after 7 days. The reinforced samples showed slightly higher water sorption than the control group. The reason was attributed to the filler particle size and distribution and interfacial bond between the filler and the resin matrix [32]. Al-mulla et al. investigated the effect of HI acrylic resin, conventional heat-cured acrylic resin and other types of resin on sorption over 28 days storage in two types of artificial saliva and distilled water. They found that the HI resin had slightly less sorption in distilled water than that in artificial saliva, but not significantly. Whereas in conventional resin, sorption in distilled water was higher than that in the artificial saliva [27]. The findings of these previous studies contradict with the results obtained in the present study, in which all groups containing (1.5, 3, 5, 7, 10 wt.%) zirconia exhibited lower sorption values compared to the control group in both storage media.
Solubility represents the mass of the materials dissolved from the polymer. The only soluble materials present in denture base resins are residual or unreacted monomers, plasticisers and initiators that are most likely leached over the immersion time [13]. This study showed that the lowest mean value of solubility was found in the group containing 3 wt.% of ZrO 2 stored in AS (−3.03 ± 0.95 µg/mm 3 ), whereas the mean value of the control group was −2.32 ± 0.89 µg/mm 3 , both of which were lower than the British Standard (EN ISO) specification No. 20795 (solubility value should not exceed 1.6 µg/mm 3 for heat-cured acrylic resin). However, the groups stored in DW had higher solubility compared to the groups stored in AS and the recommended value by the British Standard. The highest mean value of solubility was 8.01 ± 5.48 µg/mm 3 in the control group and lowest mean value of 3.78 ± 2.64 µg/mm 3 was for the group containing 3 wt.% ZrO 2 .
The solubility values obtained in this study for specimens stored in DW were higher than those reported in previous studies (ranging from 0.03 to 2.68 µg/mm 3 ) [5,7,10,13,14]. However, specimens stored in AS showed lower solubility values compared to the earlier mentioned studies that evaluated the solubility of different types of acrylic resin. This difference in values could be attributed to the amount of water sorption over a long time being high enough to offset the mass lost by the diffusion of residual monomer [19]. In addition, Silva et al. highlighted that an increase in the amount of water absorbed could lead to an increase in the capacity for dissolution [33]. Pfeiffer et al. reported that water sorption and solubility of polymers depended on the homogeneity of the material. The more homogeneous a material is, the less water it absorbs and the less soluble it is [14]. In the present study, this might be the reason for higher solubility in DW compared to that in the artificial saliva.
To overcome the limitations of the current in vitro study, further investigation should be designed to evaluate residual monomer and thermal expansion for HI PMMA nanocomposite denture base using artificial saliva to provide additional simulation of the oral environment more accurately. The specimen size should be increased to 10 specimens per group in order to obtain a better statistical distribution.

Clinical Implications
The HI PMMA nanocomposite denture base impregnated with less than 10 wt.% of zirconia did not make any significant difference in sorption and solubility for a long period of time (180 days) compared to the control group.

Conclusions
Within the limitations of this in vitro study, the following conclusions can be drawn. HI PMMA resin reinforced with zirconia nanoparticles with a range of concentrations (1.5, 3, 5, 7 and 10 wt.%) showed lower sorption compared to the control group (0 wt.% zirconia) in both distilled water (DW) and artificial saliva (AS) for up to 90 days but was not statistically significant. All nanocomposite groups in DW showed lower solubility than the control group after 180 days of storage but was not statistically significant. However, the nanocomposite with 10 wt.% of ZrO 2 showed significantly higher solubility in AS compared to the control group. Therefore, a low concentration of zirconia (1.5 to 5 wt.%) in PMMA will not significantly affect the sorption and solubility properties of the nanocomposite.

Conflicts of Interest:
The authors declare no conflict of interest.