Power brushing and chemical denture cleansers induced color changes of pre-polymerized CAD/CAM denture acrylic resins

Denture wearers are advised to follow the protocol of using both mechanical and chemical hygiene methods. In this study, the in-vitro color stability of heat-cured, light-cured and newly developed pre-polymerized CAD/CAM acrylic resin base materials were evaluated after exposure to mechanical brushing and chemical denture cleansers. Two polymethyl methacrylate (PMMA) (heat-cured, and pre-polymerised CAD/CAM) and one urethane dimethacrylate based resin denture base material were subjected to mechanical brushing, followed by immersion in chemical denture cleansers (Corega, 5.25% Sodium hypochlorite (NaOCl), and 0.2% chlorhexidine gluconate (CHG)) and thermal-cycling to simulate one-year of normal prosthesis use. Baseline and final color measurements were determined and the difference in color was calculated using bench-top UV light visible spectrophotometer. The highest (29.69 ± 1.84) and lowest (19.03 ± 8.78) mean ΔE was observed with light-cured and CAD/CAM materials immersed in 0.2% CHG, respectively. Tukey’s post-hoc test showed that heat cured and light-cured resins immersed in either of the denture cleansers showed no significant differences (p > 0.05) in the mean ΔE values. On the contrary, CAD/CAM materials immersed in either of the denture cleansers demonstrated significant differences in the mean ΔE values (p ≤ 0.05). A statistically significant interaction between the combination of materials and denture cleansers (F = 4.890; p = 0.001) was observed. The color stability of the pre-polymerized CAD/CAM acrylic discs is comparatively better than the conventional acrylic resin materials. The changes in the color values of all the tested materials were above the clinically acceptable range, regardless of the type of denture cleanser used.


Introduction
Acrylic resins have been widely used in removable dental appliances, maxillofacial appliances, and surgical guides since their introduction to dentistry in the 1930s [1].Acrylic resins provide a number of advantages, including low cost, ease of handling, mechanical and physical characteristics, and a pleasing appearance.On the other hand, it conveys some disadvantages as allergic reactions, color change, abrasion, and porosity [2,3].
The mixing ratios of resin components, polymerization device, polymerization time and technician are the factors to be considered during the fabrication of conventional PMMA resins [4].Recently, CAD/CAM PMMA based polymer blocks have been produced to be used as denture base material [4][5][6][7], with the claim that it has better mechanical properties than conventional resins [8].Previous studies have shown that CAD/CAM PMMA based polymer blocks have reduced residual monomer release, hence, improved optical properties, and color stability [9,10].The CAD-CAM denture base has the least color change because it can be well polished, has no porous structure, has less water absorption, and decreased wear [10].
The color and esthetic property of the denture base is one of the prime properties considered in the denture, in order to meet the patients' expectations [11,12].Ideally, it should match the color and appearance of the surrounding tissues [13].There are multiple factors affecting the color stability of denture base materials such as water absorption, stain accumulation, intrinsic pigments degradation, ingredients dissolution, food, beverages, chemical disinfectants and surface roughness [14,15].Furthermore, color is influenced by the resin matrix composition and its polymerization process [16,17].
Denture cleaning methods can vary from mechanical by manual brushing, chemical by the use of household bleach or commercially available cleansers, or a combination of both mechanical and chemical.Although mechanical cleaning is an effective and easy means of plaque control, it can cause wear of the denture base materials, resulting in defects on the surface of the acrylic resin denture, which promotes biofilm growth and pigmentation [18,19].On the other hand, chemical cleaning methods have a variety of options in regard to the active agent as; enzymes, hypochlorite solutions, and peroxide solutions, with the superiority of hypochlorite solutions and peroxide solutions over the enzymes [20][21][22][23].Cleansers are favoured by removable denture patients, due to their ease of handling, affordability, and cost-benefits [24,25].The cleansers have been shown to be effective in reducing biofilm formation on complete dentures [26][27][28].
Chlorhexidine is an antiseptic agent with a broad spectrum of various antimicrobial activities including bacteria, viruses, and yeast species [29][30][31][32].Chlorhexidine gluconate is a water-soluble molecule with a physiological pH and dissociability, thereby allowing the release of positively charged chlorhexidine which is attracted by negative charges of bacteria [24].Thus, chlorhexidine has been a common antiseptic of choice for disinfection of dentures infected by Candida Albicans.However, chlorhexidine solution has been shown to negatively affect the hardness and roughness of acrylic resins, as well as, the inducing of brownish discolouration of the acrylic base [33].Chlorhexidine gluconate at a 0.2% concentration, has been successfully used as an antiseptic oral rinse in the treatment of denture stomatitis, while the 2.0% suspension is used as overnight denture disinfection [30,33,34].
Sodium hypochlorite has shown to be effective in opposition to various microbes and has an effect on microorganisms penetrating 3mm of the depth of surfaces [35,36].They have also be demonstrated to be more effective than brushing against some specific microorganisms [37,38].
The sodium perborate in the effervescent tablets is a chemical soak-type product that swiftly decomposes in water to produce an alkaline peroxide solution.This peroxide solution then releases oxygen, allowing for mechanical cleaning by oxygen bubbles in addition to chemical cleaning [39].Corega/Polident (GlaxoSmithKline plc) is composed of various active agents including sodium bicarbonate, citric acid, sodium perborate monohydrate, potassium peroxymonosulfate, sodium benzoate, sodium lauryl sulfoacetate, peppermint flavor, subtilisin [36].
Until now, there is little evidence regarding the effect of denture cleansers on the color stability of newly developed pre-polymerized CAD/CAM acrylic resins.The aim of the present in-vitro study was to evaluate and compare the color stability of conventional heat and light-cured and pre-polymerized CAD/CAM acrylic resins after being subjected to mechanical brushing, immersed in chemical denture cleansers, and thermal-cycled.The hypothesis was that the color changes of pre-polymerized CAD/CAM acrylic resins would demonstrate superior color stability compared to conventional acrylic resins.
Sample size was determined using G * Power v. 3.1.9.3 freeware (Heinrich-Heine-Universität Düsseldorf, Germany).The test determined a minimum of six samples per group at a 0.6 effect size (f), 0.8 power, and α=0.05.The sample size was, however, increased to ten to compensate for any specimen loss during the study.

Specimen preparation
Heat cured PMMA acrylic resin discs were fabricated following the flasking and pressure-pack method [40].Following deflasking, a steam jet (Wassermann Dental-Maschinen, Hamburg, Germany) was used to clean the discs, and surplus flash was removed with carbide cutters (Black Hawk Cutter, GmbH & CIE, Berlin, Germany).Light cured resin discs were fabricated using a silicon putty mold lined with a separating medium.This was followed by finger pressure compaction of baseplate acrylic resin into the mold, and then air-barrier coated (Eclipse ABC, Dentsply Trubyte, USA) to prevent the polymerization inhibition from oxygen.The specimens were then polymerized for 10 min using a visible light curing unit (Eclipse Processing Unit, Dentsply Trubyte) operating at a wavelength of 400 to 500 nm [41].The pre-polymerized CAD/CAM specimens were designed with Zenotec CAD software (Wieland Digital Denture, Ivoclar Vivadent, Liechtenstein) succeeded by milling using Zenotec select ion (Wieland Digital Denture Ivoclar Vivadent, Liechtenstein).
All the specimens were then engraved with a number (1-30) and a notch for orientation purposes before finishing and polishing (figure 1(a)).The specimens were finished with sequential use of waterproof silicon carbide paper at 300 rpm under water cooling.The polishing was done using pumice in a compact unit (Derotor, London, England) that included a polishing lathe, a 45-mm polishing brush, and a pleated buff nettle cloth (Renfert GmbH, Industrie-Gebiet, Hilzingen, Germany).Finally, the specimens were cleaned with a plain toothbrush under running water followed by a steam jet before baseline color measurements.The specimens were further allocated into three groups as per the chemical denture cleansers used (n=10).

Mechanical brushing
After baseline color measurements and allocation, the specimens were exposed to mechanical brushing using an electric brush (Oral-B PRO 1000, Leicester, United Kingdom) as per the manufacturer's recommendations.The electric brush was fixated on a customized stainless steel holder (figure 1(b)) for standardization purposes of both pressure and direction.The CrossAction brush head had bristles at 16°angulations with 3D cleaning involving combined action of 20,000 pulsations, and 8,800 rotations per minute, respectively.Brushing was performed for 60 min that was equivalent to one year of oral use [42].A constant force of 2N was applied by means of weights that pressed onto the head of the toothbrush throughout the brushing process.

Immersion in denture cleanser solutions
Ten specimens from each group were completely immersed in chemical solutions containing either one denture cleanser tablet (Corega, GSK, Brentford, United Kingdom) dissolved in 250 ml water, or 5.25% Sodium hypochlorite (NaOCl), or 0.2% Chlorhexidine Gluconate (CHG) (figure 1(c)).The immersion process simulated the overnight or nocturnal immersion of 8 h a day, and the immersion process was repeated for a period of 2,880 h simulating one year of prosthesis use.For each immersion cycle, the resin discs were rinsed under tap water for 10 s, dried with absorbent paper, and immersed in a fresh solution.

Artificial ageing by thermal-cycling
The specimens were thermal-cycled in a thermo-cycler device (Huber 1100, SD Mechatronik, Feldkirchen-Westerham, Germany).The specimens were soaked alternately in hot (55 °C) and cold (5 °C) water baths with a holding time of the 30s, and a transfer time of 10s.A total of 10,000 cycles used in this study simulated one year of prosthesis use [43].

Color measurements
Finally, all the specimens were subjected to final color measurement in a manner similar to the baseline measurements.The color measurements at baseline and after treatments for all the resin discs were performed by a single operator for the purpose of standardization.At all-time during the color measurements, the specimens were cleaned and dried using an absorbent paper napkin.The factors studied are the type of resin base material and the type of chemical denture cleanser.The study outcome was the color change (ΔE) observed from baseline to final measurements.The color measurement of the specimens was recorded by a bench-top UV light visible spectrophotometer (Color Eye 7000A, X-Rite, Grand Rapid, Michigan, USA) in the 3-dimensional Commission Internationale de l'Eclairege L * a * b * (CIELab) color space system (figure 1(d)) in the wavelength range of 360-740 nm.The CIELAB coordinates (L * , a * and b * ) of the acrylic resin discs were measured relative to D65 standard light source illuminant corresponding to average daylight [44].The mean of the L * a * b * values at baseline and final color measurements for each specimen was determined and the color changes were determined using the following equation (equation ( 1)): Where ΔE represents the amount of color change, ΔL * coordinate represents lightness and darkness on a scale of 0 (black) to 100 (white); Δa * and Δb * indicates chromatic scale: Δa * coordinate represents redness (positive direction) or greenness (negative direction), and Δb * coordinate represents yellowness (positive direction) or blueness (negative direction) of the surface.

Statistical analysis
Data were analyzed using SPSS 24.0 version (IBM Inc., Chicago, USA) software for windows.Mean and SD was used to describe the quantitative outcome variables (color, ΔE).A two-way analysis of variance was used to quantify the effect of type of chemical cleansers and material type on ΔE values.Post-hoc Tukey's test was used for pair-wise mean comparison of ΔE among the three types of chemicals and materials (α<0.05).

Results
The mean and standard deviations of ΔE values are presented in figure 2. The highest (29.69±1.84)and lowest mean (19.03±8.78)ΔE was observed with light-cured materials and CAD/CAM materials immersed in 0.2% CHG, respectively.Among the materials tested, Tukey's post hoc test showed that heat-cured resins immersed in either of the denture cleansers did not show any significant differences (p>0.05) in the mean ΔE values.Similarly, light-cured resins also showed non-significant changes in mean ΔE values for the denture cleansers used.On the contrary, CAD/CAM materials in any of the denture cleansers demonstrated significant ΔE values between the tested cleansers (p0.05).Table 1 presents the mean±SD ΔE values with respect to the denture cleansers used.The mean ΔE values with denture cleansing tablet (Corega) dissolved in 250 ml water with CAD/CAM Acrylic resin discs was statistically significantly higher compared with the mean ΔE values with the other two types of cleansers with the same CAD/CAM acrylic resins.There was a significant difference between mean ΔE values of the two denture cleansers, 0.2% CHG and 5.25% NaOCl with CAD/CAM acyclic resins.
Also, there was a statistically significant difference in the mean ΔE values between the denture cleanser (Corega) and the other two cleansers with light-cured acrylic resins.Moreover, the mean ΔE values of denture cleansing tablet (Corega) is significantly lower than the mean ΔE values of 5.25% NaOCl and 0.2% CHG but no difference was seen between these two chemicals with light-cured Acrylic resins.The mean ΔE values of three denture cleansers are not significantly different from each other, with the use of heat-cured acrylic resins.
The multiple pair-wise comparisons of mean ΔE among the three resins showed a statistically significant difference between heat and light-cured resins (p=.007),and CAD/CAM and light-cured resins (p=0.001).On the contrary, no significant difference was observed between CAD/CAM and heat-cured resins (p=1.00)(table 2).
The pairwise comparison of mean ΔE values showed non-significant differences among the denture cleansers used (p>0.05)(table 3).
Two-way analysis of variance (ANOVA) for the effect of the material type and denture cleansers on the ΔE values is presented in table 4.There was a statistically significant difference in the model with one outcome variable, ΔE and two covariates; material type and denture cleansers type (F=4.476;p<0.0001).In the model, material type is statistically significant (F=7.892;p=0.001)but denture cleansers type is not statistically significant (F=1.733;p=0.177).Hence the mean difference of ΔE is statistically significant among the material type but not across the denture cleansers used.However, there was a statistically significant interaction between the combination of materials and denture cleansers (F=4.890;p=0.001).

Discussion
In the current study, the newly developed pre-polymerized CAD/CAM acrylic resins were compared with conventional heat polymerized, and light polymerized acrylic resins.The tested denture resin materials showed significantly different ΔE values following exposure to combined methods of denture cleansing.The study hypothesis that the color stability of newly developed pre-polymerized CAD/CAM acrylic resins would demonstrate superior color stability compared to conventional acrylic resins was partially rejected.Among the three denture cleansers used, the CAD/CAM resin material showed significantly higher values when immersed in Corega.On the contrary, when immersed in CHG and NaOCl, the CAD/CAM materials showed significantly lower ΔE values compared to conventional denture resin materials.
In the assessment of color stability, visual evaluation is a very subjective procedure in terms of the physiology and psychology of the evaluator.Contrarily, the spectrophotometer used in the present study not only abolishes the subjectivisms but also allows identification of even the minor color alterations [45].The CIELab is a color system with a scale that contains all colors visible to the human eye, and it's used to evaluate perceptual color changes in dental materials [46].Instrumental color value readings also have an advantage over subjective visual color readings since instrumental values are objective, quantitative, and more quickly available [47].
In dentistry, the CIELab 50:50% perceptibility threshold (PT) is reported to be ΔE=1.2,whereas the 50:50% acceptability threshold (AT) is reported to be ΔE=2.7 [48].All denture base materials in this study showed significantly high ΔEab values that were clinically unacceptable after mechanical cleansing, and immersion in chemical cleansing agents.There are intrinsic and extrinsic factors that might lead to color change of denture base materials [3,14].These factors include physio-chemical change, the residual monomer used, water sorption and the surface roughness [10].Among the materials, pre-polymerized PMMA blocks demonstrated better color stability when immersed in CHG and NaOCl.This is due to the fact that these materials are known to be highly condensed resin that has less porosity, superior polishability, and less water absorption compared to conventional acrylic resins because they are manufactured under extreme heat and pressure conditions [5].The outcome of this study is in agreement with previous studies that have demonstrated better color stability of CAD/CAM compared to conventionally fabricated acrylic resins [10,16,17].
Contrary to CAD/CAM materials, light-cured acrylic resin materials demonstrated significantly higher color changes compared to other study materials.This is consistent with the outcome of the studies by Zuo et al [3], Dayan et al [10] and Hollis et al [41] who demonstrated clinically imperceptible color changes with lightcured acrylic resin materials.The authors concluded that it could be due to the high water sorption tendencies of the light-cured resins as compared to the other materials [3,10].The authors also demonstrated that color changes are of varying degrees and is increased with prolonged storage in chemical cleanser solutions.During prolonged immersion, the monomer leaches out and water is absorbed [12].The urethane dimethacrylate (UDMA) based Eclipse light-cured material used in this study is also prone to hygroscopic expansion due to two hydrophilic urethane groups within its molecular structure [49].However, few authors report that water absorption by denture base acrylics tends to stabilize approximately after 28 days [5,50].These findings were taken into consideration in the current study, which resulted in significant color change in the light-cured acrylic group.The immersion process in this study simulated the overnight or nocturnal immersion of 8h a day.Accordingly, 24h corresponded to three immersions of 8 h and the immersion process was repeated for a period of 2,880 h simulating one year of prosthesis use.This also could be one of the possible reasons for imperceptible color changes of all denture base acrylic resin materials in our study.Denture base polymers are prone to color-shift if the cleansing solutions are not handled rightly [51].The whitening of the denture materials can be attributed to the increased temperature of the water applied in the solution [39,52,53].Studies have demonstrated that the number of microbes on dentures after chemical disinfection is of a fewer count compared to brushing [36].However, brushing showed to be more efficacious at removing plaque [36,54].Therefore, it is recommended to take advantage of both mechanical and chemical cleansing methods [36,55].However, the applied cleansing method must be based on its effectiveness against the microorganisms as well as the characteristics of the denture base materials [56].
We used power toothbrushes with a rotation action in this study because they have been shown to be superior, with findings showing greater plaque removal, as compared to manual brushes.Furthermore, patients have shown that power toothbrushes are well received and thus have the ability to increase compliance [57].These findings suggest that some special-needs patient populations, such as the elderly and disabled, may benefit from the use of powered toothbrushes [58].Routinely, brushing with toothpaste for 2 min twice a day is recommended, which means a given dental surface may only be in contact with the toothbrush for a maximum of 5 s twice daily.Subsequently, in this study, one hour of brushing equals a year of life for a tooth surface [59].
Several studies have examined the effects of chemical cleansers on the physico-chemical properties of denture base materials [28,36,52,60,61].Among the chemical denture cleansers used, Corega demonstrated increased staining of the CAD/CAM denture materials.The possible reason for the increased stainability of Corega solutions could be related to the deleterious combination of oxidation and strong alkaline solution [62].However, the conventional materials immersed in either CHG and NaOCl showed significantly higher color changes compared to Corega solution.While NaOCl is used for disinfection and plaque control, it has been stated to have drawbacks due to the possibility of whitening through oxidation reaction, which is considered the most significant disadvantage [63].Furthermore, the concentration of NaOCl and immersion time could also affect the color change [64].
Similar to NaOCl, the staining potential of CHG is also dependent on its concentration which ranges between 0.2% and 0.12%.However, Bagis et al report that the staining effect of CHG can be reversed by means of conventional cleaning regimens using bicarbonate or abrasive prophylaxis paste [65].Furthermore, the differences in the ingredient in the solution, such as sodium carbonate and percarbonate could also contribute to significant color changes of materials immersed in different solutions.However, contrary to the findings of the present study, Sato et al [60] did not reveal any color changes in the denture base acrylic materials with the use of chemical agents.This may be due to the shorter immersion period (equivalent to 30 days) as well as the lack of instrumental color values detection.The color changes in denture base materials are time-dependent and increase with prolonged immersion [3,10].
This study has a few limitations.First, the fact that it is an in-vitro study, the results prerequisites that need to be tested in in-vivo trials, especially considering the cleansing effect of saliva on the color changes.Nevertheless, in-vivo studies are considered to be more difficult to conduct.Although the color change of denture base materials examined in an in-vitro methodology may not be as valid as those obtained through in-vivo methods, they can provide useful enlightenment for clinical practice.In the face of the limitations of this study, it gives valuable evidence in regard to the color change of recently introduced pre-polymerized CAD / CAM denture base acrylic and the effect of various, commonly used cleansing agents.Exposure to different agents may cause various degrees of color change.Furthermore, the effectiveness of polishing in clinical practice is operatordependent, and different polishing techniques may produce varied results.Further research into the effect of different polishing processes and immersion in different staining solutions on the optical characteristics and surface roughness of denture base mater should be conducted based on the findings of this study.

Conclusions
Within the limitations of this in-vitro study, the color stability of the pre-polymerized CAD / CAM acrylic discs is comparatively better than the conventional acrylic resin material.The color changes of all the tested materials were clinically imperceptible, regardless of the type of denture cleanser used.Among the materials tested, the color stability of the light-cured acrylic resin was low.All chemical disinfectants used in this study had an effect on color stability, with the Corega tablets having the most significant effect.

Figure 1 .
Figure 1.Study Procedure: (a) Numbered and notched acrylic resin specimen; (b) mechanical brushing with customized holder and electric toothbrush; (c) Specimen stored in containers; (d) Specimen placed in the port of the spectrophotometer for L * a * b * readings.

Figure 2 .
Figure 2. Mean and SD of the ΔE values ( * significant difference between Corega and CHG groups; ** significant difference between Corega and NaOCl groups: *** significant difference between Corega and NaOCl groups of the CAD/CAM materials) (p0.05).

Table 1 .
Comparison of mean ΔE values of the three types of denture cleansers across the acrylic resin type.significantly higher than heat-cured resins but not different from light-cured resins; no difference between heat and light-cured resins; c Significantly higher than CAD/CAM resins but not different from heat-cured resins; no difference between heat cured and CAD/CAM resins; d Significantly higher than CAD/CAM resins but not different from heat-cured resins; no difference between heat cured and CAD/CAM resin.
a statistically significant (Tukey's post-hoc test); b

Table 2 .
Pair-wise comparison of the effect of the resin material type on the mean ΔE values.
*Statistically significant at p.05.

Table 3 .
Pair-wise comparison of the effect of the denture cleansers type on the mean ΔE values.

Table 4 .
Two-way ANOVA for the effect of the material type and denture cleansers on the ΔE values.
* Statistically significant at p.05.