Determination of the effective anticandidal concentration of denture cleanser tablets on some denture base resins

Abstract Objective Although the effectiveness of chemical cleansing against Candida albicans biofilm has been shown, the effective concentration of denture cleanser tablets has not been studied. The aim of this study was to assess the effect of three denture materials against Candida albicans biofilm and to determine effective concentrations of denture cleanser tablets. Material and methods The surface-roughness of Acron-hi™, QC-20™ and Deflex™ (n=45 per resin) resins was standardized by using a profilometer and their contact angle or surface free energy was calculated. C. albicans biofilm was formed on all three resins and were treated with Polident 3 min™, Corega™ and Fittydent™ cleanser solutions at various concentrations and both resin-biofilm and cleanser-biofilm interest were determined by using a MTT protocol according to the European Committee on Antimicrobial Susceptibility Testing's antifungal susceptibility testing (AFST-EUCAST). Scanning electron microscopy was used to compare the efficacy of different resin materials against C. albicans biofilm. Anticandidal activity and surface free energy statistical parameters were calculated by using 3-way and 1-way ANOVA, respectively (p<0.05). Results Polident 3 min™ and Corega™ tablets significantly inhibited (p<0.05) the proliferation of C. albicans against all denture resins at 27-37 mg/mL. Scanning electron microscopy results indicated that there was no significant difference among resin specimens regarding biofilm formation on dentures. We failed to find a significant relationship between surface free energy and the anticandidal effect of resin types. However, the polarity value of the resins was statistically associated with their anticandidal activity. Conclusions The polarity of the resins, the concentrations of tablets and the chemical content of the cleanser may directly affect C. albicans biofilm formations. Polident 3 min™ and Corega™ tablets should be suggested for patients who use any denture resin types, whereas the Fittydent™ tablet should only be proposed for those who use Deflex™, when two tablets are dropped into 150 mL water.


Introduction
Denture stomatitis is a common infection of the oral mucosa in denture wearers and Candida albicans is the most significant etiological agent of denture stomatitis 15,20 . C. albicans is an obstinate infection agent which is difficult to eliminate once it has been colonized as a complex biofilm formation 6,8,14,15 . The surface of acrylic resin denture base provides an ideal environment for microorganisms and biofilm formation, thus the development of C. albicans in such places 4,6,9,14,15,20,24,27,29 . The risk of denture stomatitis increases in the presence of poor oral and denture hygiene, misfit prosthesis and night wear of removable dentures 4,14,20,24 . It has been found that repeated inhalation and ingestion of microorganisms adhering to the mucosa and denture base can be a reason for various infections in patients with immune deficiency or in those receiving treatment 20 . Therefore, oral and denture hygiene is very important to remove microorganisms. Two methods are recommended to remove denture biofilm: mechanical or chemical, or a combination of both. Although the efficiency of mechanical methods in removing denture biofilm or microorganisms has been clearly shown, some people do not have the ability to apply sufficient denture hygiene 14,21 . This is especially the case for patients with limited motor capacity who have difficulty in cleaning the prosthesis with mechanical methods. To use unsuitable toothbrush with a dentifrice may also lead to surface roughness, which allows more microbial colonization 14 . The effectiveness of chemical cleansing to control C. albicans biofilm is shown in many studies, and denture cleansers are recommended for reducing biofilm formation on the dentures for these patients 4,6 .
These cleansers are available as commercial products, and they usually include alkaline peroxides 19,23 , sodium hypochlorite 5,29 , acids 29 , enzymes 19 , and neutral enzymatic peroxides solution [4][5][6]15,19 . Effervescent tablets yielding an alkaline peroxide dilution with water are the preferred denture cleansers 3,7,13,16,18,22 because they can easily provide enough cleansing without causing damage to surface resins 26 . These effervescent tablets act differently as mechanisms against microbial flora. For example, Polident 3 min™, one of the cleanser effervescent tablets, achieves chemical cleaning by using the release of oxygen from a neutral enzymatic peroxide solution [4][5][6]15,19 . However, the biofilm layer often cannot be completely removed from the resin surface and a number of viable cells remain on resins 29 . According to our knowledge, three significant factors -resin types with physicochemical features, cleanser types and cleanser concentrations -can be suggested to explain this situation.
PMMA, one of the resins, is the most commonly used denture base material due its favourable mechanical, physical and aesthetic properties.
However, it has some disadvantages, such as low flexural and impact strength 10 . Therefore, alternative materials, such as polyamide thermoplastic resin and chemical modification of PMMA with high impact resin, have been developed to achieve better mechanical properties of denture base materials. High impact acrylic resin has a high resistance against unexpected falls 25 . Also, polyamide thermoplastic resin is more elastic than PMMA. Polyamide resins are especially preferred for patients with tissue allergies to PMMA 30 .
Thus, polyamide thermoplastic resin and high impact resin are suggested for patients with a tendency to drop their prosthesis, such as elderly and handicapped denture wearers.
In addition, one of the significant physicochemical features of resin surface is the surface free energy (SFE) resulted from the asymmetry between the energies of the molecules at the surface and in the bulk of resin, since the molecules at the surface of a solidphase material are under the pressure of a one-side force, whereas in the bulk material, molecules do not have net forces due to being under equal pressure from every direction. Surface free energy (SFE) and surface roughness (Ra) both have important roles in the first adhesion of microorganisms 2,17 . Some studies showed that Ra and, to a lesser extent, SFE of resins, along with environmental conditions, are responsible for the C. albicans biofilm formation on the resin surface 2,17 .
However, the effect of the Ra on biofilm formations can be minimized and standardized by polishing resin surfaces to see the net effect from the SFE.
The effectiveness of various denture cleanser tablets in removing C. albicans biofilm formation on denture acrylic resin surfaces has been evaluated in other studies 4,6,15,29 . These studies showed a significant decrease in the amount of C. albicans after exposure to different cleansers 4,6,14,29 .
Cleanser concentrations may also play a significant role in the removal of C. albicans biofilm from resin surfaces. To the best of our knowledge, this is the first work that describes the correlation between cleanser

Specimen preparation
Two types of heat-polymerized PMMA resin and one type of thermoplastic polyamide resin were used for the fabrication of specimens (n=45 per resin). All denture base specimens were prepared according to the manufacturers' instructions. Circular wax pattern discs with dimensions of 10 mm in diameter and 2 mm in thickness were prepared using a stainless steel mould 5,6 . Wax discs were invested in denture flasks followed by a compression moulding technique for conventional heat-polymerized acrylic resin (Q-type) (QC-20, Dentsply, Addlestone, UK) and high-impact heat-polymerized acrylic resin (A-type) (Acronhi, Kemdent, Swindon, UK); then, wax discs were invested in injection flasks followed by a rapid injection technique for polyamide thermoplastic resin (D-type) (Deflex classic SR, Buenos Ares, AR) and afterwards melted with boiling water. The heat-polymerized acrylic resins were then packed into the mould, and the metal flasks were placed in a boiler unit for polymerization.
The infection flask and thermoplastic polyamide resin cartridge were placed in the device, and the resin was injected into the mould. All flasks were allowed to cool down for 2 h. All specimens were immersed in distilled water for 24 h for residual monomer release 28 .
Following this, specimens were labelled on one surface.
Respectively, one side of each specimen was ground wet with 600, 800 and 1,000 grit emery paper to standardize surface roughness, which was measured using a profilometer (Taylor Hobson, Surtronic 25, Leicester, UK). Evaluation length and range were calibrated at 1.25 mm and 100 µm, respectively. Three readings were made for each specimen, and a mean value was calculated. For all resins, surface roughness and expressed in µg/mL at 95% confidence intervals.

Determination of the minimum inhibitory concentration
The minimum inhibitory concentration (MIC) of tablets was examined using C. albicans growing in the

Statistical analysis
The statistical significance of differences was determined by the three-way analysis of variance (three-way ANOVA) followed by Tukey's test. Data that did not show homogeneity variance were analysed by the non-parametric Kruskal-Wallis test.
The SPSS for Windows computer program was used for statistical analyses. Results of SFE were reported as mean values±SD of three independent assays, and differences among groups were considered to be significant at p<0.05.

Results
Anticandidal effects of the three cleansing tablets against C. albicans biofilm were initially screened using the MTT viability assay. According to MTT test results,  on the A-, D-and Q-type resins, respectively (Table   1A). However, Polident 3 min™ anticandidal activity was strongest on the Q-type denture ( Table 1A. After an incubation time, the inhibition zone (optically clear) was produced by each cleansing solution, and the lowest concentration at which there  is "*" indicating significant difference among groups).
The plot of the mean "viability" score for each combination of groups of "resins" and "tablets" are plotted in a line graph at all concentrations, as shown in Figure 3.     According to the contact angle results, the wettability value of these resins was in the following order: Q-type (70.59) > D-type (80.56) > A-type (81.93) (Table 3D).

Discussion
The null hypothesis that denture base material type, chemical cleanser type, different concentration of chemical cleanser solution and polarity of resin would not interfere with C. albicans biofilm growth was rejected.  (Table 1).
That is why the Fittydent™ tablet had only anticandidal effects against biofilm on the D-type resin with the same administrative concentrations. In addition, the MTT analysis indicated that the polyamide resin with low polarity (Table 4D)   whereas other studies reported no correlation at al 9,27 . Likewise, in this study, we failed to find a strong correlation between SFE and C. albicans adhesion.
It is speculated that low polarity, low SFE value and low wettability may lead to a significantly increased anticandidal effect. However, we found that merely the polarity feature of resins may alter its anticandidal affect. The SEM images substantially confirmed our speculations about resin types used in this study.
For example, C. albicans biofilm layers on A-and Q-type resins were covered a much larger area and presented a higher level of growth than D-type resin.
However, even though D-type resin showed rouger surface than the others, it can be pretty smooth and slippery following the surface deburring and polishing prosses. In addition, according to SEM presentations for all resins, we failed to find a significant difference between C. albicans forms such as yeast and hyphal formation. showed that the type of resin of denture base affects the amount of C. albicans biofilm layers colonization, as observed in this study 4,6 . Murata, et al. 19 (2010) reported that the influence of neutral enzymatic denture cleanser on the surface properties was less than that of alkaline peroxide denture cleanser due to the neutral enzymatic denture cleanser containing less peroxide. However, none of the denture cleanser tablet concentrations were able to remove C. albicans biofilm completely in up to 25 mg/mL concentrations (approximately 1½ tablet). Most studies were conducted to remove C. albicans biofilm formation on the denture base resins of PMMAs via denture cleanser tablets 15,29 , whereas, to the best of our knowledge, a few studies evaluated the efficacy of denture cleansers on thermoplastic polyamide resin 4,6 . One of the thermoplastic polyamide resin studies demonstrated smaller C. albicans growth on the PMMA surface than on the thermoplastic polyamide resin 4 . They found that the residual monomer was released from the PMMA, and they were putting this forward as a serious theory.
Therefore, in this study, specimens were soaked in distilled water for 24 h after polymerization to reduce the residual monomer. The cytotoxic effects of the acrylic resins remained at high levels within the first 24 h following polymerization 28 . The water immersion method was suggested to reduce the level of residual monomer 11 because this toxic effect is reduced in a time-dependent manner 29 . Another study determined that the cleanser tablets tested were more effective for PMMA resin than for thermoplastic polyamide resin 6 . This result was inconsistent with our findings.
The reason we applied the surface roughness process to the resins using a profilometer was because of the varying study findings for both resins.

Conclusion
We have clearly demonstrated that the polarity of resins and the chemical content of the cleanser may affect C. albicans biofilm adhesion. Also, the results clearly describe a high anticandidal effect that is directly dependent on the concentrations of tablets.
Our finding suggested that the Polident 3 min™ and Corega™ tablets are suitable for patients who use any denture resin types, whereas the Fittydent™ tablet should only be advised for D-type resin users, and each cleanser solution should be prepared by two tablets and with 150 mL water. In summary, it HAYRAN Y, SARIKAYA I, AYDIN A, TEKIN YH