Effect of denture-coating composite on Candida albicans biofilm and surface degradation after disinfection protocol

Abstract

Introduction: Denture stomatitis is the most common pathology affecting denture wearers and its main cause is colonisation of dentures with Candida albicans. Objective: This study investigated the effectiveness of two commercial composite surface sealants (Biscover® LV and Surface Coat®) to reduce C. albicans biofilm colonisation on denture resin, as well as their surface integrity after disinfection cycles with 1% sodium hypochlorite solution. Methods: Heat-cured acrylic resin specimens were manufactured (10 mm × 10 mm × 1 mm). The specimen surfaces were mechanically polished to simulate rough or smooth denture surfaces. Four surface-treatment groups were tested: smooth surfaces [0.3 μm of mean roughness (Ra)]; rough surfaces (3 μm of Ra); rough surfaces treated with Biscover® LV; and rough surfaces treated with Surface Coat®. Specimens of each group were randomly divided to undergo immersion in distilled water or 1% sodium hypochlorite for 30 or 90 cycles each. Specimens of all groups in each immersion solution were tested using a crystal violet (CV) staining assay for biofilm quantification and by scanning electron microscopy for visual analyses of surface integrity and biofilm structure. CV assay data were analysed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test (P < 0.05). Results: The effectiveness and surface integrity of Biscover® LV-treated surfaces were similar to those of smooth surfaces, whereas Surface Coat®-treated surfaces presented a similar performance to rough surfaces in all solutions and cycles. Conclusion: These results suggest the possibility of clinical use of Biscover® LV for denture coating on surfaces in which mechanical polish is not indicated, such as the fitting surface.

INTRODUCTION

Denture stomatitis (DS) is the most common disorder among denture wearers, affecting up to 70% of this population1., 2.. DS has a multifactorial aetiology, comprising causes such as mucosal trauma, aging of denture material, residual monomer allergy, poor oral hygiene and Candida albicans infection, the latter two being its main causes1., 2., 3., 4., 5..

DS develops when C. albicans colonises the denture-fitting surface, mostly because of certain features of the denture acrylic resin (such as roughness, hydrophobicity and electrostatic forces) that promote microorganism adhesion, and subsequently forms a biofilm2., 6., 7.. Because of the high resistance of C. albicans biofilm to antifungal agents and the persistent contact of the infected denture with the corresponding mucosa, management of the disease is difficult8., 9., 10..

Therefore, much interest has been paid to the development and research of methods and products to prevent denture colonisation with microorganisms6., 11., 12., 13., 14.. Composite surface sealants used as denture-coating materials may be a potential alternative.

Surface sealants are unfilled, low-viscosity resins that reduce surface roughness as the uncured resin wets the surface, eliminating irregularities and the oxygen-inhibition layer after polymerisation15., 16.. The new generation of surface sealants (Biscover® LV; Bisco, Schaumburg, IL, USA and Surface Coat® ; Kuraray, Tokyo, Japan) were initially developed for coating the composite surface and are also used for coating provisional restorations¹⁷. An in vivo study of provisional restoration coating investigated a self-cured acrylic resin coated with Biscover® LV for its anti-adhesion properties and found lower adhesion of plaque compared with non-coated specimens¹⁸. However, to the best of our knowledge, despite the manufacturer’s indication, there are no reports on research evaluating Biscover® LV or any new-generation surface sealants for use as a denture coating.

Regardless of the promising features of surface sealants, biofilm formation probably will not be fully eliminated from denture surfaces with a surface sealant alone. Therefore, association between surface sealant application and mechanical and chemical cleansing is strongly recommended. Elderly people comprise the main group of denture wearers and naturally present decreased motor and mental functions, leading to a greater need for chemical solutions that assist mechanical removal of denture plaque¹⁹. Denture immersion in sodium hypochlorite is a well-stablished method of disinfection20., 21.. In addition, disinfection with 1% sodium hypochlorite for 10 minutes seems to be a very effective protocol22., 23., 24..

Therefore, this study aimed to evaluate C. albicans biofilm formation on coated (Biscover® LV and Surface Coat®) surfaces and to observe its modification and colonisation after immersion in 1% sodium hypochlorite for 30 and 90 cycles of disinfection.

MATERIAL AND METHODS

Specimen fabrication

A total of 280 specimens of denture base heat-cured acrylic resin (Lucitone 550; Dentsply International Inc., Chicago, IL, USA) were made (10 mm × 10 mm × 1 mm) according to the manufacturer’s instructions. All specimens had both sides ground in a horizontal polisher (ER 27000; Erios, São Paulo, SP, Brazil). Two-hundred and ten specimens were randomly selected to simulate the inner portion of the denture base, which has an average surface roughness (Ra) of 3 μm (achieved by polishing the specimens with 120-grit silicon carbide paper for 20 seconds). Seventy specimens were polished to simulate coated surfaces and the denture’s outer portion, which have an Ra of 0.3 μm (achieved by polishing with 600- and 1,200-grit silicon carbide paper for 30 seconds each). The most polished group (0.3 μm Ra) served as the control group, in which microorganisms adhere less than on the specimens with rougher surfaces². After polishing, the specimens were immersed in distilled water for 48 hours at 37 °C to allow release of residual monomers²⁵. To ensure the surface roughness of specimens (210 specimens with 3 μm Ra and 70 specimens with 0.3 μm Ra), a profilometer was used (Surftest SJ-301; Mitutoyo Corporation, Kanagawa, Japan).

Denture coatings

The coatings were applied on 140 of the 210 specimens with 3 μm Ra. The specimens were randomly selected and half (n = 70) were coated with Biscover® LV and the other half (n = 70) with Surface Coat®, according to the manufacturer’s instructions. Then, the layers were light cured for 20 seconds (Biscover® LV) or 30 seconds (Surface Coat®).

Distribution of groups

There were four surface-treatment groups: G03, uncoated specimens with 0.3 μm of Ra; GBL, specimens coated with Biscover® LV; GSC, specimens coated with Surface Coat®; and G3, uncoated specimens with 3 μm of Ra. In each surface-treatment group, specimens were randomly divided to undergo immersion in distilled water for 30 or 90 cycles (W30 and W90, respectively) or in 1% sodium hypochlorite for 30 or 90 cycles (S30 and S90, respectively) (n = 14). For each surface treatment/immersion solution/cycle group, 12 specimens were assigned to quantitative analysis [crystal violet (CV) staining] and the remaining two specimens were used for qualitative assay (scanning electron microscopy).

Disinfection protocol

The disinfection protocol was based on studies that confirmed disinfection of a denture with 10 minutes of immersion in 1% sodium hypochlorite22., 24.. Thus, 10 minutes of immersion represented one cycle. This study evaluated specimens after 30 and 90 cycles of immersion in the cleansing solution, simulating 10 minutes of daily soaking²⁴. After the exposure period for each group, the specimens exposed to 30 cycles of immersion (W30 and S30) were washed in distilled water for 30 minutes and the specimens exposed to 90 cycles of immersion (W90 and S90) were washed in distilled water for 90 minutes, simulating 1 minute of washing for each cleansing cycle. The washing process was repeated three times. The same cleansing and washing processes were performed in the groups that had distilled water as the immersion solution. The groups that were not assigned to any immersion solution (0) did not undergo immersion. After the disinfection process, the specimens were sterilised with ethylene oxide.

Biofilm formation

After coating with artificial saliva, as described by Hahnel et al.²⁶, specimens were inoculated with 1 mL suspension of C. albicans (SC 5314), of 10⁷ cells/mL, and incubated in an orbital shaker at 75 rpm for 90 minutes at 37 °C⁹. Subsequently, the specimens were gently washed three times with phosphate buffer solution, placed individually in 24-wells plate with 1 mL of Trypic Soy Broth medium for biofilm growth and incubated in an orbital shaker at 75 rpm for 24 hours at 37 °C9., 22..

CV staining assay

To quantify C. albicans biofilm, a CV staining method was performed. The biofilm was fixed by immersion in 99% methanol for 15 minutes at 15 °C. Supernatants were then removed and specimens were air-dried. Subsequently, the specimens were dyed with 0.02% CV solution for 20 minutes²⁷. Next, excess CV solution was removed by washing the specimens three times with sterilised Milli-Q water. Afterwards, the specimens were immersed in 95% methanol to release bound CV stain²⁸. The supernatants were collected and 100 μL of solution was placed in each of three 96-well tissue culture plates. Absorbance was measured using a spectrophotometer (Fluorstar Optima; BMG LABTECH GmbH, Ortenberg, Germany) at 595 nm²⁸. To quantify the absorbance only of the CV released from the specimens, the absorbance values of 95% methanol were measured and deducted from the values obtained for the wells.

Scanning electron microscopy analysis

Scanning electron microscopy was used to observe biofilm structure and surface integrity on inoculated and non-inoculated specimens, respectively. The inoculated specimens of each group were fixed with osmium tetroxide vapour in a fume hood and then kept in a desiccator for 72 hours²⁹. Finally, both inoculated and non-inoculated specimens were fixed on stubs using adhesive tape, sputter coated with gold and evaluated by scanning electron microscopy. Six images were taken of each specimen at 50× magnification. Magnifications of up to 1000× were taken to visualise details.

Statistical analysis

The results of the CV staining assay were calculated as mean and standard deviation. Data obtained by the CV staining assay were analysed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test (P < 0.05) (Statistica, Statsoft Inc., Oklahoma, USA). Scanning electron microscopy images were evaluated descriptively.

RESULTS

CV staining assay

There were no significant differences in absorbance values between G03 and GBL groups and between G3 and GSC groups, when evaluating the same test solutions and number of cycles. However, when comparing these two sets of groups, it was observed that G03 and GBL had significantly lower values than G3 and GSC (Table 1).

Table 1.

Comparison of absorbance values among surface treatment groups

Immersion solution and immersion cycles	Surface treatment group
	G03	GBL	GSC	G3
0	0.013 ± 0.004aA	0.091 ± 0.048aA	0.287 ± 0.124aB	0.42 ± 0.218aB
W30	0.025 ± 0.011aA	0.089 ± 0.069aA	0.367 ± 0.136abB	0.471 ± 0.173aB
S30	0.055 ± 0.045aA	0.145 ± 0.071abA	0.514 ± 0.238bB	0.585 ± 0.257abB
W90	0.052 ± 0.029aA	0.174 ± 0.063bA	0.437 ± 0.193abB	0.487 ± 0.204aB
S90	0.12 ± 0.093bA	0.186 ± 0.066bA	0.643 ± 0.209bB	0.756 ± 0.244bB

Values are given as mean absorbance and standard deviation. Different lower case letters in columns and capital letters in rows indicate statistical significance (P < 0.05). Surface treatment groups: G03, uncoated specimens with 0.3 μm of average surface roughness (Ra); G3, uncoated specimens with 3 μm of Ra; GBL, specimens coated with Biscover® LV; GSC, specimens coated with Surface Coat®. Immersion solution and immersion cycles: 0, no immersion; S30, immersion in 1% sodium hypochlorite for 30 cycles; S90, immersion in 1% sodium hypochlorite for 90 cycles; W30, immersion in distilled water for 30 cycles; W90, immersion in distilled water for 90 cycles.

When comparing all immersion and cycle groups (W30, S30, W90 and S90) with the non-immersion group (0) of each surface treatment group, it became apparent that G03 and G3 showed a significant difference only in S90, GBL showed differences in W90 and S90, and GSC showed differences in both sodium hypochlorite groups (S30 and S90).

As seen in Table 1, immersion in 1% sodium hypochlorite for 30 cycles (S30) for all surface treatments did not show a significant difference compared with groups in which specimens were immersed in distilled water for 30 cycles (W30). However, when comparing the two immersion solution groups for 90 cycles (S90 and W90), only G03 and G3 were different.

Scanning electron microscopy analysis

Scanning electron microscopy images of group 0 (no immersion) showed smoother and more regular surfaces of GBL and GSC compared with G03 and G3. The same feature was apparent after immersion in distilled water (W30 and W90). However, after immersion in 1% sodium hypochlorite, there was an increase in the irregularities and spots of degradation in the surface sealant groups, although G03 and G3 showed deeper grooves and also degradation aspects on the surface (Figure 1). All inoculated surface treatment specimens presented similar features, namely a multilayered biofilm with blastopores, pseudohyphae and hyphal structure (Figure 2).

Figure 1.

Scanning electron microscopy images (100× magnification) presenting specimens of each surface treatment after immersion and cycles in solution. Horizontal axis, surface treatment groups: G03, specimens uncoated with 0.3 μm of average surface roughness (Ra); G3, specimens uncoated with 3 μm of Ra; GBL, specimens coated with Biscover® LV; GSC, specimens coated with Surface Coat®. Vertical axis, immersion solution and immersion cycles: 0, no immersion; S30, immersion in 1% sodium hypochlorite for 30 cycles; S90, immersion in 1% sodium hypochlorite for 90 cycles; W30, immersion in distilled water for 30 cycles; W90, immersion in distilled water for 90 cycles.

Figure 2.

Scanning electron microscopy image (100× magnification) of the inoculated specimen.

DISCUSSION

In recent years there have been an increasing number of attempts to modify denture surfaces, aiming at reduction or full elimination of biofilm development6., 11., 12., 13., 14.. Several coating materials have been suggested for this purpose; however, the effectiveness, biocompatibility and degradation over time, and the cleansing methods of these products, have not yet achieved optimal results. Low-viscosity resins, such as Biscover® LV and Surface Coat®, have been developed for covering composite to provide polishing and wear protection, for coating provisional restorations to polish and to decrease biofilm adhesion18., 30., 31.. However, despite the manufacturers’ indications, the literature indicates that these surface sealants were never tested for their use as a denture coating. In this regard, in the present investigation, Biscover® LV and Surface Coat®, used as denture coatings, were tested for quantification of C. albicans biofilm, even after 30 and 90 cycles of disinfection with 1% sodium hypochlorite. Two evaluation methods were used: the CV staining assay for quantification of the biofilm’s total biomass; and scanning electron microscopy for qualitative analysis of inoculated and non-inoculated surfaces.

In this study, when comparing surface treatment groups exposed to 30 or 90 cycles in each immersion solution, it was observed that, although the initial Ra of GSC was similar to that of G03 (0.3 μm), the CV staining results of GSC were similar to those of G3, which had significantly higher initial Ra values. Conversely, the CV staining results of GBL were similar to those of G03. When evaluating scanning electron microscopy surface images of GBL and GSC specimens that did not undergo treatment in any immersion solution, it was observed that both surfaces showed layer integrity. Therefore, the difference between CV results for both coated groups could be explained by other contributing factors in biofilm formation besides surface roughness, such as hydrophobicity, electrostatic forces and salivary protein adsorption, features that are strongly connected to the chemical nature of the biomaterial surface.

Surface free-energy might influence the composition of the acquired salivary pellicle and consequently the surface hydrophobicity25., 26.. Therefore, materials that present higher surface energy are more hydrophilic³², leading to reduced adhesion of C. albicans as a result of its preference for hydrophobic surfaces33., 34., 35., 36., 37.. This occurs because of the surface exposure of monomeric units and its interaction with the hydrophobic domains of salivary proteins, forming strong hydrophobic bonds³⁸; depending on the availability of the salivary proteins, this increases C. albicans adhesion39., 40..

Davidi et al.⁴¹ elucidated some of the characteristics that might explain the decrease in adhesion of microorganisms to a self-cured acrylic resin when coated with Biscover® LV. Surface roughness, protein adsorption, antibacterial activity and hydrophobicity were evaluated. The results showed no differences in these features between uncoated and Biscover® LV-coated surfaces, except for protein adsorption, where there was an insignificant difference. The authors affirmed that, as protein adsorption influenced the acquired pellicle, biofilm formation was decreased. They hypothesised that because the adsorption of salivary proteins is mainly dependent on electrostatic forces and material hydrophobicity, and the results did not show any difference in coating hydrophobicity, the adsorption of protein may rely on a difference in the electrostatic nature of the surface. As post-cycle roughness, hydrophobicity and electrostatic forces were not evaluated in our study, we attributed GBL results to the low adsorption of salivary proteins, as elucidated by Davidi et al.⁴¹.

Concerning the performance of the GSC group, which had similar Ra as G03 and GBL yet similar CV results as G3, it can be assumed that material features related to its chemical nature may have resulted in these findings. Although both products are low-viscosity resins, they have distinct compositions. Biscover® LV is a dipentaerythritol pentaacrylate compound, an intricate molecular monomer with functional, polymerisable and cross-linking groups, which makes it more resistant to degradation⁴². Surface Coat®, according to the manufacturer, consists of stabilised methyl methacrylate, a cross-linking monomer that presents, though its radical reaction, advantages such as fast cure, insipidity, translucency and lower sensitivity to oxygen inhibition. However, the layer formed is vulnerable to degradation of the ester group when exposed to an aqueous medium⁴². Nonetheless, as there are no data in the literature on the surface characteristics caused by Surface Coat®’s chemical nature, the real reasons for these results cannot be given.

Our results show that immersion in water produced an increase in biofilm quantification only for coated specimen groups, whereas immersion in 1% sodium hypochlorite increased the CV results for all groups, indicating the higher potential of 1% sodium hypochlorite for surface modification. Both immersion solutions used in this study can affect the integrity of the resin. Water and water solutions can penetrate the polymer resin and swell the network. In addition, water can act as a plasticiser and soften the polymeric chains, leading to hydrolytic degradation43., 44..

Regarding sodium hypochlorite, the majority of studies report no difference in acrylic resin Ra values after immersion in this solution43., 45., 46., 47.. However, a previous study with longer immersion periods was able to identify an increase in Ra after 8 hours of immersion for 180 days²⁰. As our research did not assess possible causes of biofilm quantity, such as roughness, surface free-energy and electrostatic forces, it cannot be specified which surface characteristic was modified to provide a better environment for fungal growth.

Briso et al.⁴⁸ tested composites coated with Biscover® LV and immersed for 5 weeks in soft drinks and hydrochloric acid. The results revealed that coated specimens did not show a significant difference in roughness before and after immersion. Conversely, non-coated composites showed an increase in roughness after exposure to the solutions, indicating that the coating material was able to withstand an acid challenge. In our study, the CV staining results indicated higher surface modification of GBL and GSC, as the absorbance values were higher in W90 and S90 groups compared with the non-immersion group (group 0), and for non-coated groups the difference was noticeable only in the S90 group. As noted in scanning electron microscopy images (Figure 1), GSC seemed to have a higher degree of degradation compared with GBL, as the layer below (acrylic resin) is more evident in the GSC group.

The present results show that Biscover® LV, when used as a heat-cured acrylic resin coating, is effective in decreasing the formation of the C. albicans biofilm when compared with untreated rough surfaces. Also, Biscover® LV-coated surfaces showed similar degradation to uncoated acrylic resin after disinfection cycles with 1% sodium hypochlorite, indicating that Biscover® LV may be effective in denture coating aiming to prevent denture stomatitis; however, long-term clinical research should be performed before any such recommendation is made.

Acknowledgements

This study was supported by Foundation for the Support and Evaluation of Graduate Education (CAPES) and São Paulo Research Foundation (FAPESP-grant 2010/07932-8).

Conflict of interest

The authors declare no conflict of interest.