Complete denture hygiene solutions: antibiofilm activity and effects on physical and mechanical properties of acrylic resin

Abstract Appropriated denture hygiene is a predictive factor for longevity of rehabilitation treatment and maintenance of the oral mucosal health. Although, disinfectant solutions are commonly used as denture cleansers, the impact of these solutions on acrylic resin-based dentures remain unclear. Objective To evaluate, in vitro, the antibiofilm activity of complete denture hygiene solutions and their effects on physical and mechanical properties of acrylic resin. Methodology For antibiofilm activity measurement acrylic resin specimens were contaminated with Candida albicans, Candida glabrata and Streptococcus mutans. After biofilm growth, the specimens were assigned to the hygiene solutions: Distilled water (Control); 0.2% Sodium hypochlorite (SH); Efferdent Power Clean Crystals (EPC) and 6.25% Ricinus communis (RC). The viability of microorganisms was evaluated by agar plate counts. In parallel, physical, and mechanical properties of the acrylic resin were evaluated after simulating a 5-year period of daily immersion in the previously mentioned solutions. The changes in surface roughness, color, microhardness, flexural strength, impact strength, sorption and solubility were evaluated. Data were compared by ANOVA followed by the Tukey test or Kruskal-Wallis followed by the Dunn test depending on the distribution (α=0.05). Results Regarding antibiofilm action, SH eliminated all microorganisms while EPC and RC exhibited moderate action against S. mutans (p=0.001) and C. glabrata (p<0.001), respectively. Relative to effects on the physical and mechanical properties of the acrylic resin, RC led to higher values of color change (p=0.030), hardness (p<0.001), surface roughness (p=0.006) and flexural strength (p<0.001). Moreover, RC induced the highest values of changes in solubility (p<0.001). EPC promoted greater changes in surface morphology, whereas immersion in SH retained the initial appearance of the acrylic resin surface. All hygiene solutions reduced the impact strength (p<0.05). Conclusion SH presented the most effective antibiofilm activity. In addition, changes on properties were observed after immersion in RC, which were considered within acceptable limits.


Introduction
The denture biofilm must be removed daily means of proper cleaning, since it can lead to local and systemic diseases. 1,2 Sodium hypochlorite and alkaline peroxide solutions are widely indicated for denture biofilm control, in short or long-time immersions, either associated with mechanical methods such as brushing, or not. 3 These solutions must be effective without being deleterious to the materials of which the prosthetic device is made. In addition, the type of denture cleanser, manufacturer's instructions and period of use/immersion must be considered.
In vitro studies have shown the effectiveness of 1% and 0.5% sodium hypochlorite, used in different immersion periods, relative to their ability to remove biofilm and antimicrobial action against Candida spp. [4][5][6] In vivo studies have confirmed that 1%, 0.5% and 0.25% sodium hypochlorite solutions were efficacious for removing biofilm from denture surfaces, in addition to exhibiting significant antimicrobial activity against Streptococcus mutans, Candida spp. and gram negatives bacteria. [7][8][9][10][11] However, adverse effects on acrylic resin-based dentures have been reported after applying 1% and 0.5% sodium hypochlorite in routine hygiene practice. [12][13][14][15][16][17][18][19] Randomized clinical studies have shown the effectiveness of 0.2% sodium hypochlorite, with reduction in biofilm levels, notable antimicrobial action against Candida spp. and remission of denture stomatitis, without significant changes in color, surface roughness, and flexural strength of the acrylic resinbased dentures. [20][21][22] Therefore, this solution should be evaluated against different microorganisms of which the denture biofilm is composed, such as Candida spp. and S. mutans, together with its effect on other relevant acrylic resin properties. Although Ricinus communis solutions have been used as denture cleansers, 6,7,10,20,35 scientific data in the literature are controversial, and up to now, no ideal concentration has been established. A 2% solution showed moderate ability to remove denture biofilm, and effective action in reducing C. albicans and S. mutans on the surface of a complete denture reliner. 7,35 A 3.3% mouthwash resulted in remission of denture stomatitis; however, it was unable to reduce Candida spp. 36 Solutions at 8% and 10% led to a decrease in microbial load of C. albicans, Candida glabrata, and S. mutans, and remission of denture stomatitis, with moderate action on biofilm removal. 6,10,11,20,21 In addition, the deleterious effects of these solutions on acrylic resin-based dentures properties were classified as being clinically acceptable. 15,18,21 A previous study showed that the minimum inhibitory concentration of R. communis necessary to inhibit the growth of microorganisms such as C. albicans and C. glabrata was 6.25%, 22 but studies about its safety relative to effects on resin properties have not yet been conducted.
The literature has shown the importance of using chemical cleansers in denture hygiene and the feasibility of using diluted hypochlorite, [6][7][8][9][10][11][20][21][22] peroxide-based solutions 19,22,24,26,28,32 and R. communis solutions 6,7,10,20,22,35 in patients' daily hygiene. Thus, studies involving these solutions should be developed, to indicate a safe protocol for patients' health, which do not promote adverse effects on acrylic resinbased dentures. This, in turn, is one of the main factors that will ensure the long-term durability of oral rehabilitation. Therefore, in this study, in vitro analysis was performed to evaluate the antibiofilm activity of 0.2% sodium hypochlorite, Efferdent peroxide-based solution and 6.25% R. communis solution against monospecies biofilms composed of C. albicans, C. glabrata and S. mutans. In parallel, the effects on physical and mechanical properties of thermally activated acrylic resin were evaluated by simulating an estimated period of use of a complete denture. The null hypothesis was that there would have no difference on both antimicrobial action and effects on properties of acrylic resin regarding the use of the

Experimental Design
In this study, the ability of the hygiene solutions to remove C. albicans, C. glabrata and S. mutans mature biofilm, grown on acrylic resin surface, was evaluated by simulating a single short daily period of immersion.
In addition, denture cleanser effects on physical and mechanical properties of acrylic resin were analyzed by simulating a 5-year period of daily immersion. of the resulting suspension were seeded, the number of colonies was registered, and the CFU/ml value was calculated.
After hygiene procedure, two specimens of each group were fixed with 2.5% glutaraldehyde for 60 min and subsequently dehydrated in a graded ethanol series (30%, 50%, 70%, 90% and 100%). Effect on physical and mechanical properties of acrylic resin

Surface roughness
The roughness of rectangular specimens was evaluated with a rugosimeter (Surftest SJ-201P; Mitutoyo, Tokyo, Japan) (n=20) and a 3D laser confocal microscope (OLS4000; Olympus Tokyo, Japan) (n=3). Using the rugosimeter, three readings were performed (4 mm in length) for each specimen, and the cut-off value was 0.8 mm at a speed of 0.5 mm/s. The roughness of each specimen was calculated by the arithmetic mean of three measurements (μm).
Values within 0.20 µm were considered clinically acceptable. 17,21 For analysis under the 3D laser confocal microscope, the specimens were placed in a parallel position and 3 random images were captured. The images were obtained with a 5× objective, at a final magnification of 108×, and the mean roughness of each image (Sa) was calculated.

Color Change
The color measurements (n=20) were made on circular specimens by using a colorimeter (Color-guide 45/0; BYK-Gardner, Geretsried, Germany) as previously described. 17 The CIELAB color scale was used to calculate change in color of each specimen using the The results were expressed in J/m.

Sorption and solubility
Sorption and solubility tests were conducted on disc-shaped specimens (n=20) in accordance with specification ANSI/ADA No.12/1975. 38

Antibiofilm activity
Antibiofilm activity was solution dependent. SH reduced the counts of C. albicans, C. glabrata and S. mutans biofilms on acrylic resin surfaces to zero.
Compared with the control group, immersion in EPC exhibited an evident reduction in S. mutans biofilm (p=0.001) and immersion in RC promoted favorable antibiofilm activity against C. glabrata (p<0.001). Table   1 exhibits Log 10 (CFU+1) for the different microorganisms.

Surface roughness
Surface roughness was also solution dependent.
After immersion in RC a significant increase in surface roughness was observed (p<0.05). Higher surface roughness values were identified for both evaluation methods, i.e., by rugosimeter (ΔRa) and under 3D laser confocal microscope (Sa). The method of 3D laser confocal microscope seemed to be more sensitive for evaluating the surface roughness. ΔRa (µm) and Sa (µm) values are shown in Table 2. Three-dimensional laser confocal microscopy images are presented in Figure 2. Although a significant alteration could be identified after data analysis (ΔRa and Sa), the roughness surface did not vastly change among the groups (0.00-0.03 µm).

Sorption and solubility
For sorption, no changes were observed after immersion in all hygiene solutions (p=0.666) ( Table 2).
However, for solubility, immersion in RC contributed to greater weight loss than immersion in EPC (p<0.001) ( Table 2).

Discussion
In this study, hygiene solutions were evaluated   with regard to antibiofilm activity against C. albicans, C. glabrata and S. mutans that are microorganisms related to denture biofilm, 1 and adverse effects on relevant properties of the acrylic resin-based dentures. [15][16][17]19 The solutions were applied in short cycles (20 minutes), as a routinely recommended period of immersion, and were not associated with any other hygiene methods, to avoid the synergism  of the present study complement these findings, since we observed no changes in microhardness, sorption, solubility and surface morphology. Thre were changes only in impact strength, but within the values established by ISO 1567. 15,37 Thus, it can be inferred that this solution is effective and can be indicated as a safe denture cleanser in short immersions.
EPC was effective against S. mutans, in agreement with a previous study, 29 but ineffective against C. color changes classified as "noticeable", "considerable" and "very". 14, 16,34 Therefore, these findings may be related to the different compositions of the cleansers.
Regarding surface morphology, after immersion in EPC, the acrylic resin acquired a rougher exterior surface. The exact mechanism that alkaline peroxides damage the acrylic resin surface is unclear. It has been proposed that the higher peroxide content and release of oxygen can promote hydrolysis and surface decomposition. 39 RC showed antibiofilm activity against C. glabrata, with a significant decrease in microbial load; however, in agreement with Badaró, et al. 10 (2017), it was not effective against C. albicans and S. mutans. Randomized clinical studies have demonstrated moderate efficacy in biofilm removal of RC at 2%, 8% and 10%, 7,10,21,35 effectiveness of remission of denture stomatitis at 3.3%, 8% and 10% 10,20,36 and antimicrobial action at 2% and 10%. 6,10 However, Candida spp. have been shown to be more resistant to these solutions, with reports of moderate action of solutions at 2 and 10% 6 and ineffectiveness of solutions at 3.3% and 8%. 20,36 These results differed from the findings of this study, since the concentration of 6.25% was effective against C. glabrata. Thus, the concentration of RC seems to be a determining factor for effectiveness, so that the ideal amount of water is essential to allow the breakdown of sugar molecules in the cell walls and inactivation of ribosome that promotes cell death. 20,22 Regarding the adverse effects, in addition to the decrease in impact  15,18,21 Changes in surface roughness have also been reported with concentrations at 2% and 10%, as well as decrease in microhardness and flexural strength at 2% concentration. 15,18 However, when conducted at 8%, no change in the properties of surface roughness and flexural strength was identified. 21 Therefore, obtaining an ideal concentration of RC is important, not only to guarantee its effectiveness against the denture biofilm, but also to prevent changes in the properties of the acrylic resin-based dentures. According to results obtained, RC can be indicated as a denture cleanser, since it demonstrated antibiofilm action against C. glabrata, without showing significant changes in the properties of the acrylic resin.
A limitation of this study were the non-reproducibility of the oral environment. In the oral cavity, the denture to be considered is the importance of simulating the 8-hour immersion period, since it is recommended and routinely used by complete denture wearers. 3

Conclusion
Based on the experimental conditions of our study, the 0.2% sodium hypochlorite solution was effective against the three tested microorganisms, while Efferdent and 6.25% R. communis solutions showed moderate antibiofilm activity against S. mutans and C. glabrata, respectively. Furthermore, solutions did not significantly alter the acrylic resin properties, after a simulation of five years, as they were considered within acceptable limits.