The effect of nanodiamonds on candida albicans adhesion and surface characteristics of PMMA denture base material - an in vitro study

Abstract Candida albicans is the main causative pathogen of denture stomatitis, which affects many complete denture patients. Objective: To evaluate the effect of different concentrations of nanodiamonds (NDs) added to polymethyl methacrylate (PMMA) denture base material on Candida albicans adhesion as well as on surface roughness and contact angle. Methodology: Acrylic resin specimens sized 10×10×3 mm3 were prepared and divided into four groups (n=30) according to ND concentration (0%, 0.5%, 1%, 1.5% by wt). Surface roughness was measured with a profilometer, and the contact angle with a goniometer. The effect of NDs on Candida albicans adhesion was evaluated using two methods: 1) slide count and 2) direct culture test. Analysis of variance (ANOVA) and Tukey's post hoc test were used in the statistical analyses. Results: Addition of NDs decreased the Candida albicans count significantly more than in the control group (p<0.05), with a lowest of 1% NDs. Addition of NDs also significantly decreased the surface roughness (p<0.05), but the contact angle remained the same. Incorporation of NDs into the PMMA denture base material effectively reduced Candida albicans adhesion and decreased surface roughness. Conclusion: PMMA/NDs composites could be valuable in the prevention of denture stomatitis, which is considered one of the most common clinical problems among removable denture wearers.


Introduction
Edentulism increases in old age, 1 and in such cases, a conventional complete denture is commonly the treatment of choice. Denture bases are constructed from metal and/or acrylic resin. Acrylic resin, however, is more frequently used due to its ease of construction and repair, aesthetics and low cost, despite the material's drawbacks of high surface roughness and low strength. 2 A study shows that denture stomatitis (DS) affects more than 70% of patients wearing complete dentures. 3 Many factors, such as poor oral hygiene, poor fitting dentures, roughness, porous denture surfaces and systemic diseases, are associated with DS, of which Candida albicans is considered the main causative pathogen. 3 Studies found that hydrophobicity and surface roughness of denture bases affect the primary attachment and colonization of Candida albicans. 3,4 Therefore, to reduce Candida albicans adherence, the denture base material should be hydrophilic, smooth and less porous. 3,4 Examples of ways to reduce the incidence of DS are mechanical cleansing, chemical disinfection, antifungal agents, surface modifications, and/or incorporating antimicrobial agents in the denture base material. [5][6][7][8] Conventional methods used to clean dentures are usually effective at eliminating plaque accumulation. 8 Their application, however, may be challenging for older patients, particularly those with physical disabilities or in need of nursing care. Oral antifungal agents are effective in the treatment of DS, but have toxic side effects and may lead to the development of resistant strains. In addition, DS recurrence commonly occurs with their use. 9 Antimicrobial effect of chemical disinfectants is related to their proper use according to the preparation guidelines and immersion time. 10 Morever, studies found that their use adversely affect the physical properties of denture cleansers, leading to increased surface roughness, color changes and reduced flexural strength. 10,11 Therefore, many studies have investigated the effect of adding antimicrobial/antifungal agents to denture base resin in an attempt to reduce microbial and/or fungal adhesion and thereby prevent DS. [12][13][14] Surface roughness (Ra) and hydrophobicity are important properties of the denture base material that influence plaque, microbial adhesion and, subsequently, DS. 15,16 A rough denture surface provides more area for microbial adhesion. In addition, it protects entrapped microorganisms from shearing forces during denture cleaning, making their removal difficult even when using antimicrobial agents. 17,18 High hydrophobicity of denture surfaces increases Candida albicans adhesion due to the hydrophobic interaction between it and the denture base resin. 18 To avoid increased microbial colonization, Ra should be inferior to 0.2 μm. 16 Recently, the addition of nanoparticles to polymethyl methacrylate (PMMA) has attracted attention because they enhance the mechanical and physical properties of the resin as well as its antimicrobial resistance. 19 Antimicrobial effect is related to the surface energy of nanoparticles and chemical reactivity. 20   conditions. 28 After ultrasonic cleaning, the specimens were incubated for one week at 37°C in distilled water, which was changed daily to reduce accumulation of residual monomers. 29

Surface roughness test
A non-contact optical interferometric profilometer (Contour Gt-K1 optical profiler; Bruker Nano, Inc., Tucson, AZ) was used to measure the Ra of the specimens at a 0.01 mm resolution. The specimens (approximate area 0.43×0.58 mm) were scanned with a standard camera at 20× at five sites, and the average for each specimen was calculated. A software package (Vision64, Bruker Nano) was used to analyze the acquired images. Pit characteristics were determined, and the Ra value of each specimen was calculated.

Microbiology test.
The specimens were sterilized with 70% alcohol, then cleaned ultrasonically with sterilized distilled water. 29 The sterilized specimens were soaked in artificial saliva (

Results
In comparison to the control group, the addition of NDs significantly reduced the surface roughness (p<0.05) (  with no significant difference between 0.5% NDs and 1% NDs groups (p=0.396). Significant differences were found between the 0.5% NDs and 1% NDs groups and the 1.5% NDs group (p<0.05), which presented the highest Ra value of the NDs groups. Figure 2 shows the color parameter representing the average   Figure 3D shows loosely-attached clusters of NDs on the surface of specimens.   Surface roughness has been shown to be directly related to the number of microorganisms deposited on the denture surface. 31 A denture base with a higher Ra provides a greater surface area for colonization. 31 The present study indicated a significant decrease in the      37,38 Similarly, reduced hydrophobicity of denture base resin was reported with silver nanoparticles, but microbial adhesion was unaffected. 39 Hashem, et al. 40 (2017) suggested that the addition of titanium oxide nanoparticles to PMMA improves its wettability. However, Kim, et al. 41 (2019) found an increase in the water contact angle with increasing carbon nanotube content in PMMA. Their study demonstrated that an increase in the contact angle increases the material's surface hydrophobicity, enhancingCandida albicans adhesion. 42 Several studies tested the effect of the contact angle on Candida albicans adhesion and reported varied findings. 29,43 Some authors found that the contact angle significantly affected Candida albicans adhesion, 43,44 while others discovered an association between them. 29,45 Murat, et al. 29 (2019) and Serrano-Granger, et al. 45 (2005) identified no association between contact angle and Candida albicans adhesion, but found a significant correlation with surface roughness, which agree with the results of the present study. Researchers also suggested that the antibacterial effect is linked to the presence of partially oxidized and negatively charged surface functions, especially acid anhydride groups, which enhance the NDs' antibacterial activities. 26 Moreover, NDs have been described among anti-adhesive nanoparticles that inhibit biofilm formation. 27,47 Generally, adding nanoparticles improves overall performance of PMMA/filler nanocomposites. 19,30,32 The addition of NDs has demonstrated to improve mechanical properties of PMMA. 28 Clinically, NDs could be added to PMMA due to their potential to reduce Candida albicans adhesion. Unfortunately, a noticeable color change was observed in the tested specimens, particularly at higher concentrations of NDs. To overcome this problem, the denture could be

Conclusion
The study found that the addition of NDs to PMMA acrylic denture base material significantly decreased Candida albicans adhesion and surface roughness, with the lowest values observed at 1% NDs and 0.5% NDs, respectively, while no significant effect was observed on the contact angle. The addition of NDs to denture base resin could be a possible contributor to DS prevention.