INTRODUCTION

Polymethylmethacrylate (PMMA) has been used for the fabrication of complete denture bases for many decades. It has advantages such as ease of handling and repair, low cost, low weight, and acceptable aesthetics (^{Fathi et al., 2017}; ^{Shakeri et al., 2019}). However, one of the major disadvantages of PMMA is the material porosity and surface roughness, which combined with the oral environment, allows for pathogenic biofilm attachment to the denture base. Thus, an acidic environment favorable to the growth and development of yeasts such as Candida albicans is created, leading to oral infections such as denture stomatitis (DS) (^{Dhir et al., 2007}; ^{Albin-Ameer et al., 2020}).

The treatment of DS commonly involves systemic and topical medication, repackaging, hygiene, and disinfection instructions (^{Cuéllar-Cruz et al., 2012}). However, systemic drugs can cause nephrotoxic and hepatotoxic effects (^{Samet et al., 2007}). Topical agents such as nystatin and miconazole are widely used for the treatment of DS. However, they have an unpleasant taste that prevents the daily use of the complete denture. Moreover, their use prolongs treatment as the drug must be in direct contact with the mucosa (^{Lalla & Dongari-Bagtzoglou, 2014}). Prolonged use of disinfectants such as sodium hypochlorite 1 % and chlorhexidine digluconate 2 % can cause damage to the physical properties of acrylic resin, thus decreasing its longevity (^{Felton et al., 2011}; ^{Peracini et al., 2017}). Hence, studies are now focused on new approaches to improve the denture base surface in order to control and/or prevent biofilm formation without compromising oral health and material properties.

Authors have reported the coating of PMMA surfaces (^{Ali et al., 2013}; ^{Távora et al., 2019}) with cyanoacrylate-based adhesives as beneficial against C. albicans development. Ali et al. studied the effects of octyl-cyanoacrylate adhesives coated on plates of heat-cured acrylic resin and reported complete inhibition of fungal adhesion. Among cyanoacrylate adhesives, ethyl cyanoacrylate has shown promising results against C. albicans. It stands out because of its low cost and bactericidal effect. Távora et al. studied C. albicans growth on relining acrylic resin coated with ethyl-cyanoacrylate (Superbonder) and verified that this adhesive reduced the initial fungal development.

In this context, the possibility of applying cyanoacrylate-based adhesives as coatings on acrylic resins was considered, thus modifying its surface properties, reducing roughness, and increasing hydrophobicity (Távora et al.). However, no study has assessed its long-term efficacy. Therefore, the inhibitory effect of two ethyl cyanoacrylate adhesives on C. albicans biofilm was evaluated after 7, 14, and 30 days in this study.

MATERIAL AND METHOD

Fabrication of specimens. A total of 90 circular specimens (10 x 2 mm) were fabricated using a heat- polymerized acrylic resin (Vipi Cril Plus, Vipi Indústria, Comércio, Exportação e Importação de Produtos Odontológicos, Pirassununga, SP, Brazil) following the manufacturer’s instructions. Subsequently, one surface of each specimen was roughened using a polishing machine (PFL, Fortel Indústria e Comércio, São Paulo, SP, Brazil) with 120-grit sandpaper (Norton Abrasivos, São Paulo, SP, Brazil) for 15 s. The surface roughness of all specimens was measured using a roughness tester (Surftest SJ-301, Mitutoyo Corporation, Kanagawa, Japan). Readings were taken at four different positions on the roughened surface (^{Rahal et al., 2004}) and standardized to 2-3 µm to simulate the inner surface of a complete denture (^{Silva et al., 2016}). Subsequently, the specimens were immersed in 2 mL of distilled water at 37 °C for 48 h to allow the release of the residual monomer (ISO 1567, International Organization for Standardization, 1988) and were further sterilised using ethylene oxide (Acecil, Central de Esterilização de Comércio e Indústria Ltda., Campinas, São Paulo, Brazil) (^{Almeida et al., 2018}). Finally, the specimens were randomly distributed into three groups: control or without coating (GC), coated with conventional ethyl cyanoacrylate (ECAc, G1), and coated with ethyl cyanoacrylate gel (ECAg, G2) (Table I).

Surface treatment of specimens. The experimental specimens were coated with 50 µL of adhesive (ECAc or ECAg), which was equally distributed across the surface using a disposable brush tip (Disposable Brush Tips/60, 3M ESPE, St Paul, Minnesota, USA). Afterwards, the specimens were dried at room temperature for 40 min. This procedure was repeated to obtain a second layer of coating (Távora et al.).

Yeast strain and growth conditions. Initially, C. albicans (strain SC5413) were incubated in tryptic soy broth (TSB) (Accumidia manufactures Inc., Lansing, MI, USA) with 1 % chloramphenicol (Quemicetina Succinato, Carlo ErbaR, Milano, Mi, Italy) at 30 ºC for 24 h under aerobic conditions. Afterwards, the suspension was centrifuged at 5000 rpm for 10 min at 22 °C. Cells were harvested and washed with phosphate-buffered saline (PBS, pH 7.2) and standardised to 1 x 107 cell/mL-1 (Almeida et al.).

Biofilm development. Specimens were exposed to 1 mL of artificial saliva for 2 h at 37 °C using a 24-well tissue culture plate (Cell Culture Plate, Nest Biotech Co., Ltd., China) (Silva et al.). Subsequently, the specimens were immersed in 1 mL of the previously standardized C. albicans cell suspension and incubated for 90 min at 37 °C at 75 rpm. The non- adherent cells were removed from the specimens by washing with 1 mL of PBS. Finally, the specimens were immersed in 1 mL of Roswell Park Memorial Institute solution (RPMI-1640, GibcoR, Grand Island, NY, USA) for 7, 14, and 30 days at 37 °C at 75 rpm. During these periods, the medium was changed at 48-hour intervals (Almeida et al.).

Viable cell count (CFU/mL). For all groups, three independent experiments were performed to measure the number of viable C. albicans cells adhering to the specimens. After each period (7, 14, and 30 days), the specimens were removed from the culture plate and washed with PBSin another 24-well tissue culture plate. Afterwards, the biofilm was removed from the surface of the specimens with a sterilized cell scraper and the cells were stored in 1 mL of PBS, and the suspension was serially diluted (10 -1 to 10-4). Subsequently, 50 µL of these dilutions were inoculated into a Sabouraud agar plate and incubated for 48 h at 37 °C. After this period, the mean values obtained indicated the growth of biofilms in CFU/mL and all colonies were counted and expressed as CFU/mL (Almeida et al.).

Statistical methods. Statistical analysis was performed using IBM SPSS Statistics for Windows, Version 23. Additionally, to compare biofilm formation between groups and their correlation with coating material performance, a one-way ANOVA test was used, followed by the Tukey’s correction test for multiple comparisons (P=0.05).

RESULTS

According to the results, cyanoacrylate adhesives showed a marked inhibitory effect on microbial biofilm formation with statistically significantly lower values compared to the control group: ECAc (P=0,00) and ECAg (P=0,00) (Table I).

Considering the evaluated periods, the cyanoacrylate groups did not show statistically significant differences. However, the control group showed a significant increase in the colony count during the 14- and 30-day periods (P<0.05) (Table I).

Table I Mean values and Standard Deviation (SD) in number of Colony-Forming Units (CFU) of Candida albicans over the evaluation periods of 7, 14 and 30 days. 

^{GC (control group), ECAc (ethyl-cyanoacrylate), and ECAg (ethyl- cyanoacrylate formulation gel). Different lowercase letters at the end of the lines indicate a statistical difference between the experimental periods (P<0.05). Different capital letters at the end of the lines indicate a statistical difference between the groups (P<0.05)}.

DISCUSSION

The cyanoacrylate adhesives evaluated in this study have been successfully used in surgical procedures, both in medicine and dentistry (^{Thakeb et al., 1995}; ^{Pascual et al., 2016}) for the control of hemorrhages (^{Al-Bawardy et al., 2016}). Recently, cyanoacrylates and fibrin biopolymers have also been studied for use as denture coating adhesives (Ali et al.; ^{Soon et al., 2015}; Távora et al.).

This study established that ECAc and ECAg showed promising long-term results in inhibiting C. albicans biofilm formation. These findings agree with other studies reported in the literature. Távora et al. verified the lower adhesion of C. albicans to acrylic resin treated with different cyanoacrylate adhesives, including conventional ethyl cyanoacrylate and formulation gel. Similarly, Ali et al. highlighted the inhibitory effects of octyl cyanoacrylate coated prostheses on C. albicans biofilms after 24 h of exposure. This may be associated with the changes in surface energy and roughness (the main factors for the development of C. albicans biofilm on dentures) of acrylic resins that represent niches for C. albicans biofilm adhesion. Moreover, the cyanoacrylate adhesives tested in this study contained the compound methyl 2-cyanoacrylate (C5H5NO2), which could be another important factor in inhibiting C. albicans biofilm development (Soon et al.).

In addition, some authors attribute this microbial reduction to the release of formaldehyde and cyanoacetate compounds during its degradation. These by-products could trigger antifungal activity because of their cytopathic effect (^{Chen et al., 2007}; ^{De Melo et al., 2013}) that tends to decrease after 14 days and remains low for 28 days (Soon et al.).

The degradation of cyanoacrylates is related to the length of the side chain; the smaller the side chain, the faster the degradation (^{Tseng et al., 1990}; ^{Trott, 1997}; ^{Sohn et al., 2016}). In a previous study, the inhibitory effect of two short-chain adhesives was evaluated, with evidence indicating that they degrade rapidly. Thus, it was suggested that with the decrease in the cytopathic reaction, C. albicans could adapt or colonize the surface as the coating gradually degraded. However, this was not observed in the present study, as the development of the C. albicans biofilm significantly reduced for up to 30 days after the application of ethyl cyanoacrylate coating.

The cyanoacrylate groups presented no statistically significant difference at all evaluated periods, although the ECAc presented a slightly better performance. This may be associated with the low viscosity of the ECAc liquid, which seeps in filling up the porosities and cracks, thus providing a smooth and regular surface.

The results obtained in this study are promising as cyanoacrylate adhesives showed long-term inhibitory effects on C. albicans biofilm formation; however, the lack of information regarding their resistance to biological changes and sustainability against chemical and mechanical challenges (commonly caused during the disinfection of a complete denture) warrants further investigation.