Antifungal effect and characterization of denture PMMA impregnated with chitosan

Ⅰ. INTRODUCTION

Denture try-on inevitably compromises salivary flow related with hygienic effect and encourages biofilm formations on both the prosthetic surface and adjacent mucosa (Yildirim et al., 2005). Chronic denture stomatitis is an erythematous pathogenic condition of denture-bearing mucosa and one of the major etiological factors in this pathogenesis is the presence of numerous yeasts, usually Candida albicans (C. albicans) on the fitting surface of the denture (Boscato et al., 2009; Paranhos et al., 2009). Despite the use of antifungal agents to cure denture stomatitis, infection often recurs and drug tolerance has been observed in majority of cases (Chandra et al. 2001). Chemical-based oral disinfectants such as sodium hypochlorite or glutaraldehyde has been prescribed, however, their bleaching agents may interfere with the esthetics of the prostheses or might be irritant to oral mucosa due to their toxic vapors (Chassot et al., 2006). Microwave disinfection of denture base in a 60 Hz for 5 minutes was suggested to kill colonized fungi, however, the repeated irradiations significantly affected the hardness of material itself (Dixon et al., 1999). Studies for the antimicrobial denture materials incorporated with silver nanoparticles (Wady et al., 2012; Monteiro et al., 2012; Nam et al., 2012) have been reported, the significant discolorations of nanocomposites discouraged the clinical application (Chladek et al., 2011; Nam, 2014). To overcome denture-related inflammatory complications, performative and latent antimicrobial denture bases are highly requested and mandatory (Saito et al., 1997), however, they have not been available in market yet.

Recently, higher interests are focusing on synthesis of biomaterials holding antimicrobial properties in dental field. Chitosan, a polysaccharide biopolymer derived from naturally occurring chitin, displays unique polycationic, chelating, and bacterial or fungal inhibiting properties due to the presence of active amino and hydroxyl functional groups (Wang et al., 2006; Yi et al., 2005). Due to its antibacterial property, chitosan has been blended with other polymers such as alginate, hydroxyapatite, hyaluronic acid, calcium phosphate, PMMA (Hu et al., 2003). Chitosan also has a high resistance to heat due to its intramolecular hydrogen bonds (Onishi and Machida, 1999) and PMMA is strongly attached onto the chitosan surface (Wieckiewicz et al., 2017), therefore, PMMA denture might be appropriate as a possible carrier to apply chitosan to the oral mucosa.

Present study demonstrates the synthesis of a modified PMMA denture base incorporated with chitosan and determines whether the modified denture acrylic exhibits antifungal properties. Moreover, we also examine whether the chitosan additives influence the physico-mechanical properties of the pristine PMMA denture. The hypothesis is that chitosan-PMMA denture acrylic complex creates an antifungal activity and result in stable physical properties for the clinical application.

Ⅱ. MATERIALS AND METHODS

1. Fabrication of chitosan-PMMA Denture acrylic (CPD)

The specimens for test were prepared as following. Chitosan (low molecular type, 448869, Sigma–Aldrich Co. USA) with degree of deacetylation and molecular weights are 75% and 50,000 - 190,000 Da respectively, was used without further purifications (Figure 1). Primarily, chitosan powders were homogenously blended into pristine PMMA powder (Vertex-SC^Ⓡ, Vertex-Dental B.V., Netherlands) according to different mass ratios of 1.0 , 3.0 and 5.0 % [weight (w)%] respectively. Unmodified PMMA group was designated as the control (0 % of chitosan). Blended powders (chitosan-PMMA) were then mixed with MMA (methyl methacrylate: Vertex-SC^Ⓡ, Vertex-Dental B.V., Netherlands) liquid at designated powder/liquid ratio of 1.7 g/ml. When mixtures became a dough-staged, they were packed into two type of custom moulds by disc (20 mm diameter × 2 .0 mm depth) and rectangular (25 mm × 2 mm × 2 mm) specifications. The mixtures in moulds were sandwiched between two glass plates under 10 kg static pressure then cured following the manufacturer’s instructions. Cured samples were trimmed and immersed for 120 hours in sterilized distilled water to leach excess residual monomer then finished for 1 hour in distilled water using ultrasonic cleaner. The morphological surface characterization of CPD was examined by SEM (Scanning Electron Microscopy; S-4200 FESEM, Hitachi, Japan).

Figure 1.

Constitutional formula chitosan (deacetylated chitin, Poly[D-glucosamine]) used in this experiment with low molecular weight (50,000-190,000 Da)

Ⅲ. RESULT

The surface textures of 5.0 % CPD were similar to those of the control (× 1000) in roughness and porosity (Figure 2). The mean DC (%) and flexural strength (MPa) with standard deviations for tested samples are given in Table 1. The highest mean DC value was observed in control (73.2 %), however, all of CPD groups (1.0 – 5.0 %) did not differ statistically (P > 0.05) to control group with values ranged 67.4 to 71.2 %. In flexural strength, CPD groups also expressed no significant differences (P > 0.05) from control with values ranged 72.3 to 76.7 MPa. Between CPD groups, no statistically significant difference was observed in both mechanical tests respectively (P > 0.05). The mean values of color difference for of CPD are shown in table 2. ΔE^* were detected as 2.1 - 2.4 (1.0 % CPD), 5.2 - 5.6 (3.0 % CPD), 7.3 - 7.9 (5.0 % CPD) at 24 and 168 hours respectively. The higher chitosan added, the significantly greater ΔE* were expressed (P < 0.05) and no statistically significant difference was observed between two intervals (P > 0.05). The antifungal effects of CPD were demonstrated as CFU after 24 hours of incubation (Table 3). When compared with control, a significant fungal inhibition was shown at CPD 5.0 % with the reduction rate 40.9 % (p < 0.05) and CPD of 1 .0 % and 3 .0 % did not exhibited significant antifungal effects (p > 0.05) (Fig. 4).

Figure 2.

Comparative SEM images of control (left) and 5.0 % CPD (right), microscopic surface texture of CPD is similar to those of the control (x 1.000)

Figure 3.

Photographs of color changings according to chitosan doses after 168 hour from onset of curing. When compared to pristine, the colors of CPD become paler as the concentration of chitosan increased

Figure 4.

4. Mean viable cells (CFU/100 μL) of C. albicans of each group after 24 hour incubation. Different letter indicates a statistical difference (p < 0.05)

Ⅳ. DISCUSSION

It is important for CPD to evaluate the conversion degree relating to the amount of residual monomer as chitosan added. Chitosan particles could act as heterogeneous nuclei or impurities and this may influence resin curing processes or jeopardize PMMA matrix which eventually compromise the sufficient flexural strength to resist fracture. In this study, DC of denture PMMA was not influenced by chitosan impregnations and liquid monomer could diffuse to reactive radical ends of polymer regardless of chitosan maximally blended by 5.0 %. Some reports showed that combining with additives, such as fibers (Narva et al., 2005) and whiskers (Niu et al., 2010), improved the mechanical properties of polymers. Other reported the addition of silver zinc zeolite results in a significant decrease in the flexural and impact strengths of denture acrylic (Casemiro et al. (2008). Usually, the manufacturer determines the optimal P/L ratio to satisfy its minimal flexural strength by more than 60 MPa. Though values of flexural strength of CPD were decreased as chitosan dose increased, chitosan played non-negative effect in physical function of PMMA. All of CPD samples exhibited no significant differences from control expressing minimally 72.3 MPa in 5.0 % (Table 1). Color stability is another important clinical behavior for denture base since it may provide cosmetic service of this material. Despite many studies determining the efficacy of resin composites loading various antimicrobial materials (Ahn et al., 2009; Wady et al., 2012; Monteiro et al., 2012), there have been little information regarding the color stability influenced by additives. Generally, if color difference (ΔE*) were significantly greater than 2.69, the perceptibility threshold (Chang et al., 2009), the clinical applications should be limited. When compared to control, 1.0 % CPD groups were solely detected within the perceptibility threshold by 2.1 to 2.4 at the time of 24 and 168 hours elapsed from the onset of curing. CPD become paler and slight darker as the concentration of chitosan increased (Fig. 3) and an early discoloration within 24 hours from the onset of curing were expressed by 5.2 and 7.3 in 3.0 % and 5.0 % respectively (Table 2). Oxidative reaction or plasmon effect (Bohren and Huffman, 2007) by metal nanoparticles is known to influence the color instability of resin (Chladek et al., 2011; Nam, 2014), unique color of chitosan powder might contribute to this discoloration. Further studies are still needed to stabilize the color through alternative blending or chemical regulation of chitosan when added into the denture PMMA. In antifungal assay, a small volume of fungal suspension (100 μL) was used because immersing samples in a large suspension volume could not reproduce in denture fitting on gingival mucosa (Baehni and Takeuchi, 2003). 5.0 % CPD statistically inhibited biofilm formation by 40.9 % on its surface compared to control groups (Table 3), providing a promising new strategy for combating denture stomatitis. The knowledge have already been acquired for the antimicrobial effect of chitosan against fungi, (Wang et al., 2006; Yi et al., 2005), however, few information about the antimicrobial effect after their incorporation to solid acrylic resin bulk. Furthermore, the mechanism of antimicrobial denture composite has not been fully determined yet. Chitosan is reported to exhibit a strong interaction with negative charges on the bacterial cell surface and it showed better antibacterial activity with higher concentration in the biofilm prevention and susceptibility assays (Peng et al., 2011). Plastic surface generally possesses variable negative surface charges; similarly, all living bacterial cells (including yeasts) possess a net negative surface charge (Klotz et al., 1985). It could be speculated that chitosan may alter the physicochemical interactions or modify the polarity of PMMA surface which induce the anti-adherent power against the negatively charged fungal cell. Nevertheless, the present results cannot jump to the conclusion, because the experiment was an in vitro pattern, short-term analysis with a sole fungal strain selection. Further investigations including unknown interfacial factors, in vivo tests, tests of other strains, and long-term observations are needed to clarify the antifungal mechanism.

Table 1.

Results of degree of conversion and flexural strength. Data were expressed as mean values with standard deviation (SD)

Table 2.

The color stability of CPD as to control at the time of 24 and 168 hours elapsed from the onset of curing process. ΔE* means the color difference from control

Table 3.

Mean viable cells (CFU/100 μL) in fungal suspension according to each group after 24-hour incubation. Antifungal effect was expressed as reduction rate (%) compared with control

Ⅴ. CONCLUSION

In present study, self-polymerizing PMMA denture with 5.0 w% chitosan powders significantly inhibited the fungal adherence with proper physico-mechanical characters except color stability as compared to pristine PMMA. For clinical applications of chitosan-PMMA denture acrylic complex as a new bio-denture material, color stability, long-term antifungal effect and the verified antimicrobial mechanism still need to be investigated.

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