Novel Dental Poly (Methyl Methacrylate) Containing Phytoncide for Antifungal Effect and Inhibition of Oral Multispecies Biofilm

Abstract

Despite the many advantages of poly (methyl methacrylate) (PMMA) as a dental polymer, its antifungal and antibacterial effects remain limited. Here, phytoncide was incorporated into PMMA to inhibit fungal and biofilm accumulation without impairing the basic and biological properties of PMMA. A variable amount of phytoncide (0 wt % to 5 wt %) was incorporated into PMMA, and the basic material properties of microhardness, flexural strength and gloss were evaluated. In addition, cell viability was confirmed by MTT assay. This MTT assay measures cell viability via metabolic activity, and the color intensity of the formazan correlates viable cells. The fungal adhesion and viability on the PMMA surfaces were evaluated using Candida albicans (a pathogenic yeast). Finally, the thickness of saliva-derived biofilm was estimated. The flexural strength of PMMA decreased with increasing phytoncide contents, whereas there were no significant differences in the microhardness and gloss (p > 0.05) and the cell viability (p > 0.05) between the control and the phytoncide-incorporated PMMA samples. The amounts of adherent Candida albicans colony-forming unit (CFU) counts, and saliva-derived biofilm thickness were significantly lower in the phytoncide-incorporated PMMA compared to the control (p < 0.05). Hence, it was concluded that the incorporation of appropriate amounts of phytoncide in PMMA demonstrated antifungal effects while maintaining the properties, which could be a possible use in dentistry application such as denture base resin.

Keywords: antifungal; dental polymer; oral biofilm; phytoncide; poly (methyl methacrylate).

Figure 1

Chemical characterization of poly (methyl methacrylate) (PMMA)-based materials. Fourier transform infrared (FTIR) spectra of control, 1.25% PT, 2.5% PT, 3.75% PT and 5% PT.

Figure 2

Comparison of microhardness (A) and flexural strength (B) among different groups of PMMA samples. Same lowercase alphabetical letters above the bar graph indicate there are no significant differences between the groups (e.g., there are no difference between group with ‘a’ and another group with ‘a’ above the bar) (p > 0.05). Difference in lowercase alphabetical letters above the bar graph indicate significant differences between the groups (e.g., there are differences between the group with ‘a’ and the group with ‘b’.) (p < 0.05).

Figure 3

Representative images of PMMA samples; control, 1.25% PT, 2.5% PT, 3.75% PT and 5% PT (A), comparison of gloss values between different groups of PMMA samples (B). Same lowercase alphabetical letters above the bar graph indicate there are no significant differences between the groups (e.g., there are no differences between group with ‘a’ and another group with ‘a’ above the bar) (p > 0.05). Differences in lowercase alphabetical letters above the bar graph indicate significant differences between the groups (e.g., there are differences between the group with ‘a’ and the group with ‘b’.) (p < 0.05).

Figure 4

Representative microscopic images of L929 cells on the surfaces of PMMA samples; control, 1.25% PT, 2.5% PT, 3.75% PT and 5% PT at a magnification of 20× (A), comparison of cell viability among different groups of PMMA samples (B). Same lowercase alphabetical letters above the bar graph indicate there are no significant differences between the groups (e.g., there are no differences between group with ‘a’ and another group with ‘a’ above the bar) (p > 0.05). Differences in lowercase alphabetical letters above the bar graph indicate significant differences between the groups (e.g., there are differences between the group with ‘a’ and the group with ‘b’.) (p < 0.05).

Figure 5

Representative live–dead staining images of C. albicans attached on the surfaces of PMMA samples; control, 1.25% PT, 2.5% PT, 3.75% PT and 5% PT at a magnification of 5000× (A), Colony-forming units (CFU) counts derived from fungi attached on the surfaces of PMMA samples (B). Same lowercase alphabetical letters above the bar graph indicate there are no significant differences between the groups (e.g., there are no differences between group with ‘a’ and another group with ‘a’ above the bar) (p > 0.05). Differences in lowercase alphabetical letters above the bar graph indicate significant differences between the groups (e.g., there are differences between the group with ‘a’ and the group with ‘b’.) (p < 0.05).

Figure 6

Representative saliva-derived biofilm accumulation images of C. albicans attached on the surfaces of PMMA samples; control, 1.25% PT, 2.5% PT, 3.75% PT and 5% PT at a magnification of 5000× (A), Biofilm thickness derived from fungi attached on the surfaces of PMMA samples (B), Live–Dead assay quantified as the live and dead cells in equivalent surface areas (C). Same lowercase alphabetical letters above the bar graph indicate that there are no significant differences between the groups (e.g., there are no differences between the group with ‘a’ and another group with ‘a’ above the bar) (p > 0.05). Differences in lowercase alphabetical letters above the bar graph indicate significant differences between the groups (e.g., there are differences between the group with ‘a’ and the group with ‘b’.) (p < 0.05).