Effects of two peroxide enzymatic denture cleaners on Candida albicans biofilms and denture surface

Solutions of Clene® (Bitec global group, Japan), Polident® (PTI Royston LLC, USA), 3% sodium bicarbonate (NaHCO₃, Tianjin Lisheng Pharmaceutical Co. LTD, China) and phosphate-buffer Saline (PBS, Gibco® Invitrogen™, USA) were prepared at the recommended concentrations by adding one tablet or sachet in 200 ml filter-sterile tap water at room temperature (RT).

C. albicans strain ATCC 90028 (School of Stomatology, Peking University, China) was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (HyClone, Logan, USA) at 37 °C for 48 h, and the primary culture was passaged and grown for another 24 h. The yeast cells were harvested by centrifuging the broth cultures (Labofuge 400 R, Function-line, Heraeus Instruments, Bensheim, Germany) at 3000 rpm for 10 min. The cell pellets were washed twice with PBS, and re-suspended at the final density of 10⁵ CFU/ml for evaluating antifungal and anti-adhesion effects on the PMMA acrylic resin base [11], and 10⁶ CFU/ml for evaluating anti-biofilm and anti-adhesion effects on cell culture plates [12].

One milliliter of the C. albicans suspension was dispensed in five separate sterile test tubes each, and respectively treated with 9 ml Clene®, Polident®, 3% NaHCO₃, PBS and filter-sterile tap water for 30, 60 and 120 min. The tubes were sonicated at 7-W for 30s [13] and vortexed at the stipulated time points, and 1 ml aliquots were plated on Sabourad dextrose agar (SDA, Antobio Co., China) using a spiral plater (Spiral system; Interscience, Saint-Nom-La-Breeches, France). The plates were incubated at 37 °C for 24 h, and the number of colonies were counted. Three biological replicates with three technical replicates each were tested for every condition.

One hundred microliter aliquots of C. albicans suspension (10⁶ CFU/ml) were seeded in pre-sterilized 96-well cell culture plates (Corning Co., USA), and incubated for 90 min (adhesion phase) at 37 °C in an orbital shaker at 75 rpm [14]. The non-adherent cells were removed by gently washing thrice with PBS, and 100 μl RPMI-1640 was added to each well. After incubating for another 24 h, the medium was removed and the wells were washed twice with PBS, followed by addition of 100 μl each of fresh RPMI and a cleansing solution. The cells were incubated for 48 h [15] and the non-adherent cells were removed. The resultant biofilm was quantified using methyl thiazolyl tetrazolium (MTT) assay (Sigma Chemical Co., USA). Briefly, 20 μl MTT (5 mg/ml in PBS) was added to each well, and incubated for 4 h in the dark. The supernatant was aspirated and 100 μl dimethylsulfoxide (DMSO, Sigma Chemical Co., USA) was added to each well, and the optical density (OD) was measured at 490 nm after 30 mins. The experiment was repeated at least three times. Each experiment was set up in triplicate with three replicates per sample.

C. albicans suspension (10⁶ CFU/ml) was seeded in 96-well cell culture plate, and incubated with 200 μl of the different solutions for 90 mins (adhesion phase) at 37 °C in an orbital shaker at 75 rpm. After gently washing thrice with PBS to remove the non-adherent cells, the biofilm was quantified by MTT assay as described above. All experiments were performed in duplication of three independent sessions.

Forty-five circular heat-cured PMMA resin Vertex Rapid Simplified (Vertex-Dental B.V.: Zeist, Netherlands) plates (diameter 10 mm and thickness 2 mm) were fabricated, and sanded with 360, 400 and 600-grit abrasive papers (Carbimet, Buehler, Lake Bluff, IL, USA) under refrigeration to remove the scratches formed during grinding. After buffing with a polishing cloth and 1 μm diamond suspension (Metadi diamond suspension, Buehler, Lake Bluff, IL, USA), the disks were sonicated in filter-sterile tap water in a 900-W microwave oven for 3 mins at 360-W, and for another 3 mins at 810-W after a 4 mins interval [14]. The disks were then washed with a high-pressure water spray to eliminate any surface contaminants, and finally sterilized with 95% ethanol. The prepared disks were first immersed in artificial saliva [15] (0.02% calcium chloride, 0.11% sodium phosphate, 0.17% sodium bicarbonate and 0.2% sodium azide) for 24 h at 37 °C, and then transferred to 40 ml of the respective cleansing solution/filter-sterile tap water. After a 4-week incubation with daily change of solution, the disks were taken out, washed with PBS, and immersed in filter-sterile tap water for 24 h at 37 °C [9]. To measure C. albicans adhesion, 20 ml C. albicans suspension (10⁵ CFU/ml) was added to each tube and incubated for 90 mins. After washing with PBS to remove non-adherent cells, the disks were transferred to polypropylene tubes containing 3 ml sterile PBS each, and sonicated at 7-W for 30s [13]. The resulting single-cell suspension was plated on SDA, cultured at 37 °C for 24 h, and the number of colonies were counted by using a spiral plater. All experiments were performed with three independent sections on three independent sessions.

Forty-five circular heat-cured PMMA base disks were fabricated as described above, immersed in artificial saliva for 24 h at 37 °C, and then in 40 ml of the respective cleansing solution/ filter-sterile tap water for 8 h. The disks were removed and air dried, and the surface morphology micropatterns were analyzed by scanning electron microscopy (SEM). The roughness of the surface was measured in 3 different areas by the stylus method (Surftest SJ-400, Mitutoyo Industry, Japan), and the mean roughness was calculated. All experiments were performed with three independent sections on three independent sessions.

All denture cleansers decreased the growth of C. albicans, evaluated in terms of the number of colonies, in a time-dependent manner compared to filter-sterile tap water (Fig. 1 and Table 1). The inhibitory effects of Clene® and Polident® were significantly higher compared to that of 3% NaHCO₃ (P < 0.05) and PBS (P < 0.05), while no significant differences were observed between the two commercial cleansers, or between 3% NaHCO₃ and PBS. As shown in the Table 1, Clene®and Polident® decreased fungal growth by approximately 98 and 100% respectively, while 3% NaHCO₃ and PBS achieved only 67.54 and 51.30% reduction.

Compared to filter-sterile tap water (OD₄₉₀ = 0.09 ± 0.02), all reagents inhibited C. albicans biofilm formation to some extent. However, no significant differences were detected among Clene® (OD₄₉₀ = 0.06 ± 0.00), Polident® (OD₄₉₀ = 0.05 ± 0.00), 3% NaHCO₃ (OD₄₉₀ = 0.06 ± 0.01), and PBS (OD₄₉₀ = 0.06 ± 0.01) (Fig. 2).

Clene® (OD₄₉₀ = 0.11 ± 0.03) prevented adhesion of C. albicans compared to filter-sterile tap water (OD₄₉₀ = 0.17 ± 0.05). Interestingly, Polident® (OD₄₉₀ = 0.25 ± 0.12) promoted adhesion of C. albicans, resulting in dense cellular layers compared to that seen in the Clene®, 3% NaHCO₃ (OD₄₉₀ = 0.12 ± 0.02), PBS (OD₄₉₀ = 0.14 ± 0.03) and filter-sterile tap water (P < 0.05) groups. Compared to PBS and filter-sterile tap water group, 3% NaHCO₃ did not significantly inhibit the adhesion of C. albicans. Furthermore, no significant differences were seen between Clene® and 3% NaHCO₃ (Fig. 3).

Compared to PBS and filter-sterile tap water, Clene®, Polident® and 3% NaHCO₃ significantly reduced C. albicans adhesion on the PMMA base surface (P < 0.05) (Fig. 4).

The surface roughness of the PMMA base disks was not significantly affected even after overnight immersion in the different cleansing reagents (Table 2), and no significant differences were observed between the different groups on PMMA base surface morphology (Fig. 5).