Efficacy of Citric Acid Denture Cleanser on C. Albicans
Efficacy of Citric Acid Denture Cleanser on C. Albicans
This in vitro study used a randomized, blinded design. C. albicans biofilms were developed for 72 h on PMMA resin specimens (n = 168) and then randomly assigned to 1 of 3 cleansing treatments (CTs): purified water, used as a control (CTC); 1:5 dilution of citric acid denture cleanser (CT5); or 1:8 dilution of citric acid denture cleanser (CT8). Residual biofilms adhering to specimens were collected and quantified at two different time points: immediately after cleaning treatments (ICT group) or 48 h after the cleaning, when residual biofilm recolonization (RT group) would occur. Residual biofilms were analyzed by determining the number of viable cells (CFU/mL). Biofilm architecture was evaluated by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The study factors were denture cleanser treatments and evaluation periods (ICT or RT). The response variables were the number of viable cells and the architecture of C. albicans residual biofilms. A scheme of the experimental design is illustrated in the Additional file 1 http://www.biomedcentral.com/1472-6831/14/77/suppl/S1.
Disc-shaped specimens (10 mm diameter, 2 mm thickness, and 219.8 mm area) of microwave-polymerized PMMA (Onda Cryl; Artigos Odontológicos Clássico Ltd, São Paulo, Brazil) were fabricated according to the manufacturer's instructions. After polymerization, the discs were immersed in purified water at 37°C for 48 h for residual monomer release. The PMMA specimens were then ground with a horizontal polisher (model APL-4; Arotec, São Paulo, Brazil) and using progressively finer aluminum oxide papers (320-, 400-, and 600-grit) to standardize surface roughness at 0.34 ± 0.02 μm. Before developing the biofilm, the discs were ultrasonically cleaned (Thornton T 740; Thornton-Inpec Eletrônica Ltda, Vinhedo, Brazil) with 70% alcohol and sterilized ultra-purified water (20 min) to remove contaminants and artifacts from the surface. The absence of contamination was confirmed by immersing a sample of the specimens in sterile culture media.
The specimens were randomly assigned to 1 of the 3 treatment groups, which were further divided within the evaluated time points: immediately after treatments (ICT) and 48 h after cleaning and residual biofilm recolonization (RT). The number of specimens in each group (n = 12) was determined with preliminary tests, which confirmed the sample size yielded an adequate power (80%) for detecting statistically significant differences.
Before developing the biofilm assays, clean PMMA specimens were coated with saliva to mimic the oral cavity environment. Human whole saliva was donated by a healthy volunteer, who provided written informed consent. The Research and Ethics Committee of Piracicaba Dental School, State University of Campinas, approved this study. The saliva sample was collected at the same time of day, for each experiment, and the collection volume was limited to 50 mL per collection period.
Human whole saliva was collected by masticatory stimulation with flexible film (Parafilm M; American Can Co, Neenah, WI). Saliva was clarified by centrifugation at 3,800 g for 10 min at 4°C. The supernatant was collected and sterilized by filtration (22 μm) for immediate use. For each disc, a salivary pellicle was formed on the surface after incubation for 30 min, at 37°C and 75 rpm, in an orbital shaker.
A loopful of yeast culture of C. albicans (ATCC 90028) was reactivated and incubated for 24 h at 37°C. Afterwards, cells were harvested, suspended in Yeast Nitrogen Base (YNB) broth (Becton Dickinson, Franklin Lakes, NJ) supplemented with 100 mM glucose. Cell density was spectrophotometrically (Spectronic 20; Bausch & Lomb, Rochester, NY) standardized to a 0.25 optical density at 520 ηm, which corresponded to 1 × 10 CFU/mL inoculum.
PMMA saliva-coated discs were placed vertically in polystyrene 24-well culture plates. Subsequently, 2 mL of the standardized cell suspension (1 × 10 CFU/mL of C. albicans in YNB supplemented with glucose 100 mM) was added to each well. Biofilm was developed at 37°C, and under 75 rpm in an orbital shaker, for 72 h, to allow biofilm maturation. The medium was changed every 24 h. Biofilm assays were performed in triplicate in 3 independent experiments.
After the biofilm growth for 72 h, the specimens were randomly assigned to 1 of 3 CTs overnight (8 h). Citric acid cleanser (CURADEN BDC 105, Curaprox, Swiss) was diluted in purified water, according to the manufacturer's instructions, in 1:5 or 1:8 solutions, which are recommended for weekly or daily use, respectively. Each specimen was placed in a sterile beaker containing 8 mL of the treatment solution.
Following cleanser treatments (8 h), specimens were removed and washed twice in 2 mL of sterilized phosphate-buffered saline (PBS) solution (pH 7.4). The residual biofilms adhering to the specimens were immediately collected (ICT) or allowed to grow under the same conditions for 48 h (RT group).
Residual biofilms were disrupted and adhered microorganisms were removed from specimens by sonication (7 W for 30 s). Sonicated solutions were serially diluted in PBS and plated (20 μL) in triplicate on Sabouraud Dextrose agar. The plates were incubated at 37°C under aerobic conditions for 48 h. CFU were counted using a stereomicroscope, and the results are expressed in colony-forming units per mL (CFU/mL).
Specimens with attached biofilms were rinsed with sterile PBS and placed in 1% osmium tetroxide for 1 h. Specimens were subsequently washed in purified water, dehydrated in a series of ethanol washes (70% for 10 min, 95% for 10 min, and 100% for 20 min) and air-dried in a desiccator before sputter coating with gold. Next, specimens were mounted on aluminum stubs and coated with gold. The biofilm surface features were visualized with SEM (JSM 5600LV, JEOL, Tokyo, Japan) at 1,500× in a high-vacuum mode at 15 kV.
Biofilms formed on PMMA surfaces were stained using the Live/Dead BacLight Viability kit, comprising SYTO-9 and propidium iodide (PI). Before the CLSM examinations, specimens were protected from light and incubated at 37°C for 20 min. Images of stained biofilms were captured using a CLSM system (LEICA – TCS SP5, Leica Microsystems, Wetzlar, Germany). A series of images were obtained at 1-μm intervals in the z section for a three-dimensional view of the biofilm. At least, five representative optical fields were examined for each specimen.
COMSTAT software was used to analyze CLSM images. The architecture properties of biofilms analyzed by COMSTAT included the biovolume (μm/μm), average thickness (μm), and roughness coefficient.
Data were analyzed using statistical software (SAS v. 9.0; SAS Institute, Inc, Cary, NC) with a significance level fixed at 5%. The assumptions of equality of variances and normal distribution of errors were evaluated for each variable. When normality was violated, the data were logarithmically transformed. Factors interfering in the response variables (C. albicans viable cells, CFU/mL), bio-volume (μm3/μm2), average thickness (μm) and roughness coefficient) were analyzed with two-way ANOVA (type and evaluation period). Post-hoc comparisons were performed using Tukey's Honestly Significant Difference (HSD) test.
Methods
Experimental Design
This in vitro study used a randomized, blinded design. C. albicans biofilms were developed for 72 h on PMMA resin specimens (n = 168) and then randomly assigned to 1 of 3 cleansing treatments (CTs): purified water, used as a control (CTC); 1:5 dilution of citric acid denture cleanser (CT5); or 1:8 dilution of citric acid denture cleanser (CT8). Residual biofilms adhering to specimens were collected and quantified at two different time points: immediately after cleaning treatments (ICT group) or 48 h after the cleaning, when residual biofilm recolonization (RT group) would occur. Residual biofilms were analyzed by determining the number of viable cells (CFU/mL). Biofilm architecture was evaluated by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The study factors were denture cleanser treatments and evaluation periods (ICT or RT). The response variables were the number of viable cells and the architecture of C. albicans residual biofilms. A scheme of the experimental design is illustrated in the Additional file 1 http://www.biomedcentral.com/1472-6831/14/77/suppl/S1.
Resin Specimens
Disc-shaped specimens (10 mm diameter, 2 mm thickness, and 219.8 mm area) of microwave-polymerized PMMA (Onda Cryl; Artigos Odontológicos Clássico Ltd, São Paulo, Brazil) were fabricated according to the manufacturer's instructions. After polymerization, the discs were immersed in purified water at 37°C for 48 h for residual monomer release. The PMMA specimens were then ground with a horizontal polisher (model APL-4; Arotec, São Paulo, Brazil) and using progressively finer aluminum oxide papers (320-, 400-, and 600-grit) to standardize surface roughness at 0.34 ± 0.02 μm. Before developing the biofilm, the discs were ultrasonically cleaned (Thornton T 740; Thornton-Inpec Eletrônica Ltda, Vinhedo, Brazil) with 70% alcohol and sterilized ultra-purified water (20 min) to remove contaminants and artifacts from the surface. The absence of contamination was confirmed by immersing a sample of the specimens in sterile culture media.
The specimens were randomly assigned to 1 of the 3 treatment groups, which were further divided within the evaluated time points: immediately after treatments (ICT) and 48 h after cleaning and residual biofilm recolonization (RT). The number of specimens in each group (n = 12) was determined with preliminary tests, which confirmed the sample size yielded an adequate power (80%) for detecting statistically significant differences.
Salivary Pellicle Formation on Specimens
Before developing the biofilm assays, clean PMMA specimens were coated with saliva to mimic the oral cavity environment. Human whole saliva was donated by a healthy volunteer, who provided written informed consent. The Research and Ethics Committee of Piracicaba Dental School, State University of Campinas, approved this study. The saliva sample was collected at the same time of day, for each experiment, and the collection volume was limited to 50 mL per collection period.
Human whole saliva was collected by masticatory stimulation with flexible film (Parafilm M; American Can Co, Neenah, WI). Saliva was clarified by centrifugation at 3,800 g for 10 min at 4°C. The supernatant was collected and sterilized by filtration (22 μm) for immediate use. For each disc, a salivary pellicle was formed on the surface after incubation for 30 min, at 37°C and 75 rpm, in an orbital shaker.
Inoculum and Growth Conditions
A loopful of yeast culture of C. albicans (ATCC 90028) was reactivated and incubated for 24 h at 37°C. Afterwards, cells were harvested, suspended in Yeast Nitrogen Base (YNB) broth (Becton Dickinson, Franklin Lakes, NJ) supplemented with 100 mM glucose. Cell density was spectrophotometrically (Spectronic 20; Bausch & Lomb, Rochester, NY) standardized to a 0.25 optical density at 520 ηm, which corresponded to 1 × 10 CFU/mL inoculum.
Biofilm Assay
PMMA saliva-coated discs were placed vertically in polystyrene 24-well culture plates. Subsequently, 2 mL of the standardized cell suspension (1 × 10 CFU/mL of C. albicans in YNB supplemented with glucose 100 mM) was added to each well. Biofilm was developed at 37°C, and under 75 rpm in an orbital shaker, for 72 h, to allow biofilm maturation. The medium was changed every 24 h. Biofilm assays were performed in triplicate in 3 independent experiments.
Treatment Protocols
After the biofilm growth for 72 h, the specimens were randomly assigned to 1 of 3 CTs overnight (8 h). Citric acid cleanser (CURADEN BDC 105, Curaprox, Swiss) was diluted in purified water, according to the manufacturer's instructions, in 1:5 or 1:8 solutions, which are recommended for weekly or daily use, respectively. Each specimen was placed in a sterile beaker containing 8 mL of the treatment solution.
Following cleanser treatments (8 h), specimens were removed and washed twice in 2 mL of sterilized phosphate-buffered saline (PBS) solution (pH 7.4). The residual biofilms adhering to the specimens were immediately collected (ICT) or allowed to grow under the same conditions for 48 h (RT group).
Viable Cell Quantification From Residual Biofilm
Residual biofilms were disrupted and adhered microorganisms were removed from specimens by sonication (7 W for 30 s). Sonicated solutions were serially diluted in PBS and plated (20 μL) in triplicate on Sabouraud Dextrose agar. The plates were incubated at 37°C under aerobic conditions for 48 h. CFU were counted using a stereomicroscope, and the results are expressed in colony-forming units per mL (CFU/mL).
Biofilm Architecture Analysis: SEM
Specimens with attached biofilms were rinsed with sterile PBS and placed in 1% osmium tetroxide for 1 h. Specimens were subsequently washed in purified water, dehydrated in a series of ethanol washes (70% for 10 min, 95% for 10 min, and 100% for 20 min) and air-dried in a desiccator before sputter coating with gold. Next, specimens were mounted on aluminum stubs and coated with gold. The biofilm surface features were visualized with SEM (JSM 5600LV, JEOL, Tokyo, Japan) at 1,500× in a high-vacuum mode at 15 kV.
Biofilm Architecture Analysis: CLSM
Biofilms formed on PMMA surfaces were stained using the Live/Dead BacLight Viability kit, comprising SYTO-9 and propidium iodide (PI). Before the CLSM examinations, specimens were protected from light and incubated at 37°C for 20 min. Images of stained biofilms were captured using a CLSM system (LEICA – TCS SP5, Leica Microsystems, Wetzlar, Germany). A series of images were obtained at 1-μm intervals in the z section for a three-dimensional view of the biofilm. At least, five representative optical fields were examined for each specimen.
COMSTAT software was used to analyze CLSM images. The architecture properties of biofilms analyzed by COMSTAT included the biovolume (μm/μm), average thickness (μm), and roughness coefficient.
Statistical Analysis
Data were analyzed using statistical software (SAS v. 9.0; SAS Institute, Inc, Cary, NC) with a significance level fixed at 5%. The assumptions of equality of variances and normal distribution of errors were evaluated for each variable. When normality was violated, the data were logarithmically transformed. Factors interfering in the response variables (C. albicans viable cells, CFU/mL), bio-volume (μm3/μm2), average thickness (μm) and roughness coefficient) were analyzed with two-way ANOVA (type and evaluation period). Post-hoc comparisons were performed using Tukey's Honestly Significant Difference (HSD) test.
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