Statement of problem
As Candida albicans biofilm formation is associated with severe local and systemic infections in denture-wearing patients, its prevention or reduction becomes an essential factor in the health of this population.
The purpose of this in vitro study was to investigate whether 2 photopolymerized coatings of poly(acrylic acid) (PAA) and poly(itaconic acid) (PIA) can effectively reduce the adhesion of C albicans on denture base acrylic resin surfaces.
Material and methods
The surface of the polymethyl methacrylate (PMMA) denture base was modified through photopolymerization of a thin film of PAA or PIA. The polymeric coatings were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), contact angle goniometry (CA), and surface roughness measurement (Ra). For biological evaluation, the coated PMMA surfaces were tested in a C albicans biofilm dynamic formation model, observed by confocal laser scanning microscopy (CLSM), and quantified by the number of colony-forming units (CFUs). The cytotoxicity of the polymeric coatings was also evaluated by using a lactic dehydrogenase-based (LDH) test. For statistical analysis, ANOVA and the nonparametric Kruskal–Wallis test were used (α=.05).
The PMMA resin base surfaces coated with PAA and PIA had an inhibitory effect on C albicans growth, the wettability of the coated surface, and the average roughness. The PAA and PIA coatings had no statistically significant cytotoxic effect on periodontal ligament fibroblasts.
PMMA acrylic resin base material was superficially modified through the incorporation of carboxylic acid groups by using PAA and PIA coatings that reduced the adherence of C albicans biofilm by 90%.
Acrylic acid or itaconic acid coatings over conventional polymethyl methacrylate reduced the formation of C albicans biofilm. Their clinical use may have an impact on the prevention and reduction of oral and systemic infections in denture-wearing patients.
Since the latter half of the 19th century, polymers have been used extensively for fabricating removable dentures. Although vulcanized rubber, polystyrene (PS), polyvinyl alcohol (PVA), and polyamides have been used in denture base fabrication, none has proved to perform better than polymethyl methacrylate (PMMA). Nevertheless, one of the major disadvantages of PMMA is the biofilm attachment of pathogen microorganisms, being an ideal reservoir that promotes the growth and dispersion of yeasts such as Candida albicans to the polymer surface.
In an effort to add antimicrobial or antifungal activity to PMMA substrates, the use of additives such as nystatin, miconazole, chlorhexidine, modified monomers, nanoparticles, or different nanocomposites, including silver, zirconium dioxide, and aluminum borate, as well as polymeric coatings, has been reported. All of these approaches have led to changes in the topography of the PMMA surface at micrometer (μm) and nanometer (nm) levels, which can significantly affect the hydrophobicity, wettability, adsorption, and adhesive properties. Thus, modifications of the topography and physicochemical properties of surfaces are of great interest for the biomedical industry. The technological processes used to change the physicochemical properties of a material surface can be classified by whether a layer of new material is applied on the surface (coating deposition) or the surface itself is changed by exposing it to physical or chemical agents, such as abrasive particles, gases, plasmas, or wet chemicals. The antibacterial and antifungal activity of an acidic surface has been previously reported in acrylic resin–treated films.
The purpose of this in vitro study was to assess the effect of photopolymerized coatings of poly(acrylic acid) (PAA) and poly(itaconic acid) (PIA) on C albicans biofilm formation as compared with a polymethyl methacrylate (PMMA) denture resin surface. The null hypothesis was that the different surface treatments would have no influence on C albicans biofilm formation.
Material and methods
Specimens were divided into 6 groups, 4 of them with polymeric coatings and 2 as control ( Table 1 ). A total of 200 PMMA resin disks (13 mm to 14 mm in diameter; 4 mm to 5 mm in thickness) were made from 2 commercially available acrylic resins (Lucitone 199 [Dentsply Sirona] and ProBase Hot [Ivoclar Vivadent AG]) by using the conventional process for denture fabrication according to the manufacturers' instructions. Acrylic and itaconic acids of 99% purity and benzophenone (99%) (Sigma-Aldrich) were used without further purification.
|Specimen Code||Surface Composition||Modifying Agent % (w/w)|
|LT-PAA ∗||Lucitone 199 coated with poly(acrylic acid)||90 AA ∗ /10 MMA ∗|
|PB-PAA||ProBase Hot coated with poly(acrylic acid)||90 AA/10 MMA|
|LT-PIA ∗||Lucitone 199 coated with poly(itaconic acid)||1 IA ∗ /99 MMA|
|PB-PIA||ProBase Hot coated with poly(itaconic acid)||1 IA/99 MMA|
The PMMA disks were completely covered with a drop of a solution containing the modifying agents, acrylic acid (AA) or itaconic acid (IA), dissolved in methyl methacrylate (MMA) monomer and with benzophenone as the photoactivator. The surface of the PMMA probes (disks) was modified by placing the PAA- and PIA-coated PMMA disks in an ultraviolet light (UV) chamber provided with a 300-W microwave-initiated fusion 300S UV lamp (Fusion UV Systems, Inc). The PMMA surfaces were exposed for 30 minutes at a 30-cm distance from the radiation source. All characterization measurements based on 3 repetitions for each specimen were recorded by a single operator (L.D.A.), and the average values were calculated.
To identify the morphology of the polymeric coatings on the PMMA probes, all specimens were transversally cut at room temperature by using a microtome (MICROM HM335E; Thermo Scientific), and the surfaces were examined by scanning electron microscopy (SEM) (JSM-7401F; JEOL) at ×25, ×100, ×200, ×500, and ×1000 magnifications. Images were obtained at the 50- to 100-μm scale, with the voltage at 15 kV. All PMMA specimens were chemically characterized with FTIR (Nicolet NEXUS 470; Thermo Fisher) at 2.5-μm to 50-μm wavelength and at 4000 to 200 cm −1 function frequency. This analysis was made to qualitatively identify the chemical composition of coated and uncoated surfaces by using the characteristic functional group signals on the PAA and PIA coatings compared with those on the PMMA controls.
For XPS analysis, a PHI-VersaProbe II instrument (Physical Electronics) was used in a vacuum chamber at 2×10 −8 mTorr, with a monochromatic X-ray source with an aluminum anode and 1486.6 eV of radiation energy. The general spectrum (survey) was obtained with 117.4 eV of pass energy, and the analysis region was of 1400 to 0 eV in binding energy. High-resolution spectra were obtained for C1s and O1s signals on each specimen, with 11.75 eV of pass energy. The deconvolution of the spectra was calculated through Gaussian curve adjustment, without restriction on position and area, although the full width at half maximum (FWHM) value remained fixed for each adjusted curve.
The wettability measurements of the surfaces were made by using a goniometer (GBX Scientifique). Distilled water (7 μL) was dropped on the PMMAs disk surfaces at 2 different times, first or initial measurement (degrees) and second or final measure (degrees), 5 minutes after the drop hysteresis, and average values for contact angles were calculated for each specimen. The roughness of the coated and uncoated PMMA specimens was measured by using a profilometer (Alpha-Step D-600 model; KLA-Tencor) to evaluate topography and to verify the average roughness value (Ra in μm). The cutoff length was 100 μm, the length was 3 mm, the force was 1.9×10 −5 N, and the stylus speed was 0.05 mm/s.
C albicans biofilm adhesion was evaluated in a dynamic model by using a CDC bioreactor (CBR 90–1 model; BioSurface Technologies Corp). To prepare the inoculum, the yeast C albicans ATCC 90028 was inoculated in yeast extract, peptone, and dextrose (YPD) broth (1% yeast extract, 2% peptone, 2% dextrose) and incubated for 48 hours at 37 °C. After this period, the cells were washed twice with sterile phosphate buffered saline solution (PBS) (pH 7.2) agitated and centrifuged at 2500 rpm for 5 minutes. For the CDC bioreactor system, a C albicans suspension was standardized to a concentration of 0.2 to 1×10 2 cells/mL in YPD culture broth. A primary incubation period of 16 hours under orbital agitation at 80 rpm was followed by an additional 24 hours of continuous flow at a rate of 1.8 mL/min of YPD broth and was regulated by a peristaltic pump (Tubing Pumps ATP-3200 model; Automatic Science Instruments LTD) and 60-rpm constant agitation.
The viability of the C albicans biofilm cells adhering to PMMA specimen surfaces was evaluated with fluorescent staining (LIVE/DEAD yeast viability kit; Molecular Probes) incubated for 15 to 30 minutes in the dark at 30 °C before examination by confocal laser scanning microscopy (CLSM) (DMI 4000B; Leica). The stained biofilm specimens were examined by using an argon ion laser with a 480-nm excitation wavelength and 520- and 650-nm emission wavelengths. The colony-forming units (CFUs) were quantified on the biofilm recovered from the PMMA disks after rubbing the surface of each specimen with a sterilized cell scraper (TRP of 240-mm length), transferring the units to YPD culture broth, and incubating them at 37 °C for 1 hour. After the incubation period, the specimens were serially diluted (10 −1 to 10 −7 ) onto Sabouraud dextrose agar plates and incubated at 37 °C for 24 hours. The log CFU/mL was calculated by the same examiner (L.D.A.).
To evaluate the possible cytotoxicity of the polymeric coatings, an in vitro toxicology assay was carried out by using a lactic dehydrogenase–based (LDH) kit (Sigma-Aldrich). This method measured either the number of cells from the total cytoplasmic lactate dehydrogenase level or membrane integrity as a function of the amount of cytoplasmic LDH released into the medium. Previously, eluted specimens (100 μL) had been obtained from the polymeric coatings and placed in a presterilized, polystyrene, flat-bottomed, 96-well microtiter plate. Each well was seeded with periodontal ligament fibroblasts (from a primary cell culture at 80% of confluence), and the plate was incubated at 37 °C for 24 hours. The absorbance values were measured by using a microtiter plate reader (Multiskan system; Thermo Fisher Scientific) at 490 nm.
The nonparametric Kruskal-Wallis test was used to compare roughness among groups of specimens with each polymer coating. Contact angle goniometry values were analyzed statistically by ANOVA and the Wilcoxon test. C albicans biofilm formation (yeast viability assay and CFU assay) between the coated and uncoated specimens in each group were analyzed by the nonparametric Kruskal-Wallis test (α=.05).
Different compositions (w/w) of the modifying agents (acrylic and itaconic acids) mixed with the corresponding commercial acrylic resins (Lucitone 199 and ProBase Hot) of lower or higher concentrations of PAA and PIA than the ones shown in Table 1 were tested to prepare the coatings. At concentrations other than those listed, irregular surfaces were obtained because of the cross-linking reaction with UV light. The chemical composition, morphology, and successful incorporation of the polymeric coatings into the PMMA disks were confirmed by FTIR, XPS, and SEM analyses. The SEM micrographs of the PMMA disks were smooth and dense ( Fig. 1 A). A crosslinked poly(MMA-AA) copolymer formed on the modified surface, which was homogeneously distributed and had 2 polymeric phases because of the incorporation of the modifying agents. In a transversal section of a modified PMMA specimen (LT-PAA), the PAA coating was observed to be compact and well adhered to the PMMA substrate ( Fig. 1 B).
From the analysis of the uncoated control PMMA disks, the infrared spectrum obtained showed the expected signals and absorbance peaks, a strong signal at 1721 cm −1 corresponding to the carbonyl group (C=O). In the spectra obtained from the PAA and PIA coatings, broader signals were observed for the complete spectrum, and 2 different weak signals at 1625 cm −1 and 1658 cm −1 were identified, corresponding to the characteristic peaks of the carboxylic groups of PAA and PIA ( Fig. 2 ).
The XPS analysis confirmed the elemental chemical composition of the polymeric coatings on the PMMA disks. The spectra of both control and treated specimens showed intense signals from photoejection of electrons from the core orbitals of C1s and O1s on the surface at approximately 290 and 530 eV ( Fig. 3 , left side). High-resolution spectra of each region revealed the presence of more than 1 signal (for example, Fig. 3 , center) from the presence of C or O in different bonding environments. Three resolved signals were seen, corresponding to C singly bonded only to C and H, C singly bonded to one O atom, and C bonded to 2 O atoms (as in an ester group), whereas quaternary carbons (C q ) have a higher binding energy than those bonded to H. Both the C1s and O1s curves were fitted to 4 and 2 Gaussian curves, respectively, where the binding energy and area of each Gaussian curve varied but not the width.
Comparison of the structures of PMMA, PAA, and PIA revealed 4 types of C in PMMA (2 C-C, 1 Cq, 1 CO 2 , and 1 CO), only 2 types in PAA (2 C-C and 1 CO 2 ), and 3 types in PIA (2 C-C, 1 Cq, and 2 CO 2 ). The relative areas of the signals were not directly proportional to the number of the different C atoms present because the different types are detected, with somewhat different sensitivities in the XPS. Also, since the signals from C-C and Cq were incompletely resolved, the fitting of 2 Gaussian curves to this peak was inexact; their total should correlate with the number of carbon atoms not bonded to oxygen.
The results of the deconvolution data are summarized in Table 2 . When a PMMA group is substituted by an AA moiety, there should be a reduction in the number of C-C and Cq atoms, whereas for IA, there should be minimal change. This is supported in the total percentage of C-C atoms versus the percentages of C-O and CO 2 atoms. Similar results were observed for the PB acrylic resin.
|Surface||% C-C||C-C (B.E.)||% Cq||Cq (B.E.)||% C-O||C-O (B.E.)||% C-COO||C-COO (B.E.)|
LT and PB acrylic resin controls showed hydrophobic surfaces with angles of 80.8 ±0.94 and 81.7 ±0.91 degrees, respectively, as expected for PMMA disks, whereas all polymeric coated specimens showed hydrophilic surfaces (according to their contact angles, Table 3 ). This observation could be attributed to the presence of polar groups such as the carboxylic acids, previously identified by FTIR and XPS. The contact angle decreased considerably in the specimens with PIA (36.8 ±6.0 degrees), indicating highly hydrophilic surfaces that may be associated with a greater number of carboxylic groups on the surfaces.
|Surface||Initial (Degrees)||Final (Degrees)|
|LT||80.8 (0.94)||72.3 (0.64)|
|PB||81.7 (0.91)||73.6 (0.79)|
|LT-PAA||77.7 (6.38)||71.1 (5.33)|
|PB-PAA||74.7 (4.43)||66.9 (2.89)|
|LT-PIA||62.9 (6.85)||50.4 (10.92)|
|PB-PIA||36.8 (6.0)||16.8 (3.8)|
Average roughness value (Ra micrometers) was calculated for all groups of specimens ( Fig. 4 ). A statistically significant difference ( P <.05) on the PAA coatings existed between both clinical PMMA specimens, and the larger surface roughness was observed on PB-PAA (Ra=1.445 ±0.28 μm), which may be associated with the agglomerations previously observed through SEM. This rougher surface directly affects the wettability, resulting in a hydrophilic surface according to the results mentioned previously.
The dynamic formation of biofilms on the PMMA disks observed through CLSM showed, as expected, the presence of numerous C albicans yeast colonies on the LT ( Fig. 5 A) control surface. The LT-PAA coatings showed a reduction of 50% to 60% on the formed biofilm ( Fig. 5 B); meanwhile, on the LT-PIA coatings, the biofilm reduction was of 80% to 90% ( Fig. 5 C). Biofilms observed on the PB ( Fig. 5 D) control surfaces presented more filamentous structures (pseudohyphae or hyphae), the PB-PAA coatings showed a reduction of 50% to 60% on the formed biofilm ( Fig. 5 E), and on the PB-PIA coatings, the biofilm reduction was 80% to 90% ( Fig. 5 F). Thus, the major reductions in the C albicans biofilm were obtained in the PIA acrylic resin–coating groups.
The CFU quantified in the recovered biofilms indicated a reduction of C albicans counts in all surface-coated groups ( Fig. 6 ). The greatest reduction (≥4 log) was obtained with the PB-PIA acrylic resin coating, in which biofilm formation was approximately 4.80×10 4 CFU as compared with the control group (2.22×10 7 CFU). The PAA coatings also showed important biofilm reductions of 3.89×10 5 CFU for LT acrylic resin and 4.16×10 5 CFU for PB acrylic resin. These results confirm those revealed through CLSM, where a reduction in C albicans biofilm viability was observed on all polymeric coatings. The lactic dehydrogenase (LDH) activity was assayed by 2 methods: total LDH (method 1), which provided a measure of total cell numbers, and LDH release (method 2), which assessed the membrane integrity of cells as a function of the amount of LDH leakage into the medium. For both LDH methods, no significant inhibition of cell growth (cytotoxicity) occurred ( Fig. 7 ), the highest cell proliferation was observed in the PIA coating for both clinical trademarks, and the cytotoxic effect was determined by the highest amount of LDH released.