This article was downloaded by: [University of Connecticut] On: 10 January 2015, At: 11:54 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Biofouling: The Journal of Bioadhesion and Biofilm Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gbif20
In vitro evaluation of adherence of Candida albicans, Candida glabrata, and Streptococcus mutans to an acrylic resin modified by experimental coatings a
a
a
Fernanda Emiko Izumida , Eduardo Buozi Moffa , Carlos Eduardo Vergani , Ana Lúcia a
a
Machado , Janaína Habib Jorge & Eunice Teresinha Giampaolo
a
a
Department of Dental Materials and Prosthodontics, Araraquara Dental School, Universidade Estadual Paulista, UNESP, São Paulo, Brazil Published online: 01 Apr 2014.
Click for updates To cite this article: Fernanda Emiko Izumida, Eduardo Buozi Moffa, Carlos Eduardo Vergani, Ana Lúcia Machado, Janaína Habib Jorge & Eunice Teresinha Giampaolo (2014) In vitro evaluation of adherence of Candida albicans, Candida glabrata, and Streptococcus mutans to an acrylic resin modified by experimental coatings, Biofouling: The Journal of Bioadhesion and Biofilm Research, 30:5, 525-533, DOI: 10.1080/08927014.2014.894028 To link to this article: http://dx.doi.org/10.1080/08927014.2014.894028
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Biofouling, 2014 Vol. 30, No. 5, 525–533, http://dx.doi.org/10.1080/08927014.2014.894028
In vitro evaluation of adherence of Candida albicans, Candida glabrata, and Streptococcus mutans to an acrylic resin modified by experimental coatings Fernanda Emiko Izumida, Eduardo Buozi Moffa, Carlos Eduardo Vergani, Ana Lúcia Machado, Janaína Habib Jorge and Eunice Teresinha Giampaolo* Department of Dental Materials and Prosthodontics, Araraquara Dental School, Universidade Estadual Paulista, UNESP, São Paulo, Brazil
Downloaded by [University of Connecticut] at 11:54 10 January 2015
(Received 29 October 2013; accepted 4 February 2014) This study evaluated the effect of experimental coatings, containing zwitterion or hydrophilic monomers, on the adherence of Candida albicans, Candida glabrata, and Streptococcus mutans to an acrylic resin. Acrylic samples (smooth or rough surfaces) were left untreated (control) or coated with one of the following experimental coatings: 3-hydroxypropylmethacrylate (HP) or sulfobetaine methacrylate (S), at concentrations of 25, 30, or 35%. Half of the specimens were coated with saliva. The adhesion test was performed by incubating specimens in C. albicans, C. glabrata, and S. mutans suspensions at 37°C for 90 min. The number of adhered microorganisms was determined by metabolic activity (XTT) and by cell viability (CFU). All coated specimens exhibited lower absorbance and CFU values compared to control specimens. Saliva and roughness did not promote microorganism adherence. An XPS analysis confirmed the modification in the chemical composition of the coatings in the experimental samples. These experimental coatings significantly reduced the adherence of C. albicans, C. glabrata and S. mutans to acrylic resin. Keywords: acrylic resin; saliva; Candida albicans; Candida glabrata; Streptococcus mutans
Introduction Removable dentures (RD) provide edentulous patients with the rehabilitation of masticatory and esthetic functions (Dhir et al. 2007). However, one consequence of the continual use of RD is the adhesion of microorganisms and biofilm formation (Lazarin et al. 2012) in the base of the prosthesis. Candida species are opportunistic pathogens that are frequently isolated from the oral cavity and its biofilms are often associated with oral candidosis (Gomes et al. 2011; Hahnel et al. 2012; Lazarin et al. 2012). Although a majority of oral candidosis cases are attributed to C. albicans (Park et al. 2003; Dhir et al. 2007; Lazarin et al. 2012, 2013), an increasing number of infections are caused by non-C. albicans Candida (NCAC) species (Silva et al. 2011), Candida glabrata, which has been isolated from RD and palatal mucosa (Gomes et al. 2011). This yeast species is considered an emerging fungal pathogen, which exhibits superior adhesion to acrylic resin surfaces compared to C. albicans (de Freitas Fernandes et al. 2011). Furthermore, studies have shown that the adhesive interactions between Candida species and oral bacteria such as Streptococcus mutans play a crucial role in microbial colonization of denture acrylic, which may lead to denture stomatitis (Millsap et al. 1999). Bacteria facilitate the adherence of Candida cells to the denture base and mucosa, mainly by extracellular polymer *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
production as well as increased acidity, which creates optimal environmental conditions for yeast growth. Systemic alterations (Tylenda et al. 1989), poor oral hygiene (Wilson 1998) and the surface characteristics of acrylic resin, such as hydrophobicity and surface roughness, have played a role in the development and maintenance of denture stomatitis. It has been demonstrated that moderately hydrophobic surfaces support cell adhesion whereas hydrophilic surfaces demonstrate limited cell adhesion (Long et al. 2003). In the case of surface roughness, it can be seen that rough surfaces provide a large surface area and may act as niches for microorganisms, thus favoring adhesion (Waters et al. 1997; Moura et al. 2006; Zamperini et al. 2010). Furthermore, other factors, such as the salivary pellicle, may also alter the surface characteristics of the substratum involved in the adhesion process (Park et al. 2003). It has been demonstrated that the salivary pellicle may regulate specific interactions between cellular adhesion of C. albicans and receptors in the pellicle (Yildirim et al. 2006). Studies have shown that surface modification could be considered a promising approach to avoid denture stomatitis (Redding et al. 2009; Lazarin et al. 2012, 2013) because this modification could reduce the adhesion of microorganisms to the substratum (Lowe et al. 2000; West et al. 2004; Cheng et al. 2007; Lazarin et al. 2012,
Downloaded by [University of Connecticut] at 11:54 10 January 2015
526
F.E. Izumida et al.
2013). In this way, the use of coatings could reduce protein adsorption and adhesion of microorganisms. Liquid adhesives can flow easily over the entire surface of the substratum and come into contact with all the small rough areas that may be present (Özden et al. 1999). Moreover, coatings could be developed with antimicrobial agents, such as chlorhexidine or nystatin (Redding et al. 2009) or with other compounds, as such zwitterionic and hydrophilic polymers. These additives could enhance antibiofilm and antifungal activity (Redding et al. 2009). Polymeric coatings have been applied to the surfaces of materials to reduce adhesion of microorganisms (Lowe et al. 2000). Zwitterionic polymers, such as sulfobetaines and phosphatidylcholines have been used to reduce protein adsorption and microorganism adherence to a variety of medical devices (Lowe et al. 2000; Hasegawa et al. 2001; Konno et al. 2001). The ability to prevent protein adsorption may be due to the fact that the polymers have both a positively and a negatively charged group within the molecule but have an overall neutral charge (West et al. 2004; Lazarin et al. 2012). The hydrophilic nature of the head group of the polymer promotes the formation of a strongly bound hydration layer on the material surface, inhibiting protein adsorption (Long et al. 2003). Additionally, the percentage of free water in the water phase of a zwitterionic-polymer hydrogel was higher than in conventional hydrogels, which could prevent conformational changes of the proteins so that they do not attach to or easily detach from the surfaces (Nakabayashi and Williams 2003). Although the initial application of zwitterionic polymers has been to promote the compatibility of blood catheters, recent studies have demonstrated the ability of sulfobetaine, coated into polymethyl methacrylate (PMMA) surfaces, in reducing adhesion of Pseudomonas aeruginosa, Staphylococcus epidermidis, and C. albicans (Lowe et al. 2000; West et al. 2004; Cheng et al. 2007). As a result of these beneficial characteristics, sulfobetaine polymers could be used to modify the denture base surfaces, promoting a reduction in microbial adherence and consequently preventing denture stomatitis (Lazarin et al. 2012, 2013). In addition, other non-toxic hydrophilic monomers (Lippens et al. 2013), such as polyvinylpyrralidone, polyacrylamide, poly(ethylene glycol) (PEG), parylene, and hydroxypropyl methacrylate (HPMA), have been evaluated to modify the biomaterial surface and reduce protein adsorption and microbial adherence (Ishihara et al. 1998; Çagavi et al. 2004; Cringus-Fundeanu et al. 2007; Zhao et al. 2010; Lazarin et al. 2013). Covalent bonds occur in the hydrogels between the hydrophilic polymers and the biomaterial. When these hydrogels are hydrated, they form a water jacket around the material, which makes it perfectly smooth and hydrophilic, preventing bacterial colonization (Çagavi et al. 2004). However, the hydration state of these polymers is
achieved via hydrogen bonding, which is considered weaker than the ionic solvation found in zwitterionic polymers. Furthermore, the free water of hydrophilic polymers is lower than that of zwitterionic polymers (Ishihara et al. 1998; Lazarin et al. 2012). Therefore, the aim of this study was to evaluate the effectiveness of two experimental coatings, containing zwitterionic or hydrophilic monomers, in three different concentrations (25, 30 and 35%) on the adherence of C. albicans, C. glabrata and S. mutans to an acrylic resin. Additionally, the effects of surface roughness and the salivary pellicle were also evaluated. Material and methods Specimen preparation A total of 576 circular specimens (10 × 2 mm) of a microwave-cured denture base acrylic resin (Vipi Wave; VIPI Indústria e Comércio Exportação e Importação de Produtos Odontológicos Ltda, Pirassununga, SP, Brazil) were fabricated using a steel mold with a breakaway compartment. The metallic matrices were placed in dental flasks, sandwiched between two glass plates that were smooth or blasted with aluminum oxide, whose surface roughness was ~3.0 μm. These two types of glass plates were used to obtain, respectively, smooth and rough surfaces that simulate the outer and inner surface of the prosthesis. For each specimen, the acrylic resin Vipi Wave was mixed according to the manufacturer’s instructions at a mixing ratio of 1 g of powder to 0.47 ml of liquid, and packed into the molds. A hydraulic press (PM 2000, Vipi Delta, Pirassununga, SP, Brazil) was used for packing the denture base resin at 9806,65 N, maintained for 30 min. Next, specimens were polymerized in a 500 W domestic microwave oven (Brastemp da Amazonia SA, Manaus, AM, Brazil) for 20 min at 20% power, followed by 5 min at 90% power. The flasks were bench cooled overnight. They were then removed from the flask and the excess resin was trimmed with a bur (Maxi-Cut; Lesfilsde August Malleifer SA, Ballaigues, Switzerland). Surface roughness measurements The surface roughness values (Ra) were measured using a surface roughness profilometer (SJ 400, Mytutoyo Corporation, Tokyo, Japan), with a resolution of 0.01 μm, an interval (cut-off length) 0.8 mm; a transverse length 2.4 mm; a stylus speed 0.5 mm s−1, and a 5 μm active Diamond stylus tip radius. Four measurements were performed for each sample and the average reading was designated as the intact Ra (μm) value for each specimen. Experimental photopolymerized coatings After roughness measurements were taken, the specimens were randomly distributed into seven groups of 36
Downloaded by [University of Connecticut] at 11:54 10 January 2015
Biofouling samples each (18 specimens with smooth surfaces and 18 with rough surfaces). Samples of the control group (C) did not receive any surface treatment. For the experimental groups, 5 μl of each experimental photopolymerized coating were applied to all samples with a soft brush as a thin layer. Two coating formulations were evaluated: one coating contained hydrophilic monomers, namely 2-hydroxypropyl methacrylate (HPMA) (HP) and the other coating contained a zwitterionic monomer (sulfobetaine methacrylate) (S). The concentrations of monomers used were 25, 30, and 35% mol fraction of the total composition, resulting in six experimental coatings (HP25; HP30; HP35; S25; S30; S35). The coatings additionally contained the monomer methyl methacrylate, two crosslinking agents (tri-ethylene glycol dimethacrylate – TEGDMA and bisphenol-A-glycidyl methacrylate – Bis-GMA) and an initiator agent (4-methyl benzophenone). For the S coating, amino propyl methacrylate was also added. The concentrations of the components of the coatings are presented in Table 1. The application of the six coatings on both surfaces of the specimens was performed in a sterile laminar flow chamber, followed by a 4 min polymerization on each side in an EDG oven (Strobolux; EDG, São Carlos, SP, Brazil). Propane sultone was brushed on the S coating sample surfaces, followed by storage in an oven at 80°C for 2 h. Finally, all specimens were stored in distilled water at room temperature for 48 h for residual monomer elimination (American National Standard 1975 [reaffirmed 1999]; Moura et al. 2006). After application of the coatings to the specimen surfaces, surface roughness values (Ra) were measured as described previously. Sterilization of specimens Prior to the microbiological tests, samples were packed in sealed sterile plastic bags with sterile distilled water and ultrasonicated for 20 min. Then all specimen surfaces were exposed to ultraviolet light in a laminar flow chamber for 20 min for sterilization (Sheridan et al. 1997). Table 1.
Saliva collection and assay Saliva (20 ml per person) was donated by 15 healthy individuals, aged 20 to 35 years. The saliva was collected in 50 ml Falcon tubes on ice. Then, the saliva was pooled, homogenized, and subjected to centrifugation at 10,000 × g for 5 min at 4°C (Moura et al. 2006). The saliva supernatant was immediately stored at –70°C until use. This research was approved by the Ethics Committee of Araraquara Dental School, UNESP – Univ Estadual Paulista (012/2010) and all volunteers were informed about the objectives of the study and signed an informed consent form. In order to verify the effect of exposure to saliva on microorganism adhesion, half of the samples in each group were preconditioned with saliva. For pre-conditioning, the specimens were individually exposed to 2 ml of saliva at room temperature for 30 min (Waters et al. 1997; Zamperini et al. 2010; Zamperini et al. 2012).
Adherence assay S. mutans ATCC 25175, C. albicans ATCC 90028, and C. glabrata ATCC 2001 were used. To prepare the inoculum, a loopful of the stock cultures of the yeasts were streaked onto Sabouraud dextrose agar medium with chloramphenicol (SDA) and incubated at 37°C for 48 h, in an aerobic atmosphere. For S. mutans, a loopful was seeded on Mitis Salivarius bacitracin (MSB – Difco Laboratories, Detroit, MI, USA) and incubated at 37°C in a pCO2 atmosphere of 10% for 48 h. The specimens (n = 9) were placed into 24 well plates (one specimen per well). Two loopfuls of these young cultures were transferred to 20 ml of a yeast nitrogen base (YNB) medium with 50 mmol–1 glucose for the yeasts, and to 20 ml of brain heart infusion (BHI) broth for S. mutans, and incubated at 37°C for 21 h for Candida species and for 18 h in an atmosphere of 10% pCO2 for S. mutans. Cells of the resultant cultures were harvested, washed twice with sterile PBS (100 mM NaCl, 100 mM NaH2PO4, pH 7.2) at 5,000 g for 5 min, and resuspended in a sterile BHI broth. Cell suspensions were spectrophotometrically standardized to 1 × 107 cells ml−1 for Candida and
Composition of experimental coatings (in millimoles).
Coating (Code) P25 P30 P35 M25 M30 M35
527
Component MMA 61.0 56.0 51.0 61.0 56.0 51.0
TEGDMA 5.0 5.0 5.0 5.0 5.0 5.0
Bis – GMA 6.0 6.0 6.0 6.0 6.0 6.0
MPB 3.0 3.0 3.0 3.0 3.0 3.0
HPMA (P) 25.0 30.0 35.0
Sulfobetaine (S)
25.0 30.0 35.0
Abbreviations: MMA = methylmetacrilate; TEGDMA = tri-ethyleneglycoldimethacrylate; Bis – GMA = bisphenol-A-glycidylmethacrylate; MPB = 4methyl-benzophenone; HPMA = 2-hydroxypropyl methacrylate.
528
F.E. Izumida et al.
Downloaded by [University of Connecticut] at 11:54 10 January 2015
1 × 108 cells ml−1 for S. mutans (Pereira-Cenci et al. 2008). The absorbance for Candida species was 0.6 at 520 nm, and for S. mutans was 0.1 at 600 nm. An aliquot of 0.7 ml of cell suspension of each microorganism was added to each well and incubated for 90 min at 37°C (Chandra et al. 2001), under agitation (75 rpm), using an orbital shaker, in an atmosphere of 10% pCO2. Non-adherent cells were removed from the specimens by gently washing them twice with 2 ml of PBS. Forceps were used to hold each specimen during this step. Negative controls were sterile specimens immersed in BHI broth with no cells added. Experiments were repeated three times with individual samples in triplicate. XTT assay To determine the number of adherent cells of yeasts and bacteria, nine specimens from each experimental condition were evaluated by an XTT reduction assay, which is based on the metabolic activity of the cells (Ramage et al. 2011). For the XTT assay, two solutions were used: XTT solution and menadione. The XTT solution (Sigma, MO, USA) was prepared in ultra-pure water at a final concentration of 1 mg ml−1. The solution was filter sterilized and stored at -70°C until use. The menadione solution (Sigma, St Louis, MO, USA) was prepared in 0.4 mM acetone, immediately before each assay. All samples were individually removed, washed and transferred to new sterile 24-well plates containing 2 ml of the following solution in each well: 1,580 μl of PBS supplemented with 200 mM glucose, 400 μl of XTT, and 20 μl of menadione. The plates were incubated for 3 h in the dark at 37°C (da Silva et al. 2008). The solution from each well was transferred to a tube and centrifuged at 5,000 g for 1 min. The supernatant was then transferred to a 96-well microplate, and the color change was measured using a microplate reader (Thermo Plate – TP Reader, Nanshan District, Shenzhen, Guangdong, China) at 492 nm. Colony-forming unit (CFU) count The number of cultivable cells was determined by counting the colony forming units (CFU) following the adherence phase of the experiment. Briefly, after washing, the specimens were transferred to Falcon tubes containing 4.5 ml of PBS and vigorously vortexed for 1 min to detach the microorganisms from the samples (Mima et al. 2008). Then, to determine the number of microorganisms in the 10−2, 10−3, 10−4 and 10−5 dilutions, replicate aliquots (10 μl) of the suspension were transferred to plates of two selective media, CHRO-Magar (Difco) for isolation of Candida species and Mitis Salivarius (Difco) for identification of S. mutans. CHRO-Magar plates were incubated aerobically and Mitis Salivarius plates were incubated in 10% CO2 incubator at 37°C for 48 h. After incubation, bacterial
and yeast colony counts of each plated specimen were quantified using a Phoenix CP 600 Plus digital colony counter (Phoenix Indústria e Comércio de Equipamentos Científicos Ltda, Araraquara, Brazil). The counts were repeated by the same examiner for accuracy. The total CFU per unit area (Log CFU cm−2) was then calculated. X-ray photoelectron spectroscopy analysis (XPS) The characterization of the specimen surfaces after coating application was confirmed by XPS, using a UNI-SPECS UHV spectrometer (SPECS Surface Nano Analysis GmbH, Berlin, Germany). In the analysis, the Mg Kα line (E=1253.6 eV) was used and the analyzer pass energy was set to 10 eV. Shirley’s method (Shirley 1972) was used to subtract the inelastic background of the electron core-level spectra of C 1s, O 1s, and N 1s, whose binding energies were corrected using the hydrocarbon component of the polymer fixed at 285.0 eV. The ratio of relative peak areas corrected by sensitivity factors of the corresponding elements was used to determine the composition of the surface layer. Voigt profiles were applied to fit the spectra without placing constraints. The width at half maximum (FWHM) ranged between 1.6 eV and 2.0 eV, and the accuracy of the peak positions was ± 0.1 eV. One specimen from the control group (without surface treatment) and one specimen treated with the two coatings at 35% concentration were analyzed. Statistical analysis The data on the effect of coating treatments at different concentrations on the adhesion of microorganisms, determined by two methods, XTT and colony forming units (CFU), were subjected to four-way analysis of variance (ANOVA): surface roughness (smooth or rough); coating (S or HP); concentration (25, 30 or 35%) and saliva (present or absent), including controls without the coating application. The Tukey test for multiple comparisons of means was adopted to supplement these analyses. A significance of 1% was used for all analyses. The statistical program used was Statistica 6.0 Statsoft (Statistica 6.0, Statsoft, Tulsa, Okla, USA). Results The mean roughness values (Ra, μm) and standard deviations (SD) of all experimental groups, before and after application of coatings, are listed in Table 2. These results show that the acrylic surface became more regular after the coating application. The mean surface roughness of smooth specimens became similar to the rough specimens (p > 0.01), except for HP at all concentrations (p < 0.01). The summary of the analysis of variance of the absorbance for XTT and for CFU of C. albicans, C. glabrata and S. mutans are presented in Tables 3 and 4.
Biofouling Table 2. Mean roughness values (Ra, µm) and SDs of all experimental groups, before and after application of coatings. Surface
Coating and concentration (%)
Smooth
Downloaded by [University of Connecticut] at 11:54 10 January 2015
Rough
Control S 25 30 35 HP 25 30 35 Control S 25 30 35 HP 25 30 35
Before 0.13 0.14 0.17 0.15 0.12 0.17 0.16 2.08 1.97 2.38 2.20 2.09 2.25 2.14
After c
(0.07) (0.06)c (0.08)c (0.10)c (0.04)c (0.09)c (0.07)c (0.43)d (0.32)d (0.23)d (0.29)d (0.11)d (0.18)d (0.19)d
0.04 0.04 0.04 0.04 0.05 0.05
(0.00)a (0.01)a (0.01)a (0.01)a (0.01)ab (0.01)a
0.05 0.05 0.06 0.07 0.08 0.08
(0.01)ab (0.01)ab (0.01)ab (0.03)b (0.03)b (0.03)b
Different letters indicate statistically significant difference at 5%.
The adherence of C. albicans, C. glabrata, and S. mutans, determined by the XTT assay, is shown in Table 3. It can be seen that saliva did not have any effect on microorganism adherence for all experimental conditions. There were no significant differences between the control groups, smooth or rough (p > 0.01). Higher absorbance values were found for the control groups compared with the coated groups (p < 0.01). For all smooth experimental specimens, there were no statistically significant differences (p > 0.01) in the mean absorbance values among the coated groups. There was a decrease in cell viability compared with the control group (mean 66.7%; range 62–72%). Regarding rough specimens, HP 30 showed the highest absorbance mean values compared to the other coated groups (p < 0.01). All negative controls exhibited no metabolic activity (data not shown).
529
The microbial counts for C. albicans, C. glabrata, and S. mutans are shown in Table 4 (log cfu cm−2, means and SDs). The three analyses of variance showed significant effects only in surface roughness. Although the global mean of the log cfu cm−2 for the rough surfaces was higher than for the smooth surface, the values are closer. The CFU were always above 90% compared to the control group (100%). The surface composition evaluated by the XPS analysis is shown in Table 5. It can be seen that with the HP coating application, there was a decrease in the percentage of C 1s and an increase in the percentage of O 1s and Si 2p. A peak for phosphorous also appeared. For the S coating, an increase in C 1s and a decrease in O1s were observed. Additionally, a peak of sulfur was also found. The results of the interactions from the 4-way ANOVA are shown in Supplementary Tables S1–S4 [Supplementary material is available via a multimedia link on the online article webpage]. Discussion Sealers or coatings are traditionally used on dental materials to protect the material surface from degradation and the external stresses involved in mechanical and chemical factors, which could increase the surface roughness and porosity (Mainieri et al. 2011). The development of coatings or sealants with antimicrobial and anti-adherent potential is attracting great interest in the scientific community (Amano et al. 2010). Recently, Molino et al. (2013) observed that surface modification reduced interfacial protein adsorption, with the complete inhibition of adhesion and colonization by primary mouse myoblasts. Since polymer coatings can easily be applied to denture surfaces, this study evaluated the effectiveness of two photopolymerized coatings in an attempt to reduce the
Table 3. Mean absorbance values (XTT assay – 492 nm) and SDs of all experimental groups (n = 9), according to the conditions (smooth or rough surface), coating type (S or HP) and presence or absence of saliva. Surface roughness Smooth
Rough
Coating and concentration (%) Control S 25 30 35 HP 25 30 35 Control S 25 30 35 HP 25 30 35
Saliva absent 1.78 1.17 1.17 1.23 1.14 1.17 1.19 1.88 1.39 1.20 1.18 1.13 1.38 1.29
(0.20) (0.21) (0.18) (0.22) (0.18) (0.22) (0.20) (0.12) (0.14) (0.20) (0.20) (0.22) (0.17) (0.13)
Saliva present 1.68 (0.17) 0.97 (0.19) 1.04 (0.26) 1.26 (0.17) 1.19 (0.20) 1.18 (0.18) 1.12 (0.22) 1.63 (0.25) 1.24 (0.23) 1.20 (0.21) 1.24 (0.20) 1.11 (0.22) 1.37 (0.08) 1.21 (0.12)
Global 1.73 1.07 1.10 1.25 1.17 1.17 1.15 1.76 1.32 1.20 1.21 1.12 1.38 1.25
(0.19)d (0.20)a (0.22)ab (0.20)abc (0.19)abc (0.20)abc (0.21)abc (0.20)d (0.19)bc (0.20)abc (0.20)abc (0.22)ab (0.13)c (0.12)abc
CV (%)* 100.0 61.9 63.9 72.2 67.6 68.0 66.6 100.0 75.0 68.2 68.9 63.7 78.4 71.3
*Cell viability compared with control group. Groups with the same letters in the columns did not differ significantly at 1% (saliva had no effect).
530
F.E. Izumida et al.
Table 4. Mean of CFU counts and SDs of all experimental groups (n = 9), according to the conditions (smooth or rough surface), coating type (S or HP) and presence or absence of saliva. Coating
Concentration (%)
Smooth surface as S HP
Downloaded by [University of Connecticut] at 11:54 10 January 2015
ps S HP Global CV (%)* Rough surface as S HP ps S HP
Microorganism C. albicans
C. glabrata
Control 25 30 35 25 30 35 Control 25 30 35 25 30 35
5.63 4.96 4.86 5.11 5.16 5.04 4.85 5.24 4.89 4.77 5.08 5.12 5.01 4.88 4.98 91.5
(0.35) (0.38) (0.51) (0.39) (0.31) (0.62) (0.69) (0.30) (0.28) (0.42) (0.38) (0.29) (0.25) (0.49) (0.44)
6.42 6.05 5.95 6.13 6.21 6.17 6.09 6.41 6.00 6.28 6.12 6.18 6.03 6.02 6.10 95.1
(0.36) (0.17) (0.33) (0.37) (0.13) (0.25) (0.15) (0.26) (0.28) (0.24) (0.23) (0.23) (0.18) (0.13) (0.24)
6.53 6.04 5.98 6.09 6.32 6.10 6.07 6.43 6.01 6.22 6.08 6.15 6.03 6.01 6.09 94.0
(0.35) (0.28) (0.13) (0.18) (0.20) (0.15) (0.31) (0.36) (0.22) (0.19) (0.17) (0.22) (0.25) (0.30) (0.22)
Control 25 30 35 25 30 35 Control 25 30 35 25 30 35
5.76 5.23 5.11 5.22 5.14 5.24 5.15 5.41 5.17 4.98 5.22 5.13 5.14 5.07 5.15 92.2
(0.42) (0.23) (0.28) (0.25) (0.24) (0.26) (0.29) (0.19) (0.24) (0.43) (0.35) (0.20) (0.32) (0.57) (0.32)
6.44 6.33 6.17 6.29 6.20 6.38 6.21 6.41 6.25 6.08 6.29 6.20 6.21 6.17 6.23 97.0
(0.25) (0.45) (0.17) (0.14) (0.38) (0.31) (0.26) (0.29) (0.26) (0.25) (0.31) (0.16) (0.17) (0.20) (0.27)
6.56 6.34 6.14 6.30 6.19 6.34 6.20 6.46 6.25 6.06 6.26 6.18 6.20 6.10 6.21 95.5
(0.33) (0.18) (0.23) (0.34) (0.16) (0.26) (0.25) (0.32) (0.17) (0.49) (0.27) (0.24) (0.10) (0.18) (0.26)
Global CV(%)*
S. mutans
as = absence of saliva; ps = presence of saliva. *Cell viability compared with the control group smooth or rough. Note: ANOVA showed only the lower global mean of the smooth surface compared with the rough surface for all microorganisms (p ≤ 0,001).
Table 5. Elemental surface composition (at.%) of the groups measured by XPS. Groups Elements (at.%) C 1s O 1s Si 2p P 2p S 2p
Control
HP35
S35
72.54 27.45 0.2 -
66.98 28.03 5.81 0.56 -
78.97 12.63 2.7 0.78
at.% = atomic percentage.
adherence of C. albicans, C. glabrata, and S. mutans to the surface of a denture base resin. Although the XTT method is routinely used to quantify Candida biofilms (da Silva et al. 2008; Jin et al.
2004), it is possible that it could also be used on other biofilms, such as S. mutans biofilm (Gobor et al. 2011). The major advantage of the XTT assay is that manual cell detachment is not necessary and the attendant loss in cell numbers leading to a high sign noise ratio does not occur with this technique. This is especially true in the early adherent phase of the biofilm, where the cell numbers are relatively small (Jin et al. 2004). In this study, two types of surface roughness, smooth and rough, that simulate the outer and inner surfaces of a prosthesis were used. Data for surface roughness obtained by the XTT method showed that, in the control group, there were no significant differences between the smooth and the rough groups. The overall Ra values for the experimental conditions of this study ranged from 0.14 μm to 3.80 μm. These values were lower than those
Downloaded by [University of Connecticut] at 11:54 10 January 2015
Biofouling reported by Zissis et al. (2000), who found mean values ranging from 0.7 to 4.4 μm for denture base materials, and Richmond et al. (2004), who observed an arithmetical mean roughness ranging from 1.36 to 9.43 μm. Several studies have suggested that an increase in roughness facilitated microorganism retention (Lazarin et al. 2012, 2013; Moura et al. 2006; Waters et al. 1997). However, in this study, smooth or rough surfaces did not differ statistically in the adherence of C. albicans, C. glabrata, and S. mutans. The differences in these results could be attributed to the methodology used. In the studies cited above the retention test was performed by incubating specimens in C. albicans suspensions. In the present study, the adhesion of cells in the initial stages of biofilm formation was investigated to simulate the initial adhesion that occurs during biofilm development. In the retention process, cells are retained in the irregularities of the surface of the material, while the adhesion of fungal cells occurs from adhesion proteins that bind to the surface (Klis et al. 2001). The results of the present study are in agreement with those found in other studies (Moura et al. 2006; Zamperini et al. 2010, 2011). However, Zamperini et al. (2010, 2011) stated that other studies should be conducted using specimens with prepared surfaces that cover a wide range of roughness values to verify the real effect of surface roughness in microbial adherence. The application of photopolymerized coatings resulted, generally, in the absence of significant differences between the smooth and rough surfaces. The reason for these results is that the acrylic surface became more regular after the coating application (Table 2). The coating layer promotes a uniform surface since the irregularities and porosities of acrylic surface are recovered by the coatings. XPS analysis demonstrated that the coated surfaces showed changes in chemical composition, confirming the findings of this study. The carbon and oxygen composition changed in coated surfaces and for the S coating, a peak of sulfur also occurred. In this study, for all coated specimens, smooth or rough, the XTT assay showed that the adherence of all coated groups was significantly lower compared with the control. The CFU analysis confirmed this finding, although it was not statistically significant. Gobor et al. (2011) stated that the disruption of biofilms caused by serial dilution and the subsequent counting of CFU are not considered reliable, since it cannot be confirmed that each colony came from a single cell. It has been stated that the surface modification with coatings containing zwitterionic and hydrophilic groups could prevent the adherence of microorganisms to PMMA surfaces (Ishihara et al. 1998; Çagavi et al. 2004; Cheng et al. 2007; Cringus-Fundeanu et al. 2007; Zhao et al. 2010). Sulfotebaine, a member of the zwitterionic betaine family
531
of compounds which occurs widely in nature (West et al. 2004), presented in the S coating, has received greater attention, as one of the most representative structures in the well-identified class of polymeric zwitterionic materials (Yuan et al. 2003) due to its blood compatibility, protein adsorption, and reduction in microorganism adherence (Lowe et al. 2000; Hasegawa et al. 2001; Konno et al. 2001). The ability of zwitterions to reduce the adherence of microorganisms could be related to their hydration ability (Cheng et al. 2007). Water molecules bind to the hydrophobic part of the polymer through ionic solvation, which is very strong, leading perhaps to a thermodynamic hydration barrier to polymer condensation. Furthermore, a reduction in microbial adherence also results from the longer chains and higher densities of the zwitterions (Cheng et al. 2007). Thus, the effectiveness of a nonfouling zwitterionic coating is due not only to the nature of the nonfouling group, but also to the surface packing of the nonfouling group (Cheng et al. 2007). The HP coating, a hydrophilic polymer, binds water molecules via hydrogen bonding (Cheng et al. 2007), creating a water jacket around the material (Çagavi et al. 2004). In addition to surface hydration, the prevention of protein adsorption also occurs by physical aspects of the surface, such as surface roughness (Zhao et al. 2010). Zhao et al. (2010) stated that HPMA, which is a neutral hydroxyl-rich monomer, has abundant –OH functional groups that can be further functionalized via ester NHS groups for ligand immobilization. The introduction of the dual functionality of antifouling and ligand immobilization in the same material can greatly enhance its potential use in biomedical applications. The results found in this study are in agreement with other studies that also showed a reduction in the adherence of S. epidermidis, P. aeruginosa, S. aureus, and C. albicans by zwitterionic or hydrophilic polymers (Lowe et al. 2000; West et al. 2004; Cheng et al. 2007; Lazarin et al. 2012, 2013). For all experimental conditions, the salivary pellicle did not influence the adherence of C. albicans, C. glabrata, and S. mutans. Saliva is an exocrine secretion produced by different glands and consists of water, electrolytes, and proteins (Zamperini et al. 2012). The adhesive interaction between oral yeasts and bacteria depends on, amongst other things, the presence of specific salivary proteins, such as mucins, proline-rich proteins and calcium, which serve as a molecular bridge to mediate the adhesion of yeasts and bacteria to resin beads (Milsap et al. 1999). However, it has been shown that saliva also possesses antimicrobial properties, due to the nature of the substratum on the composition of the acquired pellicle, which could alter the initial adherence (Lazarin et al. 2012). Thus, the scientific literature is unclear regarding the effect of saliva on microbial
Downloaded by [University of Connecticut] at 11:54 10 January 2015
532
F.E. Izumida et al.
adherence and suggests that this divergence could be related to the differences in saliva collection and its manipulation (Park et al. 2003).The results of this study are in agreement with those found by Lazarin et al. (2013), who concluded that the interactions between C. albicans, the substratum, and saliva are complex, and factors such as the physico-chemical properties of the substrata (and conditioning film), and the cells may influence this process. In conclusion, the experimental photopolymerized coatings promoted surface modification in the denture base acrylic resin Vipi Wave. In addition, the XTT assay confirmed that these coatings were able to reduce the adherence of C. albicans, C. glabrata and S. mutans. Despite promising results, the effect of these coatings in reducing multi-species biofilm formation, and their resistance to methods of denture cleansing, such as toothbrushing, should be evaluated before clinical use. Acknowledgments The authors would like to thank to the São Paulo Research Foundation (São Paulo, SP, Brazil) for the scholarship granted to the first author (#2010/00545-9) and for financial support (#2012/01528-6). They also thank Mr Jörg Erxleben for preparing the coatings used in this study, Marlise I. Klein for microbiological assistance, and Prof. Peter Hammer for support with the XPS analysis.
References Amano D, Ueda T, Sugiyama T, Takemoto S, Oda Y, Sakurai K. 2010. Improved brushing durability of titanium dioxide coating on polymethylmethacrylate substrate by prior treatment with acryloxypropyl trimethoxysilane-based agent for denture application. Dent Mater J. 29:97–103. American National Standard. 1975. Specifications nº12 for denture base polymer. Chicago: American Dental Association. (Reaffirmed, 1999). Çagavi F, Akalan N, Celik H, Gür D, Güçiz B. 2004. Effect of hydrophilic coating on microorganism colonization in silicone tubing. Acta Neurochir. 146:603–610. Chandra J, Mukherjee PK, Leidich SD, Faddoul FF, Hoyer LL, Douglas LJ, et al. 2001. Antifungal resistance of candidal biofilms formed on denture acrylic in vitro. J Dent Res. 80:903–908. Cheng G, Zhang Z, Chen S, Bryers JD, Jiang S. 2007. Inhibition of bacterial adhesion and biofilm formation on zwitterionic surfaces. Biomaterials. 28:4192–4199. Cringus-Fundeanu I, Luijten J, van der Mei HC, Busscher HJ, Schouten AJ. 2007. Synthesis and characterization of surfacegrafted polyacrylamide brushes and their inhibition of microbial adhesion. Langmuir. 23:5120–5126. Dhir G, Berzins DW, Dhuru VB, Periathamby AR, Dentino A. 2007. Physical properties of denture base resins potentially resistant to Candida adhesion. J Prosthodont. 16:465–472. da Silva WJ, Seneviratne J, Parahitiyawa N, Rosa EA, Samaranayake LP, Del Bel Cury AA. 2008. Improvement of XTT assay performance for studies involving Candida albicans biofilms. Braz Dent J 19:364–369.
de Freitas Fernandes FS, Pereira-Cenci T, da Silva WJ, Filho AP, Straioto FG, Del Bel Cury AA. 2011. Efficacy of denture cleansers on Candida spp. biofilm formed on polyamide and polymethyl methacrylate resins. J Prosthet Dent. 105:51–58. Gobor T, Corol G, Ferreira LE, Rymovicz AU, Rosa RT, Campelo PM, Rosa EA. 2011. Proposal of protocols using D-glutamine to optimize the 2,3-bis(2-methoxy-4-nitro-5sulfophenly)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay for indirect estimation of microbial loads in biofilms of medical importance. J Microbiol Methods. 84:299–306. Gomes PN, da Silva WJ, Pousa CC, Narvaes EA, Del Bel Cury AA. 2011. Bioactivity and cellular structure of Candida albicans and Candida glabrata biofilms grown in the presence of fluconazole. Arch Oral Biol. 56:1274–1281. Hahnel S, Rosentritt M, Bürgers R, Handel G, Lang R. 2012. Candida albicans biofilm formation on soft denture liners and efficacy of cleaning protocols. Gerodontology. 29: 383–391. Hasegawa T, Iwasaki Y, Ishihara K. 2001. Preparation and performance of protein-adsorption-resistant asymmetric porous membrane composed of polysulfone/phospholipid polymer blend. Biomaterials. 22:243–251. Ishihara K, Nomura H, Mihara T, Kurita K, Iwasaki Y, Nakabayashi N. 1998. Why do phospholipid polymers reduce protein adsorption? J Biomed Mater Res. 39:323–330. Jin Y, Samaranayake LP, Samaranayake Y, Yip HK. 2004. Biofilm formation of Candida albicans is variably affected by saliva and dietary sugars. Arch Oral Biol. 49:789–798. Klis FM, de Groot P, Hellingwerf K. 2001. Molecular organization of the cell wall of Candida albicans. Med Mycol. 39:1–8. Konno T, Kurita K, Iwasaki Y, Nakabayashi N, Ishihara K. 2001. Preparation of nanoparticles composed with bioinspired 2-methacryloyloxyethyl phosphorylcholine polymer. Biomaterials. 22:1883–1889. Lazarin AA, Zamperini CA, Vergani CE, Wady AF, Giampaolo ET, Machado AL. 2012. Candida albicans adherence to an acrylic resin modified by experimental photopolymerised coatings: an in vitro study. Gerodontology. 30. doi: 10.1111/j.1741-2358.2012.00688.x. [Epub ahead of print] Lazarin AA, Machado AL, Zamperini CA, Wady AF, Spolidorio DM, Vergani CE. 2013. Effect of experimental photopolymerized coatings on the hydrophobicity of a denture base acrylic resin and on Candida albicans adhesion. Arch Oral Biol. 58:1–9. Lippens E, De Smet N, Schauvliege S, Martens A, Gasthuys F, Schacht E, Cornelissen RJ. 2013. Biocompatibility properties of surface-modified poly(dimethylsiloxane) for urinary applications. Biomater Appl. 27:651–660. Long SF, Clarke S, Davies MC, Lewis AL, Hanlon GW, Lloyd AW. 2003. Controlled biological response on blends of a phosphorylcholine-based copolymer with poly(butyl methacrylate). Biomaterials. 24:4115–4121. Lowe AB, Vamvakaki M, Wassall MA, Wong L, Billingham NC, Armes SP, Lloyd AW. 2000. Well-defined sulfobetaine-based statistical copolymers as potential antibioadherent coatings. J Biomed Mater Res. 52:88–94. Mainieri VC, Beck J, Oshima HM, Hirakata LM, Shinkai RS. 2011. Surface changes in denture soft liners with and without sealer coating following abrasion with mechanical brushing. Gerodontology. 28:146–151. Millsap KW, Bos R, van der Mei HC, Busscher HJ. 1999. Adhesion and surface-aggregation of Candida albicans
Downloaded by [University of Connecticut] at 11:54 10 January 2015
Biofouling from saliva on acrylic surfaces with adhering bacteria as studied in a parallel plate flow chamber. Antonie Van Leeuwenhoek. 75:351–359. Mima EG, Pavarina AC, Neppelenbroek KH, Vergani CE, Spolidorio DM, Machado AL. 2008. Effect of different exposure times on microwave irradiation on the disinfection of a hard chairside reline resin. J Prosthodont. 17:312–317. Molino PJ, Zhang B, Wallace GG, Hanks TW. 2013. Surface modification of polypyrrole/biopolymer composites for controlled protein and cellular adhesion. Biofouling. 29:1155– 1167. Moura JS, da Silva WJ, Pereira T, Del Bel Cury AA, Rodrigues Garcia RC. 2006. Influence of acrylic resin polymerization methods and saliva on the adhesion of four Candida species. J Prosthet Dent. 96:205–211. Nakabayashi N, Williams DF. 2003. Preparation of non-trombogenic materials using 2-methacryloyloxyethyl phosphorylcholine. Biomaterials. 24:2431–2435. Özden N, Akaltan F, Suzer S, Akovali G. 1999. Time-related wettability characteristic of acrylic resin surfaces treated by glow discharge. J Prosthet Dent. 82:680–684. Park SE, Periathamby AR, Loza JC. 2003. Effect of surfacecharged poly(methyl methacrylate) on the adhesion of Candida albicans. J Prosthodont. 12:249–254. Pereira-Cenci T, Deng DM, Kraneveld EA, Manders EM, Del Bel Cury AA, Ten Cate JM, Crielaard W. 2008. The effect of Streptococcus mutans and Candida glabrata on Candida albicans biofilms formed on different surfaces. Arch Oral Biol. 53:755–764. Ramage G, Jose A, Coco B, Rajendran R, Rautemaa R, Murray C, Lappin DF, Bagg J. 2011. Commercial mouthwashes are more effective than azole antifungals against Candida albicans biofilms in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 111:456–460. Redding S, Bhatt B, Rawls HR, Siegel G, Scott K, Lopez-Ribot J. 2009. Inhibition of Candida albicans biofilm formation on denture material. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 107:669–672. Richmond R, Macfarlane TV, Mccord JF. 2004. An evaluation of the surface changes in PMMA biomaterial formulations as a result of toothbrush/dentifrice abrasion. Dent Mater. 20:124–132. Sheridan PJ, Koda S, Ewoldsen NO, Lefebvre CA, Lavin MT. 1997. Cytotoxicity of denture base resins. Int J Prosthodont. 10:73–77. Shirley DA. 1972. High-resolution X-ray photoemission spectrum of the valence bands of gold. Phys Rev B. 5:4709–4713.
533
Silva S, Negri M, Henriques M, Oliveira R, Williams DW, Azeredo J. 2011. Adherence and biofilm formation of nonCandida albicans Candida species. Trends Microbiol. 19:241–247. Tylenda CA, Larsen J, Yeh CK, Lane HC, Fox PC. 1989. High levels of oral yeasts in early HIV-1 infection. J Oral Pathol Med. 18:520–524. Waters MG, Williams DW, Jagger RG, Lewis MA. 1997. Adherence of Candida albicans to experimental denture soft lining materials. J Prosthet Dent. 77:306–312. West SL, Salvage JP, Lobb EJ, Armes SP, Billingham NC, Lewis AL, Hanlon GW, Lloyd AW. 2004. The biocompatibility of crosslinkable copolymer coatings containing sulfobetaines and phosphobetaines. Biomaterials. 25:1195– 1204. Wilson J. 1998. The etiology diagnosis and management of denture stomatitis. Br Dent J. 185:380–384. Yildirim MS, Kesimer M, Hasirci N, Kiliç N, Hasanreisoğlu U. 2006. Adsorption of human salivary mucin MG1 onto glow-discharge plasma treated acrylic resin surfaces. J Oral Rehabil. 33:775–783. Yuan J, Zhang J, Zhu J, Shen J, Lin SC, Zhu W, Fang JL. 2003. Reduced platelet adhesion on the surface of polyurethane bearing structure of sulfobetaine. J Biomater Appl. 18:123–135. Zamperini CA, Machado AL, Vergani CE, Pavarina AC, Giampaolo ET, da Cruz NC. 2010. Adherence in vitro of Candida albicans to plasma treated acrylic resin. Effect of plasma parameters, surface roughness and salivary pellicle. Arch Oral Biol. 55:763–770. Zamperini CA, Machado AL, Vergani CE, Pavarina AC, Rangel EC, Cruz NC. 2011. Evaluation of fungal adherence to plasma-modified polymethylmethacrylate. Mycoses. 54: 344–351. Zamperini CA, Schiavinato PC, Pavarina AC, Giampaolo ET, Vergani CE, Machado AL. 2012. Effect of human whole saliva on the in vitro adhesion of Candida albicans to a denture base acrylic resin: a focus on collection and preparation of saliva samples. J Investig Clin Dent. 3:1–4. Zhao C, Li L, Zheng J. 2010. Achieving highly effective nonfouling performance for surface-grafted poly (HPMA) via atom-transfer radical polymerization. Langmuir. 26:17375– 17382. Zissis AJ, Polyzois GL, Yannikakis SA, Harrison A. 2000. Roughness of denture materials: a comparative study. Int J Prosthodont. 13:136–140.
Biofouling: The Journal of Bioadhesion and Biofilm Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gbif20
In vitro evaluation of adherence of Candida albicans, Candida glabrata, and Streptococcus mutans to an acrylic resin modified by experimental coatings a
a
a
Fernanda Emiko Izumida , Eduardo Buozi Moffa , Carlos Eduardo Vergani , Ana Lúcia a
a
Machado , Janaína Habib Jorge & Eunice Teresinha Giampaolo
a
a
Department of Dental Materials and Prosthodontics, Araraquara Dental School, Universidade Estadual Paulista, UNESP, São Paulo, Brazil Published online: 01 Apr 2014.
Click for updates To cite this article: Fernanda Emiko Izumida, Eduardo Buozi Moffa, Carlos Eduardo Vergani, Ana Lúcia Machado, Janaína Habib Jorge & Eunice Teresinha Giampaolo (2014) In vitro evaluation of adherence of Candida albicans, Candida glabrata, and Streptococcus mutans to an acrylic resin modified by experimental coatings, Biofouling: The Journal of Bioadhesion and Biofilm Research, 30:5, 525-533, DOI: 10.1080/08927014.2014.894028 To link to this article: http://dx.doi.org/10.1080/08927014.2014.894028
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Biofouling, 2014 Vol. 30, No. 5, 525–533, http://dx.doi.org/10.1080/08927014.2014.894028
In vitro evaluation of adherence of Candida albicans, Candida glabrata, and Streptococcus mutans to an acrylic resin modified by experimental coatings Fernanda Emiko Izumida, Eduardo Buozi Moffa, Carlos Eduardo Vergani, Ana Lúcia Machado, Janaína Habib Jorge and Eunice Teresinha Giampaolo* Department of Dental Materials and Prosthodontics, Araraquara Dental School, Universidade Estadual Paulista, UNESP, São Paulo, Brazil
Downloaded by [University of Connecticut] at 11:54 10 January 2015
(Received 29 October 2013; accepted 4 February 2014) This study evaluated the effect of experimental coatings, containing zwitterion or hydrophilic monomers, on the adherence of Candida albicans, Candida glabrata, and Streptococcus mutans to an acrylic resin. Acrylic samples (smooth or rough surfaces) were left untreated (control) or coated with one of the following experimental coatings: 3-hydroxypropylmethacrylate (HP) or sulfobetaine methacrylate (S), at concentrations of 25, 30, or 35%. Half of the specimens were coated with saliva. The adhesion test was performed by incubating specimens in C. albicans, C. glabrata, and S. mutans suspensions at 37°C for 90 min. The number of adhered microorganisms was determined by metabolic activity (XTT) and by cell viability (CFU). All coated specimens exhibited lower absorbance and CFU values compared to control specimens. Saliva and roughness did not promote microorganism adherence. An XPS analysis confirmed the modification in the chemical composition of the coatings in the experimental samples. These experimental coatings significantly reduced the adherence of C. albicans, C. glabrata and S. mutans to acrylic resin. Keywords: acrylic resin; saliva; Candida albicans; Candida glabrata; Streptococcus mutans
Introduction Removable dentures (RD) provide edentulous patients with the rehabilitation of masticatory and esthetic functions (Dhir et al. 2007). However, one consequence of the continual use of RD is the adhesion of microorganisms and biofilm formation (Lazarin et al. 2012) in the base of the prosthesis. Candida species are opportunistic pathogens that are frequently isolated from the oral cavity and its biofilms are often associated with oral candidosis (Gomes et al. 2011; Hahnel et al. 2012; Lazarin et al. 2012). Although a majority of oral candidosis cases are attributed to C. albicans (Park et al. 2003; Dhir et al. 2007; Lazarin et al. 2012, 2013), an increasing number of infections are caused by non-C. albicans Candida (NCAC) species (Silva et al. 2011), Candida glabrata, which has been isolated from RD and palatal mucosa (Gomes et al. 2011). This yeast species is considered an emerging fungal pathogen, which exhibits superior adhesion to acrylic resin surfaces compared to C. albicans (de Freitas Fernandes et al. 2011). Furthermore, studies have shown that the adhesive interactions between Candida species and oral bacteria such as Streptococcus mutans play a crucial role in microbial colonization of denture acrylic, which may lead to denture stomatitis (Millsap et al. 1999). Bacteria facilitate the adherence of Candida cells to the denture base and mucosa, mainly by extracellular polymer *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
production as well as increased acidity, which creates optimal environmental conditions for yeast growth. Systemic alterations (Tylenda et al. 1989), poor oral hygiene (Wilson 1998) and the surface characteristics of acrylic resin, such as hydrophobicity and surface roughness, have played a role in the development and maintenance of denture stomatitis. It has been demonstrated that moderately hydrophobic surfaces support cell adhesion whereas hydrophilic surfaces demonstrate limited cell adhesion (Long et al. 2003). In the case of surface roughness, it can be seen that rough surfaces provide a large surface area and may act as niches for microorganisms, thus favoring adhesion (Waters et al. 1997; Moura et al. 2006; Zamperini et al. 2010). Furthermore, other factors, such as the salivary pellicle, may also alter the surface characteristics of the substratum involved in the adhesion process (Park et al. 2003). It has been demonstrated that the salivary pellicle may regulate specific interactions between cellular adhesion of C. albicans and receptors in the pellicle (Yildirim et al. 2006). Studies have shown that surface modification could be considered a promising approach to avoid denture stomatitis (Redding et al. 2009; Lazarin et al. 2012, 2013) because this modification could reduce the adhesion of microorganisms to the substratum (Lowe et al. 2000; West et al. 2004; Cheng et al. 2007; Lazarin et al. 2012,
Downloaded by [University of Connecticut] at 11:54 10 January 2015
526
F.E. Izumida et al.
2013). In this way, the use of coatings could reduce protein adsorption and adhesion of microorganisms. Liquid adhesives can flow easily over the entire surface of the substratum and come into contact with all the small rough areas that may be present (Özden et al. 1999). Moreover, coatings could be developed with antimicrobial agents, such as chlorhexidine or nystatin (Redding et al. 2009) or with other compounds, as such zwitterionic and hydrophilic polymers. These additives could enhance antibiofilm and antifungal activity (Redding et al. 2009). Polymeric coatings have been applied to the surfaces of materials to reduce adhesion of microorganisms (Lowe et al. 2000). Zwitterionic polymers, such as sulfobetaines and phosphatidylcholines have been used to reduce protein adsorption and microorganism adherence to a variety of medical devices (Lowe et al. 2000; Hasegawa et al. 2001; Konno et al. 2001). The ability to prevent protein adsorption may be due to the fact that the polymers have both a positively and a negatively charged group within the molecule but have an overall neutral charge (West et al. 2004; Lazarin et al. 2012). The hydrophilic nature of the head group of the polymer promotes the formation of a strongly bound hydration layer on the material surface, inhibiting protein adsorption (Long et al. 2003). Additionally, the percentage of free water in the water phase of a zwitterionic-polymer hydrogel was higher than in conventional hydrogels, which could prevent conformational changes of the proteins so that they do not attach to or easily detach from the surfaces (Nakabayashi and Williams 2003). Although the initial application of zwitterionic polymers has been to promote the compatibility of blood catheters, recent studies have demonstrated the ability of sulfobetaine, coated into polymethyl methacrylate (PMMA) surfaces, in reducing adhesion of Pseudomonas aeruginosa, Staphylococcus epidermidis, and C. albicans (Lowe et al. 2000; West et al. 2004; Cheng et al. 2007). As a result of these beneficial characteristics, sulfobetaine polymers could be used to modify the denture base surfaces, promoting a reduction in microbial adherence and consequently preventing denture stomatitis (Lazarin et al. 2012, 2013). In addition, other non-toxic hydrophilic monomers (Lippens et al. 2013), such as polyvinylpyrralidone, polyacrylamide, poly(ethylene glycol) (PEG), parylene, and hydroxypropyl methacrylate (HPMA), have been evaluated to modify the biomaterial surface and reduce protein adsorption and microbial adherence (Ishihara et al. 1998; Çagavi et al. 2004; Cringus-Fundeanu et al. 2007; Zhao et al. 2010; Lazarin et al. 2013). Covalent bonds occur in the hydrogels between the hydrophilic polymers and the biomaterial. When these hydrogels are hydrated, they form a water jacket around the material, which makes it perfectly smooth and hydrophilic, preventing bacterial colonization (Çagavi et al. 2004). However, the hydration state of these polymers is
achieved via hydrogen bonding, which is considered weaker than the ionic solvation found in zwitterionic polymers. Furthermore, the free water of hydrophilic polymers is lower than that of zwitterionic polymers (Ishihara et al. 1998; Lazarin et al. 2012). Therefore, the aim of this study was to evaluate the effectiveness of two experimental coatings, containing zwitterionic or hydrophilic monomers, in three different concentrations (25, 30 and 35%) on the adherence of C. albicans, C. glabrata and S. mutans to an acrylic resin. Additionally, the effects of surface roughness and the salivary pellicle were also evaluated. Material and methods Specimen preparation A total of 576 circular specimens (10 × 2 mm) of a microwave-cured denture base acrylic resin (Vipi Wave; VIPI Indústria e Comércio Exportação e Importação de Produtos Odontológicos Ltda, Pirassununga, SP, Brazil) were fabricated using a steel mold with a breakaway compartment. The metallic matrices were placed in dental flasks, sandwiched between two glass plates that were smooth or blasted with aluminum oxide, whose surface roughness was ~3.0 μm. These two types of glass plates were used to obtain, respectively, smooth and rough surfaces that simulate the outer and inner surface of the prosthesis. For each specimen, the acrylic resin Vipi Wave was mixed according to the manufacturer’s instructions at a mixing ratio of 1 g of powder to 0.47 ml of liquid, and packed into the molds. A hydraulic press (PM 2000, Vipi Delta, Pirassununga, SP, Brazil) was used for packing the denture base resin at 9806,65 N, maintained for 30 min. Next, specimens were polymerized in a 500 W domestic microwave oven (Brastemp da Amazonia SA, Manaus, AM, Brazil) for 20 min at 20% power, followed by 5 min at 90% power. The flasks were bench cooled overnight. They were then removed from the flask and the excess resin was trimmed with a bur (Maxi-Cut; Lesfilsde August Malleifer SA, Ballaigues, Switzerland). Surface roughness measurements The surface roughness values (Ra) were measured using a surface roughness profilometer (SJ 400, Mytutoyo Corporation, Tokyo, Japan), with a resolution of 0.01 μm, an interval (cut-off length) 0.8 mm; a transverse length 2.4 mm; a stylus speed 0.5 mm s−1, and a 5 μm active Diamond stylus tip radius. Four measurements were performed for each sample and the average reading was designated as the intact Ra (μm) value for each specimen. Experimental photopolymerized coatings After roughness measurements were taken, the specimens were randomly distributed into seven groups of 36
Downloaded by [University of Connecticut] at 11:54 10 January 2015
Biofouling samples each (18 specimens with smooth surfaces and 18 with rough surfaces). Samples of the control group (C) did not receive any surface treatment. For the experimental groups, 5 μl of each experimental photopolymerized coating were applied to all samples with a soft brush as a thin layer. Two coating formulations were evaluated: one coating contained hydrophilic monomers, namely 2-hydroxypropyl methacrylate (HPMA) (HP) and the other coating contained a zwitterionic monomer (sulfobetaine methacrylate) (S). The concentrations of monomers used were 25, 30, and 35% mol fraction of the total composition, resulting in six experimental coatings (HP25; HP30; HP35; S25; S30; S35). The coatings additionally contained the monomer methyl methacrylate, two crosslinking agents (tri-ethylene glycol dimethacrylate – TEGDMA and bisphenol-A-glycidyl methacrylate – Bis-GMA) and an initiator agent (4-methyl benzophenone). For the S coating, amino propyl methacrylate was also added. The concentrations of the components of the coatings are presented in Table 1. The application of the six coatings on both surfaces of the specimens was performed in a sterile laminar flow chamber, followed by a 4 min polymerization on each side in an EDG oven (Strobolux; EDG, São Carlos, SP, Brazil). Propane sultone was brushed on the S coating sample surfaces, followed by storage in an oven at 80°C for 2 h. Finally, all specimens were stored in distilled water at room temperature for 48 h for residual monomer elimination (American National Standard 1975 [reaffirmed 1999]; Moura et al. 2006). After application of the coatings to the specimen surfaces, surface roughness values (Ra) were measured as described previously. Sterilization of specimens Prior to the microbiological tests, samples were packed in sealed sterile plastic bags with sterile distilled water and ultrasonicated for 20 min. Then all specimen surfaces were exposed to ultraviolet light in a laminar flow chamber for 20 min for sterilization (Sheridan et al. 1997). Table 1.
Saliva collection and assay Saliva (20 ml per person) was donated by 15 healthy individuals, aged 20 to 35 years. The saliva was collected in 50 ml Falcon tubes on ice. Then, the saliva was pooled, homogenized, and subjected to centrifugation at 10,000 × g for 5 min at 4°C (Moura et al. 2006). The saliva supernatant was immediately stored at –70°C until use. This research was approved by the Ethics Committee of Araraquara Dental School, UNESP – Univ Estadual Paulista (012/2010) and all volunteers were informed about the objectives of the study and signed an informed consent form. In order to verify the effect of exposure to saliva on microorganism adhesion, half of the samples in each group were preconditioned with saliva. For pre-conditioning, the specimens were individually exposed to 2 ml of saliva at room temperature for 30 min (Waters et al. 1997; Zamperini et al. 2010; Zamperini et al. 2012).
Adherence assay S. mutans ATCC 25175, C. albicans ATCC 90028, and C. glabrata ATCC 2001 were used. To prepare the inoculum, a loopful of the stock cultures of the yeasts were streaked onto Sabouraud dextrose agar medium with chloramphenicol (SDA) and incubated at 37°C for 48 h, in an aerobic atmosphere. For S. mutans, a loopful was seeded on Mitis Salivarius bacitracin (MSB – Difco Laboratories, Detroit, MI, USA) and incubated at 37°C in a pCO2 atmosphere of 10% for 48 h. The specimens (n = 9) were placed into 24 well plates (one specimen per well). Two loopfuls of these young cultures were transferred to 20 ml of a yeast nitrogen base (YNB) medium with 50 mmol–1 glucose for the yeasts, and to 20 ml of brain heart infusion (BHI) broth for S. mutans, and incubated at 37°C for 21 h for Candida species and for 18 h in an atmosphere of 10% pCO2 for S. mutans. Cells of the resultant cultures were harvested, washed twice with sterile PBS (100 mM NaCl, 100 mM NaH2PO4, pH 7.2) at 5,000 g for 5 min, and resuspended in a sterile BHI broth. Cell suspensions were spectrophotometrically standardized to 1 × 107 cells ml−1 for Candida and
Composition of experimental coatings (in millimoles).
Coating (Code) P25 P30 P35 M25 M30 M35
527
Component MMA 61.0 56.0 51.0 61.0 56.0 51.0
TEGDMA 5.0 5.0 5.0 5.0 5.0 5.0
Bis – GMA 6.0 6.0 6.0 6.0 6.0 6.0
MPB 3.0 3.0 3.0 3.0 3.0 3.0
HPMA (P) 25.0 30.0 35.0
Sulfobetaine (S)
25.0 30.0 35.0
Abbreviations: MMA = methylmetacrilate; TEGDMA = tri-ethyleneglycoldimethacrylate; Bis – GMA = bisphenol-A-glycidylmethacrylate; MPB = 4methyl-benzophenone; HPMA = 2-hydroxypropyl methacrylate.
528
F.E. Izumida et al.
Downloaded by [University of Connecticut] at 11:54 10 January 2015
1 × 108 cells ml−1 for S. mutans (Pereira-Cenci et al. 2008). The absorbance for Candida species was 0.6 at 520 nm, and for S. mutans was 0.1 at 600 nm. An aliquot of 0.7 ml of cell suspension of each microorganism was added to each well and incubated for 90 min at 37°C (Chandra et al. 2001), under agitation (75 rpm), using an orbital shaker, in an atmosphere of 10% pCO2. Non-adherent cells were removed from the specimens by gently washing them twice with 2 ml of PBS. Forceps were used to hold each specimen during this step. Negative controls were sterile specimens immersed in BHI broth with no cells added. Experiments were repeated three times with individual samples in triplicate. XTT assay To determine the number of adherent cells of yeasts and bacteria, nine specimens from each experimental condition were evaluated by an XTT reduction assay, which is based on the metabolic activity of the cells (Ramage et al. 2011). For the XTT assay, two solutions were used: XTT solution and menadione. The XTT solution (Sigma, MO, USA) was prepared in ultra-pure water at a final concentration of 1 mg ml−1. The solution was filter sterilized and stored at -70°C until use. The menadione solution (Sigma, St Louis, MO, USA) was prepared in 0.4 mM acetone, immediately before each assay. All samples were individually removed, washed and transferred to new sterile 24-well plates containing 2 ml of the following solution in each well: 1,580 μl of PBS supplemented with 200 mM glucose, 400 μl of XTT, and 20 μl of menadione. The plates were incubated for 3 h in the dark at 37°C (da Silva et al. 2008). The solution from each well was transferred to a tube and centrifuged at 5,000 g for 1 min. The supernatant was then transferred to a 96-well microplate, and the color change was measured using a microplate reader (Thermo Plate – TP Reader, Nanshan District, Shenzhen, Guangdong, China) at 492 nm. Colony-forming unit (CFU) count The number of cultivable cells was determined by counting the colony forming units (CFU) following the adherence phase of the experiment. Briefly, after washing, the specimens were transferred to Falcon tubes containing 4.5 ml of PBS and vigorously vortexed for 1 min to detach the microorganisms from the samples (Mima et al. 2008). Then, to determine the number of microorganisms in the 10−2, 10−3, 10−4 and 10−5 dilutions, replicate aliquots (10 μl) of the suspension were transferred to plates of two selective media, CHRO-Magar (Difco) for isolation of Candida species and Mitis Salivarius (Difco) for identification of S. mutans. CHRO-Magar plates were incubated aerobically and Mitis Salivarius plates were incubated in 10% CO2 incubator at 37°C for 48 h. After incubation, bacterial
and yeast colony counts of each plated specimen were quantified using a Phoenix CP 600 Plus digital colony counter (Phoenix Indústria e Comércio de Equipamentos Científicos Ltda, Araraquara, Brazil). The counts were repeated by the same examiner for accuracy. The total CFU per unit area (Log CFU cm−2) was then calculated. X-ray photoelectron spectroscopy analysis (XPS) The characterization of the specimen surfaces after coating application was confirmed by XPS, using a UNI-SPECS UHV spectrometer (SPECS Surface Nano Analysis GmbH, Berlin, Germany). In the analysis, the Mg Kα line (E=1253.6 eV) was used and the analyzer pass energy was set to 10 eV. Shirley’s method (Shirley 1972) was used to subtract the inelastic background of the electron core-level spectra of C 1s, O 1s, and N 1s, whose binding energies were corrected using the hydrocarbon component of the polymer fixed at 285.0 eV. The ratio of relative peak areas corrected by sensitivity factors of the corresponding elements was used to determine the composition of the surface layer. Voigt profiles were applied to fit the spectra without placing constraints. The width at half maximum (FWHM) ranged between 1.6 eV and 2.0 eV, and the accuracy of the peak positions was ± 0.1 eV. One specimen from the control group (without surface treatment) and one specimen treated with the two coatings at 35% concentration were analyzed. Statistical analysis The data on the effect of coating treatments at different concentrations on the adhesion of microorganisms, determined by two methods, XTT and colony forming units (CFU), were subjected to four-way analysis of variance (ANOVA): surface roughness (smooth or rough); coating (S or HP); concentration (25, 30 or 35%) and saliva (present or absent), including controls without the coating application. The Tukey test for multiple comparisons of means was adopted to supplement these analyses. A significance of 1% was used for all analyses. The statistical program used was Statistica 6.0 Statsoft (Statistica 6.0, Statsoft, Tulsa, Okla, USA). Results The mean roughness values (Ra, μm) and standard deviations (SD) of all experimental groups, before and after application of coatings, are listed in Table 2. These results show that the acrylic surface became more regular after the coating application. The mean surface roughness of smooth specimens became similar to the rough specimens (p > 0.01), except for HP at all concentrations (p < 0.01). The summary of the analysis of variance of the absorbance for XTT and for CFU of C. albicans, C. glabrata and S. mutans are presented in Tables 3 and 4.
Biofouling Table 2. Mean roughness values (Ra, µm) and SDs of all experimental groups, before and after application of coatings. Surface
Coating and concentration (%)
Smooth
Downloaded by [University of Connecticut] at 11:54 10 January 2015
Rough
Control S 25 30 35 HP 25 30 35 Control S 25 30 35 HP 25 30 35
Before 0.13 0.14 0.17 0.15 0.12 0.17 0.16 2.08 1.97 2.38 2.20 2.09 2.25 2.14
After c
(0.07) (0.06)c (0.08)c (0.10)c (0.04)c (0.09)c (0.07)c (0.43)d (0.32)d (0.23)d (0.29)d (0.11)d (0.18)d (0.19)d
0.04 0.04 0.04 0.04 0.05 0.05
(0.00)a (0.01)a (0.01)a (0.01)a (0.01)ab (0.01)a
0.05 0.05 0.06 0.07 0.08 0.08
(0.01)ab (0.01)ab (0.01)ab (0.03)b (0.03)b (0.03)b
Different letters indicate statistically significant difference at 5%.
The adherence of C. albicans, C. glabrata, and S. mutans, determined by the XTT assay, is shown in Table 3. It can be seen that saliva did not have any effect on microorganism adherence for all experimental conditions. There were no significant differences between the control groups, smooth or rough (p > 0.01). Higher absorbance values were found for the control groups compared with the coated groups (p < 0.01). For all smooth experimental specimens, there were no statistically significant differences (p > 0.01) in the mean absorbance values among the coated groups. There was a decrease in cell viability compared with the control group (mean 66.7%; range 62–72%). Regarding rough specimens, HP 30 showed the highest absorbance mean values compared to the other coated groups (p < 0.01). All negative controls exhibited no metabolic activity (data not shown).
529
The microbial counts for C. albicans, C. glabrata, and S. mutans are shown in Table 4 (log cfu cm−2, means and SDs). The three analyses of variance showed significant effects only in surface roughness. Although the global mean of the log cfu cm−2 for the rough surfaces was higher than for the smooth surface, the values are closer. The CFU were always above 90% compared to the control group (100%). The surface composition evaluated by the XPS analysis is shown in Table 5. It can be seen that with the HP coating application, there was a decrease in the percentage of C 1s and an increase in the percentage of O 1s and Si 2p. A peak for phosphorous also appeared. For the S coating, an increase in C 1s and a decrease in O1s were observed. Additionally, a peak of sulfur was also found. The results of the interactions from the 4-way ANOVA are shown in Supplementary Tables S1–S4 [Supplementary material is available via a multimedia link on the online article webpage]. Discussion Sealers or coatings are traditionally used on dental materials to protect the material surface from degradation and the external stresses involved in mechanical and chemical factors, which could increase the surface roughness and porosity (Mainieri et al. 2011). The development of coatings or sealants with antimicrobial and anti-adherent potential is attracting great interest in the scientific community (Amano et al. 2010). Recently, Molino et al. (2013) observed that surface modification reduced interfacial protein adsorption, with the complete inhibition of adhesion and colonization by primary mouse myoblasts. Since polymer coatings can easily be applied to denture surfaces, this study evaluated the effectiveness of two photopolymerized coatings in an attempt to reduce the
Table 3. Mean absorbance values (XTT assay – 492 nm) and SDs of all experimental groups (n = 9), according to the conditions (smooth or rough surface), coating type (S or HP) and presence or absence of saliva. Surface roughness Smooth
Rough
Coating and concentration (%) Control S 25 30 35 HP 25 30 35 Control S 25 30 35 HP 25 30 35
Saliva absent 1.78 1.17 1.17 1.23 1.14 1.17 1.19 1.88 1.39 1.20 1.18 1.13 1.38 1.29
(0.20) (0.21) (0.18) (0.22) (0.18) (0.22) (0.20) (0.12) (0.14) (0.20) (0.20) (0.22) (0.17) (0.13)
Saliva present 1.68 (0.17) 0.97 (0.19) 1.04 (0.26) 1.26 (0.17) 1.19 (0.20) 1.18 (0.18) 1.12 (0.22) 1.63 (0.25) 1.24 (0.23) 1.20 (0.21) 1.24 (0.20) 1.11 (0.22) 1.37 (0.08) 1.21 (0.12)
Global 1.73 1.07 1.10 1.25 1.17 1.17 1.15 1.76 1.32 1.20 1.21 1.12 1.38 1.25
(0.19)d (0.20)a (0.22)ab (0.20)abc (0.19)abc (0.20)abc (0.21)abc (0.20)d (0.19)bc (0.20)abc (0.20)abc (0.22)ab (0.13)c (0.12)abc
CV (%)* 100.0 61.9 63.9 72.2 67.6 68.0 66.6 100.0 75.0 68.2 68.9 63.7 78.4 71.3
*Cell viability compared with control group. Groups with the same letters in the columns did not differ significantly at 1% (saliva had no effect).
530
F.E. Izumida et al.
Table 4. Mean of CFU counts and SDs of all experimental groups (n = 9), according to the conditions (smooth or rough surface), coating type (S or HP) and presence or absence of saliva. Coating
Concentration (%)
Smooth surface as S HP
Downloaded by [University of Connecticut] at 11:54 10 January 2015
ps S HP Global CV (%)* Rough surface as S HP ps S HP
Microorganism C. albicans
C. glabrata
Control 25 30 35 25 30 35 Control 25 30 35 25 30 35
5.63 4.96 4.86 5.11 5.16 5.04 4.85 5.24 4.89 4.77 5.08 5.12 5.01 4.88 4.98 91.5
(0.35) (0.38) (0.51) (0.39) (0.31) (0.62) (0.69) (0.30) (0.28) (0.42) (0.38) (0.29) (0.25) (0.49) (0.44)
6.42 6.05 5.95 6.13 6.21 6.17 6.09 6.41 6.00 6.28 6.12 6.18 6.03 6.02 6.10 95.1
(0.36) (0.17) (0.33) (0.37) (0.13) (0.25) (0.15) (0.26) (0.28) (0.24) (0.23) (0.23) (0.18) (0.13) (0.24)
6.53 6.04 5.98 6.09 6.32 6.10 6.07 6.43 6.01 6.22 6.08 6.15 6.03 6.01 6.09 94.0
(0.35) (0.28) (0.13) (0.18) (0.20) (0.15) (0.31) (0.36) (0.22) (0.19) (0.17) (0.22) (0.25) (0.30) (0.22)
Control 25 30 35 25 30 35 Control 25 30 35 25 30 35
5.76 5.23 5.11 5.22 5.14 5.24 5.15 5.41 5.17 4.98 5.22 5.13 5.14 5.07 5.15 92.2
(0.42) (0.23) (0.28) (0.25) (0.24) (0.26) (0.29) (0.19) (0.24) (0.43) (0.35) (0.20) (0.32) (0.57) (0.32)
6.44 6.33 6.17 6.29 6.20 6.38 6.21 6.41 6.25 6.08 6.29 6.20 6.21 6.17 6.23 97.0
(0.25) (0.45) (0.17) (0.14) (0.38) (0.31) (0.26) (0.29) (0.26) (0.25) (0.31) (0.16) (0.17) (0.20) (0.27)
6.56 6.34 6.14 6.30 6.19 6.34 6.20 6.46 6.25 6.06 6.26 6.18 6.20 6.10 6.21 95.5
(0.33) (0.18) (0.23) (0.34) (0.16) (0.26) (0.25) (0.32) (0.17) (0.49) (0.27) (0.24) (0.10) (0.18) (0.26)
Global CV(%)*
S. mutans
as = absence of saliva; ps = presence of saliva. *Cell viability compared with the control group smooth or rough. Note: ANOVA showed only the lower global mean of the smooth surface compared with the rough surface for all microorganisms (p ≤ 0,001).
Table 5. Elemental surface composition (at.%) of the groups measured by XPS. Groups Elements (at.%) C 1s O 1s Si 2p P 2p S 2p
Control
HP35
S35
72.54 27.45 0.2 -
66.98 28.03 5.81 0.56 -
78.97 12.63 2.7 0.78
at.% = atomic percentage.
adherence of C. albicans, C. glabrata, and S. mutans to the surface of a denture base resin. Although the XTT method is routinely used to quantify Candida biofilms (da Silva et al. 2008; Jin et al.
2004), it is possible that it could also be used on other biofilms, such as S. mutans biofilm (Gobor et al. 2011). The major advantage of the XTT assay is that manual cell detachment is not necessary and the attendant loss in cell numbers leading to a high sign noise ratio does not occur with this technique. This is especially true in the early adherent phase of the biofilm, where the cell numbers are relatively small (Jin et al. 2004). In this study, two types of surface roughness, smooth and rough, that simulate the outer and inner surfaces of a prosthesis were used. Data for surface roughness obtained by the XTT method showed that, in the control group, there were no significant differences between the smooth and the rough groups. The overall Ra values for the experimental conditions of this study ranged from 0.14 μm to 3.80 μm. These values were lower than those
Downloaded by [University of Connecticut] at 11:54 10 January 2015
Biofouling reported by Zissis et al. (2000), who found mean values ranging from 0.7 to 4.4 μm for denture base materials, and Richmond et al. (2004), who observed an arithmetical mean roughness ranging from 1.36 to 9.43 μm. Several studies have suggested that an increase in roughness facilitated microorganism retention (Lazarin et al. 2012, 2013; Moura et al. 2006; Waters et al. 1997). However, in this study, smooth or rough surfaces did not differ statistically in the adherence of C. albicans, C. glabrata, and S. mutans. The differences in these results could be attributed to the methodology used. In the studies cited above the retention test was performed by incubating specimens in C. albicans suspensions. In the present study, the adhesion of cells in the initial stages of biofilm formation was investigated to simulate the initial adhesion that occurs during biofilm development. In the retention process, cells are retained in the irregularities of the surface of the material, while the adhesion of fungal cells occurs from adhesion proteins that bind to the surface (Klis et al. 2001). The results of the present study are in agreement with those found in other studies (Moura et al. 2006; Zamperini et al. 2010, 2011). However, Zamperini et al. (2010, 2011) stated that other studies should be conducted using specimens with prepared surfaces that cover a wide range of roughness values to verify the real effect of surface roughness in microbial adherence. The application of photopolymerized coatings resulted, generally, in the absence of significant differences between the smooth and rough surfaces. The reason for these results is that the acrylic surface became more regular after the coating application (Table 2). The coating layer promotes a uniform surface since the irregularities and porosities of acrylic surface are recovered by the coatings. XPS analysis demonstrated that the coated surfaces showed changes in chemical composition, confirming the findings of this study. The carbon and oxygen composition changed in coated surfaces and for the S coating, a peak of sulfur also occurred. In this study, for all coated specimens, smooth or rough, the XTT assay showed that the adherence of all coated groups was significantly lower compared with the control. The CFU analysis confirmed this finding, although it was not statistically significant. Gobor et al. (2011) stated that the disruption of biofilms caused by serial dilution and the subsequent counting of CFU are not considered reliable, since it cannot be confirmed that each colony came from a single cell. It has been stated that the surface modification with coatings containing zwitterionic and hydrophilic groups could prevent the adherence of microorganisms to PMMA surfaces (Ishihara et al. 1998; Çagavi et al. 2004; Cheng et al. 2007; Cringus-Fundeanu et al. 2007; Zhao et al. 2010). Sulfotebaine, a member of the zwitterionic betaine family
531
of compounds which occurs widely in nature (West et al. 2004), presented in the S coating, has received greater attention, as one of the most representative structures in the well-identified class of polymeric zwitterionic materials (Yuan et al. 2003) due to its blood compatibility, protein adsorption, and reduction in microorganism adherence (Lowe et al. 2000; Hasegawa et al. 2001; Konno et al. 2001). The ability of zwitterions to reduce the adherence of microorganisms could be related to their hydration ability (Cheng et al. 2007). Water molecules bind to the hydrophobic part of the polymer through ionic solvation, which is very strong, leading perhaps to a thermodynamic hydration barrier to polymer condensation. Furthermore, a reduction in microbial adherence also results from the longer chains and higher densities of the zwitterions (Cheng et al. 2007). Thus, the effectiveness of a nonfouling zwitterionic coating is due not only to the nature of the nonfouling group, but also to the surface packing of the nonfouling group (Cheng et al. 2007). The HP coating, a hydrophilic polymer, binds water molecules via hydrogen bonding (Cheng et al. 2007), creating a water jacket around the material (Çagavi et al. 2004). In addition to surface hydration, the prevention of protein adsorption also occurs by physical aspects of the surface, such as surface roughness (Zhao et al. 2010). Zhao et al. (2010) stated that HPMA, which is a neutral hydroxyl-rich monomer, has abundant –OH functional groups that can be further functionalized via ester NHS groups for ligand immobilization. The introduction of the dual functionality of antifouling and ligand immobilization in the same material can greatly enhance its potential use in biomedical applications. The results found in this study are in agreement with other studies that also showed a reduction in the adherence of S. epidermidis, P. aeruginosa, S. aureus, and C. albicans by zwitterionic or hydrophilic polymers (Lowe et al. 2000; West et al. 2004; Cheng et al. 2007; Lazarin et al. 2012, 2013). For all experimental conditions, the salivary pellicle did not influence the adherence of C. albicans, C. glabrata, and S. mutans. Saliva is an exocrine secretion produced by different glands and consists of water, electrolytes, and proteins (Zamperini et al. 2012). The adhesive interaction between oral yeasts and bacteria depends on, amongst other things, the presence of specific salivary proteins, such as mucins, proline-rich proteins and calcium, which serve as a molecular bridge to mediate the adhesion of yeasts and bacteria to resin beads (Milsap et al. 1999). However, it has been shown that saliva also possesses antimicrobial properties, due to the nature of the substratum on the composition of the acquired pellicle, which could alter the initial adherence (Lazarin et al. 2012). Thus, the scientific literature is unclear regarding the effect of saliva on microbial
Downloaded by [University of Connecticut] at 11:54 10 January 2015
532
F.E. Izumida et al.
adherence and suggests that this divergence could be related to the differences in saliva collection and its manipulation (Park et al. 2003).The results of this study are in agreement with those found by Lazarin et al. (2013), who concluded that the interactions between C. albicans, the substratum, and saliva are complex, and factors such as the physico-chemical properties of the substrata (and conditioning film), and the cells may influence this process. In conclusion, the experimental photopolymerized coatings promoted surface modification in the denture base acrylic resin Vipi Wave. In addition, the XTT assay confirmed that these coatings were able to reduce the adherence of C. albicans, C. glabrata and S. mutans. Despite promising results, the effect of these coatings in reducing multi-species biofilm formation, and their resistance to methods of denture cleansing, such as toothbrushing, should be evaluated before clinical use. Acknowledgments The authors would like to thank to the São Paulo Research Foundation (São Paulo, SP, Brazil) for the scholarship granted to the first author (#2010/00545-9) and for financial support (#2012/01528-6). They also thank Mr Jörg Erxleben for preparing the coatings used in this study, Marlise I. Klein for microbiological assistance, and Prof. Peter Hammer for support with the XPS analysis.
References Amano D, Ueda T, Sugiyama T, Takemoto S, Oda Y, Sakurai K. 2010. Improved brushing durability of titanium dioxide coating on polymethylmethacrylate substrate by prior treatment with acryloxypropyl trimethoxysilane-based agent for denture application. Dent Mater J. 29:97–103. American National Standard. 1975. Specifications nº12 for denture base polymer. Chicago: American Dental Association. (Reaffirmed, 1999). Çagavi F, Akalan N, Celik H, Gür D, Güçiz B. 2004. Effect of hydrophilic coating on microorganism colonization in silicone tubing. Acta Neurochir. 146:603–610. Chandra J, Mukherjee PK, Leidich SD, Faddoul FF, Hoyer LL, Douglas LJ, et al. 2001. Antifungal resistance of candidal biofilms formed on denture acrylic in vitro. J Dent Res. 80:903–908. Cheng G, Zhang Z, Chen S, Bryers JD, Jiang S. 2007. Inhibition of bacterial adhesion and biofilm formation on zwitterionic surfaces. Biomaterials. 28:4192–4199. Cringus-Fundeanu I, Luijten J, van der Mei HC, Busscher HJ, Schouten AJ. 2007. Synthesis and characterization of surfacegrafted polyacrylamide brushes and their inhibition of microbial adhesion. Langmuir. 23:5120–5126. Dhir G, Berzins DW, Dhuru VB, Periathamby AR, Dentino A. 2007. Physical properties of denture base resins potentially resistant to Candida adhesion. J Prosthodont. 16:465–472. da Silva WJ, Seneviratne J, Parahitiyawa N, Rosa EA, Samaranayake LP, Del Bel Cury AA. 2008. Improvement of XTT assay performance for studies involving Candida albicans biofilms. Braz Dent J 19:364–369.
de Freitas Fernandes FS, Pereira-Cenci T, da Silva WJ, Filho AP, Straioto FG, Del Bel Cury AA. 2011. Efficacy of denture cleansers on Candida spp. biofilm formed on polyamide and polymethyl methacrylate resins. J Prosthet Dent. 105:51–58. Gobor T, Corol G, Ferreira LE, Rymovicz AU, Rosa RT, Campelo PM, Rosa EA. 2011. Proposal of protocols using D-glutamine to optimize the 2,3-bis(2-methoxy-4-nitro-5sulfophenly)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay for indirect estimation of microbial loads in biofilms of medical importance. J Microbiol Methods. 84:299–306. Gomes PN, da Silva WJ, Pousa CC, Narvaes EA, Del Bel Cury AA. 2011. Bioactivity and cellular structure of Candida albicans and Candida glabrata biofilms grown in the presence of fluconazole. Arch Oral Biol. 56:1274–1281. Hahnel S, Rosentritt M, Bürgers R, Handel G, Lang R. 2012. Candida albicans biofilm formation on soft denture liners and efficacy of cleaning protocols. Gerodontology. 29: 383–391. Hasegawa T, Iwasaki Y, Ishihara K. 2001. Preparation and performance of protein-adsorption-resistant asymmetric porous membrane composed of polysulfone/phospholipid polymer blend. Biomaterials. 22:243–251. Ishihara K, Nomura H, Mihara T, Kurita K, Iwasaki Y, Nakabayashi N. 1998. Why do phospholipid polymers reduce protein adsorption? J Biomed Mater Res. 39:323–330. Jin Y, Samaranayake LP, Samaranayake Y, Yip HK. 2004. Biofilm formation of Candida albicans is variably affected by saliva and dietary sugars. Arch Oral Biol. 49:789–798. Klis FM, de Groot P, Hellingwerf K. 2001. Molecular organization of the cell wall of Candida albicans. Med Mycol. 39:1–8. Konno T, Kurita K, Iwasaki Y, Nakabayashi N, Ishihara K. 2001. Preparation of nanoparticles composed with bioinspired 2-methacryloyloxyethyl phosphorylcholine polymer. Biomaterials. 22:1883–1889. Lazarin AA, Zamperini CA, Vergani CE, Wady AF, Giampaolo ET, Machado AL. 2012. Candida albicans adherence to an acrylic resin modified by experimental photopolymerised coatings: an in vitro study. Gerodontology. 30. doi: 10.1111/j.1741-2358.2012.00688.x. [Epub ahead of print] Lazarin AA, Machado AL, Zamperini CA, Wady AF, Spolidorio DM, Vergani CE. 2013. Effect of experimental photopolymerized coatings on the hydrophobicity of a denture base acrylic resin and on Candida albicans adhesion. Arch Oral Biol. 58:1–9. Lippens E, De Smet N, Schauvliege S, Martens A, Gasthuys F, Schacht E, Cornelissen RJ. 2013. Biocompatibility properties of surface-modified poly(dimethylsiloxane) for urinary applications. Biomater Appl. 27:651–660. Long SF, Clarke S, Davies MC, Lewis AL, Hanlon GW, Lloyd AW. 2003. Controlled biological response on blends of a phosphorylcholine-based copolymer with poly(butyl methacrylate). Biomaterials. 24:4115–4121. Lowe AB, Vamvakaki M, Wassall MA, Wong L, Billingham NC, Armes SP, Lloyd AW. 2000. Well-defined sulfobetaine-based statistical copolymers as potential antibioadherent coatings. J Biomed Mater Res. 52:88–94. Mainieri VC, Beck J, Oshima HM, Hirakata LM, Shinkai RS. 2011. Surface changes in denture soft liners with and without sealer coating following abrasion with mechanical brushing. Gerodontology. 28:146–151. Millsap KW, Bos R, van der Mei HC, Busscher HJ. 1999. Adhesion and surface-aggregation of Candida albicans
Downloaded by [University of Connecticut] at 11:54 10 January 2015
Biofouling from saliva on acrylic surfaces with adhering bacteria as studied in a parallel plate flow chamber. Antonie Van Leeuwenhoek. 75:351–359. Mima EG, Pavarina AC, Neppelenbroek KH, Vergani CE, Spolidorio DM, Machado AL. 2008. Effect of different exposure times on microwave irradiation on the disinfection of a hard chairside reline resin. J Prosthodont. 17:312–317. Molino PJ, Zhang B, Wallace GG, Hanks TW. 2013. Surface modification of polypyrrole/biopolymer composites for controlled protein and cellular adhesion. Biofouling. 29:1155– 1167. Moura JS, da Silva WJ, Pereira T, Del Bel Cury AA, Rodrigues Garcia RC. 2006. Influence of acrylic resin polymerization methods and saliva on the adhesion of four Candida species. J Prosthet Dent. 96:205–211. Nakabayashi N, Williams DF. 2003. Preparation of non-trombogenic materials using 2-methacryloyloxyethyl phosphorylcholine. Biomaterials. 24:2431–2435. Özden N, Akaltan F, Suzer S, Akovali G. 1999. Time-related wettability characteristic of acrylic resin surfaces treated by glow discharge. J Prosthet Dent. 82:680–684. Park SE, Periathamby AR, Loza JC. 2003. Effect of surfacecharged poly(methyl methacrylate) on the adhesion of Candida albicans. J Prosthodont. 12:249–254. Pereira-Cenci T, Deng DM, Kraneveld EA, Manders EM, Del Bel Cury AA, Ten Cate JM, Crielaard W. 2008. The effect of Streptococcus mutans and Candida glabrata on Candida albicans biofilms formed on different surfaces. Arch Oral Biol. 53:755–764. Ramage G, Jose A, Coco B, Rajendran R, Rautemaa R, Murray C, Lappin DF, Bagg J. 2011. Commercial mouthwashes are more effective than azole antifungals against Candida albicans biofilms in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 111:456–460. Redding S, Bhatt B, Rawls HR, Siegel G, Scott K, Lopez-Ribot J. 2009. Inhibition of Candida albicans biofilm formation on denture material. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 107:669–672. Richmond R, Macfarlane TV, Mccord JF. 2004. An evaluation of the surface changes in PMMA biomaterial formulations as a result of toothbrush/dentifrice abrasion. Dent Mater. 20:124–132. Sheridan PJ, Koda S, Ewoldsen NO, Lefebvre CA, Lavin MT. 1997. Cytotoxicity of denture base resins. Int J Prosthodont. 10:73–77. Shirley DA. 1972. High-resolution X-ray photoemission spectrum of the valence bands of gold. Phys Rev B. 5:4709–4713.
533
Silva S, Negri M, Henriques M, Oliveira R, Williams DW, Azeredo J. 2011. Adherence and biofilm formation of nonCandida albicans Candida species. Trends Microbiol. 19:241–247. Tylenda CA, Larsen J, Yeh CK, Lane HC, Fox PC. 1989. High levels of oral yeasts in early HIV-1 infection. J Oral Pathol Med. 18:520–524. Waters MG, Williams DW, Jagger RG, Lewis MA. 1997. Adherence of Candida albicans to experimental denture soft lining materials. J Prosthet Dent. 77:306–312. West SL, Salvage JP, Lobb EJ, Armes SP, Billingham NC, Lewis AL, Hanlon GW, Lloyd AW. 2004. The biocompatibility of crosslinkable copolymer coatings containing sulfobetaines and phosphobetaines. Biomaterials. 25:1195– 1204. Wilson J. 1998. The etiology diagnosis and management of denture stomatitis. Br Dent J. 185:380–384. Yildirim MS, Kesimer M, Hasirci N, Kiliç N, Hasanreisoğlu U. 2006. Adsorption of human salivary mucin MG1 onto glow-discharge plasma treated acrylic resin surfaces. J Oral Rehabil. 33:775–783. Yuan J, Zhang J, Zhu J, Shen J, Lin SC, Zhu W, Fang JL. 2003. Reduced platelet adhesion on the surface of polyurethane bearing structure of sulfobetaine. J Biomater Appl. 18:123–135. Zamperini CA, Machado AL, Vergani CE, Pavarina AC, Giampaolo ET, da Cruz NC. 2010. Adherence in vitro of Candida albicans to plasma treated acrylic resin. Effect of plasma parameters, surface roughness and salivary pellicle. Arch Oral Biol. 55:763–770. Zamperini CA, Machado AL, Vergani CE, Pavarina AC, Rangel EC, Cruz NC. 2011. Evaluation of fungal adherence to plasma-modified polymethylmethacrylate. Mycoses. 54: 344–351. Zamperini CA, Schiavinato PC, Pavarina AC, Giampaolo ET, Vergani CE, Machado AL. 2012. Effect of human whole saliva on the in vitro adhesion of Candida albicans to a denture base acrylic resin: a focus on collection and preparation of saliva samples. J Investig Clin Dent. 3:1–4. Zhao C, Li L, Zheng J. 2010. Achieving highly effective nonfouling performance for surface-grafted poly (HPMA) via atom-transfer radical polymerization. Langmuir. 26:17375– 17382. Zissis AJ, Polyzois GL, Yannikakis SA, Harrison A. 2000. Roughness of denture materials: a comparative study. Int J Prosthodont. 13:136–140.
