Accepted Manuscript Title: In vitro effect of Paullinia cupana (guaran´a) on hydrophobicity, biofilm formation, and adhesion of Candida albicans’ to polystyrene, composites, and buccal epithelial cells Author: Ermelinda Matsuura Janine Silva Ribeiro Godoy Patr´ıcia de Souza Bonfim-Mendonc¸a Jo˜ao Carlos Palazzo de Mello Terezinha Inez Estivalet Svidzinski Andr´e Gasparetto Sandra Mara Maciel PII: DOI: Reference:
S0003-9969(14)00149-6 http://dx.doi.org/doi:10.1016/j.archoralbio.2014.05.026 AOB 3199
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
12-11-2013 21-3-2014 28-5-2014
Please cite this article as: Matsuura E, Godoy JSR, Bonfim-Mendonc¸a PS, Mello JCP, Svidzinski TIE, Gasparetto A, Maciel SM, In vitro effect of Paullinia cupana (guaran´a) on hydrophobicity, biofilm formation, and adhesion of Candida albicans’ to polystyrene, composites, and buccal epithelial cells., Archives of Oral Biology (2014), http://dx.doi.org/10.1016/j.archoralbio.2014.05.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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In vitro effect of Paullinia cupana (guaraná) on hydrophobicity, biofilm formation, and adhesion of Candida albicans’ to polystyrene, composites, and buccal epithelial cells.
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Running title: Paullinia cupana (Guarana) in Dentistry
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Authors Ermelinda Matsuuraa
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Janine Silva Ribeiro Godoyb Patrícia de Souza Bonfim-Mendonçab
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João Carlos Palazzo de Melloc Terezinha Inez Estivalet Svidzinskib
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André Gasparettoa
Department of Dentistry, State University of Maringá, Av. Mandacaru 1550, Maringá, PR,
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Brazil, CEP 87080-000 b
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a
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Sandra Mara Maciela
Department of Clinical Analysis, State University of Maringá, Av. Colombo, 5790, bloco
T20 sala 203, Maringá, PR, Brazil, CEP 87020-900 c
Departament of Pharmacy and Pharmacology, State University of Maringá Av. Colombo,
5790, bloco K80, Maringá, PR, Brazil, CEP 87020-900
*Corresponding author: Sandra Mara Maciel Universidade Estadual de Maringá, Av. Mandacaru 1.550, Maringá, PR, Brazil, CEP 87080000. Telephone: +55 43 91165306, Fax: +055 44 30319051.
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E-mail address:
[email protected]
Disclosures:
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This manuscript, or any part of it, has not been submitted or published and will not be submitted elsewhere for publication.
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This manuscript has not been funded by any source, has no commercial associations, current and within the past five years, that might pose a potential, perceived or real
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conflict of interest.
Abbreviations:
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BEC: Buccal epithelial cells CFU: colony forming units
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CSH: Cell surface hydophobicity
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CHLOR: Chlorhexidine
GUAR: Guaraná (Paullinia cupana)
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INOC: Inoculum
MIC: Minimum inhibitory concentration OD: Optical density
PBS: Phosphate buffered saline
SDA: Sabouraud's dextrose agar TB: Test-bodies
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In vitro effect of Paullinia cupana (guaraná) on hydrophobicity, biofilm formation, and adhesion of Candida albicans’ to polystyrene, composites, and buccal epithelial cells.
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ABSTRACT Objective: In vitro evaluation of the effect of guaraná (GUAR) on cell surface hydrophobicity
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(CSH), on biofilm formation, and on adhesion of C. albicans to polystyrene, to composite resins, and to buccal epithelial cells (BEC).
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Materials and methods: Lyophilized aqueous extract of GUAR was tested on C. albicans ATCC (90028). The effect of GUAR was evaluated by examining the CSH of C. albicans, as
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determined by Microbial Adhesion to Hydrocarbons test, by assessing biofilm production and
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through adhesion assays (microplates of polystyrene, BEC and composites). One nanoparticle (Z350®) and two microhybrid (LLis®, Opallis®) composites were tested. Scanning electron
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microscopy (SEM) was used to analyze adhesion of C. albicans composites. Assays were
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performed in triplicate and the results analyzed by Chi-Square test, Kruskal-Wallis test and Dunn’s Multiple Comparison post hoc test at 5% significance level.
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Results: GUAR did not inhibit growth of C. albicans at any concentration, but it reduced adhesion to polystyrene surface (p < 0.001). Exposure to GUAR did not change CSH and biofilm formation, but it increased adhesion of C. albicans to the nanoparticle composite (p = 0.042) and reduced its adhesion to BEC (p < 0.001). SEM confirmed an aggregatory pattern of adhesion of C. albicans to composites. Conclusion: GUAR increased the adhesion of C. albicans to the surface of the nanoparticle composite. However, it reduced the adhesion of C. albicans to BEC and to polystyrene, which reveals its potential use in prevention of oral diseases. Keywords: Candida albicans, Guarana (Paullinia cupana), adhesion, biofilm, composite resin.
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1. Introduction
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Candida is a commensal microorganism present in normal human microbiota,1-4 considered
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an opportunistic agent as under certain circumstances it can become pathogenic, causing
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approximately 80% of all fungal infections.3-6 These infections can range from superficial
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lesions to critical and invasive systemic disseminations, with mortality rate between 40% and
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60%.7
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Candida albicans is the most prevalent of the Candida species in human oral
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cavity.1,5,6,8,9 The capacity of this yeast to adhere to any oral substrate, the first and essential
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stage in biofilm formation, is one of the main reasons for its pathogenic character.10,11 An
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important predisposing factor to infections16 is that C. albicans can adhere both to biotic
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surfaces, such as teeth or mucosa,1,12 and to abiotic surfaces, such as acrylic denture base, 1-
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3,5,8,9,13
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hydrophobicity (CSH) contributes to the ability of C. albicans to adhere to inert surfaces.13,17,18
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Stomatitis caused by C. albicans affects about 67% of elderly denture wearers with poor oral
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hygiene,1,10,13,19 which may explain why a considerable body of research focuses on the
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adhesion of the yeast to prosthetic materials based on acrylic resin.1-3,5,8,9,13 However, yeast
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infections and their sequelae have increasingly been found in groups other than prostheses
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wearers, particularly patients under immunosuppressive conditions.4,10 Direct restorative
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materials should also be assessed as potential adhesion surfaces and reservoirs of C. albicans in
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the oral cavity.10 Nevertheless, few studies have examined the adhesion of this yeast to
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composite resins.10,15,20,21
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orthodontic metal braces,14 and surfaces of dental restorations. 10,15 Cell surface
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The use of oral chlorhexidine (CHLOR), despite its known benefits, should be
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carefully monitored because of its side effects.22 Research on natural products has increased
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in recent years due to the search for more affordable substances with improved
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pharmacological activity and lower toxicity.25 Several herbal extracts have been tested in
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Dentistry.21,25 There are several native Brazilian plants potentially medicinal,23,25 although many of
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them have not been scientific validated.23 Paullinia cupana, known as guaraná (GUAR), is a
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native plant of central Amazon. The seed extract is used in the preparation of stimulating
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beverages, as it is rich in caffeine and also contains flavonoids (catechins and epicatechins)
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and tannins.26 Both in vitro and in vivo studies show different properties of GUAR, such as
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antioxidants, anti-amnesic, stimulant, adaptogenic, antidepressant, anti-stress,27,28 and
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antimicrobial. 25 Studies on its antimicrobial property are of great interest to Medical Sciences
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in general and to Dentistry in particular.
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The aim of this study was to evaluate in vitro effect of GUAR extract on CSH, on
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biofilm formation, and on adhesion of C. albicans to buccal epithelial cells (BEC), to
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polystyrene surfaces, and to composite resins.
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2. Materials and methods
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The study was approved by the Ethics Committee in Research Involving Human Participants
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(Protocol
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Resolution196/96 CNS.
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#408/2009)
and
all
procedures
were
performed
in
accordance
with
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2.1 C. albicans strain, GUAR extract and controls
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C. albicans (ATCC 90028) was obtained from the Medical Mycology Laboratory of the State
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University of Maringá (Maringá-PR, Brazil). It was reactivated 24 hours before each
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experiment in YPD (1% yeast extract, 2% peptone and 2% dextrose, Difco, USA) dissolved
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in distilled water at pH 6.5. It was then cultivated for 24 hours in YPD agar (1% yeast extract,
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2% peptone, 2% dextrose, 2% agar) and resuspended in phosphate buffered saline (PBS, 2.7
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mM KCl and 157 mM NaCl, in 10 mM of potassium phosphate buffer, pH 7.2). The
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concentration of the inoculum (INOC) was adjusted using a Neubauer haemocytometer
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(Sigma, USA).
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Seeds of Paullinia cupana were obtained in the region of Alta Floresta city (MT,
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Brazil). The crude extract was prepared with acetone/water (70:30, v/v) using Ultra-Turrax
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UTC115KT (IKA Works, Wilmington, NC, USA). Organic solvent was eliminated on a
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rotatory evaporator under reduced pressure (Rotavapor, R-200, Büchi, Switzerland) and the
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material was lyophilized to yield the crude extract (EBPC; patent pending PI0006638-9).
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EBPC was partitioned with ethyl acetate, which resulted in an ethyl-acetate fraction and an
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aqueous fraction, 28,29 the latter being controlled according to Klein et al.30 The aqueous
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fraction powder was suspended in ultrapure sterile distilled water to a final concentration of
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10 mg/mL.
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A solution of CHLOR 0.125% (chlorhexidine gluconate 2%, FGM, Brazil) was used as positive control and PBS as negative control.
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2.2 Minimum inhibitory concentration (MIC) determination
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MIC was determined by using broth microdilution method in accordance with the M27-A3
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document of the Clinical and Laboratory Standards Institute (2008),31 with some adjustment
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for natural products. GUAR was ten-fold diluted from a 10 mg/ml concentration and
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compared to INOC 5 x 105 colony forming units per milliliter (CFU/ml). Control CHLOR
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was diluted from a 2% concentration
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2.3 Cell surface hydrophobicity (CSH)
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CSH was determined according to Microbial Adhesion to Hydrocarbons32,33 with the
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following modifications: either GUAR (350uL, 10 mg/mL), CHLOR (0.125%) or PBS were
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added to the test tubes containing the INOC (350uL; 1-2 x 105 CFU/ml). One hundred ml of
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each suspension was transferred to a 96-well non coated polystyrene microplate (Nunclon
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Delta, Nunc A/S, Roskilde, Denmark) and the initial optical density (OD) was measured by a
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microplate reader at 620 nm (Expert plus - ASYS, UK). One hundred and fifty µL of n-
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hexadecane (Sigma Chemical Co, U.S.) was added to the remaining 600 µL of each
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suspension. The tubes were agitated for 3 min (Vortex AP56, Phoenix, Brazil) and left to
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stand for 15 min. One hundred ml of the aqueous phase was transferred to another microplate
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and the final OD reading was performed.
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2.4 Adhesion to Buccal Epithelial Cells (BEC)
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C. albicans was grown in yeast nitrogen base (YNB, Difco, USA) supplemented with 50 mM
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galactose and incubated at 37°C for 24 hours. It was rinsed three times with PBS and, for
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adjusting the INOC, resuspended in the same buffer.
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BEC were collected from the oral mucosa of a healthy female volunteer and deposited
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in a test tube with 10 ml of PBS. The suspension was rinsed three times with PBS by
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centrifugation at 300 rpm for 10 min. In order to obtain and to adjust the BEC suspension, the
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sediment was resuspended in PBS according to the method described by Irie et al. (2006).34
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Two hundred and fifty µL of either GUAR, CHLOR or PBS were added to plastic test
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tubes containing both BEC (500 µL; 105 cel/ml) and INOC (250 µL; 1 - 2 x 105). The tubes
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were incubated for 1 hour at 37°C under 70-rpm agitation. The suspended cells were rinsed
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five times with PBS by centrifugation at 100 rpm for 10 min, yielding sediment of BECs with
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and without adhered yeast. Smears on glass slides were prepared from these suspensions,
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which were fixed with crystal violet followed by Papanicolaou stain. Adhesion of C. albicans to BEC was evaluated using a light microscope (Nykon,
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TMD, Nippon Kogaku Inc - 40 X) by a single examiner, who observed fields that showed no
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overlapping of BEC or artifacts of staining. Yeast was considered adhered when there was a
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clear interaction with BEC, as evidenced by a bright halo around them. Two parameters were
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used in the evaluation of adhesion: the percentage of BEC that had yeast adhered to them and
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the number of yeast adhered per cell. All results were expressed as means of the repetitions of
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each test.
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2.5 Adhesion to polystyrene
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Adhesion to polystyrene was determined using the method described by Raut et al. (2010),32
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with the following modifications: each of the 96 wells of the non coated polystyrene
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microplate received the INOC (50 µL; 1 - 2 x 107) and, separately, GUAR (50 µL), CHLOR
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and PBS, then incubated for 90 min at 37°C under continuous agitation at 50 rpm. In order to
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remove the yeast that was loosely adhered to the surface of the wells, or not adhered to them,
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the microplate was rinsed three times with PBS.
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The adhered yeast were observed in inverted microscope (Olympus CK 40-20 X) and
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counted visually by a single examiner. Mean values were calculated based on the evaluation
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of twenty fields of view from each well.
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2.6 Biofilm formation
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Biofilm production was evaluated using the method described by Shin et al. (2002)35 with the
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following modifications: the 96 wells of the non coated polystyrene microplate were prepared
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with INOC (20 µL; 1 - 2 x 107 CFU/ml) in 160 µL Sabouraud Dextrose Broth (SDB, Difco,
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USA) supplemented with 8% glucose and 20 µL of either of the experimental component—
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GUAR, CHLOR or PBS. The microplate was incubated for 24 hours at 37°C under
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continuous agitation at 50 rpm (Shaker NT712), rinsed twice with distilled water and OD was
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measured in the microplate reader at 405 nm. To quantify biofilm production, the percent
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transmittance (%T) was calculated by subtracting the %T from the values of the negative
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control (no biofilm production) and interpreted according to the following scale: Negative
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(%T < 5), 1+ (%T 5 to 20), 2+ (%T 20 to 35), 3+ (%T 35 to 50), 4+ (%T ≥ 50).
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2.7 Adhesion to composite resins
Adhesion assays to composite were performed according to the technique of counting CFU as
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described by Samaranayake and MacFarlane (1980).9
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Test-bodies (TBs) were prepared with one nanoparticle composite (Z350®) resin and two microhybrid composites (LLis®, Opallis), as shown in Table 1.
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Table 1
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Each composite was inserted in a circular metal matrix (10 mm diameter, 2 mm thick)
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and compressed manually between two glass slides, each slide covered by sheets of acetate
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film. The composites were light-cured for 40 seconds on both sides (LED Radii, SDI
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Equipment Dental Products, Australia) at 650 mW/cm², as measured with a curing radiometer
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(Demetron Research Corp.).
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The technique of counting CFU was modified as follows: hydrated and sterilized TBs
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were placed in a 24-well non coated polystyrene microplate (Nunclon Delta, Nunc A/S,
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Roskilde, Denmark), followed by INOC (500 µL; 1 - 2 x 105) and 500 µL of either GUAR,
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CHLOR or PBS. The microplates were incubated for 60 min at 37°C under continuous
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agitation at 50 rpm. The TBs were then rinsed three times with PBS, transferred to test tubes
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with PBS (2 ml) and 1 g of glass beads (1-2 mm diameter, Roni Alzi Scientific Glass) and
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agitated for 1 min on a tube shaker in order to remove the yeast that was strongly adhered to
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the surfaces of the composites. Petri plates containing SDA were inoculated with this
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suspension and then incubated at 37°C for 18 hours. CFU counting was expressed as the
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means of C. albicans adhered per mm2.
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2.8 Ultrastructural analysis of C. albicans on the surface of composites and adhesion
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analysis using scanning electron microscopy (SEM)
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The microhybrid composite LLis® and the nanoparticle composite Z350® were selected for
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the study, as C. albicans showed the same pattern of adhesion to both microhybrid composites
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Opallis® and LLis®. After the adhesion assays, the TBs were fixed in Karnovsky solution
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(2.5% glutaraldehyde, 2% paraformaldehyde in 0.2M cacodylate buffer, pH 7.2) and kept at
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4°C for 24 hours. They were dehydrated in a series of increasing ethanol concentrations (80%,
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90%, 95% and 100%) and brought to its critical point using liquid CO2 (Bal-tec – CPD 030
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Critical Point Dryer, Balzers, 40 Liechtenstein). They were mounted on an aluminum stub and
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sputter-coated with gold (Shimadzu IC-50 - Shimadzu Biotech, Japan). The specimens were
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then examined with a SEM (Shimadzu Biotech, Japan) and, for characterization of the cells
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after exposure to GUAR, CHLOR and PBS, the images were captured at different
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magnifications and in different regions.
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2.9 Statistical Analysis
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Data were analyzed using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA,
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USA). Normality of distribution was verified using Kolmogorov-Smirnov test, which
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revealed that most of the data were not normally distributed. Results are expressed as mean
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and standard deviation (M±SD) and percentages. Statistical analysis used Chi-square,
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Kruskal-Wallis test and post hoc Dunn Multiple Comparison test, with significance level at
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