Universidade Estadual de Campinas Faculdade de Odontologia de Piracicaba rea de Prtese Dental

Juliana Silva Moura

Cirurgi-dentista

Aderncia de Candida spp. a resinas acrlicas: mtodo de polimerizao e presena ou no de saliva

Tese de Doutorado apresentada Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas para obteno do Ttulo de Doutor em Clnica Odontolgica rea de Prtese Dental

Piracicaba 2005

Juliana Silva Moura

Aderncia de Candida spp. a resinas acrlicas: mtodo de polimerizao e presena ou no de saliva

Tese de Doutorado apresentada Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas para obteno do Ttulo de Doutor em Clnica Odontolgica rea de Prtese Dental

Orientadora: Profa. Dra. Altair Antoninha Del Bel Cury Co-orientadora: Profa Dra Renata C. Matheus Rodrigues Garcia Banca examinadora do exame de qualificao: Prof. Dr.Marcelo Ferraz Mesquita a a Prof . Dr . Cnthia Pereira Machado Tabchoury Dra. Adriana Franco Paes Leme

Piracicaba 2005 ii

FICHA CATALOGRFICA ELABORADA PELA BIBLIOTECA DA FACULDADE DE ODONTOLOGIA DE PIRACICABA Bibliotecrio: Marilene Girello CRB-8a. / 6159

M865a

Moura, Juliana Silva. Aderncia de Candida spp. a resinas acrlicas: mtodo de polimerizao e presena ou no de saliva. / Juliana Silva Moura. -- Piracicaba, SP : [s.n.], 2005. Orientadores: Altair Antoninha Del Bel Cury, Renata Cunha Matheus Rodrigues Garcia. Tese (Doutorado) Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba. 1. Candida albicans. 2. Candida tropicalis. 3. Candida glabrata. 4. Candida dubliniensis. 5. Resinas acrlicas. 6. Saliva. I. Del Bel Cury, Altair Antoninha. II. Garcia, Renata Cunha Matheus Rodrigues. III. Universidade Estadual de Campinas. Faculdade de Odontologia de Piracicaba. IV. Ttulo. (mg/fop)

Ttulo em ingls: Adherence assay of Candida spp. on acrylic resins: polymerization methods and presence of saliva Palavras-chave em ingls (Keywords): 1. Candida albicans. 2. Candida tropicalis. 3. Candida glabrata. 4. Candida dubliniensis 5. Acrylic resins. 6. Saliva rea de concentrao: Prtese Dental Titulao: Doutor em Clnica Odontolgica Banca examinadora: Renata Cunha Matheus Rodrigues Garcia, Rosena Santos da Rosa, Viviane Maia Barreto de Oliveira, Cnthia Pereira Machado Tabchoury, Clia Marisa Rizzatti Barbosa Data da defesa: 22/11/2005

iii

.... ~..,
UNICAMP

UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE ODONTOLOGIA DE PIRACICABA

A Comisso Julgadora dos trabalhos de Defesa de Tese de DOUTORADO, em sesso pblica realizada em 22 de Novembro de 2005, considerou a candidata JULIANA SILVA MOURA aprovada.

PROFa. DRa.

~.IJ{ CW\.(,

KO-\'Dl

~ (()g V~

PROF. DR. VIVIANE MAIA BARRETO DE OLIVEIRA

P:OF~~Ra.-C::PE~IRA~~~Y

Dedico este trabalho aos meus pais, Joo Bernardino de Moura e Vera Lcia Pereira Moura, ao meu irmo, Joo Ricardo Silva Moura e ao meu noivo, Bruno Franco Jufer, pela demonstrao de amor e compreenso pelos momentos de ausncia.

Agradecimentos especiais

minha orientadora, Profa. Dra. Altair A. Del Bel Cury, de quem sempre me lembrarei pelo exemplo de vida, dedicao ao trabalho e incentivo constante pesquisa.

vi

Agradecimentos Especiais

Profa.

Dra.

Renata

Cunha

Matheus

Rodrigues Garcia, pela co-orientao e amizade.

Aos colaboradores deste trabalho, Wander Jos da Silva e Tatiana Pereira, e pela os

competncia,

companheirismo

conhecimentos compartilhados.

vii

Agradecimentos A Universidade Estadual de Campinas na pessoa de seu Magnfico Reitor, Prof. Dr. Jos Tadeu Jorge. Faculdade de Odontologia de Piracicaba UNICAMP,

representada por seu Diretor Prof. Dr. Thales da Rocha Mattos Filho. Ao CNPq, pelo financiamento desta pesquisa. Ao Coordenador de Ps-Graduao Prof. Dr. Pedro Luiz Rosalen. Ao Coordenador do Programa de Ps-graduao em Clnica Odontolgica Prof. Dr. Roger William Moreira.

Ao laboratrio de Bioqumica Oral da Faculdade de Odontologia de Piracicaba, na pessoa dos professores Dr. Jaime A. Cury e Dra. Cnthia P. M. Tabchoury pelo uso de suas instalaes. Ao laboratrio de Farmacologia da Faculdade de Odontologia de Piracicaba, na pessoa do Prof. Dr. Pedro Luiz Rosalen, pelo uso de suas instalaes. Dona Joselena Casati Lodi, pelo carinho. Aos amigos especiais que fiz durante os meus cinco anos de vida em Piracicaba, Adriana, Blanca Liliana, Daniela, Edwin, Emilena, Fernanda, Leonardo, Manoel, Margarete, Noli, Patrcia, Paulo, Priscila, Ricardo, Rosena e Viviane, pelos momentos felizes.

viii

Agradecimentos A todos os colegas do curso de Ps-Graduao em Clnica Odontolgica e aos amigos do Curso de Ps-graduao em Odontologia, pela agradvel convivncia. Ao corpo docente do Programa de Ps-Graduao em Clnica Odontolgica da Faculdade de Odontologia de Piracicaba UNICAMP pelos conhecimentos transmitidos, fundamentais minha formao. s funcionrias da Secretaria de Ps-Graduao rica Alessandra Pinho Sinhoreti e Raquel Q. Marcondes Cesar Sacchi da Secretaria de PsGraduao em Clnica Odontolgica, Mnica Aparecida Barnab e do Departamento de Prtese e Periodontia, Shirley Rosana Sbravatti Moreto e Juliana de Souza. Ao tcnico do Laboratrio de Bioqumica Oral, Jos Alfredo da Silva, pela presteza. bibliotecria Marilene Girello, pelo auxlio durante a formatao desta tese. funcionria do Comit de tica em Pesquisa desta instituio, Leni Ceclia Rosilho.

ix

SUMRIO

RESUMO................................................................................................... 1

ABSTRACT...............................................................................................

1 INTRODUO GERAL.........................................................................

2 CAPTULO.............................................................................................

3 CONCLUSO GERAL...........................................................................

29

REFERNCIAS........................................................................................

30

ANEXO...................................................................................................... 36

RESUMO O objetivo deste trabalho foi avaliar a influncia do mtodo de polimerizao e a presena da saliva humana na aderncia de Candida spp. s superfcies de resinas acrlicas. Duzentas e cinquenta e seis amostras retangulares (2,5 x 1,2 x 0,2 cm), confeccionadas com resinas polimerizadas por banho de gua ou microondas foram avaliadas para rugosidade e energia livre de superfcie e em seguida utilizadas para o ensaio de aderncia de Candida spp. Para este propsito, as amostras foram aleatoriamente divididas em 8 grupos por resina, sendo quatro expostos durante 30 min a saliva humana. Em seguida, as amostras foram posicionadas verticalmente em tubos de plsticos estreis contendo meio de cultura Sabouraud e uma entre quatro suspenses de Candida: Candida albicans, Candida tropicalis, Candida dubliniensis e Candida glabrata (1 a 5 x 106 clulas / mL). A contagem das clulas aderidas foi realizada em microscpio ptico com 400 x de aumento. Os dados de rugosidade superficial e energia de superfcie foram submetidos a ANOVA um fator e teste t. No houve diferena estatstica significante para rugosidade (p > 0,05), enquanto maiores valores de energia livre de superfcie foram encontrados para a resina polimerizada por banho de gua (p < 0,05). Para aderncia de Candida spp., os testes Wilcoxon-Mann-Whitney, Kruskal Wallis e Qui-quadrado demonstraram no haver influncia dos valores de rugosidade e energia livre de superfcie na aderncia de Candida, enquanto houve uma diminuio geral na contagem de microrganismos em grupos expostos saliva (p < 0,05). Concluiuse que a saliva foi capaz de reduzir a aderncia total de Candida, enquanto no houve correlao destes valores com rugosidade e energia de superfcie.

ABSTRACT The aim of this work was to evaluate the influence of polymerization methods and presence of human clarified saliva on Candida spp. adherence to acrylic resins surfaces. Two hundred and fifty six rectangular acrylic resins samples (2.5 x 1.2 x 0.2 cm) polymerized by water bath or microwave were evaluated for surface roughness and surface free energy and used in an adherence assay for Candida spp. For this purpose, acrylic samples were randomly divided into 8 groups for each resin, where 4 were exposed to saliva. For the adherence assay, the samples were placed vertically on test tubes containing yeast Sabouraud broth medium and one of the four Candida suspensions: Candida albicans, Candida tropicalis, Candida dubliniensis and Candida glabrata (1 to 5 x 106 cells / mL). Adhered yeasts were counted using an optic microscope at 400 x. Data from surface roughness and surface free energy were submitted to one-way ANOVA ant t test and Candida spp. adherence values were evaluated by the Wilcoxon-Mann-Whitney, Kruskal Wallis and Chi-square tests. No statistical significance was found for roughness (p > 0.05), while higher surface free energy values were found for water bath resin (p < 0.05). Roughness and surface free energy did not influence Candida adherence, while saliva generally decreased yeast counts (p < 0.05). It was concluded that polymerization methods did not interfere with adherence assay while saliva was capable of reducing Candida spp. adherence, while roughness and free energy did not influence the adherence rates.

1 INTRODUO GERAL Diferentes espcies de Candida so geralmente comensais, sendo detectadas em at 71% da populao saudvel. No entanto, o desequilbrio da microflora normal ou comprometimento do sistema imunolgico podem ocasionar o crescimento do nmero de clulas, causando a colonizao da mucosa e de materiais dentrios, levando ao aparecimento de doenas (Naglik et al., 2004). Um tipo de manifestao a estomatite prottica que pode estar presente em at 65% dos usurios de prtese (Budtz-Jorgensen et al., 1975; Arendorf & Walker, 1987), sendo que Candida albicans, Candida glabrata e Candida tropicalis podem se encontrar associadas (Budtz-Jorgensen et al., 1975; Dar-Odeh & Shehabi, 2003). Outra espcie patognica a Candida dubliniensis, causadora de candidase orofarngea, principalmente em pacientes infectados com HIV (Ramage et al., 2001). A estomatite prottica mas pode acarretar irritao na mucosa, inchao, sensao de queimao ou dor, halitose, paladar desagradvel e xerostomia (Webb et al.1998). A etiologia desta condio multifatorial, incluindo trauma causado por desajustes em prteses removveis, ms condies de higiene bucal, alergia e reao de irritao primria resina acrlica, dieta, pH da placa dental e fatores sistmicos, incluindo fatores predisponentes (Arendorf et al., 1987; Budtz-Jorgensen, 1990; Webb et al., 1998). O uso de prteses removveis parece aumentar a contagem de Candida na cavidade oral. Desta forma, o crescimento de clulas em materiais utilizados em prtese tem sido extensivamente estudado. Entre os fatores intrnsecos de um material que podem interferir na aderncia de microrganismos est a energia livre de superfcie, que indica a capacidade de molhamento de um slido, sendo mensurada pelo ngulo de contato formado entre o sistema slido / lquido (Sipahi et al., 2001). Estudos tm demonstrado que substratos com altos nveis de energia de superfcie induzem maior aderncia microbiana (Minagi et al., 1985; van Dijk et al., 1987). Ainda, caractersticas superficiais,

como imperfeies, microporosidades e rugosidade excessiva podem alojar microrganismos (Radford et al., 1998; Verran & Maryan, 1997), dificultando a sua remoo por limpeza mecnica ou qumica (Budtz-Jorgensen, 1990; Quirynen et al., 1990; Quirynen & Bollen, 1995; Bollen et al., 1997; Radford et al., 1999), facilitando a aderncia dos mesmos. Apesar da composio qumica similar, as resinas polimerizadas por microondas e por banho de gua diferem pelo ciclo de polimerizao e temperatura, o que pode interferir nos nveis porosidade e liberao de monmero residual, resultando em maiores reas de reteno de microrganismos e inflamao da mucosa, respectivamente (Azzarri et al., 2003; Lai et al., 2004). O papel da saliva durante o processo de aderncia de Candida spp. no est completamente esclarecido (Nikawa et al., 2000a). O efeito de limpeza da saliva e componentes salivares, como lisozima, histatina, lactoferrina, calprotectina e sIgA interagem com as espcies de Candida, diminuindo a aderncia das mesmas superfcies bucais (Ueta et al., 2000; Cannon et al., 2001; Tanida et al., 2001; Dodds et al., 2004; Elguelzabal et al., 2004), enquanto componentes como mucina (Edgerton et al., 1993; Nikawa & Hamada, 1990; Nikawa et al., 1993; Dodds et al., 2004) e estaterina (Johansson et al., 2000) facilitam a adsoro de Candida albicans em resina acrlica e materiais resilientes embebidos em saliva (Nikawa et al., 2000 b). Entretanto, estudos sobre a influncia da saliva na aderncia de Candida so contraditrios; enquanto Koseki et al. (2004) encontraram maior prevalncia de Candida albicans, Candida glabrata, Candida tropicalis e Candida krusei em pacientes com diminuio do fluxo salivar, incluindo pacientes com Sndrome de Sjgren e Sndrome de Stevens-Johnson, San Milln et al. (2000) e Ramage et al. (2004) verificaram que a saliva aumentou a aderncia inicial de Candida albicans resina, mas o efeito foi inibitrio (San Milln et al., 2000) ou mnimo (Ramage et al., 2004) aps certo perodo de tempo, enquanto Jin et al. (2004) verificou que a saliva, sem adio de glicose, no interferiu nos valores de aderncia.

Ainda, redues nos nveis de aderncia de Candida albicans resina acrlica na presena de saliva foram verificadas por Saramanayake et al. (1980), Radford et al. (1999), Bosch et al. (2003) e Elguelzabal et al. (2004) e a materiais resilientes por Waters et al. (1997), enquanto um aumento na aderncia de Candida albicans ocorreu na presena de saliva de acordo com Vasilas et al. (1992), Millsap et al. (1999) e Nikawa et al. (2000b), quando houve a adio de bactrias. Estes resultados indicam que ainda no h um consenso sobre o papel da saliva na aderncia de Candida a superfcies de resina acrlica. Embora caractersticas superficiais sejam importantes para determinar a energia livre de superfcie e a composio da pelcula adquirida e consequentemente influenciar na aderncia de Candida, existem poucos estudos que comparem o efeito de diferentes mtodos de processamento neste processo. A literatura sobre aderncia de diferentes espcies de Candida, alm da Candida albicans, em resinas acrlicas in vitro tambm escassa (Minagi et al., 1985; Luo & Samaranayake, 2002). Assim, a proposta deste estudo foi avaliar a influncia dos mtodos de polimerizao da resina acrlica - polimerizada por banho de gua ou microondas - e a presena de saliva humana na aderncia clulas de Candida albicans, Candida tropicalis, Candida dubliniensis e Candida glabrata.

2 CAPTULO Adherence assay of Candida spp. on acrylic resins: polymerization methods and presence of saliva Juliana Silva Moura, Wander Jos da Silva, Tatiana Pereira, Renata Cunha Matheus Rodrigues Garcia, Altair A. Del Bel Cury* Department Prosthodontic and Periodontology, School of Dentistry, University of Campinas UNICAMP, Av. Limeira, n 901, Bairro Areio Piracicaba, SP, Brazil. Address of the authors: Av. Limeira, n 901, Bairro Areio, Piracicaba, SP, CEP 13.414-903 *Corresponding author: Dra. Altair A. Del Bel Cury Av. Limeira, n 901, Bairro Areio, Piracicaba, SP, CEP 13.414-903 Phone number: + 55 (19) 3412-5295 Fax number: + 55 (19) 3412-5218 Piracicaba e-mail: altcury@fop.unicamp.br KEY-WORDS Candida adherence; Candida albicans; Candida tropicalis; Candida dubliniensis; Candida glabrata; acrylic resins; saliva Partially supported by Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico (CNPq), Brazil.

ABSTRACT The aim of this work was to evaluate the influence of polymerization methods and presence of human clarified saliva on Candida spp. adherence to acrylic resins surfaces. Two hundred and fifty six rectangular acrylic resins samples (2.5 x 1.2 x 0.2 cm) polymerized by water bath or microwave were evaluated for surface roughness and surface free energy and used in an adherence assay for Candida spp. For this purpose, acrylic samples were randomly divided into 8 groups for each resin, where 4 were exposed to saliva. For the adherence assay, the samples were placed vertically on test tubes containing yeast Sabouraud broth medium and one of the four Candida suspensions: Candida albicans, Candida tropicalis, Candida dubliniensis and Candida glabrata (1 to 5 x 106 cells / mL). Adhered yeasts were counted using an optic microscope at 400 x. Data from surface roughness and surface free energy were submitted to one-way ANOVA ant t test and Candida spp. adherence values were evaluated by the Wilcoxon-Mann-Whitney, Kruskal Wallis and Chi-square tests. No statistical significance was found for roughness (p > 0.05), while higher surface free energy values were found for water bath resin (p < 0.05). Roughness and surface free energy did not influence Candida adherence and saliva generally decreased yeast counts (p < 0.05). It was concluded that polymerization methods did not interfere with adherence assay while saliva was capable of reducing Candida spp. adherence, while roughness and free energy did not influence the adherence rates.

INTRODUCTION Candida-associated denture stomatitis affects up to 65% of denture users (1, 2), in which Candida albicans, Candida glabrata and Candida tropicalis have been isolated from such cases (1, 3). Another species associated with oral diseases is Candida dubliniensis, commonly found in HIV-infected patients (4). An important factor when determining Candida colonization is the nature of the material placed in the oral cavity, because it might interfere with the components adsorbed to form an acquired pellicle (5, 6). Surface free energy is an important property, because it indicates the ease with which saliva spreads over a surface (7, 8, 9). In addition, surface characteristics, such as microtips, microporosities and roughness, may harbor microorganisms that are difficult to remove by mechanical or chemical cleansing (10, 11, 12, 13, 14), thus increasing the adherence of microorganisms in vitro (15, 16). Despite of the similar chemical composition, microwave and water bath resins are different concerning the polymerization cycle and temperature, which could interfere on porosity and residual monomer release (17, 18). The role of saliva during the adherence and colonization process of Candida spp. is not clear (19). It is well known that innate defenses, such as the flushing effect of saliva, and anti-candida salivary components, such as lysozyme, histatins, lactoferrin, calprotectin, sIgA interact with the Candida spp., decreasing its adherence to oral surfaces (20, 21, 22, 23), while components in whole saliva including mucins (23, 24, 25, 26, 27), statherin (28) and proline-richproteins (21) have been reported to adsorb to Candida albicans, increasing its adherence to saliva-coated resins and resilient materials (19, 29). However, studies about the influence of whole saliva on Candida adherence are contradictory. Koseki et al. (30) found increased prevalence of Candida albicans, Candida glabrata, Candida tropicalis and Candida krusei in patients with reduced salivary flow rates. As for in vitro studies, San Milln et al. (31) and Ramage et al. (32) affirmed that saliva increased Candida albicans adherence to resin in the beginning, but its effect was minimal (32) or inhibitory

(31) after a certain period of time, while Jin et al. (33) found no effect of saliva on adherence of Candida albicans to surfaces. In contrast, the adherence of Candida albicans increased in the presence of saliva according to Vasilas et al. (34) and according to Millsap et al. (35). Nonetheless, a reduction in the levels of Candida albicans adherence to acrylic resins or resilient materials in the presence of saliva was found by many authors (14, 36, 37, 38, 39). These results are indicative of a lack of consensus in the literature about the role of saliva in Candida adherence to acrylic surfaces. Moreover, although surface characteristics are important to determine the composition of an acquired pellicle and surface free energy, and therefore dictate Candida spp. colonization, there are few studies comparing the effects of different types of acrylic resins on this process. There are also few studies about adherence of different Candida spp., other than Candida albicans, to acrylic resins in vitro, indicating different levels of adherence (7, 40). Therefore, the purpose of this study was to evaluate the effect of saliva on the adherence of Candida albicans, Candida tropicalis, Candida dubliniensis and Candida glabrata to acrylic resins polymerized by water bath or microwave and correlate the data with surface roughness and surface free energy values. MATERIALS AND METHODS Experimental design For this experiment, two hundred and fifty six rectangular samples (2.5 x 1.2 x 0.2 cm) were made using water bath or microwave polymerized resins, according to manufacturers directions. After finishing and polishing, surface roughness and surface free energy measurements were made. Then, the samples were randomly divided into two groups exposed or not to human saliva and submitted to the adherence assay, being exposed for two hours to one of the four Candida suspensions: Candida albicans, Candida tropicalis, Candida dubliniensis and Candida glabrata [1 to 5 x 106 cells / mL] and the adhered cells were counted using an optic microscope at 400 x. The statistical analysis was

made considering three factors polymerization method, exposure or not to saliva and Candida spp. -, and three depended variables roughness and surface free energy, considering the polymerization method (two groups, n = 128) and adherence rates, considering the interactions between the type of polymerization, exposure or not to saliva and Candida spp. (sixteen groups, n = 16). Preparation of acrylic samples The acrylic resins used in this study are listed in Table 1. All specimens were fabricated using a sheet of wax matrix. The sheets were flasked in Type III dental stone (Herodent Soli-Rock; Vigodent, Rio de Janeiro, Brazil) using a standard metal dental flask (Uraby; DLC, So Paulo, Brazil) or plastic flask (Onda Cryl; Artigos Odontolgicos Clssico Ltd, So Paulo, Brazil) for water bath and microwave polymerization, respectively. After the gypsum has set completely, the flasks were boiled for 5 min to soften and eliminate the wax. Water bath polymerized resin was then packed and the metal flasks were placed in a polymerizing unit (Termotron P-100; Termotron Equipamentos Ltd, Piracicaba, Brazil) filled with water at 75C for 9 h. To process microwave acrylic resin, the plastic flasks were placed in a microwave oven (AW-42 Continental Eletrodomsticos Ltd, Manaus, Brazil) and polymerized for 3 min at 360 W, followed by 4 min rest, then 3 min at 810 W, according to manufacturers instructions. All flasks were allowed to bench cooling for 2 h and the samples were removed. Specimens were ground using progressively smoother aluminum oxide papers (grit 320, 400 and 600) in a horizontal polisher (model APL-4; Arotec, So Paulo, Brazil). For mechanical polishing, a brush disc with pumice slurry and a felt cone with chalk powder were used (41). They were also ultrasound cleansed (Thornton T 740, Thornton-Inpec Eletrnica LTDA, Vinhedo, Brazil) for 20 min and then immersed in distilled water at 37oC for 12 h for residual monomer release (42). After these procedures, roughness and

10

angulations for surface free energy were measured and the samples were randomly divided into two groups exposed or not to saliva. Table 1. Acrylic resins Material Jet Clssico Basic Chemical Composition Liquid: methylmethacrylate monomer Powder: polymethylmethacrylate Onda Cryl Liquid: methylmethacrylate monomer Powder: polymethylmethacrylate Microwave Clssico Artigos Odontolgicos, LTDA, So Paulo, Brazil Polymerization Technique Water bath Clssico Artigos Odontolgicos, LTDA, So Paulo, Brazil Manufacturer

Surface roughness Surface roughness (Ra) of the acrylic samples was measured using a profilometer (Surfcorder SE 1700 Kozaka Industry, Kozaka, Japan) with a 0.01 m resolution, calibrated at sample length of 0.8 mm, 2.4 mm percussion of measure, and 0.5 mm / s. Three readings were taken for each sample and a mean value was calculated (16). Surface free energy Surface free energy (erg . cm -2) was calculated with the deposition of 15 L of distilled water (in triplicate) on each acrylic resin specimen. The image of each sessile drop was captured using a digital camera (Mavika CD 350, Sony, Tokyo, Japan) immediately after its deposition and the mean value of 11

contact angles were measured using Autocad 2005 (Auto Desk, Sankt Augustin, USA) (7). Hence, surface free energy was calculated using cos of contact angles, according to Neumann et al. (43) and Gindl et al. (44). After surface roughness and surface free energy measurements, the contaminants presented in the acrylic samples were removed by initial sonication, for 20 min, further deposition on sterile recipients with sterile distilled water for one week and then sonicated again, for another 20 min (40). Microorganisms and growth conditions Four strains were used in this study (Table 2). Candida cells were reactivated from their original culture (Sabouraud agar, Difco, Sparks, MD, USA), at 4C, in 10 mL yeast medium (Sabouraud agar) for 24 h at 35C in aerobic atmosphere (NCCLS, 2002). These were then harvested, suspended in NaCl 0.9% and agitated for 15 s. After this procedure, the cell concentration was measured spectrophotometrically (Bausch & Lomb Spectronic 20, San Pablo, CA, USA) at 530 nm, using the pattern 0.5 of the Mc Farland scale, assuring a concentration of 1 to 5 x 106 cells / mL (34, 35, 45). Table 2. Strains used in the current study Candida specie Candida albicans Candida tropicalis Candida dubliniensis Candida glabrata Isolate number ATCC 90028 ATCC 750 CD 36 ATCC 9950

Human saliva collection and preparation for the adherence assay Human whole saliva was collected from one healthy volunteer (27), aged 27, who had not used antibiotics, mouth rinses or any other medication known to affect salivary composition and flow, in the past 3 months, and who signed a written informed consent, previously approved by the Ethics Committee

12

of Piracicaba Dental School, UNICAMP (Protocol number 116 / 2004). Saliva was collected during masticatory stimulation with Parafilm M (American Co., Greenwich, Ct, USA) in an ice-chilled Erlenmeyer tube and clarified by centrifugation at 10 000 g for 5 min at 4C (34, 35). The supernatant was collected and put into sterile Petri dishes, where half of the acrylic samples were placed with the polished surface facing down for 30 min (6) to form an acquired pellicle, while the other half remained not exposed to any liquid (control) (15, 24, 32, 35). After this period, the acrylic samples were removed from the Petri dishes and used in the adherence assay. Adherence assay Using an aseptic technique, the acrylic samples were individually placed, in a vertical position (15, 24, 36, 37, 40, 46), into sterile test tubes containing 5 mL of Sabourauds broth (Difco, Sparks, MD, USA) and inoculated with 5 mL of one of the four Candida spp. suspensions (31) and the whole assembly was incubated for 2 h at 35C to promote yeast adherence (19, 24, 26, 27, 34). After this period, acrylic samples were removed, and gently washed for 75 s with phosphate buffered saline solution (PBS), to remove non-adhered yeast cells, washed with ethanol at 80 %, stained for 1 min with crystal violet and washed with PBS (7, 14, 39). Then, the adhered yeasts were counted using a light microscope, as described below. Yeast counts Adherent yeast cells in 15 fields of view in each sample (0.25 mm2 per field) were enumerated and the results were expressed as cells / mm2 of material, using a light microscope (Leitz Ortholux, Leitz Wetzlar, Germany) at 400 x magnification. The majority of the attached yeasts were at the blastospore stage, some with daughter cells and only a very few with hyphae or pseudohyphae. Thus, the following parameters were used to standardize the counts: 1) A budding yeast was considered as a unit cell if the daughter was smaller than the mother cell and 2) a hyphum was counted as a single cell (36).

13

Statistical analysis All the data were analyzed using SPSS version 10 and MINITAB version 13 programs, being first checked for normality, using the AndersonDarling method; variance homogeneity, using the F maximum or Levene test; and absence of outliers, using the Box-plot graphic. Surface roughness and surface free energy values were evaluated using one-way ANOVA and t test, considering the water bath and microwave energy polymerized acrylic resin groups (n = 128). Surface roughness data were transformed using natural logarithm and the t test could be applied. WilcoxonMann-Whitney test was used to verify the distribution of resins with regard to roughness and surface free energy, considering the interaction related to the type of resin, whether or not exposed to saliva and Candida spp. For Candida spp. adherence, Wilcoxon-Mann-Whitney test was applied considering the interaction related to the type of resin and adherence values, while the interaction between saliva and Candida spp. adherence was analyzed using Kruskall Wallis and Chi-square tests (47). All tests were conducted at 95% level of confidence. RESULTS Surface roughness results for both acrylic resins were analyzed by Wilcoxon-Mann-Whitney test, indicating that none of the groups received resins with different roughness values (Table 3). Moreover, no statistically significant differences were found for roughness values at 5 % (p > 0.05) (Table 4) by the t test. For surface free energy, Wilcoxon-Mann-Whitney test also showed no statistical differences between samples that were further exposed or not to saliva and to different Candida spp. (Table 3). The t test indicated significant differences of surface free energy between the two acrylic resins, with the water

14

bath resin showing the highest contact angle and lowest surface free energy values (Table 4). Table 3. Interactions (p values) between polymerization method, surface roughness, surface free energy, presence of saliva, general Candida adherence and species of Candida. Source Polymerization method * roughness General Candida adherence * roughness Saliva * roughness Polymerization method * surface free energy General Candida adherence * surface free energy Saliva * surface free energy Polymerization adherence Saliva * general Candida adherence General Candida adherence * Candida species 0.00301 < 0.00001 method * general Significance (p values) 0.0953 0.246 0.193 0.001 0.222 0.659 Candida 0.425

Table 4. Mean values and standard deviations of surface roughness (Ra) in m, and surface free energy (erg . cm -2) (n = 128). Acrylic resin Water bath Microwave Surface roughness 0.1123 ( 0.0239) A 0.1166 ( 0.0244) A Surface free energy 40.87 ( 2.75) A 39.74 ( 2.69) B

Means followed by capital letters differ vertically at 5 %. The presence or absence of saliva and the different Candida species significantly influenced these values (Table 3), while adherence counts were not influenced by the polymerization method (Table 3). Therefore, samples polymerized by water bath and microwave resins were analyzed separately, constituting eight groups for each acrylic resin (Table 5). The presence of saliva 15

influenced Candida spp. counts, causing a general decrease on adherence values. However, this influence was different for each specie (Table 5). Table 5. Median of adherence (cells / mm2) of Candida albicans, Candida tropicalis, Candida dubliniensis and Candida glabrata on water bath and microwave polymerized acrylic resins in the presence or absence of saliva (n = 16). Acrylic resin Water bath Strain Candida albicans Candida tropicalis Presence of saliva 28.19 Aa 44.22 ABa Absence of saliva 56.75 Ab 113.12 Bb 37.87 Aa 89.16 Ba 72.44 ABb 100.00 Bb 62.94 Aa 79.34 ABa

Candida dubliniensis 70.37 Bb Candida glabrata Microwave Candida albicans Candida tropicalis 76.31 Ca 44.75 Aa 73.44 Aa

Candida dubliniensis 51.09 Aa Candida glabrata 62.00 Aa

Means followed by capital letters differ vertically at 5% in each polymerization method. Means followed by lower case letters differ horizontally at 5%.

16

DISCUSSION Candida spp. are generally ubiquitous commensal microorganisms that are usually resident as part of an individuals normal mucosa microflora and can be detected in up to 71% of the healthy population. However, if the balance of the normal flora is disrupted or the immune defenses are compromised, Candida yeasts can invade mucosal surfaces and cause diseases (48), one of these being candida-associated denture stomatitis (1). Candida albicans is the most prevalent yeast isolated from the human body, while other Candida spp. are usually regarded as opportunists (3, 49). To ensure clinical relevance, the adherence assay in this study was conducted on materials routinely used in prosthodontic procedures (15, 34): water bath and microwave acrylic resins, which have similar chemical compositions, but vary on polymerization mechanisms (17, 18). Therefore, water bath and microwave polymerized acrylic resins were evaluated for surface roughness, contact angle and surface free energy before the adherence assay, to investigate if possible differences caused by the polymerization method would interfere in the yeast counts. Surface roughness did not differ statistically between the resins (p > 0.05), while higher contact angle and lower surface free energy values were found in the microwave polymerized acrylic resin (p < 0.001) (Table 4). According to the results of the present study, polymerization method, surface roughness and surface free energy did not interfere with general Candida spp. adherence (Table 3), although interspecies adherence variations occurred between water bath and microwave polymerized resins (Table 5). The presence of saliva also interfered on adherence levels (Tables 3 and 5). The similar resin roughness results may explain why the polymerization method did not interfere with general Candida spp. adherence (Table 3), since roughness is known to be a crucial factor in the entrapment of microorganisms and their protection from shear forces (11, 13, 16, 34). In this study, roughness results were below 0.2 m, in which no further reduction in

17

bacterial accumulation could be expected (13). Therefore, these similar roughness results would provide similar retention conditions for both resins. As for surface free energy, although statistical differences were found, these differences were too small to influence the interaction between polymerization type and adherence rates (Table 3). However, it might be possible that the higher adherence of Candida tropicalis, Candida glabrata and Candida dubliniensis when compared to Candida albicans could be explained by the surface free energy values, since hydrophobic microorganisms seems to adhere more to acrylic surfaces and although there are no studies about hydrophobicity of Candida tropicalis and Candida dubliniensis, Luo & Samaranayake (40) affirmed that Candida glabrata is more hydrophobic than Candida albicans. The statistically significant differences found for surface free energy are in accordance with Sipahi et al. (9), who affirmed that water bath acrylic resin is more wettable than the microwave polymerized acrylic resin, due to acid-base interactions. The presence of saliva generally decreased the number of yeasts per mm2 (Table 3) but did not interfere with Candida glabrata adherence values, while Candida dubliniensis adhered more to saliva-coated water bath resin (Table 5). This general decrease in yeast adherence in the presence of saliva is in accordance with in vitro studies conducted by Samaranayake et al. (37), Radford et al. (15), Millsap et al. (35), Bosch et al. (38) and Elguezabal et al. (27). These authors suggest that whole saliva decreases the adherence of Candida albicans to acrylic resin, and also with Waters et al. (39), as regards resilient materials and with Ramage et al. (32), who verified greater adherence of Candida dubliniensis to saliva-coated polystyrene and acrylic, when compared with a control. However, the results of the present study do not agree with Jin et al. (33), who verified that the presence or absence of saliva did not interfere with Candida albicans adherence as much as the presence of glucose in the growth media, and with Vasilas et al. (34), Nikawa et al. (26), Nikawa et al. (19) and

18

Nikawa et al. (29), who affirmed that the salivary pellicle promotes Candida albicans colonization on tissue conditioning materials or acrylic resins. These conflicting results are due to the different protocols used in each study, including different incubation periods, use of filtered or whole saliva, unstimulated or stimulated saliva, different temperatures and the presence or absence of nutrients that interfere either in cell viability and adherence capacity (15, 23, 33, 36, 37, 39, 40, 50). Although one healthy saliva donor was used in an attempt to standardize saliva quality, this may have influenced the results, since individual variations interfere in the composition of saliva and therefore in its capacity to adhere to inert substances and yeasts (6, 19, 27). In addition, the nature of the substrate may influence the composition of the pellicle and the formation of pellicle, that in turn, may mask the surface properties of the dental materials (6). It is cited in the literature that saliva decreases surface roughness (15) and surface free energy (8, 9, 51) of acrylic resins, which may explain the general decrease of Candida spp. in samples coated with saliva. Saliva proteins can facilitate or difficult Candida spp. adherence; mucins, statherin and proline-rich-proteins interact with Candida cells, increasing their adsorption to oral surfaces (23, 24, 25, 26, 27, 28), while lysozyme, histatins, lactoferrin, calprotectin and sIgA decrease Candida spp. adherence to oral surfaces, either blocking their access to important nutrients or by attaching to their adhesins (20, 21, 22, 23). Clinical studies conducted by Ueta et al. (22), Tanida et al. (21), Bosch et al. (38) and Koseki et al. (30), demonstrated that patients with low salivary flow rates and altered saliva compositions due to stress or systemic problems presented higher Candida spp. counts, when compared with saliva from patients with normal salivary flow, indicating the regulatory role of saliva on inhibiting Candida spp. adherence. The ability of Candida spp. to attach to oral surfaces is a critical prerequisite for their survival in the hostile oral environment, contributing to their virulence, as well as hyphal formation, phenotypic switching and extracellular hydrolytic enzyme production (48, 52). Although this attribute has been

19

extensively studied in Candida albicans, there have been few studies comparing different species. In this study, Candida tropicalis adhered more than the other yeasts, followed by Candida glabrata, Candida dubliniensis and Candida albicans (Table 5). These results are in accordance with Minagi et al. (7), who verified greater adherence values of Candida tropicalis when compared with Candida albicans, in samples covered with saliva or not and with Luo & Samaranayake (40), who found interspecies variation between Candida glabrata and Candida albicans. These different adherence results may be explained by the complexity and phenotypic heterogeneity of the Candida spp. population within the oral cavity (32), expressed in different hidrophobicity (34, 40, 52), secretion of extracelular proteinases (48, 52), hyphal formation (48) and thigmotropism (53), that directly influence Candida spp. adherence rates. Results found in this study showed that saliva interferes with Candida spp. adherence, but it varied according to the species. Therefore, considering the limitations of this study and the controversial results in literature, further studies are necessary to elucidate why saliva decreases the adherence of some Candida spp. but not of others. CONCLUSION Results found in this study suggest that: 1) Saliva interferes on adherence values, since it was responsible for a general decrease in adherence of Candida spp. However, isolated results indicate that Candida albicans and Candida tropicalis counts decreased in the presence of saliva, while Candida dubliniensis increased in the presence of saliva for heat polymerized resin and Candida glabrata were not influenced by saliva. 2) The polymerization method did not influence on Candida spp. adherence values.

20

ACKNOWLEDGEMENTS We thank CNPq, which partially supported this study, Fundao Oswaldo Cruz for donating Candida spp., the Laboratory of Biochemistry and Laboratory of Pharmacology of Piracicaba Dental School.

21

REFERENCES 1 Budtz-Jorgensen E, Stenderup A, Grabowski M. An epidemiologic study of yeasts in elderly denture wearers. Community Dent Oral Epidemiol. 1975;3: 115-9. 2 Arendorf TM & Walker DM. Denture stomatitis: a review. J Oral Rehabil. 1987;14: 217-27. 3 Dar-Odeh NS & Shehabi AA. Oral Candidosis in patients with removable dentures. Mycoses 2003;46: 187-91. 4 Ramage G, Walle KV, Wickes BL, Lopez-Ribot J. Biofilm formation by Candida dubliniensis. J Clin Microbiol. 2001;39(9): 3234-40. 5 Edgerton M & Levine MJ, Characterization of acquired pellicle from healthy and stomatitis patients. J Prosthet Dent. 1992;68: 683-91. 6 Gcke R, Gerath F, von Shwanewede H. Quantitative determination of salivary components in the pellicle on PMMA denture base material. Clin Oral Invest. 2002;6: 227-35. 7 Minagi S, Miyake S, Inagaki K, Tsuru H, Suginaka H. Hydrophobic interaction in Candida albicans and Candida tropicalis adherence to various denture base resin materials. Infec Immun. 1985;47(1): 11-14. 8 van Dijk J, Herkstroter F, Busscher H, Weerkamp A, Jansen H, Arends J. Surface free energy and bacterial adherence. An in vivo study in beagle dogs. J Clin Periodontol. 1987;14(5): 300-4. 9 Sipahi C, Anil N, Bayramli E. The effect of acquired salivary pellicle on the

22

surface free energy and wettability of different denture base materials. J Dent. 2001;29: 197-204. 10 Budtz-Jorgensen E. Etiology, pathogenesis, therapy, and prophylaxis of oral yeast infections. Acta Odontol Scand. 1990;48: 61-9. 11 Quirynen M, Marechal M, Bussher HJ, Weerkamp AH, Darius PL, van Steenbergue D. The influence of surface free energy and surface roughness on early plaque formation. An in vitro study in man. J Clin Periodontol. 1990;17: 138-44. 12 Quirynen M, Bollen CM. The influence of surface roughness and surface free energy on supra- and subgingival plaque formation in man. A review of the literature. J Clin Periodontol. 1995;22(1): 1-14. 13 Bollen CML, Lambrachts P, Quirynen M. Comparison of surface roughness of oral hard materials to the threshold surface roughness for bacterial plaque retention: a review of the literature. Dent Mater. 1997;13: 258-69. 14 Radford DR, Challacombe SJ, Walter JD. Denture plaque and adherence of Candida albicans to denture-base materials in vivo and in vitro. Crit Rev Oral Biol Med. 1999;10(1): 99-116. 15 Radford DR, Sweet SP, Challacombe SJ, Walter JD. Adherence of Candida albicans to denture-base materials with different surface finishes. J Dent. 1998;26: 577-83. 16 Verran J & Maryan CJ. Retention of Candida albicans on acrylic resin and silicone of different surface topography. J Prosthet Dent. 1997;77: 535-9. 17 Azzarri MJ, Cortizo MS, Alessandrini JL. Effect of the curing conditions on

23

the properties of an acrylic denture base resin microwave-polymerized. J Dent. 2003;31:463468. 18 Lai CP, Tsai MH, Chen M, Chang HS, Tay HH. Morphology and properties of denture acrylic resins cured by microwave energy and conventional water bath. Dent Mater. 2004;20: 13341. 19 Nikawa H, Jin C, Hamada T, Murata H. Interactions between thermal cycled resilient denture lining materials, salivary pellicles and Candida albicans in vitro. Part I. Effects on fungal growth. J Oral Rehabil. 2000; 27: 41-51. 20 Cannon RD & Chaffin WL. Colonization is a crucial factor in oral candidiasis. J Dent Edu. 2001;65(8): 785-7. 21 Tanida T, Ueta E, Tobiume A, Hamada T, Rao F, Osaki T. Influence of aging on candidal growth and adherence regulatory agents in saliva. J Oral Pathol Med. 2001;30: 328-35. 22 Ueta E, Tanida T, Doi S, Osaki T. Regulation of Candida albicans growth and adherence by saliva. J Lab Clin Med. 2000;136(1): 66-73. 23 Dodds MWJ, Johnson DA, Yeh CK. Health benefits of saliva: a review. J Dent. 2004;33: 223-33. 24 Edgerton M, Scannapieco FA, Reddy MS, Levine MJ. Human

submandibular-sublingual saliva promotes adherence of Candida albicans to Polymethylmethacrylate. Infec Immun. 1993;61(6): 2644-52. 25 Nikawa H & Hamada T. Binding of salivary or serum proteins to Candida albicans in vitro. Arch Oral Biol. 1990;35: 571-3.

24

26

Nikawa H, Hayashi S, Nikawa Y, Hamada T. Interactions between denture lining material, protein pellicles and Candida albicans. Arch Oral Biol. 1993;38: 631-4.

27

Eguelzabal N, Maza JL, Ponton J. Inhibition of adherence of Candida albicans and Candida dubliniensis to a resin composite restorative dental material by salivary secretory IgA and monoclonal antibodies. Oral Dis. 2004;10: 81-6.

28

Johansson I, Bratt P, Hay DI, Shluckebier S, Stromberg N. Adherence of Candida albicans, but not Candida krusei, to salivary sthaterin and mimicking host molecules. Oral Microbiol Immunol. 2000;15: 112-8.

29

Nikawa H, Jin C, Hamada T, Makihira S, Kumagai H, Murata H. Interactions between thermal cycled resilient denture lining materials, salivary and serum pellicles and Candida albicans in vitro: Part II. Effects on fungal colonization. J Oral Rehabil. 2000;27: 124-30.

30

Koseki M, Maki Y, Matsukubo T, Ohashi Y, Tsubota K. Salivary flow and its relationship to oral signs and symptoms in patients with dry eyes. Oral Dis. 2004;10: 75-80.

31

San Milln R, Elguezabal N, Regulez P, Moragues MD, Quindos G, Ponton J. Effect of saliva secretory IgA on the adherence of Candida albicans to polystyrene. Microbiology 2000;146: 2105-12.

32

Ramage G, Tomsett K, Wickes BL, Lopey-Ribot JL, Redding SW. Denture stomatitis: a role for Candida biofilms. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2004;98: 53-9.

25

33

Jin Y, Samaranayake LP, Samaranayake Y, Yip HK. Biofilm formation of Candida albicans is variably affected by saliva and dietary sugars. Arch Oral Biol. 2004;49: 789-98.

34

Vasilas A, Molina l, Hoffman M, Haidaris CG. The influence of morphological variation on Candida albicans adherence to denture acrylic in vitro. Arch Oral Biol. 1992;37(8): 613-22.

35

Millsap KW, Bos R, van der Mei HC, Busscher HJ. Adherence and surfaceaggregation of Candida albicans from saliva on acrylic surfaces with adhering bacteria as studied in a parallel plate flow chamber. Antonie Van Leeuwenhoek 1999;75(4): 351-9.

36

Samaranayake LP & Mac Farlane TW. An in vitro study of the adherence of Candida albicans to acrylic surfaces. Arch Oral Biol. 1980; 25: 6039.

37

Samaranayake LP, Mc Courtie J, Mac Farlane TW. Factors affecting the in vitro adherence of Candida albicans to acrylic surfaces. Arch Oral Biol. 1980;25(8-9): 611-5.

38

Bosch JA, Turkenburg M, Nazmi K, Veerman ECI, Geus EJC, Amerongen AVN. Stress as a determinant of saliva-mediated adherence and coadherence of oral and non oral microorganisms. Psychosom Med. 2003;65: 604-12.

39

Waters MGJ, Willians DW, Jagger RG, Lewis MAO. Adherence of Candida albicans to experimental denture soft lining materials. J Prosthet Dent. 1997;77(3): 306-12.

40

Luo G & Samaranayake LP. Candida glabrata, an emerging fungal

26

pathogen, exhibits superior relative cell surface hydrophobicity and adherence to denture acrylic surfaces compared with Candida albicans. APMIS 2002;110(9): 601-10. 41 Braun KO, Mello JA, Rached RN, Del Bel Cury AA. Surface texture and some properties of acrylic resins submitted to chemical polishing. J Oral Rehabil. 2003;30(1): 91-8. 42 Oliveira VM, Leon BL, Del Bel Cury AA, Consani, S. Influence of number and position of flasks in the monomer release, Knoop hardness and porosity of a microwave-cured acrylic resin. J Oral Rehabil. 2003;30(11): 1104-8. 43 Neumann AW, Good RJ, Hope CJ, Sejpal M. An equation-of-state approach to determine surface tensions of low-energy solids from contact angles. J Coll Interf Sci. 1974;49(2): 291-305. 44 Gindl M, Sinn g, Gindl W, Reiterer A, Tschegg S. A comparison of different methods to calculate the surface free energy of wood using contact angle measurements. Coll Surf. 2001;181: 279-87. 45 NCCLS, Reference method for broth dilution antifungal susceptibility testing of yeasts; Approved Standard- Second Edition. NCCLS Document M27-A2 (ISBN 1-56238-469-4). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2002. 46 Ellepola ANB, Samaranayake LP. Adherence of oral Candida albicans isolates to denture acrylic following limited exposure to antifungal agents. Arch Oral Biol. 1998;43: 999-1007. 47 Pontes ACF, Corrente JE. The use of nonparametric contrasts in one-way layouts and random block designs. J Nonpar Stat. 2005;17(3): 335-46.

27

48

Naglik J, Albrecht A, Bader O, Hube B. Candida albicans proteinases and host/pathogen interactions. Cell Microbiol. 2004;6(10): 915-26.

49

Cannon RD, Holmes AR, Mason AB, Monk BC. Oral Candida: Clearance, Colonization, or Candidiasis? Journal of Dental Research 1995;74(5): 115261.

50

Chandra J, Mukherjee PK, Leidich SD, Faddaoul FF, Hoyer LL, Douglas LJ et al. Antifungal resistance of Candidal Biofilms formed on denture acrylic in vitro. J Dent Res. 2001;80(3): 903-8.

51

de Jong, de Boer P, Busscher HJ, van Pelt AWJ, Arends J. Surface free energy changes of human enamel during pellicle formation. Caries Res. 1984;18: 408-15.

52

Segal E. Candida, still number one what do we know and where are we going from here? Mycoses 2005;48(Suppl.1): 3-11.

53

Nikawa H, Nishimura H, Hamada T, Makihira S, Saramanayake LP. Relationship between thigmotropism and Candida biofilm formation in vitro. Mycopathologia 1999;144: 125-9.

28

3 CONCLUSO GERAL O papel da saliva como intermediria no processo de aderncia de Candida spp. a superfcie acrlica no foi totalmente esclarecido, uma vez que resultados conflitantes so encontrados na literatura; enquanto alguns estudos apontam para um aumento da aderncia na presena de saliva, outros comprovam o papel inibidor da mesma. Ainda, a literatura atribui que maiores valores de rugosidade e menores valores de energia livre de superfcie influenciem positivamente no nmero de clulas aderidas resina acrlica. Neste estudo, o objetivo foi verificar a influncia da saliva na aderncia de quatro espcies de Candida em resina polimerizada por banho de gua ou por microondas, sendo a mesma responsvel pela diminuio do nmero total de Candida spp. aderida. No entanto, resultados isolados indicaram que a concentrao de Candida albicans e Candida tropicalis diminuram na presena de saliva, enquanto a contagem de Candida dubliniensis aumentou na presena de saliva para a resina polimerizada por banho de gua e a contagem de Candida glabrata no foi influenciada pela presena da mesma. Outro objetivo deste estudo foi verificar se o mtodo de polimerizao por banho de gua ou microondas - interfere na aderncia de Candida spp. Os resultados demonstraram no haver correlao entre as duas variveis. Ainda, no houve correlao entre os valores de rugosidade e energia livre de superfcie e os valores de aderncia. Estudos posteriores so necessrios para elucidar por que a saliva diminui a aderncia de algumas espcies de Candida, mas no de outras.

29

REFERNCIAS* Arendorf TM & Walker DM. Denture stomatitis: a review. J Oral Rehabil. 1987; 14: 217-27. Azzarri MJ, Cortizo MS, Alessandrini JL. Effect of the curing conditions on the properties of an acrylic denture base resin microwave-polymerized. J Dent 2003; 31: 463468 Bollen CML, Lambrachts P, Quirynen M. Comparison of surface roughness of oral hard materials to the threshold surface roughness for bacterial plaque retention: a review of the literature. Dent Mater. 1997; 13: 258-69. Bosch JA, Turkenburg M, Nazmi K, Veerman ECI, Geus EJC, Amerongen AVN. Stress as a determinant of saliva-mediated adherence and coadherence of oral and non oral microorganisms. Psychosom Med. 2003; 65: 604-12. Budtz-Jorgensen E, Stenderup A, Grabowski M. An epidemiologic study of yeasts in elderly denture wearers. Community Dent Oral Epidemiol. 1975; 3: 1159. Budtz-Jorgensen E. Etiology, pathogenesis, therapy, and prophylaxis of oral yeast infections. Acta Odontol Scand. 1990; 48: 61-9. Cannon RD & Chaffin WL. Colonization is a crucial factor in oral candidiasis. J Dent Edu. 2001; 65(8): 785-7. Dar-Odeh NS & Shehabi AA. Oral Candidosis in patients with removable dentures. Mycoses 2003; 46: 187-91.

De acordo com a norma utilizada na FOP/Unicamp, baseada no modelo Vancouver. Abreviatura dos peridicos em conformidade com o Medline.

30

Dodds MWJ, Johnson DA, Yeh CK. Health benefits of saliva: a review. J Dent. 2004; 33: 223-33. Edgerton M & Levine MJ, Characterization of acquired pellicle from healthy and stomatitis patients. J Prosthet Dent. 1992; 68: 683-91. Edgerton M, Scannapieco FA, Reddy MS, Levine MJ. Human submandibularsublingual saliva promotes adherence of Candida albicans to Polymethylmethacrylate. Infect Immun. 1993; 61(6): 2644-52. Eguelzabal N, Maza JL, Ponton J. Inhibition of adherence of Candida albicans and Candida dubliniensis to a resin composite restorative dental material by salivary secretory IgA and monoclonal antibodies. Oral Dis. 2004; 10: 81-6. Gcke R, Gerath F, von Shwanewede H. Quantitative determination of salivary components in the pellicle on PMMA denture base material. Clin Oral Invest. 2002; 6: 227-35. Jin Y, Samaranayake LP, Samaranayake Y, Yip HK. Biofilm formation of Candida albicans is variably affected by saliva and dietary sugars. Arch Oral Biol. 2004; 49: 789-98. Johansson I, Bratt P, Hay DI, Shluckebier S, Stromberg N. Adherence of Candida albicans, but not Candida krusei, to salivary sthaterin and mimicking host molecules. Oral Microbiol Immunol. 2000; 15: 112-8. Koseki M, Maki Y, Matsukubo T, Ohashi Y, Tsubota K. Salivary flow and its relationship to oral signs and symptoms in patients with dry eyes. Oral Dis. 2004; 10: 75-80.

31

Lai CP, Tsai MH, Chen M, Chang HS, Tay HH. Morphology and properties of denture acrylic resins cured by microwave energy and conventional water bath. Dental Maters 2004; 20: 133141 Luo G & Samaranayake LP. Candida glabrata, an emerging fungal pathogen, exhibits superior relative cell surface hydrophobicity and adherence to denture acrylic surfaces compared with Candida albicans. APMIS. 2002; 110(9): 601-10. Millsap KW, Bos R, van der Mei HC, Busscher HJ. Adherence and surfaceaggregation of Candida albicans from saliva on acrylic surfaces with adhering bacteria as studied in a parallel plate flow chamber. Antonie Van Leeuwenhoek 1999; 75(4): 351-59. Minagi S, Miyake S, Inagaki K, Tsuru H, Suginaka H. Hydrophobic interaction in Candida albicans and Candida tropicalis adherence to various denture base resin materials. Infec Immun. 1985; 47(1): 11-4. Naglik J, Albrecht A, Bader O, Hube B. Candida albicans proteinases and host/pathogen interactions. Cell Microbiol. 2004; 6(10): 915-26. Nikawa H & Hamada T. Binding of salivary or serum proteins to Candida albicans in vitro. Arch Oral Biol. 1990; 35: 571-3. Nikawa H, Hayashi S, Nikawa Y, Hamada T. Interactions between denture lining material, protein pellicles and Candida albicans. Arch Oral Biol. 1993; 38: 631-4. Nikawa H, Jin C, Hamada T, Murata H. Interactions between thermal cycled resilient denture lining materials, salivary pellicles and Candida albicans in vitro. Part I. Effects on fungal growth. J Oral Rehabil. 2000a; 27: 41-51.

32

Nikawa H, Jin C, Hamada T, Makihira S, Kumagai H, Murata H. Interactions between thermal cycled resilient denture lining materials, salivary and serum pellicles and Candida albicans in vitro: Part II. Effects on fungal colonization. J Oral Rehabil. 2000b; 27: 124-30. Quirynen M, Marechal M, Bussher HJ, Weerkamp AH, Darius PL, van Steenbergue D. The influence of surface free energy and surface roughness on early plaque formation. An in vitro study in man. J Clin Periodontol. 1990; 17: 138-44. Quirynen M, Bollen CM. The influence of surface roughness and surface free energy on supra- and subgingival plaque formation in man. A review of the literature. J Clin Periodontol. 1995; 22(1): 1-14. Ramage G, Walle KV, Wickes BL, Lopez-Ribot J. Biofilm formation by Candida dubliniensis. J Clin Microbiol. 2001; 39(9): 3234-40. Ramage G, Tomsett K, Wickes BL, Lopey-Ribot JL, Redding SW. Denture stomatitis: a role for Candida biofilms. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2004; 98: 53-9. Radford DR, Sweet SP, Challacombe SJ, Walter JD. Adherence of Candida albicans to denture-base materials with different surface finishes. J Dent. 1998; 26: 577-83. Radford DR, Challacombe SJ, Walter JD. Denture plaque and adherence of Candida albicans to denture-base materials in vivo and in vitro. Crit Rev Oral Biol Med. 1999; 10(1): 99-116.

33

Samaranayake LP, Mc Courtie J, Mac Farlane TW. Factors affecting the in vitro adherence of Candida albicans to acrylic surfaces. Arch Oral Biol. 1980; 25(8-9): 611-5. San Milln R, Elguezabal N, Regulez P, Moragues MD, Quindos G, Ponton J. Effect of saliva secretory IgA on the adherence of Candida albicans to polystyrene. Microbiology 2000; 146: 2105-12. Sipahi C, Anil N, Bayramli E. The effect of acquired salivary pellicle on the surface free energy and wettability of different denture base materials. J Dent. 2001; 29: 197-204. Tanida T, Ueta E, Tobiume A, Hamada T, Rao F, Osaki T. Influence of aging on candidal growth and adherence regulatory agents in saliva. J Oral Pathol Med. 2001; 30: 328-35. van Dijk J, Herkstroter F, Busscher H, Weerkamp A, Jansen H, Arends J. Surface free energy and bacterial adherence. An in vivo study in beagle dogs. J Clin Periodontol. 1987; 14(5): 300-4. Vasilas A, Molina l, Hoffman M, Haidaris CG. The influence of morphological variation on Candida albicans adherence to denture acrylic in vitro. Arch Oral Biol. 1992; 37(8): 613-22. Verran J, Maryan CJ. Retention of Candida albicans on acrylic resin and silicone of different surface topography. J Prosthet Dent. 1997; 77: 535-9. Waters MGJ, Willians DW, Jagger RG, Lewis MAO. Adherence of Candida albicans to experimental denture soft lining materials. J Prosthet Dent. 1997; 77(3): 306-12.

34

Ueta E, Tanida T, Doi S, Osaki T. Regulation of Candida albicans growth and adherence by saliva. J Lab Clin Med. 2000; 136(1): 66-73. Webb BC, Thomas CJ, Willcox MDP, Harty DWS, Knox KW. Candida-associated denture stomatitis. Aetiology and management: a review. Part 2. Oral diseases caused by candida species. Aust Dent J. 1998; 43(3): 160-6.

35

ANEXO 1 Figuras

Figura 1

Figura 2

Figuras 1 e 2. Leitura de rugosidade superficial com perfilmetro (Surfcorder SE 1700 (Kosaka Industry, Kosaka, Japo).

Figura 3. Fotografia de 15l de gua destilada deionizada para mensurao do ngulo de contato.

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Figura 4. Mensurao do ngulo de contato por meio do programa AUTOCAD 2005, para obteno da energia livre de superfcie.

Figura 5. Esquema de distribuio dos campos para a leitura em microscpio ptico de clulas aderidas nas amostras.

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Figura 6

Figura 7

Figuras 6 e 7. Fotografias de clulas de Candida albicans aderidas resina acrlica em 400 x de aumento.

Figura 8

Figura 9

Figuras 8 e 9. Fotografias de clulas de Candida albicans aderidas resina acrlica em 200 x de aumento.

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ANEXO 2 Submisso do artigo revista Journal of Oral Rehabilitation

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ANEXO 3 Certificado de aprovao do Comit de tica em Pesquisa

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ANEXO 4 Resultados Tabela 6. Grupos divididos de acordo com o tipo de resina acrlica, espcie de Candida e exposio ou no a saliva
Grupo MAS MA MTS MT MDS MD MGS MG HAS HA HTS HT HDS HD HGS HG Resina Microondas Microondas Microondas Microondas Microondas Microondas Microondas Microondas Termopolimerizvel Termopolimerizvel Termopolimerizvel Termopolimerizvel Termopolimerizvel Termopolimerizvel Termopolimerizvel Termopolimerizvel Espcie de Candida C. albicans C. albicans C. tropicalis C. tropicalis C. dubliniensis C. dubliniensis C. glabrata C. glabrata C. albicans C. albicans C. tropicalis C. tropicalis C. dubliniensis C. dubliniensis C. glabrata C. glabrata x x x x x x x x x x x x x x Exposta saliva x x No exposta saliva

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Tabela 7. Mdias e desvio padro da rugosidade superficial (m) das amostras de resina acrlica (n = 16).
GRUPO MAS 0,099 0,096 0,094 0,093 0,102 0,071 0,085 0,104 0,107 0,144 0,115 0,143 0,078 0,090 0,098 0,121 MDIA Desvpad 0,102 0,02 MA 0,117 0,090 0,118 0,104 0,110 0,083 0,112 0,102 0,081 0,116 0,089 0,142 0,174 0,107 0,134 0,126 0,113 0,02 MTS 0,112 0,119 0,102 0,121 0,121 0,108 0,092 0,088 0,092 0,106 0,092 0,096 0,084 0,111 0,080 0,103 0,102 0,01 MT 0,108 0,099 0,080 0,087 0,118 0,107 0,116 0,123 0,112 0,144 0,076 0,120 0,076 0,086 0,104 0,093 0,103 0,02 MDS 0,141 0,103 0,108 0,083 0,114 0,112 0,104 0,104 0,099 0,115 0,085 0,117 0,104 0,113 0,138 0,163 0,113 0,02 MD 0,119 0,094 0,102 0,088 0,107 0,087 0,086 0,095 0,172 0,105 0,098 0,080 0,086 0,100 0,110 0,147 0,105 0,02 MGS 0,133 0,128 0,105 0,121 0,201 0,160 0,128 0,136 0,099 0,116 0,111 0,176 0,107 0,154 0,151 0,109 0,133 0,03 MG 0,133 0,169 0,152 0,121 0,126 0,141 0,129 0,129 0,108 0,126 0,156 0,110 0,130 0,118 0,088 0,115 0,128 0,02 HAS 0,127 0,104 0,155 0,106 0,131 0,121 0,103 0,104 0,082 0,107 0,109 0,148 0,129 0,123 0,150 0,107 0,119 0,02 HA 0,131 0,137 0,103 0,129 0,136 0,113 0,150 0,149 0,109 0,173 0,110 0,134 0,088 0,117 0,123 0,104 0,125 0,02 HTS 0,124 0,096 0,103 0,121 0,117 0,108 0,124 0,098 0,145 0,127 0,143 0,140 0,195 0,108 0,100 0,114 0,123 0,03 HT 0,143 0,149 0,148 0,121 0,099 0,091 0,084 0,125 0,121 0,116 0,126 0,181 0,095 0,138 0,092 0,113 0,121 0,03 HDS 0,119 0,091 0,102 0,122 0,094 0,126 0,118 0,108 0,106 0,121 0,117 0,086 0,118 0,129 0,118 0,088 0,110 0,01 HD 0,105 0,308 0,122 0,117 0,125 0,129 0,156 0,130 0,070 0,186 0,188 0,076 0,093 0,171 0,088 0,101 0,135 0,06 HGS 0,087 0,092 0,095 0,100 0,113 0,092 0,094 0,089 0,135 0,103 0,096 0,094 0,106 0,121 0,085 0,079 0,099 0,01 HG 0,130 0,101 0,098 0,117 0,085 0,144 0,125 0,091 0,096 0,156 0,074 0,128 0,135 0,114 0,125 0,113 0,114 0,02

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Tabela 8. Mdias e desvio padro do ngulo de contato de gua destilada sobre a superfcie de resina acrlica, em graus (n = 16).
GRUPO MAS 66,670 74,334 71,214 70,962 72,931 72,103 70,230 71,678 76,101 70,670 68,195 66,452 68,412 67,541 69,185 71,947 MDIA Desvpad 70,54 2,78 MA 70,236 64,242 62,524 67,498 69,197 70,851 67,122 67,854 75,903 71,095 71,914 69,045 61,987 72,211 72,309 73,517 69,22 3,85 MTS 65,296 71,999 69,274 66,855 64,674 61,727 70,780 68,416 76,657 73,402 69,634 70,705 71,914 72,509 72,070 73,816 69,98 3,84 MT 66,026 69,354 69,542 70,159 73,378 65,678 70,943 69,600 74,100 75,834 69,349 78,949 74,262 67,982 57,272 72,163 70,29 5,09 MDS 66,534 72,103 69,555 63,510 72,320 72,868 73,008 68,090 66,463 74,268 72,872 71,394 70,270 67,140 71,214 69,594 70,08 3,11 MD 63,904 69,340 67,372 71,016 65,916 66,182 71,482 69,164 71,640 73,035 66,347 79,834 69,631 71,028 75,078 65,159 69,76 4,04 MGS 73,813 73,654 73,384 67,641 67,707 65,164 64,056 63,855 67,391 67,583 56,739 64,585 66,469 63,301 72,820 60,052 66,76 4,73 MG 75,933 75,220 70,803 70,656 59,309 66,667 70,470 63,695 66,152 64,375 54,984 63,739 69,753 73,116 66,131 64,989 67,25 5,75 HAS 71,756 58,204 70,630 73,349 69,629 71,072 71,191 67,371 70,037 71,600 69,548 77,180 67,258 71,871 76,933 66,320 70,25 4,42 HA 75,268 74,336 65,613 73,504 67,622 72,010 70,143 69,797 67,164 77,624 74,741 72,294 72,547 69,447 79,826 61,279 71,45 3,94 HTS 74,689 70,791 72,545 74,617 77,363 73,741 67,894 67,299 67,545 70,502 69,894 71,868 69,793 81,218 48,690 64,534 70,19 7,15 HT 79,210 66,269 68,780 70,573 70,386 70,307 64,987 73,527 71,843 71,740 80,270 69,061 76,898 65,878 69,025 73,540 71,39 4,58 HDS 71,651 68,264 65,543 71,191 69,492 66,021 66,918 75,956 69,892 69,767 76,094 74,940 76,120 72,851 47,841 68,662 69,45 6,98 HD 79,397 68,954 66,019 68,868 70,545 74,332 70,905 71,298 75,554 66,096 74,218 70,564 57,880 67,683 61,664 68,740 69,54 5,42 HGS 79,028 71,863 71,168 75,945 76,121 72,917 74,071 72,178 69,503 66,062 70,796 75,908 70,150 64,593 69,862 71,902 72,00 3,86 HG 79,282 70,851 77,381 71,473 72,905 65,876 69,521 69,480 73,648 67,182 69,391 70,189 71,707 72,090 71,351 66,388 71,17 3,45

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Tabela 9. Mdias e desvio padro de clulas aderidas por 0,26 mm2 (n = 16).

GRUPO

MAS 2,13 5,80 20,00 8,73 3,93 5,20 5,13 3,27 7,80 15,27 7,67 6,87 8,53 3,27 27,27 27,27

MA 9,47 9,13 6,47 9,00 5,80 20,13 3,53 13,40 27,33 8,67 47,73 49,07 9,60 44,40 18,20 35,87 19,86 15,88

MTS 21,27 3,60 7,20 5,73 6,60 10,67 6,93 4,00 8,27 2,80 12,00 6,40 13,67 4,87 10,47 8,80 8,33 4,78

MT 126,73 45,80 141,93 119,33 61,60 91,20 21,60 27,53 49,93 23,53 8,80 53,53 5,20 11,80 102,40 11,67 56,41 45,99

MDS 1,73 17,53 1,73 1,00 6,07 47,27 9,73 8,27 2,67 13,33 2,60 8,87 15,60 22,53 26,53 10,93 12,28 12,48

MD 38,20 6,47 16,00 6,67 7,33 17,40 23,80 46,80 13,00 4,87 6,13 20,47 7,00 72,00 2,07 7,07 18,45 19,39

MGS 2,47 16,20 130,53 16,93 23,47 24,73 4,60 7,53 46,47 5,33 3,00 2,93 4,33 25,07 22,47 17,47 22,10 32,36

MG 24,67 39,93 14,07 5,73 27,00 17,33 34,53 42,13 10,87 11,33 21,27 6,40 25,93 10,33 28,20 8,07 20,49 11,74

HAS 12,67 4,33 2,33 5,67 19,07 4,73 1,33 2,47 4,53 8,00 6,13 2,33 2,93 9,80 9,80 3,07 6,20 4,80

HA 12,80 10,13 6,07 7,27 7,33 30,87 21,87 3,20 5,13 5,73 16,53 18,93 21,20 21,47 7,60 17,80 13,37 8,22

HTS 8,20 2,33 0,27 1,53 5,60 2,47 11,00 8,13 8,73 20,67 10,40 16,80 10,07 13,33 16,07 19,60 9,70 6,01

HT 129,33 106,00 136,13 108,27 69,07 112,40 35,33 193,87 29,20 47,67 32,20 35,87 87,47 21,40 25,47 59,73 76,84 51,72

HDS 91,33 19,20 48,53 10,80 6,00 4,13 41,47 10,27 58,53 6,87 11,73 22,20 27,73 9,73 3,93 20,80 24,58 25,06

HD 4,67 17,40 5,53 20,33 8,93 1,40 9,80 5,53 39,53 10,20 5,93 3,07 2,20 1,80 2,73 11,07 9,38 10,05

HGS 9,33 7,40 12,07 10,20 88,00 5,93 13,33 23,93 25,73 16,53 13,53 16,67 24,73 56,67 33,60 31,20 24,30 21,94

HG 15,20 24,73 37,13 54,13 37,53 56,47 20,33 11,53 54,13 10,53 34,00 24,27 53,27 19,60 10,53 15,27 29,92 17,11

MDIA Desvpad

9,88 6,96

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