BIBLIOTECA DIGITAL DE TESES E DISSERTAÇÕES UNESP RESSALVA Alertamos para Capítulo 5 e 7 enviados originalmente em inglês pelo autor no arquivo original. JAMIL AWAD SHIBLI ETIOLOGIA, PROGRESSÃO E TRATAMENTO DAS PERI-IMPLANTITES Tese apresentada à Faculdade de Odontologia de Araraquara, Universidade Estadual Paulista "Júlio de Mesquita Filho", como parte dos requisitos para a obtenção do título de Doutor do Curso de Pós-Graduação em Odontologia, Nível de Doutorado, Área de Periodontia. Orientador: Prof. Dr. Elcio Marcantonio Junior Araraquara 2003 UNIVERSIDADE ESTADUAL PAULISTA - UNESP FACULDADE DE ODONTOLOGIA CÂMPUS DE ARARAQUARA 2 SUMÁRIO 1. PREFÁCIO…………………………………………………………………...….3 2. AGRADECIMENTOS..................................................................................5 3. INTRODUÇÃO E JUSTIFICATIVA.....................……………………….......7 4. PROPOSIÇÃO………………………………………………………………...14 5. ESTUDOS EM ANIMAIS (Artigos I a VI).................................................15 6. ESTUDO EM HUMANOS (Artigo VII) ...................................................214 7. ESTUDOS IN VITRO (Artigos VIII e IX)................................................250 8. DISCUSSÃO GERAL.............................................................................288 9. CONCLUSÕES……………………………….......………………………....296 10. PERSPECTIVAS FUTURAS..................................................................297 11. REFERÊNCIAS BIBILIOGRÁFICAS.....................................................298 3 1. PREFÁCIO Esta tese é constituída pelos seguintes artigos: I . Shibli JA, Martins MC, Lotufo RFM, Marcantonio Jr. E. Microbiologic and radiographic analysis of ligature-induced peri-implantitis with different dental implant surfaces (Parcialmente aceito para publicação na International Journal of Oral and Maxillofacial Implants) II. Shibli JA, Martins MC, Jordan SF, Araujo MB, Haraszthy VI, Zambon JJ, Marcantonio Jr. E. Detection of periodontal pathogens in ligature-induced peri- implantitis. An experimental study in dogs (Submetido ao Journal of Periodontology) III. Shibli JA, Martins MC, Jordan SF, Haraszthy VI, Zambon JJ, Marcantonio Jr. E. Progression of experimental chronic peri-implantitis. Clinical and microbiological evaluation in a canine model (Finalizado para o envio ao Clinical Oral Implants Research) IV. Shibli JA, Martins MC, Theodoro LH, Lotufo RF, Garcia VG, Marcantonio Jr. E. Lethal photosensitization in microbiological treatment of ligature-induced peri- implantitis: a preliminary study in dogs. Journal of Oral Science 2003, (in press) V. Shibli JA, Martins MC, Nociti Jr. FH, Garcia VG, Marcantonio Jr. E. Treatment of ligature-induced peri-implantitis by lethal photosensitization and guided bone regeneration: a preliminary histologic study in dogs. J Periodontol 2003, 74; 338- 345. 4 VI. Shibli JA, Martins MC, Nociti Jr. FH, Marcantonio Jr. E. Guided bone regeneration and lethal photosensitization in treatment of ligature-induced peri- implantitis in different dental implants surfaces. A histomorphometrical study in dogs. (Finalizado para submissão ao Journal of Periodontology) VII. Shibli JA, Jordan SF, Haraszthy VI, Zambon JJ, Marcantonio Jr. E. Host response and microbiological evaluation of peri-implantitis in patients with periodontal diseases (Submetido ao Journal of Periodontology) VIII. Shibli JA, Silverio KG, Martins MC, Marcantonio Jr. E., Rossa Jr. C. Effect of air-powder system in titanium surface on fibroblast adhesion and morphology Implant Dent 2003, 12 (in press) IX. Shibli JA, Marcantonio E, d’Avila S, Guastaldi AC, Marcantonio Jr. E. Analysis of failed dental implant surfaces. (Finalizado para submissão - International Journal of Oral and Maxillofacial Implants) 5 2- AGRADECIMENTOS `A Deus, pelos ensinamentos dos quais espero ser merecedor Aos meus pais Awad e Sabah, e irmãos Sami e Samira pelo apoio incondicional e por manter a chama da perseverança sempre acesa. À querida Susana, amor da minha vida, incentivadora de todas as horas e a melhor companheira que um homem poderia imaginar. Ao meu mestre e tutor, Prof. Dr. Elcio Marcantonio Jr., por não me privar da oportunidade de desfrutar dos seus conhecimentos e compartilhar de sua grande amizade e companherismo. A minha querida tutora, Profa. Dra. Rosemary Adriana C. Marcantonio, responsável pelo meu ingresso na carreira acadêmica. Ao Prof. Dr. Ricardo Samih Georges Abi-Rached, pelo exemplo de dedicação, honestidade e austeridade. Ao grande mestre Prof. Dr. Elcio Marcantonio pelas maravilhosas horas de aprendizado não somente acadêmico, mas também pelo exemplo de ser humano. Aos Profs. Drs. Joseph J. Zambon e Violet I, Haraszthy pela agradável e produtiva estada no departamento de Biologia Oral da SUNY at Buffalo, Buffalo- NY, EUA. À minha amiga e sócia, Marilia Compagnoni Martins, pelo longo convívio, amizade, e principalmente na confiança de um objetivo alcançado. 6 À incentivadora, Profa. Dra. Maria Regina Sposto, pelo exemplo de dedicação a carreira acadêmica e exemplo de austeridade. A todos os meus colegas da Pós Graduação, em especial, Solange Alonso Vergani, Roberto Andrade Acevedo, Micheline Trentin, Rodrigo Rego, Rogério Margonar, Carlos Augusto Nassar e Patrícia Nassar pela amizade e companheirismo. Ao Prof. Dr. Francisco Humberto Nociti Jr. pela presteza competência com que desempenhou o processamento histológico Ao Prof.Dr. Valdir Gouveia Garcia e Profa. Letícia Helena Theodoro pela orientação e utilização do laser de baixa intensidade Aos amigos e mestrandos Fernando Salimon Ribeiro, Vagner Samy Lemo e Maurício Ribeiro Costa pela inestimável ajuda no tratamento dos cães. A todos os docentes e amigos da Disciplina de Periodontia da Faculdade de Odontologia de Araraquara-UNESP As minhas amigas Regina Lúcia e Fernanda pelos sempre valiosos préstimos e palavras de carinho Ao amigo Daniel pela ajuda na parte laboratorial de informática Aos funcionários do Departamento de Diagnóstico e Cirurgia da Faculdade de Odontologia de Araraquara-UNESP As funcionárias do setor de Pós-Graduação da Faculdade de Odontologia de Araraquara-UNESP 7 INTRODUÇÃO E JUSTIFICATIVA A necessidade de um tecido saudável ao redor dos implantes osseointegrados é essencial para obtenção de sucesso a longo prazo. (El Askary et al.15 1999; Shibli et al.72 2003). Vários autores têm estudado o papel do biofilme bacteriano no desenvolvimento da peri-implantite em humanos (Mombelli et al.44 1987; Mombelli et al.45 1988; Mombelli et al.47 1995; Lee et al.39 1999; Listgarten & Lai,41 1999) e também em modelos animais por meio da utilização de ligaduras, juntamente com a cessação dos procedimentos de higienização bucal (Akagawa et al.41993; Lang et al.37 1993; Shou et al.69 1996; Hanisch et al.28 1997; Eke et al.14 1998; Tillmanns et al.77 1998). O papel do biofilme bacteriano na falência dos implantes osseointegrados sob função tem atraído a atenção de muitos pesquisadores. No entanto algumas controvérsias em relação às condições que propiciam um maior risco para a peri-implantite, tais como a influência da macroestrutura e microestrutura, tipos de superfícies, pacientes tratados periodontalmente, pacientes com doença periodontal crônica, microbiota, histofisiologia do tecido peri-implantar e oclusão, ainda não estão totalmente esclarecidos (Quirynen et al.63 2002, van Steenberghe et al.79 1999; Esposito et al.18 1998, Mombelli51 2002, Mombelli & Lang,50 1998, Shibli & Marcantonio73 2002). Segundo Ellen16 (1998), os modelos de periodontite e peri- implantite induzidos por ligadura têm sido usados por diversas razões: confirmar a seqüência de alterações na composição bacteriana (aeróbios Gram-positivos) antes da ocorrência de lesões destrutivas (anaeróbia Gram-negativa); confirmar 8 o efeito exacerbado da Porphyromonas gingivalis quando super-infectante na microbiota periodontal ou peri-implantar; testar a importância da infecção experimental e trauma oclusal (Isidor,32 1997; Hurzeler et al.30 1998) na progressão das lesões periodontais e peri-implantares e prover lesões reprodutíveis que possam ser tratadas de acordo com vários protocolos: debridamento das lesões utilizando jatos abrasivos e/ou curetas de teflon (Hurzeler et al.,29 1997; Wetzel et al.82 1999) associados a antimicrobianos e antibióticos (Ericsson et al.17 1996); cirúrgicos, RTG, lasers de baixa intensidade (Haas et al.25 1997, Haas et al.26 2000) e associações. (Persson et al.59 1996; Grunder et al.23 1993; Jovanovic et al.34 1993) No entanto, os tratamentos desses defeitos peri-implantares, por serem de origem bacteriana, são de difícil prognóstico, já que pesquisas buscando otimizar o processo de osseointegração (Buser et al.8 1991; Buser et al.9 1998), têm desenvolvido micro e ultraestruturas (plasma spray de titânio, superfícies modificadas por meio de ácidos e superfícies jateadas com óxidos), que dificultam o debridamento e detoxificação tanto da superfície do implante quanto da superfície peri-implantar. No entanto, ainda são escassos os estudos que visam esclarecer qual tipo de superfície de implante osseointegrado é mais ou menos favorável à progressão e tratamento da peri-implantite (Esposito et al.18 1998; Esposito et al.19 1998; Shibli et al.71 2003; Shibli et al.72 2003). Algumas importantes diferenças entre os diversos tipos de superfícies que recobrem os implantes osseointegrados parecem influenciar a adsorção e colonização bacteriana (Nakazato et al.52 1989; Gatewood et al.20 1993; 9 Quirynen et al.62 1993; Bollen et al.7 1996; Rimondini et al. 1997; Ichikawa et al.31 1998; Rasperini et al.64 1998; Steinberg et al.76 1998). Sabe-se, também, que com o aumento da rugosidade da superfície do implante há uma maior dificuldade para os procedimentos de higienização/manutenção da saúde dos tecidos peri-implantares assim como no tratamento dos defeitos ósseos peri- implanteres (Persson et al.60 2001; Persson et al.61 2001). A presença de porosidades e irregularidades, inerentes a superfícies de implantes tratadas com ácidos ou jateadas com titânio plasma spray e hidroxiapatita, funcionariam como nichos perfeitos para a proliferação bacteriana, desde que expostas ao meio bucal, tendo assim uma rota direta para a infecção do tecido ósseo peri- implantar (Siegrist et al.74 1991; Gatewood et al.20 1993; Bollen et al.7 1996; Ichikawa et al.31 1998), enquanto uma diminuição da rugosidade da superfície dos implantes abaixo de Ra de 0,2µm poderia retardar a maturação do biofilme supra e subgengival (Bollen et al.71995). De forma semelhante à doença periodontal, a peri-implantite é resultante do desequilíbrio hospedeiro-microrganismo que pode manifestar por meio de uma série de mudanças inflamatórias levando a duas síndromes distintas: mucosite peri-implantar que é uma lesão confinada aos tecidos moles peri- implantares e peri-implantite que envolve, além dos tecidos moles, o tecido ósseo adjacente ao implante osseointegrado (Mombelli & Lang,49 1998; Tonetti & Schmid,80 1994; Lie et al.39 1995). Enquanto, não há um consenso sobre a microbiota presente na doença peri-implantar, na doença periodontal, microrganismos, atuando isolados ou em 10 combinações, como Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Porphyromonas gingivalis, Prevotella intermedia, Campylobacter rectus, Fusobacterium nucleatum, Prevotella nigrescens, Peptostreptococcus micros, Eubacterium nodatum e Treponema spp. são freqüentemente associadas a doença (Zambon,84 1996; Socransky & Haffajee,75 2002); enquanto Porphyromonas gingivalis e Prevotella intermedia estão diretamente associadas à indução e progessão das doenças periodontais. (Renvert et al.65 1996; Zambon84 1996; Socransky & Haffajee,75 2002) Uma clara relação entre acúmulo do biofilme dental e mudanças inflamatórias superficiais (mucosite peri-implantar) tem sido demonstrada em estudos envolvendo modelos animais (Abrahamsson et al.2 1988; Berglundh et al.6 1992, Schou et al.70 2002) e humanos (Bollen et al.7 1996). Alguns autores têm utilizado modelos animais induzindo a peri-implantite por meio de ligaduras ao redor dos implantes (Akagawa et al.41993; Lang et al.37 1993, Schou et al.69 1996; Hanisch et al.28 1997; Eke et al.141998; Tillmanns et al.77 1998; Nociti et al.53 2001, Schou et al.70 2002) demonstrando a importância do biofilme bacteriano no processo de falência desses implantes. Tonetti & Schmid80 (1994) chegaram a afirmar que esses estudos indicam, inequivocamente, que a peri-implantite induzida por ligadura é resultado da ação de uma microbiota patogênica. Alguns trabalhos (Abrahamsson et al.21998; Berglundh et al.6 1992) evidenciaram tal afirmação, no qual o acúmulo de biofilme sem a presença de ligaduras não induziu à peri-implantite, mas a uma mucosite peri-implantar, já que as ligaduras poderiam levar à uma modificação 11 tecidual ou a um trauma mecânico (Esposito et al.19 1998), sem no entanto haver um desequilíbrio “verdadeiro” entre a relação hospedeiro-parasita. Neste contexto, ainda não está bem demonstrado que a presença de periodontopatógenos necessariamente tem como conseqüência a destruição do tecido peri-implantar (Mombelli et al.47 1995, Zambon84 1996), entretanto a detecção de periodontopatógenos poderia evidenciar um aumento no risco para a progressão das doenças peri-implantares. A correlação entre a profundidade da bolsa peri-implantar e a presença de espiroquetas e bastonetes móveis Gram-negativos (Papaioannou et al.57 1995) sugerem que a profundidade da bolsa peri-implantar, que depende também da espessura e qualidade da mucosa peri-implantar (Warrer et al.81 1995, Toljanic et al.78 2001; Schou et al.70 2002), proporciona um ambiente adequado para o crescimento bacteriano. Outra evidência indireta sobre o importante papel dos microrganismos na falência dos implantes pode ser observado nos estudos que analisaram a natureza do tecido peri-implantar (Sanz et al.66 1991) ou a composição do fluido crevicular peri-implantar (Apse et al.5 1986). Jepsen et al.33(1996) sugeriram, ainda, que as enzimas proteóliticas de origem bacteriana seria um indicador importante no diagnóstico e monitoramento dos implantes ao longo da fase de manutenção. Os achados microbiológicos sugerem que a infeção peri-implantar é, possivelmente, a causa da perda tardia dos implantes, ou seja, após estarem sob função mastigatória. Os microrganismos mais associados com a doença 12 peri-implantar são as espiroquetas, bastonetes, organismos Gram-negativos facultativos ou anaeróbios estritos, tais como Porphyromonas gingivalis , Prevotella intermedia e Actinobacillus actinomycetemcomitans . Estas bactérias podem lesar os tecidos peri-implantares de diferentes maneiras: invasão e destruição direta dos tecidos peri-implantares por meio da liberação de enzimas, subprodutos e fatores de reabsorção óssea, evasão das defesas do hospedeiro, indução de uma reação inflamatória mediada pelo sistema imune do hospedeiro ou ainda uma combinação dos fatores citados. Similaridades nos componentes encontrados nos fluidos gengival e peri-implantar demonstraram que existe um mecanismo análogo que controla a resposta imune e inflamatória ao redor de dentes e implantes (Adonogianaki et al.31995). Bactérias como a Capnocytophaga spp. estão associadas ao desenvolvimento de gengivites e podem participar da transição entre gengivite e periodontite, sendo menos importante no estabelecimento da doença periodontal e até nas doenças peri-implantares (Mombelli & Mericske-Stern,46 1990). Porphyromonas gingivalis e Prevotella intermedia parecem estar associadas à progressão e indução da doença periodontal (Renvert et al.65 1996). Treponema denticola, uma das principais representantes do grupo das espiroquetas, o Fusobacterium nucleatum e Campylobacter rectus foram também associados às doenças periodontal e peri-implantar. Eikenella corrodens tem papel pouco esclarecido sobre a sua participação na patogênese da peri-implantite (Papaioannou et al.58 1996). Bacteroides forsythus tem sido relatado em poucos estudos já que, somente técnicas de biologia molecular tais como a reação de 13 polimerase em cadeia (PCR) e a hibridização de sondas de DNA parecem detectar sua presença, pois a cultura falha na grande maioria das vezes (Danser et al.10 1997). Os diferentes métodos utilizados para detecção dos microrganismos presentes na peri-implantite (cultura, PCR e sondas de DNA), poderiam justificar os diferentes resultados encontrados na literatura. A resposta local do hospedeiro frente à infecção bacteriana levanta algumas questões que necessitam ser discernidas e respondidas. Será que a perda de inserção e perda óssea ocorrem com maior velocidade no implante de superfície lisa quando compara ao implante de superfície tratada? A diferente histofisiologia do tecido peri-implantar aumenta as condições de risco frente o acúmulo do biofilme bacteriano? Existe uma correlação entre obtenção de re- osseointegração e superfície de implante? Esta superfície apresenta alguma característica especial em implantes falidos? Está associado ao biofilme bacteriano? 14 PROPOSIÇÃO GERAL O objetivo geral deste trabalho é avaliar o modelo de estudo animal, etiologia e tratamento das peri-implantites. Objetivos Específicos • Analisar clínica e microbiologicamente a peri-implantite induzida por ligadura em diferentes superfícies de implantes osseointegrados (Artigos I, II e III) • Avaliar, microbiologicamente, o efeito da utilização do laser de baixa intensidade associado a agente fotossensibilizador no tratamento das peri-implantites (Artigo IV) • Analisar o potencial de re-osseointegração de defeitos peri-implantares após a utilização de laser de baixa intensidade associado a agente fotossensibilizador e Regeneração Óssea Guiada (ROG) (Artigos V e VI) • Comparar a resposta do hospedeiro frente as deonças periodontal e peri- implantar (Artigo VII) • Verificar a biocompatibilidade da superfície de titânio após a utilização de jato de bicarbonato por meio de cultura de células assim como a presença de contaminantes na superfície de implantes falidos removidos precocemente ou após estarem sob função (Artigos VIII e IX) 15 CAPITULO 5 - MICROBIOLOGIC AND RADIOGRAPHIC ANALYSIS OF LIGATURE-INDUCED PERI-IMPLANTITIS WITH DIFFERENT DENTAL IMPLANT SURFACES Jamil Awad SHIBLI1 - DDS, MS, Department of Periodontology, Dental School at Araraquara, State University of São Paulo (UNESP), Araraquara, SP, Brazil. Marilia Compagnoni MARTINS1 - DDS, MS Department of Periodontology, Dental School at Araraquara, State University of São Paulo (UNESP), Araraquara, SP, Brazil. Roberto Fraga Moreira LOTUFO2 – DDS, MS, PhD, Department of Periodontology, Dental School of São Paulo, University of São Paulo State (USP), São Paulo, SP, Brazil. Elcio MARCANTONIO JR.3 - DDS, MS, PhD, Professor of Department at Periodontology, Dental School of Araraquara, State University of São Paulo (UNESP), Araraquara, SP, Brazil. Correspondence: Elcio Marcantonio Jr. Departamento de Periodontia Faculdade de Odontologia de Araraquara -UNESP R. Humaitá, 1680 14801-903 Araraquara - SP, Brasil Fax: ++55 16 201-6314 e-mail: elciojr@foar.unesp.br 16 MICROBIOLOGIC AND RADIOGRAPHIC ANALYSIS OF LIGATURE-INDUCED PERI-IMPLANTITIS IN DIFFERENT DENTAL IMPLANT SURFACES ABSTRACT PURPOSE: The goal of this study was to evaluate microbiota and radiographic peri- implant bone loss associated with ligature-induced peri-implantitis. METHOD AND MATERIALS: In 6 dogs, 36 dental implants with four different surfaces (9 CPTi, 9 TPS, 9 HA, and 9 acid-etched) were placed in the edentulous mandibles. After 3 months with optimal plaque control, abutment connection was performed. On day 0, 20, 40, and 60 after cotton ligature placement, both microbiologic samples and periapical radiographs were obtained. The presence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia/nigrescens, Campylobacter spp., Capnocytophaga spp., Fusobacterium sp., beta-hemolytic Streptococcus and Candida spp. was evaluated culturally. RESULTS: P. intermedia/nigrescens was detected in 13.89% at baseline and 100% of implants at other periods. P. gingivalis was not detected at baseline; but after 20 and 40 days it was detected in 33.34% and 60 days, 29.03% of dental implants. Fusobacterium spp. was detected in all periods. Streptococci were detected in 16.67% at baseline and 83.34; 72.22 and 77.42% of implants respectively at 20, 40, and 60 days. Campylobacter spp. and Candida spp. were detected in low proportions. The total viable count analysis showed no significant differences among surfaces (P=0.831), although significant difference was observed after ligature placement (P<0.0014). However, there was no significant qualitative difference, in spite of the difference among the periods. The peri-implant bone loss was not significant among the dental implant surfaces (P=0.908). 17 CONCLUSIONS: These data suggest that with ligature-induced peri-implantitis both time period and periodontal pathogens affect all surfaces equally after 60 days. KEY WORDS: Dental implants; ligature-induced/peri-implantitis; microbiology/periodontal pathogens; periapical radiograph; dogs; dental implants/surfaces. 18 INTRODUCTION Healthy soft and hard peri-implant tissue around dental implants is essential for long term success.1,2 The relationship between different dental implant surfaces and bacterial biofilm in peri-implantitis development has not been completely studied. Cross-sectional microbiologic studies of dental implants with clinically healthy marginal peri-implant tissues in humans3-8 and animals9,10 have demonstrated a scattered submucosal microbiota dominated by facultative Gram- positive cocci and rods. In contrast, failing dental implants have been associated with periodontal pathogens such as fusobacteria, spirochets, Actinobacillus actinomycetemcomitans, the black-pigmented species Porphyromonas gingivalis and Prevotella intermedia and Campylobacter rectus.3,11-13 These bacterial shifts have been reported to be caused by peri-implant bone loss resulting in osseointegration failure.10,14 The importance of microbiologic factors for the development and progression of pathologic conditions in the tissues supporting dental implants is controversial. In addition, studies seeking to determine which implant surface (microstructure) or coating is more favorable for the progression of the peri-implantitis are scarce. Therefore, the aims of this study were: (1) to identify, by culture tests, the presence of periodontal pathogens and (2) to evaluate peri-implant bone loss by standardized radiography in ligature-induced peri-implantitis in dogs with endosseous implants having different surfaces. 19 MATERIAL AND METHODS Animals and Anesthesia Six adult, systemically healthy, male mongrel dogs were used. Dogs were 2- years old with an average weight of 18Kg. Animal selection, management, and surgical protocol routines were approved by the Dental School at Araraquara Institutional Animal Care and Use Committee. All surgical and clinical procedures, as well as the removal of microbial samples, were performed under general anesthesia accomplished by 0.05mg/Kg of subcutaneous preanesthesia sedation (atropine sulphate) and intravenous injection of chlorpromazine and thiopental. Oral prophylaxis was performed up to 2 weeks before teeth extraction. All mandibular premolars were then extracted creating an edentulous ridge and both the mandibular quadrants and the alveoli were allowed to heal for a period of 3 months. The maxillar premolars were extracted to avoid occlusion trauma interference. Plaque control was instituted during the healing period by scrubbing daily with 0.12% chlorhexidine, scaling and root planing once a month, until ligature placement (Fig. 1). Implant Design Thirty-six dental implants with four different surfaces involving three different implant systems were used in this study. Nine commercially pure titanium implants (CPTi, 3i Implants Innovations, Palm Beach Gardens, FL, USA), nine titanium plasma sprayed implants (TPS, Esthetic plus ITI, Straumann AG, Waldenburg, Switzerland), nine hydroxyapatite (HA, Calcitek, Sulzer Medica, Carlsbad, CA, USA) and nine hybrid surface–machined titanium in the three first threads and acid- etched in other threads (Acid, Osseotite-3i Implants Innovations, Palm Beach 20 Gardens, FL, USA) were used. All implants had lengths of 10mm and diameters of 3.75mm (except TPS which had a diameter of 4.1mm) (Fig.2). Surgical Procedures Under aseptic surgical conditions, the dental implants were placed after preparation of a full thickness flap. The recipient sites were prepared using original instruments for each dental implant surface, according to the surgical techniques indicated by each implant manufacturer. The implants were randomly distributed among the dogs so that each dental implant surface was represented at least once in each animal (TABLE 1). The implants were placed at the bone level and a cover screw was screwed onto the implant, including the TPS dental implant which had been modified in technique placement as indicated by the manufacturer. The flaps were sutured with single interrupted sutures to submerge all implants. Antibiotic coverage with potassium and sodium benzyl penicillin was given once a week, for 2 weeks, to avoid post-surgical infection. Paracetamol was given for pain control medication, and the sutures were removed after 10 days. A soft diet was instituted post-surgically. After a healing period of 3 months, healing abutment connections were made, according to the indication of each dental implant system. After 45 days and healing of the soft tissue, cotton floss ligatures were placed in a submarginal position around the dental implants and sutured in peri-implant mucosa to hold the ligatures in position. The positions of the ligatures were checked twice a week. Peri-implant bone loss was accelerated by tying further ligatures at 20-day intervals for a period of 60 days, or until the implants had a loss of about 40% of radiographic bone height.15 21 Microbial Samples Peri-implant microbial samples were obtained with paper points immediately before the ligature placement and 20, 40 and 60 days after ligature placement from the mesio-distal sites of all dental implants, as described by Slots et al.16 Supramucosal debridement at the sample site was initially performed with a sterile plastic curette and dry gauze after isolation from saliva using cotton tips/wool and suction. Four sterile paper points were subsequently inserted into the peri- implant sulci, as far apical as possible, for a period of 20 seconds, at the baseline and immediately after removal of the ligature at 20, 40 and 60 days. The paper points and cotton floss ligatures were removed and placed into to a 3mL vials containing VMGA III anaerobic transport medium.17 All samples were collected by the same operator and coded by an assistant for blind identification. The microbiologic procedures were initiated within 24 hours. The samples were centrifuged for 60s and serially diluted 10-fold in peptonated water to between 10-1 and 10-6 for quantitative evaluation of CFU/ml and to obtain isolated colonies for qualitative identification. Aliquots of 0.1 ml of the dilutions were plated onto Enriched Tryptic Soy Agar (ETSA)18 and Tryptic Soy- Serum-Bacitracin-Vancomycin agar (TSBV)19 in a standardized manner. ETSA plates were incubated in anaerobic jars containing an atmosphere with mixed gas (85% N2, 10% H2, 5%CO2) at 37oC for 7 to 10 days. TSBV agar plates were incubated in a 5% CO2 atmosphere for 5 days at 37oC. The bacterial species were identified from anaerobic cultures based on gram-stain, aerotolerance, colony morphology, esculin hydrolysis,20 [alpha]-glucosidase and N-benzoyl-DL-arginine-2- naphthylamide (BANA) hydrolysis,21 oxidase and catalase activities. Total viable count (TVC) and cultivable microbiota, including Porphyromonas gingivalis, 22 Prevotella intermedia/nigrescens, Fusobacterium spp., Capnocytophaga spp., beta- hemolytic Streptococcus, Campylobacter spp. and Actinobacillus actinomycetemcomitans, detection were performed based on colony morphology and positive catalase tests.19 Candida spp. identification was also performed. Radiographs Baseline periapical radiographs were taken at the time of ligature placement, 20, 40 and 60 days after ligature-induced peri-implantitis to evaluate changes in bone levels. The standardized radiographs were obtained with a customized occlusal index fabricated from a film holder by affixing a silicone bite block made of polyvinyl siloxane putty impression material. A dental x-ray machine equipped with a 35-cm-long cone was used to expose the periapical intraoral film (Agfa Dentus, Size 0, Agfa Gevaert, Mortsel, Belgium). Exposure parameters were 70 kilivolt (peak), 15 mA, and ¼ second at a focus-to- sensor distance of 37cm. The linear distance between a fixed point in the abutment and the first visible bone-to-implant contact was determined at the mesial and distal of each implant digital image. The mesial and distal values were averaged to obtain a mean implant value. Relative peri-implant bone loss was measured to avoid interference by the different dental implant macrostructures used in this study. All measurements were made independently by 2 of the authors. If discrepancies were of 0.5mm or less, the mean value of the 2 measurements was used. In situations with greater discrepancies, the images were analyzed again and discussed until consensus was reached. Data Analysis The TVC were transformed into colony forming units per mL (CFU/mL) using predetermined conversion factors to account for dilution and the size of the 23 evaluated surface on the plate. Data were then analyzed for dental implant surface and time of ligature placement and relative bone loss via nonparametric analysis of variance (Kruskal-Wallis test) with alpha 0.05. Differences between groups were assessed by the Dunn test. Microorganism analysis was performed after logarithmic transformation. All test were stratified according to dog (unit of analysis), i.e., n=6. RESULTS Microbiologic Analysis Microbiological data were available for analysis from 36 sites/implants in 6 dogs (6 sites per animal). Five implants (2CPTi, 1HA and 2 acid-etched) did not receive ligatures within 40 days of ligature-induction since they demonstrated 40% bone loss, therefore at 60 days, just 31 implants were analyzed. Therefore, hundred thirty-nine microbiological samples were analyzed over the experimental period. TABLE 2 summarizes the positive samples for each dental implant surface at all times for each microorganism. A. actinomycetemcomitans and Capnocytophaga spp. could not be detected for any of the dental implants in this study. In the TVC, there were no statistically significant differences between the dental implant surfaces (P=0.813). However, after the ligature placement, statistically significance was observed among the periods (P<0.0014) (Fig. 3a). The measurements taken following ligature breakdown increased. The TPS and acid surface were observed, on average, to be less colonized. In relation to time, the baseline was statistically significantly different in relation to the other times. Porphyromonas gingivalis was not detected at baseline. At other times low colonization was detected: 12 dental implants (4CPTi, 2TPS, 4HA, and 2 acid- etched) were colonized at days 20 and 40. At day 60, the number of positive implants decreased, 2CPTi and 1HA did not receive ligatures because there was a 24 40% peri-implant bone loss. There was no significant quantitative difference among the dental implant surfaces (P=0.704), neither between days 0 and 20, 40, and 60 (P>0.05) (Fig. 3b). Prevotella intermedia/nigrescens was detected at baseline on 5 dental implants (2CPTi, 2HA, and 1 acid-etched). At days 20, 40, and 60 all implants were colonized. Quantitative difference was not significant among implant surfaces (P=0.877); significance was observed between day 0 and days 20, 40, and 60 (P<0.0033) (Fig. 3c). Fusobacterium spp. was identified on 4 dental implants (2CPTi and 2HA) at baseline. At days 20, 40, and 60, respectively, 24 (6CPTi, 7TPS, 6HA, and 5 acid- etched); 27(6CPTi; 8TPS; 7HA, and 6 acid-etched), and 25 (5CPTi, 7TPS, 7HA, and 6 acid-etched) dental implants were colonized by Fusobacterium spp. Significant difference was observed between day 0 and days 20, 40, and 60 (P<0.047), except for CPTi surface (P=0.143). There was no significant difference between the different dental implant surfaces (P=0.375) (Fig.3d). Seven dental implants (3CPTi, 1TPS, and 3HA) were beta-hemolytic Streptococcus positive at baseline. At the other times, 30 (7CPTi, 8TPS, 7HA, and 8 acid-etched), 26 (6CPTi, 6TPS, 6HA, and 8 acid-etched), and 24 (6CPTi, 6TPS, 6HA, and 6 acid-etched) dental implants were colonized at days 20, 40, and 60, respectively. Differences among the dental implant surfaces were not observed (P=0.993), although significant difference between times was demonstrated for CPTi and acid surfaces (P<0.0284) (Fig.3e). Campylobacter spp. was not identified at baseline. However, it was detected at days 20, 40, and 60, in 4 (2CPTi, 1TPS, and 1 acid-etched), 6(4CPTi, 1TPS, and 1 acid-etched), and 3 (1CPTi, 1TPS, and 1 acid-etched) dental implants, 25 respectively. Therefore significant difference was not observed among dental implant surfaces (P=0.425), so periods either (P>0.05) (Fig.3f). Candida spp. was isolated at only 6 dental implants (2CPTi, 1TPS, and 1 acid-etched) at day 20. Radiographic Analysis At the start of ligature-induced peri-implantitis, the linear distance between the fixed point and first relative peri-implant bone loss was measured to avoid interference from the different macrostructures of the dental implants utilized in this study. The radiographically measured mean bone loss was observed at days 20, 40, and 60 (Figs 4a and 4b). No dental implant exhibited peri-implant radiolucencies at baseline. Significant difference was not found between surfaces (P=0.908), despite the fact that the relative means of the TPS (1.79+1.52mm) and the acid-etched surface (1.62+1.32mm) were lower than those of the HA (1.94+1.59mm) and CPTi (2.09+1.70mm) implants among the periods (Fig. 5). Significant differences (P<0.005) were found between baseline and the other time points. DISCUSSION In this study, it was observed that ligature-enhanced bacterial biofilm accumulation around different dental implant surfaces resulted in rapid peri-implant tissue breakdown. Radiographically significant peri-implant bone loss was established within 60 days. Tissue breakdown around different dental implant surfaces was accomplished by bacterial shift in a relatively short period (20 days), in agreement with Schou et al9 and Nociti et al.22 Other reports evaluated just the microbiota for longer periods: Hanisch et al10 for 10 months and Tillmanns et al14 for 3 months. 26 However, all reports found similar microbiota before and after ligature placement. The increase in radiographic bone loss takes place between days 0 and 60 in dogs, not because of mechanical trauma from the ligature, but as result of peri-implant microbiota. These data are in accordance with those of Zappa & Polson;23 Schou et al.24 However,Tonetti25 disagreed with this statement, and further studies could answer this question. Persson et al26 reported that 6 weeks after ligature placement, about 20% bone loss was observed. Ligature-placement was (8 weeks) able to rapidly induce significant peri-implant bone loss, comparable with the studies of Hanisch et al10, Tillmanns et al14, Lang et al27, and Hurzeler et al.28 Some important differences between the types of surfaces that affect dental implant microstructure and ultrastructure seem to influence the adsorption and the bacterial colonization. Statistical difference was not observed for TVC, although lower counts for TPS and acid-etched could be observed. Several studies29,30 have shown that the presence and density of periodontal pathogens were influenced more by oral status than by the implant surface characteristics. Although this study used ligatures to facilitate bacterial colonization, TVC means were significantly higher in CPTi than in the TPS surface. It is speculated that the smooth surface present on the neck of TPS dental implants could explain these data. In the case of an acid-etched surface, which has a machined surface on the first three threads and a treated surface on the other threads, the difference in results compared with CPTi surface could be explained by oxide present after acid treatment. The presence of different oxides could influence the affinity of LPS (bacterial lipopolissacaride) for these components.31 In addition, the sample size and the short period evaluated in present study could be explain these microbiologic and radiographic data. 27 The P. gingivalis and P. Intermedia/nigrescens, in this study, were associated with the induction and progression of the peri-implantitis, as well as with periodontal diseases.32 Fusobacterium spp. and Campylobacter spp. which were also identified on some dental implant surfaces have also been associated with peri-implant diseases, according to Papaioannou et al33, Macuch & Tanner34, and Mombelli et al.35 The greatest increase in bone loss was accomplished by the detection of these microorganisms. These microbiologic results confirm those of Mombelli et al3, Mombelli et al35, Mombelli et al36, Shou et al9, Hanisch et al10, Tillmanns et al14, Lee et al7, and Listgarten & Lai37. The presence of beta-hemolytic Streptococcus agrees with the results of Hanisch et al10, although this bacteria was not found in the same proportion. This bacterium was detected on five implants (62.5%) at 10 months in the study by Hanisch et al10 in comparison to 30 implants (83.3%) at 20 days after ligature placement in the present study. The absence of this microorganism in the buccal cavity or at low frequency38 indicates the existence of a low pH resulting from the induced peri-implantitis39. Leonhardt et al40 reported the presence of Candida sp. in association with failing implants, in accordance with the present data. The absence of A. Actinomycetemcomitans and Capnocytophaga sp. is not in accordance with the reports of Renvert et al41, Shou et al9, Hanisch et al10 and Tillmanns et al14. The difference between the results of this study and the aforementioned studies is possibly related to diet, time of evaluation, marginal inflammation, ligature materials, use of clorhexidine and antibiotics, and different microbiologic methods (culture media, PCR and DNA probes). In the present investigation, the association between increased viable counts of periodontal pathogens and peri-implant bone loss was evident. Thus, within the 28 limits of this study, it can be concluded that: (1) there was no quantitative significant statistical difference, considering the TVC on the different implant surfaces, without qualitative difference; (2) there was a bacterial shift at 20 days after the ligature placement, and (3) these data suggest that coating dental implant surfaces were as susceptible as smooth surfaces to ligature-induced peri-implantitis in 60 days. ACKNOWLEDGMENTS The authors wish to thank Dr. Izabel Yoko Ito for invaluable assistance in preparing this manuscript. We also to thank Drs. Carlos Nassar, Patricia Nassar, Rodrigo Rego, and Susana d’Avila for assistance in surgical and clinical procedures. This study was supported by grants FAPESP 98/10100-0 and 1999/03026-1. 29 REFERENCES 1. 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J Clin Periodontol 1996; 23: 310-319. 33 ILLUSTRATIONS Fig. 1 Extraction Implant abutment 1st. 2nd. 3rd. 4th. -225 -135 -45 0 20 40 60 days Connection Plaque control (chlorhexidine 0.12%) placement Microbiologic and radiographic exams 34 Figure 2 35 Fig. 3a Fig. 3b CPTi TPS HA Acid 0.0 2.5 5.0 7.5 10.0 * * ** *** p=0.813 *p=0.0015; **p=0.0014; ***p=0.0024 TVC (lo g 10 CFU ) CPTi TPS HA Acid 0.0 2.5 5.0 7.5 Por ph yromonas gin givalis (lo g 10 CFU) p=0.704 ns ns ns ns ns=Non-significant p>0.05 36 Fig. 3c Fig. 3d CPTi TPS HA Acid 0.0 2.5 5.0 7.5 10.0 Prevotella intermedia/nigrescens (lo g 10 CFU ) * ** * *** p=0.877 *p=0.002; **p=0.003;***p=0.0008 CPTi TPS HA Acid 0.0 2.5 5.0 7.5 10.0 Fusobacteriums pp. (log 10 CFU ) * ** *** **** p=0.375 *p=0.143; **p=0.018; ***p=0.028; ****p=0.047 ns * * *** ns=Non significant p>0.05; *p=0.018; **p=0.028; ***p=0.047 37 Fig. 3e Fig. 3f CPTi TPS HA Acid 0.0 2.5 5.0 7.5 10.0 * ns ns ** p=0.993 ns=Non-significant p>0.05 *p=0.028;**p=0.013 β Hemolitic Stre ptococcus (lo g 10 CFU) CPTi TPS HA Acid 0.0 2.5 5.0 7.5 p=0.401 ns ns ns ns ns=Non-significant p>0.05 Cam pylobacterspp. (lo g 10 CFU ) 38 Fig. 4a Fig. 4b 39 Fig. 5 CPTi TPS HA Acid 0 1 2 3 4 5 * *** p=0.908 ** *** *p=0.005; **p<0.0001; ***p=0.0001 Relative Bone Loss (mm ) 40 Legends Fig. 1. Outline of the experiment. Ligatures were placed around the implants on day 0, and were changed every 20 days, when microbiologic and radiographic procedures were performed. Animals n=6; implants n=36. Fig. 2. Dental implant surfaces used in this experiment, from left to right: titanium- plasma sprayed (TPS); hydroxyapatite (HA); acid-etched surface in three first threads and machined surface in others threads (acid); commercially pure titanium (CPTi). Fig. 3a. Mean and standard deviation of Total Viable Count (TVC) of different dental implant surfaces at baseline and 20, 40, and 60 day-period. Fig. 3b. Mean and standard deviation of P. gingivalis of different dental implant surfaces at baseline and 20, 40, and 60 day-period. Fig. 3c. Mean and standard deviation of P.intermedia/nigrescens of different dental implant surfaces at baseline and 20, 40, and 60 day-period. Fig. 3d Mean and standard deviation of Fusobacterium spp. of different dental implant surfaces at baseline and 20, 40, and 60 day-period. Fig. 3e Mean and standard deviation of beta-hemolytic Streptococcus of different dental implant surfaces at baseline and 20, 40, and 60 day-period. Fig. 3f. Mean and standard deviation of Campylobacter spp. of different dental implant surfaces at baseline and 20, 40, and 60 day-period. Fig.4a. Periapical radiograph taken at baseline. Fig. 4b. Periapical radiograph taken at 60 days after ligature-placement. Fig.5. Mean and standard deviation of radiographic bone loss of different dental implant surfaces at 20, 40, and 60 day-period. 41 Table 1- Distribuition of dental implants with different surfaces in 6 dogs. Animal Right jaw side PM2 PM3 PM4 Left jaw side PM2 PM3 PM4 1 CPTi Acid TPS TPS HA Acid 2 CPTi TPS HA HA Acid CPTi 3 HA Acid CPTi CPTi TPS HA 4 TPS HA Acid Acid CPTi TPS 5 HA Acid CPTi CPTi TPS HA 6 TPS HA Acid Acid CPTi TPS CPTi- Comercially pure titanium; TPS – titanium plasma sprayed; HA – hydroxyapatite; Acid – acid-etched surface PM2, PM3, PM4 – Mandibular premolars 42 Table 2 - Microorganism detected during the experiment. TIME (DAYS) MICROORGANISM 0 CPTi TPS HA Acid 20 CPTi TPS HA Acid 40 CPTi TPS HA Acid 60 CPTi1 TPS HA2 Acid3 P. gingivalis 0 0 0 0 4 2 4 2 4 2 4 2 2 2 3 2 P.intermedia/ nigrescens 2 0 2 1 9 9 9 9 9 9 9 9 7 9 8 7 Campylobacter spp. 0 0 0 0 2 1 0 1 4 1 0 1 1 1 0 1 Fusobacterium spp. 2 0 3 0 6 7 6 5 6 8 7 6 5 7 7 6 beta-hemolític Streptococcus 3 1 3 0 7 8 7 8 6 6 6 8 6 6 6 6 Candida spp. 0 0 0 0 2 1 2 1 0 0 0 0 0 0 0 0 (1n=7; 2n=8; 3n=7 – Dental implants excluded because of 40% radiographic bone loss at period 40 day) 43 CAPITULO 5 - DETECTION OF PERIODONTAL PATHOGENS IN LIGATURE-INDUCED PERI- IMPLANTITIS. AN EXPERIMENTAL STUDY IN DOGS. Jamil Awad SHIBLI∗ †, Marilia Compagnoni MARTINS*, Shawn F. JORDAN ‡, Marcelo W.B. ARAUJO§, Violet I. HARASZTHY‡, Joseph J. ZAMBON†||, Elcio MARCANTONIO JR.* Correspondence Address: Elcio Marcantonio Jr. Departamento de Periodontia Faculdade de Odontologia de Araraquara –UNESP R. Humaitá, 1680 14801-903 Araraquara - SP, Brasil Fax: ++55 16 201 6314 e-mail: elciojr@foar.unesp.br Sources of support: FAPESP (grants no 98/10100-0, 00/02433-1 and 1999/03026-1) and CAPES Running Title: Periodontal pathogens in ligature-induce peri-implantitis ∗ Department of Periodontology, Dental School of Araraquara, State University of Sao Paulo (UNESP), Araraquara, SP, Brazil. † Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo, Buffalo, NY ‡ Department of Restorative Dentistry, School of Dental Medicine, State University of New York at Buffalo, Buffalo, NY § Department of Social and Preventive Medicine, School of Medicine, State University of New York at Buffalo, Buffalo, NY | |Department of Periodontics and Endodontics, School of Dental Medicine, State University of New York at Buffalo, Buffalo, NY 44 DETECTION OF PERIODONTAL PATHOGENS IN LIGATURE-INDUCED PERI- IMPLANTITIS. AN EXPERIMENTAL STUDY IN DOGS. ABSTRACT Background: The purpose of this study was to evaluate the attachment loss around different dental implant surfaces by means of microbiological, clinical and radiographic analysis. Methods: In 6 male mongrel dogs, a total of 36 dental implants of four different surface coating (9 hydroxyapatite-HA, 9 titanium plasma-sprayed-TPS; 9 acid- etched surface; 9 commercially pure titanium surface-CPTi) were inserted after 3 months healing period of mandibular premolars extraction. After 3 months with optimal plaque control, abutment connection was performed. Microbiological samples were taken at 0, 20, 40, and 60 days after cotton ligature placement. The presence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Bacteroides forsythus, Prevotella intermedia, Fusobacterium nucleatum, Fusobacterium nucleatum ss vicentii, Campylobacter rectus, Eikenella corrodens, Neisseria spp., Treponema spp. and spirochetes were assessed by DNA probes in a checkerboard assay and analysis of amplified 16S rDNA by means polymerase chain reaction. Results: No differences among the four dental implants surfaces were observed for microbiological features except for the higher detection of Neisseria spp. at acid surface (p=0.003). Clinical attachment levels and radiographic bone loss were not statistically significant among dental implant surfaces (p>0.05), however all surfaces were suceptible to ligature induced peri-implatitis over time (p<0.001). 45 Correlation analysis revealed a statistical significance among P. gingivalis, P. intermedia and B. forsythus with CAL to acid, CPTi and TPS surface (p<0.05). P. gingivalis and B. forsythus presented significant correlation for CPTi surface with bone loss. Conclusions: The detection of periodontal pathogens was associated to peri- implant tissue breakdown. In addition, it can be concluded that different dental implants surfaces presented similar rate of attachment loss after bacterial shift induced by cotton floss ligatures at 60 days. Key Words: Dental implants/microbiology; peri-implantitis/biofilm; peri- implantitis/etiology; dental implants/microstructure; dogs. 46 INTRODUCTION The potential cause of osseointegration failure is the biofilm accumulation.1-3 Several animal studies have shown that the peri-implantitis is a condition characterized by soft tissue inflammation, bleeding and suppuration, presented both rapid clinical attachment loss and vertical/horizontal bone loss.4-7 These studies compare the clinical, histological and microbiological changes around dental implants, after plaque accumulation by means of ligature placement. The placement of ligature nearly always resulted in a marginal bone loss destruction. The peri-implant bone loss generally is associated with the presence of periodontal pathogens such as Porphyromonas gingivalis, Prevotella intermedia, Prevoltella nigrescens, Bacteroides forsythus, Campylobacter rectus and Actinobacillus actinomycetemcomitans.8-11 Special attention has been focused on a presence of periodontal pathogens in diseased dental implants sites without considering the microstructure of dental implant. However, there is scarce the information about what microorganism are more suitable for each different dental implant surface. Recently,numerous studies have investigated the osseointegration around different dental implant coatings such as titanium plasma-sprayed (TPS), hydroxyapatite (HA), and commercially pure titanium surface treated with acids12-16. These studies seeking the better surface for osseointegration and consequently, a higher long- term favorable prognosis as well as utilize these implants to immediate loading17,18. Therefore, the purpose of the present study was to identify the periodontal pathogens associated with ligature-induced peri-implantitis in dogs, at different dental implant surfaces. 47 MATERIAL AND METHODS Dental implant surfaces In this study, thirty-six dental implants with four different surfaces were used. Nine hydroxyapatite-HA¶, nine titanium plasma sprayed implants-TPS#, nine hybrid surface–machined titanium in the three first screws and acid-etched in other screws-Acid**, and nine commercially pure titanium implants-CPTi††, were used. All implants had lengths of 10mm and diameters of 3.75mm (except TPS which had a diameter of 4.1mm). Animals Institute of Animal Care and Use Committee of the Dental School of Araraquara, approved this protocol. Six adult, male mongrel dogs were used. At the beginning of the study, the dogs were 2-years old with an average weight of 18Kg. Surgeries Extraction Extractions were carried out in an operatory room under general anesthesia and sterile conditions utilizing 0.05mg/Kg of subcutaneous pre-anesthesia sedation (atropine sulphate) and intravenous injection of chlorpromazine (0.2mL/Kg body weight) and 4% thiopental-Na solution (0.5mL/Kg body weight). The surgical site was disinfected with 0.2% chlorhexidine and subsequently, 2% lidocaine HCl with epinifrine 1:100,000 was given as local anesthesia, and all 4 mandibular premolars were extracted creating an edentulous ridge and both the mandibular quadrants ¶ Calcitek , Sulzer Medica, Carlsbad, CA # Esthetic plus ITI, Straumann AG, Waldenburg, Switzerland ** Osseotite-3i Implants Innovations, Palm Beach Gardens, FL †† 3i Implants Innovations, Palm Beach Gardens, FL 48 and the alveoli were allowed to heal for a period of 3 months. The maxillary premolars were also extracted to avoid occlusion trauma interference. Oral prophylaxis was performed up to 2 weeks before teeth extraction. Plaque control was instituted during the healing period by scrubbing daily with 0.12% chlorhexidine, scaling and root planning once a month, until ligature placement (Fig. 1). Dental implant placement Under aseptic surgical conditions, all dental implants were placed over the full thickness flap. Three implant sites were prepared per mandibular quadrant using original instruments for each dental implant system, according to the surgical techniques indicated by each implant manufacturer. A distance of approximately 10mm between dental implant centers was maintained to avoid comunication among the further bone defects. The implants were randomly distributed among the dogs so that each dental implant surface was represented at least once in each animal (Table 1). The implants were placed at the bone level and a cover screw was screwed onto the implant, including the TPS dental implant surface due to a modification in technique insertion indicated by the manufacturer. The flaps were sutured with single interrupted sutures to submerge all implants. Antibiotic therapy with potassic and sodic benzilpenicilin (24.000UI/Kg) was startedand continued once a week, for 2 weeks, to avoid post-surgical infection. Paracetamol was given for pain control medication, and the sutures were removed after 10 days. 49 Experimental Peri-implantitis After a healing period of 3 months, healing abutment connections were installed, according to the indication of each dental implant system. After soft tissue healing of 2-months, cotton floss ligatures were placed in a submarginal position around dental implants and sutured in peri-implant mucosa to hold the ligatures in position. The positions of the ligatures were checked twice a week. Tying further ligatures at 20-day intervals for a period of 60 days accelerated peri-implant bone loss. Microbiological Procedures Peri-implant microbial samples were taken from the mesio-distal site with paper points immediately before the ligature placement and 20, 40 and 60 days after ligature placement. Supra-mucosal debridment at the sample site was initially performed with a sterile plastic curette and dry gauze after isolation from saliva using cotton rolls. The area was carefully dried and the bacterial sample was collected with 4 sterile paper points gently inserted into the peri-implant sulcus, as far apical as possible and left for 20 seconds. The paper points were placed in sterile transport vials and storaged at freezer until to be processed. All samples were collected by the same examiner and coded by an assistant to keep the blindness of the study. Periodontal Pathogens Detection Analysis of Amplified 16S rDNA The PCR-amplified 16S rDNA using digoxigenin-labed species-specific oligonucleotide probes19,20 were tested for Actinobacillus actinomycetemcomitans (CACTTAAAGGTCCGCCTACGTGCC), Porphyromonas gingivalis (GCAGTTTCA 50 ACGGCAGGGCTGAACG), Prevotella intermedia (GGTCCTTATTCGAAGGGTAA ATGC), Bacteroides forsythus (CGTATCTCATTTTATTCCCCTGTA), Campylobacter rectus (CAAGCTACTTAATCTCCGTTCGAC), Fusobacterium nucleatum (GGTTTCCCCGAAGGGACATGAAAC); Fusobacterium nucleatum ss vincetii (ACTTCACAGCTTTGCGACTCTCTGTTC), Eikenella corrodens (ACC GTCAGCAAAAAGTGGTATTAGCAC), Treponema spp.(GGCAGTAGGGGTTGC GCTCGTT), Neisseria spp. (CCTCTGTACCGACCATTGTATGAC) and for spirochetes (CGACTTTGCATGSTTAARAC). The oligonucleotide probes were 3’-end labeled with digoxigenin-11-ddUTP§§. The DNA was tranferred overnight onto membranes using 10x standart salt phosphate EDTA buffer (SSPE). Hybridization with digoxigenin-labeled oligonucleotide probes was performed at 45oC. The hybrydized membranes were washed twice with hight salt solution (2xSCC[0.15M NaCl plus 0.015 trisodium citrate, pH 7.0] in 0.1% sodium dodecyl sulphate) at room temperature and then twice with a low salt solution (0.1xSCC in 0.1% sodium dodecyl sulphate) at 45oC. The membranes were reacted with anti- digoxigenin alkaline phosphatase conjugate. The color reaction was produced with 5-bromo-4chloro-3-indolyl phosphate and nitroblue tetrazolium salts. Polymerase Chain Reaction of 16S rDNA In addition to DNA probes in checkerboard assay, the PCR-amplification of conserved region of 16S ribossomal DNA were also tested for periodontal pathogens including A. actinomycetemcomitans (primer 1-ATTGGGGTTTAGCCC TGGTG and Rev16s-ACGTCATCCCCACCTTCCTC), P. gingivalis (primer1-TG §§ Genius 5, Boehringer Mannheim, Indianapolis,IN. 51 TAGATGACTGATGGTGAAAACC and Rev16s-ACGTCATCCCCACCTTCCTC) and B. forsythus (primer 1-TACAGGGGAATAAAATGAGATACG and Rev 16s- ACGTCATCCCCACCTTCCTC). All these PCR primers were obtained commercially. Between 30 to 100ng of genomic DNA was added to the PCR mixture wich contained 1µmol/L of the primers, 2.5U of Taq polymerase¶¶ in 1x buffer and 0.2mmol/L of dCTP, dGTP, dATP, and dTTP¶¶ in a total volume of 50µL. The amplification was performed for 30 cycles of 30 seconds at 95oC, 30 seconds at 55oC and 30 seconds at 72oC in thermocycler##. Positive and negative controls were included with each set. The negative control includes all the PCR reagents except for the sample DNA. The positive control contained all the PCR reagents together with positive controls for A. actinomycetemconitans; P.gingivalis and B. forsythus. Twenty µL of each PCR reaction mixture was electroforesed in 1.5% (A. actinomycetemconitans and B. forsythus) and in 2% (for P.gingivalis) agarose gel in TBE buffer and the amplification products were visualized under 302nm ultraviolet light, on ethidium bromide-stained gels. Clinical examination After microbiological sampling, the clinical attachment level was recorded at baseline and 20, 40, and 60 days after ligature tissue-breakdown. A single pre- calibrated examiner carried out the clinical exams. The attachment levels were  Gibco BRL, Grand Island, NY ¶¶ Perkin-Elmer,Norwalk,CT ## GeneAmp PCR System 9600, Perkin-Elmer, Norwalk,CT. 52 registered using a force-controlled calibrated periodontal probe∗ ∗ ∗ with a constant probing force of 0.20N and a probe-tip diameter of 0.4mm. The distobuccal, midbuccal, mesiobuccal, mesiolingual, midlingual, and distolingual were measured. Probing depth (PD) and the distance between gingival margin (GM) and the fixed point in the abutment surface (FP) was recorded. CAL was then calculated according the formula: PD- (GM-FP). Radiographs Intraoral radiographs were taken at the time of ligature placement, 20, 40 and 60 days after ligature-induced peri-implantitis to evaluate changes in bone levels. The standardized radiographs were taken with a customized occlusal index fabricated from a sensor holder by placing a silicone bite block made of polyvinyl siloxane impression putty material. A dental x-ray machine equipped with a 35-cm-long cone was used to expose the periapical intra-oral sensor†††. Exposure parameters were 70 kilivolt (peak), 15 mA, and 1/4 second at a focus-to-sensor distance of 37cm. The linear distance between the fixed point in abutment and the first visible bone-to-implant contact was determined mesial and distal of each implant digital image. The mesial and distal values were averaged to obtain a mean implant value. Relative peri-implant bone loss was measured to avoid interference by the different dental implant macrostructures used in this study. ∗ ∗ ∗ Florida Probe, Computerized Probe Inc, Gainesville, FL ††† CDR- Computed Dental Radiography – Schink Tecnologis Inc.,USA 53 All measurements were made independently by 2 examiners. If discrepancies were of 0.5mm or less, the mean value of the 2 measurements was used. In situations with greater discrepancies, the images were analyzed again until desired measurements were reached. Data Analysis Data management and calculation were done using statistical software‡‡‡. Analysis of variance, using comparison of several proportions (contingency table) was used to compared the distribuition of different bacteria for each type of implant, and also to compare bacterial colonization at different points.21 The clinical attachment loss and Radiographical bone loss were compared by means paired t-test (2-tailed). To determine the correlations of microbiological features on CAL and vertical bone loss, a Pearson Correlation was determined. All test were stratified according to dog (unit of analysis), i.e., n=6. Level of significance was set at 0.05. RESULTS Clinical and microbiological data were available for analysis from 36 sites/implants in 6 dogs (6 sites per animal). Hundred forty-four microbiological samples were analyzed over the experimental period. None implant was lost due to ligature- induced peri-implantitis. ‡‡‡ SPSS-Statistical Package for the Social Sciences version 10.1, Chicago, IL. 54 Microbiological features Figures 2 to 12 summarize the prevalence and incidence of target periodontal pathogens at each period. All target periodontal pathogens (Table 2) were found in all animals in varied proportions. A. actinomycetemcomitans was not identified at baseline and 20 days. Although a higher prevalence have been detected to HA-coated surface, no statistically difference was observed among the surfaces (p>0.05). P. gingivalis was not detected at baseline in HA-coated surface. The positive sites percentage range between 36,12% for HA-coated surface to 25% for CPTi. However statisticall significance was not achieved among the different dental implants surfaces (p>0.05). B. forsythus was detected only in acid surface at baseline and 20 days. The higher detection over time was found in acid surface (13.88% of the samples). The same occurred to C. rectus: the higher percentages were observed in acid and HA- coated surface (22.23% of the samples). For both microorganisms significant difference was not observed. F. nucleatum and F. nucleatum ss vicentii were detected in higher percentages at baseline and 20 days for all surfaces. At 40 and 60 days their detection was lower for all differente dental implant surfaces (p>0.05). E. corrodens and spirochetes were detected in lower porcentage at baseline, 20, 40 and 60 days. However E. corredens shown more positive sites to CPTi surface (11,12% of the samples), even though not significant (p>0.05). 55 Treponema spp. and Neisseria spp. were detected in all surfaces at baseline and 20 days. At 40 days both bacteria were not detected in TPS surface. A statistical significance was observed among the positive detection sites for Neisseria spp. at acid surfaces (p=0.003). Clinical Attachment Level After ligature-induced tissue breakdown, all dental implants were associated with a continuous increase of clinical attachment loss. Differences in clinical attachment loss among the different implant surfaces were not statistically significant (p>0.05). The lower attachment loss was presented by TPS surface (3.87+1.69) and the higher to CPTi (5.16+1.53) (Table 3). Over time, all surfaces exhibited statistically significant attachment loss when compared to baseline recordings (p<0.001 for HA, Acid and CPTi and p=0.02 for TPS). Radiographic Analysis At baseline, none implant surface exhibited peri-implant radiolucencies. The means of relative bone loss for all dental implants surfaces are presented in Table 4. The Ha-coated surface showed the higher bone loss measurement (4.20+0.47mm) and the lower for TPS surface (3.50+0.97mm). However statistical significance was no assessed for different dental implants surfaces (p>0.05). When the bone levels were compared between baseline and 60 days after ligature placement, a statistically significant difference (p<0.0001) was found, demonstrating a clear effect of biofilm accumulation over time. 56 Correlations Correlation analysis revealed a high correlation of P. gingivalis (r=0.991; p=0.032) and B. forsythus (r= 0.948; p=0.004) with CAL to Acid surface. P. intermedia was statistically correlated with CAL measurements to CPTi surface (r=0.853; p=0.031). P. gingivalis was also correlated with CAL in TPS surface (r=0.831; p=0.040) A consistent correlation was noted for CPTi surface to vertical bone loss measurements with the detection of P. gingivalis (r= 0.952; p=0.003) and B. forsythus (r=0.997; p=0.006). DISCUSSION The ligature-induced peri-implant tissue breakdown has been evaluated in canines, 6,7,22 microswine23 and non-human primates.4,5,24,25 The acute inflammatory tissue response to biolfim accumulation seemed to represent a localized lesion, comparable to that encountered in periodontal as well as in peri-implant diseases. The objective of this study was to evaluate, by means microbiological and clinical analysis, the ligature-induced peri-implantitis around different dental implant surfaces. A significant clinical attachment loss associated to bacterial shift was observed after placing cotton floss ligatures around dental implants and ceasing plaque control procedures. A significant radiographic bone loss was also observed around all dental implants after short period (60 days). These features were in accordance with Schou et al.26 and Nociti et al.22 Other studies reported clinical and microbiological evaluations for longer periods: Hanisch et al.24 for 10 months and Tillmanns et al.6,7 for 3 and 6 months. However, all reports agreed on bacterial shift after ligature-placement resulting in attachment and bone loss. 57 The different coating surfaces could to influence the bacterial adsorption.27,28 Physical and chemical factors can affect the attachment of biofilms to a hard surface. The roughness of the surface can increase surface area and hence increase the colonization29. Roughness also provides protection from shear forces and increases the difficulty of cleaning methods. Further, Quirynen and Bollen29 have shown that supragingival plaque formation, after initial colonization has occurred, was faster on a roughened surface. The initial colonization of an intra- oral hard surface starts from surface irregularities such as cracks, grooves, or abrasion defects and subsequently spreads out from these areas as a relatively even monolayer of cells.30 The roughness of different dental implant surfaces can work like grooves for initial pathogens adhesion. The chemical composition of dental implant surface also acts on a bacterial colonization since it may contain beneficial or detriment components.31 Those events probably occurred after ligature-induced breakdown peri-implant tissues. This may explain the higher detection of Neisseria spp. at acid surfaces observed in our investigation. The smoother surface (CPTi) showed the lowest positive detection number for the target microorganisms at all periods (Table 2). However, the data of the present study did not show any statistical difference for microbiological features among the different dental implant surfaces in a 60 days time period. The non-statistical difference for detection frequencies for periodontal pathogens among the different surfaces may be explained by the small sample size used in this study. In relation to clinical and radiographic data, no relevant differences among HA, TPS, Acid and CPTi surfaces were found, similar to microbiological findings. There 58 was not a agreement shown in previously published studies, where HA-coated surface and TPS implants were associated with substantially more failures resulting from peri-implantitis.32,33,34 However, in two studies conducted by Tillmanns et al.6,7, differences were not found among the HA, TPS and CPTi surfaces in clinical, microbiological and histological aspects. Probably the different results obtained for HA surface are due to the short period of time utilized in our methodology. In this study the most severe radiographic evidence of bone loss was observed for HA surface, although statistically significance was not observed (p>0.05). The detection of A. actinomycetemcomitans was similar to that shown by Ong et al.35; Hanisch et al.24; Eke et al.25; Schou et al.26; Tillmanns et al.7 However, only Tillmanns et al.7 evaluated the bacterial shift in canine model. Nociti et al.22, analyzed the clinical and microbiological status of periodontitis and peri-implantitis induced in dogs, but they did not find A. actinomycetemcomitans using polymerase chain reaction. This result may have occurred due to time ligature-induced breakdown tissues (30 days) used by Nociti et al.22 when compared with our methodology. The A. actinomycetemcomitans detection in our study occurred after 40 days of ligature-induced peri-implantitis. The detection of P. gingivalis, P. intermedia and F. nucleatum confirm previous studies that analyzed dental implants with peri-implant diseases in animals7,22,24,25,26,36 and humans.10,11,37-39 The canine peri-implantitis model used in this study found similar results about microbiological features at periodontal diseases40,41 and peri-implant diseases22,24,26 related in literature. 59 The detection of F. nucleatum ss vicentii, C. rectus and E. corrodens, has also been associated both failing and ailing implants.11,39 The spirochetes have been associated with failing implants.37,42 However, its detection was lower in all surfaces. The use of chlorhexidine at plaque control phase may have reduced the potential sources from that spirochetes could colonize.24 The presence of A. actinomycetemcomitans, P. gingivalis and B. forsythus were strongly associated with periodontal disease status, disease progression and unsuccessful therapy.43 A statistical correlation found between P. gingivalis and CAL/VBL in this investigation was in agreement with that statement. F. nucleatum, C. rectus, P. intermedia and various spirochetes have also been implicated in causing periodontal diseases, although the evidence for their causative role is less expressive.44 The progression of clinical attachment loss and vertical bone loss observed in our study were statistically associated with target periodontal pathogens detection such as P. gingivalis and P. intermedia, after ligature placement. This study applied DNA probes in a checkerboard assay and amplification of 16S rDNA by means of polymerase chain reaction, which unlike cultural methods that have been used, does not require anaerobic methods for sample viability and species characterization. The molecular biology techniques are be able to detect species that may be at low relative proportions, comparing to other species in samples, but are above the probe assay threshold. Some periodontal pathogens 60 such as B. forsythus are rarely reported in peri-implantitis due to its difficult to be cultivated45. In previous study conducted by our group, there was no identification of A. actinomycetemcomitans using culture media, probably due to low prevalence of this bacterium in samples from ligature-induced peri-implantitis in dogs. These techniques are more sensitive than culture, for which sensitivities are low as 50 bacteria for PCR, 103 for DNA probe and 2x105 for culture.46,47,48 In this investigation, the microbiological samples positive for A. actinomycetemcomitans , P. gingivalis and B. forsythus were also assessed by PCR of 16S rDNA. Several investigations49-52 have related the existence of high probability of false-positive results with the use of whole genomic DNA probes, principally for A. actinomycetemcomitans. Dental implants with peri-implantitis thus reveal a complex microbiota encompassing conventional periodontal pathogens. These features confirm the bacterial shifts associated to attachment loss detected in this animal study after the induction of experimental peri-implantitis. Species such as A. actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum are often isolated from failing sites, but can also be detected around healthy peri- implant sites. 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Humaitá, 1680 14801-903 Araraquara - SP, Brasil Fax: ++55 16 201 6314 e-mail: elciojr@foar.unesp.br 68 FIGURE LEGENDS 1. Outline of the experiment. Animals n=6, dental implants n=36. 2. Frequency distribuition of Actinobacillus actinomycetemcomitans around different dental implant surfaces before ligature placement (baseline) and after 20, 40 and 60 days. 3. Frequency distribuition of Porphyromonas gingivalis around different dental implant surfaces before ligature placement (baseline) and after 20, 40 and 60 days. 4. Frequency distribuition of Bacteroides forsythus around different dental implant surfaces before ligature placement (baseline) and after 20, 40 and 60 days. 5. Frequency distribuition of Prevotella intermedia around different dental implant surfaces before ligature placement (baseline) and after 20, 40 and 60 days. 6. Frequency distribuition of Campylobacter recuts around different dental implant surfaces before ligature placement (baseline) and after 20, 40 and 60 days. 7. Frequency distribuition of Fusobacterium nucleatum around different dental implant surfaces before ligature placement (baseline) and after 20, 40 and 60 days. 8. Frequency distribuition of Fusobacterium nucleatum ss vicentii around different dental implant surfaces before ligature placement (baseline) and after 20, 40 and 60 days. 9. Frequency distribuition of Eikenella corrodens around different dental implant surfaces before ligature placement (baseline) and after 20, 40 and 60 days. 69 10. Frequency distribuition of Treponema spp. around different dental implant surfaces before ligature placement (baseline) and after 20, 40 and 60 days. 11. Frequency distribuition of Neisseria spp. around different dental implant surfaces before ligature placement (baseline) and after 20, 40 and 60 days. 12. Frequency distribuition of spiroquetes around different dental implant surfaces before ligature placement (baseline) and after 20, 40 and 60 days. 70 Table 1- Randow distribuition of different implant surfaces in six animals. Animal Right jaw side PM2 PM3 PM4 Left jaw side PM2 PM3 PM4 1 TPS HA Acid Acid CPTi TPS 2 HA Acid CPTi CPTi TPS HA 3 TPS HA Acid Acid CPTi TPS 4 HA Acid CPTi CPTi TPS HA 5 CPTI TPS HA HA Acid CPTi 6 CPTi Acid TPS TPS HA Acid HA – hydroxyapatite; TPS – titanium plasma sprayed; Acid – acid-etched surface; CPTi- Comercially pure titanium PM2, PM3, PM4 – Mandibular premolars 71 TIME (DAYS) Periodontal Pathogens 0 HA TPS Acid CPTi 20 HA TPS Acid CPTi 40 HA TPS Acid CPTi 60 HA TPS Acid CPTi A. actinomycetemcomitans 0 0 0 0 0 0 0 0 3 4 0 3 4 1 3 1 P. gingivalis 0 1 2 1 3 3 0 1 5 5 3 4 5 3 5 3 P. intermedia 0 0 1 0 0 0 0 0 1 1 1 0 2 1 2 1 B. forsythus 0 0 1 0 0 0 1 0 1 1 1 1 3 2 2 1 C. rectus 0 0 0 0 2 1 2 0 3 3 2 3 3 3 3 3 F. nucleatum 2 2 4 2 5 6 5 5 1 2 0 0 3 3 3 2 F. nucleatum ss vicentii 3 3 1 2 2 3 3 4 0 1 0 1 1 1 1 1 E. corrodens 1 2 1 0 2 1 1 2 0 0 0 1 0 1 0 1 Neisseria spp. 2 3 2 2 6 2 6 1 3 0 5 1 0 0 3 0 Treponema spp. 5 6 6 5 4 4 1 2 2 0 0 3 2 0 0 0 Spirochetes 0 0 0 0 1 1 1 2 1 0 0 0 0 1 2 0 Total of positive bacteria detection 13 17 18 12 25 21 20 17 20 17 12 17 22 16 24 13 Table 2. Distribuition of implants being positive for indicated periodontal pathogen at baseline, 20, 40, and 60 days. 72 Table 3 – Mean+SD of clinical attachment levels, measured in mm, at baseline and 20, 40 and 60 days after ligature placement. Surface Baseline 20 days 40 days 60 days Clinical attachment loss HA 7.47+0.48 10.25+0.84* 11.52+0.60* 12.11+0.60* 4.64+0.80‡ TPS 7.60+1.35 9.75+0.93* 9.78+0.93* 11.49+1.27† 3.87+1.69‡ Acid 8.08+0.53 11.03+0.90* 11.68+0.96* 12.75+0.98* 4.66+1.13‡ CPTi 8.24+0.80 11.37+1.06* 12.22+1.18* 13.40+1.20* 5.16+1.53‡ * Significantly different from baseline p<0.0001 †Significantly different from baseline p=0.002 ‡Not significant among the surfaces p>0.05 73 Table 4. Crestal bone levels at baseline and 20, 40 and 60 days after ligature placement and crestal bone loss measured in mm from implant healing abutment fixed point (mean+SD) Surface Baseline 20 days 40 days 60 days Crestal bone loss HA 2.01+0.46 3.62+0.29* 4.65+0.84* 6.22+0.50* 4.20+0.47† TPS 2.50+0.61 3.85+0.95* 4.62+0.90* 6.00+0.70* 3.50+0.97† Acid 2.36+0.54 3.64+0.17* 5.19+0.51* 6.06+0.27* 3.70+0.57† CPTi 2.40+0.51 4.12+0.72* 5.20+0.71* 6.32+0.33* 3.92+0.61† * Significantly different from baseline p<0.0001 †Not significant among the surfaces p>0.05 74 Figure 1 Extraction Dental implants placement Abutment connection Microbial sampling and clinical/ radiographical measurements 0 Plaque control – chlorhexidine 0.12% daily -225 -135 -45 0 20 40 60 days 75 Fig. 2 Fig.3 Frequency of Actinobacillus actinomycetemcomitans detection in ligature-induced peri-implantitis 0 20 40 60 0 25 50 75 100 HA TPS Acid CP Time(days) p os iti ve s ite s (% ) Frequency of Porphyromonas gingivalis detection 0 20 40 60 0 25 50 75 100 HA TPS Acid CP Time(days) po si tiv e si te s (% ) 76 Fig.4 Fig.5 Frequency of Bacteroides forsythus detection 0 20 40 60 0 10 20 30 40 HA TPS Acid CP Tme (days) po si tiv e si te s (% ) Frequency of Prevotella intermedia detection 0 20 40 60 0 10 20 30 40 HA TPS Acid CP Time(days) po si tiv e si te s (% ) 77 Fig.6 Fig.7 Frequency of Campylobacter rectus detection 0 20 40 60 0 10 20 30 40 50 HA TPS Acid CP Time (days) po si tiv e si te s (% ) Frequency of Fusobacterium nucleatum detection 0 20 40 60 0 25 50 75 100 HA TPS Acid CP Time(days) po si tiv e si te s (% ) 78 Fig. 8 Fig. 9 Frequency of Fusobacterium nucleatum vicentii detection 0 20 40 60 0 10 20 30 40 50 HA TPS Acid CP Time(days) po si tiv e si te s (% ) Frequency of Eikenella corrodens detection 0 20 40 60 0 10 20 30 40 HA TPS Ac