UNIVERSIDADE ESTADUAL PAULISTA Faculdade de Odontologia de Araraquara UNESP CELSO EDUARDO SAKAKURA O efeito da ciclosporina –A na osseointegração ARARAQUARA 2005 UNIVERSIDADE ESTADUAL PAULISTA Faculdade de Odontologia de Araraquara P UNES CELSO EDUARDO SAKAKURA ‘ O efeito da ciclosporina –A na osseointegração Tese apresentada à Faculdade de Odontologia de Araraquara da Universidade Estadual Paulista, como parte dos requisitos para a obtenção do título de Doutor em Odontologia – Área de PERIODONTIA. Orientadora: Prof.a Dr.a Gulnara Scaf Co-orientador: Prof. Dr. Elcio Marcantonio Junior ARARAQUARA 2005 DADOS CURRICULARES CELSO EDUARDO SAKAKURA Nascimento: 24. 11. 1975 – São Paulo - SP Filiação: Akira Sakakura Tereza Maeda Sakakura 1994-1997 Curso de Graduação em Odontologia Faculdade de Odontologia de Bauru – USP 1998 – 2000 Curso de Especialização em Implantodontia Hospital de Reabilitação de Anomalias Crâniofaciais – USP 2000 – 2002 Curso de Pós-graduação em Periodontia, nível Mestrado Faculdade de Odontologia de Araraquara – UNESP 2002-2005 Curso de Pós-graduação em Periodontia, nível Doutorado Faculdade de Odontologia de Araraquara – UNESP Dedico este trabalho... ...À minha esposa Cristiana, pelo amor, apoio, carinho, paciência e compreensão nos muitos momentos de minha ausência. ...Aos meus pais, Akira e Tereza, meus amigos verdadeiros, pelo carinho, amor, paciência pelas “palmadas” e por todas as vezes, em que abriram mão de seus sonhos, para a realização dos meus. Agradecimento especial... ...À Deus, Agradeço-te por tudo que tens feito na minha vida: pelos momentos alegres, tristes, vitórias e derrotas, pelas portas abertas e fechadas, mas, principalmente pela salvação em Cristo Jesus. Agradecimentos especiais... ...À minha orientadora Prof.a. Dra. Gulnara Scaf, pela sua dedicação, competência e sabedoria. ...Ao meu orientador Prof. Dr. Elcio Marcantonio Júnior, pela confiança, respeito e conhecimentos transmitidos. ...À Prof.a. Dra. Maria Lúcia Rubo de Rezende pela amizade, incentivo, apoio e exemplo na prática docente. ...À Prof.a. Dra. Ann Wenzel pela confiança, hospitalidade e conhecimentos transmitidos durante a minha estada na Universidade de Aarhus, Dinamarca Agradecimentos À Faculdade de Odontologia de Araraquara, da Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP, nas pessoas de sua Diretora Prof.a. Dra. Rosemary Adriana Chiérici Marcantonio e seu Vice-Diretor, Prof. Dr. José Cláudio Martins Segalla; Ao Coordenador do curso de Pós-Graduação em Periodontia, Prof Dr. Elcio Marcantonio Marcantonio Júnior, e ao Ex-Coordenador Prof Dr. Joni Augusto Cirelli, pela dedicação e esforço empreendidos na administração deste curso; Aos Professores do Curso de Pós-Graduação da Faculdade de Odontologia de Araraquara – UNESP, pela atenção dedicada; Aos Professores do Departamento de Periodontia da Faculdade de Odontologia de Araraquara: Adriana C. Marcantonio, Elcio Marcantonio Júnior, Carlos Rossa Júnior, Joni Augusto Cirelli, José Eduardo Sampaio, Benedito Egbert Corrêa, Silvana e Ricardo Samih G. Abi Rached, pela amizade e conhecimentos transmitidos. À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES, pela concessão das bolsa de estudos; À Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP, pelo apoio financeiro a este estudo; Aos Funcionários da Biblioteca da Faculdade de Odontologia de Araraquara – UNESP, pela gentileza e eficiência com que sempre me atenderam; Aos funcionários da seção de Pós-Graduação e da Biblioteca, pela dedicação e eficiência com que sempre me atenderam; Aos Funcionários do Departamento de Periodontia, Dona Maria do Rosário, Dona Teresa, Zezé, Claudia, e especialmente, Regina, pela dedicação, respeito, competência com que sempre me ajudaram; Aos meus colegas Rogério Margonar, Juliana A. N. D. Morais, Rafael Sartori, Rafael Faeda, Gibson Pilatti, Beatriz V. Lopes, Rhené Christiansen, Erik Gotfredsen por participarem nas diversas etapas deste trabalho; Aos meus colegas de turma: Rogério, Luiz, Rodrigo, Cliciane, Esmeralda, Karina, Marinela, Cris, Ricardo e Zé Marcos, pela convivência e amizade; Aos meus parceiros de clínica: Rogério, Fernando, Josiane pela paciência e companheirismo. À todos os colegas e amigos do curso de Pós- Graduação de Mestrado e Doutorado nas diversas áreas, pela amizade conquistada, especialmente àqueles com que tive oportunidade de conviver; Aos meus irmãos César e Patrícia, pelo amor, apoio constante, amizade e incentivo; Aos meus parentes dentistas Sérgio, Cecília, Harumi e Pricila pelo estímulo e exemplo na profissão. Aos meus irmãos de fé Gerson, Adriano, Lincoln, Paulo Renato, Sonia, Andréia, Adriana, Karen, Mauro, Carminda, Nilton, Maurício... e tantos outros, pelo apoio espiritual e incentivo que sempre me deram; ...À todos que direta ou indiretamente contribuíram para o desenvolvimento deste trabalho; Muito Obrigado. PREFÁCIO Esta tese é constituída pelos seguintes artigos: I. Sakakura, C.E., Margonar, R., Holzhausen, M., Nociti Jr, F.H., Alba Jr, R.C., Marcantonio Jr, E. Influence of cyclosporin a therapy on bone healing around titanium implants. A histometric and biomechanic study in rabbits. Publicado no Journal of Periodontology, volume 74, p. 976-81, 2003. II. Sakakura, C.E., Lopes B.M.V., Margonar, R., Nociti Jr, F.H., Pilatti G.L. Marcantonio Jr, E. Ciclosporin-A and bone density around titanium implants: a histometric study in rabitts. Trabalho finalizado e pronto para ser submetido para publicação no Journal of Oral Maxillofacial Implants. III. Sakakura, C.E., Margonar, R., Sartori, R., Morais, J.A.D.M., .Marcantonio Jr, E. The influence of cyclosporin-a on mechanical retention of dental implants integrated in bone. A study in rabbits. Submetido para publicação no Journal of Periodontology. IV. Sakakura, C.E., Marcantonio Jr, E., Wenzel A., Scaf G. The influence of cyclosporin a on quality of bone around integrated dental implants. A radiographic study in rabbits. Trabalho aceito pela revista Clinical Oral Implant Research. 2 SUMÁRIO RESUMO------------------------------------------------------------------------------------ INTRODUÇÃO ---------------------------------------------------------------------------- REVISÃO DA LITERATURA---------------------------------------------------------- CICLOSPORINA-A-------------------------------------------------------------------- OSSEOINTEGRAÇÃO----------------------------------------------------------------- Conceito--------------------------------------------------------------------- Avaliação da Osseointegração------------------------------------------- SUBTRAÇÃO RADIOGRÁFICA DIGITAL--------------------------------------------- Conceito--------------------------------------------------------------------- Interferência (Noise)------------------------------------------------------- Precisão da SRD----------------------------------------------------------- X-poseIT-------------------------------------------------------------------- PROPOSIÇÃO------------------------------------------------------------------------------ ESTUDO I----------------------------------------------------------------------------------- ESTUDO II---------------------------------------------------------------------------------- ESTUDO III--------------------------------------------------------------------------------- ESTUDO IV--------------------------------------------------------------------------------- DISCUSSÃO-------------------------------------------------------------------------------- CONCLUSÃO------------------------------------------------------------------------------ REFERÊNCIAS---------------------------------------------------------------------------- ABSTRACT--------------------------------------------------------------------------------- 3 4 6 6 9 9 9 14 14 15 17 18 20 21 42 56 70 91 93 94 104 3 SAKAKURA C.E. O efeito da ciclosporina-A na osseointegração. 2005. 104 f. Tese (Doutorado em Periodontia) – Faculdade De Odontologia de Araraquara, Universidade Estadual Paulista, Araraquara, 2005 RESUMO Agentes imunossupressores provocam alterações severas no metabolismo ósseo mineral podendo resultar em osteopenia. Tais alterações podem ser prejudiciais no processo e manutenção da osseointegração. Este estudo teve o objetivo de avaliar a influência da administração de ciclosporina –A (CSA) na osseointegração de implantes de titânio, através da avaliação: durante a cicatrização óssea ao redor de implantes dentais (Estudo I); da densidade óssea ao redor implantes dentais (Estudo II); da retenção do implante após a cicatrização óssea de implantes dentais (Estudo III) e radiográfica da qualidade óssea ao redor de implantes dentais já osseointegrados (Estudo IV). Os resultados permitiram concluir que a administração de CSA durante a cicatrização óssea resulta em diminuição da osseointegração e da densidade óssea ao redor do implante dental. Ainda, a administração de CSA após o período de cicatrização óssea ao redor do implante reduz a sua retenção mecânica ao tecido ósseo e promove a diminuição da qualidade e da densidade óssea radiográfica ao redor do implante dental. PALAVRAS CHAVE: osseointegração, ciclosporina-a, radiografia digital, subtração radiográfica 4 INTRODUÇÃO Os implantes osseointegrados (IO) revolucionaram a reabilitação de desdentados parciais e totais. Quando o conceito de osseointegração foi introduzido por Per-Ingvar Branemark (Branemark et al. 1977) foi possível alcançar um alto índice de sucesso nessa modalidade de tratamento e diversos estudos demonstraram excelente prognóstico de longo prazo (Jemt et al. 1989; Adell et al. 1990; Block & Kent, 1994; Lazzara et al.,1996). Inicialmente, os IO foram indicados para reabilitações totais (Adell et al. 1981) e posteriormente foram utilizados com sucesso na reabilitação de desdentados parciais (Zarb and Schmitt 1993a). Gradativamente, as suas indicações foram ampliadas e hoje a restauração unitária com IO é uma modalidade estabelecida de tratamento (Zarb & Schimitt 1993b). A integração do implante dental (ID) ao tecido ósseo é considerada um fator fundamental no tratamento com IO. A obtenção previsível dessa integração é diretamente dependente das propriedades da camada de óxido de titânio do ID e da qualidade e quantidade do tecido ósseo receptor. Portanto, fatores que alterem a qualidade e a quantidade do tecido ósseo podem influenciar na obtenção previsível da osseointegração. Dentre estes fatores, os mais importantes são tabagismo, discrasias sangüíneas, osteoporose, radioterapia, alterações psicológicas, alcoolismo, diabetes mellitus não controlada e uso de medicamentos imunossupressores (Balshi & Wolfinger 1999; Margonar et al. 2004; van Steenberghe et al. 2003). A presença destas alterações ou hábitos pode contra-indicar o tratamento com IO (van Steenberghe et al. 2003). A ciclosporina-A (CSA) é uma droga imunossupressora utilizada amplamente na prevenção de rejeição de aloenxertos, bem como no tratamento de doenças de natureza 5 autoimune (Guslandi & Tittobello 1992; Julian et al. 1991; Cayco et al. 2000). Dentre os efeitos adversos da CSA sobre o periodonto, a ocorrência de crescimento gengival é o fenômeno mais relatado na literatura (Seymour & Jacobs,1992; Pilatti & Sampaio 1997). Por outro lado, a administração da CSA também provoca profundas alterações no metabolismo ósseo, resultando em osteopenia (Buchinsky et al. 1996; Movsowitz et al. 1989; Julian et al. 1991; Cayco et al. 2000). Pacientes submetidos ao tratamento com CSA, após transplantes renais ou cardíacos, freqüentemente apresentam osteopenia e conseqüentemente aumento na freqüência de fraturas femorais e vertebrais (Julian et al. 1991; Cayco et al. 2000). A relação entre CSA e osseointegração é ainda pouco conhecida. Considerando a sua influência nas alterações no metabolismo ósseo, e prováveis modificações na quantidade e qualidade óssea do leito receptor de ID, torna-se relevante investigar esse tema. 6 REVISÃO DA LITERATURA CICLOSPORINA-A A ciclosporina (CSA) é um decapepitídio extraído do fungo Tolypocladium inflatum (Zocher et al. 1986). Essa droga se liga a proteínas intracelulares chamadas ciclofilinas, formando um complexo de calcineurin A e B, cálcio e calmodulina. Esses complexos interferem na produção de receptores para a interleucina-1 nas células T "helper" e bloqueiam a síntese de interleucina-2; evitam a produção de receptores para a interleucina-2 na superfície das células T indifrenciadas, bloqueando a produção de mais células T "helper", células T citotóxicas e células T "killer" ( Daley & Wysocki, 1984). A CSA tem sido empregada no tratamento de uma vasta gama de doenças do sistema imunológico como diabetes mellitus tipo I, doença de Behcet, artrite reumatóide, lupus eritematoso sistêmico, doença de Crohn, colite ulcerativa, psoríase e oftalmopatia de Graves (Guslandi & Tittobello 1992; Seymour & Jacobs 1992; Franchi et al. 2004; Pozzilli et al. 1995). Mas sem dúvida, sua maior aplicação se dá nos transplantes de órgãos, onde o seu emprego diminui os índices de rejeição e aumentou drasticamente a sobrevida dos aloenxertos (Julian et al. 1991; Cayco et al. 2000) Como toda droga, a CSA apresenta efeitos colaterais, atualmente bem conhecidos. Dentre os efeitos adversos da CSA sobre o periodonto, a ocorrência de crescimento gengival é o fenômeno mais reportado na literatura, sendo que fatores como idade, dosagem, concentração plasmática e tempo de utilização da droga, grau de inflamação gengival e de controle da placa bacteriana parecem influenciar significativamente na sua severidade (Seymour & Jacobs 1992; Boltchi et al. 1999). Outro importante efeito colateral 7 da CSA é a osteopenia (Movsowitz et al. 1988; Movsowitz et al. 1989; Julian et al. 1991; Cayco et al. 2000). Os efeitos da CSA no tecido ósseo são controversos. Nos estudos in vitro a CSA inibe a reabsorção óssea estimulada pelo paratormônio, prostaglandina E2, 1-25-dihidroxi vitamina D3 e interleucina-I (Stewart et al. 1986; Klein et al. 1994; Sasagawa et al. 1989; Klaushofer et al. 1987). Esses efeitos diretos da CSA são dependentes de sua ação imunossupressora, uma vez que o emprego dos análogos sem efeito imunossupressor não demonstrou a mesma ação (Horowitz et al. 1984). Por outro lado, estudos in vivo realizados em ratos têm demonstrado que o uso dessa droga induz a um alto metabolismo ósseo, resultando em severa osteopenia (Schlosberg et al. 1989; Movsowitz et al. 1988; Fu et al. 1999). Esse aumento no metabolismo ósseo é caracterizado pelo desequilíbrio entre a reabsorção e a formação óssea (Schlosberg et al. 1989; Movsowitz et al. 1988; Fu et al. 1999; Nassar et al. 2004), onde a taxa de reabsorção suplanta a taxa de formação resultando em perda óssea. Estudos em pacientes transplantados submetidos à terapia imunossupressora com CSA reportam que ocorrem perdas ósseas severas com aumento do risco de fraturas femorais e vertebrais típicas de osteoporose (Julian et al. 1991). Especificamente sobre o osso alveolar, FU et al. (1999) observaram inibição da formação e aumento da reabsorção óssea alveolar em sítios acometidos por periodontite. Por outro lado, Nassar et al. (2004) relataram que a CSA diminui a perda óssea inicial em sítios acometidos por doença periodontal induzida em ratos em razão das suas propriedades inibidoras do sistema imune. Especificamente sobre a osseointegração, Duarte el al (2003) relataram diminuição da formação óssea dentro das espiras dos implantes quando os animais foram submetidos a 14 dias de administração de CSA, entretanto não constataram nenhuma alteração no contato osso-implante. 8 A ação da CSA no metabolismo ósseo parece ser dependente da dose administrada. Doses não imunossupressivas (5mg/kg via oral) parecem não provocar efeitos deletérios no esqueleto ósseo dos ratos, entretanto doses de 15 a 30 mg/kg via oral produzem efeitos severos. Quanto maior a dose e o tempo de administração, mais evidente é a osteopenia nos animais (Movsowitz et al. 1988; Pozo et al. 1995). Outro fator que pode influenciar na severidade da osteopenia causada pela CSA é a idade. Animais mais jovens podem exibir uma tendência a osteopenia mais severa em função de um maior metabolismo ósseo presente na fase de crescimento (Katz et al. 1994). Entretanto, estudo recente conduzido por Spolidorio et al. (2004) demonstrou que a osteopenia observada em osso alveolar provocada por CSA não é dependente da idade. 9 OSSEOINTEGRAÇÃO CONCEITO O conceito de osseointegração foi originariamente definido por Brånemark et al. (1969) como sendo o contato direto do tecido ósseo vital com a superfície de um implante em plena função ao nível da microscopia óptica. Esses autores descreveram um tipo de fixação do implante ao osso, sem a interposição de tecido fibroso e que podia ser usado para a reabilitação de pacientes desdentados com muita previsibilidade. Posteriormente, Brånemark et al. (1985) acrescentaram que essa união deveria ser uma conexão direta, estrutural e funcional entre o osso vivo organizado e a superfície de um implante em função. Entretanto, quando a interface osso-implante é analisada por meio de microscopia eletrônica, dificilmente observa-se um real contato entre o osso e o metal, pois freqüentemente existe uma camada de proteoglicanas de 20 a 40 nm e de substância fundamental amorfa separando o osso do implante. A obtenção da osseointegração é um fator fundamental, mas não pode ser considerada como o único critério para o sucesso do tratamento com IO. O conceito de sucesso na implantodontia atual envolve resultados estéticos, fonéticos, funcionais, sistêmicos e uso clínico mínimo de 10 anos (Albrektsson & Lekholm 1989; Esposito et al. 2005). AVALIAÇÃO DA OSSEOINTEGRAÇÃO Brånemark et al. (1969) foram os primeiros autores a relatar o contato direto entre o osso e o implante sem interposição de tecido mole ou fibroso. Esse fenômeno, o qual eles chamaram de osseointegração, foi obtido em cães que tiveram seus dentes extraídos e 10 receberam posteriormente parafuso de titânio com 4mm de diâmetro e 10 mm de comprimento. Após um período de cicatrização de 3 a 4 meses, esses parafusos receberam estruturas protéticas semelhantes aos dentes antigos. As análises radiográficas e histológicas indicaram estabilidade das próteses e ainda foi observado que cada parafuso era capaz de suportar 100 kg de carga na mandíbula e 30 kg a 50 kg na maxila. Além disso, os autores relataram que ao tentar separar os implantes do osso ocorria fratura óssea ao redor do parafuso e a interface osso-implante permanecia intacta. A avaliação da interface osso-implante ganhou um novo impulso após a introdução de um método apresentado por Donath & Breuner (1982). Este método constitui no corte e desgaste para avaliação histológica de peças contento osso, dentes, implantes cerâmicos e metálicos sem a necessidade de descalcificação prévia ao corte. Os autores modificaram uma máquina de serra para madeira acoplando um sistema de refrigeração a água, permitindo o corte de blocos de resina contendo o espécime. O desgabste desse bloco de resina era feito por meio de uma lixadeira que trabalhava automaticamente até atingir a espessura programada. Com esse equipamento os autores conseguiram cortes que variavam de 5 a 10 µm de espessura com qualidade para avaliação por meio de microscópio óptico. A análise da interface osso-implante por meio de cortes não descalcificados tornou- se o padrão ouro para avaliação da osseointegração de ID. Inúmeros autores desde então utilizaram esse método para investigações sobre a osseointegração. Além da análise histológica das lâminas realizada de maneira tradicional, a análise do contato osso-implante e da formação óssea dentro das roscas do implante é realizada por meio de programas de computador que permitem quantificar a extensão do osso em contato com o metal. Nesse caso, a osseointegração é definida como a porção do tecido ósseo em contato com o metal do implante. Entretanto, não são raras as vezes em que se observam 11 segmentos do implante ou metal que não estão em contato com o osso, mas também não apresentam nenhum tipo de tecido mole interposto. Isso é considerado como uma superfície osseointegrada, em virtude desse espaço ter sido provocado pela contração da peça resultado do processo de desidratação ou de polimerização da resina1. Dependendo do tipo do osso (cortical ou trabecular) em análise e do animal (ratos, coelhos ou cães) usado para obtenção dos espécimes, têm se adotado as três ou as quatro melhores roscas consecutivas para inclusão na análise histomorfométrica (Johansson & Albrektsson 1987; Carlsson et al. 1989; Stenport et al. 2001; Ellingsen et al. 2004). Particularmente, os pesquisadores de Gotemburgo (Suécia) adotam essa metodologia na análise de espécimes obtidos de coelhos, fundamentados na característica do osso tibial desses animais. As três ou quatro primeiras roscas do implante correspondem à passagem do implante na cortical tibial do animal. Quando se adota a análise das roscas de todo o implante, freqüentemente obtém-se um valor muito baixo, por vezes não verossímil, pois se inclui na análise todo o espaço medular da tíbia do coelho, que originalmente é preenchida não por osso, mas sim por tecido medular. Por outro lado, outros autores (Cordioli et al. 2000; Sul et al. 2002) têm utilizado a análise de todas as roscas do implante com o argumento que são mais representativos. Com a inclusão de todas as roscas pode se correr o risco de diluir o efeito biológico pesquisado, portanto alguns autores têm sugerido a análise segmentada, ou seja, tanto das roscas referentes à cortical quanto à trabecular (Ellingsen et al. 2004; Duarte et al. 2005). Além disso, quando se estuda possível efeito de alterações sistêmicas no processo de osseointegração, torna-se válido analisar de forma segmentada a passagem cortical e a região trabecular do implante, uma vez que esses dois tipos de osso 1 Comunicação pessoal – Bernd Franke, Presidente da Three Roll Mills Precision Cutting and Grinding Units (EXAKT). Alemanha. 12 apresentam diferenças na velocidade de seu metabolismo (Krejci 1996; Kanis 1996; Duarte et al. 2005) Nos cortes não descalcificados também há a possibilidade de se realizar a avaliação da densidade óssea ao redor do implantes. Nociti Jr et al. (2002) propuseram a avaliação do tecido ósseo imediatamente adjacente ao implante em uma faixa restrita de 500 µm paralela ao longo eixo do implante, como uma área de possível influência do implante. Rezende (1991) e Stenport et al. (2001) descreveram uma análise similar ao de Nociti Jr et al. (2002), mas utilizando a avaliação da densidade óssea imediatamente ao redor do implante por meio da imagem espelho das roscas do implante produzida pelo programa de computador. Um outro parâmetro muito utilizado na literatura para avaliar a osseointegração é a mensuração do torque necessário para desrosquear/remover o implante que está supostamente osseointegrado. Esse teste foi introduzido por Carlsson et al. (1989) e consiste na adaptação de um torquímetro ao implante para mensurar a força máxima necessária para remover o implante do leito ósseo, ou seja, a resistência da osseointegração. Assim, esse método tem sido utilizado para avaliar o comportamento de diferentes superfícies (Cordioli et al. 2000; Li et al. 2002; Bernard et al. 2003; Elingsen et al. 2004), bem como os efeitos de alterações sistêmicas na osseointegração (Fujimoto et al. 1998; Johnsson et al. 2000; Stenport et al. 2001; Narai & Nagata 2003. Assim como a análise histométrica, comparações entre os diversos estudos ficam comprometidas em função da grande variabilidade dos animais e dos examinadores. Além disso, a tíbia do coelho apresenta padrões ósseos distintos. Na região próxima da articulação do joelho, o tecido ósseo é cortical e trabecular; na região mais distal, é somente cortical o que pode gerar diferenças entre os resultados de torque de remoção. Entretanto, 13 podemos notar padrões de comportamento semelhantes como o aumento do torque de remoção com o passar do tempo, o que demonstra a consolidação óssea ao redor do ID (Rezende (1991); Johansson & Albrektsson 1987; Li et al. 2002; Ellingsen et al. 2004). 14 SUBTRAÇÃO RADIOGRÁFICA DIGITAL (SRD) CONCEITO DE SRD A subtração radiográfica digital foi introduzida na Odontologia na década de oitenta por Webber et al. (1982), Grondahl et al. (1983) e Hausmann et al. (1985). Consiste em uma operação de subtração, na qual as estruturas que não apresentaram mudanças entre um exame e outro são eliminadas, evidenciando as estruturas que apresentaram mudanças. A imagem radiográfica digital é representada por uma matriz matemática, na qual cada casa representa um pixel, cujo valor pode variar de 0 a 255 (tons de cinza em um sistema de 8 bits). Quando duas imagens radiográficas são subtraídas, o computador realiza uma operação de subtração entre uma matriz (imagem inicial) e outra (imagem final) gerando uma terceira matriz (imagem subtraída), sendo que o valor zero representa ausência de mudança e um valor diferente de zero representa mudança, podendo ser um valor positivo ou negativo. Com o objetivo de gerar uma imagem subtraída de fácil leitura, os programas de SRD acrescentam automaticamente aos valores subtraídos o valor de 128, o que representa um tom médio de cinza (Figura 1-Estudo IV) . Assim, uma perfeita subtração de um sítio sem alteração óssea deverá mostrar ausência das estruturas anatômicas, resultando em uma imagem cujo nível de cinza deverá ser 128. Os pixels de uma região onde houve ganho de densidade deverão apresentar nível de cinza superior a 128. Pixels que apresentaram perda da densidade devem apresentar valores inferiores a 128 (Christgau et al. 1998a; Christgau et al. 1998b). Pode se resumir a operação de SRD em uma fórmula muito simples: 15 n1-n2 = n3+128, sendo: n1- imagem inicial n2 – imagem final n3 – imagem subtraída 128 – tom médio de cinza INTERFERÊNCIA (“NOISE”) O processo de padronização das imagens é relevante na SRD. Para a obtenção de uma imagem subtraída com validade é imperativo que as duas imagens a serem subtraídas apresentem a mesma projeção geométrica e densidade radiográfica (Weber et al. 1982; Grondahl et al. 1983; Hausmann et al. 1985; Wenzel & Sewerin 1991). Diversos dispositivos para a padronização de séries radiográficas foram descritos previamente, geralmente baseados em métodos de moldagem dos dentes e fixação filme- paciente-tubo de raios-X (Nery et al. 1985; Rudolph & White 1988). Além disso, todo cuidado deve ser empregado no ajuste do tempo de exposição, quilovoltagem e miliamperagem do aparelho de raios-X, no processamento e na digitalização do filme radiográfico (Weber et al. 1982; Grondahl et al. 1983; Hausmann et al. 1985; Wenzel and Sewerin 1991; Christgau et al. 1998a; Schou et al. 2003). Pequenos descuidos ocorridos nos diversos passos de padronização da imagem podem gerar resultados na imagem subtraída que não são originários do processo doença/tratamento ocorrido no período compreendido entre as duas imagens radiográficas. Esse resultado falso-positivo ou negativo conceitua-se como interferência da imagem (Wenzel & Sewerin, 1991). Os programas de SRD devem ser capazes de compensar pequenas interferências que inevitavelmente ocorrem na obtenção de duas imagens padronizadas. Programas de SRD 16 prévios proporcionavam uma sobreposição manual, e pequenos desajustes geométricos eram corrigidos com movimentos rotacionais e translacionais (Wenzel 1989). O ajuste geométrico disponível nos programas atuais é baseado em pontos de referência que permitem um ajuste matemático e automático entre a imagem inicial e a final, facilitando a obtenção de uma melhor sobreposição das imagens⏐ Apesar da correção de pequenas interferências oriundas das inevitáveis diferenças entre a imagem inicial e final, ainda é preciso caracterizar a origem da diferença presente na imagem subtraída, ou seja, se a mudança ocorreu devido a uma alteração fisiológica do tecido ósseo ou ao tratamento instituído. Segundo Wenzel & Sewerin (1991) uma imagem subtraída homogenea é aquela que apresenta em seus pixels uma pequena variação nos tons de cinza. O uso do histograma da região de controle (RC) da imagem subtraída fornece a amplitude de variação dos tons de cinza na imagem subtraída. Histogramas de RC com grande amplitude de tons de cinza podem significar que há grande quantidade de interferência. De qualquer forma, toda imagem subtraída apresenta interferência que não pode ser evitada e isso deve ser considerado na avaliação quantitativa das alterações na densidade óssea. Nesses casos recomenda-se considerar como interferência um ou mais desvios-padrão observados no histograma da RC da imagem subtraída. Schou et al. (2003), avaliando regeneração óssea ao redor de defeitos peri-implantares em macacos encontraram um desvio - padrão de 10 níveis de cinza nas regiões de controle e consideraram como alteração óssea de ganho/perda 128 mais ou menos dois desvios-padrão (20 níveis de cinza). Schropp et al. (2003) avaliando a remodelação óssea pós-extração dentária também utilizaram dois ⏐ Comunicação Pessoal - Haiter-Neto F & Wenzel A. Noise in subtraction images made from pairs of bitewing radiographs. A comparison between two subtraction programs. Artigo aceito pela revista Dentomaxillofac Radiol. 17 desvios-padrão como interferência na análise por SRD. Entretanto, segundo Wenzel2 não existe um número de desvio-padrão correto a ser adotado e deverá ser definido a critério do pesquisador dependendo da natureza de cada experimento, podendo apresentar variação nas magnitudes das interferências. PRECISÃO DA SRD Mensurações realizadas em radiografias convencionais e digitais demonstram alterações ósseas somente em um plano (ex. altura óssea), por outro lado, a SRD pode proporciona informações a respeito das alterações ósseas em até três parâmetros (altura, densidade óssea e área). Christgau et al. (1998b) demonstraram em estudo in vitro alta correlação entre aumento de espessura e mudança na densidade óssea detectada pela SRD. Neste estudo, foram encontradas altas correlações lineares que variaram de r2= 0,89 a 0,99 para osso cortical e de r2 = 0,61 a 0,86. Nesse estudo o limite de detecção da SRD foi um aumento de 200 µm para cortical e 500 µm para medular na espessura óssea enquanto que para radiografia convencional foi respectivamente 600 µm e 2850 µm comprovando a alta sensibilidade da SRD em detectar alterações na espessura óssea. As alterações na massa de cálcio também são detectadas pela SRD. Christgau et al. (1998a), em estudo in vitro, demonstraram uma alta correlação linear entre alterações de cálcio na massa óssea por pixel e mudanças na densidade óssea detectada por SRD. Uma média de 0,1-0,15 mg de cálcio foi o mínimo de perda necessária para a SRD detectar. Assim, a SRD pode ser aplicada com o objetivo de detectar discretas alterações em tecido duro como osso, esmalte e dentina (Nummikoski et al. 1992; Jeffcoat et al. 1992; 2 Comunicação pessoal – Dr. Ann Wenzel, PhD, Dr. Odont, Head and Chair, Professor of Radiology Department, Aarhus University, Denmark 18 Schropp et al. 2003). Na Odontologia a SRD é utilizada quando o objetivo é detectar alterações sutis de forma mais precoce possível. Na Periodontia e Implantodontia a SRD é utilizada para o diagnóstico de perdas ósseas iniciais provocadas pelas lesões periodontais proporcionando uma intervenção mais precoce (Moreland et al. 1992; Jeffcoat et al. 1992). Essa técnica também é utilizada na avaliação dos resultados de terapias regenerativas como enxertos ósseos e regeneração óssea e tecidual guiada proporcionando não somente resultados qualitativos, mas também quantitativos (Christgau et al. 1996; Christgau et al. 1998a; Christgau et al. 1998b), bem como no estudo da remodelação óssea em humanos (Schropp et al. 2003) X-POSEIT Este programa de subtração radiográfica está baseado no posicionamento de ilimitados pontos de referência com o objetivo de permitir o alinhamento das duas imagens a serem subtraídas (Figura 1 – Estudo IV). Neste programa, existe uma ferramenta que permite verificar a precisão do alinhamento dos pontos de referência, possibilitando correções para um melhor alinhamento (rotação, translação, tamanho, distorção perspectiva). O programa permite, ainda, a correção gama automática com o objetivo de corrigir a densidade e o contraste das radiografias. A colocação dos pontos de referência em ambas radiografias certamente é a operação que consome mais tempo no processo de subtração das imagens. O programa também oferece a possibilidade de se definir várias regiões de interesse e/ou controle que podem ser desenhadas com auxílio do “mouse”. Tanto os pontos de referências, como as regiões de interesse/controle podem ser apagadas e refeitas. 19 Neste programa há a possibilidade de se definir, em nível de cinza, a interferência provocada pelo processo de exposição e processamento da radiografia. Isso é feito por meio das opções do programa, na qual se pode definir quantos desvios - padrão serão considerados na subtração. Nas imagens subtraídas, as regiões que sofreram perda ou ganho ósseo aparecem, respectivamente, mais escuras e mais claras que a imagem subtraída. O programa permite que essas alterações sejam coloridas de acordo com a necessidade do pesquisador (Figura 1C – Estudo IV). Todos os dados quantitativos referentes ao nível de cinza e à área, tanto da região de ganho, perda ou com ausência de alterações, são automaticamente exportados para um banco de dados para futura análise estatística. As imagens também podem ser copiadas e enviadas para outras pessoas ou programas. 20 PROPOSIÇÃO O objetivo desta tese foi avaliar a influência da ciclosporina-A na osseointegração. Os objetivos específicos desta tese foram: 1. Avaliar histometricamente e biomecanicamente a influência da CSA durante a cicatrização óssea de implantes dentais (Estudo I) 2. Avaliar histometrricamente a influência da CSA na densidade óssea ao redor de implantes dentais (Estudo II) 3. Avaliar a influência da CSA na retenção do implante após a cicatrização óssea de implantes dentais (Estudo III) 4. Avaliar radiograficamente a influência da CSA na qualidade óssea ao redor de implantes dentais com osseointegração estabelecida (Estudo IV) 21 ESTUDO I Artigo intitulado “Influence Of Cyclosporin A Therapy On Bone Healing Around Titanium Implants. A Histometric And Biomechanic Study In Rabbits” Publicado no Journal of Periodontology, volume 74, p. 976-81, 2003. 22 INFLUENCE OF CYCLOSPORIN-A THERAPY ON BONE HEALING AROUND TITANIUM IMPLANTS. A HISTOMETRIC AND BIOMECHANIC STUDY IN RABBITS. Celso E. Sakakura, D.D.S., M.S. * Rogério Margonar, D.D.S., M.S. * Marinella Holzhausen, D.D.S., M.S. * Francisco H. Nociti Jr., D.D.S., M.S., Ph.D. † Rodolfo Candia Alba Jr., D.D.S.‡ Elcio Marcantonio Jr., D.D.S., M.S., Ph.D. * * Departament of Periodontology – Dental School of Araraquara, State University of São Paulo (UNESP) Araraquara, São Paulo, Brazil. † Dept. of Prosthodontics and Periodontics, Division of Periodontics, School of Dentistry at Piracicaba, UNICAMP, São Paulo, Brazil. ‡ Private Practice Author to whom correspondence should be sent: Elcio Marcantonio Jr, DDS, PhD, Departamento de Cirurgia e Diagnóstico, Disciplina de Periodontia; Faculdade de Odontologia de Araraquara, UNESP Rua Humaitá, 1680, Centro, 14801-903, Araraquara, SP, Brazil,. Phone/Fax: 55 (16) 201- 6314, e-mail: elciojr@foar.unesp.br Financial support: FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo, grant no. 1999/09672-2 and CAPES. Running title: Immunosuppression and Titanium implants – Study in Rabbits 23 ABSTRACT Background: Immunosuppressive agents may induce severe changes on bone metabolism. The purpose of the present study was to evaluate the influence of the administration of cyclosporin A (CsA) on the bone tissue around titanium implants. Methods: Eighteen New Zealand rabbits were randomly divided into 2 groups with nine animals each. The test group (CsA) received daily subcutaneous injection of CsA (10mg/kg bodyweight) and the control group (CTL) received saline solution by the same route of administration. Three days after the beginning of therapy, 2 implants (7.0 mm in lenght and 3.75 mm in diameter) were inserted bilaterally at the region of the tibial methaphysis. After 4, 8 and 12 weeks the animals were sacrificed and the biomechanical tests and the histometrical procedures, consisting of the determination of the percentages of bone-implant contact and bone area within the limits of the implant threads, were performed. Results: Intergroup analysis showed that the removal torque and the percentage of bone contact with the implant surface for CsA group were significantly lower than those of the CTL group at the experimental period of 12 weeks (28.5 and 39.2 N.cm, p=0.01; 7.76 and 18.52%, p=0.02; respectively). Conclusion: The data of the present study suggest that long term administration of cyclosporin-A may negatively influence bone healing around dental implants. Key words: Dental implants / osseointegration; cyclosporin A / Systemic conditions; histometric. 24 INTRODUCTION Cyclosporin-A (CsA) is a potent immunosuppressive agent produced by the fungi Cylindrocarpon lucidum 1 that has been widely used to prevent organ rejection after allograft transplantation and in the treatment of several autoimmune diseases like pemphigus vulgaris, Behcet's disease, lichen planus, psoriasis and diabetes mellitus. 2- 4 Cyclosporin-A acts on the immune system by selectively suppressing the helper T cells, inhibiting the macrophage production of IL-2 and IL-1 and decreasing the cytotoxic T lymphocytes proliferation and differentiation. 2, 5-10 The use of this drug can lead to a number of side effects, being the gingival overgrowth the most reported effect in the dental literature. 11, 12 Other adverse effects have also been reported following CsA use: nephrotoxicity, hepatotoxicity, hypertension and osteoporosis. 3, 13, 14 The effects of cyclosporin A on the bone tissue appear contradictory. Some studies in rats 6, 7, 15, 16, 17, 18 have showed that this drug leads to a high bone turnover, resulting in severe osteopenia. Furthermore, bone loss has been reported in patients receiving CsA immunotherapy after transplantation. 2, 3, 4, 13, 14 The effects of CsA particularly on the alveolar bone have been reported by Fu et al. (1999) 15 who showed decreased bone formation and increased osteoclasia in rats. On the other hand, some in vitro studies have showed that CsA could have a protective action on the bone by inhibiting the bone resorption stimulated by parathyroid hormone, interleukin-1 (IL-1), prostaglandin E2, 1,25-dihydroxyvitamin D3, osteoclast-activating factor, thrombin and lipopolysaccharides. 5, 8, 19, 20 There is little information in the literature regarding the effects of immunosuppressive agents on the osseointegration process. Duarte et al. (2001)22 showed that immunosuppressive therapy with CsA and nifedipine may influence bone healing around titanium implants by 25 decreasing the bone area within the limits of the threads of the implant. The aim of the present study was to investigate the influence of immunosuppressive therapy with CsA alone on the biomechanical retention and osseointegration of commercially pure (CP) titanium implants in the rabbit tibia. MATERIALS AND METHODS ANIMALS Eighteen New Zealand white rabbits, with 9 to 12 months old (3500-4500 g) were used in the study. The animals were housed in individual cages, fed by a standard laboratory diet and given tap water ad libitum. The experiment was approved by the Institutional Experimentation Committee of the School of Dentistry of Araraquara, São Paulo, Brazil. EXPERIMENTAL PROTOCOL After a 2-week acclimatization period, the animals were randomly divided into two groups, a test (CsA) and a control (CTL) group, with nine animals each. The CsA group received a daily subcutaneous immunosuppressive dose of 10-mg/kg bodyweight Cyclosporin-A∗, whereas the CTL group received saline solution (NaCl 0.9%) by the same route of administration. The administration of the drugs began three days before the implants placement and lasted until 3 animals were killed per group, at 4, 8 and 12 weeks postoperative. ∗ Sandimmum®, Novartis Pharma AG, Switzerland. 26 IMPLANT SURGERY The animals were anesthetized by intramuscular injections of a combination of ketamine† (0.35 mg/kg bodyweight) and xylazine‡ (0.5mg/kg bodyweight). The region of the tibial metaphysis was cleansed with iodine surgical soap. Incisions of approximately 3 cm in length were performed bilaterally at the internal side of the hind-leg, just below the knee. After gentle dissection, the bone surface of the tibial metaphysis was exposed. Unicortical implant beds were prepared by using a progressive sequence of spiral drills under generous saline cooling. Two implants§, (7mm in length and 3,75 mm in diameter) one with a modified head to enable fixation of the torque meter and the other with a conventional head, were placed in each leg. The soft tissues were sutured in separate layers and the animals received a single intramuscular injection of antibiotic | (0.1 ml/kg bodyweight of an association of Penicillin with Streptomycin.) postoperatively. REMOVAL TORQUE After four, eight and twelve weeks, the implants were surgically exposed under general anesthesia. The specially designed key connected the modified head implant with the torque manometer. ¶ An anticlockwise movement was performed in order to remove the implant. The maximal torque necessary for manual removal of each implant was measured in Newton centimeters. † Francotar®; Virbac do Brasil Ltda, Brazil. ‡ Rumpum ® Bayer S.A. São Paulo, Brazil § Master Screw®, Conexão, São Paulo, Brazil. | Pentabiótico®, Wyeth-Whitehall Ltda, São Paulo, Brazil ¶ 15-BTG, Tonich, Japan. 27 HISTOMETRIC PROCEDURE After the animals were killed, the conventional implants with surrounding tissue in each tibia were removed and fixed in 4% neutral formalin for 48 hours. Undecalcified sections were prepared by a technique previously described by Donath & Breuner (1992).23 Subsequently, the sections were stained as follows 24: i) the slide-containing specimen was placed in a vessel containing Stevenel’s blue preheated to, and maintained at, 60 °C for 15 minutes; ii) the specimen was rinsed in distilled water at 60 °C and air dried; iii) a small amount of alizarin red S was placed onto the specimen surface at room temperature for 5 minutes. Then, it was washed thoroughly in running distilled water to remove excess stain and air dried. The percentage of bone contact with the implant surface and the bone area formed within the threads were measured in both sides of the implant, at the three first threads. The mean of both sides of the implant were considered. STATISTICAL ANALYSIS The differences in the removal torque, in the percentage of bone contact with the implant surface or in the percentage of bone area within the threads of the implant among groups (CsA and CTL) and, among the different experimental periods (4, 8 or 12 weeks) in the same group were tested by the non-parametric Mann Whitney and Wilcoxon tests (p < 0.05). Whether the immunosuppressive therapy would influence body weight was evaluated by intergroup analysis of the difference between final and initial weight using paired t-test. 28 RESULTS CLINICAL OBSERVATIONS The rabbits in the CsA group developed some side effects like hair and mustache growth. A significant (p< 0,01) weight loss was also observed in the animals of the CsA group (- 994 g and - 67g, for CsA and CTL, respectively; see Fig. 1). REMOVAL TORQUE VALUES The intragroup analysis (Table 1) revealed significant differences only in the CTL group between the periods of 4 and 8 weeks (15.8 + 5.0 N.cm and 32.3 + 8.5 N.cm, respectively; p = 0.02) and between the periods of 4 and 12 weeks (15.8 + 5.0 N.cm and 39.2 + 5.8 N.cm , respectively; p = 0.02). When data were compared between CTL and CsA groups (Table 1), a significant difference was found at the experimental period of 12 weeks (39.2 + 5.8 N.cm and 28.5 + 5.7 N.cm, respectively; p=0.01). HISTOMETRIC RESULTS Intragroup analysis (Table2 and 3) showed significant difference regarding the percentage of bone contact with the implant surface only in the CsA group between the experimental periods of 4 and 12 weeks (17.08 + 7.95% and 7.76 + 6.36%, respectively; p= 0.02). With regard to the percentage of bone area within the threads of the implant, there were significant differences in the CTL group between the experimental periods of 4 and 8 weeks (48.35 + 6.2% and 66.57 + 8.1%, respectively; p = 0.04) and, between 4 and 12 weeks (48.35 + 6.2% and 71.18 + 8.76%, respectively; p = 0.02). 29 Intergroup analysis (Tables 2 and 3) showed significant difference regarding the percentage of bone contact with the implant surface only in the experimental period of 12 weeks (7.76 + 6.30% and 18.52 + 5.3%, for CsA and CTL groups, respectively; p=0.02). With regard to the percentage of bone area within the threads of the implant, there were no significant differences between groups in any of the experimental periods. DISCUSSION The resistance of the titanium implant to the removal force can be correlated to the degree of contact between mineralized bone and irregularities on the implant surface.25, 26 Thus, the removal torque of an implant is influenced by the mechanical properties of the adjacent bone (quality and quantity) and by the degree of bone-implant contact.25, 27 Bone remodeling occurs progressively, increasing the degree of bone-implant contact, consequently increasing the torque needed to remove the implant. 25- 28 The results of the present study for the CTL group were similar to the findings of previous studies that observed an increase in the removal torque values with time (Table 1), 25- 28 which suggests that an increase in the degree of bone-implant contact (Table 2 and Fig. 2). On the other hand, the CsA group did not show a similar behavior. There were no significant increases in the removal force (Table1), degree of bone-implant contact (Table2) and percentage of bone area within the threads of the implant (Table 3) between observation periods. In fact, it was observed a decrease in the bone-implant contact evaluation (Table 2), which suggests a poor osseointegration process (Fig. 3), with low implant retention to the bone (see torque values – Table1). 30 Low values for the evaluated parameters obtained for the CsA group, mainly at the longest period (12 weeks), are probably due to the low percentage of bone-implant contact28 (Table 2 and Fig. 6b). These alterations observed in the bone tissue of the CsA group animals can be explained by the action of cyclosporin-A. It is known that the immune system actively participates in bone mineral metabolism and that the T lymphocytes play a critical role in the development of CsA-induced osteopenia.29 This is not surprising as the T cell is the traditional target of CsA and naturally occurring T lymphocyte perturbations are implicated in the development of primary osteoporosis in humans. 29 Besides, an in vitro study 30 corroborates with this result, by describing the necessity of thymus-derived lymphocytes presence for the production of the osteoclast-activating factor. T lymphocytes suppression results in a high bone metabolism state, where the bone formation is supplanted by the bone resorption, leading to a decrease in the trabecular bone volume.6, 7, 29 Under these conditions, a decreased percentage of bone contact with the implant surface was observed. The precise mechanism of action of CsA on bone tissue is still not well understood. It is known that these bone alterations correlate with immunosuppressive mechanisms and are mediated by cytokines.9, 10, 21, 29, Moreover, possible CsA effects on osteoblasts and osteoclasts are not rejected and may result in a secondary phenomenon, leading to a high bone remodeling state with exceeding bone resorption.9 Evaluating the results obtained for control and test groups during the three experimental periods, it was observed that test group showed minimum increases for torque parameter (Table 1) and percentage of bone within the threads of the implant (Table 3) and, a decrease in the parameter percentage of bone contact with the implant (Table 2). Between the periods of 4 and 12 weeks, the values for the control group supplanted those of the test group (Tables 1, 2 and 3), which suggests that the effects of CsA administration on the osseointegration process turned evident only after an administration period longer than 4 weeks. This result is 31 supported by Movsowitz et al (1988)7 who showed that the effect of CsA on bone is dependent on the duration and dose of CsA. Similarly, Duarte et al. (2001) 22 have showed that, after CsA administration for two weeks, no changes in the percentage of bone contact with the implant were detected in the experimental periods of 2 or 6 weeks. The changes were limited to a lower bone formation within the limits of the threads of the implant which, according to Johansson & Albrektsson (1987),28 is not fundamental for implant anchorage, being the bone-implant contact the most important factor for higher removal torque. Considering that the CsA effects are more severe after longer administrations,7 we can understand the reason why the differences between control and test group turned significant only in the last experimental period of our study (Tables 1 and 2). Another important aspect to be observed when comparing the values for removal torque and the values for bone implant contact in the test group is that the results seem to be contradictory. Contrary to our expectations, the decrease in the percentage of bone implant contact with time, although not being significant, was not accompanied by a decrease in the removal torque values. A possible explanation for this fact may be the difference in the anatomic location of implants in the tibia. The implants designed for the biomechanical test were placed close to the knee joint, whereas those designed for the histological analysis were placed in a more distal position. Whether the difference in the porosity or in the architecture of the bone would promote different results remains to be investigated. Therefore, future studies should be considered in order to evaluate this topic. The rabbit was the animal model used in the present investigation not only because of its small size but also because several studies are available relating its use in the study of osseointegration.25-28 However, according to Gratwohl et al.,31 rabbits treated with immunosuppressive doses of CsA for prolonged periods of time may develop a clinically distinct toxic syndrome characterized by loss of weight, reduced food and water intake and 32 reduced movements. One can speculate that this side effect could have interfered with the results of the test group. However, because there is little information in the literature, we can not determine how much of the observed effects were due to the possible toxicity of CsA. The choice for the experimental periods of 4, 8 and 12 weeks aimed to evaluate the influence of CsA administration on the whole osseointegration process, specially on the period related to the maturation of woven bone to lamellar bone, which lasts 6 weeks in rabbits. 28 In order to reach a successful osseointegration, this critical period should occur without healing disturbances. Therefore, the influence of CsA could be evaluated in the most critical period of bone healing in the rabbit. In the present study, the subcutaneous route of administration of CsA was chosen because it can promote more consistent immunologic levels than oral route once CsA is poorly absorbed from the gastrointestinal tract in the rabbit.31 The dosage of CsA (10mg/kg/day body weight) used in the present study was equal to the one used by Duarte et al.22 and it can promote serum levels of CsA ranging from 100 to 400ng/ml which are able to sustain an allograft transplantation in rabbits.31 Similarly, serum levels of CsA ranging from 100 to 400 ng/ml are described in patients receiving immunotherapy after renal transplantation.32 Besides, higher dosages could lead to systemic complications and could cause nephrotoxicity, impairing the results. We have concluded that CsA administration, for periods of time greater than 4 weeks, can decrease the osseointegration process of commercially pure (CP) titanium implants inserted in rabbit tibia. However, further investigations are necessary in order to evaluate the effect of CsA on implants which have already suffered osseointegration. 33 ACKNOWLEDGMENTS This study was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) grant no. 1999/09672-2 and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). The authors greatly appreciated the assistance of Dr. Beatriz Maria Valério Lopes in surgical phase. We also thank Solange Aranha for English review. REFERENCES 1. Ruegger A, Kuhn M, Lichti H, et al. Cyclosporin A, a Peptide Metabolite from Trichoderma polysporum (Link ex Pers.) Rifai, with a remarkable immunosuppressive activity Helv Chim Acta. 1976; 59:1075-92. 2. Cayco A, Wysolmerski J, Simpson C, et al. Posttransplant bone disease: evidence for a high bone resorption state. Transplantation 2000; 70: 1722-1728. 3. Julian BA, Laskow DA, Dubovsky J, Dubovsky EV, Curtis JJ, Quarles LD. Rapid loss of vertebral mineral density after renal transplantation. N Engl J Med 1991; 325: 544-50. 4. Vedi S, Greer S, Skingle SJ, et al. Mechanism of bone loss after liver transplantation: a histomorphometric analysis. J Bone Miner Res 1999; 14: 281-287. 5. Mccauley LK, Rosol TJ, Capen CC. Effects of cyclosporin A on rat osteoblasts (ROS 17/2.8 Cells) in vitro. Calcif Tissue Int 1992; 51: 291-297. 6. Movsowitz C, Epstein S, Ismail F, Fallon M, Thomas S. Cyclosporin A in the oophorectomized rat: unexpected severe bone resorption. J Bone Miner Res 1989; 4: 393-398. 34 7. Movsowitz C, Epstein S, Fallon M, Ismail F, Thomas S. Cyclosporin-A in vivo procedures severe osteopenia in the rat: effect of dose and duration of administration. Endocrinol 1988; 123: 2571-2577. 8. Orcel P, Denne MA, De Vernejoul MC. Cyclosporin-A in vitro decreases bone resorption, osteoclast formation, and the fusion of cells of the monocyte-macrophage lineage. Endocrinol 1991;128:1638-1646. 9. Rucinski B, Liu CC, Epstein S. Utilization of cyclosporine H to elucidate the possible mechanisms of cyclosporine A – Induced osteopenia in the rat. Metabolism 1994; 43:1114 – 1118. 10. Sasagawa K, Fushibayashi S, Okano K, et al. Different inhibitory actions of immunomodulating agents and immunosuppressive agents on bone resorption of mouse calvaria. Int J Immunopharmac 1989; 11: 953-959. 11. Rateitschak-Pluss EM, Hefti A, Lortscher R, Thiel G. Initial Observation that cyclosporin A induces gingival enlargement in man. J Clin Periodontol 1983;10: 237-46. 12. Seymour RA, Jacobs DJ. Cyclosporin and the gingival tissues. J Clin Periodontol 1992;19: 1-11. 13. Shane E, Rodino MA, McMahon DJ. et al. Prevention of bone loss after heart transplantation with antiresorptive therapy: a pilot study. J Heart Lung Transplant 1998; 17: 1089-1096. 14. Thiébaud D, Krieg MA, Gillard-Berguer D, Jacquet AF, Goy JJ, Burckhardt P. Cyclosporine induces high bone turnover and may contribute to bone loss after heart transplantation. Eur J Clin Invest 1996; 26: 549-555. 15. Fu E, Hsieh Y-D, Nieh S, Wikesjö, Liu D. Effects of cyclosporin A on Alveolar bone: an experimental study in the rat. J Periodontol 1999; 70:189-194. 35 16. Katz I, Li M, Joffe I, et al. Influence of age on cyclosporine A-induced alterations in bone mineral metabolism in the rat in vivo. J Bone Miner Res 1994; 9: 59-67. 17. Klein L, Lemel MS, Wolfe MS, Shaffer J. Cyclosporin A does not affect the absolute rate of cortical bone resorption at the organ level in the growing rat. Calcif Tissue Int 1994; 55: 295-301. 18. Schlosberg M, Movsowitz C, Epstein S, Ismail F, Fallon M, Thomas S. The effect of cyclosporin A administration and its withdrawal on bone mineral metabolism in the rat. Endocrinol 1989; 124: 2179-2184. 19. Stewart PJ, Green OC, Stern PH. Cyclosporine A inhibits calcemia hormone-induced bone resorption in vitro. J Bone Miner Res 1986; 1: 285-291. 20. Chowdhury MH, Shen V, Dempster, DW. Effects of cyclosporine A on chick osteoclast in vitro. Calcif Tissue Int 1991; 49: 275-279. 21. Klaushofer K, Hoffmann O, Stewart PJ, et al. Cyclosporine A inhibits bone resorption in cultured neonatal mouse calvaria. J Pharmacol Exp Ther 1987; 243: 584-590. 22. Duarte PM, Nogueira Filho GR, Sallum EA, Sallum AW, Nociti Junior FH. The effect of an immunosuppressive therapy and its withdrawal on bone healing around titanium implants. A histometric study in rabbits. J Periodontol 2001; 72:1391-7. 23. Donath K, Breuner G. A method for study of undecalcified bones and teeth with attached soft tissue. The sage-Scliff (sawing and grinding) technique. J Oral Pathol 1982; 11: 318- 326. 24. Maniatopoulos C, Rodrigues A, Deporter DA, Melcher AH. An improved method for preparing histological sections of metallic implant Int J Oral Maxillofac Implants 1986; 1: 31- 37. 36 25. Sennerby L, Thomsen P, Ericson LE. A morphometric and biomechanic comparison of titanium implants inserted in rabbit cortical and cancellous bone. Int J Oral Maxillofac Implants 1992; 7: 62-71. 26. Carlsson L, Röstlund T, Albrektsson B, Albrektsson T. Removal torques for polished and rough titanium implants. Int J Oral Maxillofac Implants 1988; 3: 21-24. 27. Ivanoff C-J, Sennerby L, Lekholm U. Influence of mono- and bicortical anchorage on the integration of titanium implants. A study in the rabbit tibia Int Oral Maxillofac Surg 1996; 25: 229-235. 28. Johansson C, Albrektsson T. Integration of screw implants in the rabbit: A 1-yr follow-up of removal torque of titanium implants. Int J Oral Maxillofac Implants 1987; 2: 69-75. 29. Buchinsky FJ, Ma Y, Mann GN, et al. T lynphocytes play a critical role in the development of cyclosporin A–induced osteopenia. Endocrinology 1996; 137: 2278-2285. 30. Horowitz M, Vignery A, Gershon RK, Baron R. Thymus-derived lymphocytes and their interaction with macrophages are required for production of osteoclast-acting factor in mouse. Proc Natl Acad Sci USA 1984; 81: 2181-2185. 31. Gratwohl A, Riederer I, Graf E, Speck B. Cyclosporine toxicity in rabbits. Lab Anim 1986; 20: 213-220. 32. Boltchi FE, Rees TD, Iacopino AM. Cyclosporine A - induced gingival overgrowth: A comprehensive review. Quintessence int 1999; 30: 775-783. 37 TABLES Table 1 – Mean and standard deviation of removal torque (N.cm) for each experimental group in each experimental period. Experimental groups CTL CsA p value 4 weeks 15.8 + 5.0 21.8 + 5.1 0.09 8 weeks 32.3 + 8.5† 24.7 + 2.3 0.12 12 weeks 39.2 + 5.8‡ 28.5 + 5.7 0.01* *Significant difference at p<0.05, Mann-Whitney test. † p=0.02, significant difference between the periods of 4 and 8 weeks in the CTL group, Wilcoxon test. ‡p=0.02, significant difference between the periods of 4 and 12 weeks in the CTL group, Wilcoxon test. Table 2 – Mean and standard deviation of bone contact (%) with the implant surface for each experimental group according to experimental period. Experimental groups CTL CsA p value 4 weeks 9.76 + 4.7% 17.08 + 7.95% 0.26 8 weeks 13.08 + 3.77% 12.5 + 3.66% 0.74 12 weeks 18.52 + 5.3% 7.76 + 6.30%† 0.02* *Significant difference at p<0.05, Mann-Whitney test. †p= 0.02, significant difference between the periods of 4 and 12 weeks in the CsA group, Wilcoxon test. 38 Table 3 – Mean and standard deviation of bone area (%) within the limits of the implant threads for each experimental group according to experimental period. † p= 0.02, significant difference between the periods of 4 and 8 weeks in the CTL group, Wilcoxon test. Experimental groups CTL CsA p value 4 weeks 48.35 + 6.2% 46.27 + 6.17 % 0.52 8 weeks 66.57 + 8.1%† 51.07 + 10.34 % 0.26 12 weeks 71.18 + 8.7%‡ 59.50 + 5.47 % 0.1 ‡ p= 0.02, significant difference between the periods of 4 and 12 weeks in the CTL group, Wilcoxon test. 39 FIGURES Figure 1 – Mean and standard deviation of the initial and final body weight (Kg) for CTL and CsA groups. p<0.01* 43.8 33.7 0 1 2 3 4 5 B od y w ei gh t ( K g) p>0.40 Initial Final CTL CsA * Significance difference at p<0.05, T-test. 40 Figure 2a Figure 2b I Figure 2 – Photomicrograph illustrating the histological aspect of the bone formed within the limits of the threads of an implant for CTL group, 12 weeks / Original magnification = 25x (Figure 2a). Note the direct bone contact with the implant surface / Original magnification =100x. Stevenel’s blue and alizarin red S (Figure 2b). B/I 41 Figure 3a Figure 3b I Figure 3 Photomicrograph illustrating the histological aspect of the bone formed within the limits of the threads of an implant for CsA group, 12 weeks / Original magnification = 25x (Figure 3a). Note the small extension of bone contact with the implant surface / Original magnification = 100x (Figure 3b). Stevenel’s blue and alizarin red S. 42 ESTUDO II Artigo intitulado “Ciclosporin-A and bone density around titanium implants: a histometric study in rabitts”. Submetido para avaliação no Journal of Periodontology 43 CYCLOSPORIN-A AND BONE DENSITY AROUND TITANIUM IMPLANTS: A HISTOMETRIC STUDY IN RABITTS Celso Eduardo Sakakura, DDS, MS* Beatriz M.V. Lopes, DDS, MS* Rogério Margonar DDS, MS* Francisco Humberto Nociti Júnior, DDS, MS, PhD† Gibson Luiz Pilatti, DDS, MS, PhD& Elcio Marcantonio Júnior, DDS, MS, PhD* * Department of Periodontology – Araraquara Dental School, São Paulo State University (UNESP) Araraquara, São Paulo, Brazil. † Department of Prosthodontics and Periodontics, Division of Periodontics, Piracicaba Dental School, UNICAMP, São Paulo, Brazil. & Department of Periodontology – Ponta Grossa Dental School, Paraná State University (UEPG) Ponta Grossa, Paraná, Brazil. Author to whom correspondence should be sent: Elcio Marcantonio Jr, DDS, PhD, Departamento de Cirurgia e Diagnóstico, Disciplina de Periodontia; Faculdade de Odontologia de Araraquara, UNESP Rua Humaitá, 1680, Centro, 14801-903, Araraquara, SP, Brazil,. Phone/Fax: 55 (16) 3301- 6378, e-mail: elciojr@foar.unesp.br 44 Financial support: FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo, grant no. 1999/09672-2 and CAPES. 45 ABSTRACT Background: Cyclosporine A (CsA) is an immunosuppressive agent commonly used to prevent organ transplantation rejection. It has been demonstrated that CsA may negatively affect osseointegration around dental implants. Therefore, the aim of this study was to evaluate the influence of CsA administration on bone density around titanium dental implants. Materiais e Métodos: Twelve New Zealand rabbits were randomly divided into 2 groups with six animals each. The test group (CsA) received daily subcutaneous injection of CsA (10mg/kg bodyweight) and the control group (CTL) received saline solution by the same route of administration. Three days after the beginning of immunosuppressive therapy, one machined dental implant (7.0 mm lenght and 3.75 mm in diameter) was inserted bilaterally at the region of the tibial methaphysis. After 4 and 8 weeks the animals were sacrificed and the histometrical procedures were performed and the bone density was mensured at first four threads. Results: Bone density showed a statistically significant increase from the 4-week to the 8 week-period in the control group (37.41% + 14.85 versus 58.23% + 16.38 – p < 0.01). However, bone density consistently decreased in the test group over the time (46.31% + 17.38 versus 16.28 + 5.08 – p <0.05). In the 8-week period, there was a statistically significant difference in bone density between the control and the test group (58.23 + 16.38 e 16.28 + 5.08 – p= 0.001). Conclusions: Within the limits of this study, long-term CsA administration may reduce bone density around titanium dental implants during the osseointegration process. Key-words: dental implant; bone density; cyclosporin-a; immunosupression 46 INTRODUCTION The principle of osseointegration is based on intimate bone-implant contact. The bone volume and quality are fundamental factors to achieve osseointegration during the healing and maintained over the years under load conditions1. Several systemic diseases such as diabetes mellitus 2,3, osteoporosis4,5. radiotherapy6, smoking habits7 and some drugs therapy8,9 can induce alterations on bone metabolism leading in a poor quality and impairing the healing of bone tissue.. Cyclosporine A (CsA) is an immunosuppressive agent commonly used to prevent organ transplantation rejection and treat other immunologic diseases10. CsA acts on immune system inducing T-helper lymphocytes suppression. This mechanism may affect bone tissue, since immune system, particularly T-lymphocytes, play a critical role on bone remodeling11-14. Some animal studies have demonstrated that this drug leads to a high bone turnover, resulting in unbalance of resorption and formation, leading to osteopenia12-14. Studies15-17 in transplanted patients receiving CsA therapy showed high incidence of osteoporosis confirming this deleterious effect on bone metabolism in human. The influence of CsA in osseointegration has been studied by Duarte et al 20018 and Sakakura et al (2003)9 and it has been shown that CsA may negatively affect osseointegration, reducing the bone-to-implant contact and bone formation within implants threads. In other hand, there are no studies reporting the influence of CsA on bone density, which may be considered an important indicator of the quality of bone tissue formed around dental implants. Therefore, the aim of this study was to evaluate the influence of CsA administration on bone density around titanium dental implants. 47 MATERIALS AND METHODS ANIMALS Twelve New Zealand white rabbits, with 9 to 12 months of age (3500-4500 g) were used in the study. The animals were housed in individual cages, fed by a standard laboratory diet and given tap water ad libitum. The experiment was approved by the Institutional Experimentation Committee of the Araraquara Dental School, São Paulo, Brazil. EXPERIMENTAL PROTOCOL After a 2-week acclimatization period, the animals were randomly divided into two groups, a test (T) and a control (CTL) group, with six animals each. The T group received a daily subcutaneous immunosuppressive dose18 of 10-mg/kg bodyweight Cyclosporin-A∗, whereas the CTL group received saline solution (NaCl 0.9%) by the same route of administration. The administration of the drugs began three days before the implants placement and lasted until the day of sacrifice ( 4 and 8 weeks). IMPLANT SURGERY The animals were anesthetized by intramuscular injections of a combination of ketamine† (0.35 mg/kg bodyweight) and xylazine‡ (0.5mg/kg bodyweight). The region of the tibial metaphysis was cleansed with iodine surgical soap. Incisions of approximately 3 ∗ Sandimmum®, Novartis Pharma AG, Switzerland. † Francotar®; Virbac do Brasil Ltda, Brazil. ‡ Rumpum ® Bayer S.A. São Paulo, Brazil 48 cm in length were performed bilaterally at the internal side of the hind-leg, just below the knee. After gentle dissection, the bone surface of the tibial metaphysis was exposed. Unicortical implant beds were prepared by using a progressive sequence of spiral drills under generous saline cooling. One implant§, (7mm length and 3,75 mm in diameter), was placed in each leg. The soft tissues were sutured in separate layers and the animals received a single intramuscular injection of antibiotic | (0.1 ml/kg bodyweight of an association of Penicillin with Streptomycin.) postoperatively. HISTOMETRIC PROCEDURE After the animals were killed, the conventional implants with surrounding tissue in each tibia were removed and fixed in 4% neutral formalin for 48 hours. Undecalcified sections were prepared by a technique previously described by Donath & Breuner (1992)19 Subsequently, the sections were stained as follows: i) the slide-containing specimen was placed in a vessel containing Stevenel’s blue preheated to, and maintained at, 60 °C for 15 minutes; ii) the specimen was rinsed in distilled water at 60 °C and air dried; iii) a small amount of alizarin red S was placed onto the specimen surface at room temperature for 5 minutes. Then, it was washed thoroughly in running distilled water to remove excess stain and air dried. The bone density (i.e, proportion of mineralized bone in a 500 µm-wide zone lateral to the implant.) tissue was measured in both sides of the implant, at the first four threads. The mean of both sides of the implant was considered for statistical analysis (Figure 2). § Master Screw®, Conexão, São Paulo, Brazil. 3| Pentabiótico®, Wyeth-Whitehall Ltda, São Paulo, Brazil 49 STATISTICAL ANALYSIS Since data were normally distributed, as demonstrated by Kolmogorov and Smirnov test, unpaired t test was used to access difference in bone density between the groups in each experimental period (4-week and 8-week period). Paired t test was used to compare differences in bone density between the 4-week and the 8-week period in each experimental group separately. The level of statistical significance was set to α = 0.05. RESULTS A statistically significant increase in bone density between the 4-week and the 8- week period could be seen in the control group (37.41% + 14.85 versus 58.23% + 16.38 – p < 0.01). However, bone density consistently decreased in the test group along the time (46.31% + 17.38 versus 16.28 + 5.08 – p <0.05). In the 8-week period, there was a statistically significant difference in bone density between the control and the test group (58.23 + 16.38 e 16.28 + 5.08 – p= 0.001). No statistically significant difference could be found between test and control groups in the 4-week period (46.31% + 17.38 versus 37.41% + 14.85) (Figure 1). 50 DISCUSSION The increase of bone density around dental implants over time could be seen in control group showing the bone quality improvement. However, in the T group, a statistically significant decrease in bone density was observed around the implants from the 4-week to the 8-week period (46.31% + 17.38 e 16.28 + 5.08 – p <0.05). This suggests that CsA administration during 8-week period may have negatively affected the bone quality around dental implant. The possible reasons for explain these results are related with the mechanism of imunossupression caused by CSA. It is known that the immune system actively participates in bone mineral metabolism and that the T lymphocytes play a critical role in the development of CsA-induced osteopenia.20 This is not surprising as the T cell is the traditional target of CsA, and naturally occurring T lymphocyte perturbations are implicated in the development of primary osteoporosis in humans. 20 Besides, an in vitro study21 corroborates with this result, by describing the necessity of thymus-derived lymphocytes presence for the production of the osteoclast-activating factor. The lymphocytes suppression results in a high bone metabolism state, where the bone formation is supplanted by the bone resorption, leading to a decrease in the trabecular bone volume.12, 13, 20 Under these conditions, a decrease in bone density around dental implant was observed despite of the osteoinduction properties of titanium oxide on the implant surface. The precise mechanism of action of CsA on bone tissue is still not well understood. It is known that these bone alterations correlate with immunosuppressive mechanisms and are mediated by cytokines.14, 20, 22, 23 Moreover, possible CsA effects on osteoblasts and osteoclasts should not be disregarded, which may result in a secondary phenomenon, leading to a high bone remodeling state with exceeding bone resorption.14 51 The side effects of CsA on bone tissue seem to be time and dose-dependent. Higher doses and long time administration may lead to severe alterations in bone metabolism.13 The percentage of bone tissue formed at the first four threads was used to access bone density due to the close proximity to the cortical bone of the tibial metaphysis. Despite the fact that CsA exerts its effect mainly on trabecular bone, bone density was dramatically reduced in the cortical bone, as demonstrated in this study. This suggests that in areas where trabecular bone predominates, such as in the maxilla, bone density around dental implants may be severely damaged. Fu et al24 (1999) found an increased alveolar bone resorption in rats receiving CsA, with greater bone loss in sites affected by periodontitis. Although it could be demonstrated a negative side effect of CsA on cortical bone healing around dental implants corroborating the previous findings8,9, further studies should be developed in order to investigate the effects of this drug on trabecular bone and around osseointegrated functionally loaded dental implants. Within the limits of this study, long- term CsA immunesuppression may reduce bone density around titanium dental implants during the osseointegration process. CONCLUSIONS Within the limits of this study, long-term CsA immunosuppression may reduce bone density around titanium dental implants during the osseointegration process. 52 REFERENCES 1. Albrektsson T. Bone tissue response. In: Brånemark P-I, Zarb G, Albrektsson T, eds. Tissue integrated prosthesis osseointegration in clinical dentistry. Chicago: Quintessence, 1985: 129-144. 2. Margonar R, Sakakura CE, Holzhausen M, Pepato MT, Alba Jr RC, Marcantonio Jr E. The Influence of Diabetes Mellitus and Insulin Therapy on Biomechanical retentionaround Dental Implants: A Study in Rabbits. Implant Dentistry.2003, 4: 333-339. 3. Takeshita F, Murai K, Iyama S, et al.Uncontrolled diabetes hinders bone formationaround titanium implants in rat tibiae. A light and fluorescence microscopy,and image processing study. J Periodontol.1998;69:314–320. 4. Dao TT, Anderson JD, Zarb GA. Is osteoporosis a risk factor for osseointegration of dental implants? Int J Oral Maxillofac Implants. 1993: 8; 137-144. 5. Duarte PM, Cesar-Neto JB, Sallum AW, Sallum EA, Nociti FH Jr. Effect of estrogen and calcitonin therapies on bone density in a lateral area adjacent to implants placed in the tibiae of ovariectomized rats. J Periodontol. 2003;74(11):1618-24. 6. Granström G. Radiotherapy, osseointegration and hyperbaric oxygen therapy. Periodontol 2000 2003: 33; 145-162. 7. Cesar-Neto JB, Duarte PM, Sallum EA, Barbieri D, Moreno H Jr, Nociti FH Jr. A comparative study on the effect of nicotine administration and cigarette smoke inhalation on bone healing around titanium implants. J Periodontol. 2003 Oct;74(10):145-9. 53 8. Duarte PM, Nogueira Filho GR, Sallum EA, Sallum AW, Nociti Junior FH. The effect of an immunosuppressive therapy and its withdrawal on bone healing around titanium implants. A histometric study in rabbits. J Periodontol 2001; 72:1391-7. 9. Sakakura CE, Margonar R, Holzhausen M, Nociti Jr FH, Alba Jr RC, Marcantonio Jr E. influence of cyclosporin a therapy on bone healing around titanium implants. A histometric and biomechanic study in rabbits. J Periodontol. 2003; 74: 974-979. 10. Ruegger A, Kuhn M, Lichti H, et al. Cyclosporin A, a Peptide Metabolite from Trichoderma polysporum (Link ex Pers.) Rifai, with a remarkable immunosuppressive activity Helv Chim Acta. 1976; 59:1075-92. 11. Mccauley LK, Rosol TJ, Capen CC. Effects of cyclosporin A on rat osteoblasts (ROS 17/2.8 Cells) in vitro. Calcif Tissue Int 1992; 51: 291-297. 12. Movsowitz C, Epstein S, Ismail F, Fallon M, Thomas S. Cyclosporin A in the oophorectomized rat: unexpected severe bone resorption. J Bone Miner Res 1989; 4: 393-398. 13. Movsowitz C, Epstein S, Fallon M, Ismail F, Thomas S. Cyclosporin-A in vivo procedures severe osteopenia in the rat: effect of dose and duration of administration. Endocrinol 1988; 123: 2571-2577. 14. Rucinski B, Liu CC, Epstein S. Utilization of cyclosporine H to elucidate the possible mechanisms of cyclosporine A – Induced osteopenia in the rat. Metabolism 1994; 43:1114 – 1118. 15. Cayco A, Wysolmerski J, Simpson C, et al. Posttransplant bone disease: evidence for a high bone resorption state. Transplantation 2000; 70: 1722-1728. 54 16. Julian BA, Laskow DA, Dubovsky J, Dubovsky EV, Curtis JJ, Quarles LD. Rapid loss of vertebral mineral density after renal transplantation. N Engl J Med 1991; 325: 544-50. 17. Vedi S, Greer S, Skingle SJ, et al. Mechanism of bone loss after liver transplantation: a histomorphometric analysis. J Bone Miner Res 1999; 14: 281-287. 18. Gratwohl A, Riederer I, Graf E, Speck B. Cyclosporine toxicity in rabbits. Lab Anim 1986; 20: 213-220. 19. Donath K, Breuner G. A method for study of undecalcified bones and teeth with attached soft tissue. The sage-Scliff (sawing and grinding) technique. J Oral Pathol 1982; 11: 318-326. 20. Buchinsky FJ, Ma Y, Mann GN, et al. T lynphocytes play a critical role in the development of cyclosporin A–induced osteopenia. Endocrinology 1996; 137: 2278-2285. 21. Horowitz M, Vignery A, Gershon RK, Baron R. Thymus-derived lymphocytes and their interaction with macrophages are required for production of osteoclast-acting factor in mouse. Proc Natl Acad Sci USA 1984; 81: 2181-2185. 22. Sasagawa K, Fushibayashi S, Okano K, et al. Different inhibitory actions of immunomodulating agents and immunosuppressive agents on bone resorption of mouse calvaria. Int J Immunopharmac 1989; 11: 953-959. 23. Klaushofer K, Hoffmann O, Stewart PJ, et al. Cyclosporine A inhibits bone resorption in cultured neonatal mouse calvaria. J Pharmacol Exp Ther 1987; 243: 584-590. 24. Fu E, Hsieh Y-D, Nieh S, Wikesjö, Liu D. Effects of cyclosporin A on alveolar bone: an experimental study in the rat. J Periodontol 1999; 70:189-194. 55 FIGURES 0 10 20 30 40 50 60 Control Test 4 weeks 8 weeks Figure 1 – Mean (%) and standard deviation for bone density around implants for control and test groups at 4 and 8 weeks post surgery. 500 µm Figure 2 –Schematic illustration demonstrating the region of bone density analysis. 56 ESTUDO III Artigo intitulado: “The influence of cyclosporin-a on mechanical retention of dental implants integrated to the bone. A study in rabbits”. Submetido ao Journal of Periodontology, onde está sob processo de revisão 57 THE INFLUENCE OF CYCLOSPORIN-A ON MECHANICAL RETENTION OF DENTAL IMPLANTS INTEGRATED TO THE BONE. A STUDY IN RABBITS Celso E. Sakakura, DDS, MS, PhD* Rogerio Margonar, DDS, MS, PhD* Rafael Sartori, DDS, MS student* Juliana Najarro Dearo de Morais, DDS, MS, PhD student* Elcio Marcantonio Junior DDS, MS, PhD, Assistance Professor* AUTHOR TO WHOM CORRESPONDE SHOULD BE SENT (can be published): Elcio Marcantonio Jr, DDS, PhD, Departamento de Cirurgia e Diagnóstico, Disciplina de Periodontia; Faculdade de Odontologia de Araraquara, UNESP Rua Humaitá, 1680, Centro, 14801-903, Araraquara, SP, Brazil,. Phone/Fax: 55 (16) 3301- 6314, e-mail: elciojr@foar.unesp.br This study was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) Grant # 2003/04253-9 and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). Disclosure - The authors claim to have no financial interests in any company or any of the products described in this manuscript. * * Departament of Periodontology – Dental School of Araraquara, State University of São Paulo (UNESP) Araraquara, São Paulo, Brazil. 58 Numbers of figures – 2 Running Title: the influence of cyclosporin-a on dental implant mechanical retention 59 ABSTRACT: Background: Immunosuppressive agents may induce severe changes on bone metabolism and may impair the osseointegration process during the implant healing. No data are available concerning the influence of cyclosporin A on healed bone around dental implants. The aim of this study was evaluate the influence of cyclosporine-A administration in the mechanical retention of bone integrated to dental implants. Material and Methods: Eighteen female New Zealand Rabbits were submitted to an implant surgery. Each animal received one commercial dental implant of 10mm x 3.75mm. After 12 weeks of undisturbed healing period, 6 animals were randomly sacrificed and the removal torque was performed (Group A). Besides, another 6 animals were submitted to a daily injection of cyclosporine-A in a 10mg/Kg dosage (Group B) and the other 6 animals received saline solution as a control (Group C). After 12 weeks of cyclosporine-A administration both group B and C were sacrificed and submitted to a removal torque test. Results: the removal torque results were 30.5 (±9.8) N.cm for group A; 50.16 (±17.5) N.cm for group B; 26 (±7.8) N.cm for group C. The statistical analysis showed significant difference between group A and B (p<0.05) and between B and C (p<0.01). Conclusion: The cyclosporine-A administration may impair the mechanical retention of dental implants integrated to the bone. Key Words: Cyclosporin A, Osseointegration, Dental Implant, Removal Torque 60 INTRODUCTION Cyclosporin A is a potent immunosuppressive drugs used to treat patients who received organ transplantation.1 Besides, this drug might be used for the treatment of type 2 diabetes, psoriasis, malaria, multiple sclerosis, rheumatoid arthritis, sarcoidosis and several other immunologic diseases.2 The long term use of cyclosporin A is related of several side-effects which included nephrotoxicity, neurologic disturbances, hypertension and gingival overgrowth.1-3 Another side-effect reported in allogenic organ transplantation is osteoporosis, and the use of cyclosporin A associate with other immunosuppressive drugs such as steroidal anti-inflammatory may be responsible to its pathogenesis.4-6 Several studies both in animals7-8 and humans4-6 have been reported that cyclosporin A increase the bone turnover producing higher resorption than formation, increasing the incidence of bone fractures. 5,6 There are few studies reporting some relationship between cyclosporin A and dental implants. Duarte et al.9showed negative impact in bone formation within implant threads when the animals where submitted to a short-time cyclosporin A administration. Sakakura et al.10 reported also negative influence in bone-to-implant contact and removal torque values when the cyclosporin was administered for longer periods. These previous studies were designed to assess the influence of cyclosporin-A administration during the bone healing immediately after implant placement. There are, however, no data available on the effects of cyclosporin on bone already healed around dental implants. Thus, the aim of this study was evaluate the influence of cyclosporine-A administration in the mechanical retention of bone integrated to dental implants. 61 MATERIALS AND METHODS ANIMALS Eighteen New Zealand white rabbits, with 9 to 12 months old (3500-4500 g) were used in the study. The animals were housed in individual cages, fed by a standard laboratory diet and given tap water ad libitum. The experiment was approved by the Institutional Experimentation Committee of the School of Dentistry of Araraquara, São Paulo, Brazil. EXPERIMENTAL PROTOCOL After a 2-week acclimatization period, the animals were submitted into an implant surgery. After a 12 weeks of implant healing 6 animals were randomly sacrificed (Group A) and other 6 animals were randomly selected and submitted into a daily subcutaneous immunosuppressive dose of 10-mg/kg11 bodyweight Cyclosporin-A∗ (group C), whereas the 6 other animals received saline solution (NaCl 0.9%) by the same route of administration (Group B). IMPLANT SURGERY The animals were anesthetized by intramuscular injections of a combination of ketamine† (0.35 mg/kg bodyweight) and xylazine‡ (0.5mg/kg bodyweight). The region of the tibial metaphysis was cleansed with iodine surgical soap (figure 1A). Incisions of approximately 3 cm in length were performed at the internal side of the hind-leg, just below the knee. After gentle dissection, the bone surface of the tibial metaphysis was exposed. Bicortical implant beds were prepared by using a progressive sequence of spiral drills under generous saline ∗ Sandimmum®, Novartis Pharma AG, Switzerland. † Francotar®; Virbac do Brasil Ltda, Brazil. ‡ Rumpum ® Bayer S.A. São Paulo, Brazil 62 cooling. One implants with machine surface4 (10mm in length and 3,75 mm in diameter) with a modified head to enable fixation of the torque meter were placed (figure 1B). The soft tissues were sutured in separate layers (figure 1C) and the animals received a single intramuscular injection of antibiotic | (0.1 ml/kg bodyweight of an association of Penicillin with Streptomycin.) postoperatively. REMOVAL TORQUE Immediately after the animals sacrifice the tibias were surgically removed and the implants were submitted into a removal torque test. The specially designed key were connected the modified head implant with the torque-gauge wrech¶. An anticlockwise movement was performed in order to remove the implant and the maximal torque value necessary for manual removal of each implant was measured in Newton centimeters. STATISTICAL ANALYSIS To test the differences in body weight and torque values between the three groups the Mann Whitney test was used. The value of P<0.05 was consider the limit for a significant difference. 4 Master, Conexão Sistemas de Prótese Ltda, São Paulo, Brazil. | Pentabiótico®, Wyeth-Whitehall Ltda, São Paulo, Brazil ¶ 15-BTG, Tonich, Japan. 63 RESULTS CLINICAL FINDINGS The healing was uneventful in all rabbits after implant placement. However, the remaining animals subjected to cyclosporine A administration experienced a significant weight loss (p<0.05). The mean and standard deviation were 4216 ± 728g, 4783 ± 432g and 3266 ± 531g, respectively group A, B and C TORQUE VALUES All the implants showed values compatible with osseointegration status. The removal torque and the standard deviation results were 30.5(±9.8) N.cm group A; 50.16(±17.5) N.cm for group B; 26(± 7.8) N.cm for group C. The statistical analysis showed significant difference between group A and B (p<0.05) and between B and C (p<0.01) (Figure 2). DISCUSSION The present study evaluated the influence o cyclosporin-A administration on mechanical retention of dental implants already osseointegrated. The compound administration began after 12 weeks after implant placement, at this time it was supposed that all implants were osseointegrated, since the 6 animals were randomly selected and submitted to a removal torque assessment (group A) showing similar torque values reported in previous studies10,12,13 The others remaining six animals (group B) receiving saline solution showed 24 weeks after implant placement an improvement in mechanical retention comparing to group A (12 weeks). However, in group C, which the animals were subjected to a compound 64 administration during 12 weeks showed lower mechanical retention values. Indeed, the torque values were almost half than values of group B and even lower than group A. These findings suggested that the continuing processes of bone attachment into the surface of implant were impaired, probably because the cyclosporin-A administration might result in dramatic alteration in bone metabolism. These alterations can be explained by the action of cyclosporin-A. It is known that the immune system actively participates in bone mineral metabolism and that the T lymphocytes play a critical role in the development of CsA-induced osteopenia.14 This is not surprising as the T cell is the traditional target of CsA and naturally occurring T lymphocyte perturbations are implicated in the development of primary osteoporosis in humans. 14 Besides, an in vitro study 15 corroborates with this result, by describing the necessity of thymus-derived lymphocytes presence for the production of the osteoclast-activating factor. T lymphocytes suppression results in a high bone metabolism state, where the bone formation is supplanted by the bone resorption, leading to a decrease in the trabecular bone volume.7, 8, 14 Under these conditions, a decreased percentage of bone contact with the implant surface could be expected resulting in a lower mechanical retention of implant to the bone10. The precise mechanism of action of CsA on bone tissue is still not well understood. It is known that these bone alterations correlate with immunosuppressive mechanisms and are mediated by cytokines.14 Moreover, possible CsA effects on osteoblasts and osteoclasts are not rejected and may result in a secondary phenomenon, leading to a high bone remodeling state with exceeding bone resorption.15 The fixture mechanical retention is directly dependent of amount of bone attached into implant surface. Implants showing larger area of implant-to-bone contact shows greater removal torque values .10,12,13, 17,18 Regarding to implants subjected to a cyclosporin-A administration and considering the bone as one of the most dynamic tissues in the body, we 65 can speculate that the bone attached to implant surface might be resorbed during the cyclosporin A administration, or there was mineral contents loosening leading in lower bone strength, resulting in lower mechanical retention. This confirmed the hypothesis that even bone already healed around dental implants may be affected by CsA. This fact should be considered in the clinical situation for patients who are candidates for implant placement and who had implants and began CsA treatment. However, there is no data regarding neither if such bone alterations could impair the prostheses supporting nor if this bone alterations remain for a long periods or is only a transitional phenomena. Therefore, within limits of this study we conclude that the cyclosporin A administration might impair the mechanical retention of dental implants integrated to the bone. ACKNOWLEDGMENTS The authors greatly appreciated the assistance of Celso Luiz Borsato for technical support in the Animal Laboratory. REFERENCES 1. Faulds D, Goa KL, Benfield P. Cyclosporin. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in immunoregulatory disorders. Drugs. 1993 Jun;45(6):953-1040. Review. Erratum in: Drugs 1993 Sep;46(3):377. 2. Adams D, Davies G. Gingival hyperplasia associated with cyclosporin A. A report of two cases. Br Dent J. 1984 Aug 11;157(3):89-90. 66 3. Seymour RA, Ellis JS, Thomason JM. Risk factors for drug-induced gingival overgrowth. J Clin Periodontol. 2000 Apr;27(4):217-23. Review. 4. Cayco A, Wysolmerski J, Simpson C, et al. Posttransplant bone disease: evidence for a high bone resorption state. Transplantation 2000; 70: 1722-1728. 5. Julian BA, Laskow DA, Dubovsky J, Dubovsky EV, Curtis JJ, Quarles LD. Rapid loss of vertebral mineral density after renal transplantation. N Engl J Med 1991; 325: 544- 50. 6. Vedi S, Greer S, Skingle SJ, et al. Mechanism of bone loss after liver transplantation: a histomorphometric analysis. J Bone Miner Res 1999; 14: 281-287. 7. Movsowitz C, Epstein S, Ismail F, Fallon M, Thomas S. Cyclosporin A in the oophorectomized rat: unexpected severe bone resorption. J Bone Miner Res 1989; 4: 393-398. 8. Movsowitz C, Epstein S, Fallon M, Ismail F, Thomas S. Cyclosporin-A in vivo procedures severe osteopenia in the rat: effect of dose and duration of administration. Endocrinol 1988; 123: 2571-2577. 9. Duarte PM, Nogueira Filho GR, Sallum EA, Sallum AW, Nociti Junior FH. The effect of an immunosuppressive therapy and its withdrawal on bone healing around titanium implants. A histometric study in rabbits. J Periodontol 2001; 72:1391-7. 10. Sakakura CE, Margonar R, Holzhausen M, Nociti FH Jr, Alba RC Jr, Marcantonio E Jr. Influence of cyclosporin A therapy on bone healing around titanium implants: a histometric and biomechanic study in rabbits. J Periodontol. 2003 Jul;74(7):976-81. 11. Gratwohl A, Riederer I, Graf E, Speck B. Cyclosporine toxicity in rabbits. Lab Anim 1986; 20: 213-220. 67 12. Ivanoff C-J, Sennerby L, Lekholm U. Influence of mono- and bicortical anchorage on the integration of titanium implants. A study in the rabbit tibia Int Oral Maxillofac Surg 1996; 25: 229-235. 13. Johansson C, Albrektsson T. Integration of screw implants in the rabbit: A 1-yr follow-up of removal torque of titanium implants. Int J Oral Maxillofac Implants 1987; 2: 69-75. 14. Buchinsky FJ, Ma Y, Mann GN, et al. T lynphocytes play a critical role in the development of cyclosporin A–induced osteopenia. Endocrinology 1996; 137: 2278- 2285. 15. Horowitz M, Vignery A, Gershon RK, Baron R. Thymus-derived lymphocytes and their interaction with macrophages are required for production of osteoclast-acting factor in mouse. Proc Natl Acad Sci USA 1984; 81: 2181-2185. 16. Rucinski B, Liu CC, Epstein S. Utilization of cyclosporine H to elucidate the possible mechanisms of cyclosporine A – Induced osteopenia in the rat. Metabolism 1994; 43:1114 – 1118. 17. Cordioli G, Majzoub Z, Piattelli A, Scarano A. Removal torque and histomorphometric investigation of 4 different titanium surfaces: an experimental study in the rabbit tibia. Int J Oral Maxillofac Implants. 2000 Sep-Oct;15(5):668-74. 18. Klokkevold PR, Nishimura RD, Adachi M, Caputo A. Osseointegration enhanced by chemical etching of the titanium surface. A torque removal study in the rabbit. Clin Oral Implants Res. 1997 Dec;8(6):442-7. 68 FIGURES A B C A Figure 1 –A) Rabbit Tibia Metaphysis; B) Dental implant installed with modified head to fit in torque meter gouge; C) Tissues sutured. 30.5 50.17 26 0 10 20 30 40 50 60 70 80 Groups A B C Figure 2 – Removal torque values (N.cm) for group A, B and C 69 ESTUDO IV Artigo intitulado “The Influence Of Cyclosporin A On Quality Of Bone Around Integrated Dental Implants. A Radiographic Study In Rabbits” Trabalho aceito pelo Clinical Oral Implants Research. 70 THE INFLUENCE OF CYCLOSPORIN A ON QUALITY OF BONE AROUND INTEGRATED DENTAL IMPLANTS. A RADIOGRAPHIC STUDY IN RABBITS Celso Eduardo Sakakura, DDS, MSc, PhD5 Elcio Marcantonio Junior, DDS, MSc, PhD∗ Ann Wenzel, DDS, PhD, Dr. Odont.6 Gulnara Scaf, DDS, MSc, PhD∗ RUNNING-TITLE: Influence of cyc