UNIVERSIDADE FEDERAL FLUMINENSE FACULDADE DE ODONTOLOGIA
PERIODONTITE CRÔNICA COMO FATOR DE RISCO AO DESENVOLVIMENTO DE PERI-IMPLANTITE: ESTUDO DA ASSOCIAÇÃO GENÉTICA NA VIA
MMP13-TIMP2-TGFB3
Niterói 2014
FACULDADE DE ODONTOLOGIA
PERIODONTITE CRÔNICA COMO RISCO AO DESENVOLVIMENTO DE PERI-IMPLANTITE: ESTUDO DA ASSOCIAÇÃO GENÉTICA NA VIA
MMP13-TIMP2-TGFB3
ROBERTO GONÇALVES JÚNIOR
Dissertação apresentada à Faculdade de Odontologia da Universidade Federal Fluminense, como parte dos requisitos para obtenção do título de Mestre, pelo Programa de Pós-Graduação em Odontologia.
Área de Concentração: Clínica
Odontológica
Orientadora: Profa. Dra. Priscila Ladeira Casado.
Co-orientador: Prof. Dr. José Mauro Granjeiro
Niterói 2014
G635 Gonçalves Junior, Roberto
Periodontite crônica como risco ao desenvolvimento de peri- implantite: estudo da associação genética na via mmp13-timp2-tgfb3 / Roberto Gonçalves Junior; orientadora: Profa. Dra. Priscila Ladeira Casado, co-orientador:Prof. Dr. José Mauro Granjeiro. – Niterói : [s.n.], 2014.
42 f.
Dissertação (Mestrado em Clínica Odontológica) – Universidade Federal Fluminense, 2014.
Bibliografia: f. 23-29.
1. Periodontite crônica. 2. Peri-implante. 3. Polimorfismo genético. Casado, Priscila Ladeira [orien.]. II. Granjeiro, José Mauro [co-orient.]. III. Título.
Profa. Dra. Priscila Ladeira Casado
Instituição: Faculdade de Odontologia da Universidade Federal Fluminense
Decisão: _________________________Assinatura:_________________________ Profa. Dra. Eliane dos Santos Porto Barboza
Instituição: Faculdade de Odontologia da Universidade Federal Fluminense
Decisão: _________________________Assinatura:_________________________ Prof. Dr. Diego Pinheiro Aguiar
Instituição: Instituto Nacional de Traumatologia e Ortopedia
.
Dedico este trabalho primeiramente a Deus, que junto com os enigmas do corpo humano nos deu a inteligência para decifrá-los, e a meus pais, Sueli e Roberto, a quem honro pelo esforço com o qual me mantiveram na universidade me permitindo condições de galgar êxito em minha trajetória profissional.
A Deus por abrir portas, as quais me trouxeram até aqui.
A meus pais, por serem canais de bênção em minha vida, me ensinando os reais valores da vida e me mostrando a importância do conhecimento.
A Professora Priscila Ladeira Casado por ter dispensado a mim grande atenção e paciência, se tornando a figura mais notória em minha formação, iniciando-me na
vida científica.
Ao Professor Doutor José Mauro Granjeiro, por acreditar no nosso trabalho, por toda contribuição e incentivo. Pessoa admirável e exemplo a ser seguido.
Ao Lucas Carneiro Costa, Marcos Alexandre da Fonseca e Renata Barboza Coelho por toda amizade e companheirismo, pois sem eles a caminhada teria sido muito
mais árdua e menos prazerosa.
E a todos os professores e funcionários da UFF que foram direta ou indiretamente responsáveis por minha formação.
Gonçalves RJ. Periodontite crônica como risco ao desenvolvimento de peri-implantite: estudo da associação genética na via mmp13-timp2-tgfb3 [dissertação]. Niterói: Universidade Federal Fluminense, Faculdade de Odontologia; 2014.
Indivíduos suscetíveis à periodontite crônica (PC) apresentam risco elevado ao desenvolvimento de peri-implantite (PI). Ambas as doenças são multifatoriais, apresentando similaridades em sua patofisiologia e perfil poligênico. A MMP-13
(metaloproteinase da matriz 13/ colagenase 3) é uma potente enzima colagenolitica
e gelatinolitica, tendo sua expressão induzida por TGFB-3 (fator de crescimento transformador beta tipo 3), em fibroblastos gengivais humanos, e inibida por TIMP-2 (inibidor tecidual de metaloproteinase tipo 2). O objetivo deste estudo foi correlacionar a ocorrência associada de peri-implantite (PI) em humanos com história de periodontite crônica (PC) e sua interação com alterações nos genes do eixo MMP13,TIMP2 e TGFB3. Cento e sessenta e três voluntários que receberam implante dentário, foram submetidos aos exames oral e radiográfico para diagnóstico de histórico de periodontite crônica e/ou peri-implantite. Os voluntários foram divididos em 4 grupos: controle (sem PI e sem PC, n=72), PC (com PC e sem PI, n=28), PI (com PI e sem PC, n=28) e doença (com PC e PI, n=35). O teste do qui-quadrado correlacionou os genótipos nas regiões dos genes MMP13 rs2252070, TIMP2 rs7501477 e TGFB3 rs2268626, considerando a interação entre PC e PI. Os resultados mostraram que voluntários com PC possuem 3.2 vezes mais chances de desenvolver PI (p=0.0004) comparados aos sem PC. Nenhuma associação significativa foi observada no eixo MMP13, TIMP2 e TGFB3 e o desenvolvimento de PC e PI. Sendo assim, concluiu-se que a PC é um fator de risco ao desenvolvimento de PI, no entanto, não há associação entre polimorfismos para MMP13–TIMP2– TGFB3 e o desenvolvimento comórbido de PI e PC.
Palavras-chave: peri-implantite, periodontite crônica, polimorfismo genético, metaloproteinase 13, inibidor tecidual de metaloproteinase-2, fator de crescimento transformador beta
Gonçalves RJ. Chronic periodontitis as a risk for the development of peri-implantitis: a study of genetic association via the MMP13-TIMP2-tgfb3 [dissertation]. Niterói: Universidade Federal Fluminense, Faculdade de Odontologia; 2014.
Subjects susceptible to chronic periodontitis (CP) show a high risk for the development of peri-implantitis (PI). Both diseases are multifactorial, presenting similarities in their pathophysiology and polygenic profile. MMP-13 (matrix metalloproteinases 13/ collagenase 3) is a collagenolytic enzyme, which expression is induced by TGFB-3 (transforming growth factor type 3) in human gingival fibroblasts and inhibited by TIMP-2 (tissue inhibitor of metalloproteinase type 2). The aim of this study was to associate the occurrence of peri-implantitis (PI) in subjects with history of chronic periodontitis (CP) and its interaction with polymorphisms in MMP13-TIMP2-TGFB3 gene pathway. One hundred and sixty-three volunteers underwent dental implant placement were submitted to oral and radiographic examination in order to identify past history of CP or presence of PI. Volunteers were divided into 4 groups: control (without PI and CP, n = 72), PC (with CP and without PI, n = 28), PI (with PI and without CP, n = 28) and disease (with CP and PI, n = 35). The chi-square correlated genotypes in specific regions of MMP13 rs2252070, rs7501477 and TIMP2 TGFB3 rs2268626 genes, considering the interaction between CP and PI. The results showed that volunteers with CP have 3.2 times more chance of developing PI (p=0.0004) compared to those without PC. No significant association was observed in genes MMP13-TIMP2-TGFB3 with CP or PI. Based on our results, we concluded that CP is a risk factor for PI development, however, there is no association of both diseases with polymorphisms in the MMP13-TIMP2- TGFB3 pathway.
Keywords: peri-implantitis, chronic periodontitis, genetic polymorphism, matrix metalloproteinases 13, tissue inhibitor of metalloproteinases-2, transforming growth factor beta
1 - INTRODUÇÃO
O aumento do índice de insucesso em implantodontia está relacionado a
presença de doença peri-implantar (DPI).30 Sabe-se que as principais causas de DPI
são devido à falha biomecânica,7 sendo os principais fatores de risco a periodontite,
má higiene bucal, tabagismo e diabetes mellitus. 30,9 A taxa de sucesso após
instalação de implantes em pacientes sem história de periodontite é de 96,5%, em
contraste com 90.5% em pacientes com histórico de periodontite.40
A etiologia microbiana da DPI está intimamente relacionada à microbiota presente na periodontite crônica. No entanto, a resposta imune individual ao insulto bacteriano é a responsável pela evolução ou paralisação da doença, incluindo um
perfil de resposta regulada por diferentes citocinas.22 Elevação dos níveis destas
moléculas imunorreguladoras, tais como IL-1-b (interleucina-1 beta), IL-6 (interleucina-6), TNF (fator de necrose tumoral) e PGE2 (prostraglandina E2) tem sido observada em sítios com periodontite em comparação aos saudáveis.8,10,15,18,19,47
O aumento da expressão destas citocinas pode resultar na destruição dos tecidos mineralizados e/ou não mineralizadas, através da indução autócrina ou parácrina, estimulando outros mediadores biológicos, tais como as metaloproteinases de matriz (MMPs),21 uma família composta de 23 enzimas
capazes de degradar proteínas da matriz extracelular (MEC), incluindo colágeno desnaturado.11,12
Dentre elas a MMP-13 (colagenase-3) representa o terceiro membro da subfamília de colagenase das MMPs, sendo uma potente enzima gelatinolítica e colagenolítica,26 expressa por diferentes tipos de celulares, tais como fibroblastos em
diferentes locais anatómicos, condrócitos, osteoblastos, células da polpa dentária e células endoteliais.37,41,44,49,53
A atividade destas MMPs é principalmente controlada por inibidores teciduais, conhecidos como TIMPs, através de um domínio N-terminal. O equilíbrio entre MMPs e TIMPs determina o grau de degradação da MEC, em conjunto com hormônios, produtos oncogênicos, citocinas pró e anti-inflamatórias e fatores de crescimento.51
Dentre os principais fatores de crescimento envolvidos na regulação da MEC no tecido periodontal, TGF-b (fator de crescimento transformador beta) tem demonstrado bloquear ou iniciar a diferenciação e migração celular, aumentando
sua expressão após a instalação dos implantes endósseos.44 TGF-b3 tem um papel
central na proliferação e diferenciação de fibroblastos, estimulando a deposição de colágeno.35
O fator de crescimento transformador beta é capaz de induzir a expressão de MMP-13 em fibroblastos gengivais humanos, durante a cicatrização, através da ativação MAPK (MAPK - do inglês mitogen-activatedproteinkinases) que ativa P38, e complexo AP-1 (proteína ativadora tipo-1), sugerindo que MMP-13 desempenha um papel importante na rápida remodelação do colágeno da MEC durante a reparação de feridas periodontais.37,38
Sabe-se que o processo inflamatório possui características que são coordenadas pelo padrão genético. Por isso, estudos explorando o padrão genético individual como forma de justificar a comum etiologia da periodontite e peri-implantite tem sido o foco de trabalhos recentes, mas possuem ainda resultados limitados e conflitantes através da análise de regiões específicas em um único gene.10,13,25,32
Considerando que a regulação da homeostase é exercida pela MMP-13 e seus reguladores nos tecidos mineralizados e não-mineralizados, o objetivo deste estudo foi correlacionar a ocorrência associada de peri-implantite com história de periodontite crônica com alterações na sequencia dos genes para MMP13,TIMP2 e TGFB3. Nossa hipótese foi que os polimorfismos nestes genes podem ter correlação com ambas as doenças e que, o histórico de periodontite crônica é um fator de risco ao desenvolvimento de peri-implantite. Acreditamos que futuros métodos diagnósticos e terapêuticos desenvolvidos em implantodontia poderão basear-se nas características genéticas do indivíduo, sendo protetora ou destrutiva de acordo com a identificação das regiões polimórficas e história de periodontite, prevendo o resultado da reabilitação de implante de longo prazo.
2.1. Seleção dos Voluntários
Cento e sessenta e três indivíduos, apresentando 587 implantes osseointegrados endósseos, foram selecionados para este estudo transversal, ao longo de um ano, na Clínica de Odontologia da Faculdade de Odontologia da Universidade Federal Fluminense e Universidade Veiga de Almeida, ambas localizados no Rio de Janeiro, Brasil. Os procedimentos clínicos foram conduzidos de acordo com as recomendações do Comitê de Ética em Pesquisa das Universidades (número de matrícula 80627 e 286.354/2013). O termo de consentimento livre e esclarecido foi obtido de todos os participantes. Os voluntários responderam a uma ficha de anamnese pessoal, médica e odontológica. Os critérios de exclusão foram a falha do implante (perda óssea patológica, a mobilidade do implante ou perda do implante) antes do período de osseointegração (3 meses para mandíbula e seis meses para maxilar), periodontite crônica ativa, uso de bifosfonato, gravidez/lactação, ausência de radiografia pré-operatória, instalação do implante imediatamente após exododntia ou pela técnica de um estágio, utilização de enxerto
ósseo prévio, exposição precoce do implante6. Os critérios de inclusão foram: pelo
menos um implante endósseo osseointegrado instalado, radiografia do pós-operatório imediato, mostrando o nível vertical de osso ao redor do implante para comparar o nível ósseo após o período de osseointegração, radiografia periapical mostrando o status periodontal antes da colocação do implante, e acompanhamento anual clínico e radiográfico. Todos os implantes foram instalados pela técnica de dois estágios cirúrgicos1, em locais que anteriormente mostraram qualidade e
quantidade de osso favorável.
Todas as regiões peri-implantares foram avaliadas clínica e radiologicamente. O exame clínico peri-implantar consistiu de inspeção visual e palpação, análise da inflamação da mucosa, profundidade de sondagem, sangramento à sondagem e sangramento espontâneo em quatro superfícies (mesial, vestibular, distal e lingual/ palatina), presença de placa, fenótipo peri-implantar, mobilidade do implante (qualquer mobilidade durante o teste de mobilidade), período de osseointegração e configuração da plataforma do implante. Radiografia periapical convencional, utilizando a técnica do paralelismo, foi utilizada para avaliar a presença de perda óssea vertical, ao redor dos implantes através da medição da altura do osso peri-implantar comparando-o com a radiografia inicial realizada imediatamente após a instalação do implante. De acordo com as características clínicas e radiográficas periimplantares, os voluntários foram caracterizados como tendo sítios saudáveis -sem sinais clínicos de inflamação na mucosa peri-implantar e -sem sinais de perda óssea patológica- ou peri-implantite com sinais clínicos de inflamação na mucosa peri-implantar e sinais radiográficos de perda óssea patológica de pelo menos uma região. Perda óssea fisiológica foi caracterizada considerando a perda óssea normal de um milímetro durante o primeiro ano após a colocação do implante e 0,2 mm para os anos subsequentes.41 De acordo com o período de integração óssea dos
implantes, a quantidade total de perda óssea foi calculada com base na diferença entre a radiografia pós-operatória imediata e radiografia diagnóstica (no momento do exame peri-implantar). Se a perda óssea total foi superior a 1 mm e 0,2 milímetros por ano, o voluntário foi diagnosticado com peri-implantite.
2.3. Diagnóstico de Periodontite Crônica
Os voluntários foram caracterizados como saudáveis (sem histórico de periodontite crônica) ou com periodontite crônica (com histórico de periodontite crônica).
O diagnóstico de periodontite crônica foi estabelecido com base em parâmetros radiográficos, incluindo a análise radiográfica periapical mostrando
reabsorção óssea horizontal antes da instalação do implante e exame clínico e da história dental pregressa, de forma a diferenciar periodontite crônica da periodontite agressiva.
O diagnóstico e a classificação de periodontite crônica foi baseado em Armitage.4 Todos os voluntários diagnosticados com periodontite crônica foram
tratados e estavam sob cuidados com acompanhamento regular.
Baseado no estado peri-implantar e periodontal, os voluntários foram divididos nos seguintes grupos: saudáveis - grupo controle (sem histórico de periodontite crônica e peri-implantite, n = 72); e grupos teste: doença (com histórico de periodontite crônica e peri-implantite, n = 35); periodontite crônica (história periodontite crônica sem peri-implantite, n = 28) e peri-implantite (com peri-implantite sem periodontites crônicas, n = 28).
2.4. Análise do Polimorfismo Genético
O DNA genómico foi obtido a partir de amostras de saliva, como descrito anteriormente.28 A quantidade e a pureza do DNA foram determinadas por
espectrofotômetro (Nanodrop® 1000, Thermo Scientific, Wilmington, EUA). Apenas as amostras de DNA que apresentaram relação A260nm/A280nm maior que 1.8 foram utilizadas. Todos os examinadores no laboratório não tinham conhecimento do trabalho. A análise para a presença do polimorfismo nos genes MMP-13 (rs2252070), TIMP-2 (rs7501477) e TGFB3 (rs2268626) (Tabela 1) foram realizadas a partir da reação em cadeia de polimerase em tempo real utilizando o ensaio tipo
TaqMan36 (Agilent Technologies, Mx3005P Stratagene). Primers, sondas e master
mix foram fornecidas pela Applied Biosystems (Foster City, Califórnia., EUA).
As variáveis nominais foram expressas como frequências e porcentagens. Para calcular a significância das variáveis nominais entre os grupos, foi realizado o teste do qui-quadrado (χ2). As variáveis numéricas foram expressas em média e desvio padrão. O teste de Shapiro-Wilk avaliou a distribuição entre as variáveis, e a análise de variância (ANOVA), teste t ou Mann-Whitney foram utilizados para comparação das médias entre os grupos, quando as variáveis apresentaram distribuição normal ou não-normal. As diferenças na prevalência dos genótipos e
alelos entre os grupos foram analisadas pelo teste do qui-quadrado de Pearson (χ2)
feito após constatado o equilíbrio de Hardy-Weinberg. Valores de p<0,05 foram considerados estatisticamente significativos. As análises estatísticas foram realizadas utilizando Stata 11.1 (StataCorp, Texas EUA).
Chronic periodontitis is a risk factor for peri-implantitis: a study of polygenetic association by MMP13-TIMP2-TGFB3 pathway
Abstract: Subjects susceptible to chronic periodontitis (CP) show a high risk for the
development of peri-implantitis (PI). Both diseases are multifactorial, presenting similarities in their pathophysiology and polygenic profile. MMP-13 (matrix metalloproteinases 13/ collagenase 3) is a collagenolytic enzyme, which expression is induced by TGFB-3 (transforming growth factor type 3) in human gingival fibroblasts and inhibited by TIMP-2 (tissue inhibitor of metalloproteinase type 2). The aim of this study was to associate the occurrence of peri-implantitis (PI) in subjects with history of chronic periodontitis (CP) and its interaction with polymorphisms in MMP13-TIMP2-TGFB3 gene pathway. One hundred and sixty-three volunteers underwent dental implant placement were submitted to oral and radiographic examination in order to identify past history of CP or presence of PI. Volunteers were divided into 4 groups: control (without PI and CP, n = 72), PC (with CP and without PI, n = 28), PI (with PI and without CP, n = 28) and disease (with CP and PI, n = 35). The chi-square correlated genotypes in specific regions of MMP13 rs2252070, rs7501477 and TIMP2 TGFB3 rs2268626 genes, considering the interaction between CP and PI. The results showed that volunteers with CP have 3.2 times more chance of developing PI (p=0.0004) compared to those without PC. No significant association was observed in genes MMP13-TIMP2-TGFB3 with CP or PI. Based on our results, we concluded that CP is a risk factor for PI development, however, there is no association of both diseases with polymorphisms in the MMP13-TIMP2- TGFB3 pathway.
Keywords: peri-implantitis, chronic periodontitis, genetic polymorphism, matrix metalloproteinases 13, tissue inhibitor of metalloproteinases-2, transforming growth factor beta
The increased failure rate in implant dentistry is associated with the
development of peri-implant disease (PID).30 It is known that the main causes of PID
are due to biomechanical failure resulting from bacterial infection and occlusal overload7, with the main risk factors being poor oral hygiene, smoking, diabetes
mellitus, as well as periodontitis.9,30 The implant success rate in volunteers without a
history of periodontitis is 96.5% whereas in volunteers with a history of periodontitis it is 90.5%.40
Microbial etiology of PID is closely related to the microbiota associated with chronic periodontitis (CP). However, the individual's immune response to bacterial insult is responsible for the development or cessation of the disease, including a profile of the immune response regulated by different cytokines. 22 Increased levels of
immunoregulatory molecules, such as IL-1-b (interleukin-1 beta), IL-6 (interleukin-6), TNF (tumor necrosis factor) and PGE2 (prostaglandin E2), have been observed in periodontitis and peri-implantitis sites when compared with healthy sites.8,10,15,18-20,47
The overexpression of these cytokines can result in the destruction of mineralized and/or non-mineralized tissues through autocrine or paracrine induction,
stimulating other biological mediators such as matrix metalloproteinases (MMPs)21
family, composed of 23 enzymes capable of degrading most extracellular matrix proteins (ECM), including native and denatured collagen.11,12
MMP-13 (collagenase-3) is the third member of collagenase subfamily of MMPs and it is a potent gelatinolytic and collagenolytic enzyme26 expressed by
different cell types, such as fibroblasts in different anatomical sites, chondrocytes, osteoblasts, dental pulp cells, and endothelial cells.37,41,44,49,53
The activity of these MMPs is primarily controlled by tissue inhibitors, known as TIMPs, which have an N-terminal domain capable of inhibiting MMPs. The balance between MMPs and TIMPs determines the degree of degradation of ECM, together with hormones, oncogenic products, pro- and anti-inflammatory cytokines and growth factors.51
Among the main growth factors involved in the regulation of ECM in the periodontal tissue, TGF-b has shown to block or initiate cell differentiation and
migration, becoming overexpressed after placement of endosseous implants.44
TGF-b3 has a central role in fibroblastic proliferation and differentiation, stimulating collagen deposition.35
TGF-b is capable of inducing the expression of MMP-13 in human gingival fibroblasts in wound healing through cascade of mitogen-activated protein kinases (MAPK), p38 and complex AP-1 (activating protein-1), suggesting that MMP-13 plays an important role in the rapid remodeling of the extracellular matrix collagen during repair of periodontal lesions.37,38
Knowing that the inflammatory process is controlled by the genetic trait, recent studies have focused on the individual genetic pattern to justify the common etiology of periodontitis and peri-implantitis, but have yielded conflicting results and limited analyses of specific regions in a single gene.10,13,25,32
Considering that homeostasis regulation is exerted by MMP-13 and its regulators in the mineralized and non-mineralized tissues, the aim of this study was to associate the occurrence of peri-implantitis (PI) in subjects with history of chronic periodontitis (CP) and its interaction with polymorphisms in MMP13-TIMP2-TGFB3 gene pathway. Our hypothesis is that polymorphisms in these genes have correlation with CP and PI, and that CP history is a risk factor for PI development. We believe that future diagnostic and therapeutic methods developed in implant dentistry could be based on the genetic characteristics, either protective or destructive, of the individual according to the identification of the polymorphic regions and history of periodontitis to predict the outcome of long-term implant rehabilitation.
Methods
Volunteer Selection
One hundred and sixty-three individuals, presenting 587 osseointegrated endosseous implants, were selected for this cross-sectional study over the course of one year from the volunteers’ pool at the Dental Clinic of the School of Dentistry at the Fluminense Federal University and Veiga de Almeida University, both located in Rio de Janeiro, Brazil. The clinical procedures were conducted in accordance with the recommendations from the University Research Ethics Committee (Registration number 80627 and 286.354/2013). Informed consent was obtained from all participants. Volunteers answered a personal, medical, and dental history anamnesis, and their smoking habit and alcohol consumption were recorded. The
exclusion criterion was implant failure (pathologic bone loss, implant mobility or implant loss) before the osseointegration period (3 months for mandible and six months for maxillary), biphosphonate use, pregnancy and/or lactation in female volunteers, no preoperative radiography, one stage or immediate implant placement, concurrent bone grafting needed, early implant exposure during osseointegration
period6,non-treated periodontitis, non-compliance with study protocol. The inclusion
criteria were as follows: at least one osseointegrated endosseous implant, immediate postoperative radiography showing the vertical bone level around implant to compare bone level after the osseointegration period, periapical radiography showing periodontal status before implant placement, and annual follow-up clinical and radiographic examinations. All implants were placed in a submerged healing modality
(two-stage concept)1 in sites that had previously showed favorable bone quality and
quantity.
Diagnosis of peri-implantitis
All peri-implant regions were clinically and radiographically evaluated. Clinical examination of the peri-implant sites consisted of visual inspection and palpation, analysis of mucosa inflammation, probing pocket depth, bleeding on probing and spontaneous bleeding in four surfaces (mesial, buccal, distal and lingual/palatine), presence of plaque, peri-implant phenotype, implant mobility (any mobility during percussion test), osseointegration period, and implant platform configuration. Conventional periapical radiography, using the paralleling technique, was used to assess the presence of vertical bone loss around the implants by measuring the height of peri-implant bone and comparing it with the initial radiography immediately taken after implant placement. According to the clinical and radiographic characteristics of the peri-implant sites, volunteers were characterized as having healthy sites - no clinical signs of inflammation in the peri-implant mucosa and no signs of bone loss – or peri-implantitis with radiographic signs of pathologic bone loss in at least one region. Physiological bone loss was characterized considering the normal bone loss of 1 mm during the first year following implant placement and 0.2
mm for subsequent years.41 According to the implant osseointegration period, the
postoperative radiography and diagnosis radiography (at the moment of peri-implant examination). If total bone loss was higher than 1 mm and 0.2 mm per year, the volunteer was diagnosed with peri-implantitis.
Diagnosis of Chronic Periodontitis
Volunteers were characterized as healthy (without history of chronic periodontitis) or having chronic periodontitis (with history of chronic periodontitis). The diagnosis of chronic periodontitis was established on the basis of radiographic parameters, including periapical radiographic analysis showing horizontal bone resorption before implant placement and assessment of clinical examination and past dental history to differentiate chronic from aggressive periodontitis.
Diagnosis and classification of generalized chronic periodontitis was based on
Armitage.4 All volunteers diagnosed with chronic periodontitis had been treated and
were under regular follow-up care.
Based on peri-implant and periodontal status, volunteers were divided into the following groups: healthy – control group (no history of chronic periodontitis and peri-implantitis, n = 72); and test groups: disease (with a history of chronic periodontitis and peri-implantitis, n = 35); chronic periodontitis (chronic periodontitis history without peri-implantitis, n= 28) and peri-implantitis (with peri-implantitis without chronic periodontitis, n = 28).
Single Nucleotide Polymorphism Genotyping
Genomic DNA was obtained from saliva samples as previously described.28
The amount and purity of the DNA was determined by the spectrophotometer instrument (Nanodrop® 1000, Thermo Scientific, Wilmington, USA). Only DNA samples presenting A260nm/A280nm ratio greater than 1.8 were used. All examiners at the laboratories were blinded to the group assignment. Analysis for the presence of the polymorphism in MMP-13 (rs2252070), TIMP-2 (rs7501477) and TGFB3 (rs2268626) (Table 1) genes were performed from the reactions of real-time polymerase chain reactions using the Taqman assay36 (Agilent Technologies,
Stratagene Mx3005P). Primers, probes and the universal master mix were provided by Applied Biosystems (Foster City,Calif., USA).
Statistical Analyses
Nominal variables were expressed as frequencies and percents. To access
the significance of nominal variables among the groups, the chi-square (χ2) test was
performed. Continuous variables were expressed as mean and standard deviation. After the Shapiro-Wilk test evaluated the distribution among variables, analysis of variance (ANOVA), t-test or Mann-Whitney tests were used to compare means among the groups, when the variables presented normal or non-normal distribution. Differences in the prevalence of genotypes and alleles among the groups were analyzed using the Pearson chi-square (χ2) test after fitting for Hardy-Weinberg
equilibrium. Values of p<0.05 were considered statistically significant. Statistical analyses were performed using STATA 11.1 (StataCorp, Texas USA).
Results
One hundred sixty-three volunteers were evaluated over the course of one year. The sample consisted of 110 (67,5%) women and 53 (32,5%) men, with mean age 55,4 ±13,2 years. No difference was found among the test groups (diseased; chronic periodontitis only; peri-implantitis only) when compared with the control group regarding the ethnic group, age, gender and smoking habits. In contrast, when comparing with the control volunteers the prevalence of diabetes showed a higher incidence (8.6%) in the diseased group (p=0.06) (Table 2).
Taking into account the peri-implant status (Table 3), plaque accumulation around implant was highly prevalent in all test groups compared with control (p<0.01). In addition, spontaneous bleeding was significantly higher in the Diseased group (p=0.01) and the Peri-implantitis group (p=0.004) as well as the presence of bleeding on probing (p<0.0001). A tendency for higher probing pocket depth was also observed in the diseased group (p=0.05). Considering the platform type of implant, there was no difference among groups. However, volunteers with history of CP
showed 3.2 times more chance of developing PI than volunteers without CP history (Table 4).
Genotype distributions were within Hardy-Weinberg equilibrium analyses (data not shown). All genotypes and allele frequencies were compared between control group and each test group to further clarify the results. Genotypes and allele frequencies for MMP-13 (rs2252070), TGFb3 (rs2268626) and TIMP-2 (rs7501477) showed no association with test groups (Table 5). When adjusted by age (data not shown), the results were similar for all genotype association.
Discussion
Based on the previously established association between peri-implantitis and chronic periodontitis,9 the aim of this study was to investigate the correlation of
genetic information contained in the DNA, considering, the MMP-13, TIMP-2 and TGFb3 axis, involved in the homeostasis of ECM, with the development of peri-implantitis in volunteers with a history of chronic periodontitis. Our results showed that, despite the clinical differences observed among groups, polymorphisms in the TGFB3, MMP-13 and TIMP-2 genes have no association with both diseases.
Our work also showed the association among several risk factors for periodontitis, previsouly established in the literature, such as diabetes17 and plaque
accumulation,30 in volunteers with peri-implantitis. This association may indicate that,
as in periodontitis, external factors directly influence the spread of peri-implantitis, irrespective of history of chronic periodontitis.
There is a predominance of anaerobic bacteria in peri-implant plaque accumulation, as well as CP, such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Treponema denticola and Tannerella forsythenses,23,31 which cause apical spread of infection and culminate in the loss of
surrounding bone through similar mechanisms that destroy ECM. This exaggerated immune response, mediated by Th1 or Th2 cells, will determine the stability or progression of the lesion.44 However, considering that the peri-implant tissue has
anatomical differences and particularities that anatomically distinguish it from the periodontium, the presence of pathogens in the peri-implant tissue results in the disruption of the mucosal seal, considered the only physical barrier against bacterial
penetration, leading to the rapid extension of the pathological pocket to the bone tissue.24
The peri-implant mucosa is less capable of recovering from lesions associated
with plaque than the gingiva.16,29 However, the majority of volunteers who receive
implants have a history of periodontitis, and, therefore, a destructive inflammatory tissue response as a consequence of microbiota changes. This immune response can conduct to the higher incidence of peri-implant bone loss (peri-implantitis) observed in subjects with CP, as demonstrated in our research.
This excessive destruction of connective tissue components plays a role in the loss of functional tissue architecture.21 Within this context, the equilibrium in the
activity of MMPs controlled primarily by protein tissue inhibitors (TIMPs) represent a
crucial aspect for the maintenance of homeostasis of the ECM 50 in the peri-implant
soft tissue in response to the presence of pathogens. MMP-13 has a high ability to digest type II collagen and its gelatinase activity is 40-fold greater than MMP-1 and
MMP-8.28 The presence of protein derived from P. gingivalis is capable of decreasing
the amount of TIMP-2, a potential regulator of MMP-13, suggesting that this pathway may be a pathological mechanism that promotes tissue destruction. Furthermore, TIMP-2 has erythroid- and mitogenic-potentiating activity, but its overexpression reduces the growth of tumor cells,34,56 which justifies its reduction being associated
with tissue destruction of ECM by MMPs.
Instead, TGF-b can induce the expression of MMP-13 in human gingival
fibroblasts reducing the expression of TIMP-2.14 Therefore, changes in the levels of
these molecules may offer greater protection or susceptibility to peri-implantitis. Genetic modifications can be associated to a greater or lesser degree with the production of these molecules, or interfere in their physiology.10
Polymorphism in the MMP-13 gene was previously associated with the development of colorectal cancer, aneurysm of the abdominal aorta, tooth agenesis
and caries.3,28,33,51 In addition, polymorphisms in the TGFb-3 gene were associated
with non-syndromic cleft lip and development of tooth agenesis.2,47 Some studies
have shown a clear association between various genetic polymorphisms, including IL-6 gene, with PID,10,25,32 and periodontitis,5,39,45 but so far no studies have correlated
the presence of comorbid history of CP with PID and polymorphisms in genes associated with homeostasis of the ECM.
For the first time, this study evaluated the biological relationship between CP and PI considering polygenic profile in MMP-13, TIMP-2 and TGFb-3 pathway. Despite we found no genetic predisposition associated with both diseases, we affirmed that CP is a risk factor for PI development. In order to investigate the central role of this pathway in periodontitis and peri-implantitis pathogenesis, we encourage futures studies analyzing gene expression and protein levels from gingival and bone tissues in a bigger sample size. Our researches believe that novel methods such as gene therapy (with local or systemic applications) and tissue engineering can be developed on the basis of genetic response.
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Table 1. Characteristics from polymorphic regions.
Gene Chromosome SNP ID SNP Type Base Change*
TGFb3 Ch14 Rs2268626 Intron, C/T
TIMP2 Ch17 Rs7501477 Intergenic G/T
TGFB3 Ch11 Rs2252070 Intergenic C/T
Table 2. Baseline characteristics from volunteers included in the study.
Total
(n=163) Healthy(n=72) Diseased(n=35) PeriodontitisChronic Only (n=28) Peri-ImpIantitis Only (n=28) p-value (CI) n (%) n (%) n (%) N (%) n (%) Healthy X Diseased Healthy X Chronic Periodontitis Only Healthy X Peri-ImpIant Disease Only Ethnic group Whites 141(86.5) 63(87.5) 33(94.3) 21(75.0) 24(85.7) 0.27 (2.36;0.43-16.83) 0.12 (0.43; 0.12-1.48) 0.81 (0.86; 0.21-3.69) Non-Whites 22(13.5) 9(12.5) 2(5.7) 7(25.0) 4(14.3)
Age (years) (t-test) 55.4 ±13.2 51,7 ±14.4 60.8 ±09.92 58.8 ±11.92 54.5 ±12.30 0.99 0.98 0.80 Sex Female 110(67.5) 45(62.5) 23(65.7) 21(75.0) 21(75.0) 0.74 (1.15; 0.462.92) (1.80; 0.62-0.23 5.40) 0.23 (1.80; 0.62-5.40) Male 53(32.5) 27(37.5) 12(34.3) 7(25.0) 7(25.0) Smoking habit Non-smoking 146(89.5) 65(90.2) 30(85.7) 26(92.8) 27(96.4) 0.38 (1.75; 0.42-7.20) (0.81; 0.10-4.90)0.80 (0.39; 0.023.56)0.37 Smoking 14(8.5) 7(9.8) 5(14.3) 2(7,2) 1(3,6) Diabetes Yes 6(3.7) 1(1.3) 3(8.6) 1(3.6) 1(3.6) 0.06 (6.66; 0.58-172.93) 0.48 (2.63; 0.0-100.49) 0.48 (2.63; 0.0-100.49) No 157(96.3) 71(98.7) 32(91.4) 27(96.4) 27(96.4)
Table 3. Peri-implant status of the studied population.
Peri-implant status Healthy(n=72) Diaeased(n=35)
Chronic Periodontiti s Only (n=28) Peri-ImpIantitis Only (n=28) p-value n (%) n (%) n (%) n (%) Healthy X Diseased Healthy X Chronic Periodontitis Only Healthy X Peri-ImpIant Disease Only Bleeding on probing *Presence 9 (12.5) 20 (57.1) 5 (17.9) 14 (50.0) <0.0001 (9.33; 3.23-27.82) 0.48 (1.52; 0395.71) <0.0001 (7.00; 2.27-2.14) Absence 63 (87.5) 15 (42.9) 23 (82.1) 14 (50.0) Spontaneous bleeding *Presence 0 (00.0) 3 (8.6) 0 (00.0) 3 (10.7) 0.01 (RR 3.25; 2.44-4.34) 0 0.004 (RR 3.88; 2.77-5.44) Absence 72 (100.0) 32 (91.4) 28 (100.0) 25 (89.3)
Probing Pocket Depth (mm) 1.64 ±0.77 2.27 ±1.19 1,44 ±0.52 2.28 ±0.75 0.05 0.36 0.17 Peri-implant plaque *Presence 2 (2.8) 10 (28.6) 6 (21.4) 5 (17.9) <0.0001 (14.00; 2.59-99.93) (9.55; 1.56-74.38)0.002 (7.61; 1.18-61.37)0.007 Absence 70 (97.2) 25 (71.4) 22 (78.6) 23 (82.1) Peri-implant phenotype *Thin 29 (40.2) 16 (45.7) 17 (60.7) 11 (39.2) 0.57 (1.26; 0.513.10) 0.06 (2.32; 0.87-6.27) 0.94 (0.97; 0.36-2.60) Thick 43 (59.8) 19 (54.3) 11 (39.3) 17 (60.8) Implant Mobility 0 (00.0) 1 (02.8) 0 (00.0) 2 (7.1) 0.14 (RR 3.12; 2.36-4.11) 0.0 0.04 (RR 3.18; 2.40-4.22) Implant- platform Type
External Hexagon 35 (48.6) 19 (54.3) 14 (50.0) 16 (57.1) 0.49 0.99 0.85 Internal Hexagon 5 (6.9) 4 (11.4) 2 (7.1) 1 (3.6)
Morse Cone 29 (40.3) 12 (34.3) 11 (39.3) 10 (35.7) Others 3 (4.2) 0 (00.0) 1 (3.6) 1 (3.6)
Implant region *Upper 41 (56.9) 17 (48.6) 9 (32.1) 17 (60.7) 0.41 (0.71; 0.29-1.73) (0.36; 0.13-0.98)0.02 (1.17; 0.44-3.13)0.73 Lower 31 (43.1) 18 (51.4) 19 (67.9) 11 (39.3) Edentulism *Parcial 62 (86,1) 27 (77,1) 21 (75,0) 25 (89,3) 0.24 (0.54; 0.17-1.72) (0.48 0.14-1.63)0.18 (1.34; 0.30-0.67 6.76) Total 10 (13,9) 8 (22,9) 7 (25,0) 3 (10,7) Osseointegration period (months) 34.71 ±31.62 43.40 ±51.49 30.85 ±23.42 31.17 ±25.21 0.67 0.97 0.81
Table 4. Correlation between history of chronic periodontitis and peri-implantitis. Healthy Peri-implant tissue (n) Peri-implantitis (n)
p-value Odds ratio (CI)
Without history of CP 72 28 0.0004 3.21 (1.58-6.59)
With history of CP 28 35
Table 5. Allele and genotype frequencies of SNP markers. Healthy Diseased Chronic
Periodontitis Only Peri-Implantitis Only p value (OR; CI) (n=72) (n=35) (n=28) (n=28) Healthy X Diseased Healthy X Chronic Periodontitis Only
Healthy X Peri-ImpIant Disease Only n (%) n (%) n (%) n (%) MMP13 rs2252070 CC 7 (9.72) 5(14.28) 2(07.14) 1(3.57) 0.42 0.63 0.59 CT 26(36.11) 12(34.28) 9(32.14) 11(39.28) TT 36(50.00) 18(51.42) 17(60.71) 15(53.57) CT+TT 62(86.11) 30(85.71) 26(92.85) 26(92.85) 0.53 (0.68; 0.17-2.72) 0.64 (1.47; 0.25-11.01) 0.30 (2.94;0.33-66.6) C 40(27.77) 22(31.42) 13(23.21) 13(23.21) 0.71 (1.12; 0.57-2.19) 0.41 (1.35;0.62-2.96) 0.49 (1.29;0.59-2.83) T 98(68.05) 48(68.57) 43(76.78) 41(73.21) TGFB3 rs2268626 0.58 (0.77;0.26-2.19) CC 0(00.00) 0(00.00) 0(00.00) 0(00.00) 1.0 (1.00;0.39-2.55) 0.21 (0.52;0.16-1.61) CT 46(63.88) 23(65.71) 20(71.42) 22(78.57) TT 24(33.33) 12(34.28) 8(28.57) 6(21.42) C 46(31.94) 23(32.85) 20(35.71) 22(39.28) 1.0 (1.00;0.52-1.93) 0.70 (0.88;0.44-1.78) 0.39 (0.76;0.38-1.51) T 94(65.27) 47(67.14) 36(64.28) 34(60.71) TIMP2 rs7501477 GG 51(70.83) 27(77.14) 19(67.85) 21(75.00) 0.58 0.67 0.66 GT 17(23.61) 8(22.85) 7(25.00) 7(25.00) TT 2(2.77) 0(00.00) 0(00.00) 0(00.00) GT+TT 19(26.38) 8(22.85) 7(25.00) 7(25.00) 0.63 (0.80;0.28-2.25) 0.98 (0.99;0.32-3.02) 0.82 (0.89;0.29-2.70) G 119(82.62) 62(88.57) 45(86.5) 49(87.50) 0.47 (0.73;0.28-1.87) 0.07 (0.88;0.32-2.38) 0.65 (0.81;0.29-2.18) T 21(14.58) 8(11.42) 7(13.5) 7(12.50)
4 - CONCLUSÕES
- A periodontite crônica é um fator de risco para peri-implantite.
- Polimorfismos na via MMP13-TGFB3-TIMP2 não estão associados com o desenvolvimento de peri-implantite.
ANEXO B - APROVAÇÃO DO COMITÊ DE ÉTICA EM P ESQUISA
ANEXO C - Termo de consentimento livre e esclarecido
TERMO DE CONSENTIMENTO LIVRE E ESCLARECIDO
Projeto: Periodontite crônica como risco ao desenvolvimento de peri-implantite: estudo da associação genética na via MMP13-TIMP2-TGFB3
Pesquisador Responsável: Dra. Priscila Ladeira Casado – Universidade Federal do Rio de Janeiro Telefones para contato: (21) 8190-7074/ 2629-9255
Nome do
Voluntário:_________________________________________________________________________ Idade:__________anos R.G.________________
O Sr.(a) está sendo convidado a participar do projeto de pesquisa “Periodontite crônica como risco ao desenvolvimento de peri-implantite: estudo da associação genética no eixo mmp13-timp2-tgfb3” de responsabilidade da pesquisadora Dra. Priscila Ladeira Casado, que objetiva correlacionar a ocorrência associada de peri-implantite com história de periodontite crônica e sua interação com alterações nos genes para MMP13-TIMP2-TGFB3, através da avaliação clinica e radiográfica da gengiva e da coleta de saliva
Esses procedimentos não são prejudiciais. Ao contrário disto, beneficiarão a reabilitação da boca ao utilizar implantes dentários. O Sr. (a) receberá qualquer informação (respostas e esclarecimentos) a dúvidas acerca dos procedimentos, se haverão riscos, benefícios, e sobre o seu tratamento. Sua identidade se manterá em caráter confidencial incluindo as informações contidas em seu prontuário que puderem interferir na sua privacidade. A região estudada poderá ser fotografada, mas o seu rosto não aparecerá em nenhuma foto de modo a te identificar. O Sr. (a) tem liberdade de retirar seu consentimento a qualquer momento e deixar de participar do estudo sem que isso traga prejuízo em posteriores tratamentos que possa vir a necessitar dentro desta Instituição ou em outras. Não são previstos danos neste estudo, mas a Instituição está disponível para qualquer tratamento médico em caso de danos diretamente causados pela pesquisa.
Todo material coletado poderá ser utilizado como fonte para esta pesquisa, podendo também ser utilizado como fonte para futuras pesquisas na Unidade de Pesquisa Clinica da Universidade Federal Fluminense.
Eu, ___________________________________, RG no _____________________ declaro ter sido
informado e concordo em participar, como voluntário, do projeto de pesquisa acima descrito.
Rio de Janeiro, ________de________________de________.
Assinatura do Voluntário:_____________________________________________________________ Assinatura do Cirurgião-dentista que obteve o consentimento: _______________________________ Testemunha:________________________________________
ANEXO D - : Ficha Clínica
Ficha clínica Data: ____/_____/_____
Banco DNA: ____________________
Nome: Iniciais: Nasc: Endereço: Telefone: Sexo Etnia:
Nacionalidade: Legenda: A – saúde B – peri-implantite C – Periodontite crônica GRUPO: _________ Exame clínico geral
Instituição da coleta: ___________________________________________________ Tipo de coleta: ( ) Saliva ( ) Biópsia / regiões: ______________________ AUDIT: (valor) ________________________________________________________ Medicação em uso: _____________________________________________________ Doenças sistêmicas:______________________________________________
Edentado: ( ) parcial ( ) total
CPOD
Anomalias dentárias Agenesias
Quantidade de implante total:
Fenótipo periodontal: ( ) Espesso ( ) Fino
Tabagista: ( ) Sim ( ) Não
Etilista: ( ) Sim ( ) Não
Diabetes: ( ) Sim ( ) Não
História de periodontite: ( ) Sim ( ) Não
Tipo de periodontite: ( ) Crônica ( ) Agressiva
ANEXO E - Avaliação Clínico-Radiográfica Avaliação Clinico-radiográfica Região Tipo do Implante Fabricante Tamanho do Implante Tipo de prótese Sangramento espontâneo Sangramento à sondagem Cor da mucosa Edema Pus PCS – v/p/m/d V p v p V p v p v p M d m d M d m d m d Fenótipo peri-implantar (F/E) Roscas expostas (S/N) Placa Peri-implantar Mobilidade Tempo de osseointegração (meses) Exposição do implante (classificar/ legenda) Nomenclatura Assinatura/carimbo do cirurgião-dentista: ___________________________
