UNIVERSIDADE FEDERAL FLUMINENSE FACULDADE DE ODONTOLOGIA
A osseointegração inadequada do implante endósseo tem
correlação com baixos níveis do fator de crescimento epidérmico e
seu receptor na mucosa peri-implantar
Niterói 2015
UNIVERSIDADE FEDERAL FLUMINENSE FACULDADE DE ODONTOLOGIA
A osseointegração inadequada do implante endósseo tem
correlação com baixos níveis do fator de crescimento epidérmico e
seu receptor na mucosa peri-implantar
MARCOS ALEXANDRE DA FONSECA
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 Orientador: Profa Dra. Priscila Ladeira Casado.
Co-orientador: Prof. Dr. José Mauro Granjeiro
Niterói 2015
FICHA CATALOGRÁFICA
F 676 Fonseca, Marcos Alexandre Lima Silva da
A osseointegração inadequada do implante endósseo tem correlação com baixos níveis do fator de crescimento epidérmico e seu receptor na mucosa peri-implantar / Marcos Alexandre Lima Silva da Fonseca ; tador: Profª Drª Priscila Ladeira Casado, co-orientador: Profº Drº José Mauro Granjeiro. – Niterói: [s.n.], 2015.
54 f.; il.
Inclui gráficos e tabelas.
Dissertação (Mestrado em Clínica Odontológica) – Universidade ral Fluminense, 2015.
Bibliografia: f.27-30.
1.Fator de crescimento epidérmico. 2. Receptor do fator de to epidérmico. 3. Osseointegração. 4. Cicatrização. I. Casado, Priscila deira [orien.]. II. Granjeiro, José Mauro [co-orien.]. III. Título.
BANCA EXAMINADORA
Profa. Dra. Priscila Ladeira Casado
Instituição: Faculdade de Odontologia da Universidade Federal Fluminense
Decisão: _________________________Assinatura:_________________________
Profa. Dra. Julie Calixto Lobo
Instituição: Faculdade de Odontologia da Universidade Federal Fluminense
Decisão: _________________________Assinatura:_________________________
Prof. Dra. Mônica Diuana Calasans Maia
Instituição:Faculdade de Odontologia da Universidade Federal Fluminense
Dedico este trabalho a Deus, pois sem ele não teria forças para essa longa caminhada, aos meus pais, Jorge Roberto “in memorian " e Wanda pelo exemplo, dedicação e amor incondicional, à minha amada esposa, Fabiana Helena, por todo amor e compreensão, e ao meu filho, Antônio Roberto por ser minha maior motivação na vida.Quero compartilhar esta conquista com vocês.
AGRADECIMENTOS
A Deus , por colocar pessoas especiais no meu caminho …
Aos meus pais, pela capacidade de acreditar e investir em mim, com amor e dedicação e por tornarem a minha felicidade a principal meta de suas vidas.
À minha amada esposa , amiga e companheira, pelo incentivo e apoio incondicional, mesmo nos momentos de ausência para elaboração deste trabalho.
À minha irmã e meus afilhados, pelo carinho e incentivo para realização deste trabalho.
Ao meu filho, pela motivação essencial à conclusão deste trabalho. À minha dedicadíssima Professora e Orientadora Priscila Ladeira Casado, por transformar minha vida profissional, pelo incentivo e apoio incondicional, respeito e
admiração pela sua competência profissional, minha gratidão plena por todos ensinamentos e esforços para realização deste trabalho.
Ao meu Co-Orientador, Doutor José Mauro Granjeiro, pela credibilidade e contribuição ao nosso trabalho. Exemplo de competência acadêmica. Ao Lucas Carneiro Costa, pela amizade, incentivo, orações, pelo companheirismo
nesta jornada de trabalho.
À Doutora Mônica Diuana Calasans Maia, por toda colaboração e apoio, dedicação na coordenação deste Programa de Pós Graduação da Universidade Federal
Fluminense, sempre tão competente e solícita.
À Thays dos Santos Vieira, pelo empenho dedicado nos ensinamentos da prática laboratorial.
A toda equipe do Instituto Nacional de Traumatologia e Ortopedia e UPC, pela acolhida para o desenvolvimento laboratorial deste trabalho.
Por fim aos Participantes de Pesquisa, essenciais para realização deste trabalho Meus sinceros agradecimentos.
RESUMO
Da Fonseca MA. A osseointegração inadequada do implante endósseo tem correlação com baixos níveis do fator de crescimento epidérmico e seu receptor na mucosa peri-implantar. Tese/Dissertação Niterói: Universidade Federal Fluminense, Faculdade de Odontologia; 2015.
A inadequada osseointegração, incluindo deficiente cicatrização do tecido mole peri-implantar, pode facilitar a penetração bacteriana, promovendo o desequilíbrio na resposta do hospedeiro e favorecendo a reabsorção óssea. O objetivo deste estudo foi avaliar a associação entre a expressão dos genes EGF e
EGFR na mucosa peri-implantar com a qualidade da osseointegração do implante
endósseo. Quarenta e nove participantes de pesquisa, apresentando 59 implantes endósseos ósseointegráveis, foram recrutados para o estudo. Todos os participantes incluídos foram reabilitados com implantes em procedimento de dois estágios cirúrgicos, mostrando qualidade e quantidade de osso favorável para a instalação. A osseointegração foi avaliada 1 mês após a cirurgia de reabertura, realizada 3 meses (em mandíbula) e 6 meses (em maxila) após a instalação do implante. Os critérios para considerar a osseointegração adequada foram: imobilidade do implante durante o teste clínico; ausência de radiotransparência peri-implantar; e ausência de sinais e/ou sintomas persistentes; e ausência de sinais clínicos de inflamação. Com base nas características clínicas dos sítios, os participantes foram caracterizados como (i) tendo a cicatrização sem complicações (com a osseointegração adequada, sem mobilidade do implante, e sem qualquer sinal clínico de inflamação da mucosa) (grupo controle) ou (ii) cicatrização peri-implantar inadequada (com osseointegração inadequada caracterizada por sinais de inflamação na mucosa e/ou mobilidade de implante) (grupo teste). Os descartes gengivais foram coletados de 49 participantes de pesquisa após o período de osseointegração, durante o procedimento de reabertura do implante. O RNA total a partir das amostras gengivais foi isolado utilizando o reagente Trizol®. A reação de transcrição reversa do PCR foi realizada para a síntese de DNA complementar a partir de 300 ng de RNA. Iniciadores específicos para EGF (NM_001963.4) e EGFR (NM_005228.3) foram
confeccionados com base nos dados BLAST. O Método Livak (2-ΔΔCT) foi utilizado para determinar a quantificação relativa da expressão de EGF e EGFR. Os valores foram normalizados em relação à expressão constitutiva de β-actina. Não houve diferença entre os grupos Controle e Teste quanto à raça, sexo, idade, consumo de álcool, condições médicas gerais, medicação atual, edentulismo e fenótipo periodontal. Todas as amostras de RNA apresentavam proporções A260 nm/ A280 nm superior a 1.9. EGF e EGFR mostraram significante menor expressão nos tecidos gengivais removidos de regiões com cicatrização inadequada (grupo teste). EGF apresentou expressão de RNAm com média de 44.53±79.16 e 1.33±1.02 nos grupos controle e teste, respectivamente (p=0.008). Similarmente a expressão de EGFR foi significantemente maior no grupo controle (102.03±329.57) comparado ao teste (7.85±16.04) (p=0.04). Concluímos então, que baixos níveis de EGF e EGFR tem associação com inadequada cicatrização da mucosa peri-implantar durante o período de osseointegração.
Palavras-chave: fator de crescimento epidérmico; receptor do fator de crescimento epidérmico; osseointegração; cicatrização.
ABSTRACT
Da Fonseca MA. Osseointegration failure of dental implant is associated with low EGF and EGFR expressions in the peri-implant mucosa.
Tese/Dissertação Niterói: Universidade Federal Fluminense, Faculdade de Odontologia; 2015.
Osseointegration failure can promote host response imbalance and bone resorption by bacteria infiltration. The objective of this study was to evaluate the association between the expression of EGF and EGFR genes expression in the peri-implant mucosa with the quality of osseointegration of endosseous implant. Forty-nine participants, with 59 endosseos implants, were recruited for this study. All participants included were rehabilitated with implants in two stages surgical protocol, presenting favorable bone quality and quantity. Osseointegration was evaluated one month after exposure surgery, wich was performed three months (mandible) and 6 months (maxillary) after implant placement. The criteria to consider the proper osseointegration were: implant immobility; absence of peri-implant radiolucency; and no clinical signs of inflammation. Based on clinical and radiographic characteristics of peri-implant sites, participants were characterized as (i) having healing without complications (with proper osseointegration without mobility of the implant, and without clinical signs of mucosal inflammation) (control group) or (ii) failure peri-implant healing (with inadequate osseointegration characterized by signs of mucosae inflammation and/or implant mobility) (test group). Gingival biopsies were collected from 49 participants after osseointegration period, during the exposure procedure. Total RNA from gingival samples was isolated using the Trizol® reagent. The reaction of reverse transcription of PCR was performed for the synthesis of complementary DNA from 300ng RNA using Improm-II Reverse Transcription System ™. Specific primers for EGF (NM_001963.4) and EGFR (NM_005228.3) were based on the BLAST data. The Livak method (2-ΔΔCT) was used to determine the relative quantification of the expression of EGF and EGFR. The values were normalized by relative expression of β-actin. There was no difference between control and test groups to race, sex, age, alcohol consumption, general medical
conditions, current medications, edentulism and periodontal phenotype. All RNA samples revealed proportions A260 nm / A280 nm more than 1.9. EGF and EGFR showed significant lower expression in gingival tissues removed from regions with failure healing (test group). EGF showed mRNA expression with an average of 44.53 ± 79.16 and 01.02 ± 1:33 in the control groups and test, respectively (p = 0.008). Similarly EGFR expression was significantly higher in the control group (102.03 ± 329.57) compared to the test group (7.85 ± 4.16) (p = 0:04). We conclude that low levels of EGF and EGFR are associated with inadequate healing of mucosal peri-implant during the osseointegration period.
Keywords: epidermal growth fator; receptor of epidermal growth fator; osseointegration; wound healing.
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1
–
INTRODUÇÃOO processo osteogênico requer uma coordenação cronológica e espacial cuidadosa dos sinais moleculares para conduzir a proliferação, migração e diferenciação das células mesenquimais precursoras em osteoblastos (ZAIDI, 2007). Durante a instalação do implante, a formação de osso em torno do implante de titânio é ativada quando a matriz é exposta ao fluido extracelular, liberando proteínas não colagenosas e fatores de crescimento (SCHENK et al., 1998). A osseointegração bem sucedida é determinada pela incorporação do osso trabecular e adaptação da massa óssea à carga, com aposição óssea contínua e remodelação em direção ao implante (PARITHIMARKALAIGNAN et al., 2013).
Um dos principais fatores que influenciam diretamente no processo de osseointegração é a formação do selamento mucoso peri-implantar, o qual representa a única barreira física capaz de não permitir interferências do meio externo na formação óssea (KONSTANTINIDIS et al., 2015). Dentre as moléculas responsáveis pela adequada cicatrização gengival, está o fator de crescimento epidérmico (EGF), que por meio da ligação ao seu receptor (EGFR), ativa uma cascata de eventos intracelulares capaz de estimular a proliferação das células epiteliais, fibroblastos e células endoteliais, influenciando diretamente na formação do epitélio juncional durante o período de osseointegração do implante endósseo (KONSTANTINIDIS et al., 2015).
A ausência de adequada osseointegração, incluindo deficiente cicatrização do tecido mole peri-implantar, pode facilitar a penetração bacteriana, promovendo o desequilíbrio na resposta do hospedeiro e favorecendo a inflamação (ZAIDI, 2007; KADKHODAZADEH et al., 2013). Por consequência, a atividade osteoclástica aumenta, com reabsorção óssea ativa e possível perda do implante (GIANNOPOULOU et al., 2012; HULTIN et al., 2002).
A associação entre a saúde do tecido mole peri-implantar durante o processo de osseointegração orquestrado por EGF e EGFR ainda não foi estudada, assim como a influencia da expressão dos genes EGF e EGFR no processo de osseointegração. Por isso, o objetivo deste estudo foi avaliar a associação entre a expressão dos genes EGF e EGFR na mucosa peri-implantar com a qualidade da osseointegração do implante endósseo.
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2 – MATERIAL E MÉTODOS
2.1 Participantes da Pesquisa
Quarenta e nove participantes de pesquisa, apresentando 59 implantes endósseos osseointegráveis, foram recrutados para o estudo na Especialização em Implantodontia da Universidade Veiga de Almeida, Rio de Janeiro, Brasil, durante um ano. Os procedimentos clínicos foram conduzidos de acordo com as recomendações do Comitê de Ética da Universidade Federal Fluminense sob o número de registro 80627 (Anexo A). O termo de consentimento livre e esclarecido informado foi obtido de todos os participantes (Anexo B). Os parâmetros clínicos iniciais da população estudada (Anexos C e D) são apresentados nas tabelas 1 e 2, assim como a anamnese e história médica e odontológica. Os critérios de exclusão foram: uso bifosfonato, ingestão de antibióticos e agentes anti-inflamatórios nos últimos três meses, gravidez e/ou lactantes, tabagistas, ausência de radiografia pré-operatória,participantes com doença periodontal ativa, e com exposição do parafuso de cobertura. Todos os participantes incluídos foram reabilitados com implantes de dimensões mínimas de 3,4mm de espessura e 7mm de altura, em procedimento de dois estágios cirúrgicos,mostrando qualidade e quantidade de osso favorável para a instalação,fazendo uso apenas de agentes analgésicos durante o período pós-operatório imediato (ADELL et al., 1981).
2.2 Avaliação da osseointegração
A osseointegração foi avaliada 1 mês após a cirurgia de reabertura, realizada 3 meses(em mandíbula) e 6 meses(em maxila) após a instalação do implante. Os critérios para considerar a osseointegração adequada foram: imobilidade do implante durante o teste clínico; ausência de radio transparência peri-implantar; e ausência de sinais e/ou sintomas persistentes, como dor, infecção, neuropatia, parestesia, ou comprometimento do canal mandibular (ALBREKTSSON et al.,1994),
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ausência de sinais clínicos de inflamação (sangramento espontâneo, edema, secreção purulenta, mucosa avermelhada).
O exame clínico dos tecidos peri-implantares foi realizado através de inspeção visual e palpação, análise de inflamação da mucosa, edema, sangramento espontâneo, pus, tipo plataforma do implante e mobilidade. Com base nas características clínicas dos sítios, os participantes foram caracterizados como (i) tendo a cicatrização sem complicações (com a osseointegração adequada, sem mobilidade do implante, e sem qualquer sinal clínico de inflamação da mucosa) (grupo controle) ou (ii) cicatrização peri-implantar inadequada (com osseointegração inadequada caracterizada por sinais de inflamação na mucosa e/ou mobilidade de implante) (grupo teste).
2.3 Expressão de RNA
Descartes gengivais foram coletados de 49 participantes de pesquisa após o período de osseointegração, durante o procedimento de reabertura do implante. As amostras foram imediatamente submersas no reagente para estabilização do RNA (RNA later, Qiagen, Valencia, CA) e armazenadas a -800C até a extração de RNA.
O RNA total a partir das amostras gengivais foi isolado utilizando o reagenteTrizol® (InvitrogenTM por Life Technologies, Nova Iorque, EUA),de acordo com o protocolo do fabricante. Tratamento com DNase, para digerir o DNA genômico que poderia levar a falsos resultados positivos, foi realizado usando DNA-freeDNase® (Ambion por InvitrogenTM pela Life Technologies, NY, EUA). A integridade do RNA foi confirmada ecorrida em eletroforese, gel de agarosea 1,2% corada com SYBR Stain® (InvitrogenTM pela Life Technologies, NY, EUA). A pureza do RNA foi confirmada com a razão das absorvâncias 260/280 em spectrophotômetro, e a quantidade de RNA foi estimada a 260 nm (Nanodrop® 1000, ThermoScientific, Wilmington, EUA). A reação de transcrição reversado PCR (RT-PCR) foi realizada para a síntese de DNA complementar (cDNA), a partir de 300 ng de RNA utilizando o sistema ImProm-II Transcrição Reversa System™ (Promega Corporation, Wisconsin, EUA), de acordo com o protocolo do fabricante. O controle do branco (RT-PCR, sem matriz de RNA) e as reações RT (reações de PCR sem a
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transcrição reversa) foram executadas juntamente com todos os RT-PCRs. As reações de PCR quantitativo (qPCR) foram realizadas no software MxPro-Mx3005P (Stratagene/Agilent Technologies, Wilmington, DE, EUA), utilizando o sistema de detecção rápida SYBR Green Master Mix (AppliedBiosystems, Foster City, CA, EUA) com 1,5 ul de cDNA em cada reação. qPCR foi realizada com um ativação a 95 ° C durante 10 minutos, seguido por 40 ciclos de desnaturação e prolongamento (95 ° C durante 15 segundos e 60 ° C durante 1 minuto). Iniciadores específicos para EGF (forward 5’ – TGG AAC TGC TTG GTG TTC GT - 3’ / reverso 5’ – AAC TTC ACC ACC TGC CAA CA - 3’) NM_001963.4 e EGFR (forward 5’ – CTC TTG CTG CTG GTG GTG G - 3’ / reverso 5‘ – CTC CAC AAG CTC CCT CTC CT - 3’) NM_005228.3, foram confeccionados com base nos dados BLAST (http: //blast.ddbj. nig.ac.jp/top-j.html). O Método Livak(2-ΔΔCT) foi utilizado para determinar a quantificação relativa da expressão de EGF e EGFR. Os valores foram normalizados em relação à expressão constitutiva de β-actina (para a frente 5 '- AAT TAC GAG CTG CGT GTG G - 3' / reverso 5 '- AGA GCG CAG GTA GGA TAG CA - 3') (WARA-ASWAPATI et al., 2007). Duas séries de experimentos foram realizados para cada amostra de tecido para assegurar a reprodutibilidade. Os dados são apresentados com a variação relativa de dobragem de um calibrador (pool de RNA a partir de todas as amostras). Todas as reações foram realizadas em duplicata contendo informações essenciais de acordo com Bustin et al. (2009).
2.4 Análise Estatística
O tamanho da amostra necessária para um estudo com 80% de poder foi calculado usando uma calculadora on-line em http://statpages.org/proppowr.html. As Nominais variáveis foram expressas em frequências e percentuais. Para acessar o significado das variáveis nominais entre os grupos, foi realizado o teste do qui-quadrado (χ2). As variáveis contínuas foram expressas em média e desvio padrão. O teste de Shapiro-Wilk foi utilizado para avaliar a distribuição entre as variáveis. A análise de variância foi realizada utilizando (ANOVA)/testa t-Test ou Mann-Whitney para comparação das médias entre os grupos, quando a variável estava em uma distribuição normal ou não-normal, incluindo a análise de expressão gênica após o
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cálculo pelo método 2-ΔΔCT. Valores de p<0,05 foram considerados estatisticamente significativos.As análises estatísticas foram realizadas utilizando Stata 11.1 (StataCorp, Texas EUA).
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3 – ARTIGO PRODUZIDO
The inadequate osseointegration of the endosseous implant is correlated with low levels of epidermal growth factor and your receiver in the peri-implant mucosa.
Abstract: Osseointegration failure can promote host response imbalance and bone resorption by bacteria infiltration. The objective of this study was to evaluate the association between the expression of EGF and EGFR genes expression in the peri-implant mucosa with the quality of osseointegration of endosseous peri-implant. Forty-nine participants, with 59 endosseos implants, were recruited for this study. All participants included were rehabilitated with implants in two stages surgical protocol, presenting favorable bone quality and quantity. Osseointegration was evaluated one month after exposure surgery, wich was performed three months (mandible) and 6 months (maxillary) after implant placement. The criteria to consider the proper osseointegration were: implant immobility; absence of peri-implant radiolucency; and no clinical signs of inflammation. Based on clinical and radiographic characteristics of peri-implant sites, participants were characterized as (i) having healing without complications (with proper osseointegration without mobility of the implant, and without clinical signs of mucosal inflammation) (control group) or (ii) failure peri-implant healing (with inadequate osseointegration characterized by signs of mucosae inflammation and/or implant mobility) (test group). Gingival biopsies were collected from 49 participants after osseointegration period, during the exposure procedure. Total RNA from gingival samples was isolated using the Trizol® reagent. The reaction of reverse transcription of PCR was performed for the synthesis of complementary DNA from 300ng RNA using Improm-II Reverse Transcription System ™. Specific primers for EGF (NM_001963.4) and EGFR (NM_005228.3) were based on the BLAST data. The Livak method (2-ΔΔCT) was used to determine the relative quantification of the expression of EGF and EGFR. The values were normalized by relative expression of β-actin. There was no difference between control and test groups to race, sex, age, alcohol consumption, general medical conditions, current medications, edentulism and periodontal phenotype. All RNA samples revealed proportions A260 nm / A280 nm more than 1.9. EGF and EGFR showed significant lower expression in gingival tissues removed from regions with failure healing (test group). EGF showed mRNA expression with an average of 44.53
18
± 79.16 and 01.02 ± 1:33 in the control groups and test, respectively (p = 0.008). Similarly EGFR expression was significantly higher in the control group (102.03 ± 329.57) compared to the test group (7.85 ± 4.16) (p = 0:04). We conclude that low levels of EGF and EGFR are associated with inadequate healing of mucosal peri-implant during the osseointegration period.
Keywords: epidermal growth fator; receptor of epidermal growth fator; osseointegration; wound healing.
INTRODUCTION
The osteogenic process requires a careful chronological and spatial coordination of molecular signals leading to proliferation, migration and differentiation of mesenchymal precursor cells into osteoblasts (ZAIDI, 2007). During installation of the implant, bone formation around the implant of titanium is activated when the matrix is exposed to the extracellular fluid, releasing non-collagenous proteins and growth factors (SCHENK et al., 1998). The successful osseointegration is determined by incorporation of trabecular bone and bone mass adaptation to the load, with continuous bone apposition and remodeling toward the implant (PARITHIMARKALAIGNAN et al., 2013).
One of the main factors that have directly influence the osseointegration process is the formation of peri-implant mucosal sealing, which is the only physical barrier able to not allow interference of the external environment in bone formation (KONSTANTINIDIS et al., 2015). Among the molecules responsible for the adequately gingival healing is epidermal growth factor (EGF), that through binding to its receptor (EGFR), activates a cascade of intracellular events capable of stimulating the proliferation of epithelial cells, fibroblasts and endothelial cells , directly influencing the formation of the junctional epithelium during the period of osseointegration of endosseous implant (KONSTANTINIDIS et al., 2015).
The absence of adequate osseointegration, including scarring of the peri-implant soft tissue, can facilitate bacterial penetration, promoting imbalance in the host response and favoring inflammation (ZAIDI, 2007; KADKHODAZADEH et al,
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2013.). Consequently, osteoclast activity increases with active bone resorption and possible loss of the implant (GIANNOPOULOU et al, 2012;. Hultin et al., 2002).
The association between the health of the peri-implant soft tissue during the osseointegration process orchestrated by EGF and EGFR has not been studied, as well as the influence of the expression of EGF and EGFR genes in the process of osseointegration. Therefore, the aim of this study was to evaluate the association between the expression of EGF and EGFR genes in the peri-implant mucosa to the quality of osseointegration of endosseous implant.
MATERIAL AND METHODS
Research participants
Forty-nine research participants, with 59 osseointegrated endosseous implants, were recruited for a year to the study in the Specialization in Implantology at the Universidade Veiga de Almeida, Rio de Janeiro, Brazil. The clinical procedures were conducted in accordance with the recommendations of the Ethics Committee of the Universidade Federal Fluminense under the registration number 80 627 (Annex A). The free and clear consent term was obtained from all participants (Annex B). Initial clinical parameters of the study population (Annexes C and D) are presented in tables 1 and 2, as well as medical and dental history. Exclusion criteria were: bisphosphonate use, ingestion of antibiotics and anti-inflammatory agents in the last three months pregnancy and / or lactating smokers, absence of preoperative radiographs, participants with active periodontal disease, and exposure of the cover screw . All participants included were rehabilitated with minimum dimensions of implant 3.4mm thick and 7mm in height in two-stage surgical procedure, showing quality and quantity of bone favorable for installation, making use only of analgesic agents during the post operatively immediate (ADELL et al., 1981).
Evaluation of osseointegration
The osseointegration was evaluated one month after the reopening surgery, performed 3 months (in the mandible) and 6 months (maxilla) after implant
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placement. The criteria to consider adequate osseointegration were immobility of the implant during the clinical test; absence of radio transparency peri-implant; and the absence of signs and/or persistent symptoms such as pain, infection, neuropathy, paresthesia, or compromised mandibular canal (ALBREKTSSON et al., 1994), absence of clinical signs of inflammation (spontaneous bleeding, swelling, pus, reddish mucosa ).
The clinical examination of peri-implant tissues was carried out by visual inspection and palpation, mucosal inflammation analysis, edema, spontaneous bleeding, pus, platform type of implant and mobility. Based on the clinical characteristics of the sites, participants were characterized as (i) having healing without complications (with proper osseointegration without mobility of the implant, and no clinical signs of inflammation of the mucosa) (control group) or (ii) inadequate peri-implant healing (with inadequate osseointegration characterized by signs of inflammation in the mucosa and / or mobility of implant) (test group).
RNA Expression
Gingival Descartes were collected from 49 research participants after the osseointegration period, during the reopening procedure of the implant. The samples were immediately submerged in the reagent for stabilization of RNA (RNA later, Qiagen, Valencia, CA) and stored at -800oC until RNA extraction.
Total RNA from gingival samples was isolated using the reagenteTrizol® (InvitrogenTM by Life Technologies, NY, USA) according to the manufacturer's protocol. Treatment with DNase to digest the genomic DNA which could lead to false positive results was performed using DNA freeDNase® (Ambion by InvitrogenTM by Life Technologies, NY). The RNA integrity was confirmed by electrophoresis, agarosea gel 1.2% stained with SYBR Stain® (InvitrogenTM by Life Technologies, NY). The purity of the RNA was confirmed by the ratio of absorbance at 260/280 spectrophotometer, and the amount of RNA was estimated at 260 nm (Nanodrop® 1000, ThermoScientific, Wilmington, USA). The reverse transcription PCR reaction (RT-PCR) was performed for the synthesis of complementary DNA (cDNA) from 300 ng RNA using Improm-II Reverse Transcription System ™ (Promega Corporation, Wisconsin, USA), according to the manufacturer's protocol. The background control (RT-PCR without RNA template) and the RT reactions (PCR reactions without
21
reverse transcription) was performed with all the RT-PCRs. The reactions quantitative PCR (qPCR) were performed in software MxPro-Mx3005P (Stratagene / Agilent Technologies, Wilmington, DE, USA) using the rapid detection system SYBR Green Master Mix (AppliedBiosystems, Foster City, CA) with 1,5 ul of cDNA in each reaction. qPCR was performed with an activation at 95 ° C for 10 minutes, followed by 40 cycles of denaturing and extension (95 ° C for 15 seconds and 60 ° C for 1 minute). Specific primers forEGF (forward 5’ – TGG AAC TGC TTG GTG TTC GT - 3’ / reverse 5’ – AAC TTC ACC ACC TGC CAA CA - 3’) NM_001963.4 e EGFR (forward 5’ – CTC TTG CTG CTG GTG GTG G - 3’ / reverse 5‘ – CTC CAC AAG CTC CCT CTC CT - 3’) NM_005228.3, foram were made based on the BLAST data (http: //blast.ddbj. nig.ac.jp/top-j.html). The Livak method (2-ΔΔCT) was used to determine the relative quantification of the expression of EGF and EGFR. The values were normalized relative to constitutive expression of β-actin (forward 5'- AAT TAC GAG CTG CGT GTG G - 3' / reverse 5 '- AGA GCG CAG GTA GGA TAG CA - 3') (WARA-ASWAPATI et al., 2007). Two series of experiments were performed for each tissue sample to ensure reproducibility. Data are presented with the relative variation of bending a calibrator (RNA pool from all samples). All reactions were performed in duplicate containing essential information according Bustin et al. (2009).
Statistical Analysis
The sample size required for a study with 80% power was calculated using an online calculator at http://statpages.org/proppowr.html. The Nominal variables were expressed by frequencies and percentages. To access the meaning of nominal variables between groups was performed chi-square test (χ2). Continuous variables were expressed as mean and standard deviation. The Shapiro-Wilk test was used to assess the distribution between the variables. Analysis of variance was performed using (ANOVA) / t-test or Mann-Whitney tests to compare means between groups, when the variable was in a normal or non-normal distribution, including gene expression analysis after the calculation for 2-ΔΔCT method. P values <0.05 were considered statistically significant. Statistical analyzes were performed using Stata 11.1 (Stata Corp, Texas USA).
22
Results
Clinical Results
Of the total of 49 individuals evaluated for six months, there were 36 (73.4%) women and 13 (26.6%) menwith a mean age 56.54 ± 9.8 years. There was no difference between the control and test groups as to race, sex, age, alcohol consumption, general medical conditions, current medications, tooth loss and periodontal phenotype (Tables 1 and 2).
Considering the peri-implant assessment (Table 3), a month after exposure of the implant, the test group showed a higher prevalence of red mucous membranes, swelling and pus (p <0.0001).
However, plaque build-up, the implant region and the loss of the implant were not statistically different between groups.
Participants with inadequate osseointegration (test group) had a higher prevalence of history of chronic periodontitis (p <0.0001). According to the analysis of oddsratio, participants with chronic periodontitis history have 22.5 times more likely to develop inadequate osseointegration than participants with no history of chronic periodontitis (OR22.5 CI 4.1-120.5) (Table 4).
Correlation between clinical parameters and the expression of EGF and EGFR
In order to withstand the potential association between the EGF and EGFR genes with the quality of the endosseous implant osseointegration, the expression of these genes was investigated in 49 gingival discards obtained after the osseointegration process in subjects with and without inadequate osseointegration. All RNA samples revealed proportions A260 nm / A280 nm of more than 1.9. From the total sample, EGF and EGFR were detected in 42 tissue mRNA (85.7%). EGF and EGFR showed significant lower expression in gingival tissues removed from regions with inadequate healing (test group). EGF showed mRNA expression with an average of 44.53 ± 79.16 and 01.02 ± 1:33 in control and test groups, respectively (p
23
= 0.008) (Figure 1). Similarly EGFR expression was significantly higher in the control group (102.03 ± 329.57) compared to the test (7.85 ± 4.16) (p = 0:04) (Chart 2).
Discussion
Based on the potential correlation between inadequate healing of the peri-implant tissue and the development of early peri-implant disease, the aim of this study was to evaluate the association between the expression of EGF and
EGFR genes in the peri-implant mucosa, with the quality osseointegration of
endosseous implant. Our results clearly show that EGF and EGFR genes are less expressed in the mucous corresponding to regions with inadequate healing. In contrast, regions with proper osseointegration and integrity of the peri-implant mucosa showed high expression of the same genes, consolidating the low expression of these genes as a risk factor for the inappropriate tissue regeneration around the implant and therefore the development of precocious peri-implantar disease.
The main function of the gingival epithelium is to protect the underlying structures against physical stress generated during the chewing process and act as the primary barrier to bacterial penetration (KIM et al., 2011). The gingival fibroblasts are the most numerous cells in the gingival connective tissue, responsible for the synthesis of collagen and elastic fibers, and especially the formation of granulation tissue during tissue healing (HAKKINEN et al., 2000; NEWMAN et al., 2002) .
In natural teeth, the junctional epithelium provides sealing at the base of the periodontal groove against the penetration of chemical pathogens and bacterial substances. As no cement or fiber insertion is seen in the titanium surface, a mucous sealing provides the main barrier against the spread of pathological insults to the peri-implant deep tissue. This sealing around the endosseous implants is provided by the presence of junctional epithelium, the sulcular epithelium and connective tissue, which has weak adhesion to the titanium structure made by hemidesmosomes (KAN et al, 2005; PELEGRINI et al, 2006). Destruction of the integrity of perimucosa surface of titanium leads to the extension of pathological bag directly to bone tissue.
24
These anatomical and physiological features of the peri-implant region, and the fact of cell migration and proliferation at the site of tissue injury, represented in our study by the location of the endosseous implant installation, being directed by the release of EGF and its receptor (KING et al . 2002), led us to try to understand what the association between scar profile post implant placement and the levels of this growth factor.
The gene that controls the production of EGF in the human is in the chromosome 4 and its molecule contains 53 amino acids. Specific receptors or
EGFR are present in epithelial cells of local with high and low proliferation index and
cell differentiation. In oral tissues EFG receptors are present in all epithelia. In other cells, such as fibroblasts and endothelial cells, EGF also acts as a mitogen. Molecules of EGF are found in the interstitium of the oral submucosal tissue. In saliva, the EGF is present since the salivary glands are epithelial organs. In the repair, the EGF has proved to be important and the epithelial surfaces stimulates proliferation, differentiation, organization and keratinization of the superficial layers in the regenerative process of ulceration (CONSOLARO et al., 2010).
The EGF in the saliva and in the epithelial cells stimulate peri-implant epithelial proliferation, starting the formation of peri-implant junctional epithelium closely related to the implant surface, also stimulated by the EGF present in saliva. Once that integration occurs between the implant and the epithelium, salivary EGF penetration stops or decreases drastically and the cell renewal process returns to the normal level (CONSOLARO et al. 2010). In our study, the levels of EGF/EGFR were measured immediately after the clinically determined implant osseointegration, and this growth factor and its receptor were measured directly in the peri-implant mucosa without being regarded their levels in the saliva or crevicular fluid peri-implantar. Thus, based on the findings obtained in the reopening of the implant may suggest that high levels of EGF/EGFR in the peri-implant mucosa can be correlated with the high regenerative capacity still present on the osseointegration process and maintenance of the peri-implantar mucosal sealing in healthy tissue, supplying the absence of EGF derived from saliva for this purpose, which may not have occurred in regions with inappropriate scarring where low expression of EGF/EGFR was observed.
Another important issue to consider is that, physiologically, the healing is divided into three phases: inflammation, proliferation and remodeling (SINGER,
25
1999). During this process, EGF has a central role being produced by a variety of cells, including endothelial cells and fibroblasts, with regulatory function in the final healing cascade after the formation of the hemostatic plug and its expression is altered by the expression of its receptor EGFR on target cells (HARDWICKE et al., 2008), being a dose-dependent mitogen for the fibroblasts granulation in the healing remodeling phase (TSANG et al. 2003). Therefore, studies have explored the importance of studying the EGF receptor along with EGF (BALICKI et al., 2005) to better understand the biology of this growth factor and its level after the period of acute healing phase, technically , occurs after 14 days in the gingival epithelium and 30 days in the bone tissue. Thus, our study looked not only the expression of EGF, but also of EGFR and its correlation with the clinical carcterísticas observed after the period of peri-implant healing (remodeling) previously established by Branemark et al. (1977).
Despite the EGF/EGFR be associated with the process of peri-implant saucerization (CONSOLARO, 2010), and the etiopathogenesis of cancer, especially squamous cell carcinoma, our study, in contrast, showed that high levels of EGF are associated with health peri-implant and mucosal healing properly. These results, however, have not considered the analysis of peri-implant bone. Deficient
EGF mouses altered severely the process of ossification and the recruitment of
osteoclasts and interfering in bone remodeling (WANG, 2004). Based on these findings, future studies are needed, including the analysis of bone tissue, to better understand the function of the EGF/EGFR in peri-implant tissue.
However, confirming our findings, several studies (IRWIN et al, 1991;. TAJIMA et al, 1992;. GARANT, 2003) identified high levels of EGF and EGFR in cells derived from the junctional epithelium in healthy tissues. It is speculated that
EGF/EGFR system participates in osteoblast differentiation, by stimulating the
proliferation of osteoblastic progenitors (CHIEN et al., 2000), with high levels at regenerated tissues (PARKAR et al., 2001). EGF negatively regulates ALPase activity in periodontal ligament cells, while increasing DNA synthesis and fibronectin (FUJITA et al. 2004), suggesting that EGF may act as a regulator of proliferation and periodontal regeneration.
Based on the apparent contradiction in the literature concerning levels of EGF/EGFR in healthy gingiva we can consider that these findings were conducted on studies following different clinical protocols, sample collection in
26
different tissues, including mucosa, bone and saliva, and different times of healing factors which modify the expression of this growth factor and its receptor. In our study, we sought to evaluate the expression of EGF/EGFR in situ in the peri-implant tissue, immediately after the period of clinical osseointegration, in other words, in tissue remodeling phase. Our findings, found biological support and consider the high levels of EGF and EGFR in healthy mucosa as active in tissue homeostasis and maintenance of mucosal sealing.
EGF has been explored for use in regenerative therapies, to increase
their levels in regenerating tissues, including alveolar bone, root, periodontal and tooth pulp (FURFARO et al. 2014), also decreasing the risk of anchylosis and root resorption.
This therapeutic application is extremely relevant, considering that current indices show an increasing incidence of peri-implant disease in rehabilitated participants with endosseous implants (LINDHE, 2008) .The inadequate implant osseointegration occurs due to the action of external and internal forces, being called biomechanical failures. These factors include: bacterial infection, occlusal overload, genetic factors, local factors and early exposure of the implant (BLOCK & KENT, 1990). Among one of the factors related to failures in implant dentistry is the peri-implant anatomy and bacterial invasion of the mucous sealing formed during the osseointegration period, immediately after installation and during the exhibition of the endosseous implant.
These failures arising from the peri-implant mucosal injury (mucositis) that can spread to the underlying bone (peri-implant), triggering bone loss and / or loss of the implant, affect about to 65% of the population rehabilitated with endosteal implant (KONSTANTINIDIS et al., 2015). And, according Tolstunov (2006), can be represented by two major periods characterizing the failure in the implant (1) early failures or failures during the osseointegration period occurring during the first year after the implant insertion and load initial, (2) late failures that occur usually one year after implant insertion. In our study, we consider the failures that occur early and are directly related to the early development of peri-implant disease.
This is the first study demonstrating the association between inadequate healing during the period of osseointegration and expression of
27
with the proviso peri-implant health. One reason may be related to the fact that perhaps the expression in healthy peri-implant tissue, in our study, is within the normal range to allow proper healing and formation of mucous sealing. By contrast, the group with mucosal inflammation or inadequate osseointegration may have presented these clinical features for low expression of EGF and EGFR which may have hindered the proper epithelial proliferation and peri-implant remodeling. Thus, further studies are necessary to allow better identify which levels considered "normal" for peri-implant proper healing and which EGF and EGFR levels considered "at risk" for developing injury.
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Table 1. Characteristics of the study population.
Parameters Total (n= 49) Controle (n= 32) DPI
(n= 17) P-valor Oddsratio (CI) n (%) n (%) n (%) Ethinicgroup Caucasians 26 18 8 0.75 0.69 (0.21-2.25) Non-Caucasians 23 14 9 Age (years) 56.54±9.8 55.59±10.18 58,438.68 0.17 ---Gender Female 36 25 11 0.49 0.51 (0.13-1.88) Male 13 7 6 Alcohol Consumption No 48 32 16 0.34 RR 0.33 (0.22-0.49) Yes 1 0 1
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Table 2. Participants medical clinical and dental characteristics.
Parameters Total (n= 49 ) Control (n= 32 ) DPI (n= 17)
P-value Oddsratio (CI) n (%) n (%) n (%)
Diabetes 1 1 0 0.65
---High blood pressure 4 2 2 0.60 2 (0.25-15.62)
Hypothyroidism 1 1 0 1.0 ---Current medication Anti-hypertension 5 3 2 0.57 1.28 (0.19-8.57) Hormone reposition 3 3 0 0.30 ---Antidepressant 1 1 0 1.0 ---Anti-epileptic 1 1 0 1.0 ---Clinical measurements Total Edentulism 9 5 4 0.70 1.66 (0.38-7.24) Partial Edentulism 40 27 13 Periodontal Phenotype Thick 33 21 12 0.76 1.25 (0.35-4.49) Slim 6 11 5
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Tabela 3. Peri-implant status of subjects.
Peri-implant status Total (n= 49 )
Controle (n= 32)
DPI
(n= 17) P-value OddsRatio (CI)
Redmucosae 17 0 17 <0.000 1 ---Edema 12 0 12 <0.000 1 ---Pus 1 0 1 <0.000 1 ---Mobility (presence) 1 0 1 0.35 ---Implant region Maxilla 32 20 12 0.75 1.44 (0.4-5.1) Mandible 17 12 5 Peri-implant Plaque 1 0 1 0.35 ---Implant loss 2 0 2 0.12
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Table 4.Correlation between history of chronic periodontitis and harmful peri-implant healing.
Controle (n= 32)
DPI
(n= 17)
p-value Oddsratio (CI) Without a history of periodontitis 24 2 <0.0001 22.5 (4.1-120.5) With a history of periodontitis 8 15
35
Graphic 1. EGF expression in the Test and Control groups. Note the low expression, considering the average of the mRNA determined by 2-ΔΔCT in the Test group (p = 0.008).
36
Graphic 2. EGFR expression in the Test and Control groups. Note the low expression, considering the average of the mRNA determined by 2-ΔΔCT in the Test group (p = 0.008).
54
4 – CONCLUSÕES
- Baixos níveis de EGF e EGFR tem associação com inadequada cicatrização da mucosa peri-implantar durante o período de osseointegração.
- Pacientes com histórico de periodontite apresentam maior risco para o desenvolvimento de osseointegração inadequada.
54
54
ANEXO B - Termo de consentimento livre e esclarecido
TERMO DE CONSENTIMENTO LIVRE E ESCLARECIDO
Projeto: A expressão do fator de crescimento epidêrmico e de seu receptor influencia no aspecto clínico da região peri-implantar
Pesquisador Responsável: Dra. Priscila Ladeira Casado – Universidade Federal do Rio de Janeiro Telefones para contato: (21)98190-7074
Nome do
Voluntário:_________________________________________________________________________ Idade:__________anos R.G.________________
O Sr.(a) está sendo convidado a participar do projeto de pesquisa “A expressão do
fator de crescimento epidêrmico e de seu receptor influencia no aspecto clínico da região peri-implantar” de responsabilidade da pesquisadora Dra. Priscila Ladeira Casado, que objetiva analisar a
expressão de marcadores de perda óssea na mucosa peri-implantar, através da avaliação clinica e radiográfica da gengiva e da coleta da mucosa em cima do implante, a qual seria descartada durante o procedimento cirúrgico para expor o implante à cavidade oral..
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 Paciente: _____________________________________________________________ Assinatura do Cirurgião-dentista que obteve o consentimento: ________________________________
Testemunha:________________________________________Testemunha:____________________ _____
54
Ficha clínica Data: ____/_____/_____
Banco DNA: ____________________
Nome: Iniciais: Nasc: Endereço: Telefone: Sexo Etnia:
Nacionalidade: Legenda: A – saúde B – mucosite C – peri-implantite 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 Acúmulo de placa na boca (higiene oral) ( ) Boa ( ) Ruim
54
ANEXO D -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 osseointegraçã o (meses) Exposição do implante (classificar/ legenda) Nomenclatura Assinatura/carimbo do cirurgião-dentista: ____________________________
