Reabilitação em curto período com PRGF de segunda geração geração associada a implantes: estudo clínico randomizado

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UNIVERSIDADE FEDERAL FLUMINENSE FACULDADE DE ODONTOLOGIA

PROGRAMA DE PÓS-GRADUAÇÃO EM ODONTOLOGIA DOUTORADO EM ODONTOLOGIA

REABILITAÇÃO EM CURTO PERÍODO COM PRGF DE 2a GERAÇÃO ASSOCIADA A IMPLANTES – ESTUDO CLÍNICO RANDOMIZADO

NITERÓI 2016

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UNIVERSIDADE FEDERAL FLUMINENSE FACULDADE DE ODONTOLOGIA

PROGRAMA DE PÓS-GRADUAÇÃO EM ODONTOLOGIA DOUTORADO EM ODONTOLOGIA

REABILITAÇÃO EM CURTO PERÍODO COM PRGF DE 2a_GERAÇÃO

ASSOCIADA A IMPLANTES – ESTUDO CLÍNICO RAMDOMIZADO

GUSTAVO VICENTIS DE OLIVEIRA FERNANDES

Tese apresentada à Faculdade de Odontologia da Universidade Federal Fluminense, como parte dos requisitos para obtenção do título de Doutor, pelo Programa de Pós-Graduação em Odontologia.

Área de Concentração: Biomateriais / Implantodontia

Orientadores: Prof. Dr. José Mauro Granjeiro e Profa. Dra. Mônica Diuana Calasans Maia

Niterói 2016

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BANCA EXAMINADORA

Prof. Dr. José Mauro Granjeiro

Instituição: Universidade Federal Fluminense

Decisão: _________________________Assinatura: _______________________

Profa. Dra. Mônica Diuana Calasans Maia Instituição: Universidade Federal Fluminense

Prof. Dr. Plínio Mendes Senna

Instituição: UNIGRANRIO

Prof. Dr. Márcio Baltazar Conz

Instituição: Osseocon

Prof. Dr. Antonio Carlos Canabarro Andrade Junior

Instituição: Universidade Veiga de Almeida

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RESUMO

Fernandes GVO, Calasans-Maia MD, Granjeiro JM. Reabilitação em curto período com PRGF de 2a_{geração associada a implantes – Estudo clínico ramdomizado [tese].}

Niterói: Universidade Federal Fluminense, Faculdade de Odontologia; 2016.

O objetivo deste estudo foi avaliar in vitro a segunda geração do plasma rico em fatores de crescimento (PRGF-2) e avaliar clinicamente através de estudo clínico randomizado, controlado e duplo-cego a estabilidade dos implantes dentários (duplo ataque ácido), instalados com ou sem aplicação do PRGF-2 e colocados em função em curto período de tempo. Houve a quantificação de diferentes citocinas, fatores de crescimento e quimiocinas a partir do PRGF-2 obtido utilizando o Luminex®. Sessenta e nove participantes de pesquisa foram incluídos no estudo, divididos aleatoriamente em Grupo 1 (35 implantes, controle) e Grupo 2 (34 implantes com PRGF-2, grupo experimental). Observou-se que, a partir de 27 proteínas avaliadas, somente a IL-15 (p<0,001), o PDGF-BB (p<0,05), MIP-1b (p<0,05) e eotaxina (p<0,01) tiveram relevância estatística. Na avaliação clínica, sete participantes de pesquisa do total do estudo (10,1%) perderam o implante. Destes, quatro foram do grupo controle (5,80%) e três do grupo experimental (4,3%). O Grupo 1 alcançou a média aproximada de 68 dias para a reabilitação dos participantes, com taxa de sucesso de 88,6% e 31,4 ± 6,1 N.cm para o torque médio de inserção do implante. No Grupo 2, média aproximada de 71 dias para a reabilitação, com taxa de sucesso de 91,2% e 28,0 ± 8,1 N.cm para o torque de inserção. O ISQ inicial para Grupo 1 e Grupo 2, respectivamente, foram 66,4 ± 6,0 e 65,6 ± 13,0; o ISQ final obtido antes de instalar a coroa foi de 68,1 ± 6,8 para o grupo1 e 70,2 ± 5,7 para o PRGF-2, porém sem significância estatística. Nenhuma relevância clínica foi observada para o implante instalado justaposto ao osso quando comparado entre os grupos. A sugestão de nova metodologia para reabilitação precoce (até 60 dias), em qualquer região da boca, pode ser aplicada com torque de até 34 N.cm em superfícies de duplo ataque ácido. Concluiu-se que uma nova e simplificada estratégia de produção gerou um conteúdo similar de PRGF, quando comparado com o PRGF inicial, porém sem aditivos, e para implante instalado em justaposição com o osso, não houve relevância no uso do PRGF-2.

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ABSTRACT

Fernandes GVO, Calasans-Maia MD, Granjeiro JM. Short term for rehabilitation with PRGF-2nd Generation and dental implants – Ramdomized Clinical Trial [thesis]. Niterói: Universidade Federal Fluminense, Faculdade de Odontologia; 2016.

The objective of this study was to evaluate in vitro the second generation of growth factor rich plasma (PRGF-2) and to evaluate the stability of dental implants (double acid attack) Or without application of PRGF-2 and put into operation in a short period of time. Quantification of different cytokines, growth factors and chemokines from PRGF-2 obtained using Luminex® was performed. Sixty-nine research participants were enrolled in the study, randomly divided into Group 1 (35 implants, control) and Group 2 (34 implants with PRGF-2, experimental group). It was observed that only IL-15 (p <0.001), PDGF-BB (p <0.05), MIP-1b (p <0.05) and eotaxin (p < 0.01) were statistically significant. In the clinical evaluation, seven study participants from the total study (10.1%) lost the implant. Of these, four were taken from the control group (5.80%) and three from the experimental group (4.3%). Group 1 achieved a mean of approximately 68 days for the participants' rehabilitation, with a success rate of 88.6% and 31.4 ± 6.1 N.cm for the mean insertion torque of the implant. In Group 2, a mean of approximately 71 days for the rehabilitation, with a success rate of 91.2% and 28.0 ± 8.1 N.cm for the insertion torque. The initial ISQ for Group 1 and Group 2, respectively, were 66.4 ± 6.0 and 65.6 ± 13.0; the final ISQ obtained before installing the crown was 68.1 ± 6.8 for Group 1 and 70.2 ± 5.7 for PRGF-2, but without statistical significance. No clinical relevance was found in the implant installed juxtaposed to the bone when compared between the groups. The suggestion of a new methodology for early rehabilitation (up to 60 days), in any region of the mouth, can be implemented with torque of up to 34 N.cm on acid double attack surfaces. It was concluded that a new and simplified production strategy generated a similar content of PRGF, when compared to the initial PRGF, but without additives, and implant installed in juxtaposition with the bone, there was no relevance in the use of PRGF-2.

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1. INTRODUÇÃO

Grandes avanços têm ocorrido na Implantodontia, na qual têm surgido novos materiais e técnicas, como diferentes tipos de tratamento de superfícies dos implantes1, os quais buscam reduzir o tempo de carregamento pós-cirúrgico, acelerando o crescimento ósseo e a maturação do íntimo contato entre as superfícies implante-osso2. Assim, ensaios in vitro3, in vivo1 e estudos clínicos4, buscam avaliar e aprimorar o tratamento destas superfícies e a sua influência sobre o mecanismo de integração óssea.

Uma abordagem interessante é sua associação com concentrados plaquetários. Este tem por finalidade auxiliar em tratamentos médicos e odontológicos, ajudando na cicatrização dos tecidos5. Sua citação ocorre desde a década de 50, demonstrando que as plaquetas são uma potente fonte de mitógenos presentes em todo sérum sanguíneo6. Outros estudos, in vitro, demonstraram uma tendência geral de que o material pode estimular osteoblastos7_{, fibroblastos}8 _{e células-tronco}

mesenquimais9_.

Desde então, muitos protocolos têm surgido buscando melhorar esta técnica. A variação pode ocorrer no tempo, nas forças e na centrifugação. Para técnica padrão de PRP, normalmente é utilizada o processo de centrifugação em dois passos, a qual pode variar entre 160 G a 3.000 G (primeira centrifugação), com tempo de 3 a 20 minutos. Desta maneira, o tratamento clínico tornar-se cansativo e prolongado.

A primeira matriz foi denominada cola de fibrina e foi desenvolvida para ajudar na cicatrização10_{. A segunda matriz autóloga foi desenvolvida por alguns}

pesquisadores que tentaram reforçar a preparação, modificando e combinando com um fator natural de coagulação. Estas preparações autólogas foram nomeadas misturas de plaquetas com fibrinogênio e trombina ou gelatina de plaquetas (ou espuma gel)11 e foi apresentado com sucesso na medicina12. Até este momento, a presença de plaquetas, fatores de crescimento e o potencial para a cura não foram considerados.

Em seguida, este conceito foi alterado em 1986, quando alguns pesquisadores13 desenvolveram um material eficiente, utilizando um procedimento em duas centrifugações, chamados de "fatores de cicatrização derivados de plaquetas" ou "produto derivado de plaquetas para cicatrização de feridas (PDWHF)"14.

Alguns anos mais tarde, Whitman et al.15 publicaram resultados clínicos usando concentrado plaquetário, o que chamou de "gel de plaquetas". Em 1970, este

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concentrado já havia aparecido, mas se tornou popular na década de 9016_{. Neste}

sentido, diferentes formas de preparação de PRP surgiram a partir da centrifugação do sangue convencional, com a ativação pela trombina e/ou adição de cálcio, contato com o silicato (vidro) ou aplicado como suspensão17 de plaquetas. Ainda assim, novos métodos continuam surgindo18-19.

Em seguida, uma confusão significativa ocorreu na literatura sobre concentrados plaquetários, devido a diferentes protocolos denominados "PRP" os quais não tinham uma caracterização adequada do conteúdo20 e, também, devido ao conceito de "fatores de crescimento para regeneração", o que seduziu ainda mais muitos pesquisadores21.

Neste cenário, o Plasma Rico em Fatores de Crescimento (PRGF) emergiu. Pertence ao grupo de Puro-Plasma Rico em Plaquetas (PRP-P)22, segundo a classificação atual, possui grande interesse por ter sido usado para tratamento de múltiplas patologias23,24_{, bem como na medicina regenerativa e na área de engenharia}

de tecidos25,26_{, com resultados favoráveis.}

No processo de confecção, no interior do tubo de PRGF, existe citrato trissódico (3,2% ou 3,8%), utilizado para teste de coagulação27_{como anticoagulante sanguíneo,}

e cloreto de cálcio para ativar a cascata da coagulação28. Precisa-se preservar ambos os aditivos para um melhor controle do processo.

A quantidade de citrato trissódico e cloreto de cálcio / mL no PRGF pode ser, respectivamente de 0,9 mL e 50 μL29_{ou 0,4 mL e 20 μL}30_{. Esta redução proposta na}

literatura não afetou as propriedades de anticoagulação, uma vez que a quantidade de anticoagulante no tubo esteve em excesso em relação a quantidade de cálcio do sangue total coletado31.

Em ambos os protocolos PRGF mencionados acima, o “buffy coat” foi excluído, obtendo-se uma contagem de leucócitos próximo a zero, sendo o potencial terapêutico dos dois semelhantes30. No entanto, o protocolo com menor quantidade de citrato trissódico e cloreto de cálcio encurtou o tempo de coagulação e a fase de latência30, podendo isto ser relevante do ponto de vista clínico.

Portanto, algumas dúvidas podem surgir sobre os anticoagulantes utilizados. Embora quase não aparecem problemas clinicamente, eles podem interferir com outro analito na utilização do plasma32. Além disso, citrato de sódio inibe a ativação de plaquetas, que também pode inibir a aspartato aminotransferase e a fosfatase alcalina

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pela quelação de cátions27_{, além de ser utilizado para medir os níveis plasmáticos dos}

componentes derivados das plaquetas.

Além disso, alguns ativadores da coagulação são problemáticos porque devem ser completamente misturados para permitir a granulação com todo o coágulo. Se ocorrer a formação de coágulos de fibrina solúveis, os ativadores podem interferir com a precisão do dispositivo de pipetagem ou na fase sólida de ligação em imunoensaios33.

Neste panorama, surge a segunda geração de agregados plaquetários, tal como a fibrina rica em plaquetas (PRF)34, até a fibrina rica em plaquetas avançada (a-PRF)35, a qual não utiliza material como anticoagulante, permitindo que o processo natural de coagulação ocorra.

Esses fatos conduzem para um questionamento: seria favorável a utilização de uma segunda geração do PRGF para a osseointegração de implantes para reabilitação em curto período? Assim, o objetivo deste estudo foi avaliar e caracterizar o PRGF-2, em comparação com PRGF de primeira geração, e aplicá-lo em um ensaio clínico controlado, cego e randomizado para avaliar a estabilidade dos implantes dentários, com tratamento de superfície de duplo ataque com ácido, inserido com e sem aplicação PRGF-2, colocado em função em curto período com a coroa de cerâmica final.

A hipótese nula (H0) deste trabalho é de que o material não acelere a cicatrização do implante endósseo e não se consiga realizar a reabilitação em curto tempo; a hipótese alternativa (H1) seria o sucesso desta reabilitação em curto período com acompanhamento por mais de 6 (seis) meses.

3. MATERIAIS E MÉTODOS

Este estudo clínico foi submetido e aprovado pelo Comitê de Ética em Pesquisa (número 859.788), anexo 2. Depois de explicação e concordância dos participantes, os quais foram selecionados pelo autor da pesquisa (Gustavo V. O. Fernandes), eles assinaram um Termo de Consentimento Livre e Esclarecido Informado (anexo 1). Assim, foi feito o teste das proteínas existentes nos concentrados de plaquetas (1a geração de PRGF e a 2ª geração). Depois, seguiu para o ensaio clínico, em que os pacientes fizeram, inicialmente, exames tomográfico e de sangue, além de risco cirúrgico, quando necessário (pacientes com 40 anos ou mais).

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3.1 Teste Laboratorial

A amostra de sangue, tubo de 9 mL, foi coletada em dois tubos com agulha borboleta de calibre 21 a partir da veia antecubital do paciente. Cada um dos tubos seguido por um grupo. A mesma rota e profissional estavam presentes para a coleta, para ambos os grupos. Imediatamente após coletados, no máximo, três minutos, o sangue transferido e colocado na centrífuga.

Group1 – Preparo PRGF 1ª Geração (PRGF-1)

As amostras de sangue foram centrifugadas (80-2B Centrífuga, Daiki®) a 580 G (»2.400 RPM) durante 8 minutos, à temperatura ambiente (25ºC) e respeitando a técnica original descrita por Anitua et al.29.

Grupo2 – Preparo de PRGF 2ª Geração (PRGF-2)

As amostras de sangue foram coletadas em tubos de vidro de 9 mL sem agente anti-coagulante (seco / Plano Vacutube, Biocon®, Brasil) e imediatamente centrifugadas a 2000 RPM por 10 minutos, à temperatura ambiente, com uma centrífuga de mesa (80-2B Centrífuga, Daiki®). O conteúdo sobrenadante foi coletado com uma pipeta. Este foi transferido para tubo Eppendorf onde o implante foi inserido.

Análise Luminex - Quantificação de Citocinas, Quimiocinas e Fatores de Crescimento

Vinte e sete citocinas, fatores de crescimento e quimiocinas da primeira geração de PRGF e do PRGF-2 foram quantificados e divididos em: citocinas pró-inflamatórias (IL-1β, IL-6, IL-8, TNF-a, IFN-g, IL-2, IL-7, IL-12p70, IL-15 e IL-17A), citocinas anti-inflamatórias (IL-1Ra, IL-4, IL-5, IL-9, IL-10 e IL-13), quimiocinas (eotaxina, IP-10, MCP-1, MIP-1a, MIP-1b e RANTES), fatores de crescimento angiogênicos (VEGF) e fatores de crescimento (bFGF, G-CSF, GM-CSF e PDGF-BB). Foi utilizado o kit comercial Human Cytokine 30-Plex Assay (Invitrogen, Carlsbad, Califórnia, EUA) para Luminex. Os procedimentos foram realizados de acordo com as instruções dos fabricantes.

3.2 ENSAIO CLÍNICO

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Seleção Pacientes

Pacientes de ambos os sexos foram selecionados e distribuídos de forma aleatória, método da moeda (cara e coroa), controlada e cega, de acordo com os critérios de inclusão e exclusão descritos abaixo.

3.2.2 Critérios de seleção A) Critérios de inclusão

- Necessidade de implante dentário (qualquer local da boca) - Torque máximo na instalação do implante de 34 N.cm - Aceitou a punção venosa (grupo específico)

- Aceitar e assinar o Termo de Consentimento Informado - Ausência de qualquer lesão óssea na cavidade oral

- Volume ósseo residual suficiente para a instalação de implantes (em espessura, altura e largura)

- Extração feita, pelo menos, 6 meses antes do estudo

- O implante não pode ser associado a enxertos ósseos ou região com necessidade de elevação do seio maxilar

- Condições médicas aceitáveis

- Aceitar o risco de instalação coroa protética, individualizada, em aproximadamente 60 dias, com chance de perder o implante dentário.

B) Critérios de exclusão

- Rejeição do formulário de consentimento informado, a qualquer momento durante o estudo

- Pacientes fumante; apresentação de hábitos parafuncionais; gravidez; radioterapia local na cavidade oral; quimioterapia; doenças renais, fígado e/ou sangue; imunossupressão, uso de corticosteroides ou bisfosfonato; doenças mucocutâneas envolvendo a cavidade oral

- O consumo excessivo de álcool, uso diário de acordo com a definição da OMS - Alergia(s) ou problema(s) sistêmico(s), tais como HIV, diabetes, HPV, problemas neurológicos ou outros problemas que poderiam alterar o resultado do estudo

- Volume ósseo insuficiente

- Necessidade de cirurgia para recuperar as dimensões requeridas (enxerto em bloco e enxerto sinusal)

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- Áreas de extração de dente com menos de seis meses; a necessidade de enxerto particulado por algum tipo de reparo peri-implante

- Má higiene oral, apresentando gengivite em região adjacente.

3.2.3 Avaliação inicial

Uma condição básica de saúde foi necessária para incluir o paciente no estudo. Toda a atenção foi dada aos critérios de inclusão e exclusão. Então, tomografia e exames médicos (hemograma, coagulação, glicemia de jejum, EAS, creatinina e de risco cirúrgico, quando mais de 40 anos de idade) foram solicitados.

3.2.4 GRUPOS

Seguindo distribuição aleatória, randomizado e cega, 69 pacientes foram divididos em 2 (dois) grupos:

- Grupo 1 (35 implantes, 15 homens e 20 mulheres) - implante de hexágono externo e superfície com duplo ácido ataque (Porous®) (Conexão©, São Paulo, Brasil) - grupo de controle.

- Grupo 2 (34 implantes, 9 homens e 25 mulheres) - implante de hexágono externo e superfície com duplo ácido ataque (Porous®) (Conexão©, São Paulo, Brasil) associado com PRGF 2ª geração - Grupo teste.

3.2.5 Preparo sanguíneo e das Plaquetas Ricas em Fatores de Crescimento de 2ª Geração (PRGF-2) para aplicação clínica

O sangue periférico de 34 doadores saudáveis (Grupo 2) foram coletados por meio de punção venosa com tubos de vidro secos (VacuTube, 9 mL, Ref: GD090A, Biocon), com uma agulha de borboleta de calibre 21 inserida na veia antecubital do paciente. Imediatamente, o material foi centrifugado com uma centrífuga de mesa (80-2B centrífuga, Daiki®), à temperatura ambiente de 27oC.

Na segunda geração (PRGF-2) não houve a utilização de qualquer anticoagulante ou qualquer outro material, como citrato trissódico, obtendo um PRGF puro e natural. O seu processo de preparo consistiu de uma única centrifugação, a 2000 RPM durante 10 minutos, e após isso, todo o líquido sobrenadante foi coletado, descartando-se as células vermelhas do sangue e zona de “buffy coat”. Esta parte foi

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então transferida e deixada dentro de um tubo Eppendorf, armazenados a -80°C para análise em Luminex.

3.2.6 Procedimento Cirúrgico

Todos os procedimentos cirúrgicos foram realizados pelo mesmo cirurgião (GVOF) e auxiliado por outro profissional quanto ao grupo de tratamento. Foi realizado anestesia por infiltração local (Alphacaine® 100, lidocaína a 2% com adrenalina 1:100.000, DFL©, Brasil). Uma incisão cristal foi feita e um retalho mucoperiosteal foi elevado, permitindo uma exposição adequada do osso local. Perfurações no osso foram realizadas de acordo com as recomendações do sistema de implante (Conexão©, Brasil), sob irrigação abundante, utilizando motor específico para implantologia (OsseoSet™ 100, Nobel Biocare®) e um contra-ângulo Kavo Koncept® com redução de 20:1.

Antes de instalar o implante, no grupo de PRGF-2, o mesmo foi imerso em PRGF-2 durante o período inicial de cristalização do concentrado, tempo necessário para a polimerização na superfície do implante (Figura1). Em seguida, o implante foi completamente inserido com um torquímetro digital (W&H© IA-400®) com torque máximo de 34 N.cm, não atingindo o mínimo recomendado para carga imediata, 35 N.cm mínimo37.

Os valores de torque de inserção foram registrados durante a colocação dos implantes. Depois disso, o retalho mucoperiosteal foi reposicionado para sua posição original, sendo suturado com fio de sutura de seda 3.0 (Johnson & Johnson, Ethicon®). Sobre o implante, já foi deixado o cicatrizador.

Após a cirurgia, os pacientes receberam as recomendações pós-operatórias, como aplicar gelo para diminuir o edema, evitar atividades físicas, repouso e tomar as medicações prescritas para o pós-cirúrgico, incluindo antibióticos (Amoxicilina 500 mg, ou, em caso de alergia, Clindamicina 600mg, três vezes por dia durante sete dias), anti-inflamatório (Nimesulida de 100 mg, duas vezes por dia, durante cinco dias) e gel anti-séptico oral (0,2% de gluconato de clorexidina, duas vezes por dia) durante uma semana.

3.2.7 Medidas de Estabilidade do Implante

A estabilidade do implante foi medida por frequência de ressonância pelo dispositivo Osstell®, quando inserido o transdutor (SmartPeg) no implante, obtendo o

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quociente de estabilidade do implante (ISQ, do inglês Implant Stability Quotient). A análise estatística dos dados foi realizada.

As medidas foram feitas no período pós-operatório, pós-operatório primeira semana e antes de instalar a coroa final. A Análise da Frequência de Ressonância (AFR) também pode ser utilizada para confirmar a estabilidade primária do implante no momento da cirurgia. Se há fraca estabilidade primária (menos de 20 N.cm), deve-se ter cuidado para realizar as medidas.

3.2.8 Etapa Protética

Após duas a três semanas, foi realizada uma radiografia periapical local, seguido de avaliação clínica. Em seguida, começou a fase protética (moldagem), procurando concluir a instalação da coroa metalocerâmica final próximo de 60 dias pós-cirúrgico, colocando-o em função.

3.2.9 Análise Radiográfica

Imagens radiográficas padronizadas da região foram obtidas com aparelho de raios-X (radiografia periapical, 70 kVp, 10 mA) e com filme radiográfico intraoral E-Speed Film (Kodak®). O cilindro de raios-X foi posicionado de acordo com o dispositivo colocado na boca do paciente. O mesmo operador e equipamento foram utilizados para radiografias periapicais, mantendo a padronização da distância focal entre a fonte de raios X e o filme radiográfico.

As imagens digitais foram analisadas em software ImageProPlus® (Media Cybernetics LP, Silver Spring, MD), a fim de medir a quantidade de osso perdido, depois da calibração do programa, tiradas em diferentes estágios:

I - Etapa de instalação da coroa implante; II - Após 6 meses da instalação da prótese.

A distinção entre o implante e o tecido mineralizado foi realizada para cada paciente por avaliação visual de cada imagem, e este passo também teve um avaliador externo cego para os grupos experimentais.

Assim, foi feita a análise do tecido ósseo peri-implantar para determinar o grau de radiolucência local, sendo classificado de acordo com os critérios pré-estabelecidos, observadas através da técnica de subtração:

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1 = reabsorção maior do que 0 e até 1 mm; 2 = reabsorção óssea entre 1 mm e 2 mm; 3 = reabsorção óssea superior a 2 mm.

3.2.10 Avaliação Clínica pós-cirúrgica

O paciente foi acompanhado no pré-operatório, pós-operatório e nas revisões, para análise qualitativa do estado do local. O sucesso de um implante foi clinicamente avaliado observando a falta de mobilidade e outros sinais e sintomas tais como a dor, a inflamação e supuração38. A mobilidade é a principal fator observado para se obter o sucesso do implante e o maior sinal de problemas graves no tratamento39.

Os sujeitos da pesquisa foram observados e avaliados cerca de 21 dias após a instalação da coroa, quanto ao índice de inflamação gengival, o sangramento gengival, a perda óssea ao redor do implante, a mobilidade do implante e a dor/desconforto à percussão.

- Índice de Inflamação Gengival: o índice de inflamação gengival para cada implante foi determinado de acordo com o Índice de Löe & Silness40_{. Os graus de inflamação}

gengival em torno do implante foram avaliados como se segue: 0 = Sem inflamação

1 = ligeira inflamação e alterações na cor da superfície da mucosa

2 = moderado inflamação, vermelhidão e hiperplasia gengival, sangramento e pressão 3 = inflamação, vermelhidão, hiperplasia gengival aguda e tendência a sangramento espontâneo ou ulceração

- Presença / ausência de sangramento no sulco peri-implantar (análise dicotômica): foi avaliado com sonda periodontal ao redor do implante (sonda específica para implantes)41.

- Presença / ausência de mobilidade (análise dicotômica): a mobilidade exige a presença de uma cápsula fibrosa ou perda óssea ao redor do implante. Por conseguinte, os implantes foram considerados com ou sem mobilidade. Para esta avaliação, utilizou-se 2 cabos de espelhos clínicos ou um cabo e um dedo, movendo horizontalmente o implante.

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O som do implante à percussão também foi avaliado. Um som sólido não foi indicativo de mobilidade; mas se o som for abafado/amorfo, a osseointegração não ocorreu, sugerindo a presença de tecido fibroso42.

3.2.11 A análise estatística

As médias e os desvios padrão foram submetidos ao teste t de Student utilizando o programa Instat®, definindo diferenças significativa p <0,05. Foi utilizado o software GraphPad InStat (v3.01, San Diego - Califórnia, EUA) e GraphPad Prism (v5.0, San Diego, Califórnia – EUA).

A análise estatística foi realizada para os seguintes parâmetros: Osstell® (ISQ); Nível ósseo; Índice de Inflamação Gengival; Sangramento gengival; Mobilidade do implante; Dor / desconforto.

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Artigo:

SHORT TERM FOR REHABILITATION WITH PRGF-2ND GENERATION (PRGF-2) AND DENTAL IMPLANTS – RANDOMIZED, CONTROLLED, BLINDED CLINICAL TRIAL

Gustavo Vicentis de Oliveira Fernandes1,*, Paulo Emílio Corrêa Leite2, Carlos Fernando de Almeida Barros Mourão3, Gutemberg Alves4, Mônica Diuana Calasans Maia4, José Mauro Granjeiro2,4,5

1_{PhD in Dentistry (Fluminense Federal University)}

2_{INMETRO (National Institute of Metrology Quality and Technology)} 3_{PhD student in Dentistry (Fluminense Federal University)}

4_{Adjunct Professor (Fluminense Federal University)}

5_{Senior Researcher on Metrology and Quality (INMETRO)}

*Corresponding author: Gustavo V. O. Fernandes - Universidade Federal Fluminense / Faculdade de Odontologia, Programa de Pós-Graduação em Odontologia (PPGO) - Rua Mário Santos Braga, nº 30 - Campus Valonguinho, Centro, Niterói, RJ, Brazil - CEP 24040-110

Phone: +55 21 98131-5840 / E-mail: gustfernandes@gmail.com

Revista:

COIR (Clinical Oral Implant Research)

INTRODUCTION

Nowadays, great attention has been given to accelerate the patient treatment, as enhancing the process of implant osseointegration. Thus, if the healing of bone around implant occur quickly, it could allow a minor time for rehabilitation. Besides, to reduce the anxious and stress between first surgical and finalization, short time using any provisory tooth and rapid reconstruction of the smile (esthetic) and chew (function). With that objective, many types of implant surface treatment are performed1 which they are aim of researches and have as objective to reduce the time for loading after surgery, quickening the bone growth and maturation on close contact with the implant surface, and allow patient rehabilitation in a shorter time2. So, in vitro assays3, in vivo1 and clinical studies4, looking for assess the surface treatment and its influence

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on the osseointegration mechanism.

An interesting approach is the association of platelet concentrate which has gained great attention for assisting in medical and dentistry treatments, helping on tissue healing for many years5. Its report has occurred since, at least, 50’s decades which demonstrated that the platelets are a potent source of mitogens present in whole blood serum6. Other studies published, in vitro, demonstrated a general tendency from that material to stimulate osteoblasts7, fibroblasts8and bone mesenchymal stem cells (BMSCs)9.

Since then, many in-house protocols have surged pursuing to improve the technique. The variation occurs in time, forces and step for centrifugation. For PRP standard technique, normally is used two-step centrifugation process which has varied between 160 G to 3,000 G (first centrifugation), with time from 3 to 20 minutes. On this way, the clinical treatment become tiring and prolonged.

The first matrix was termed fibrin glues and was developed to help on healing10_.

The second autologous matrix was developed by some researchers which tried to enhanced the preparation, modifying and combining it with a natural factor of coagulation. These autologous preparations were named platelet-fibrinogen-thrombin mixtures or gelatin platelet (gel foam)11 and was presented with success in medicine12. Until this moment, the presence of platelet, growth factors and the potential for healing were not considered yet. Then, this concept changed in 1986, when some researches13_{developed an efficient material, using a two-step centrifugation}

procedure and named “platelet-derived wound healing factors” or “platelet-derived wound healing formula (PDWHF)”14.

Some years later, Whitman et al.15 published clinical results using platelet concentrate, what termed “platelet gel”. In 1970s, already had appeared but it had become popular on the 1990s16. On this hand, different ways of preparing PRP have emerged from conventional blood centrifugation, activation by thrombin and/or calcium addition, by silicate (glass) contact or applied as platelet suspension17. Even so, the methodology continues broadening18-19.

Then, a significant confusion in the literature happened about platelet concentrates with different protocols named “PRP” which sometimes do not have a proper characterization of the material content20 and also, the concept of “regeneration throughout growth factors” has seduced still more many researches21.

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Pure Platelet-Rich Plasma (P-PRP)22_{group and brings large interest to be used in}

multiple pathologies23,24, as well as in the regenerative medicine and in the engineering tissue area25,26 with favorable results.

On the procedure, inside the PRGF tube, there are trisodium citrate (3.2% or 3.8%), which is preferred for coagulation testing27, as blood anticoagulant and calcium chloride to activate the coagulation cascade28. They need to preserve both additives for a best control of the process. The quantity of trisodium citrate and calcium chloride/mL PRGF can be, respectively, 0.9 mL and 50 μL29 or 0.4 mL and 20 μL30. The reduction on that different protocols does not affect the properties of anticoagulation, since the amount of anticoagulant in the tube is in excess in relation to whole blood calcium31.

In both PRGF protocols mentioned, the buffy coat is excluded, yielding a leukocyte count close to zero, the therapeutic potential is similar30, however the protocol with minor quantity has shortened the time of clotting and the lag phase30_.

This may be relevant from a clinical point of view.

Therefore, some questions can arise about anticoagulants used, though almost do not appear problem clinically, it may interfere with other analyte when using plasma32. Moreover, sodium citrate inhibits platelet activation, it can inhibit also aspartate aminotransferase and alkaline phosphatase by the chelation of cations27 and it is used to measure plasma levels of platelet-derived components. Furthermore, some clot activators are problematic because they must be completely mixed to allow whole pelleting with the clot. If soluble fibrin clots form, these activators can interfere with pipetting device accuracy or in solid-phase binding in immunoassays33.

Thus, emerge the second generation of platelet aggregates, like platelet-rich fibrin (PRF)34, until the advanced platelet-rich fibrin clot (a-PRF)35, which do not use anticoagulant material, permitting the natural process of coagulation.

These facts lead us for a question: Would the second generation of PRGF be favorable for the implant osseointegration and rehabilitation in shorter time? Hence, the goal of this article was introduce, assess and characterize the PRGF-2, comparing to PRGF of first generation, and apply it in a controlled, blinded and randomized clinical trial to evaluate the stability of dental implants, double acid attack, inserted with one-stage surgical protocol with or without PRGF-2 application, put in function in early period with final ceramic crown.

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3. MATERIALS AND METHODS

This clinical trial followed Declaration of Helsinki and the CONSORT check list. Besides, was submitted and approved by Ethics Research Committee (number 859.788), according index 2. After explanation and agreement of the patients, they signed an Informed Consent Form (index 1). Then, firstly, follow to platelet concentrate assay (PRGF 1st_{and 2}nd_{Generation). After, followed to clinical assay, in which the}

patients made tomography and blood exams, besides surgical risk when necessary.

3.1 LABORATORIAL ASSAY

Blood samples, two tubes, were taken with a 21-gauge butterfly needle from the patient’s antecubital vein. Each tube followed for one group. The same route and professional were present to harvest, for both groups. Immediately after collected, maximum three minutes, the blood was token and put on the centrifuge.

Group 1 - PRGF 1st Generation Preparation

Blood samples were centrifuged at 580 G (2,400 RPM) for 8 minutes at room temperature in the 80-2B Centrifuge (Daiki®), respecting the original technique described by Anitua et al.46.

Group 2 - PRGF 2nd Generation Preparation

Blood samples were collected in 9-mL glass-coated tubes without anti-clotting agent (Dry/Plan Vacutube, Biocon®, Brazil) and immediately centrifuged at 2,000 RPM for 10 minutes at room temperature with a table centrifuge (80-2B Centrifuge, Daiki®). The liquid overflow, on top of the tube, was harvested using a pipette. This content, was transferred to Eppendorf tube where the implant was put inside.

Luminex Analysis – Cytokine, Chemokines and Growth Factors quantification Twenty-seven different cytokines, growth factors and chemokines from first generation of PRGF and PRGF-2 were quantified, divided in: pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-a, IFN-g, IL-2, IL-7, IL-12p70, IL-15 and IL-17A), anti-inflammatory cytokines (IL-1RA, IL-4, IL-5, IL-9, IL-10 and IL-13), chemokines (eotaxin, IP-10, MCP-1, MIP-1a, MIP-1b and RANTES), angiogenic growth factors (VEGF) and growth factors (bFGF, G-CSF, GM-CSF and PDGF-BB).

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Commercial Luminex kit used was Human Cytokine 30-plex Assay (Invitrogen, Carlsbad, California, USA). Procedures were performed according to the manufacturers’ instructions.

3.2 CLINICAL TRIAL 3.2.1 PATIENTS

Sample Size (Statistical analysis)

Firstly, to verify the “n” of subjects, it was tested the null hypothesis (H0) against the alternative hypothesis (Ha), where H0: e = 0 e Ha: e ¹ 0, with significance level of 5% (a = 0,05) and power of test of 80% (1-b = 0,80), considering the unknown standard deviation aggregated of the population difference between the population means and so, selecting the value found from samples published by Hauser et al. (2013)36, where the value of the aggregate variance of the difference between the groups means were s2_{= 0.34mm}2_{, getting n1 = n2 = 8, according model presented by Chow et al. (2008)}37_.

Thus, according to the statistical calculation of sample number, the quantity of sites to be consider as total sample were at least 16, eight for each group, with random selection.

Patients selection

Patients, both genders, were selected were chosen and distributed randomly and blinded, coursing inclusion and exclusion criteria.

3.2.2 Selection criteria a) Inclusion criteria

- necessity of dental implant (any place of the mouth) - maximum implant torque of 34 N.cm

- accepted the venipuncture (specific group) - to accept and sign the Informed Consent Form - absence of any lesions in the oral cavity

- sufficient residual bone volume (thick, height and wide) for implant installation - extraction at least 6 months prior to the study

- the implant cannot be associated to bone grafts or region with necessity of sinus lift - favorable basic medical conditions

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- to accept the risk of prosthetic crown installation, individualized, within approximately 60 days (chance of lose the dental implant)

b) Exclusion criteria

- Rejection of the Informed Consent Form at any time during the study

- Smoker patients, parafunctional habits presented, pregnancy, localized radiotherapy of the oral cavity, antitumor chemotherapy, liver, blood, and/or kidney diseases, immunosuppression, current corticosteroid or bisphosphonate use, mucocutaneous diseases involving the oral cavity

- Excessive consumption of alcohol, daily use

- Allergy(ies) or issue(s) systemic(s) such as HIV, diabetes, HPV, neurological problems or other issues that prevent the treatment

- Insufficient bone volume

- Need of surgery to recover the required dimensions (block graft and sinus graft) - Areas of tooth extraction with less than six months, the need for particulate graft for some kind of peri-implant repair

- Poor oral hygiene, presenting gingivitis on adjacent sites.

3.2.3 Initial Patient Assessment

Basic condition of healthy was necessary to include the patient on the study. All attention was given to inclusion and exclusion criteria. Then, tomography and medical exams (complete blood count, coagulation, glucose, creatinine and surgical risk when more than 40 years-old) were requested.

3.2.4 GROUPS

Following randomized and blinded distribution, the patients (n=69) were divided in 2 (two) groups:

Group 1 (35 implants, 15 men e 20 women) – implant with external hexagon and surface with double acid attack (Porous®) (Conexão©, São Paulo, Brazil) – Control Group.

Group 2 (34 implants, 9 men e 25 women) – implant with external hexagon and surface with double acid attack (Porous®) (Conexão©, São Paulo, Brazil) associated with PRGF 2nd generation – Test Group.

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3.2.5 Blood and Platelet-Rich Growth Factors 2nd_{Generation (PRGF-2)}

preparation to clinical application

Peripheral blood from 34 healthy male and female donors (Group 2) were collected using blood collection plain tubes (VacuTube, 9 mL, Ref: GD090A, Biocon),

with a 21-gauge butterfly needle from the patient’s antecubital vein. Immediately, the material was centrifuged with a table centrifuge (80-2B centrifuge, Daiki). The room temperature was »27o_C.

The second generation (PRGF-2) was evidenced due to no use of any anticoagulant or any supporting material, likely trisodium citrate, obtaining a pure and natural PRGF. Its preparation procedure consisted of one centrifugation step, at 2,000 RPM for 10 minutes, and after it, the whole liquid contends on top were collected, discarding the red blood cells and buffy coat zone. This part harvested was transferred and dropped off inside an Eppendorf, which samples were stored at -80oC to Luminex analysis.

3.2.6 Surgical Procedure

All surgical procedures were performed by the same surgeon (GVOF) and assisted by other professional blinded, carried out with local infiltration anesthesia (Alphacaine® 100, Lidocaine 2% with Adrenaline 1:100,000, DFL©, Brazil). A crestal incision was made and a mucoperiosteal flap was elevated, permitting an adequate exposition of the local bone. Implant osteotomies were prepared according to the recommendations of the implant system (Conexão©, Brazil), under abundant irrigation, using specific motor for implantology (Osseoset™100, Nobel Biocare®) and a Kavo Koncept® contra-angle with reduction 20:1.

Before to install the implant, to PRGF-2 group, that was immersed on PRGF-2 for three minutes, necessary time for initial polymerization over implant’s surface (Fig. 1). Then, the related implant was thoroughly inserted with a digital screwdriver (W&H© IA-400®) and maximum torque of 34 N.cm, does not achieving the minimum recommended for immediate loading, 35 N.cm minimum threshold54.

After that, the mucoperiosteal aps were brought into their original positions. Healing caps were left uncovered, and aps were sutured with Johnson & Johnson, Ethicon® Sutures Mersilk 3/0.

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Fig. 1 – A. Implant immerged inside the Eppendorf tube with liquid PRGF-2 yet; B. Implant after 3 minutes removed from the tube; C. Digital screwdriver (W&H© IA-400®) used to install the dental implant.

After surgery, patients received the post operatory advises, which as to apply cold compresses to decrease edema, avoid physical activity, rest at least three days and take the medications prescribed for postsurgical, including antibiotics (Amoxicillin 500 mg capsule; or, if allergic, Clindamycin tablet, three times per day for seven days), anti-inflammatory (Nimesulide tablet 100 mg, two times per day for five days) and antiseptic oral gel (0.2% chlorhexidine gluconate two times per day) for a week.

3.2.7 Implant Stability Measurements

The patient’s implant was measured throughout resonance frequency by Osstell® device when connected with the transducer (SmartPeg), obtained the Implant Stability Quotients (ISQ).

Three periods were determined for analysis: on the immediate post-operatory, post-operative first week and before to install the final crown. Resonance Frequency Analysis (RFA) could also be used to confirm primary implant stability at the time of

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surgery. If poor primary stability is achieved (less than 20 N.cm), it may be done.

3.2.8 Prosthetic Stage

After two weeks, it was done a local periapical radiography, followed of clinical evaluation. Then, it has begun the prosthetic stage (molding), searching conclude close 60 days postsurgical, installing the final metal ceramic crown above of dental implant, functionally.

3.2.9 Radiographic Analysis

Standardized radiographic images of the region were obtained using the X-ray device (periapical radiography) (70 kVp, 10 mA) and an intraoral radiographic film E-Speed Film (Kodak®). The X-ray cylinder was then positioned according to the positioner placed in the mouth of the patient. The same operator and equipment were used for periapical radiographs while maintaining standardization of the focal distance between the X- ray source and the radiographic film.

The digital images were analyzed in ImageProPlus® software (Media Cybernetics LP, Silver Spring, MD) in order to measure the amount of bone lost, after prior calibration program through radiographs taken at different stages, after installation step of the implant crown and after 6 months of installation of the prosthesis.

The distinction between the implant and the mineralized tissue was performed for each patient by visual assessment of each image, and this step also aided by external evaluator, who did not know which group the patient belonged.

Thus, it was done the analysis of peri-implant bone tissue to ascertain whether or not local radiolucency. This peri-implant radiolucency was classified according to the following pre-established criteria, observed through the image subtraction technique, following 0 for no peri-implant radiolucency; 1 for resorption greater than 0 and up to 1 mm; 2 for bone resorption between 1 mm and 2 mm; and 3 for bone resorption higher than 2 mm.

3.2.10 Postsurgical Clinical Evaluation

The patient was followed up routinely in the preoperative, postoperative and reviews, for qualitative analysis of local state. The success of an implant was clinically evaluated and matched to the lack of mobility and other signs and symptoms such as

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pain, inflammation and suppuration38_{. Mobility is the main success of the implant and}

endpoint is the first sign of failure of treatment39.

Postoperatively, about 21 days after final crown installation and review period were observed and/or evaluated Gingival Inflammation Index, Gingival Bleeding, bone loss around the implant, mobility of the implant and Pain/Discomfort to percussion.

- Gingival Inflammation Index: the gingival inflammation index for each implant was determined according to the Löe & Silness Index40. The degrees of gingival inflammation around the implant was evaluated as follows, 0 for no inflammation; 1 for mild inflammation and changes in surface color of the mucosa; 2 for moderate inflammation, redness and gingival hyperplasia, bleeding and pressure; 3 for acute inflammation, redness, gingival hyperplasia and tendency to spontaneous bleeding or ulceration.

- Presence / absence of bleeding peri-implant on sulcus (dichotomous analysis): was found to pass the periodontal probe around the implant (specific probe for implants)41.

- Presence / absence mobility (dichotomous analysis): the mobility requires the presence of a fibrous capsule tissue surrounding the implant. Therefore, the implants were considered with or without mobility. For this evaluation, it was used 2 cables clinical mirrors or one cable and a finger, moving horizontally the implant. The sound of the implant to percussion was also evaluated. A solid sound will not be indicative of implant mobility and suggested its osseointegration/juxtaposition without problem; but, if the sound is muffled/amorphous, the osseointegration was not occurred suggesting that the implant was surrounded by fibrous tissue42.

3.2.11 Statistical analysis

Means and standard deviations were subjected to Student’s t-test using the Instat® program, defining significant differences p<0.05. It was used the GraphPad InStat (v.3.01, San Diego – California, USA) and GraphPad Prism (v.5.0, San Diego, California – USA).

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Statistical analysis was performed for the following parameters: Osstell® (ISQ); periapical radiography; Index of Gingival Inflammation; Gingival Bleeding; Implant mobility; Pain / Discomfort.

4. RESULTS

4.1 PROTEIC QUANTIFICATION - LUMINEX

From 27 proteins quantified by Luminex kit, in which 16 had level not detectable by device’s sensibility. They are pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-a, IL-2, IL-7, IL-12p70 and IL-17A), anti-inflammatory cytokines (IL-1RA, IL-4, IL-9 and IL-13), chemokines (MCP-1 and MIP-1a) and growth factors (G-CSF and GM-CSF). Luminex plate reliability was tested, using the same PRGF sample, through Elisa assay, obtaining the same result, confirming the good test performance.

Among the analyzed proteins were detected: IP-10, Eotaxin, RANTES and MIP-1b, VEGF, PDGF-BB, bFGF, IFN-g, IL-15, IL-5 and IL-10. The Table 1 show the statistical results of those elements detected.

Table 1. Mean of PRGF for Protein and Statistical analysis (Student t-test).

Proteins Observed PRGF-1 (pg/mL) PRGF-2 (pg/mL) One-tailed (p value) Two-tailed (p value) IL-5 2.3 ± 0.3 2.2 ± 0.4 0.2 0.5 IL-15*** 0.1 ± 0.04 0.5 ± 0.1 0.0001 0.0003 IL-10 0.5 ± 0.5 0.5 ± 0.3 0.5 1.0 IP-10 1,761.2 ± 1,076.0 2,290.6 ± 1,266.5 0.2 0.3 PDGF-BB* 18,322.8 ± 11,186.3 4,474.6 ± 1,525.8 0.01 0.02 VEGF 77.2 ± 119.3 5.1 ± 4.6 0.05 0.1 Basic-FGF 11.6 ± 1.0 11.8 ± 1.0 0.3 0.6 Eotaxin** 538.7 ± 160.4 186.7 ± 41.9 0.001 0.003 RANTES 1,526.9 ± 333.6 1,242.1 ± 537.0 0.09 0.2 IFN-g 50.6 ± 9.4 46.5 ± 10.1 0.2 0.4 MIP-1b* 139.8 ± 74.1 209.3 ± 29.2 0.02 0.04 * p<0.05; ** p<0.01; *** p<0.001; nsd = no significative difference

Applying Student t-test, we can observe that only IL-15 (p<0.001), PDGF-BB (p<0.05), MIP-1b (p<0.05) and Eotaxin (p<0.01) had statistical relevance. From the pro-inflammatory cytokines, IL-15 level was found 7 times more in PRGF-2 than PRGF-1 (control group). PDGF-BB, growth factor, was detected 4.09 times greater in the PRGF-1 than PRGF-2. Of chemokines, MIP-1b was slightly higher in the second generation and eotaxin was in the first generation (2.9 times).

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1 (2. 86% ) 0 6 (17. 14% ) 18 (51. 43% ) 3 (8. 57% ) 0 5 (14. 29% ) 2 (5. 71% ) I N C I S I VE C AN I N E P R EMOL AR MOL AR 4.2 CLINICAL TRIAL

In the clinical evaluation, it was initially observed that seven patients (7) of the total study (10.1%) had the implant lost. Of those, four were in the control group (5.8%) and three in the experimental group (4.3%). Only one patient lost the implant before installing the crown (Group 2).

The teeth involved on the study follow on the graphic below (Graph 1). The largest number of installed implant is for posterior region (premolars and molars), 88.6% in Group 1 and 76.5% in Group 2, which is the area that suffers the greatest impact on chewing and thus has a higher risk of losing implant early.

Group 1. Number of participants in each group of teeth.

Group 2. Number of participants in each group of teeth.

Graph 1. Percentage of region that received implant. To the left, all values representing Mandible and to the right, Maxilla (per group).

This was confirmed when was verified the collected and shown data, in the tables below (Tables 3 and 4), in which all lost implants were in posterior region.

Group 1

The implant loss was observed in 4 patients (11.4%, percentage per group), where were all posterior teeth (regions 4, 14, 19 and 30) on function, only 3 had pain. The local inflammation and bleeding were clinically observed only in one patient in this group (2.9%), which lost the implant after 120 days (4 months) from the surgery.

Seven other patients also experienced pain, either on site or soft tissue due to non-stabilization of the implant. Of these 10 patients with pain symptoms, in nine

1 (2. 94% ) 1 (2. 94% ) 5 (14. 71% ) 15 (44. 12% ) 5 (14. 71% ) 1 (2. 94% ) 4 (11. 76% ) 2 (5. 88% ) I N C I S I VE C AN I N EP R EMOL AR MOL AR

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(25.7%) the score was 1, and one patient (2.9%), the score was 3 - there was no loss of this implant. In such cases, a longer period was necessary to ensure treatment and osseointegration.

Table 3 – Group 1, clinical evaluation.

TOOTH PLATELETS

(MIL / MM3₎ BONE _LOST _INFLAMMATIONLOCAL BLEEDING PAIN

A1 ₁₀ ₂₃₂ ₀ ₀ ₀ ₁ A2 ₅ ₁₉₇ ₂ ₀ ₀ ₁ A3 ₄ ₂₀₈ ₀ ₀ ₀ ₁ A4 ₂₁ ₁₈₉ ₁ ₀ ₀ ₀ A5 ₃₀ ₂₂₅ ₂ ₀ ₀ ₀ A6 ₂₁ ₃₃₈ ₁ ₀ ₀ ₀ A7 ₁₄ ₁₅₄ ₁ ₀ ₀ ₀ A8 ₃ ₂₉₉ ₀ ₀ ₀ ₁ A9 ₁₉ ₁₈₉ ₀ ₀ ₀ ₀ A10 ₁₉ ₃₃₈ ₂ ₁ ₁ ₁ A11 ₁₉ ₂₂₁ ₀ ₀ ₀ ₁ A12 ₂₉ ₂₆₁ ₁ ₀ ₀ ₀ A13 ₁₉ ₂₆₁ ₁ ₀ ₀ ₀ A14 ₉ ₂₀₇ ₀ ₀ ₀ ₁

A15 ₃₁ ₂₅₁ ₀ ₀ ₀ _{3 (not stabilized)}

A16 ₃₀ ₂₉₆ ₀ ₀ ₀ ₀ A17 ₁₉ ₃₁₆ ₀ ₀ ₀ ₀ A18 ₁₃ ₃₁₆ ₀ ₀ ₀ ₀ A19 ₄ ₁₇₂ ₁ ₀ ₀ ₀ A20 ₃₀ ₃₉₆ ₀ ₀ ₀ ₀ A21 ₂₉ ₂₅₈ ₁ ₀ ₀ ₀ A22 ₃₀ ₂₅₈ ₀ ₀ ₀ ₁ A23 ₁₉ ₃₃₈ ₁ ₀ ₀ ₀

A24 ₃₀ ₁₇₂ ₀ ₀ ₀ _{1 (not stabilized)}

A25 ₃₀ ₂₄₆ ₀ ₀ ₀ ₀ A26 ₃₀ ₂₈₁ ₀ ₀ ₀ ₀ A27 ₃₀ ₃₁₀ ₀ ₀ ₀ ₀ A28 ₂₉ ₂₁₆ ₀ ₀ ₀ ₀ A29 ₅ ₂₄₇ ₀ ₀ ₀ ₀ A30 ₁₉ ₂₄₇ ₀ ₀ ₀ ₀ A31 ₂₀ ₁₉₂ ₀ ₀ ₀ ₀ A32 ₂₄ ₂₇₃ ₀ ₀ ₀ ₀ A33 ₁₉ ₂₀₈ ₁ ₀ ₀ ₀ A34 ₃₀ ₂₀₈ ₀ ₀ ₀ ₀ A35 ₇ ₁₅₁ ₀ ₀ ₀ ₀ AVERAGE 247.7

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Table 4 – Clinical evaluation of Group 2. TOOTH PLATELETS (MIL / MM3) BONE LOST LOCAL INFLAMMATION BLEEDING PAIN B1 19 347 0 0 0 1 B2 19 225 2 0 0 0 B3 19 195 0 0 0 0 B4 30 387 0 1 0 0 B5 11 278 0 0 0 0 B6 27 293 0 0 0 0 B7 28 217 0 0 0 0 B8 9 270 0 0 0 1 B9 30 221 0 0 0 0 B10 30 261 1 1 0 1 B11 19 200 1 1 0 2 B12 7 246 0 0 0 0 B13 8 207 0 0 0 1 B14 30 196 0 0 0 0 B15 14 296 0 0 0 0 B16 30 316 - - - - B17 4 316 0 0 0 0 B18 10 316 1 0 0 0 B19 13 172 1 0 0 0 B20 21 258 0 0 0 0 B21 20 258 0 0 0 1 B22 19 258 2 0 0 0 B23 19 172 0 0 0 0 B24 19 246 0 0 0 0 B25 31 281 0 0 0 0 B26 20 216 2 0 0 0 B27 12 247 0 0 0 0 B28 29 192 0 0 0 0 B29 25 273 0 0 0 0 B30 13 273 0 0 0 0 B31 19 273 0 0 0 0 B32 14 395 - - - 2 B33 31 395 0 0 0 1 B34 10 151 0 0 0 0 AVERAGE 260.2

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Regarding bone loss, three patients were between 1 and 2 mm loss (8.6%) - score 2, and only one of them had the loss of the implant. Other 9 patients (25.7%) had score 1 of bone loss (up to 1 mm), with loss in only one case.

According to the study proposal, it achieves the approximate average of 68 days (SD 28.1) and median of 66 days (maximum 184 days and minimum 33 days) for rehabilitation of patients, with 88.6% success rate in Group 1.

Group 2

The implant loss was observed in 3 patients of group 2 (8.8%), where were all posterior teeth (regions 14, 20 and 30), one on function and two before placing the final crown.

The local inflammation and bleeding were clinically observed in only three patients in this group (8.8%), and none of them had the implant lost.

From three patients who lost the implant (8.8%), the question pain was observed in 2 of them. Six other patients also experienced pain, but without loss of the implant. Of those eight patients with pain symptoms, six (26.5%) the score was 1 and in two patients (5.9%) had score two.

Regarding bone loss, two patients were between 1 and 2 mm loss (5.9%) – score 2, without loss of implants. Other 4 patients (11.8%) had score 1 of bone loss (up to 1 mm), without loss of implant.

According to the study proposal, there was approximately average of 71 days (SD 27.8) and median of 69 days (maximum 141 days and minimum 29 days) for rehabilitation of patients, with 91.2% success rate in Group 2.

Table 5. Percentage of bone loss, local inflammation, pain and implant index of success per group.

BONE LOST LOCAL

INFLAMMATION

PAIN SUCCESS

INDEX Level 1 Level 2 Level 3 Level 1 Level 2 Level 3

Group 1 25.7% 8.6% 0 2.9% 25.7% 0 2.9% 88.6%

Group 2 11.8% 5.9% 0 8.8% 26.5% 5.9% 0 91.2%

P value G1xG2 (p = 0,5) G1xG2 (p = 0,3) G1xG2 (p = 0,8) -

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Of all patients, 58% were concluded in early period (until 60 days). In the Group1, the average to finalization was 69 days. Twenty-three patients (65.7%) had the final crown within 60 days (early period). The other 12 patients (34.3%) finished the treatment in conventional loading period (after 60 until 120 days).

Group 2 received approximate average of 71 days to complete rehabilitation. Among those, 17 patients (50%) were completed within 60 days, early loading. The other 50% of patients ended in conventional loading period (after 60 until 120 days).

The reasons for delayed complete treatment, within 60 days was the patient missed the consultation, delayed return of prosthesis from dental laboratory, need of prosthesis adjustments by the dental laboratory, lower primary stability of the implant, for patient and treatment’s security and integrity.

4.4 Insertion Dental Implant and Screw Torque

The mean insertion torque values of the implants on Group 1 (Control) was 31.4 ± 6.1 N.cm and 28.0 ± 8.1 N.cm for the PRGF-2 Group. There was no significant difference between groups in insertion torque values (p=0.5).

The mean of torque values of the final screw on Group 1 (Control) was 30.3 ± 4.3 N.cm and 30.6 ± 4.0 N.cm for the PRGF-2 Group. There was no significant difference between groups in insertion torque values (p=0.8).

4.5 ISQ Value

Immediate postsurgical ISQs for Group 1 were 66.4 ± 6.0 and 65.6 ± 13.0 for PRGF-2. At the end of the first week, this mean ISQ was confirmed. These results were statistically no significant (p=0.8).

The mean ISQs before install the crown was 68.1 ± 6.8 for the Group 1 and 70.25 ± 5.71 for the PRGF-2. This difference between groups was not significant (p=0.2).

For the group 1, the statistical analysis by period also has no difference (p=0.3). Group 2 presented neither statistical difference too (p=0.06).

Mean ISQs in the control group increased by 1.7 units comparing from the first measurement to the final. Mean ISQs in the treatment group increased 4.6 units, achieving result 2.7 times bigger than group 1.

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Graph2. INITIAL ISQ of the groups. Colors proposals by SENNERBY (2013)43.

Graph3. FINAL ISQ of the groups. Colors proposals by SENNERBY (2013)43.

5. DISCUSSION

The use of new and promising protocols of platelet concentrates preparation, well-defined, it is still something that researchers are establishing. However, several alternatives have appeared, leading the parameters frequently seem to be empirical. Thus, contrasting results have been reported on literature44,45.

The use of P-PRPs seem to be the most frequently tested, in vitro and in vivo. So, the choice for PRGF to develop this work was based on the results obtained for years23-26,28-30. Then, our aim was simplifying the technical process without affecting

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the protein contends when comparing with the first generation. Besides, there is a claim for the production of a natural platelet concentrate without bovine thrombin, considering the first PRGF, an unnatural condition.

Thus, enabling PRGF obtained by the new protocol (PRGF-2, no additive – Second Generation) to remain in the same categories that the former protocol, in terms of the different classifications of PRP.

Besides that, a comparative and unprecedented evaluation of cytokines, chemokines and growth factors presents in PRGF29 and PRGF-2 was necessary. Verifying this literature gap, we quantified 27 proteins by Luminex methodology, in which chemokine quantification detected only IP-10, Eotaxin, RANTES and MIP-1b, the same for growth factors VEGF, PDGF-BB, bFGF, pro-inflammatory cytokines IFN-g and IL-15 and anti-inflammatory cytokines IL-5 and IL-10.

Great importance has the PDGF-BB due to its function as growth factor. Osteogenesis is intimately linked with angiogenesis during bone modeling and remodeling46 and this factor promote angiogenesis and hence bone formation. Possible, it appeared high in the first generation due to time to preparation, RPM or G force and the k-factor related to the centrifugation, which suggests that PRGF-2 with a higher k-factor had lower efficiency on the sedimentation47_.

It was observed that PDGF increases bone formation around dental implants and consequently the osseointegration. In this study, though the existing quantity on the PRGF-1 was 4.09 times greater, the quantity of PDGF-BB on the PRGF-2 suggest to be sufficient to trigger the local osteogenesis too. Other important point, on the systemic field, how higher the level of that protein, higher the risk may induce the formation of tumor by stimulation of angiogenic cells49, becoming the high values obtained a contrast of success.

The interleukin 15 (IL-15) presented extremely significance level at PRGF-2 compared to PRGF-1, being favorable and important for defense / protection, which activate cells of the innate immune system whose principal role is to kill virally infected cells50, assisting on the healing of the local tissue.

Like IL-15, MIP-1b (macrophage inflammatory protein 1-beta) also was greater in PRGF-2 (p<0.05) and it is crucial for immune responses towards infection and inflammation51, activate human granulocytes (neutrophils, eosinophils and basophils), induce the synthesis and release of other pro-inflammatory cytokines such as 1,

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IL-6 and TNF-a from fibroblasts and macrophages. Then, this founds suggested that PRGF-2 can an excellent defense property, and this was sight on the clinical results, when used PRGF-2 associated to implant.

On this way, follow the PRGF-2 for the clinical evaluation together with the implant. The null hypothesis (H0) of this work is the material does not speed healing of the endosteal implant and cannot perform the restoration in a short time. However, the alternative hypothesis (H1) would be the success of rehabilitation in a short period mainly when associate with PRGF-2.

When achieve the primary implant stability, there is avoidance of micromotion during early healing, favoring the osseointegration. Then, implants were originally left unloaded for 3 to 4 months in the mandible and 6 to 8 months in the maxilla52.

The addition of proteins to the implant surface has been approached to enhance bone-implant contact (BIC). Thus, experimental studies have shown that cell adhesion molecules or bone morphogenetic proteins could enhance and increase osteoblastic differentiation together with functional integration of implants53.

Even so, many restrictions are encountered to clinical diagnostic methods, primarily to determine the implant stability and to confirm its osseointegration. The clinical examination and imaging exam54_{, do not reflect confidence in the quality and}

thus ensuring the survival rate of the implants55_.

Albrektson et al.79 proposed the following success criteria for the implant osseointegration: a) individual immobility of the implant before to connect to the prosthesis; b) lack of radiolucency peri-implant, demonstrated by radiography; c) annual bone loss of 0.2 mm, after the first year of use of implants; d) absence of signs and symptoms irreversible and/or persistent (as pain, infection, neuropathy, paresthesia or violation of the mandibular canal). All of them items were observed in this study and their correlation did not define the implant success or fail, but helped on the evaluation.

So, quantitative methods have been proposed to assist in the evaluation, as well as the prognosis of this treatment57, even if there is little scientific evidence. Among these techniques to measure, we find in the literature: the insertion torque and removal of implant58, the Periotest59 and frequency analysis resonance60,61, known commercially as Osstell® (Integration Diagnostics, Goteborg, Sweden).

The Osstell® is the less invasive and more efficient device62 using a piezo effect for producing a deflection in implant wherein the transducer is fitted directly on or over

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the implant and the prosthetic intermediate is stimulated vibrated by sinusoidal waves. Thus, there was obtained a value of the resonance frequency of which is converted to an Implant's Stability Quotient (ISQ) for evaluation of the installed implant63. The decrease of these values are related to alterations at the bone-implant contact / interface (BIC) and consequently a potential failure of the implant64, as cited on the literature with zone of established43 (Graphs2 and 3 was done applying this concept associating with our obtained results).

Three different zones measured with Osstell were proposed by Senneby43, being of great stability (“green zone”, ISQ equal or above than 70), risk zone (“red zone”, ISQ equal or below of 55) and intermediary zone (“yellow zone”, ISQ between 55 to 70). This methodology was applied on graphs 2 and 3.

A clinical study found65 obtained success rate of 100% of the implants placed in patients, with a mean ISQ of 76.9 ± 0.8. In the first six weeks, the ISQ values increased significantly in the groups. Bornstein et al.66_{recommended that values <65}

ISQ must wait an additional healing time and can be measured again 3 weeks for admeasurement. Other authors preferred values ≥ 70 ISQ for single tooth and early loading. In another study67_{, the authors affirmed that no loss occurred when the ISQ}

values were greater than 60, and that 19% of the implants that had been lost ISQ < 60. Others68 correlated micromotion with ISQ measures and found that when ISQ value was between 60 and 70, the micromoviment reduced by 50%.

Comparing with the literature, we found the same, with great chance of success if ISQ was > 60. Only 2 patients who lost the implant had inferior value. Others participants (n=5) had different reasons. Slightly major ISQ was found in the PRGF-2 group but without statistical significance.

Understand the term early load is necessary. Then, there was an agreement for this terminology according to time of loading and for the implant loading (occlusal loading and nonocclusal loading) in the Consensus Meeting (2002). This is the goal of the interest of this work, then following terminology was described. On the early loading, the prosthesis is attached at a second procedure, between 1 and 90 days. The time of loading is started after some days/weeks; On the delayed loading, the prosthesis is attached at a second procedure begins within 90 days69.

Two others Consensus was performed two years later (2004) and 200770, and defined early loading as the application of load between 1 and 90 days, keeping the