UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE ODONTOLOGIA DE PIRACICABA
EDNA ZAKRZEVSKI PADILHA
PIRACICABA 2017
RESPOSTA MECÂNICA E IMUNO-HISTOQUÍMICA DO
LIGAMENTO PERIODONTAL DE RATOS COM TRAUMA
OCLUSAL DENTAL
MECHANICAL AND IMMUNOHISTOCHEMISTRY RESPONSE
OF PERIODONTAL LIGAMENT OF RATS WITH DENTAL
EDNA ZAKRZEVSKI PADILHA
RESPOSTA MECÂNICA E IMUNO-HISTOQUÍMICA DO
LIGAMENTO PERIODONTAL DE RATOS COM TRAUMA
OCLUSAL DENTAL
MECHANICAL AND IMMUNOHISTOCHEMISTRY RESPONSE OF
PERIODONTAL LIGAMENT OF RATS WITH DENTAL
TRAUMATIC OCCLUSION
Tese apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para obtenção do título de Doutora em Biologia Buco-Dental, na Área de Anatomia.
Thesis presented to the Piracicaba Dental School of the University of Campinas in partial fulfillment of the requirements for the degree of Doctor in Dental Biology, in the Anatomy area.
Orientador: Prof. Dr. Felippe Bevilacqua Prado Co-orientador: Prof. Dr. Alexandre Rodrigues Freire
ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA PELA ALUNA EDNA ZAKRZEVSKI PADILHA E ORIENTADA PELO PROF. DR. FELIPPE BEVILACQUA PRADO.
PIRACICABA 2017
Agência(s) de fomento e nº(s) de processo(s): CAPES
Ficha catalográfica
Universidade Estadual de Campinas
Biblioteca da Faculdade de Odontologia de Piracicaba Marilene Girello - CRB 8/6159
Padilha, Edna Zakrzevski,
P134r PadResposta mecânica e imuno-histoquímica do ligamento periodontal de ratos com trauma oclusal dental / Edna Zakrzevski Padilha. – Piracicaba, SP : [s.n.], 2017.
PadOrientador: Felippe Bevilacqua Prado. PadCoorientador: Alexandre Rodrigues Freire.
PadTese (doutorado) – Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba.
Pad1. Ligamento periodontal. 2. Oclusão dentária traumática. 3. Imuno-histoquímica. 4. Análise de elementos finitos. I. Prado, Felippe
Bevilacqua,1980-. II. Freire, Alexandre Rodrigues,1985-. III. Universidade Estadual de Campinas. Faculdade de Odontologia de Piracicaba. IV. Título.
Informações para Biblioteca Digital
Título em outro idioma: Mechanical and immunohistochemistry response of periodontal
ligament of rats with dental traumatic occlusion
Palavras-chave em inglês:
Periodontal ligament Dental occlusion traumatic Immunohistochemistry Finite element analysis
Área de concentração: Anatomia
Titulação: Doutora em Biologia Buco-Dental Banca examinadora:
Felippe Bevilacqua Prado [Orientador] Roberta Okamoto
Mariza Akemi Matsumoto Eduardo Daruge Junior
Eduardo César Almada Santos
Data de defesa: 23-02-2017
Programa de Pós-Graduação: Biologia Buco-Dental
DEDICATÓRIA
Dedico este trabalho aos professores e verdadeiros mestres Dr. Alexandre Rodrigues Freire, Dra. Ana Cláudia Rossi e Dr. Felippe Bevilacqua Prado, desbravadores dessa linha de pesquisa.
AGRADECIMENTOS
À Deus, eterno Pai.
À Universidade Estadual de Campinas, na pessoa do Magnífico Reitor Prof. Dr. José Tadeu Jorge.
À Faculdade de Odontologia de Piracicaba (FOP-UNICAMP), na pessoa do Senhor Diretor, Prof. Dr. Guilherme Elias Pessanha Henriques.
A Coordenadoria de Pós Graduação, na pessoa da Senhora Coordenadora Prof. Dr.ª Cinthia Pereira Machado Tabchoury.
À Equipe Técnica da Coordenadoria de Pós-graduação, em nome da Ana Paula Carone, da Érica Alessandra Pinho Sinhoreti e Leandro Viganó sempre dispostos a ajudar.
À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pelo apoio financeiro oferecido para realização desta pesquisa.
Ao programa de pós-graduação em Biologia Buco-Dental, na figura da coordenadora Profª. Dr.ª Maria Beatriz Duarte Gavião.
Ao orientador Prof. Dr. Felippe Bevilacqua Prado, pelo acolhimento, questionamentos e contribuições para minha formação profissional. Ao co-orientador Prof. Dr. Alexandre Rodrigues Freire, por ser indispensável para a realização deste trabalho e à Prof.ª Ana Cláudia Rossi, pelo seu respeito incondicional com as pessoas, por sua capacidade de dialogar, mas, sobretudo, pelos períodos de convivência que me ensinaram humanidade e profissionalismo. Agradeço pela humildade, sabedoria e convicção que, sem nenhuma formalidade, contribuíram para que eu me fortalecesse e compreendesse melhor minha vida.
Aos professores Dr. Eduardo Daruge Júnior, Dr. Eduardo César Almada Santos e Dr. Alan Roger dos Santos Silva pelos questionamentos e contribuições no exame de qualificação da tese. E aos professores participantes da banca examinadora Roberta Okamoto, Mariza Akemi Matsumoto, Eduardo Daruge Júnior, Eduardo César Almada Santos como titulares, e aos professores suplentes Paulo Vitor Farago, Leonardo Perez Faverani e Pedro Duarte Novaes.
À minha família, que me mostraram que pela fé tudo é possível e sempre acreditaram em mim.
Ao meu amado esposo, Fabrício Rutz da Silva, por compartilhar os momentos de pós-graduação comigo e sempre incentivar, apoiar e acreditar nas minhas convicções.
Aos colegas e amigos que a FOP me proporcionou, pela amizade, apoio e convivência, sem as quais, a vida perderia muito do seu encanto.
Aos meus alunos, pela confiança e convivência que possibilitaram um significativo aprendizado pessoal.
Aos professores que desde o início me ensinaram e incentivaram a continuar estudando, da Escola Municipal Professor Sebastião Antunes Ferreira, Colégio Estadual Francisco Neves Filho, Colégio São Vicente de Paulo, e em especial à profa. Jáima Pinheiro de Oliveira na Universidade Estadual do Centro-Oeste.
A todos os familiares e amigos que de forma direta e indireta colaboraram para a realização deste estudo.
RESUMO
A homeostase do ligamento periodontal e, consequentemente, a manutenção do espaço periodontal, é atingida através da adaptação do ligamento periodontal às forças aplicadas e a mecanismos celulares que regulam a promoção e supressão da formação de osso, cemento e ligamento periodontal. O presente estudo buscou investigar se um trauma oclusal nos molares superiores em ratos resulta em alterações de expressão proteicas e mecânicas no ligamento periodontal. Foram utilizados 50 ratos machos, 40 no grupo experimental, sendo distribuídos em 4 subgrupos (n=10) de acordo com o período de eutanásia: 7, 14, 21 e 28 dias pós aplicação de resina fotopolimerizável no 1º molar superior, e 10 no grupo controle que foram eutanasiados após 28 dias. Após a eutanásia, as cabeças dos 50 ratos foram destinadas para análise imuno-histoquímica. Os anticorpos primários utilizados foram a Osteoprotegerina (OPG) e Receptor activator of nuclear factor kappa-B ligand (RANKL). Foi avaliada a expressão das imunomarcações pela atribuição de diferentes “scores” variando entre marcações leve, moderada, intensa, de acordo com a área das células imunomarcadas para cada período estudado. Para a análise de elementos finitos, uma cabeça do grupo controle e uma do grupo experimental foram escaneadas em um microtomógrafo para a construção da geometria 3D utilizada na simulação computacional. A análise foi realizada para simular uma mordida posterior com a associação entre a ação da musculatura mastigatória de elevação da mandíbula do rato e a força de mordida de 20N aplicada sobre o primeiro molar superior. As deformações relacionadas às alterações na morfologia foram avaliadas pelo cálculo das deformações mínimas principais. A análise imunohistoquímica apresentou respostas diferentes quanto à expressão de OPG e RANKL, com oscilação na intensidade da marcação de ambas as proteínas no grupo experimental. Entre os grupos controle e experimental, a expressão de OPG foi semelhante no grupo controle e no período de 7 dias com contato prematuro, apresentando predomínio de atividade leve; nos demais períodos houve expressão moderada. Já a expressão de RANKL foi de leve a moderada no grupo controle e intensa no grupo experimental no período de 7, 21 e 28 dias. No grupo experimental, a oscilação na intensidade das marcações ocorreu no período de 14 dias, sendo moderada. Quanto às deformações mínimas principais localizadas no ligamento periodontal do grupo experimental pode-se observar regiões de
compressão mais elevadas que do grupo controle, ultrapassando deformações iguais a -3,3e-8 (3,3x10-10) mm/mm. Pode-se concluir que o trauma oclusal causou alterações associadas à atividade das células presentes no ligamento periodontal, ligadas à remodelação óssea, e os estímulos mecânicos, com aumento de compressão e da expressão de RANKL.
Palavras-chave: Mecanobiologia. Ligamento periodontal. Trauma oclusal. Imuno-histoquímica. Análise de elementos finitos.
ABSTRACT
Periodontal ligament homeostasis is achieved by adapting the periodontal ligament to applied forces and cellular mechanisms that regulate the promotion and suppression of bone, cementum and periodontal ligament formation. The aim of the present investigation was to evaluate the mechanical and the immunohistochemistry response of PDL of upper first molar of rats with dental traumatic occlusion. Fifty male rats (Rattus norvegicus albinos), Wistar linage, with 2 months of age (weight, 200-250g) were submitted two separate groups for the experiments. In experimental group (n=40), the rats were submitted to unilateral application (right side) of light-cured composite resin on the occlusal face of upper first molar, simulating a dental traumatic occlusion condition. Then these animals were divided into 4 subgroups (n = 10) according to the euthanasia period: 7, 14, 21 and 28 days after application of the resin. In control group (n=10) the dentition was maintained without application of resin and from the early age (two months), the animals were euthanized to 28 days. All the specimens (head of rats) were fixed in formalin. The specimens were dissected and the right maxillary was removed to decalcified and processed to imunohistochemistry analysis. Primary antibodies were used: osteoprotegerin and receptor activator of nuclear factor kappa-B ligand. The finite element analysis was set to simulate a maxillary molar biting through the rat masticatory muscles activity and the molar bite force with 20 N magnitude on the maxillary molars, which features the normal occlusal contact in physiological condition, i.e. without significant bone remodeling changes. In the experimental condition, the same forces of muscles and molar biting were applied on the resin surface, featuring the dental traumatic occlusion. The immunohistochemical analysis presented different responses regarding the OPG and RANKL expression, which there was oscilation in the intensity in both protein in the experimental group. In periodontal ligament, the control group was observed a medium compressive strain response and in the experimental group the strains presented a medium to high compressive strain response. In conclusion, dental occlusal trauma caused changes associated with the activity of cells present in the periodontal ligament, linked to bone remodeling, and mechanical stimuli.
Keywords: Mechanobiology. Periodontal ligament. Occlusal trauma. Immunohistochemistry. Finite element analysis.
LISTA DE ILUSTAÇÕES
Figure 1 - Geometry and finite element mesh of rats with normal occlusion (control group) and altered occlusion (experimental
group). 25
Figure 2 - Osteoprogeterin (OPG) expression in the PDL tissue. (C) control group; (7d) 7 days experimental group; (14d) 14 days
days experimental group. 28
Figure 3 - Osteoprogeterin (OPG) expression in the PDL tissue. (21d) 21 days experimental group; (28d) 28 days experimental group. 29 Figure 4 - RANKL expression in the PDL tissue. (C) control group; (7d) 7
days experimental group; (14d) 14 days days experimental
group. 31
Figure 5 - RANKL expression in the PDL tissue. (21d) 21 days experimental group; (28d) 28 days experimental group. 32 Figure 6 - Minimum principal strain calculation of control group. The
negative values in the colored scale indicate compressive
strain. 34
Figure 7 - Minimum principal strain calculation of experimental group. The negative values in the colored scale indicate compressive
LISTA DE TABELAS
Table 1 - Mechanical properties of the anatomical structures.
MPA=megapascal. 26
LISTA DE ABREVIATURAS E SIGLAS
CCL2 - CC quimiocina ligante 2 CCR2 - CC quimiocina receptor 2
d - Dias
FEA - Análise de elementos finitos
FE - Elementos finitos
N - Newton
OPG - Osteoprotegerina
RANK - Receptor ativador do fator nuclear-𝜅
RANKL - Receptor ativador do ligante fator nuclear-𝜅 TRAP - Fosfatase ácida resistente ao tartarato
SUMÁRIO
1 INTRODUÇÃO 15
2 ARTIGO: MECHANICAL AND IMMUNOHISTOCHEMISTRY RESPONSE OF PERIODONTAL LIGAMENT OF RATS WITH DENTAL TRAUMATIC OCCLUSION
19
3 CONCLUSÃO 41
REFERÊNCIAS 42
ANEXOS
ANEXO 1: Comprovante de submissão do artigo científico 44 ANEXO 2: Certificação da Comissão de Ética no Uso de Animais 45
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1 INTRODUÇÃO
O ligamento periodontal é um tecido conjuntivo denso, fibroso, alojado ao redor da raiz do dente e que conecta o dente ao osso alveolar a fim de manter o suporte dental (Nanci, 2008). O ligamento periodontal atua nas funções estruturais, nutricionais e sensitivas dos dentes e também participa na função da mastigação. A manutenção das relações dentais envolve um mecanismo multifacetado viscoelástico (Wang et al., 2012) e a capacidade adaptativa do ligamento periodontal é essencial para permitir que os dentes movimentem-se para manter a função da oclusão dental (Huang et al. 2016; Ishigaki et al., 2006).
A espessura do ligamento periodontal está relacionada com as cargas a que é submetido. No caso de função mastigatória normal, ele se adapta às forças oclusais, aumentando sua espessura. Em condições hipofuncionais, com diminuição da carga oclusal, surge um ligamento periodontal mais estreito e um osso alveolar mais fino. As correlações entre o ligamento periodontal e o osso alveolar, bem como entre o ligamento periodontal e o espaço periodontal sugerem que uma redução da carga oclusal induz uma resposta simultânea nestes tecidos (Denes et al. 2013).
A perda da função, ou da homeostase em resposta à demanda funcional pode resultar em alterações. O trauma oclusal surge quando o periodonto excede sua capacidade de adaptação devido à demanda funcional e ocorrem alterações patológicas ou alterações adaptativas no periodonto, em resposta às forças oclusais (Davies et al., 2001).
O efeito das forças mecânicas na homeostase do ligamento periodontal é prejudicial na manutenção do espaço periodontal. A tendência geral após a aplicação de força nos dentes é de tentar manter a largura do espaço periodontal, um processo notável envolvendo reabsorção osteogênica controlada com precisão e deposição em locais específicos nos tecidos (Manokawinchoke et al., 2015). As forças mecânicas exercidas sobre o ligamento periodontal podem causar o aumento de compressão e induzir a produção de outros fatores bioquímicos para manter a homeostase (Pavasant e Yongchaitrakul, 2011). O trauma de oclusão pode causar reabsorção do osso adjacente. O osso é um tecido duro, com estrutura mineralizada e dinâmico, passando por contínua remodelação. As principais células que remodelam o osso são os osteoblastos, as quais depositam osso novo e os
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osteoclastos, os quais ativam a reabsorção óssea. Em uma condição saudável a atividade destas células é balanceada. Os osteoclastos tem sua origem de monócitos sob a influência de RANKL (Receptor ativador do ligante fator nuclear-kB). O RANKL liga-se ao RANK, o qual ativa a proteína intracelular Fator Nuclear - kB (NF- kB), um fator de transcrição para citocinas inflamatórias. Os osteoblastos produzem tanto RANKL e osteoprotegerina (OPG). A OPG previne a reabsorção óssea excessiva, ligando-se a RANKL impossibilitando que ele se ligue ao seu receptor RANK. Assim, a razão RANKL/OPG é um importante determinante da integridade do tecido ósseo (Miyamoto e Suda, 2003).
Estudos em modelos laboratoriais demonstraram que a presença de força compressiva constante no ligamento periodontal pode induzir a presença de RANKL (Kanzaki et al., 2002) e que a aplicação de força de tração pode regular a expressão de OPG (Tsuji et al., 2004). Em condições de trauma oclusal, Walker et al. (2008) sugerem que ambos RANKL e OPG contribuem para a estimulação da reabsorção óssea. A força oclusal como causa da inibição da aposição óssea, é um mecanismo importante para o controle da altura oclusal, e poderia atuar em sinergismo com o RANKL induzindo a reabsorção óssea para manter o equilíbrio oclusal.
Kumazawa et al. (1995) estudaram a condição de trauma oclusal em ratos adultos pela aplicação unilateral de resina composta nos molares superiores e, por meio de análise histológica, sugeriram que o ligamento periodontal apresentava áreas de compressão por alterações morfológicas em suas fibras.
Mavropoulos et al. (2004), ao investigar o efeito de um bloco de resina nos molares superiores sobre a mandíbula de ratos constatou inibição da erupção dos molares ao final de quatro semanas de experimento. No caso dos animais serem alimentados com dieta dura parece ter havido uma intrusão dos molares, após comparação com os animais mais jovens de referência. Goto et al. (2011) investigaram as relações entre a expressão de quimiocina e a formação de osteoclasto no ligamento periodontal e osso alveolar utilizando métodos imunohistológicos; tanto in vitro como in vivo as forças mecânicas durante a hiperoclusão regularam a expressão de CCL2 em tecidos periodontais, bem como a expressão do seu receptor CCR2, em pré-osteoclastos tais como monócitos/macrófagos, e de células TRAP-positivas no osso alveolar após 4 e 7 dias. Constatou-se que as forças mecânicas podem induzir CCL2 durante a hiperoclusão o que pode contribuir para a quimiotaxia e diferenciação dos
pré-17
osteoclastos, resultando na destruição dos tecidos periodontais e do osso alveolar durante o trauma oclusal.
Com a aplicação de fio de aço ligado por resina de metil-metacrilato no primeiro molar superior esquerdo, Kaku et al. (2005) observaram mobilidade do dente na direção mesiodistal após 7 dias e no sentido vestibulolingual após 9 dias. Na análise histológica e imuno-histológica da região apical do septo interradicular após 1 dia, estes autores verificaram que as fibras do ligamento periodontal foram comprimidas e estavam quase paralelas à dentina, a proteína RANKL foi observada em um pequeno número de osteoblastos; com 3 dias a compressão aumentou e no osso alveolar adjacente foram visualizados osteoclastos, a proteína RANKL foi observada tanto em osteoblastos como em osteoclastos; após 5 dias a área comprimida encontrava-se mais larga e havia a permanência de osteoclastos, a RANKL foi observado tanto em osteoblastos como em osteoclastos; aos 7 dias as compressões do ligamento periodontal começaram a se recuperar, a RANKL foi observada em poucos osteoblastos e osteoclastos e após 14 dias a largura do ligamento periodontal voltou ao nível do grupo controle e a RANKL era pouco detectável na área estudada.
Alguns estudos têm utilizado sistemas computacionais para reconstrução de imagens em três dimensões, a construção de malhas de elementos finitos, a caracterização das estruturas quanto às propriedades mecânicas, simulação de forças e o cálculo das deformações estruturais provenientes da simulação (Freire et al., 2014; Rossi et al., 2014). Assim, a simulação da mastigação pode ser realizada através de um modelo tridimensional pela utilização do método de elementos finitos, este método permite melhor compreensão da distribuição das forças oclusais na morfologia do complexo dentoalveolar (Prado et al., 2014).
Milne et al. (2009) realizaram as análises de elementos finitos e histológicas das forças geradas no ligamento periodontal sobre condições de cargas oclusal (2 N) e ortodôntica. Observaram que as áreas de pouca estimulação mecânica foram coincidentes com as regiões de perda óssea verificadas histologicamente, enquanto que em áreas de altos níveis de carga a massa óssea foi preservada.
Sabe-se que o mecanismo da degradação do ligamento periodontal por carga oclusal excessiva permanece incerto. Diante do exposto, o presente estudo buscou investigar se o aumento da altura na superfície oclusal dos molares
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superiores em ratos resulta em trauma oclusal no lado alterado e alterações de expressão proteicas e mecânicas no ligamento periodontal.
19
2 ARTIGO: MECHANICAL AND IMMUNOHISTOCHEMISTRY RESPONSE OF PERIODONTAL LIGAMENT OF RATS WITH DENTAL TRAUMATIC OCCLUSION
Artigo submetido ao periódico Journal of Periodontal Research (Anexo 1). Authors: Padilha, Edna Zakrzevski; Freire, Alexandre Rodrigues; Rossi, Ana Cláudia; Okamoto, Roberta; Prado, Felippe Bevilacqua
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ABSTRACT
The aim of the present investigation was to evaluate the mechanical and the immunohistochemistry response of PDL of upper first molar of rats with dental traumatic occlusion. Fifty male rats (Rattus norvegicus albinos), Wistar linage, with 2 months of age (weight, 200-250g) were submitted two separate groups for the experiments. In experimental group (n=40), the rats were submitted to unilateral application (right side) of light-cured composite resin on the occlusal face of upper first molar, simulating a dental traumatic occlusion condition. Then these animals were divided into 4 subgroups (n = 10) according to the euthanasia period: 7, 14, 21 and 28 days after application of the resin. In control group (n=10) the dentition was maintained without application of resin and from the early age (two months), the animals were euthanized to 28 days. All the specimens (head of rats) were fixed in formalin. The specimens were dissected and the right maxillary was removed to decalcified and processed to imunohistochemistry analysis. Primary antibodies were used: osteoprotegerin and receptor activator of nuclear factor kappa-B ligand. The finite element analysis was set to simulate a maxillary molar biting through the rat masticatory muscles activity and the molar bite force with 20 N magnitude on the maxillary molars, which features the normal occlusal contact in physiological condition, i.e. without significant bone remodeling changes. In the experimental condition, the same forces of muscles and molar biting were applied on the resin surface, featuring the dental traumatic occlusion. The immunohistochemical analysis presented different responses regarding the OPG and RANKL expression, which there was oscilation in the intensity in both protein in the experimental group. In periodontal ligament, the control group was observed a medium compressive strain response and in the experimental group the strains presented a medium to high compressive strain response. In conclusion, dental occlusal trauma caused changes associated with the activity of cells present in the periodontal ligament, linked to bone remodeling, and mechanical stimuli.
Keywords: Mechanobiology. Periodontal ligament. Occlusal trauma. Immunohistochemistry. Finite element analysis.
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INTRODUCTION
The periodontal ligament (PDL) performs the basic function to distributes the applied loads to alveolar bone complex and dissipates stress concentrations in the region where it connects to the bone [1, 2].
One of the main principles of dental treatment in clinical praticte is to impose external loads to a tooth, leading to an altered mechanical environment in the PDL surrounding the tooth and bone tissues supporting it. This altered environment induces remodelling and leads the tooth into a new position. The driving force of remodelling is the biological interaction between bone tissues and the PDL [3].
Chewing loads lead to displacements of teeth in their sockets [4, 2] with an overall non-linear response of the tooth-PDL-bone complex. The latter has no simple analytic description but has been addressed by 3D imaging and numerical simulations under variable simplifying assumptions. Naveh et al. [2] reported measurements obtained from a loading system situated within a micro computer tomography (μCT) scanner. Following the motion of a rat molar in the bone socket in 3D, they identified specific contact areas between the root surface and the jaw bone, appearing at increased loads. This resulted in a deflection of the tooth as it moves into the socket, described as a 'seesaw' tooth motion. Interestingly, tooth tilting following loading was also observed in compression on a bucco-lingual tooth slice from a pig premolar [5] both in laboratory experiments and in computer simulations. Ho et al. [6] report seemingly matching root-bone surfaces in human molars, areas that come into contact under compressive load.
The integration of the PDL with the alveolar bone and the cement provides information of the site of functional modeling in the junction areas between them since adaptive changes result from increased stresses in these areas [5]. The physiologically normal occlusal forces in mastication provide a positive stimulus to maintain the periodontium and alveolar bone in a healthy and functional condition [7,8].
Dental occlusal trauma occurs when there are excessive or non-physiological forces exerted on the tooth and it presents with normal, healthy and non-inflamed periodontal. When occlusal forces exceed the adaptive capacity of the periodontium, they cause damage to the periodontal tissues causing structural and functional changes [8-10].
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An example of dental occlusal trauma is premature contact of teeth during occlusion. In this condition, the occlusal forces originate in different and even opposite directions, which causes more complex histological changes in PDL with the presence of apposition and resorption on both sides, resulting in an enlargement of the periodontal space in radiographic images [8].
Studies in animal models have been used to evaluate the effects of occlusal trauma on the stomatognathic system [9]. According to Naveh et al. [11] in the PDL of first molars of rats there are two types of collagen networks that are organized in mesh. The scarce mesh appears in function in areas that undergo compressive loads and is located mainly in the distal aspect of the roots and in the area of furca, whereas the dense network appears in function in region of stress and is located in the mesial aspect of the roots and in direction to the crown.
The study by Milne et al. [12] showed that the association between FEA and histological analysis in rats under orthodontic forces resulted in increased compressive stresses and areas of resorption in the experimental groups. These results are associated with geometric factors, not considering the association with mechanobiology characterization, as strain data and the cellular response.
The functional mechanism of PDL mechanical and cellular response by excessive dental traumatic occlusion remains uncertain. Thus, the aim of the present investigation was to evaluate the mechanical and the immunohistochemistry response of PDL of upper first molar of rats with dental traumatic occlusion.
MATERIALS AND METHODS
This study was approved by the Ethics Committee on the Use of Animals of the Institute of Biology of the University of Campinas (Protocol CEUA/UNICAMP number 3661-1/2015) (ANEXO 2).
Sample and groups
Fifty male rats (Rattus norvegicus albinos), Wistar linage, with 2 months of age (weight, 200-250g) from CEMIB-UNICAMP were kept in collective cages (four animals / box), with temperature at 22 ± 2 ° C, controlled light cycle (12/12 h) and free access to water and feed. The rats were randomly divided into two separate groups for the experiments as described below:
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- Group 1 (n=40): unilateral application was carried out (right side) of light-cured composite resin (Fill Magic, Vigodent) on the upper first molar, simulating a dental traumatic occlusion condition. Then these animals were divided into 4 subgroups (n = 10) according to the euthanasia period: 7, 14, 21 and 28 days after application of the resin.
- Group 2 (n=10): the dentition was maintained without occlusal change (without application of resin). From the early age (two months), the animals were euthanized to 28 days.
Dental traumatic occlusion induction
The procedure was performed under general anesthesia using ketamine solution (40-87 mg/kg) and muscle relaxant xylazine (5-13 mg/kg) intraperitoneally. Once checked the sedation and anesthesia signals, the animal was placed on a plate. The induction of dental traumatic occlusion condition was carried out by unilateral application (right side) of composite resin (Fill Magic, Vigodent, USA), with 1 mm thick, on the occlusal surface of upper first molar, an experimental model which was adapted from Kumazawa et al. [13].
The euthanasia of animals was carried out on previously proposed periods (7, 14, 21 and 28 days) after application of the resin for the experimental group and 28 days after the initial age (2 months) for the control group. The animals were euthanized by anesthesia overdose (pentobarbital sodium, 100 mg/kg). The head was disjointed of the body, dissected and removed. The head was fixed in 10% formalin solution and 0.1 M phosphate buffer (pH 7.4) for 24 h at 4°C.
Imunohistochemistry analysis
All the specimens (head of rats) were fixed in formalin. The specimens were dissected and the right maxillary was removed to decalcified in ethylenediaminetetraacetic acid (EDTA, 10%) for 3 months, and then dehydrated using a series of ethanol concentrations. Tissues were washed for 24 h in running water, dehydrated through an alcohol sequence, cleared in xylene and embedded in Paraplast® (Embedding Media, McCormick Scientific, OH, USA). The slices were sectioned in a microtome and mounted on to glass slides. We obtained longitudinal seriated sections with 5-µm thick from each specimen. These slices were mounted on slides and subsequently stained with immunohistochemical procedures. After
24
these procedures, the images were captured using a conventional optical microscope (Aristoplan Leitz; Leica Microsystems, Bensheim, Germany) coupled to a camera (Leica DFC 300FX; Leica Microsystems, Heer-brugg, Switzerland) and connected to a computer with Optika View1 software (IL, USA) for capture of slices.
Endogenous peroxidase activity was inhibited by incubating the sections in hydrogen peroxide. Sections were subjected to antigen retrieval with citrate phosphate buffer (pH 6.0). Primary antibodies were used: osteoprotegerin (OPG, goat anti-OPG; Santa Cruz Biotechnology, SC21038), and receptor activator of nuclear factor kappa-B ligand (RANKL, goat anti-RANKL; Santa Cruz Biotechnology, SC7627).
Signal was detected using the immunoperoxidase method with a biotinylated anti-goat secondary antibody raised in rabbit (Pierce Biotechnology, Life Technologies Corporation, Grand Island, NY, USA), an avidin and biotin amplifier (Vector Laboratories, Burlingame, CA, USA) and diaminobenzidine (Dako, Carpinteria, CA, USA) as a chromogen. After the diaminobenzidine color reaction, sections were counter-stained with Harris hematoxylin, a counterstaining that allows to have the cytoarchitecture reference of the dental socket thirds evaluated.
All of the evaluations were performed at same conditions and by the same evaluator. The evaluation was performed by assigning different "scores" according to the area of immunostained cells present at each period studied (1 = Light, up to 25% of the area of interest with positive immunostained; 2 = moderate, until 50% of the area of interest with positive immunostained; 3 = intense, until 75% of the region of interest with positive immunostained), as previously described by Pedrosa et al. [14], Manrique et al. [15], and Ramalho-Ferreira et al. [16]. Positive markers were considered according to the marking pattern for each protein analyzed. The analysis was performed using a Nikon microscope (Eclipse 80i, Shinagawa, Tokyo, Japan), and images were captured with a 10x objective. The evaluation was conducted throughout the region of interest (ROI), composed by PDL tissue between the alveolar bone crest and the region of root bifurcation for the control, 7, 14, 21 and 28 days after traumatic occlusion induction.
25
Finite element analysis
Geometries preparation
One of the heads from control group was scanned in the Skyscan 1176 microtomography (Bruker, Kontich, Belgium). The tube current was 500 µA and peak voltage was 50 kV. Scans of each tissue piece were performed in the longitudinal plane. Image pixel size was 30 µm. The filter to correct for beam hardening was Al 0.5 mm. The rotation step was 0.2 deg.
From micro-CT images, the software Materialise MIMICS Research v18 (Materialise, Leuven, Belgium) was used for segmentation of anatomical structures through pixel marking involving bone, periodontal space (for PDL representation) and the teeth. For experimental group the resin on the upper first molar was also selected. All structures were converted to a 3D surface and, then, converted to finite element mesh (Figure 1).
Figure 1. Geometry and finite element mesh of rats with normal occlusion (control group) and altered occlusion (experimental group).
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Analysis configuration
The software Ansys v17 Structural Mechanics (Ansys, Inc., USA) was used to assign the mechanical properties and to calculate the structural strain from the simulation of molar biting. The structures were assigned as linear elastic and isotropic (table 1), whose values were determined experimentally and simulated by FEA in rats, according to the study of Cox et al. [17, 18].
Table 1. Mechanical properties of the anatomical structures. MPA=megapascal.
The analysis was set to simulate a maxillary molar biting through the rat masticatory muscles activity (table 2) [17] and the molar bite force with 20 N magnitude on the maxillary molars, which features the normal occlusal contact in physiological condition, i.e. without significant bone remodeling changes [19]. In the experimental condition, the same forces of muscles and molar biting were applied on the resin surface, featuring the dental traumatic occlusion. Restraints were applied on the posterior cut plane of the skull to keep the stability and absence of skull movements during the muscular action. The muscular forces were applied on each specific muscular insertion in rats [20].
Table 2. Masticatory muscles forces applied to the model. N=Newtons.
Structure Elastic modulus (MPa) Poisson’s ratio
Bone 19920 0.3
Teeth (molars) 30000 0.3
Periodontal ligament 50 0.4
Muscles Forces (N)
Masseter – superficial 5.95
Masseter – deep anterior 6.01
Masseter – deep posterior 11.49
Anterior zygomatic-mandibularis 1.16 Posterior zygomatic-mandibularis 1.03 Infraorbital zygomatic-mandibularis 1.94 Temporalis 9.56 Internal pterigoid 7.44 External pterigoid 2.36
27
Strain analysis in FEA
The ROI was evaluated to characterize the mechanical stimuli for control and experimental group. Qualitatively, the minimum principal strain (compressive strain) was calculated. The compressive strain calculation was performed. Compressive strain is determined by negative values.
RESULTS
The morphological changes presented an increased periodontal space associated to bone resorption in the interradicular septum and it was more visible in the 7 days period. The immunohistochemical analysis presented different responses regarding the OPG and RANKL expression, which there was oscilation in the intensity in both protein in the experimental group. Between control and experimental group, the OPG expression (Figures 2 and 3) was similar in the control group and 7 days period with premature contact, presenting light expression (score 1). In the subsequent periods there were moderate expression (score 2).
28 Figure 2 . Ost e o p rog e te ri n (OPG ) e x p ressio n in th e P DL tissu e . (C ) co n trol g rou p ; (7 d ) 7 d a y s e x p e ri m e n ta l g rou p ; (14 ) 1 4 d a y s e x p e ri m e n ta l g rou p (1 0 x o b ject iv e m a g n ificat ion ).
29 Figure 3 . Ost e o p rog e te ri n (OPG ) e x p ression in th e P DL tissu e . (21 d ) 2 1 d a y s e x p e ri m e n ta l g rou p ; (28 d ) 2 8 d a y s e x p e ri m e n ta l g rou p (10 x o bje ctiv e m ag ni fic at ion ).
30
The RANKL expression (Figures 4 and 5) was from light to moderate in the control group (score 1/2) and intense (score 3) in the experimental group following the periods 7, 21 and 28 days. In the experimental group there was oscilation in the expression intensity in the 14 days period, presenting moderate expression (score 2).
31 Figure 4 . RAN K L e x p re ssion in th e P DL tiss u e . (C ) co n tr o l g rou p ; (7d ) 7 d a y s e x p e ri m e n ta l g rou p ; (1 4 ) 1 4 d a y s e x p e ri m e n ta l g rou p (10 x o b ject iv e m a g n ific a tio n ).
32 Figure 5 . RANK L e x p ression in th e P DL tiss u e . (2 1 d ) 2 1 d a y s e x p e ri m e n ta l g rou p ; (2 8 d ) 2 8 d a y s e x p e ri m e n ta l g rou p (10 x o b ject iv e m a g n ificat io n ).
33
In the finite element analysis, the control group presented minimum and maximum values of strain ranging from -7.7e-8 (-7.7x10-8) mm/mm to 4.2e-11 (4.2x10-11) mm/mm throughout the periodontal ligament. The experimental group presented minimum and maximum values equal to -1e-7 (1x10-7) and 4.9e-11 (4.9x10-11) mm/mm, respectively. In the ROI, the control group was observed a medium compressive strain response, ranging from -1.3e-8 to -2.3e-8 mm/mm (Figure 6); in the experimental group the strains presented a medium to high compressive strain response reaching -3.6e-8 mm/mm (Figure 7).
34 Figu re 6 . M ini m u m pri ncipa l str ain calcula tio n o f c on tr ol g rou p. Th e ne g at iv e v alu es in th e colore d scale ind ic a te co m pressiv e str ain . (IS) I n te rr a d ic u lar se p tu m ; (T ) T o o th .
35 Figu re 7 . M inim u m pri ncipa l str ain calcula tio n o f ex pe rime nt al g rou p. T h e n eg at iv e v alu es in th e color ed sc ale ind ic at e co m pressiv e strain . (IS) I n te rr a d icul a r sep tu m ; (T ) Too th .
36
DISCUSSION
Mechanical loads have been reported to be involved in orthodontic tooth movement by events which transformed mechanical stresses into biological responses, and causing tissue remodeling [21-23]. The application of sufficiently intensity load on the teeth can result in change in in tensile and compressive stresses with the initial movement of the tooth [24].
The changes in the periodontal tissue morphology in condition of traumatic occlusion is associated to the RANKL expression. In samples of PDL of teeth with orthodontic treatment, the compression and tensile sides were observed and, when compared, the RANKL expression was greater in the compression side and the OPG in the traction side [25]. According Yamaguchi [26] during changes in occlusion as the orthodontic tooth movement, the PDL cells can regulate RANKL expression and induce osteoclastogenesis.
The OPG and RANKL expression determines the response in bone remodeling [27], which in the present study, the intense expression of RANKL indicated reabsorption. Previous studies [19, 28, 29] reported regions of intense bone resorption activity in short periods (from 1 day to 5 days), with greater intensity of RANKL expression in the PDL in rats under traumatic occlusion. From 7 days, according to our results, RANKL expression was localized in both the PDL and the periphery of the bone matrix and, thus, we can determine similar behaviour regarding the morphological changes from this period. These findings can be associated to PDL compression, which was observed histologically by Kaku et al. [28] study, and in Naveh et al. [2] and Walker et al. [30]. These authors found the furca region as the tooth-to-bone contact region and bone resorption, respectively, when dental occlusal forces were present.
In premature contact in the molar region, the morphological changes are characterized with a reduction of the periodontal space in the period from 1 to 5 days after induction [28], and it can be associated to compression of PDL between the root bifurcation and the interradicular septum. Similarly, from excessive force on the occlusal surface of first molar in rat, the compression in this region. These findings can be associated to the high biological response associated to bone resorption of interradicular septum. In fact, Kaku et al. [28] reported the increase in periodontal space from 7 days, whose characteristic can be also observed in our study. In this way, the option of studying the characterization of the minimum principal strain in
37
FEA located in PDL allowed the mechanical/numerical differentiation of the regions of greater compression. Furthermore, we studied the protein expression with the objective to validate the mechanical results considering the association between mechanical compressive strain, biological response and morphological changes of tissue.
The parafunctional conditions in occlusion has been studied using FEA [12, 31]. Compressive strains were reported with masticatory loading and parafunctional load mainly in the cervical and middle thirds on the buccal surface of the internal region of the PDL. Moga and Chiorean [32] concluded that FEA with increased strain gradually when verified the loss of bone height and PDL, but these strain decline from the cervical to the apical region. In the present study, in the experimental group, higher compression regions were observed than in the control group. Due to the limitations in the FEA regarding the accuracy of the characterization of the mechanical properties of the biological structures, we can be affirmed that the values found in this study do not establish a direct relation with the threshold of the mechanics of the periodontal tissues. However, it is possible to determine and localize the increase of the mechanical stimulus that, together with the immunohistochemical evaluation, allows to understand how the tissues behave in a condition of dental traumatic occlusion [12].
CONCLUSION
Dental occlusal trauma caused changes associated with the activity of cells present in the PDL, linked to bone remodeling, and mechanical stimuli, with increased compression and RANKL expression. The FEA allowed to simulate an experimental condition obtaining results compatible with biological responses.
ACKNOWLEDGMENTS
We are thanks to Coordination and Improvement of Higher Level or Education Personnel of Brazil (Capes) for the financial support.
CONFLICTS OF INTEREST
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3 CONCLUSÃO
O presente estudo permitiu concluir que:
houve aumento da expressão de RANKL no ligamento periodontal na região do terço cervical da raiz e, consequentemente, aumento da reabsorção óssea;
foi possível determinar as áreas com maior estímulo de deformação compressiva, principalmente nas regiões central e mesial;
as respostas biológicas são compatíveis com as deformações compressivas obtidas através de simulação computacional;
o aumento da altura na superfície oclusal dos molares superiores em ratos resultou em respostas biológicas com alterações no ligamento periodontal e osso alveolar circundante diante de deformações compressivas que culminaram em trauma oclusal no lado alterado.
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ANEXOS
45
