i

UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE ODONTOLOGIA DE PIRACICABA

GUSTAVO HAUBER GAMEIRO

O EFEITO DE FÁRMACOS E FATORES SISTÊMICOS NO

MOVIMENTO DENTÁRIO ORTODÔNTICO

Tese apresentada à Faculdade de Odontologia de Piracicaba, da Universidade Estadual de Campinas, para obtenção do título de Doutor em Radiologia Odontológica, Área de Concentração em Ortodontia.

Orientador:

Prof. Dr. Darcy Flávio Nouer

PIRACICABA 2008

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FICHA CATALOGRÁFICA ELABORADA PELA BIBLIOTECA DA FACULDADE DE ODONTOLOGIA DE PIRACICABA

Bibliotecário: Marilene Girello – CRB-8a. / 6159

G145e

Gameiro, Gustavo Hauber. O efeito de fármacos e fatores sistêmicos no movimento dentário ortodôntico. / Gustavo Hauber Gameiro. -- Piracicaba, SP: [s.n.], 2008. Orientador: Darcy Flávio Nouer. Tese (Doutorado) – Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba. 1. Estresse. 2. Agentes antiinflamatórios. 3. Movimentação dentária. I. Nouer, Darcy Flávio. II. Universidade Estadual de Campinas. Faculdade de Odontologia de Piracicaba. III. Título.

(mg/fop)

Título em Inglês: The effect of drugs and systemic factors on orthodontic tooth movement Palavras-chave em Inglês (Keywords): 1. Stress. 2. Anti-inflammatory agents. 3. Tooth movement Área de Concentração: Ortodontia Titulação: Doutor em Radiologia Odontológica Banca Examinadora: Darcy Flávio Nouer, José Fernando Castanha Henriques, Franco Arsati, Maria Cristina Volpato, Fernanda Klein Marcondes Data da Defesa: 19-02-2008 Programa de Pós-Graduação em Radiologia Odontológica

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DEDICO ESTE TRABALHO...

Aos meus pais

João Luís Priétto Gameiro e Mara Eunice Hauber Gameiro, que

com muito empenho, amor e dedicação tornaram possível a

concretização do meu sonho de tornar-me ortodontista.

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AGRADECIMENTOS ESPECIAIS

A Deus, que sempre ilumina e abençoa os meus caminhos.

Aos meus irmãos Augusto Hauber Gameiro e Paula Hauber Gameiro, meus cunhados

Rafael Dutra de Armas e Mariana Perozzi Gameiro e aos meus queridos afilhados

Manoela Perozzi Gameiro e Bruno Perozzi Gameiro, por compartilharem suas vidas

comigo na cidade de Piracicaba.

A Annicele Andrade, pelo apoio, compreensão e companheirismo ao longo desta jornada,

e principalmente por estar junto a mim em todos os momentos.

A minha avó Rosália Hauber e a minha segunda mãe Maria Luiza, por estarem sempre

presentes alegrando e iluminando minha vida.

viii

AGRADECIMENTOS ESPECIAIS

Às agências de fomento brasileiras:

CAPES

pelo apoio financeiro para o desenvolvimento desta pesquisa, na concessão da

Bolsa de Doutorado.

FAPESP

pelo apoio financeiro para o desenvolvimento desta pesquisa, na concessão de

Auxílio a Pesquisa.

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AGRADECIMENTOS ESPECIAIS

Ao meu orientador,

Prof. Dr. Darcy Flávio Nouer, pela orientação, apoio e estímulo à prática

ortodôntica sempre embasada nos princípios fisiológicos do sistema estomatognático,

compreendendo a importância da oclusão para a saúde oral e geral dos indivíduos.

Aos professores,

Profa. Dra. Maria Cecília Ferraz de Arruda Veiga, pelo carinho e pronta ajuda em

todos os momentos ao longo de minha vida acadêmica.

Prof. Dr. João Sarmento Pereira Neto, pela colaboração neste trabalho e pelo

constante apoio na clínica de ortodontia, sendo um belo exemplo de atenção e boa

vontade no atendimento aos pacientes.

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AGRADECIMENTOS ESPECIAIS

Aos professores da área de ortodontia da FOP/UNICAMP,

Profa. Dra. Maria Beatriz Magnani Araújo, pela paciência, atenção e carinho com

que sempre me ouviu e auxiliou durante minha estada na área de ortodontia.

Profa. Dra. Vânia Célia Vieira de Siqueira, por todos os ensinamentos que recebi

durante o curso, especialmente no primeiro ano em que fui seu estagiário na clínica de

graduação.

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AGRADECIMENTOS ESPECIAIS

Aos animais de laboratório, fundamentais para a realização deste trabalho.

"Peço a Deus que ilumine a mente humana na busca por métodos de ensino e pesquisa

que não envolvam a dor e o sofrimento dos animais.”

Gustavo Hauber Gameiro

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AGRADECIMENTOS

À Universidade Estadual de Campinas, na pessoa do seu Magnífico Reitor Prof. Dr. José

Tadeu Jorge; à Faculdade de Odontologia de Piracicaba, na pessoa do seu diretor Prof.

Dr. Francisco Haiter Neto, do Coordenador Geral da Pós-Graduação da FOP – UNICAMP

Prof. Dr. Mário Alexandre Coelho Sinhoreti, da Coordenadora do programa de Pós-

Graduação em Radiologia Odontológica da FOP-UNICAMP Profa. Dra. Gláucia Maria

Bovi Ambrosano, pela oportunidade de um crescimento científico e profissional nesta

conceituada instituição. Agradeço ainda a Profa. Gláucia por toda a atenção e ajuda

dispensada na execução das análises estatísticas desta tese.

Aos professores integrantes da banca examinadora desta tese: Prof. Dr. José Fernando

Castanha Henriques, Prof. Dr. Franco Arsati, Profa. Dra. Maria Cristina Volpato, Profa. Dra

Fernanda Klein Marcondes, Prof. Dr. Ary dos Santos Pinto, Prof. Dr. Luciano José Pereira,

e Profa. Dra. Ynara Bosco de Oliveira Lima Arsati, pela avaliação e colaboração em nosso

trabalho.

Ao Laboratório de Endocrinologia da Faculdade de Medicina de Ribeirão Preto-USP, na

pessoa da Profa. Dra. Margaret de Castro e Adriana Rossi, pela realização das dosagens

hormonais e colaboração no nosso trabalho.

Ao Professor Pedro Duarte Novaes, pelos ensinamentos e pronta ajuda fundamentais

para a realização deste trabalho.

Ao Professor Eduardo Dias de Andrade, pela valiosa amizade e preciosa contribuição

nesta pesquisa.

Á Maria Aparecida Santiago Varella e Eliene Aparecida Orsini Narvaes Romani, por toda a

assistência necessária para a realização das análises histológicas desta pesquisa.

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Aos professores Dr. Jaime Aparecido Cury, Dra. Cínthia Pereira Machado Tabchoury, Dra.

Lívia Maria Andaló Tenuta e Dra. Altair Antoniha Del Bel Cury, pela recepção e

ensinamentos recebidos na área de bioquímica.

Aos meus amigos Luciano Pereira, Leonardo Bonjardim, Maximiliano Cenci, Tatiana

Pereira-Cenci e Vander José das Neves, pelo apoio, companheirismo e sincera amizade

durante toda a caminhada.

Às amigas Ana Zilda Nazar Bergamo de Carvalho e Viviane Santíni Tamburús, pela

maravilhosa convivência e amizade durante todos os momentos do curso.

Aos companheiros Ricardo Sousa, Fábio Lourenço Romano, Meire Alves Sousa e

Vanessa Salvadego de Queiroz, pela amizade e oportunidade de aprendizado e

crescimento profissional.

À aluna de mestrado Marília Bertoldo Urtado, pelo empenho, dedicação e valiosa ajuda na realização desta pesquisa. Ao técnico Carlos Alberto Feliciano, pela colaboração, paciência e disposição durante a

utilização dos laboratórios da fisiologia.

À secretária e amiga Elisabete Casanova de Godoy, pela pronta ajuda em todos os

momentos, e pelo imenso carinho e amizade que construímos nestes anos de

convivência.

Aos Funcionários da Biblioteca da FOP – UNICAMP, pela orientação e ajuda.

A todos meus amigos e familiares, avós e avôs, tios e tias, primos e primas, sogro e

sogra, cunhados, enfim, a todos vocês que são fundamentais na minha formação.

Obrigado pelas orações, pelo carinho e pela força. A todos que direta ou indiretamente

contribuíram para a realização deste trabalho.

Meus sinceros agradecimentos.

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RESUMO

O objetivo da presente pesquisa foi avaliar a influência do estresse sistêmico e de um

antiinflamatório no movimento dentário ortodôntico. No experimento 1, os efeitos do

estresse sistêmico sobre a movimentação ortodôntica foram avaliados em ratos

estressados (por contenção) em modelos de estresse de curta (3 dias) ou longa duração

(40 dias). O grupo controle não foi submetido à contenção. O primeiro molar superior

esquerdo dos ratos foi movimentado mesialmente nos últimos 14 dias do experimento.

Logo depois, os animais foram mortos por decapitação para coleta de sangue e

mensuração da corticosterona plasmática; o movimento dentário foi quantificado e os

tecidos ao redor da raiz mesial do primeiro molar foram processados para análise

histoquímica por coloração para fosfatase ácida tartarato-resistente (TRAP). O estresse

crônico produziu maior taxa de movimentação dentária e aumentou o número de

osteoclastos em relação ao grupo controle e ao grupo submetido ao estresse de curta

duração, que não diferiram entre si. Não houve diferença entres os grupos em relação ao

número de odontoclastos e ao grau de reabsorção radicular. No experimento 2, os efeitos

da administração de celecoxibe, em regimes de curta (3 dias) e longa duração (14 dias),

foram avaliados em ratos submetidos à movimentação ortodôntica por 14 dias. Os grupos

controles receberam injeções i.p. de salina. Após análise da movimentação dentária e

análise histoquímica para avaliação dos osteoclastos, odontoclastos e grau de reabsorção

radicular, confirmou-se a ação inibitória do celecoxibe sobre a taxa de movimentação

dentária, embora a redução do número de osteoclastos não tenha sido estatisticamente

significativa em relação aos animais tratados com salina. O número de odontoclastos e o

grau de reabsorção radicular não foram afetados pelo medicamento. Os resultados destes

estudos indicam que o estresse crônico pode aumentar o número de osteoclastos e

acelerar a movimentação ortodôntica, porém este fator sistêmico não aumenta o risco à

reabsorção radicular; a administração de celecoxibe em regimes de curta e longa duração

reduziu significativamente a movimentação ortodôntica, porém seu efeito sobre a

reabsorção radicular não foi significativo. Estes experimentos fornecem evidências

científicas ao fato de que determinadas reações teciduais induzidas pelo movimento

ortodôntico podem ser influenciadas por fármacos e fatores sistêmicos.

Palavras-chave: estresse, agentes antiinflamatórios, movimentação dentária

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ABSTRACT

The aim of the present study was to evaluate the influence of systemic stress and an anti-

inflammatory on orthodontic tooth movement. In experiment 1, the effects of systemic

stress on orthodontic movement were evaluated in rats stressed (by restraint) in stress

models of short (3 days) or long duration (40 days). The control group was not submitted

to restraint. The upper left first molar of the rats was moved mesially in the last 14 days of

the experiment. Then, the animals were killed by decapitation for blood collection and

mensuration of plasmatic corticosterone; the tooth movement was quantified and the

tissues around mesial root of the first molar were processed for tartrate-resistant acid

phosphatase (TRAP) histochemistry. The chronic stress produced larger tooth movement

rate and an increased number of osteoclasts in relation to the control and short-stress

groups, which did not differ between each other. There was no difference between groups

in relation to the number of odontoclasts and root resorption degree. In experiment 2, the

effects of short (3 days) and long-term (14 days) celecoxib administration were evaluated

in rats submitted to orthodontic movement for 14 days. Control groups received saline i.p.

injections. After the exam of tooth movement and histochemical analysis for evaluation of

osteoclasts, odontoclasts and root resorption degree, the inhibitory action of celecoxib on

tooth movement rate was confirmed, although the reduction of the number of osteoclasts

was not statistically significant in relation to the animals treated with saline. The number of

odontoclasts and the root resorption degree were not affected by the drug. The results of

these studies indicate that chronic stress can increase the number of osteoclasts and

accelerate the orthodontic movement, however this systemic factor does not increase the

risk for root resorption; The short and long-term celecoxib administration reduced the

orthodontic movement, however its effect on root resorption was not significant. These

experiments provide scientific evidences to the fact that some tissue reactions induced by

orthodontic movement can be influenced by drugs and systemic factors.

Keywords: stress, anti-inflammatory agents, tooth movement

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SUMÁRIO I. INTRODUÇÃO 1 II. CAPÍTULOS 5

Artigo 1: The influence of drugs and systemic factors on orthodontic tooth movement (artigo de revisão) 7 Artigo 2: The effects of systemic stress response on orthodontic tooth movement 23 Artigo 3: Evaluation of root resorption associated with orthodontic movement in stressed rats 47 Artigo 4: The effects of short and long-term celecoxib administration on orthodontic tooth movement 63 Artigo 5: Histological analysis of orthodontic root resorption in rats treated with the cyclooxygenase-2 (COX-2) inhibitor celecoxib 81

III. CONCLUSÕES 97 IV. REFERÊNCIAS BIBLIOGRÁFICAS 99

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APÊNDICE - Resumos (em português) 101

- Dados referentes aos valores individuais da amostra 106

ANEXO - Comprovantes de aceite ou submissão dos artigos 113

- Certificados da comissão de ética na experimentação animal 118

1

I. INTRODUÇÃO

O movimento dentário ortodôntico ocorre pela aplicação prolongada de

forças mecânicas controladas ao dente. Tais forças causam zonas de pressão e

tensão no ligamento periodontal e osso alveolar, com a subseqüente remodelação

do alvéolo dental, possibilitando o movimento do dente (Meikle, 2006). O pré-

requisito para a remodelação óssea e deslocamento dentário caracteriza-se por

uma reação inflamatória envolvendo osteoclastos, osteoblastos, neuropeptídeos

(Davidovitch et al., 1988; Norevall et al., 1995), citocinas (Alhashimi et al., 2000,

2001) e alterações na inervação e vascularização local (Kvinnsland et al., 1989;

Vandevska-Radunovic et al., 1994). Os mediadores químicos sintetizados e

liberados neste processo possibilitam a intercomunicação celular responsável pela

seqüência de eventos que caracterizam o movimento dentário ortodôntico

(Krishnan & Davidovitch, 2006). Estas interações celulares podem ser reguladas

tanto por fatores mecânicos, tais como as forças aplicadas, quanto por drogas e

fatores sistêmicos, tais como hormônios e vitaminas (Gameiro et al., 2007).

Entre os fatores sistêmicos destaca-se a resposta de estresse – reação do

organismo a uma variedade de estímulos físicos e/ou psicológicos que perturbam

sua homeostase (Habib et al., 2001). Alguns estudos sugerem que os nervos

simpáticos modulam a inflamação e a remodelação óssea in vivo (Levine et al.,

1985; Sandhu et al., 1987). Osteoblastos e osteoclastos possuem receptores

adrenérgicos e glicocorticóides (Togari, 2002; Kim et al., 2006), indicando que

estas células podem ser influenciadas pelos neurotransmissores simpáticos. Além

disso, um maior número de osteoclastos, bem como uma maior atividade

osteoclástica tem sido observada após a remoção do gânglio simpático em ratos

(Cherruau et al., 2003; Haug &. Heyeraas, 2003), o que reforça a possibilidade da

resposta de estresse afetar o movimento dentário ortodôntico.

Em relação aos fármacos, os antiinflamatórios não-esteróides (AINES)

situam-se entre os mais utilizados pelos ortodontistas para o alívio da dor

(Krishnan & Davidovith, 2006; Gameiro et al., 2007). Estes fármacos inibem a

2

enzima ciclooxigenase (COX), a qual modula a transformação de prostaglandinas

a partir do ácido araquidônico na membrana plasmática celular (Leone et al.,

2007). Existem basicamente dois tipos de COX. A COX-1 (constitutiva) possui

papel importante na homeostase tecidual, enquanto a COX-2 (indutiva) relaciona-

se principalmente com o desenvolvimento da inflamação (Smith & Dewitt, 1996).

Vários estudos demonstram a efetividade dos AINES no controle da dor

ortodôntica (Polat et al., 2005; Arias et al., 2006), embora estes fármacos também

possam afetar a seqüência do movimento dentário, inibindo ou reduzindo a

inflamação e a reabsorção óssea associada (Arias et al., 2006). Assim, o uso dos

inibidores seletivos da COX-2, também conhecidos como coxibes, tem aumentado

na prática clínica (Young et al., 2006; Krishnan, 2007). Tem sido demonstrado que

alguns coxibes (celecoxibe e parecoxibe) não interferem na movimentação

dentária induzida em ratos (Jerome et al., 2005; de Carlos et al., 2007). Entretanto,

a especificidade dos coxibes pode ser responsável por diferentes efeitos destes

fármacos sobre o movimento ortodôntico (de Carlos et al., 2007). Atualmente, não

encontra-se um fármaco capaz de atender as expectativas idealizadas pelo

ortodontista, ou seja, controlar a velocidade da movimentação dentária, reduzir

eventuais desconfortos, influenciar no tempo de tratamento e tratar com

segurança pacientes que fazem uso de medicamentos para doenças crônicas.

Além disso, vislumbra-se muito em breve a ação de fármacos para diminuir a

incidência de reabsorções radiculares na prática clínica.

A administração pré-operatória (ou preemptiva) de fármacos para o

controle da dor tem sido alvo de pesquisas recentes na ortodontia (Steen et al.,

2000; Bernhardt et al., 2001; Polat et al., 2005; Young et al., 2006). Além da dose

pré-operatória, alguns autores recomendam mais duas doses pós-operatórias para

o adequado controle da dor após a ativação do aparelho orodôntico (Polat et al.,

2005). Acredita-se que baixas doses destes fármacos, administradas por um ou

dois dias, não interferirão no processo de movimentação dentária de forma

significativa. Entretanto, esta hipótese não encontra-se embasada por estudos

experimentais controlados.

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Assim, os objetivos do presente trabalho foram:

� Realizar uma revisão de literatura sobre os principais fármacos

e fatores sistêmicos capazes de afetar a biologia do movimento dentário

ortodôntico.

� Avaliar o efeito do estresse de curta e longa duração na taxa

de movimentação dentária, alterações hormonais e reabsorção radicular

após movimentação ortodôntica experimental.

� Avaliar a influência do inibidor seletivo da COX-2 celecoxibe

na taxa de movimentação dentária e reabsorção radicular associada à

aplicação de força ortodôntica experimental.

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II. CAPÍTULOS

Capítulo 1: “The influence of drugs and systemic factors on orthodontic tooth

movement”. Este artigo foi publicado no periódico Journal of Clinical Orthodontics.

Uma versão traduzida deste artigo foi publicada no periódico Ortodoncia.

Capítulo 2: “The effects of systemic stress response on orthodontic tooth

movement”. Este artigo foi submetido à publicação no periódico Archives of Oral

Biology.

Capítulo 3: “Evaluation of root resorption associated with orthodontic movement in

stressed rats”. Este artigo foi submetido à publicação no periódico The Angle

Orthodontist.

Capítulo 4: “The effects of short and long-term celecoxib administration on

orthodontic tooth movement”. Este artigo foi aceito para publicação no periódico

The Angle Orthodontist.

Capítulo 5: “Histological analysis of orthodontic root resorption in rats treated with

the cyclooxygenase-2 (COX-2) inhibitor celecoxib”. Este artigo foi submetido à

publicação no periódico Orthodontics and Craniofacial Research.

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Capítulo 1

OVERVIEW The influence of drugs and systemic factors on orthodontic tooth movement

GUSTAVO HAUBER GAMEIRO JOÃO SARMENTO PEREIRA-NETO MARIA BEATRIZ BORGES DE ARAÚJO MAGNANI DARCY FLÁVIO NOUER Abstract

The orthodontic tooth movement occurs by an inflammatory process involving osteoclasts,

osteoblasts, neuropeptides, cytokines and alterations in innervation and local

vascularization. Researches have demonstrated that bone remodeling activity can be

regulated either by local factors, such as the applied forces, or by systemic factors, such

as drugs, hormones and vitamins. This work presents the effects of drugs and systemic

factors capable of affecting bone metabolism and influencing the speed of orthodontic

tooth movement.

The non-steroidal anti-inflammatory drugs (except celecoxib), biphosphonates and sex

hormones can reduce the speed of orthodontic movement, while the corticosteroids,

relaxin, thyroid hormones, parathyroid hormone and vitamin D can increase the rate of

tooth movement. Thus, it was concluded that the dentist should be attentive to the

medications being used by patients, in order to select the best therapeutic strategy (control

of the forces and interval between visits) for each case. Moreover, acetaminophen should

be the drug of choice to relieve any possible discomfort associated with orthodontic

treatment, because it has no significant influence on the rate of tooth movement.

Key-words: orthodontic movement, anti-inflammatory drugs, systemic factors

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Introduction

Orthodontic tooth movement is induced by the prolonged application of controlled

mechanical forces to a tooth. Such forces cause pressure and tension zones in the

periodontal ligament and alveolar bone, subsequently remodeling the tooth socket and

enabling tooth movement. The prerequisite for bone remodeling and tooth displacement is

the occurrence of an inflammatory process involving osteoclasts, osteoblasts,

neuropeptides,1, 2 cytokines,3, 4 and changes in innervation and local vascularization.5, 6

Over the last few years, the discovery of new molecules and the development of new

experimental techniques have allowed orthodontic tooth movement to be studied at the

molecular level. Molecular biology studies identified the main mediators involved in the

complex process of extravasation, inflammatory cell chemotaxis and recruitment of

osteoclast and osteoblast progenitors, responsible for bone remodeling and orthodontic

movement7 (Tab. 01). This approach shows that in common with all the other biological

sciences, the methodology of tooth movement research has become progressively more

reductionist, making the subject less accessible to the clinician. Thus, the aim of this work

was to present up to date data concerning the role of drugs and systemic factors capable

of affecting bone metabolism and influencing the speed of orthodontic tooth movement.

This information is considered essential for clinical practice, to enable the professional to

consider all the factors related to the orthodontic treatment, and to select the best

individual therapeutic strategy for each case.

9

Table 1 – Factors affecting bone-remodeling process Hormones Growth factors Cytokines Colony-

stimulating factors*

Others

Parathyroid hormone Calcitonin Insulin Growth hormone Vitamin D Glucocorticoids Sex steroids Thyroid hormones

Insulin-like growth factors I & II Transforming growth factor β Fibroblast growth factor Platelet derived growth factor

Interleukin-1,4, 6,11,13 ,18 Tumor necrosis factor

Osteoclast differentiating factor

Interferon-γ Osteoprotegrin

M-CSF G-CSF

GM-CSF

Prostaglandins Leukotrienes Nitric Oxide

*Colony-stimulating factors (CSF) related to granulocytes (G-CSF), macrophages (M-CSF), or to both cell types (GM-CSF)

Effects of drugs on induced tooth movement

According to the data found in the literature, the drugs capable of influencing the rate of

orthodontic movement can be divided into four main categories:

1. Non-steroidal anti-inflammatory drugs (NSAIDs)

2. Corticosteroids

3. Biphosphonates

4. Acetaminophen

Non-steroidal anti-inflammatory drugs (NSAIDs)

The knowledge of mechanisms involved in the transduction of mechanical forces into

biological responses began in the 1970s. Harell et al., 1977 observed the synthesis of

prostaglandins in petri dishes deformed with orthodontic screws cemented to the base, on

which osteoblast-like cells had been cultured.8 In an interesting practical application of

these findings, Yamasaki et al., 1980 found that indomethacin, a non-steroidal

cyclooxygenase 1 and 2 (COX-1 and COX-2) inhibitor, reduced bone resorption and

orthodontic tooth movement in rats.9 Moreover, these authors demonstrated that the local

injection of prostaglandin E-1 and prostaglandin E-2 into the submucosa overlying

10

orthodontically treated teeth doubled the rate of tooth movement, both in monkeys10 and in

humans.11 Because prostaglandins appear to be important in the process of tooth

movement under orthodontic forces, it is suggested that the use of over-the-counter

NSAIDs by the patients during orthodontic treatment significantly alters the efficacy of

tooth movement. The action of conventional NSAIDs (aspirin12, diclofenac, ibuprofen,

indomethacin), specific COX-2 inhibitors (rofecoxib, celecoxib), and other drugs on

orthodontic tooth movement is illustrated in Tab. 02. Recently, Jerome et al., 2005

showed that Celebrex administered in rats during the application of orthodontic forces did

not interfere with tooth movement and appeared to offer some slight protection against root

resorption.13 A research study to analyze the effects of this drug in humans receiving

orthodontic treatment is now necessary.

Table 2 – Effects of drugs on induced tooth movement

Drugs Effects on bone metabolism Effects on tooth movement

Nonsteroidal anti-inflamatory drugs

Aspirin Diclofenac Ibuprofen

Indometacin Celecoxib

↓ bone resorption ↓ bone resorption ↓ bone resorption ↓ bone resorption

↓ bone resorption (in-vitro)

↓ tooth movement ↓ tooth movement ↓ tooth movement ↓ tooth movement has no influence

- Corticosteroids - Biphosphonates - Acetaminophen

↑ bone resorption- chronic use ↓ bone resorption

unproved

↑ tooth movement ↓ tooth movement has no influence

11

Corticosteroids

The increasing use of glucocorticoid therapy for many inflammatory and autoimmune

diseases indicates that in the clinical practice, an important number of orthodontic patients

can present variations from normal bone turnover because of this steroid.14 In animal

experiments that studied glucocorticoid administration and orthodontic tooth movement,

the glucocorticosteroid dose was high, which made the animals osteoporotic.15, 16, 17

However, Kalia et al., 2004 evaluated the rate of tooth movement in rats during the course

of short- and long-term therapeutic corticosteroid therapy.18 These authors demonstrated

that in acute administration of drug therapy, bone remodeling seems to slow down. On the

other hand, the tooth movement rate increased in chronic treatment. Clinically, these

results suggest that it is possible to treat patients undergoing corticosteroid therapy, with a

minimum of adverse effects. Patients who are within the short-term phase of using the

drug, may be advised to postpone orthodontic treatment, or their appliance adjustments

should be scheduled with longer intervals, as bone turnover will be delayed. In long-term

drug therapy, when the tooth movement could be accelerated, the orthodontic appliance

can be controlled as usual or more frequently.

Biphosphonates

This class of pharmacological agents selectively inhibits osteoclasts, and has been used to

treat various metabolic bone diseases associated with excessive bone resorption.19

Laboratory studies have demonstrated that the orthodontic tooth movement can be

inhibited by the topical application of biphosphonates.20, 21 Adachi et al., 1994 suggested

that the topical application of biphosphonates could be useful in orthodontic anchorage

and retention of teeth.20 In 2004, Liu and colleagues applied a biphosphonate without a

nitrogen atom (clodronate) in the sub-periosteal area of molars in rats submitted to

12

orthodontic movement for 3 weeks. The local application of clodronate not only reduced

the orthodontic movement and the number of osteoclasts, but also reduced the root

resorption.21 Further studies are necessary before clinical application of these drugs in

orthodontic therapy. However, orthodontists should be aware of the interaction of this drug.

In 2005, Schwartz reported an important case of a female orthodontic patient being

medicated with Zometa (biphosphonate) to control the bone metastases related to breast

cancer22. At the time the patient began treatment with this drug, when the premolar spaces

were about a third closed, all orthodontic movement stopped.

Acetaminophen

Acetaminophen (Paracetamol), a weak COX-1 and COX-2 inhibitor that also reduces the

urinary PG levels after systemic administration showed no effect on orthodontic tooth

movement in guinea pigs23 and rabbits24. Moreover, comparative studies25, 26 and our

clinical experience demonstrated that acetaminophen is effective for controlling

pain/discomfort associated with orthodontic treatment.

Effects of systemic factors on induced tooth movement

Sex hormones, relaxin, thyroid hormones, parathyroid hormone and vitamin D are the

main systemic factors capable of influencing the rate of orthodontic movement (Tab. 03).

13

Table 3 – Effects of systemic factors on induced tooth movement

Sex hormones

Estrogen is considered the most important hormone that affects bone metabolism in

women. It inhibits the production of cytokines involved in osteoclastic activation and bone

resorption, such as interleukin-1, tumor necrosis factor-a, and interleukin-627. In 2001,

Yamashiro and Takano-Yamamoto demonstrated an acceleration of tooth movement in

castrated female rats.28 On the other hand, Miyajima et al., 1996 reported the case of a

patient submitted to orthodontic treatment, in which reduction in the speed of tooth

movement occurred. The authors attributed the slow turnover of alveolar bone to the

patient’s menopausal status and the estrogen supplement she had been taking for 3

years29. Furthermore, these authors suggested that young women who were taking oral

contraceptives might also show a reduction in tooth movement, therefore the orthodontist

should pay special attention in these cases. Further laboratorial and clinical studies are

necessary to explain this subject. As regards the androgens, although their inhibitory effect

Hormones Effects on bone metabolism Effects on tooth movement

- Hormones Estrogen

Androgen

Relaxin

Thyroid hormones

Parathyroid hormone

- Vitamin D

↓ bone resorption ↓ bone resorption

↑ bone resorption

↑ rate of bone remodeling

↑ bone resorption

↑ bone resorption

↑ rate of bone remodeling ↑ bone resorption

↓ tooth movement unproved

↑ tooth movement

↑ tooth movement

↓ root resorption

↑ tooth movement

↑ tooth movement

14

on bone resorption has been explained,30 their influence on orthodontic tooth movement

has not been clarified.

Relaxin

Relaxin has been known for decades as a pregnancy hormone. It is released just before

childbirth to loosen the pubic symphysis, so the relaxed suture allows for the widening of

the birth canal for parturition. It has also been shown to have effects on a multitude of

physiological processes far beyond pregnancy and reproduction, including the regulation

of vasotonus, plasma osmolality, angiogenesis, collagen turnover, and renal function.31

Its effects on remodeling soft tissue, and on regulation of several mediators that stimulate

osteoclast formation increased attention from researchers in orthodontics. Liu et al., 2005

showed that administration of human relaxin might accelerate the early stages of

orthodontic tooth movement in rats.32 Stweart and colleagues used gingival injection of

relaxin to relieve the rotational memory in the connective tissues of dog maxillary second

incisors that were orthodontically rotated. The results were not significant, but authors

suggested that dose and treatment optimization might improve the response in future

studies.33 In 2000, Nicozisis and colleagues demonstrated that the presence of relaxin

abolished the integrity of sutures in vitro. These authors also suggested that relaxin might

be used as an adjunct to orthodontic therapy during or after tooth movement promoting

stability, rapid gingival tissue remodeling during space closure in extraction sites, and for

orthopedic expansion in non-growing patients by a reduction in the tension of the stretched

soft tissue envelope following orthognathic surgery, particularly the expanded palatal

mucosa.34 The question whether or not these findings hold true in clinical practice remains

to be investigated.

15

Thyroid hormones

Thyroid hormones play an essential role in the normal growth and development of

vertebrates. They are involved in bone maturation, stimulate cartilage growth and

differentiation, enhance the response to growth hormone, and stimulate bone resorption.

They act directly on bone remodeling, stimulating the action of the osteoclasts, but they

also have an indirect effect via some growth factors that are closely related to bone

metabolism, such as insulin-like growth factor I (IGF-I), produced locally in bone cells by

the action of thyroid hormones.35

According to the study of Shirazi et al., 1999 thyroid hormone administration in rats not

only increased the speed of tooth movement, but also reduced the extent of root

resorption, as seen from scanning electron micrographs.36 Loberg & Engstrom, 1994, also

reported a protective effect of thyroxin on root resorptive lesions, induced by the

application of orthodontic forces.37 In a recent study, Vasquez et al., 2002 showed that

animals treated with thyroid hormones (intra-peritoneal or oral) had significantly less force-

induced root resorptive lesions compared with a control group. The authors suggested that

the application of low doses of thyroid hormones may have a protective effect on the root

surface either during orthodontic treatment, or in patients that present spontaneous root

resorptive lesions.38 The clinical application of these drugs still needs to be clarified.

Parathyroid hormone

Parathyroid hormone (PTH) is produced by the parathyroid glands and its function is to

regulate serum calcium concentration by concerted effects on kidney and bone. In the

kidney, PTH increases renal calcium re-absorption, and stimulates the urinary phosphate

excretion. In bone, PTH can induce a rapid release of calcium, but it also mediates longer-

term changes by acting directly on osteoblasts and indirectly on osteoclasts. In

osteoblasts, PTH affects cellular metabolic activity, gene transcriptional activity, and

16

multiple protease secretion. Its effects on osteoclasts occur by the production of RANKL, a

protein that plays a crucial role in osteoclast formation and activity.39 Thus, the increase of

bone turnover induced by PTH could accelerate orthodontic movement. This was

demonstrated by studies in animals in the 1970s,40, 41 and more recently by Soma and

collaborators, who observed an acceleration of the tooth movement in rats treated with

PTH administrated systemically42 and locally.43 These results indicate that the orthodontist

should be attentive to patients treated with PTH, for example, in cases of severe

osteoporosis.44

Vitamin D

In 1988, Collins and Sinclair demonstrated that intraligamentous injections of a vitamin D

metabolite 1,25-dihydroxycholecalciferol (1,25D) caused an increase in the number of

osteoclasts and a larger tooth movement, during canine retraction with light forces in

cats.45 Similar results were observed by Takano-Yamamoto and colleagues in 1992.46

Corroborating these findings, Kale et al., 2004 observed that local application of vitamin D

enhances the rate of tooth movement in rats, and according to the authors, this effect was

due to the well balanced bone turnover induced by vitamin D.47

In addition, the stimulatory action of vitamin D on osteoblasts can also increase

stabilization of orthodontic movement. In a 1996 study by Baran and colleagues, rats

treated with vitamin D presented more bone formation on the pressure side of the

periodontal ligament after application of orthodontic forces.48 In 2004, Kawakam and

Takano-Yamamoto, also observed an increase in the mineral appositional rate on alveolar

bone, after the application of orthodontic forces in rats.49 They suggested that local

application of vitamin D could intensify the re-establishment of supporting tissue, especially

alveolar bone, after orthodontic treatment.

17

Conclusions

Orthodontists have long been observed that teeth move at different rates, and that

individuals have different responses to treatment. Some of these differences are caused

by changes in bone remodeling induced by drugs and systemic factors.

The non-steroidal anti-inflammatory drugs (except celecoxib), biphosphonates and sex

hormones can reduce the speed of orthodontic movement, while the corticosteroids,

relaxin, thyroid hormones, parathyroid hormone and vitamin D can increase the rate of

tooth movement. Therefore, clinicians should pay careful attention to the medications

being used by their patients, so that the best therapeutic strategy – including force control

and appointment intervals – can be selected for each case. Acetaminophen, which does

not have a significant influence on the rate of tooth movement, can be recommended for

controlling pain during orthodontic treatment.

REFERENCES 1. Davidovitch, Z.; Nicolay, O.F.; Ngan, P.W.; Shanfeld, J.L.: Neurotransmitters, cytokines, and the control of alveolar bone remodeling in orthodontics, Dent Clin North Am. 32:411-435, 1988. 2. Norevall, L.I.; Forsgren, S.; Matsson, L.: Expression of neuropeptides (CGRP, substance P) during and after orthodontic tooth movement in the rat, Eur J Orthod. 17:311-325, 1995. 3. Alhashimi, N.; Frithiof, L.; Brudvik, P.; Bakhiet, M.: Orthodontic movement induces high numbers of cells expressing IFN-gamma at mRNA and protein levels, J Interferon Cytokine Res. 20:7-12, 2000. 4. Alhashimi, N.; Frithiof, L.; Brudvik, P.; Bakhiet, M.: Orthodontic tooth movement and de novo synthesis of proinflammatory cytokines, Am J Orthod Dentofacial Orthop. 119:307-312, 2001. 5. Kvinnsland, S.; Heyeraas, K.; Ofjord, E.S.: Effect of experimental tooth movement on periodontal and pulpal blood flow, Eur J Orthod. 11:200-205, 1989.

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6. Vandevska-Radunovic, V.; Kristiansen, A.B.; Heyeraas, K.J.; Kvinnsland, S.: Changes in blood circulation in teeth and supporting tissues incident to experimental tooth movement, Eur J Orthod. 16:361-369, 1994. 7. Krishnan, V.; Davidovitch, Z.: Cellular, molecular, and tissue-level reactions to orthodontic force, Am J Orthod Dentofacial Orthop. 129:469.e1-32, 2006. 8. Harell, A.; Dekel, S.; Binderman, I.: Biochemical effect of mechanical stress on cultured bone cells, Calcif Tissue Res. 22:202-207, 1977. 9. Yamasaki, K.; Miura, F.; Suda, T.: Prostaglandin as a mediator of bone resorption induced by experimental tooth movement in rats, J Dent Res. 59:1635-1642, 1980. 10. Yamasaki, K.; Shibata, Y.; Fukuhara, T.: The effect of prostaglandins on experimental tooth movement in monkeys (Macaca fuscata), J Dent Res. 61:1444-1446, 1982. 11. Yamasaki, K.; Shibata, Y.; Imai, S.; Tani, Y.; Shibasaki, Y.; Fukuhara, T.: Clinical application of prostaglandin E1 (PGE1) upon orthodontic tooth movement, Am J Orthod. 85:508-518, 1984. 12. Arias O.R.; Marquez-Orozco M.C. : Aspirin, acetaminophen, and ibuprofen: their effects on orthodontic tooth movement, Am J Orthod Dentofacial Orthop. 130:364-370, 2006. 13. Jerome, J.; Brunson, T.; Takeoka, G.; Foster, C.; Moon, H.B.; Grageda, E.; Zeichner-David, M.: Celebrex offers a small protection from root resorption associated with orthodontic movement, J Calif Dent Assoc. 33:951-959, 2005. 14. Baid, S.K.; Nieman, L.K.: Therapeutic doses of glucocorticoids: implications for oral medicine, Oral Dis. 12:436-442, 2006. 15. Davidovitch, Z.; Musich, D.; Doyle, M.: Hormonal effects on orthodontic tooth movement in cats--a pilot study, Am J Orthod. 62:95-96, 1972. 16. Thompson, J.S.; Palmieri, G.M.; Eliel, L.P.; Crawford, R.L.: The effect of porcine calcitonin on osteoporosis induced by adrenal cortical steroids, J Bone Joint Surg Am. 54:1490-1500, 1972. 17. Ashcraft, M.B.; Southard, K.A.; Tolley, E.A.: The effect of corticosteroid-induced osteoporosis on orthodontic tooth movement, Am J Orthod Dentofacial Orthop. 102:310-319, 1992. 18. Kalia, S.; Melsen, B.; Verna, C.: Tissue reaction to orthodontic tooth movement in acute and chronic corticosteroid treatment, Orthod Craniofac Res. 7:26-34, 2004. 19. Fleisch, H.: Development of bisphosphonates, Breast Cancer Res. 4:30-34, 2002. 20. Adachi, H.; Igarashi, K.; Mitani, H.; Shinoda, H.: Effects of topical administration of a bisphosphonate (risedronate) on orthodontic tooth movements in rats, J Dent Res. 73:1478-1486, 1994.

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21. Liu, L.; Igarashi, K.; Haruyama, N.; Saeki, S.; Shinoda, H.; Mitani, H.: Effects of local administration of clodronate on orthodontic tooth movement and root resorption in rats, Eur J Orthod. 26:469-73, 2004. 22. Schwartz, J.E.: Ask us: Some drugs affect tooth movement, Am J Orthod Dentofacial Orthop. 127:644, 2005. 23. Kehoe, M.J.; Cohen, S.M.; Zarrinnia, K.; Cowan, A.: The effect of acetaminophen, ibuprofen, and misoprostol on prostaglandin E2 synthesis and the degree and rate of orthodontic tooth movement, Angle Orthod. 66:339-349, 1996. 24. Roche, J.J.; Cisneros, G.J.; Acs, G.: The effect of acetaminophen on tooth movement in rabbits, Angle Orthod. 67:231-236, 1997. 25. Simmons, K.E.; Brandt, M.: Control of orthodontic pain, J Indiana Dent Assoc. 71:8-10, 1992. 26. Polat, O.; Karaman, A.I.: Pain control during fixed orthodontic appliance therapy, Angle Orthod. 75:214-219, 2005. 27. Carlsten, H.: Immune responses and bone loss: the estrogen connection, Immunol Rev. 208:194-206, 2005. 28. Yamashiro, T.; Takano-Yamamoto, T.: Influences of ovariectomy on experimental tooth movement in the rat, J Dent Res. 80:1858-1861, 2001. 29. Miyajima, K.; Nagahara, K.; Iizuka, T.: Orthodontic treatment for a patient after menopause, Angle Orthod. 66:173-178, 1996. 30. Michael, H.; Harkonen, P.L.; Vaananen, H.K.; Hentunen, T.A.: Estrogen and testosterone use different cellular pathways to inhibit osteoclastogenesis and bone resorption, J Bone Miner Res. 20:2224-2232, 2005. 31. Dschietzig, T.; Bartsch, C.; Baumann, G.; and Stangl, K.: Relaxin— a pleiotropic hormone and its emerging role for experimental and clinical therapeutics, Pharmacol. Ther. 112:38-56, 2006. 32. Liu, Z.J.; King, G.J.; Gu, G.M.; Shin, J.Y.; and Stewart, D.R.: Does human relaxin accelerate orthodontic tooth movement in rats? Ann. N.Y. Acad. Sci. 1041:388-394, 2005. 33. Stewart, D.R.; Sherick, P.; Kramer, S.; and Breining, P.: Use of relaxin in orthodontics, Ann. N.Y. Acad. Sci. 1041:379-387, 2005. 34. Nicozisis, J.L.; Nah-Cederquist, H.D.; and Tuncay, O.C.: Relaxin affects the dentofacial sutural tissues, Clin. Orthod. Res. 3:192-201, 2000. 35. Wakita, R.; Izumi, T.; Itoman, M.: Thyroid hormone-induced chondrocyte terminal differentiation in rat femur organ culture, Cell Tissue Res. 293:357-364, 1998.

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36. Shirazi, M.; Dehpour, A.R.; Jafari, F.: The effect of thyroid hormone on orthodontic tooth movement in rats, J Clin Pediatr Dent. 23:259-264, 1999. 37. Poumpros, E.; Loberg, E.; Engstrom, C.: Thyroid function and root resorption, Angle Orthod. 64:389-393, 1994. 38. Vazquez-Landaverde, L.A.; Rojas-Huidobro, R.; Alonso Gallegos-Corona, M.; Aceves, C.: Periodontal 5'-deiodination on forced-induced root resorption--the protective effect of thyroid hormone administration, Eur J Orthod. 24:363-369, 2002. 39. Carmeliet, G.; Van Cromphaut, S.; Daci, E.; Maes, C.; Bouillon, R.: Disorders of calcium homeostasis, Best Pract Res Clin Endocrinol Metab. 17:529-546, 2003. 40. Kamata, M.: Effect of parathyroid hormone on tooth movement in rats, Bull Tokyo Med Dent Univ. 19:411-425, 1972. 41. Miura, F.; Kamata, M.: Proceedings: Effect of parathyroid hormone on tooth movement in rats, Calcif Tissue Res. 15:168, 1974. 42. Soma, S.; Iwamoto, M.; Higuchi, Y.; Kurisu, K.: Effects of continuous infusion of PTH on experimental tooth movement in rats, J Bone Miner Res. 14:546-554, 1999. 43. Soma, S.; Matsumoto, S.; Higuchi, Y.; Takano-Yamamoto, T.; Yamashita, K.; Kurisu, K.; Iwamoto, M.: Local and chronic application of PTH accelerates tooth movement in rats, J Dent Res. 79:1717-1724, 2000. 44. Cranney, A.; Papaioannou, A.; Zytaruk, N.; Hanley, D.; Adachi, J.; Goltzman, D.; Murray, T.; Hodsman, A.: Clinical Guidelines Committee of Osteoporosis Canada. Parathyroid hormone for the treatment of osteoporosis: a systematic review, CMAJ. 175:52-59, 2006. 45. Collins, M.K.; Sinclair, P.M.: The local use of vitamin D to increase the rate of orthodontic tooth movement, Am J Orthod Dentofacial Orthop. 94:278-84, 1988. 46. Takano-Yamamoto, T.; Kawakami, M.; Kobayashi, Y.; Yamashiro, T.; Sakuda, M.: The effect of local application of 1,25-dihydroxycholecalciferol on osteoclast numbers in orthodontically treated rats, J Dent Res. 71:53-59, 1992. 47. Kale, S.; Kocadereli, I.; Atilla, P.; Asan, E.: Comparison of the effects of 1,25 dihydroxycholecalciferol and prostaglandin E2 on orthodontic tooth movement, Am J Orthod Dentofacial Orthop. 125:607-614, 2004. 48. Baran, S.; Hamamci, O.; Akalar, M.: An investigation of the effects of the local use of 1:25 dihydroxycholecalciferol (1:25 D) on tension sites during experimental tooth movement in rats, J Marmara Univ Dent Fac. 2:557-561, 1996.

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49. Kawakami, M.; Takano-Yamamoto, T.: Local injection of 1,25-dihydroxyvitamin D3 enhanced bone formation for tooth stabilization after experimental tooth movement in rats. J Bone Miner Metab. 22:541-546, 2004.

23

Capítulo 2 The effects of systemic stress response on orthodontic tooth movement GUSTAVO HAUBER GAMEIRO DARCY FLÁVIO NOUER JOÃO SARMENTO PEREIRA-NETO MARÍLIA BERTOLDO URTADO PEDRO DUARTE NOVAES MARGARET DE CASTRO MARIA CECÍLIA FERRAZ DE ARRUDA VEIGA

Abstract

Objective: The aim of this study was to investigate whether systemic stress response

affects biological reactions induced by orthodontic tooth movement. Design: Male Wistar

rats were restrained during 1 hour a day by stress models of short (3 days) and long

duration (40 days), while control group was not submitted to restraint (n=10/group). The

upper left first molars of all rats were moved mesially during the last 14 days of the

experiment. Then, animals were killed for blood collection and mensuration of plasmatic

corticosterone; tooth movement was quantified and tissues around the first molar were

processed for tartrate-resistant acid phosphatase histochemistry. Results: The plasmatic

levels of corticosterone were significantly higher in the both stressed groups than in the

control one. The long-term stress produced the highest rate of tooth movement in relation

to the control and short-term stress groups, which did not differ between each other. Also,

the number of osteoclasts on the long-term stress group was significantly greater than on

the other two groups. Conclusions: These results indicate that persistent systemic stress

response can increase the local osteoclastic bone resorption induced by orthodontic tooth

movement, and this could include the stress response as a systemic factor capable to

interfere in orthodontic treatment.

Key Words: stress; tooth movement; systemic factors

24

1. Introduction

The term stress defines the psychophysiological reactions of the body to a variety of

emotional or physical stimuli that threaten homeostasis, and the neuroendocrine

substances secreted by these reactions are clearly involved in every organ system of the

human organism, in almost every physiologic, cellular and molecular network.[1], [2], [3] and [4]

The central components of the stress system are located in the hypothalamus and the

brainstem and include the corticotropin-releasing hormone (CRH) and the locus ceruleus-

norepinephrine/autonomic sympathetic nervous systems.5 The peripheral limbs of the

stress system are the hypothalamic–pituitary–adrenal (HPA) axis, together with the

efferent sympathetic/adrenomedullary system, and components of the parasympathetic

system.6 Central CRH and norepinephrine systems, together with peripheral secretion of

large amounts of glucocorticoids and catecholamines, affect virtually every cell in the

body.7

In orthodontic therapy, the application of controlled mechanical forces to a tooth initiate

complex biochemical interactions related to bone remodeling activity,[8], [9] and [10] which can

be regulated either by local factors, such as the applied forces, or by systemic factors,

such as drugs, hormones and vitamins.[11], [12] and [13] A growing number of studies suggest

that sympathetic nerves modulate inflammation and bone remodeling in vivo.[14], [15], [16] and

[17] Osteoblasts and osteoclasts are well equipped with adrenergic and glucocorticoid

receptors,[18] and [19] indicating that these cells are influenced by sympathetic

neurotransmitters. Some studies have also been reported increased osteoclastic activity

and osteoclast number following sympathectomy,[20], [21] and [22] which reinforces the

possibility that orthodontic tooth movement can be affected by the systemic stress

response.

25

In most of the animal experiments that studied glucocorticoid administration and

orthodontic tooth movement, the glucocorticosteroid dose was high, which made the

animals osteoporotic.[23], [24] and [25] Kalia et al26 evaluated the rate of tooth movement in rats

during the course of short and long-term therapeutic corticosteroid therapy. These authors

demonstrated that in acute administration of drug therapy, bone remodeling seems to slow

down. On the other hand, the tooth movement rate increased in chronic treatment.

Clinically, these results suggest that it is possible to treat patients undergoing

corticosteroid therapy, with a minimum of adverse effects. Patients who are within the

short-term phase of using the drug should be scheduled with longer intervals, as bone

turnover will be delayed. In long-term drug therapy, when the tooth movement could be

accelerated, the orthodontic appliance could be controlled as usual or more frequently.26

Although glucocorticoids are a well recognized factor that influences bone metabolism and

tooth movement,[23], [24], [25] and [26] little is known about the role of endogenous glucocorticoids

in determining possible alterations in orthodontic tooth movement. The synthetic derivative

of cortisol differs from the natural hormone in strength and only mimics the dynamics of a

normal adrenocortical response during stress.27 Furthermore, the stress response involves

an orchestrated interplay of several neurotransmitter systems, and not only the

glucocorticoids.[28] and [29] Therefore, the present study was designed to investigate possible

effects of systemic stress response on the osteoclastic cells and on the rate of tooth

displacement caused by orthodontic movement.

26

2. Materials and methods

2.1. Animals

Male Wistar rats (weighing 260–290 g at the beginning of experiment) obtained from

Centro Multidisciplinar de Investigação Biológica-Cemib, Unicamp, Campinas, Brazil were

used in this study. The rats were housed in groups of five and maintained in a

temperature-controlled room (23 ± 1 °C) with a 12/12 light–dark cycle (lights on at 7:00

am) and food (ground pellets) and water were available ad libitum. The body weight of

each animal was recorded once a week throughout the experiment. The study was

conducted in accordance with the ethical guidelines for investigations of experimental pain

in conscious animals.30 This research was approved by the institutional ethics committee

in animal experimentation, according to the Brazilian College of Experimentation

Guidelines.

2.2. Stress exposure

In the short-stress model, animals were stressed by restraint 1 h daily, during 3 days (on

days 11, 12 and 13 after appliance placement). In the long-stress model, animals were

stressed by restraint 1 h daily, 5 days per week for 40 days. The stress procedure in the

long-stress model consisted in 5 days of stress + 2 days of rest until 60 days (the

orthodontic force was applied in the last 14 days). These stress protocols follows the

design used by Gameiro et al.31 Restraint was carried out by placing the animal in a

plastic restraint device (adjustable in size depending on the animal's weight) for 1 h. The

area of the tube could be adjusted individually to each rat with a mobile inside wall and

the tube was held firmly in place with Velcro straps. There was a 1 cm hole in the far end

for breathing. The control group was not submitted to restraint. The animals were

27

distributed into three groups as follows: ten animals had only an orthodontic appliance

inserted (control group), whereas twenty rats received both orthodontic force and stress

procedures in short (short-stress group) or long (long-stress group) stress models

(n=10/group). For body weight comparisons and hormonal dosages, one additional group

(n=8) without stress and without appliance was also evaluated (no-appliance group).

2.3. Hormonal assays

The rats were decapitated immediately after the last stress session and the whole blood

was collected. The time interval between the stress procedure and manipulations until

sacrifice was strictly maintained similar (30 s) among the different groups. Plasma

corticosterone levels were determined by radioimmunoassay after plasma extraction

using ethanol as previously described.32

2.4. Appliance placement and measurement of tooth movement

The appliance design of this study follows that used by Leiker et al.33 Animals were first

placed under general anesthesia with xylazine (10 mg/kg) and ketamine (50 mg/kg). A

closed coil nickel-titanium spring (Sentalloy®, GAC, Ctr. Islip, NY) calibrated to provide a

force of 50 g was ligated to the maxillary first molar and connected to an orthodontic band

cement onto the incisors (Fig. 1). Previous studies have demonstrated that a 40–60 g

level of force stimulated substantial molar tooth movement in rats.[34], [35], [36] and [37] A nickel–

titanium spring was used to provide a relatively constant force level over the course of the

experiment. To limit the influence of inter-animal variation in response to metabolic stimuli,

a split-mouth design was used and the untreated contralateral side served as the control.

After 14 days of tooth movement, the rats were decapitated and the maxillae were

excised. The distance between the mesial surface of the first and the distal surface of the

28

third molar was measured bilaterally with an electronic caliper for high accuracy

(Digimatic-Mitutoyo, Telford, UK) under a dental operating microscope (D.F. Vasconcellos

S.A., São Paulo, SP, Brazil) at 16× magnification, improving the reliability of the method.

Fig.1. Experimental appliance. An active coiled spring exerted a force of approximately 50 g

in the mesial direction.

Tooth movement was estimated by subtracting the mean of the repeated measured

values from the untreated and treated sides as described by Hong et al.37 The error of the

method based on double measurements performed on 20 randomly selected animals was

estimated by using Dahlberg’s equation (S = √∑(d)2/2n), where n = number of paired

measurements and d = deviations between the 2 measurements.39 The error in

measurement was 0.02 mm and was thus considered to be of no further importance.

2.5. Tartrate-resistant acid phosphatase histochemistry

The right and left hemi-maxilla of five rats from each group were fixed in 2.5

glutaraldehyde buffered at pH 7.4 with 0.1 M sodium cacodylate for 4 h and decalcified in

neutral 10% ethylenediaminetetraacetic acid (EDTA) at 40C for at least 10 days. The

29

EDTA solution was changed twice every day. Specimens were dehydrated in graded

ethanol and embedded in JB4 historesin. For each experimental and control side three

sagittal sections (5 µm thick) were taken at 50 µm intervals. Each section should be cut

through the pulp of the roots under study.

Tartrate-resistant acid phosphatase (TRAP) staining was performed to identify and

quantify osteoclasts. After washing in 0.1 M acetate buffer (pH 5.0), histological sections

were incubated with a mixture of naphtol AS-MX phosphate as substrate and red violet LB

salt diluted in 0.1 M acetate buffer (pH 5.0) containing 50 mML tartaric acid at 37°C for 90

minutes. The slides were washed in running water for 30 minutes and counterstained with

Harris's hematoxilin for 7 min. Cover slips were mounted with Entellan before examining

the slides with a Leica Microsystems light microscope (Wetzlar, Germany).

In each section the osteoclasts were counted at the alveolar bone surface (compression

side) adjacent to the entire mesial root. Cells were considered to be osteoclasts if they

were multinucleated, TRAP positive, and located on or close to bone surfaces. The

estimate of TRAP positive cells was determined by summing the value of the TRAP

positive cells in the three sections per case. The mesial root was chosen because it is the

largest of the first molar's five roots, is in approximately the same plane as the applied

force and is most commonly evaluated in tooth movement studies.[26], [33], [40] and [41]

Cell counting was performed manually in a blinded manner, and a reproducibility error of

less than 10% was established by recounting 20 randomly selected images. These counts

were then compared with the original counts by using Dahlberg’s equation. The error for

TRAP variable was 0.62 and was thus considered to be of no further importance.

2.6. Statistical analyses

Descriptive statistics (mean, standard error) for each parameter were calculated for all

groups. Comparisons of tooth movement and plasmatic corticosterone were made using

30

analysis of variance (ANOVA) and Tukey's test. Osteoclasts number comparisons were

made using split-plot ANOVA and Tukey's test after square-root transformation. Body

weight was analyzed by repeated measures ANOVA and Tukey's test. The SAS software

(version 9.1, 2003; SAS Institute Inc., Cary, NC, USA) was used and the significance level

set at p<0.05.

3. Results

3.1. Animal status and hormonal dosages

The orthodontic appliance did not affect the growth of the animals. Except for a temporary

episode of weight loss in all animals for 1 to 2 days following appliance insertion, there

was an overall gain in body weight over weeks [p<0.0001 (Fig. 2A)]. There was no effect

of stress procedure (treatment) on body weight (p=0.8127) but there was a significant

interaction between weeks and treatment (p<0.0001). The hormonal dosage was carried

out to define the efficacy of restraint in inducing stress-like hormonal modifications. There

was a significant increase in plasma corticosterone levels after the short and long-term

stress models [p<0.0001 (Fig. 2B)]. There were no statistical differences between no-

appliance and control (appliance) groups, neither between the short and long-term

stressed groups [p>0.05 (Fig. 2B)].

3.2. Tooth movement

All appliance-treated molars in the 3 groups showed evidence of tooth movement, with the

development of spacing between the first and second molars. The amount of tooth

movement was significantly larger in the long-term stress group than in the control and

short-term stress groups [p<0.05 (Fig. 3)]. The difference between the control and short-

term stress group was not significant [p>0.05 (Fig. 3)].

31

Fig.2. (A) Body weight over the course of 8-weeks (B) Plasma corticosterone level after the

experimental period. Each column represents the mean. Error bars indicate the SD. Single

asterisk indicates significant difference from control and no-appliance groups.

Fig.3. The effects of short and long-term stress on tooth movement in rats. Each column

represents the mean. Error bars indicate the SD. Single asterisk indicates significant

difference from control and short-term stress groups.

32

3.3. TRAP cells

TRAP-positive cells were detected in all groups studied. There were significantly more

TRAP positive cells counted in the mesial (compression) region of the experimental side

than in the control side of the 3 studied groups [p<0.005 (Fig. 4)]. Comparisons between

the experimental sides revealed that there were significantly more TRAP-positive cells on

the alveolar bone surface of the long-term stress group in relation to the control and short-

term stress groups, which did not differ between each other (Fig. 4 and Fig. 5).

Fig.4. The effects of short and long-term stress on osteoclasts induced by orthodontic tooth

movement in rats. Each column represents the mean. Error bars indicate the SD. The

number of osteoclasts for three sections, selected at ten-section intervals were summed for

each sample (n=5/group). White bars: untreated side; black bars: treated side. (*) Significant

difference between untreated vs. treated sides. (#) Significant difference between treated

sides of long stress vs. control and short stress groups. ($) Significant difference between

control sides of long stress vs. control and short stress groups.

$

33

Fig.5. TRAP stained histological section (5 µm) counterstained with hematoxylin taken from

the mesial root of the maxillary first molar. Arrows indicate TRAP-positive multinucleated

osteoclasts (20× magnification) on the experimental side of a rat submitted to long-term

stress. CO: compression side alveolar bone; PL:periodontal ligament.

4. Discussion

In this study, the alveolar bone changes induced by orthodontic tooth movement in rats

submitted to short and long-term stress were evaluated. The orthodontic appliance alone

did not influence the corticosterone levels after 14 days of tooth movement (Fig. 2B).

Although orthodontic movement is known to cause inflammatory reactions in the

periodontium and dental pulp,[9], [22], [42] and [43] which will stimulate release of various

biochemical mediators causing the sensation of pain,44 the orthodontic pain usually peaks

at 24 hours after archwire placement and then decline in up to 7 days.[45], [46] and [47] Thus,

the orthodontic pain, which could be a stimulus to stress system activation, was absent

after 14 days of tooth movement, indicating that the orthodontic appliance alone was not

34

an additional stress factor in this experiment. On the other hand, the efficacy of restraint

stress models in inducing stress-like hormonal modifications was confirmed by a significant

increase in plasma corticosterone in both short and long-term stressed groups (Fig. 2B).

The restraint stress was chosen because this model is generally regarded as inducing

psychological stress on the animal.[48] and [49] Orthodontic patients may be affected by

psychological stress-related disorders in which HPA axis hyperactivity occurs, for example,

in melancholic depression,50 anorexia nervosa,51 obsessive–compulsive disorder,52 panic

anxiety,53 and chronic active alcoholism.54 The high prevalence of this disorders and the

increasing evidence about the role of systemic factors on orthodontic treatment,[11], [13] , [55]

and [56] emphasize the need for a better understanding of the influence of systemic stress

response on tissue reactions to orthodontic tooth movement.

The present study showed that the amount of tooth movement increased in rats submitted

to long-term stress (Fig. 3). This indicates that prolonged stress can increase osteoclastic

activity induced by orthodontic tooth movement, as confirmed by the elevated number of

osteoclasts observed on the experimental side in the long-stress group. The number of

osteoclasts was higher than in the short-stress and control groups, which did not differ

between each other (Fig. 4). In the short-term stress group, the number of osteoclasts on

the experimental side was similar to the control group, and this could explain the absence

of statistical difference between the rate of tooth movement when control and short-term

stress groups were compared (Fig. 3 and Fig. 4). Thus, these results suggest that

prolonged activation of the stress system is necessary to consider the stress response as

a systemic factor capable to interfere in orthodontic tooth movement.

The periodontal scientific literature contains numerous studies examining the relationship

between psychological stressors and destructive periodontal changes mediated by the

stress system.[57], [58], [59] and [60] According to these studies, stress alone does not result in

35

alveolar bone loss, but may modulate the pathophysiological processes of already present

periodontal inflammation, resulting in accelerated degradation of periodontal tissues.[58], [59]

and [60] This modulation is thought to be mediated by products of the neuroendocrine

systems that are released under stress conditions and influence the function of

neutrophils, lymphocytes and macrophages, suppressing cellular immune response and

enhancing likelihood of infection and bone resorption.[61], [62] and [63] Research on the

relationship between systemic stress and alveolar bone loss so far has not led to

unequivocal conclusions and underlying molecular mechanisms are not fully understood.

For an example, several studies have demonstrated stress-associated increases of

interleukin-1β (Il-1 β) both in humans[64], [65] and [66] and in animals.[67], [68] and [69] On the other

hand, there are also some contradictory results. Dugué et al70 and Lacey et al71 failed to

establish such a relation in humans, and other authors even found a stress-associated

reduction in lipopolysaccharide-stimulated human leukocytes in vitro.[72] and [73]

In a recent study of experimental periodontitis in rats, Nakagima et al60 found that

combined restraint stress and oral challenge with Porphyromonas gingivalis resulted in

significantly higher bone loss and osteoclasts in stressed animals than in control ones.

These results could be explained by the suppressive actions of the systemic stress on the

immune and inflammatory responses. These authors also evaluated the gene expression

profiles of Il-1 β, tumor necrosis factor-α and interferon-γ, and found low gingival cytokine

gene expression in the restrained groups. The authors reported that these cytokines were

not up-regulated under stress conditions although they are prominent cytokines in

infection-stimulated bone resorption. The discrepancy between experimental models in

their susceptibility to modulation by stress is due to the fact that several factors can

interfere in the characteristics of the stress system response. Data from Quan et al69 and

Waschul et al74 indicate that the nature of the stressor and gender effects, respectively,

36

make much of a difference in the Il-1β response to stress. In the present study we

therefore aimed to control for these possible confounders by evaluating two different stress

models, which are well established in the literature as reliable short and long-term stress

models[31] and [75] and only male rats were used. According to our knowledge, this study is

the first to demonstrate significant interactions between stress and bone metabolism on

orthodontic tooth movement. Our results indicate that restraint stress can both increase

bone turnover around the untreated teeth (Fig. 4) and also modulate the inflammatory

reactions induced in the periodontium by orthodontic force, increasing the recruitment and

activity of osteoclasts (Fig. 4), and then resulting in an increased tooth movement (Fig. 3).

Considering that the tooth movement induced by controlled mechanical force application

stimulate release of various inflammatory cytokines and neuropeptides, such as

prostaglandins, leukotriens, substance P, histamine, enkephalin, dopamine, serotonin,

glycine, glutamate, gamma-amino butyric acid, calcitonin gene-related peptide (CGRP),

vasoactive intestinal polypeptide (VIP), and neuropeptides Y (NPY), [9], [13], [17], [22] and [44]

future studies should evaluate the role of these substances on the interaction between

systemic stress and orthodontic tooth movement.

A possible explanation for the increased osteoclastic activity and tooth movement rate in

chronic stressed animals could be related to the long duration of stress in this group, which

may have lead to an increased secretion of parathyroid hormone (PTH). PTH is produced

by the parathyroid glands and its function is to regulate serum calcium concentration by

concerted effects on kidney and bone. In the kidney, PTH increases renal calcium re-

absorption, and stimulates the urinary phosphate excretion. In bone, PTH can induce a

rapid release of calcium, but it also mediates longer-term changes by acting directly on

osteoblasts and indirectly on osteoclasts. In osteoblasts, PTH affects cellular metabolic

activity, gene transcriptional activity, and multiple protease secretion. Its effects on

37

osteoclasts occur by the production of receptor activator of nuclear factor kappaB-ligand

(RANK-L), a protein that plays a crucial role in osteoclast formation and activity.76 Thus,

the increase of bone turnover induced by PTH could accelerate orthodontic movement.[77]

and [78] Although excessive glucocorticoids are a wellrecognized cause of reduced intestinal

calcium absorption,[79], [80] and [81] which represents a physiological stimulus to PTH

secretion,76 little is known about the role of endogenous glucocorticoids in determining

serum calcium and PTH secretion. The fact that restraint stress can decrease serum

concentration of calcium in rats,82 let us to infer that this could be the case in our

experiment, leading to an enhanced PTH secretion, an increased osteoclastic activity, and

an accelerated orthodontic tooth movement in the chronic stressed rats. These results

need to be reevaluated and confirmed under other experimental sets, such as the

evaluation of calcium and PTH concentrations in rats submitted to these stress models.

Also, these results in rats should be confirmed and validated in humans.

In conclusion, chronic activation of the stress system could produce significant effects on

osteoclastogenesis in response to orthodontic force application, increasing the osteoclastic

activity and the orthodontic tooth movement. Accordingly, these findings include the

systemic stress response as a systemic factor capable to interfere in orthodontic

treatment.

Acknowledgements

The authors thank Gláucia M. B. Ambrosano for statistical analyses. Thanks are due to

Maria Aparecida Santiago Varella and Eliene Aparecida Orsini Narvaes Romani for

technical assistance. This work was supported by CAPES and FAPESP, Brazil.

38

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Capítulo 3 Evaluation of root resorption associated with orthodontic movement in stressed rats GUSTAVO HAUBER GAMEIRO DARCY FLÁVIO NOUER MARIA BEATRIZ BORGES DE ARAÚJO MAGNANI PEDRO DUARTE NOVAES GLÁUCIA MARIA BOVI AMBROSANO ANNICELE DA SILVA ANDRADE MARIA CECÍLIA FERRAZ DE ARRUDA VEIGA

Objective: The aim of this study was to investigate the effects of acute and chronic

systemic stress response on orthodontically induced root resorption. Materials and

Methods: Male Wistar rats were restrained during 1 hour a day by stress models of short

(3 days) and long duration (40 days), while control group was not submitted to restraint

(n=10/group). The upper left first molars of all rats were moved mesially by a fixed

orthodontic appliance exerting 50 g force upon insertion during the last 14 days of the

experiment. Then, animals were killed for blood collection and mensuration of plasmatic

corticosterone by radioimmunoassay; the tissues around mesial root of the first molar were

processed for histological and histochemical techniques with tartrate-resistant acid

phosphatase (TRAP). The degree of root resorption and the number of odontoclasts were

evaluated, being the contralateral side of each animal serving as its control (split-mouth

design). Results: The results revealed that the plasmatic levels of corticosterone were

significantly higher in the both stressed groups than in the control one. There were no

significant differences in the degree of root resorption and in the number of odontoclasts

on the root between the 3 groups studied. Conclusion: These results indicate that

systemic stress alone can not be considered a risk factor for root resorption induced by

orthodontic tooth movement.

48

KEY WORDS: root resorption, stress, systemic factors

Introduction

Orthodontically induced inflammatory root resorption or, as it is better known, root

resorption, is one of the common, undesirable side effects of orthodontic treatment.1

Various factors relevant to root resorption are basically divided into mechanical and

biological factors, or a combination of both.2 For the mechanical factors, its generally

accepted that prolonged orthodontic treatment,3 high forces,4 extensive tooth movement,5

root torque,6 jiggling and intrusive forces2,7are associated with the onset of a clinically

evident root resorption. The main biological factors considered as risk factors for root

resorption include the individual susceptibility on a genetic basis,8 some systemic

diseases9 and anomalies in root morphology.5

The investigations about the link between systemic factors and root resorption have been

related to hormone alterations. Hypothyroidism,10 hypophosphatemia11 and

hyperparathyroidism12 have been linked to root resorption in a few case reports. However,

these hypotheses were not confirmed by controlled experimental studies and controversial

data are available concerning the effects of hormone alterations on orthodontic root

resorption. Engström et al.13 found an increased amount of root resorption in hypocalcemic

rats, and these authors suggested that this could be attributed to an increase in alveolar

bone resorption mediated by low calcium levels together with parathyroid hormone action.

On the other hand, Goldie and King14 found that rats stressed with severe calcium

deficiency and lactation displayed significantly less root resorption than unstressed

animals. Other studies also did not find increased amounts of root resorption in animals

with increased bone turnover.15,16 The discrepancy between experimental models in their

49

susceptibility to modulation by systemic factors is also evident in experiments that studied

glucocorticoid administration and orthodontic root resorption.17-19 Young rabbits treated

with osteoporotic doses of cortisone acetate injections (15 mg/kg) showed significantly

greater tendency to root resorption.17 Similar results were obtained by Verna et al.,18 using

short-term administration of therapeutic dosages of methylprednisolone, whereas low

doses of 1 mg/kg oral prednisolone in rats produced, in the contrary, significantly less root

resorption, suggesting a suppressing role of the drug on clastic activities.19

Glucocorticoids are a well recognized factor that influences bone metabolism and

orthodontic root resorption,17,20,21 but to our knowledge, the role of endogenous

glucocorticoids in determining possible alterations in orthodontic root resorption has never

been investigated. The synthetic derivative of cortisol differs from the natural hormone in

strength and only mimics the dynamics of a normal adrenocortical response during

stress.22 Furthermore, the stress response involves an orchestrated interplay of several

neurotransmitter systems, and not only the glucocorticoids.23 Therefore, the present study

was designed to investigate possible effects of systemic stress response on the number of

odontoclasts and on the degree of root resorption induced by orthodontic tooth movement.

Materials and Methods

Male Wistar rats (weighing 260–290 g at the beginning of experiment) were used in this

study. The rats were housed in groups of five and maintained in a temperature-controlled

room (23 ± 1 °C) with a 12/12 light–dark cycle and food (ground pellets) and water were

available ad libitum. The body weight of each animal was recorded once a week

throughout the experiment. The study was conducted in accordance with the ethical

guidelines for investigations of experimental pain in conscious animals.24 This research

was approved by the institutional ethics committee in animal experimentation.

50

In the short-stress model, animals were stressed by restraint 1 h daily, during 3 days (on

days 11, 12 and 13 after appliance placement). In the long-stress model, animals were

stressed by restraint 1 h daily, 5 days per week for 40 days. The stress procedure in the

long-stress model consisted in 5 days of stress + 2 days of rest until 60 days (the

orthodontic force was applied in the last 14 days). These stress protocols follows the

design used by Gameiro et al.25 Restraint was carried out by placing the animal in a

plastic restraint device (adjustable in size depending on the animal's weight) for 1 h. The

control group was not submitted to restraint. The animals were distributed into three

groups as follows: ten animals had only an orthodontic appliance inserted (control group),

whereas twenty rats received both orthodontic force and stress procedures in short (short-

stress group) or long (long-stress group) stress models (n=10/group). For body weight

comparisons and hormonal dosages, one additional group (n=8) without stress and

without appliance was also evaluated (no-appliance group).

The rats were decapitated immediately after the last stress session and the whole blood

was collected. The time interval between the stress procedure and manipulations until

sacrifice was strictly maintained similar (30 s) among the different groups. Plasma

corticosterone levels were determined by radioimmunoassay after plasma extraction

using ethanol as previously described.26

The appliance design of this study follows that used by Leiker et al.27 Animals were first

placed under general anesthesia with xylazine (10 mg/kg) and ketamine (50 mg/kg). A

closed coil nickel-titanium spring (Sentalloy®, GAC, Ctr. Islip, NY) calibrated to provide a

force of 50 g was ligated to the maxillary first molar and connected to an orthodontic band

cement onto the incisors. Previous studies have demonstrated that a 40–60 g level of

force stimulated substantial molar tooth movement in rats.28,29 A nickel–titanium spring

51

was used to provide a relatively constant force level over the course of the tooth

movement period (14 days).

The right and left hemi-maxilla of five rats from each group were processed for tartrate-

resistant acid phosphatase (TRAP) staining as described elsewhere.19 For each

experimental and control side three sagittal sections (5 µm thick) were taken at 50 µm

intervals. The slides were counterstained with Harris's hematoxilin for 7 min. Cover slips

were mounted with Entellan before examining the slides with a Leica Microsystems light

microscope (Wetzlar, Germany).

Counting of odontoclasts was performed in a selected section localized disto-apically to

the mesial root (Figure 1). The overall size of each measurement area was 1000 X 800

µm. The magnification used to view the selected area was achieved with an objective of

20X magnification. Cells were considered to be odontoclasts if they were multinucleated,

TRAP positive, and located on or close to root surface. The estimate of TRAP positive

cells was determined by summing the value of the TRAP positive cells in the three

sections per case. The mesial root was chosen because it is the largest of the first molar's

five roots, is in approximately the same plane as the applied force and is most commonly

evaluated in tooth movement studies.21,27

52

Figure 1. The area under investigation (black rectangle) where tartrate-resistant acid

phosphatase (TRAP)-positive cells were counted and root resorption score were analyzed.

The arrow indicates the direction of orthodontic force applied for 14 days.

Cell counting was performed manually in a blinded manner, and a reproducibility error of

less than 10% was established by recounting 20 randomly selected images.

The degree of root resorption was determined according to the method described by Lu et

al.30 with little modifications. Under ×10 magnification, a selected area localized disto-

apically to the mesial root (Figure 1) was examined using an image analysis system (Leica

Qwin). A grid-sheet (10X10) was superimposed in the selected area (1000 × 800 µm), and

the number of grids with or without resorption lacunae were counted. Root resorption

scores (percentage of resorption grids) were determined by dividing the number of grids

with resorption lacunae by the total number of grids along the root surface.

Comparisons of plasmatic corticosterone were made using analysis of variance (ANOVA)

and Tukey's HSD post-hoc comparison. Body weight, odontoclasts number and root

resorption score were analyzed by repeated measures ANOVA and Tukey's test. The

significance level was set at p<0.05.

53

Results

Except for a temporary episode of weight loss in all animals for 1 to 2 days following

appliance insertion, there was an overall gain in body weight over weeks [p<0.0001

(Table 1)]. There was no effect of stress procedure (treatment) on body weight (p=0.8127)

but there was a significant interaction between weeks and treatment (p<0.0001). There

was a significant increase in plasma corticosterone levels after the short and long-term

stress models [p<0.0001 (Table 2)]. There were no statistical differences between no-

appliance and control (appliance) groups, neither between the short and long-term

stressed groups [p>0.05 (Table 2)].

Table 1. Values (mean and standard deviation) for body weight (g) in the four groups at the beginning and at the end of the experiment

Group

n first week last week

no-appliance 5 290 (13.5) Aa 381 (8.17) Ba control 5 286 (8.08) Aa 368 (12.4) Ba

short stress 5 291 (14.1) Aa 369 (21.9) Ba long stress 5 298 (20.4) Aa 373 (24.3) Ba

Distinct letters (capital letters for rows and small letters for columns) indicate statistical difference (repeated measures ANOVA + Tukey, p<0.05)

Table 2. Values (mean and standard deviation) for plasmatic corticosterone (µg/dl) in the four groups at the end of the experiment

Group

n Plasmatic

corticosterone (µg/dl) no-appliance 8 2.5 (1.9) A

control 10 2.7 (2.8) A short stress 10 8.6 (4.6) B long stress 10 8.5 (2.5) B

Distinct letters indicate statistical difference (ANOVA + Tukey, p<0.05)

54

There were significantly more TRAP positive cells counted in the experimental side than in

the control side of the 3 studied groups [p<0.001 (Table 3)]. The effect of treatment (stress

procedure) was not statistically significant (p=0.2872). Also, the interaction between

treatment and side was not statistically significant (p=0.4845).

All groups exhibited significantly greater root resorption scores in the experimental side

than in the control side [p<0.0001 (Table 4)]. There was no effect of stress procedure on

root resorption score (p=0.8632) and no significant interaction between sides and stress

procedure (p=0.7979).

Table 3 .Values (mean and standard deviation) for the number of tartrate-resistant acid phosphatase (TRAP)-positive cells on root in the three groups at the control

and experimental sides

Group

n control side experimental side

control 5 1.8 (3.4) Aa 21.8 (8.3) Ba short stress 5 2.6 (4.2) Aa 23.0 (5.2) Ba long stress 5 1.0 (1.7) Aa 16.0 (8.2) Ba

Distinct letters (capital letters for rows and small letters for columns) indicate statistical difference (ANOVA + Tukey, p<0.05)

Table 4. Values (mean and standard deviation) for the root resorption score in the three groups at the control and experimental sides

Group

n control side experimental side

control 5 14.0 (19.4) Aa 49.5(11.4) Ba short stress 5 13.1 (11.1) Aa 48.5(13.1) Ba long stress 5 13.1 (7.5) Aa 42.5(19.2) Ba

Distinct letters (capital letters for rows and small letters for columns) indicate statistical difference (ANOVA + Tukey, p<0.05)

55

Discussion

In this study, the root resorption induced by orthodontic tooth movement in rats submitted

to short and long-term systemic stress were evaluated. The efficacy of restraint stress

models in inducing stress-like hormonal modifications was confirmed by a significant

increase in plasma corticosterone in both short and long-term stressed groups (Table 2).

The restraint stress was chosen because this model is generally regarded as inducing

psychological stress on the animal.31 The orthodontic appliance alone did not influence the

coricosterone levels after 14 days of tooth movement (Table 2). This was probably due to

an adaptation of the animals to the orthodontic appliance.

Psychological stress-related disorders associated with hyperactivity of the stress system

occurs, for example, in melancholic depression,32 anorexia nervosa,33 obsessive–

compulsive disorder,34 panic anxiety,35 and chronic active alcoholism.36 The high

prevalence of this disorders and the increasing evidence about the role of systemic factors

on orthodontic treatment,37 emphasize the need for a better understanding of the influence

of systemic stress response on tissue reactions to orthodontic tooth movement. We have

previously observed that chronic stressed animals showed an increased tooth movement

rate and an increased osteoclastic activity, when compared with unstressed animals

(unpublished data). In the present study, the effects of short and long-term systemic stress

on orthodontic root resorption were evaluated. As expected, root resorption was larger in

the experimental than in the control side of the 3 studied groups (Tables 3 and 4). It’s well

known that the orthodontic pressure itself can damage the outer root surface layers,

including cementoblasts and precementum layers.1 On the other hand, the relationship

between systemic factors and root resorption so far has not led to unequivocal

conclusions. The present experiment showed that systemic stress response did not affect

56

the orthodontic root resorption, as confirmed by the absence of statistical difference

between the number of odontoclasts on the experimental side of the 3 studied groups

(Table 3). Moreover, the short and long-term stress procedures were not able to alter the

root resorption scores, as observed when stressed and control groups were compared

(Table 4).

Some studies showed that exogenous administration of glucocorticoids can affect root

resorption in response to orthodontic forces. However, controversy exists as to the effects

of these drugs on this process.17-19 As noted previously, the acute administration of high

(15 mg/Kg)17 and therapeutic dosages (8 mg/Kg)18 of glucocorticoids produced an

increased orthodontic root resorption in rabbits and rats, respectively. In contrast, Ong et

al.19 reported that orthodontic root resorption in rats was inhibited by a lower dosage of

corticosteroid (1 mg/Kg). These differences may be explained by variations within animal

species studied, forces used to move teeth, duration of the experiment, dosage and time

interval of administration, and potency of the drug used. Moreover, direct comparison with

our data is impossible, because exogenous corticosteroids differ from the endogenous

hormone alterations that occur during the systemic stress response.22,23 In the present

study we therefore aimed to control for these possible confounders by evaluating two

different stress models, which are well established in the literature as reliable short and

long-term stress models.25,38 The short-term stress was chosen to mimic episodic stressful

situations. The long-term stress was chosen to mimic prolonged stressful situations i.e.

chronic psychological diseases. According to our knowledge, this study is the first to

evaluate the role of systemic stress on orthodontic root resorption. Our results indicate that

hormone alterations induced by short and long-term systemic stress were not significant to

affect orthodontic root resorption. Although systemic stress has been associated with

increased bone resorption,39,40 the present data indicate that osteoclasts and odontoclasts

57

differ in their susceptibility to modulation by endogenous hormone alterations. This

observation is consistent with previously reported findings.14,15 These results need to be

reevaluated and confirmed under other experimental sets, such as the evaluation of

calcium and parathyroid hormone concentrations in rats submitted to these stress models.

Although systemic stress activation may not predispose for root resorption, other systemic

diseases and hormonal or dietary imbalances should not be ignored.

Conclusions

In conclusion, the acute and chronic activation of the stress system did not affect the root

resorption in response to orthodontic force application. Accordingly, these findings indicate

that the systemic stress response alone should not be considered a risk factor for

orthodontic root resorption.

Acknowledgments

The authors thank Maria Aparecida Santiago Varella and Eliene Aparecida Orsini Narvaes

Romani for technical assistance. This work was supported by CAPES and FAPESP,

Brazil.

References

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3. Apajalahti S, Peltola JS. Apical root resorption after orthodontic treatment a

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4. Harris DA, Jones AS, Darendeliler MA. Physical properties of root cementum: part 8.

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5. Sameshima GT, Sinclair PM. Predicting and preventing root resorption: Part I.

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8. Al-Qawasmi RA, Hartsfield JK Jr, Everett ET, Flury L, Liu L, Foroud TM, Macri JV,

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10. Becks H. Orthodontic prognosis:evaluation of routine dentomedical examination to

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11. Tangney NJ. Hypophosphatasia: a case report and literature review. Ir Med J. 1979;

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12. Goultschin J, Nitzan D, Azaz B. Root resorption. Review and discussion. Oral Surg

Oral Med Oral Pathol. 1982; 54:586-90.

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13. Engstrom C, Granstrom G, Thilander B. Effect of orthodontic force on periodontal

tissue metabolism. A histologic and biochemical study in normal and hypocalcemic young

rats. Am J Orthod Dentofacial Orthop. 1988; 93:486-95.

14. Goldie RS, King GJ. Root resorption and tooth movement in orthodontically treated,

calcium-deficient, and lactating rats. Am J Orthod. 1984; 85:424-30.

15. Midgett RJ, Shaye R, Fruge JF Jr. The effect of altered bone metabolism on

orthodontic tooth movement. Am J Orthod. 1981; 80:256-62.

16. Verna C, Dalstra M, Melsen B. Bone turnover rate in rats does not influence root

resorption induced by orthodontic treatment. Eur J Orthod. 2003; 25:359-63.

17. Ashcraft MB, Southard KA, Tolley EA. The effect of corticosteroid-induced

osteoporosis on orthodontic tooth movement. Am J Orthod Dentofacial Orthop. 1992 ;

102:310-9.

18. Verna C, Hartig LE, Kalia S, Melsen B. Influence of steroid drugs on orthodontically

induced root resorption. Orthod Craniofac Res. 2006; 9:57-62.

19. Ong CK, Walsh LJ, Harbrow D, Taverne AA, Symons AL. Orthodontic tooth movement

in the prednisolone-treated rat. Angle Orthod. 2000; 70:118-25.

20. Davidovitch Z, Musich D, Doyle M. Hormonal effects on orthodontic tooth movement in

cats-a pilot study. Am J Orthod. 1972; 62:95-96.

21. Kalia S, Melsen B, Verna C. Tissue reaction to orthodontic tooth movement in acute

and chronic corticosteroid treatment. Orthod Craniofac Res. 2004; 7:26-34.

22. Jefferies WM. Cortisol and immunity. Med Hypotheses. 1991; 34:198-208.

23. Gameiro GH, da Silva Andrade A, Nouer DF, Ferraz de Arruda Veiga MC. How may

stressful experiences contribute to the development of temporomandibular disorders? Clin

Oral Investig. 2006; 10:261-8.

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24. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious

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26. Castro M, Figueiredo F, Moreira AC. Time-course of hypothalamic CRH and pituitary

ACTH contents, and pituitary responsiveness to CRH stimulation after bilateral

adrenalectomy. Horm Metab Res. 1995; 27:10-5.

27. Leiker BJ, Nanda RS, Currier GF, Howes RI, Sinha PK. The effects of exogenous

prostaglandins on orthodontic tooth movement in rats. Am J Orthod Dentofacial Orthop.

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28. King GJ, Keeling SD, McCoy EA, Ward TH. Measuring dental drift and orthodontic

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Orthop. 1991; 99:456–465.

29. Rody WJJ, King GJ, Gu G. Osteoclast recruitment to sites of compression in

orthodontic tooth movement. Am J Orthod Dentofacial Orthop. 2001; 120:477–489.

30. Lu LH, Lee K, Imoto S, Kyomen S, Tanne K. Histological and histochemical

quantification of root resorption incident to the application of intrusive force to rat molars.

Eur J Orthod. 1999; 21:57-63.

31. Dayas CV, Buller KM, Crane JW, Xu Y, Day TA. Stressor categorization: acute

physical and psychological stressors elicit distinctive recruitment patterns in the amygdala

and in medullary noradrenergic cell groups. Eur J Neurosci. 2001; 7:1143–1152.

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32. Bernini GP, Argenio GF, Vivaldi MS, Del Corso C, Sgro M, Franchi F, Luisi M. Effects

of fenfluramine and ritanserin on prolactin response to insulin-induced hypoglycemia in

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33. Kaye WH, Gwirtsman HE, George DT, Ebert MH, Jimerson DC, Tomai TP, Chrousos

GP, Gold PW. Elevated cerebrospinal fluid levels of immunoreactive corticotropin-

releasing hormone in anorexia nervosa: relation to state of nutrition, adrenal function, and

intensity of depression. J Clin Endocrinol Metab. 1987; 64:203–208.

34. Insel TR, Kalin NH, Guttmacher LB, Cohen RM, Murphy DL.The dexamethasone

suppression test in patients with primary obsessive-compulsive disorder. Psychiatry Res.

1982; 6:153–160.

35. Gold PW, Pigott TA, Kling MK, Kalogeras K, Chrousos GP. Basic and clinical studies

with corticotropin releasing hormone: implications for a possible role in panic disorder.

Psychiatr Clin North Am. 1988; 11:327-334.

36. Wand GS, Dobs AS. Alterations in the hypothalamic–pituitary–adrenal axis in actively

drinking alcoholics. J Clin Endocrinol Metab. 1991; 72:1290–1295.

37. Gameiro GH, Pereira-Neto JS, Magnani MB, Nouer DF. The influence of drugs and

systemic factors on orthodontic tooth movement. J Clin Orthod. 2007; 41:73-8.

38. Gameiro GH, Andrade Ada S, de Castro M, Pereira LF, Tambeli CH, Veiga MC. The

effects of restraint stress on nociceptive responses induced by formalin injected in rat's

TMJ. Pharmacol Biochem Behav. 2005; 82:338-44.

39. Takada T, Yoshinari N, Sugiishi S, Kawase H, Yamane T, Noguchi T. Effect of restraint

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63

Capítulo 4 The effects of short and long-term celecoxib administration on orthodontic tooth movement GUSTAVO HAUBER GAMEIRO DARCY FLÁVIO NOUER JOÃO SARMENTO PEREIRA-NETO VÂNIA CÉLIA VIEIRA DE SIQUEIRA EDUARDO DIAS DE ANDRADE PEDRO DUARTE NOVAES MARIA CECÍLIA FERRAZ DE ARRUDA VEIGA

ABSTRACT

Objective: To test the hypothesis that short and long-term celecoxib administration has no

effect on orthodontic tooth movement.

Materials and Methods: Male Wistar rats were submitted to short (3 days) and long-term

(14 days) celecoxib administration, while the respective control groups received

equivolumetric saline i.p. injections. The upper left first molars of all rats were moved

mesially for 14 days by a fixed orthodontic appliance exerting 50 g force upon insertion.

After the experimental period, tooth movement was quantified and tissues around the first

molar were processed for tartrate-resistant acid phosphatase (TRAP) histochemistry. The

amount of tooth movement and the number of TRAP-positive cells on the alveolar bone

surface were evaluated.

Results: The amount of tooth movement was significantly reduced in rats submitted to

short and long-term celecoxib administration, while the number of osteoclasts on the

alveolar bone did not differ between the 4 groups studied.

Conclusions: The hypothesis is rejected. Although celecoxib administration did not affect

the number of osteoclasts, the osteoclast activity might be reduced, which could explain

the inhibition of tooth movement observed in the celecoxib-treated animals. These results

64

indicate that orthodontists should be aware of patients under short and long-term therapy

with celecoxib.

KEY WORDS: Celecoxib; tooth movement; Orthodontics

INTRODUCTION

Patients undergoing orthodontic treatment usually experience some degree of pain or

discomfort.1 Surveys performed to determine the experience of orthodontic pain have rated

it as a key deterrent to orthodontic therapy and a major reason for discontinuing

treatment.2-5

The most common group of medications used in orthodontics for pain relief consists of

non-steroidal anti-inflammatory drugs (NSAIDs). 6-8 These drugs function by inhibition of

the enzyme cyclooxygenase (COX), which modulates the transformation of prostaglandins

(PGs) from arachidonic acid in the cellular plasma membrane.9 PGs, such as PGE1 and

PGE2, are important mediators of bone resorption.10,11 Two isoforms of COX have been

described: the constitutive COX-1 and the inducible COX-2. The COX-1 is considered

important in tissue homeostasis and the COX-2 is transcriptionally induced by cytokines

and appears to be important in the development of inflammation.12 Numerous studies

evaluated the pain-reducing effects of various NSAIDs, including ibuprofen,13

acetylsalicylic acid,14 and naproxen sodium15. These studies demonstrated that NSAIDs

effectively reduce pain and discomfort caused by the periodic activation of orthodontic

appliances, but these drugs may also affect the sequence of tooth movement by inhibiting

or at least by reducing the associated inflammatory and bone resorptive processes.

65

One approach to deal with this problem is the use of selective COX-2 inhibitors, also

named coxibs, which are replacing conventional NSAIDs, especially for chronic

inflammatory conditions.16,17 It has previously been shown that some coxibs (Celecoxib11,18

and Parecoxib11) do not interfere in the rate of orthodontic tooth movement. However, the

specificity of coxib can account for different effects of these drugs on tooth movement.11

One example is the Rofecoxib, a drug that can disturb the process of tooth movement.11,19

In addition, this drug has been the object of debate and even withdrawn from the market

due to reports of unwanted cardiovascular and renal side effects.20 Considering that COX-2

is upregulated when orthodontic forces are applied,11 it is possible that coxibs would

interfere in tooth movement.

It is important to point out that the preemptive or preoperative administration of analgesics,

in order to decrease postoperative pain, has become the focus of recent research in

orthodontics.15,21-23 Some authors also recommended two postoperative doses, in addition

to a preoperative dose, for complete pain control during each orthodontic appointment.15 It

was suggested that low doses administered for one or two days in the initial stages will not

affect the tooth movement process as such.8 However, this hypothesis was not

substantiated by controlled experimental studies. On this background, the present study

was designed to investigate possible effects of short and long-term celecoxib

administration on tooth movement induced by experimental orthodontic force application.

66

MATERIALS AND METHODS

Male Wistar rats (300-400 g) were used in this study. The rats were housed in groups of

five and maintained in a temperature-controlled room (23 ± 1 °C) with a 12/12 light–dark

cycle and food (ground pellets) and water were available ad libitum. The body weight of

each animal was recorded once a week throughout the experiment. The study was

conducted in accordance with the ethical guidelines for investigations of experimental pain

in conscious animals.24 This research was approved by the institutional animal

experimentation ethics committee.

The rats were randomly divided into four groups: Group I (n=9) - treated with saline i.p.

injections on days 1, 2 and 3; Group II (n=9) - treated with celecoxib (10 mg/Kg) i.p.

injections on days 1, 2 and 3. Group III (n=7) - treated with saline i.p. injections on days 1

to 14; Group IV (n=7) - treated with celecoxib (10 mg/Kg) i.p. injections on days 1 to 14.

Celecoxib (Pfizer, São Paulo, Brazil) was freshly dissolved in saline and given i.p. twice a

day in a dose of 10 mg/Kg and in a volume of 1ml/Kg. The first injection was made 2

hours before appliance placement, in order to test the preoperative use of the drug. The

control groups received equivolumetric saline injections during the same period according

to their experimental groups (Celecoxib for 3 or 14 days).

The appliance design of this study follows that used by Leiker et al25. Animals were first

placed under general anesthesia with xylazine (10 mg/kg) and ketamine (50 mg/kg). A

closed coil nickel-titanium spring (Sentalloy®, GAC, Ctr. Islip, NY) calibrated to provide a

force of 50 g was ligated to the maxillary first molar and connected to an orthodontic band

cemented onto the incisors (Figure 1a). Previous studies have demonstrated that a 40–

60 g level of force stimulated substantial molar tooth movement in rats.26-28A nickel–

67

titanium spring was used to provide a relatively constant force level over the course of the

experiment. After 14 days of tooth movement, the rats of all groups were decapitated and

the maxillae were excised.

The distance between the mesial surface of the first and the distal surface of the third

molar was measured bilaterally with an electronic caliper for high accuracy (Digimatic-

Mitutoyo, Telford, UK) under a dental operating microscope (D.F. Vasconcellos S.A., São

Paulo, SP, Brazil) at 16X magnification, improving the reliability of the method. Tooth

movement was estimated by subtracting the mean of the repeated measured values from

the untreated and treated sides (Figure 1b) as described by Hong et al.29 The error of the

method based on double measurements performed on 20 randomly selected animals was

estimated by using Dahlberg’s equation (S = √∑(d)2/2n), where n = number of paired

measurements and d = deviations between the 2 measurements.30 The error in

measurement was 0.02 mm and was thus considered to be of no further importance.

Figure 1. (a) Appliance used to move the molars mesially (arrows). (b) Indication of

measurement procedure. The distance between the mesial side of the first molar (1M) and

the distal side of the third molar (3M) was measured, and that of the treated side (X)

subtracted from the one of the untreated side (Y).

68

The left hemi-maxilla of five rats from each group were processed for tartrate-resistant acid

phosphatase (TRAP) staining as described elsewhere.31 For each sample three sagittal

sections (5 µm thick) were taken at 50 µm intervals. The slides were counterstained with

Harris's hematoxylin for 7 min. Cover slips were mounted with Entellan before examining

the slides with a Leica Microsystems light microscope (Wetzlar, Germany).

In each section the osteoclasts were counted at the alveolar bone surface (compression

side) adjacent to the entire mesial root. Cells were considered to be osteoclasts if they

were multinucleated, TRAP positive, and located on or close to bone surfaces. The

estimate of TRAP positive cells was determined by summing the value of the TRAP

positive cells in the three sections per case. The mesial root was chosen because it is the

largest of the five first molar roots, is in approximately the same plane as the applied force

and is most commonly evaluated in tooth movement studies.25,32

Cell counting was performed manually in a blinded manner, and a reproducibility error of

less than 10% was established by recounting 20 randomly selected images. These counts

were then compared with the original counts by using Dahlberg’s equation. The error for

TRAP variable was 0.64 and was thus considered to be acceptable.

Body weight was analyzed by repeated measures ANOVA and Tukey's test. The amount

of tooth movement and the number of osteoclasts were analyzed by Two-way ANOVA and

Tukey's test. The SAS software (version 9.1, 2003; SAS Institute Inc., Cary, NC, USA) was

used and the significance level set at P<.05.

69

RESULTS

There was an overall gain in body weight over weeks [p<.0001 (Table 1)]. There was no

statistical difference between groups (p=0.7632), and the interaction between groups and

weeks was also not statistically significant (p=0.1520).

The amount of tooth movement was significantly lower in the celecoxib-treated animals

than in the control ones [p=0.0009 (Table 2)]. The difference between times of treatment

was also significant (p=0.0430). The interaction between drugs and time was not

statistically significant (p=0.6025).

The number of TRAP positive cells on the alveolar bone surface did not differ between

drugs [p=0.1230 (Table 3)] neither between times of treatment (p=0.4014). Furthermore,

the interaction between drugs and times was not statistically significant (p=0.3812).

Table 1. Values (mean and standard deviation) for body weight (g) in the four groups at the beginning, after 1 week and at the end of the experiment

Group n before after 1 week after 2 weeks

I-saline for 3 days 9 357.7 (30.6) Ca 363.3 (31.7) Ba 378.4 (33.7) Aa

II – celecoxib for 3 days

9 360.5 (27.4) Ca 371.8 (27.2) Ba 376.2 (25.5) Aa

III-saline for 14 days 7 359.5 (32.8) Ca 359.7 (32.6) Ba 366.2 (33.8) Aa

IV- celecoxib for 14 days

7 369.1 (20.8) Ca 380.5 (16.8) Ba 383.0 (30.0) Aa

Distinct letters (capital letters for rows and small letters for columns) indicate statistical difference (repeated measures ANOVA + Tukey, p<0.05)

70

Table 2. Values (mean and standard deviation) for the amount of tooth movement (mm) in animals treated with saline or celecoxib

Drugs

TIME OF TREATMENT

Saline

Celecoxib

3 days 0.33 (0.12) Aa 0.23 (0.07) Ab

14 days 0.28 (0.05) Ba 0.15 (0.06) Bb

Distinct letters (capital letters for rows and small letters for columns) indicate statistical difference (Two-way ANOVA + Tukey, p<0.05)

Table 3.Values (mean and standard deviation) for the number of tartrate-resistant acid phosphatase (TRAP)-positive cells on alveolar bone surface in animals treated with saline or celecoxib

Drugs

TIME OF TREATMENT

3 days 14 days Saline 28.8 (15.3) Aa 29.0 (13.0) Aa

Celecoxib 25.0 (10.8) Aa 15.8 (4.7) Aa Distinct letters (capital letters for rows and small letters for columns) indicate statistical difference

(Two-way ANOVA, p<0.05)

DISCUSSION

There have been reports on the effectiveness of NSAIDs in relieving the pain induced by

orthodontic force activation.13-15 However, some of these drugs can interfere with tooth

movement.14 As a result, the use of selective COX-2 inhibitors is increasing, replacing

conventional NSAIDs in clinical practice,6-8,23 although the specificity of coxibs can account

for different effects of these drugs on tooth movement.11

In the present study, the amount of tooth movement and the associated bone resorption

process were evaluated in rats submitted to short and long-term celecoxib administration.

Celecoxib was chosen in this study because this drug is the most common alternative to

71

rofecoxib.33 The short-term treatment was chosen in this study to mimic the preemptive or

preoperative administration of analgesics to decrease postoperative pain, which has

become the focus of recent research in orthodontics15,21-23. Some authors also

recommended two postoperative doses, in addition to a preoperative dose, for a complete

pain control during each orthodontic appointment.15 Since the orthodontic pain will usually

last for 2 – 3 days34, we used one preemptive dose followed by postoperative doses for 2

days. The long-term treatment was chosen to mimic a situation in patients undergoing

celecoxib treatment during all days of tooth movement, which can occur in the treatment of

chronic diseases. The dose of 10 mg/Kg was chosen based on the literature

experience,35,36 and the protocol of administration (twice a day) was chosen considering

the pharmacodynamics of celecoxib.37 Our results showed that both the short and long-

term therapy with celecoxib significantly reduced the amount of tooth movement (Table 2).

Previous studies have shown that celecoxib did not interfere with tooth movement in

rats.11,18 Recently, de Carlos et al11 showed that celecoxib and parecoxib, but not

rofecoxib, are appropriate for discomfort and pain relief while avoiding interference during

orthodontic tooth movement. In addition, Jerome et al18 showed that celecoxib (Celebrex

50 mg/Kg) given to rats in their drinking water did not affect tooth movement and appeared

to offer some slight protection against root resorption. Our results did not confirm these

findings.

The present study showed that both short and long-term celecoxib administration were

able to significantly reduce the amount of orthodontic tooth movement. The differences

between the results of these studies could be due to the dosage, time interval of

administration and methodology of tooth movement analysis, which were not the same.

72

The study of de Carlos et al11 used local injections (in the maxillary gingiva, close to the

first molar) on the day of appliance placement and after 3 and 5 days. Jerome et al18 used

celecoxib given to rats in their drinking water, which made it difficult to control drug

ingestion. Since preoperative administration of analgesics for controlling orthodontic pain is

increasing,15,21-23 we wanted to mimic this situation, by administering celecoxib 2 hours

before appliance placement, followed by two more days of medication, as suggested by

clinical research.15

It is known that when an orthodontic force activates the microenvironment of periodontal

tissue, several key proinflammatory cytokines are rapidly produced to trigger a cascade of

cellular events involved in the tooth displacement.38 Therefore, the use of preoperative

analgesics, followed by 2 days of medication, can both reduce orthodontic pain15,21-23 and

the associated inflammatory and tooth movement processes, as observed in the animals

treated with the short-term therapy (Table 2). Moreover, long-term celecoxib

administration, which is commonly used to treat many different diseases,16,17 can also

reduce the rate of tooth movement, and this reduction was more evident than that

observed in the short-term therapy (Table 2).

According to our knowledge, this study is the first to examine the effects of preoperative

doses and the long-term use of coxibs on orthodontic tooth movement. The lack of

statistical significance for the number of osteclasts on the alveolar bone surface was

unexpected. Although the celecoxib-treated animals showed a reduction in osteoclasts

when compared with saline-treated animals (Table 3), these differences were not

statistically significant. This does not exclude the possibility that celecoxib administration

may affect the osteoclast activity induced by orthodontic appliance. Some studies

73

demonstrate that the size of the osteoclasts and activity of the proton pump is also related

to the ability of the individual osteoclast to resorb bone.39,40 Thus, the trend towards a

reduction in the number of osteoclasts observed in the present study indicates that our

results need to be reevaluated and confirmed under other experimental sets, including the

evaluation of osteoclastogenesis, cell fusion, acidification, and resorptive activity. Sari et

al41 showed that rofecoxib administration did not significantly affect PGE2 levels. However,

our study used celecoxib, and the drug could also affect other factors, such as interleukin-

1 and -6, which are both related to bone resorption and tooth movement.38

The significant difference in the amount of tooth movement between animals treated with

saline for 3 and 14 days (Table 2) was also unexpected. One possible explanation could

be the stress system response evoked by the two injections/day during the 14 days of

treatment. Kalia et al42 showed that the administration of glucocorticoids can reduce bone

turnover induced by orthodontic forces. Perhaps the endogenous glucocorticoids secreted

by the rats submitted to repeated injections might be enough to interfere in the tooth

movement.

CONCLUSIONS

• Orthodontists should be aware of patients under short and long-term therapy with

celecoxib, since this drug can slow down the rate of orthodontic tooth movement.

• Perhaps the use of other drugs or other administration protocols might be effective

for discomfort and pain relief while avoiding interference during tooth movement.

74

ACKNOWLEDGEMENTS

Celecoxib was kindly provided by Pfizer (São Paulo, Brazil). The authors thank Gláucia

Maria Bovi Ambrosano for statistical analyses. Thanks are due to Maria Aparecida

Santiago Varella and Eliene Aparecida Orsini Narvaes Romani for technical assistance.

This work was supported by CAPES and FAPESP, Brazil.

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Capítulo 5 Histological analysis of orthodontic root resorption in rats treated with the cyclooxygenase-2 (COX-2) inhibitor celecoxib GUSTAVO HAUBER GAMEIRO DARCY FLÁVIO NOUER JOÃO SARMENTO PEREIRA-NETO MARIA BEATRIZ BORGES DE ARAÚJO MAGNANI EDUARDO DIAS DE ANDRADE PEDRO DUARTE NOVAES MARIA CECÍLIA FERRAZ DE ARRUDA VEIGA Structured Abstract

Introduction: It has been reported that anti-inflammatory drugs used for treatment of pain

and discomfort related to orthodontic treatment could slow down tooth movement.

However, the effect of these drugs on orthodontic root resorption is not well understood.

Objectives: The aim of this study was to investigate whether the COX-2 inhibitor

celecoxib offer some protection against orthodontically induced root resorption.

Design: Male Wistar rats were divided into four groups: Groups I and II were treated with

saline and celecoxib (10 mg/Kg), respectively, for 3 days. Groups III and IV were treated

with saline and celecoxib for 14 days. The upper left first molars of all rats were moved

mesially for 14 days with 50 g of force. An area including the disto-apical aspect of the

mesial root of the first molar was processed for histological and histochemical techniques

with tartrate-resistant acid phosphatase (TRAP).

Outcome measure: The degree of root resorption was measured using an image analysis

system with a grid-sheet superimposed in the root where resorption lacunae were counted.

82

The number of TRAP-positive cells on the tooth root surface defined as odontoclasts were

also evaluated.

Results: The results revealed that there were no significant differences in the degree of

root resorption and in the number of odontoclasts on the root between the 4 groups

studied. Conclusion: The short and long-term celecoxib administration did not suppress

the root resorption in case of experimental orthodontic force application.

Key Words: root resorption, celecoxib, orthodontics

Introduction

Root resorption is a partial loss of tooth root cementum and dentin and is one of the most

frequent iatrogenic problems in orthodontic tooth movement (1-2) The cause of root

resorption is considered to be multi-factorial, and these causes are basically divided into

mechanical and biological factors, or a combination of both (2). For the mechanical factors,

it is generally accepted that prolonged orthodontic treatment, high forces, extensive tooth

movement, root torque, jiggling and intrusive forces are associated with the onset of a

clinically evident root resorption (2). The main biological factors for root resorption include

the individual susceptibility on a genetic basis (3), some systemic diseases (4) and

anomalies in root morphology (5). However, exact mechanisms of root resorption have not

yet been fully understood.

The cells responsible for root resorption are called odontoclasts, and these cells are

considered to be similar types of cells as the osteoclasts (6). Odontoclasts are attached to

the tooth root and are activated to resorb the matrix (7). Several approaches to prevent

83

root resorption during orthodontic tooth movement were reported including the

administration of L-thyroxine (8), bisphosphonates (9), doxyciline (10), echistatin, (11) and

others (6,12). However, the effects of these drugs on root resorption are still controversial.

For example, some studies revealed that the bisphosphonates, which are potent inhibitors

of bone resorption, caused a significant dose-dependent inhibition of root resorption in rats

after force application (9,13-14). On the other hand, other studies have reported increased

root resorption with bisphosphonate treatment (15-16). Controversy also exists as to the

effects of corticosteroids on orthodontic root resorption. The acute administration of high

(15 mg/Kg) and therapeutic dosages (8 mg/Kg) of glucocorticoids produced an increased

orthodontic root resorption in rabbits (17) and rats (18), respectively. In contrast, Ong et al.

(19) reported a reduced root resorption in rats treated with a lower dosage of corticosteroid

(1 mg/Kg).

Recently, Jerome et al. (20) showed that the selective COX-2 inhibitor celecoxib (Celebrex

50 mg/Kg) given to rats in their drinking water did not interfere with tooth movement and

appeared to offer some slight protection against orthodontic root resorption. However, the

method of administration might not be able to maintain significant plasma drug

concentration and the clasts cells responsible for root resorption were not quantified. On

this background, the present study was designed to investigate possible effects of the

controlled celecoxib administration on the number of odontoclasts and on the degree of

root resorption induced by orthodontic tooth movement.

84

Materials and methods

Animals

Male Wistar rats (3.5 month-old, weighing 350 g on average) were used in this study. The

rats were housed in groups of five and maintained in a temperature-controlled room

(23 ± 1 °C) with a 12/12 light–dark cycle and food (ground pellets) and water were

available ad libitum. The body weight of each animal was recorded once a week

throughout the experiment. The study was conducted in accordance with the ethical

guidelines for investigations of experimental pain in conscious animals (21). This research

was approved by the institutional ethics committee in animal experimentation.

The rats were randomly divided into four groups: Group I (n=9) - treated with saline i.p.

injections on days 1, 2 and 3; Group II (n=9) - treated with celecoxib (10 mg/Kg) i.p.

injections on days 1, 2 and 3. Group III (n=7) - treated with saline i.p. injections on days 1

to 14; Group IV (n=7) - treated with celecoxib (10 mg/Kg) i.p. injections on days 1 to 14.

The asymmetric distribution of the animals was due to the fact that two rats in group III

died under general anesthesia, and two rats in group IV had to be excluded because their

appliances were damaged during the experiment.

Drug treatment

Celecoxib (Pfizer, São Paulo, Brazil) was freshly dissolved in saline and given i.p. twice a

day in a dose of 10 mg/Kg and in a volume of 1ml/Kg. The first injection was made 2

hours before appliance placement, in order to test the preemptive or preoperative use of

the drug, which is the current trend in the orthodontic pain management (22-23). The

control groups received equivolumetric saline injections during the same period according

to their experimental groups (Celecoxib for 3 or 14 days).

85

Appliance placement

The appliance design of this study follows that used by Leiker et al. (24). Animals were

first placed under general anesthesia with xylazine (10 mg/kg) and ketamine (50 mg/kg).

A closed coil nickel-titanium spring (Sentalloy®, GAC, Ctr. Islip, NY) calibrated to provide a

force of 50 g was ligated to the upper left first molar and connected to an orthodontic band

cement onto the incisors. Previous studies have demonstrated that a 40–60 g level of

force stimulated substantial molar tooth movement in rats (25-26). A nickel–titanium spring

was used to provide a relatively constant force level over the course of the tooth

movement period (14 days).

Tartrate-resistant acid phosphatase histochemistry and evaluation of root resorption score

The left hemi-maxilla of five rats from each group were processed for tartrate-resistant acid

phosphatase (TRAP) staining as described elsewhere (19). For each sample three sagittal

sections (5 µm thick) were taken at 50 µm intervals. The slides were counterstained with

Harris's hematoxilin for 7 min. Cover slips were mounted with Entellan before examining

the slides with a Leica Microsystems light microscope (Wetzlar, Germany).

Counting of odontoclasts was performed in a selected section localized disto-apically to

the mesial root (Fig. 1). The overall size of each measurement area was 1000 X 800 µm.

The magnification used to view the selected area was achieved with an objective of 20X

magnification. Cells were considered to be odontoclasts if they were multinucleated, TRAP

positive, and located on or close to root surface (Fig. 2). The estimate of TRAP positive

cells was determined by summing the value of the TRAP positive cells in the three

sections per case. The mesial root was chosen because it is the largest of the first molar's

five roots, is in approximately the same plane as the applied force and is most commonly

evaluated in tooth movement studies (24,27).

86

Fig 1. The area under investigation (black rectangle) where tartrate-resistant acid

phosphatase (TRAP)-positive cells were counted and root resorption score were analyzed.

The arrow indicates the direction of orthodontic force applied for 14 days.

Fig 2. TRAP stained histological section taken from the disto-apical area of mesial root of the

maxillary first molar (10× magnification) (A). Details of odontoclasts cells are in (B). B, bone;

PL, periodontal ligament; R, root.

87

The root resorption scores were determined according to the method described by Lu et al.

(28) with little modifications. Under ×10 magnification, a selected area localized disto-

apically to the mesial root was examined using an image analysis system (Leica Qwin). A

grid-sheet (10X10) was superimposed in the selected area (1000 × 800 µm), and the

number of grids with or without resorption lacunae were counted (Fig. 3). Root resorption

scores (percentage of resorption grids) were determined by dividing the number of grids

with resorption lacunae by the total number of grids along the root surface (Fig. 3).

Fig 3. Schematic illustration of the evaluation of root resorption score. Root resoprtion score

= number of grids containing resorption lacunae (black circles) divided by the total number

of grids along the root surface (black squares) X 100.

88

In order to determine the random intra-individual error for the odontoclasts counts and root

resorption scores, 20 randomly chosen sections were counted again in a blinded manner

and Dahlberg’s equation was used. A reproducibility error of less than 10% was

established and the errors for odontoclasts counts and root resporption scores were 0.47

and 3.1, respectively, which were considered to be acceptable.

Statistical analysis

Body weight was analyzed by repeated measures ANOVA and Tukey's test. The

odontoclasts number and root resorption score were analyzed by Two-way ANOVA and

Tukey's test. The SAS software (version 9.1, 2003; SAS Institute Inc., Cary, NC, USA) was

used and the significance level set at p<0.05.

Results

Except for a temporary episode of weight loss in all animals for 1 to 2 days following

appliance insertion, there was an overall gain in body weight over weeks (p<0.0001).

There was no statistical difference between groups (p=0.7632), and the interaction

between groups and weeks was also not statistically significant (p=0.1520) (data not

shown).

The number of TRAP positive cells on the root surface did not differ between drugs

(p=0.7285) neither between times of treatment (p=0.3721). Also, the interaction between

drugs and times was not statistically significant (p=0.8348) (Table 1).

The values for root resorption score was similar between saline and celecoxib groups

(p=0.7607). There was no significant differences between times of treatment (p=0.8313)

and the interaction between drugs and times was also not statistically significant

(p=0.1485) (Table 2).

89

Table 1.Values (mean and standard deviation) for the number of tartrate-resistant acid phosphatase (TRAP)-positive cells on the root surface in animals treated with saline or celecoxib

Drugs

TIME OF TREATMENT

3 days 14 days Saline 11.8 (7.8) Aa 15.0 (3.0) Aa

Celecoxib 11.4 (5.3) Aa 13.4 (7.8) Aa

Means followed by the same letters (capital letters for rows and small letters for columns) do not differ significantly (Two-way ANOVA, p<0.05).

Table 2.Values (mean and standard deviation) for the root resorption score in animals treated with saline or celecoxib

Drugs

TIME OF TREATMENT

3 days 14 days Saline 40.9 (8.68) Aa 49.6 (9.15) Aa

Celecoxib 50.1 (13.6) Aa 43.6 (12.5) Aa

Means followed by the same letters (capital letters for rows and small letters for columns) do not differ significantly (Two-way ANOVA, p<0.05).

Discussion

The existing literature supports the use of non-steroidal anti-inflammatory drugs (NSAIDs)

for orthodontic pain control (29-30) even though some of these drugs can impair bone

resorption and hence the tooth movement (29). One approach to deal with this problem is

the use of selective COX-2 inhibitors, which are replacing conventional NSAIDs in the

clinical practice (23,31-32). It has previously been shown that some COX-2 inhibitors

(Celecoxib and Parecoxib) do not interfere in the rate of orthodontic tooth movement

(20,33). However, the specificity of COX inhibitors can account for different effects of these

drugs on tooth movement (33). One example is the Rofecoxib, a drug that can disturb the

90

process of tooth movement (33-34). In addition, this drug has been the object of debate

and even withdrawn from the market due to reports of unwanted side effects. Celecoxib

was the first COX-2 inhibitor introduced on the market, and it still remains so, whereas

rofecoxib and valdecoxib were withdrawn due to excess cardiovascular risk (35). To our

knowledge, the possibility of using selective COX-2 inhibitors to prevent root resorption

was reported only once (20). The present study was designed to evaluate the orthodontic

root resorption in rats treated with short and long-term celecoxib administration.

The short-term treatment was chosen in this study to simulate the administration of

preemptive or preoperative analgesics to decrease postoperative pain, which has become

the focus of recent research in orthodontics (22-23). It is assumed that preemptive

analgesia will block the afferent nerve impulses before they reach the central nervous

system, abolishing the process of central sensitization (22). Some authors also

recommended two postoperative doses, in addition to a preoperative dose, for a complete

pain control during each orthodontic appointment (30). Since the orthodontic pain will

usually last for 2 – 3 days (32), we have used one preemptive dose followed by

postoperative doses for 2 days. The long-term treatment was chosen to mimic a situation

in patients undergoing celecoxib treatment during all days of tooth movement, which can

occur in the treatment of chronic diseases (36). The dose of 10 mg/Kg was chosen based

on the literature experience (37-38), and the protocol of administration (twice a day) was

chosen considering the pharmacodynamics of celecoxib (39). Our results showed that not

only the short-term therapy but also the long-term therapy with celecoxib did not affect the

orthodontic root resorption, as confirmed by the absence of statistical difference between

the number of odontoclasts on the root surface of the 4 studied groups (Table 1).

Moreover, the short and long-term celecoxib administration were not able to alter the root

91

resorption scores, as observed when the medicated and control groups were compared

(Table 2).

Jerome et al. used celecoxib (Celebrex 50 mg/Kg) given to rats in their drinking water, and

found a significantly lower number of root lacunaes formed as consequence of orthodontic

forces (20). However, no information about the control of drug ingestion was reported, and

direct comparison with our data is impossible. Moreover, the dosage, time interval of

administration and methodology of root resorption analysis were not the same. We wanted

to simulate the short and long-term use of celecoxib in the clinical practice. Although we

have previously observed that the protocol of drug administration used in this study (with

the same dosage) was able to reduce the rate of tooth movement in rats (40), it is well

known that osteoclasts and odontoclasts differ in their susceptibility to modulation by

pharmacologic agents (13-16,18-19). In contrast to alveolar bone, which is rich in cells and

vessels, tooth cementum and dentin are largely matrix-dominant tissue with no vessels.

Thus, dental tissues may not be affected by drugs and systemic factors compared to the

alveolar bone. This observation is consistent with previously reported findings (41-42).

Conclusions

In conclusion, the results of the present investigation indicated that the short and long-term

administration of celecoxib did not reduce the odontoclasts and root resorption induced by

orthodontic force application. These results need to be reevaluated and confirmed under

other experimental sets. Perhaps the use of other COX-2 inhibitors or other drugs might be

effective in protecting from root resorption induced by orthodontic treatment. Together with

the evidence that celecoxib can slow down tooth movement, the results of the present

study do not support the prescription of this drug to orthodontic patients.

92

Acknowledgements

Celecoxib was kindly provided by Pfizer (São Paulo, Brazil). The authors thank Gláucia

Maria Bovi Ambrosano for statistical analyses. Thanks are due to Maria Aparecida

Santiago Varella and Eliene Aparecida Orsini Narvaes Romani for technical assistance.

This work was supported by CAPES and FAPESP, Brazil.

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23. Young AN, Taylor RW, Taylor SE, Linnebur SA, Buschang PH. Evaluation of preemptive valdecoxib therapy on initial archwire placement discomfort in adults. Angle Orthod 2006;76:251-9. 24. Leiker BJ, Nanda RS, Currier GF, Howes RI, Sinha PK. The effects of exogenous prostaglandins on orthodontic tooth movement in rats. Am J Orthod Dentofacial Orthop 1995;108:380-8. 25. King GJ, Keeling SD, McCoy EA, Ward TH. Measuring dental drift and orthodontic tooth movement in response to various initial forces in adult rats. Am J Orthod Dentofacial Orthop 1991;99:456–465. 26. Rody WJJ, King GJ, Gu G. Osteoclast recruitment to sites of compression in orthodontic tooth movement. Am J Orthod Dentofacial Orthop 2001;120:477–489. 27. Kalia S, Melsen B, Verna C. Tissue reaction to orthodontic tooth movement in acute and chronic corticosteroid treatment. Orthod Craniofac Res 2004;7:26-34. 28. Lu LH, Lee K, Imoto S, Kyomen S, Tanne K. Histological and histochemical quantification of root resorption incident to the application of intrusive force to rat molars. Eur J Orthod 1999;21:57-63. 29. Arias OR, Marquez-Orozco MC. Aspirin, acetaminophen, and ibuprofen: their effects on orthodontic tooth movement. Am J Orthod Dentofacial Orthop 2006;130:364-70. 30. Polat O, Karaman AI, Durmus E. Effects of preoperative ibuprofen and naproxen sodium on orthodontic pain. Angle Orthod 2005;75:791-6. 31. Gameiro GH, Pereira-Neto JS, Magnani MB, Nouer DF. The influence of drugs and systemic factors on orthodontic tooth movement. J Clin Orthod 2007;41:73-8. 32. Krishnan V. Orthodontic pain: from causes to management--a review. Eur J Orthod 2007;29:170-9. 33. de Carlos F, Cobo J, Perillan C, Garcia MA, Arguelles J, Vijande M, Costales M. Orthodontic tooth movement after different coxib therapies. Eur J Orthod 2007;18 [Epub ahead of print] 34. de Carlos F, Cobo J, Diaz-Esnal B, Arguelles J, Vijande M, Costales M. Orthodontic tooth movement after inhibition of cyclooxygenase-2. Am J Orthod Dentofacial Orthop 2006;129:402-6. 35. Antoniou K, Malamas M, Drosos AA. Clinical pharmacology of celecoxib, a COX-2 selective inhibitor. Expert Opin Pharmacother 2007;8:1719-32. 36. Rattray B, Nugent DJ, Young G. Celecoxib in the treatment of haemophilic synovitis, target joints, and pain in adults and children with haemophilia. Haemophilia 2006;12:514-7.

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37. Fabricio AS, Veiga FH, Cristofoletti R, Navarra P, Souza GE. The effects of selective and nonselective cyclooxygenase inhibitors on endothelin-1-induced fever in rats. Am J Physiol Regul Integr Comp Physiol 2005;288:R671-7. 38. Mizuno M, Sotoyama H, Narita E, Kawamura H, Namba H, Zheng Y, Eda T, Nawa H. A cyclooxygenase-2 inhibitor ameliorates behavioral impairments induced by striatal administration of epidermal growth factor. J Neurosci 2007;27:10116-27. 39. Davies NM, McLachlan AJ, Day RO, Williams KM. Clinical pharmacokinetics and pharmacodynamics of celecoxib: a selective cyclo-oxygenase-2 inhibitor. Clin Pharmacokinet 2000;38:225-42. 40. Gameiro, GH, Nouer, DF, Neto, JSP, Siqueira, VC, Andrade, ED, Novaes, PD, Veiga, MCFA. Effects of short and long-term celecoxib on orthodontic tooth movement. Angle Orthod (in press). 41. Goldie RS, King GJ. Root resorption and tooth movement in orthodontically treated, calcium-deficient, and lactating rats. Am J Orthod 1984;85:424-30. 42. Midgett RJ, Shaye R, Fruge JF Jr. The effect of altered bone metabolism on

orthodontic tooth movement. Am J Orthod 1981;80:256-62.

97

III. CONCLUSÕES

De acordo com os resultados do presente trabalho, concluiu-se que:

� A ativação prolongada da resposta de estresse aumentou o número de

osteoclastos no osso alveolar e o movimento dentário ortodôntico, porém

este fator sistêmico não representa um fator de risco para a reabsorção

radicular associada à movimentação ortodôntica.

� A administração de celecoxibe em regimes de curta e longa duração pode

reduzir significativamente o movimento dentário ortodôntico, e a indicação

deste medicamento para prevenir a reabsorção radicular não foi

comprovada neste estudo.

99

IV. REFERÊNCIAS BIBLIOGRÁFICAS

Alhashimi N, Frithiof L, Brudvik P, Bakhiet M. Orthodontic movement induces high numbers of cells expressing IFN-gamma at mRNA and protein levels. J Interferon Cytokine Res. 2000 Jan;20(1):7-12. Alhashimi N, Frithiof L, Brudvik P, Bakhiet M. Orthodontic tooth movement and de novo synthesis of proinflammatory cytokines. Am J Orthod Dentofacial Orthop. 2001 Mar;119(3):307-12. Arias OR, Marquez-Orozco MC. Aspirin, acetaminophen, and ibuprofen: their effects on orthodontic tooth movement. Am J Orthod Dentofacial Orthop. 2006 Sep;130(3):364-70. Bernhardt MK, Southard KA, Batterson KD, Logan HL, Baker KA, Jakobsen JR. The effect of preemptive and/or postoperative ibuprofen therapy for orthodontic pain. Am J Orthod Dentofacial Orthop. 2001; 120:20-7. Cherruau M, Morvan FO, Schirar A, Saffar JL. Chemical sympathectomy-induced changes in TH-, VIP-, and CGRP-immunoreactive fibers in the rat mandible periosteum: influence on bone resorption. J Cell Physiol. 2003 Mar;194(3):341-8. Davidovitch Z, Nicolay OF, Ngan PW, Shanfeld JL. Neurotransmitters, cytokines, and the control of alveolar bone remodeling in orthodontics. Dent Clin North Am. 1988 Jul;32(3):411-35. Review. de Carlos F, Cobo J, Perillan C, Garcia MA, Arguelles J, Vijande M, Costales M. Orthodontic tooth movement after different coxib therapies. Eur J Orthod. 2007 Sep 18; [Epub ahead of print] Gameiro GH, Pereira-Neto JS, Magnani MB, Nouer DF. The influence of drugs and systemic factors on orthodontic tooth movement. J Clin Orthod. 2007; 41:73-8. Habib KE, Gold PW, Chrousos GP. Neuroendocrinology of stress. Endocrinol Metab Clin North Am. 2001 Sep;30(3):695-728; Haug SR, Heyeraas KJ. Effects of sympathectomy on experimentally induced pulpal inflammation and periapical lesions in rats. Neuroscience. 2003;120(3):827-36. Jerome J, Brunson T, Takeoka G, Foster C, Moon HB, Grageda E, Zeichner-David M. Celebrex offers a small protection from root resorption associated with orthodontic movement. J Calif Dent Assoc. 2005 Dec;33(12):951-9. * De acordo com a norma da UNICAMP/FOP, baseada no modelo Vancouver. Abreviatura dos periódicos em conformidade com o Medline.

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Krishnan V, Davidovitch Z. Cellular, molecular, and tissue-level reactions to orthodontic force. Am J Orthod Dentofacial Orthop. 2006 Apr;129(4):469.e1-32. Krishnan V. Orthodontic pain: from causes to management--a review. Eur J Orthod. 2007; 29:170-9. Kvinnsland S, Heyeraas K, Ofjord ES. Effect of experimental tooth movement on periodontal and pulpal blood flow. Eur J Orthod. 1989 Aug;11(3):200-5. Leone S, Ottani A, Bertolini A. Dual acting anti-inflammatory drugs. Curr Top Med Chem. 2007;7:265-75. Levine JD, Moskowitz MA, Basbaum AI. The contribution of neurogenic inflammation in experimental arthritis. J Immunol. 1985 Aug;135(2 Suppl):843s-847s. Meikle MC. The tissue, cellular, and molecular regulation of orthodontic tooth movement: 100 years after Carl Sandstedt. Eur J Orthod. 2006 Jun;28(3):221-40. Norevall LI, Forsgren S, Matsson L. Expression of neuropeptides (CGRP, substance P) during and after orthodontic tooth movement in the rat. Eur J Orthod. 1995 Aug;17(4):311-25. Polat O, Karaman AI, Durmus E. Effects of preoperative ibuprofen and naproxen sodium on orthodontic pain. Angle Orthod. 2005; 75:791-6. Sandhu HS, Herskovits MS, Singh IJ. Effect of surgical sympathectomy on bone remodeling at rat incisor and molar root sockets. Anat Rec. 1987 Sep;219(1):32-8. Smith WL, Dewitt DL. Prostaglandin endoperoxide H synthases-1 and -2. Adv Immunol .1996; 62:167-215. Steen Law SL, Southard KA, Law AS, Logan HL, Jakobsen JR. An evaluation of preoperative ibuprofen for treatment of pain associated with orthodontic separator placement. Am J Orthod Dentofacial Orthop. 2000; 118:629–33. Togari A. Adrenergic regulation of bone metabolism: possible involvement of sympathetic innervation of osteoblastic and osteoclastic cells. Microsc Res Tech. 2002 Jul 15;58(2):77-84. Vandevska-Radunovic V, Kristiansen AB, Heyeraas KJ, Kvinnsland S. Changes in blood circulation in teeth and supporting tissues incident to experimental tooth movement. Eur J Orthod. 1994 Oct;16(5):361-9. Young AN, Taylor RW, Taylor SE, Linnebur SA, Buschang PH. Evaluation of preemptive valdecoxib therapy on initial archwire placement discomfort in adults. Angle Orthod. 2006 Mar;76(2):251-9.

101

APÊNDICE

Capítulo 1 A influência de drogas e fatores sistêmicos no movimento dentário ortodôntico GUSTAVO HAUBER GAMEIRO JOÃO SARMENTO PEREIRA-NETO MARIA BEATRIZ BORGES DE ARAÚJO MAGNANI DARCY FLÁVIO NOUER

Resumo

O movimento dentário ortodôntico ocorre por um processo inflamatório envolvendo

osteoclastos, osteoblastos, neuropeptídeos, citocinas e alterações na inervação e

vascularização local. Pesquisas demonstram que a atividade de remodelação óssea pode

ser regulada tanto por fatores locais, tais como as forças aplicadas, quanto por fatores

sistêmicos, como drogas, hormônios e vitaminas. Este trabalho apresenta os efeitos de

drogas e fatores sistêmicos capazes de afetar o metabolismo ósseo e influenciar a

velocidade do movimento dentário ortodôntico.

Os antiinflamatórios não-esteroidais (exceto celecoxibe), bifosfonatos e os hormônios

sexuais podem diminuir a velocidade do movimento ortodôntico, enquanto os

corticosteróides, relaxina, hormônios tireóideos, paratormônio e vitamina D podem

aumentar a taxa de movimentação dentária. Assim, conclui-se que o dentista deve estar

atento às medicações utilizadas pelos pacientes, a fim de selecionar a melhor estratégia

terapêutica (controle das forças e intervalo entre as consultas) para cada caso. Além

disso, o paracetamol deve ser o fármaco de escolha para alívio de possível desconforto

associado ao tratamento ortodôntico, pois não influencia significativamente na taxa de

movimentação dentária.

* este artigo foi publicado no periódico Journal of Clinical Orthodontics. Uma versão

traduzida deste artigo foi publicada no periódico Ortodoncia.

102

Capítulo 2

O efeito da resposta de estresse sistêmico sobre o movimento dentário ortodôntico GUSTAVO HAUBER GAMEIRO DARCY FLÁVIO NOUER JOÃO SARMENTO PEREIRA-NETO MARÍLIA BERTOLDO URTADO PEDRO DUARTE NOVAES MARGARET DE CASTRO MARIA CECÍLIA FERRAZ DE ARRUDA VEIGA

Resumo

Objetivo: O objetivo deste estudo foi investigar se a resposta sistêmica de estresse afeta

as reações biológicas induzidas pelo movimento dentário ortodôntico. Delineamento

experimental: Ratos machos Wistar foram imobilizados durante 1 hora por dia em

modelos de estresse de curta (3 dias) ou longa duração (40 dias), enquanto o grupo

controle não foi submetido às sessões de estresse (n=10/grupo). O primeiro molar

superior esquerdo foi movimentado mesialmente nos últimos 14 dias do experimento.

Logo depois, os animais foram mortos por decapitação para coleta de sangue e

mensuração da corticosterona plasmática; o movimento dentário foi quantificado e os

tecidos ao redor da raiz mesial do primeiro molar foram preparados para análise

histoquímica pela fosfatase ácida tartarato-resistente. Resultados: Os níveis plasmáticos

de corticosterona foram significativamente maiores nos grupos estressados, em relação

ao grupo controle. O estresse crônico produziu maior taxa de movimentação dentária em

relação aos outros dois grupos. Conclusões: Estes resultados indicam que a ativação

persistente da resposta de estresse sistêmico crônico pode aumentar a reabsorção óssea

osteoclástica local induzida pelo movimento dentário ortodôntico, e isto pode incluir a

resposta de estresse como um fator sistêmico capaz de interferir no tratamento

ortodôntico.

* Este artigo foi submetido à publicação no periódico Archives of Oral Biology.

103

Capítulo 3 Avaliação da reabsorção radicular associada ao movimento ortodôntico em ratos estressados GUSTAVO HAUBER GAMEIRO DARCY FLÁVIO NOUER MARIA BEATRIZ BORGES DE ARAÚJO MAGNANI PEDRO DUARTE NOVAES GLÁUCIA MARIA BOVI AMBROSANO ANNICELE DA SILVA ANDRADE MARIA CECÍLIA FERRAZ DE ARRUDA VEIGA

Resumo

Objetivo: O objetivo deste estudo foi investigar os efeitos da resposta de estresse

sistêmico agudo e crônico na reabsorção radicular induzida por movimento ortodôntico.

Material e métodos: Ratos machos Wistar foram imobilizados durante 1 hora por dia em

modelos de estresse de curta (3 dias) ou longa duração (40 dias), enquanto o grupo

controle não foi submetido às sessões de estresse (n=10/grupo). O primeiro molar

superior esquerdo foi movimentado mesialmente nos últimos 14 dias do experimento.

Logo depois, os animais foram mortos por decapitação para coleta de sangue e

mensuração da corticosterona plasmática por radioimunoensaio; os tecidos ao redor da

raiz mesial do primeiro molar foram preparados para análise histológica e histoquímica

pela fosfatase ácida tartarato-resistente (TRAP). O grau de reabsorção radicular e o

número de odontoclastos foram avaliados, sendo que o lado contra-lateral de cada animal

serviu como controle (estudo split-mouth) Resultados: Os resultados demonstraram que

os níveis plasmáticos de corticosterona foram significativamente maiores nos grupos

estressados, em relação ao grupo controle. Não houve diferença estatística para o grau

de reabsorção radicular e número de odontoclastos nas raízes dos 3 grupos estudados.

Conclusões: Estes resultados indicam que o estresse sistêmico por si só não pode ser

considerado um fator de risco à reabsorção radicular induzida por movimento ortodôntico.

* Este artigo foi submetido à publicação no periódico The Angle Orthodontist.

104

Capítulo 4 Os efeitos da administração de celecoxibe - em regimes de curta e longa duração - no movimento dentário ortodôntico GUSTAVO HAUBER GAMEIRO DARCY FLÁVIO NOUER JOÃO SARMENTO PEREIRA-NETO VÂNIA CÉLIA VIEIRA DE SIQUEIRA EDUARDO DIAS DE ANDRADE PEDRO DUARTE NOVAES MARIA CECÍLIA FERRAZ DE ARRUDA VEIGA

Resumo

Introdução: O objetivo deste estudo foi investigar os efeitos da administração de

celecoxibe - em regimes de curta e longa duração - no movimento dentário ortodôntico.

Materiais e métodos Ratos machos Wistar submetidos a administração de celecoxibe -

em regimes de curta (3 dias) e longa duração (14 dias), enquanto os respectivos grupos

controles receberam injeções i.p. de salina. O primeiro molar superior esquerdo dos ratos

foi movimentado mesialmente por 14 dias, com a utilização de um aparelho ortodôntico

fixo exercendo força de 50 g no momento da instalação. Após o período experimental, o

movimento dentário foi quantificado e os tecidos ao redor da raiz mesial do primeiro molar

foram processados para análise histoquímica pela fosfatase ácida tartarato-resistente

(TRAP). A quantidade de movimento dentário e o número de células TRAP-positivas na

superfície do osso alveolar foram avaliados. Resultados: O movimento dentário foi

significativamente reduzido pela administração (curta e longa-duração) de celecoxibe,

enquanto que o número de osteoclastos no osso alveolar não diferiu entre os 4 grupos

estudados. Conclusões: Embora o número de osteoclastos não tenha sido afetado pela

administração de celecoxibe, a atividade dos osteoclastos pode ter sido reduzida, o que

explica a inibição do movimento dentário observada nos animais tratados com celecoxibe.

Estes resultados indicam que os ortodontistas devem estar atentos aos pacientes sob

tratamento esporádico ou contínuo com celecoxib.

* Este artigo foi aceito para publicação no periódico The Angle Orthodontist.

105

Capítulo 5 Análise histológica da reabsorção radicular em ratos tratados com o inibidor seletivo da ciclooxigenase-2 (COX-2) celecoxibe GUSTAVO HAUBER GAMEIRO DARCY FLÁVIO NOUER JOÃO SARMENTO PEREIRA-NETO MARIA BEATRIZ BORGES DE ARAÚJO MAGNANI EDUARDO DIAS DE ANDRADE PEDRO DUARTE NOVAES MARIA CECÍLIA FERRAZ DE ARRUDA VEIGA

Resumo

Introdução: Tem sido relatado que as dorgas anti-inflamatórias utilizadas para o

tratamento da dor e desconforto relacionada ao tratamento ortodôntico podem retardar o

movimento dentário.

Objetivos: O objetivo deste estudo foi investigar se o inibidor seletivo da COX-2

celecoxibe oferece alguma proteção contra a reabsorção radicular induzida pelo

movimento dentário ortodôntico. Delineamento experimental: Ratos machos Wistar

forma divididos em quatro grupos: Grupos I e II foram tratados com saliona e celeoxibe

(10 mg/Kg), respectivamente por 3 dias. Os grupos III e IV foram tratados com salina e

celecoxibe por 14 dias. O primeiro molar superior esquerdo dos ratos foi movimentado

mesialmente por 14 dias, com uma força de 50 g. Uma área do aspecto disto-apical da

raiz mesial do primeiro molar foi processada para análise histoquímica pela fosfatase

ácida tartarato-resistente (TRAP). Métodos de avaliação: O grau de reabsorção radicular

foi avaliado com um sistema de análises de imagem com um grid superposto na raiz onde

as lacunas de reabsorção foram quantificadas. e o número de células TRAP-positivas na

superfície radicular, definidas como odontoclastos, também foram avaliados. Resultados:

Os resultados demonstraram que não houve diferença estatística no grau de reabsorção

radicular e número de odontoclastos na raiz entre os 4 grupos estudados. Conclusões: A

administração de celecoxibe - em regimes de curta e longa duração – não reduziu a

reabsorção radicular induzida pela aplicação de força ortodôntica experimental.

* Este artigo foi aceito para publicação no periódico Orthodontics and Craniofacial

Research.

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DADOS REFERENTES AOS VALORES INDIVIDUAIS DA AMOSTRA

- EXPERIMENTO 1: (CAPÍTULOS 2 E 3)

1) Peso dos animais (n=5) em gramas ao longo das 8 semanas do experimento

(capítulos 2 e 3):

controle sem 1 sem 2 sem 3 sem 4 sem 5 sem 6 sem 7 sem 8

1 283 300 318 332 340 356 346 366 2 298 322 345 363 364 378 366 380 3 292 312 324 350 360 364 368 380 4 281 304 315 334 348 352 344 366 5 279 291 306 317 323 328 330 350

média 287 306 322 339 347 356 351 368 DP 8.1 11.8 14.6 17.7 16.5 18.4 16.0 12.4

e. longa 1 315 331 350 364 366 372 362 388 2 288 302 316 330 340 346 330 346 3 323 342 365 388 400 408 385 402 4 273 289 306 323 335 342 340 350 5 292 312 330 350 363 376 364 380

média 298 315 333 351 361 369 356 373 DP 20.5 21.4 24.2 26.3 25.8 26.6 21.7 24.4

e. curta 1 278 293 306 318 330 338 328 344 2 310 330 349 370 381 392 394 404 3 288 304 322 344 355 368 350 372 4 280 302 318 336 348 358 348 364 5 303 317 333 352 360 366 354 362

média 292 309 326 344 355 364 355 369 DP 14.2 14.5 16.3 19.2 18.5 19.5 24.1 21.9

baseline 1 298 312 330 344 356 362 370 376 2 310 326 336 350 366 376 388 396 3 282 300 320 340 352 362 368 380 4 278 292 318 333 340 355 364 378 5 282 300 314 338 350 360 368 378

média 290 306 324 341 353 363 372 382 DP 13.6 13.3 9.1 6.4 9.4 7.8 9.4 8.2

Grupos: controle, estresse longa, estresse curta e baseline (sem aparelho)

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2) Corticosterona plasmática (µg/dL) após as 8 semanas do experimento

(capítulos 2 e 3):

Controle e.longa e.curta baseline n Cortic. Cortic. Cortic. Cortic.

1 0,16 7,7 8,4 0,7 2 0,4 11,8 9,1 5,5 3 0,3 12,8 2,6 1,0 4 2,2 8,4 4,8 3,2 5 2,6 9,6 17,2 2,3 6 4,6 6 4,6 4,8 7 8,6 5,4 5,5 0,9 8 2,2 7,8 9,4 1,3 9 0,16 6 14,9 10 5,4 9,6 9,4 11

média 2,7 8,5 8,6 2,5 DP 2,8 2,5 4,6 1,9

Grupos: controle, estresse longa, estresse curta e baseline (sem aparelho)

3) Movimento dentário (mm) após as 8 semanas do experimento (capítulo 2):

Grupos: controle, estresse longa e estresse curta

Controle e.longa e.curta n Mov. Mov. Mov.

1 0.22 0.62 0.34 2 0.47 0.39 0.34 3 0.50 0.48 0.40 4 0.45 0.44 0.42 5 0.25 0.34 0.16 6 0.15 0.42 0.15 7 0.22 0.53 0.29 8 0.27 0.33 0.23 9 0.43 0.47 0.41 10 0.21 0.49 0.35

média 0.32 0.45 0.31 DP 0.13 0.09 0.10

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4) Contagem dos osteoclastos (soma do no células TRAP positivas no osso

alveolar) após as 8 semanas do experimento (capítulo 2):

controle e.longa e.curta lado C lado E lado C lado E lado C lado E 3 14 6 27 3 9 8 9 3 14 9 8 2 18 6 19 1 12 1 20 13 34 3 10 6 10 10 31 2 12 média 4 14,2 7,6 25 3,6 10,2

DP 2,92 4,82 3,91 8,34 3,13 1,79

Grupos (n=5): controle, estresse longa e estresse curta; Lado C (controle) e Lado E

(experimental).

5) Contagem dos odontoclastos (soma do no células TRAP positivas na raiz)

após as 8 semanas do experimento (capítulo 3):

controle e.longa e.curta lado C lado E lado C lado E lado C lado E 0 9 0 21 0 23 0 19 0 15 2 17 8 29 4 11 1 19 0 29 1 6 10 26 1 23 0 27 0 30 média 1,8 21,8 1,0 16,0 2,6 23,0

DP 3,5 8,3 1,7 8,2 4,2 5,2

Grupos (n=5): controle, estresse longa e estresse curta; Lado C (controle) e Lado E

(experimental).

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6) Scores de reabsorção radicular (%) após as 8 semanas do experimento

(capítulo 3):

controle e.longa e.curta lado C lado E lado C lado E lado C lado E 0,0 43,7 8,3 58,3 16,6 46,2 0,0 35,7 16,6 50,0 0,0 36,4 40,0 45,5 25,0 45,5 9,1 40,0 0,0 61,5 7,7 9,1 30,0 70,0 30,0 61,5 8,3 50,0 10,0 50,0 média 14,0 49,6 13,2 42,6 13,1 48,5

DP 19,5 11,5 7,6 19,3 11,1 13,1

Grupos (n=5): controle, estresse longa e estresse curta; Lado C (controle) e Lado E

(experimental).

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- EXPERIMENTO 2: (CAPÍTULOS 4 E 5)

1) Peso dos animais (gramas) antes, 1 semana e 2 semanas após o experimento

(capítulos 4 e 5):

GRUPO I antes 1 sem 2 sem

GRUPO II antes 1 sem 2 sem

1 398 408 433 1 333 350 360 2 393 400 413 2 390 396 402 3 393 394 414 3 362 370 364 4 328 322 345 4 350 360 370 5 314 326 340 5 351 342 350 6 342 354 358 6 333 354 352 7 362 340 355 7 403 418 420 8 348 368 380 8 390 404 404 9 342 358 368 9 333 353 364

média 357,8 363,3 378,4 360,6 371,9 376,2 DP 30,6 31,7 33,7 27,4 27,2 25,6

GRUPO III antes 1 sem 2 sem

GRUPO IV antes 1 sem 2 sem

1 342 335 345 1 362 372 344 2 333 328 336 2 365 378 372 3 406 408 418 3 404 401 395 4 350 358 365 4 344 355 350 5 402 402 410 5 354 372 383 6 322 335 347 6 365 384 414 7 362 352 343 7 390 402 423

média 359,6 359,7 366,3 369,1 380,6 383,00 DP 32,9 32,7 33,8 20,8 16,8 30,1

Grupo I (n=9) - salina 3 dias; Grupo II (n=9) - celecoxib 3 dias;

Grupo III (n=7) - salina 14 dias; Grupo IV (n=7) - celecoxibe 14 dias.

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2) Movimento dentário (mm) após o experimento - 14 dias (capítulo 4):

Grupo I Grupo II Grupo III Grupo IV 0,33 0,27 0,39 0,12 0,16 0,34 0,33 0,27 0,31 0,26 0,24 0,10 0,61 0,29 0,26 0,17 0,40 0,18 0,23 0,14 0,30 0,26 0,26 0,07 0,40 0,13 0,30 0,19 0,23 0,11 0,30 0,28 média 0,34 0,24 0,29 0,15 DP 0,13 0,08 0,06 0,07

Grupo I (n=9) - salina 3 dias; Grupo II (n=9) - celecoxib 3 dias;

Grupo III (n=7) - salina 14 dias; Grupo IV (n=7) - celecoxibe 14 dias.

3) Contagem dos osteoclastos (soma do no células TRAP positivas no osso

alveolar) após o experimento - 14 dias (capítulo 4):

Grupo I Grupo II Grupo III Grupo IV 53,0 13,0 27,0 14,0 11,0 39,0 16,0 17,0 29,0 19,0 20,0 15,0 28,0 44,0 43,0 23,0 23,0 30,0 19,0 10,0 média 28,8 29,0 25,0 15,8

DP 15,3 13,1 10,8 4,8

Grupo I (n=9) - salina 3 dias; Grupo II (n=9) - celecoxib 3 dias;

Grupo III (n=7) - salina 14 dias; Grupo IV (n=7) - celecoxibe 14 dias.

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4) Contagem dos odontoclastos (soma do no células TRAP positivas na raiz)

após o experimento - 14 dias (capítulo 5):

Grupo I Grupo II Grupo III Grupo IV 22,0 18,0 18,0 12,0 13,0 12,0 15,0 17,0 4,0 12,0 4,0 7,0 4,0 15,0 10,0 25,0 16,0 18,0 10,0 6,0 média 11,8 15,0 11,4 13,4

DP 7,8 3,0 5,4 7,8

Grupo I (n=9) - salina 3 dias; Grupo II (n=9) - celecoxib 3 dias;

Grupo III (n=7) - salina 14 dias; Grupo IV (n=7) - celecoxibe 14 dias.

5) Scores de reabsorção radicular (%) após o experimento - 14 dias (capítulo 5):

Grupo I Grupo II Grupo III Grupo IV 35,7 53,8 70,0 27,3 41,7 60,0 50,0 60,0 42,9 40,0 54,5 50,0 30,8 40,0 42,9 36,4 53,8 54,5 33,3 44,4

média 41,0 49,7 50,1 43,6 DP 8,7 9,2 13,7 12,5

Grupo I (n=9) - salina 3 dias; Grupo II (n=9) - celecoxib 3 dias;

Grupo III (n=7) - salina 14 dias; Grupo IV (n=7) - celecoxibe 14 dias.

113

ANEXO

COMPROVANTE 1 (cópia da 1ª página do artigo)

114

COMPROVANTE 2 (cópia de e-mail do periódico)

Archives of Oral Biology

Title: The effects of systemic stress response on orthodontic tooth

movement

Authors: Gustavo Hauber Gameiro, Ph.D.; Darcy F Nouer, PhD; João S

Pereira-Neto; Marília B Urtado; Pedro D Novaes; Margaret Castro; Maria

Cecília F Veiga

Article Type: Original Paper

Dear Gustavo,

Your submission entitled "The effects of systemic stress response on

orthodontic tooth movement" has been received by Archives of Oral

Biology.

You may check on the progress of your paper by logging on to the Elsevier

Editorial System as an author. The URL is http://ees.elsevier.com/aob/.

Your manuscript will be given a reference number once an Editor has been

assigned.

Thank you for submitting your work to this journal. Please do not

hesitate to contact me if you have any queries.

Kind regards,

(On behalf of the Editors)

Archives of Oral Biology

115

COMPROVANTE 3 (cópia de e-mail do editor do periódico)

Dear Dr. Gameiro,

On September 27, 2007, we received your manuscript “Evaluation of root

resorption associated with orthodontic movement in stressed rats”

submitted to us for publication in The Angle Orthodontist. As is our

usual practice, I will send your manuscript out to two reviewers. It

generally takes a minimum of eight weeks for the review process to be

completed.

Please note that I have assigned a number to your manuscript #092707-459.

Thank you for the opportunity to review your work and thank you for

considering The Angle Orthodontist for your publication needs.

Sincerely,

Robert J. Isaacson, DDS, MSD, PhD

Editor

The Angle Orthodontist

116

COMPROVANTE 4 (cópia de e-mail do editor do periódico)

Dear Dr. Gameiro,

I am pleased to inform you that your manuscript "Effects of Short and

Long-term Celecoxib on Orthodontic Tooth Movement" has been accepted for

publication. We look forward to publishing your contribution in The Angle

Orthodontist.

Allen Press will e-mail instructions to you in about 6 weeks telling you

how to view a galley proof of your article on the Internet in PDF format.

These proofs will look like your article as it will appear in print. The

proofs will contain changes that occurred in the printing process

(editing, typesetting or layout).

Please print out the proofs and make ONLY PRINTER ERROR CORRECTIONS on

the paper print outs. Further editing of the content of your manuscript

is not permissable on the galley copy. Please also include a typed page

of the changes/corrections that you are requesting.

Please do not add new material as that will incur costs to the author.

After you correct the paper print out, express ship it to me at 5813

Vernon Lane, Edina, MN 55436-2239 (Phone: 952-922-9586). A quick turn-

around is important, please.

Again, congratulations on your work and thank you for your contributions

to the orthodontic literature. I look forward to seeing your article in

print. If you have any questions, please be sure to contact us.

Sincerely,

Robert J. Isaacson, DDS, MSD, PhD

Editor, The Angle Orthodontist

Professor Emeritus

University of Minnesota

Virginia Commonwealth University

117

COMPROVANTE 5 (cópia de e-mail do periódico)

18-Jan-2008

Dear Dr. Gameiro:

It is a pleasure to accept your manuscript entitled "Histological

analysis of orthodontic root resorption in rats treated with the

cyclooxygenase-2 (COX-2) inhibitor celecoxib" in its current form for

publication in the Orthodontics and Craniofacial Research. The production

manager will contact you later with regard to the actual publication.

As part of the Journal’s continued commitment to its authors, the

Editorial Office and Publisher wish to keep you informed about what will

happen next and, as the attached footer contains important information

regarding journal publication and services for authors, you may wish to

save it for future reference.

Please note that now your paper has been accepted, all queries related to

the production of your paper may be directed to the Production Office at

Blackwell. Information about the production services is given below.

Thank you for your fine contribution. On behalf of the Editors of the

Orthodontics and Craniofacial Research, we look forward to your continued

contributions to the Journal.

Sincerely,

Professor Anne Marie Kuijpers-Jagtman, DDS PhD

Editor in Chief, Orthodontics and Craniofacial Research

ocr.editor@gmail.com

118

Certificados de aprovação dos protocolos de pesquisa pela Comissão de ética na

experimentação animal (CEEA-IB-UNICAMP)

119