universidade federal de pelotas faculdade de...
TRANSCRIPT
UNIVERSIDADE FEDERAL DE PELOTAS
Faculdade de Odontologia
Programa de Pós-Graduação em Odontologia
Dissertação
Avaliação comparativa em mandíbulas e tíbias de ratos submetidos à terapia
com bisfosfonatos nitrogenados e não nitrogenados: análise imaginológica e
histomorfométrica.
Stephanie Joana Roman Martelli
Pelotas, 2018
Stephanie Joana Roman Martelli
Avaliação comparativa em mandíbulas e tíbias de ratos submetidos à terapia
com bisfosfonatos nitrogenados e não nitrogenados: análise imaginológica e
histomorfométrica.
Dissertação apresentada ao Programa de Pós-Graduação em Odontologia da Faculdade de Odontologia da Universidade Federal de Pelotas, como requisito parcial à obtenção do título de Mestre em Odontologia, área de concentração Diagnóstico Bucal.
Orientador: Prof.ª Dr.ª Ana Carolina Uchoa Vasconcelos
Co-orientadora: Prof.ª Dr.ª Melissa Feres Damian
Pelotas, 2018
Universidade Federal de Pelotas / Sistema de Bibliotecas
Catalogação na Publicação
M376a Martelli, Stephanie Joana Roman
MarAvaliação comparativa em mandíbulas e tíbias de ratos submetidos à terapia com bisfosfonatos nitrogenados e não nitrogenados : análise imaginológica e histomorfométrica / Stephanie Joana Roman Martelli ; Ana Carolina Uchoa Vasconcelos, orientadora ; Melissa Feres Damian, coorientadora. — Pelotas, 2018.
Mar71 f. : il.
MarDissertação (Mestrado) — Programa de Pós-Graduação em Diagnóstico Bucal, Faculdade de Odontologia, Universidade Federal de Pelotas, 2018.
Mar1. Bisfosfonato. 2. Osteonecrose associada ao uso de bisfosfonatos. 3. Tomografia computadorizada cone beam. 4. Histomorfometria. I. Vasconcelos, Ana Carolina Uchoa, orient. II. Damian, Melissa Feres, coorient. III. Título.
Black : D7
Elaborada por Fabiano Domingues Malheiro CRB: 10/1955
Stephanie Joana Roman Martelli
Avaliação comparativa em mandíbulas e tíbias de ratos submetidos à terapia com
bisfosfonatos nitrogenados e não nitrogenados: análise imaginológica e
histomorfométrica.
Dissertação apresentada, como requisito parcial, para obtenção do grau de Mestre em Odontologia, Programa de Pós-Graduação em Odontologia, Faculdade de Odontologia de Pelotas, Universidade Federal de Pelotas.
Data da defesa: 07/03/2018
Banca examinadora:
Prof.ª Dr.ª Ana Carolina Uchoa Vasconcelos Doutora em Estomatologia pela Pontifícia Universidade Católica do Rio Grande do Sul (PUC-RS). Prof.ª Dr.ª Ana Paula Neutzling Gomes Doutora em Patologia Bucal pela Universidade de São Paulo Prof.ª Dr.ª Caroline de Oliveira Langlois Doutora em Odontologia (área de concentração Radiologia Odontológica e Imaginologia) pela Universidade Luterana do Brasil
Prof.ª Dr.ª Sandra Beatriz Chaves Tarquinio (suplente) Doutora em Patologia Bucal pela Universidade de São Paulo Prof. ª Dr. ª Elizabete Bagordakis Pinto (suplente) Doutora em Estomatopatologia (área de concentração Patologia) pela Universidade Estadual de Campinas
Agradecimentos
Aos meus pais, Marta e Nelson, e à minha família, pelo amor, incentivo e apoio,
mesmo estando distantes.
Aos meus queridos amigos, pelo companheirismo, compreensão e pela
felicidade proporcionada pela nossa convivência. Agradeço muito por terem estado
presentes sempre, me dando suporte em todos os momentos.
À minha orientadora e “mãe”, Prof. Dra. Ana Carolina Uchoa Vasconcelos, pelo
apoio, confiança e empenho dedicado à elaboração deste e outros trabalhos. Também
pela amizade, paciência e orientação pessoal, essenciais para concluir minha
formação.
À minha co-orientadora, Prof. Dra. Melissa Feres Damian, pelos conhecimentos
transmitidos, pela disponibilidade em ajudar e pelo incentivo.
Ao Centro de Diagnóstico das Doenças da Boca da Faculdade de Odontologia
da Universidade Federal de Pelotas, em especial às professoras, pela disponibilidade
de recursos concedidos em todas as minhas pesquisas e pelo acolhimento durante
toda a minha formação.
Aos funcionários, em especial à Ivana, pela ajuda e paciência em ensinar como
realizar as atividades no laboratório. Teu suporte foi essencial à elaboração deste
trabalho.
Aos demais professores que tornaram possível minha caminhada até aqui.
Ao Programa de Pós-Graduação em Odontologia, à Faculdade de Odontologia
e à Universidade Federal de Pelotas, pela oportunidade de fazer o curso.
A tudo e a todos que contribuíram, direta ou indiretamente, para minha
formação.
Muito obrigada.
Notas Preliminares
A presente dissertação foi redigida segundo o Manual de Normas para
Dissertações, Teses e Trabalhos Científicos da Universidade Federal de Pelotas de
2013, adotando o Nível de Descrição 2 – estrutura em Capítulos, descrita no Apêndice
D do referido manual. <http://sisbi.ufpel.edu.br/?p=documentos&i=7> Acesso em:
05/12/2017.
Resumo
MARTELLI, Stephanie Joana Roman. Avaliação comparativa em mandíbulas e tíbias de ratos submetidos à terapia com bisfosfonatos nitrogenados e não nitrogenados: análise imaginológica e histomorfométrica. 2018. 71f. Dissertação (Mestrado em Odontologia) – Programa de Pós-Graduação em Odontologia. Universidade Federal de Pelotas, Pelotas, 2018.
Bisfosfonatos (BFs) são fármacos empregados na prevenção e no tratamento de doenças caracterizadas por excessiva reabsorção óssea. A osteonecrose dos maxilares associada ao uso de medicamentos (OMAM) é uma condição que se caracteriza por exposição óssea persistente no complexo maxilomandibular de pacientes expostos a terapia antirreabsortiva e/ou antiangiogênica, sem histórico de radioterapia em região de cabeça e pescoço. O presente estudo teve como objetivo realizar a avaliação comparativa da estrutura óssea de mandíbulas e tibias de ratos usuários e não usuários de BFs, por meios imaginológicos e histomorfométricos. Trinta e quatro espécimes de ratos (Rattus novergicus, linhagem Wistar) foram distribuídos em três grupos: (1) ratos tratados com ácido zoledrônico; (2) ratos tratados com clodronato; e (3) ratos que receberam solução salina. As mandíbulas e tíbias dos animais foram submetidas à tomografia computadorizada de feixe cônico (TCFC), com posterior processamento, análise e obtenção da densidade óssea (DO) por meio de Unidades Hounsfield (UH) com o software OsiriX 7.0. A partir dos espécimes foram obtidas 68 lâminas histológicas para as mandíbulas e 34 para as tíbias, coradas em hematoxilina e eosina (HE). As lâminas histológicas foram digitalizadas, e utilizando-se o programa Adobe Photoshop CC, realizou-se quantificação da proporção de volume ósseo (VO). Os dados obtidos foram analisados com os testes ANOVA e t de Student, utilizando o programa Stata 15.0. Na análise de DO, não se observou diferença significativa entre os grupos de tratamento, nem entre mandíbula e tíbia. Não houve diferença significativa quando comparados os valores de VO entre os grupos teste e controle. No entanto, na comparação entre ossos, as mandíbulas do grupo controle apresentaram maior proporção, estatisticamente significativa, que as tíbias do mesmo grupo. A partir dos resultados obtidos no presente estudo permite-se concluir que (1) a DO não é alterada pela localização óssea, nem pelo tipo de terapia administrada, e (2) a terapia com BFs nitrogenados e não-nitrogenados não foi capaz de alterar o VO, em mandíbulas e tíbias, nos grupos teste.
Palavras-chave: Bisfosfonato; Osteonecrose associada ao uso de bisfosfonatos;
Tomografia computadorizada cone beam; Histomorfometria.
Abstract
MARTELLI, Stephanie Joana Roman. Imaginological and histomorphometrical evaluation of mandibles and tibias of rats submitted to zoledronic acid and clodronate treatment. 2018. 71p. Dissertation (Master degree in Dentistry). Graduate Program in Dentistry. Federal University of Pelotas, Pelotas, 2018.
Bisphosphonates (BPs) are synthetic analogs of the inorganic pyrophosphate, used for prevention and treatment of diseases with excessive bone resorption. The medication-related osteonecrosis of the jaw (MRONJ) is a condition characterized by persistent bone exposure in the maxillofacial region of patients exposed to this class of drugs, with no history of head and neck radiotherapy. The objective of the present study was to compare, histologically and imagiologically, the bone structure of mandibles and tibiae of rats that were treated with BPs and rats that did not received BPs. Thirty-four rat especimens (Rattus novergicus, Wistar strain) were divided in 3 groups: (1) 12 mandibles and tibiae of rats treated with zoledronic acid; (2) 12 mandibles and tibiae of rats treated with clodronate; (3) control group, containing 10 mandibles and tibiae of rats that received saline solution. All individuals were exposed to cone beam computed tomography (CBCT). The images were processed and analised to obtain the bone density (BD) in Hounsfield Units (HU), using the software OsiriX 7.0. Sixty-eight histological slides were obtained from the mandibles and 34 from the tibiae, stained with hematoxilin and eosin (HE). The histological sections were scanned, and using the software Adobe Photoshop CC, the proportion of bone volume (BV) was quantified. The statistical analysis was made using ANOVA and Student’s t-test, performed by the software Stata 15.0. There was no statistically significant difference comparing the BD neither among the drug groups, nor between mandible and tibia. The comparison of BV among BPs and control group was not significant different. However, comparing the two bones, the mandibles in control group presented higher BV than tibiae of the same group. Based on the results, it is possible to conclude that (1) BD is not altered by bone type, neither by the kind of drug administered, and (2) the therapy with zoledronic acid and clodronate did not affect the BV in mandibles and tibias of tests groups.
Keywords: Bisphosphonate; Bisphosphonate-related osteonecrosis of the jaw; Cone-
beam computed tomography; Histology.
Lista de Abreviaturas e Siglas
AAOMS American Association of Oral and Maxillofacial Surgeons
ATP Adenosina Trifosfato
BD Bone Density
BF Bisfosfonato
BP Bisphosphonate
BRONJ Bisphosphonate-Related Osteonecrosis of the Jaws
BV Bone Volume
CBCT Cone Beam Computed Tomography
CD Células Dendríticas CL Clodronate
DICOM Digital Imaging and Communications in Medicine
DO Densidade Óssea
ELISA Enzyme-Linked Immunosorbent Assay
FDA Food and Drugs Administration
FDF Farnesildifosfato
FOV Field of view – Campo de Visão
GGDF Geranilgeranildifosfato
HE Hematoxilina e Eosina
HU Hounsfield Units
ITQ Inibidores de Tirosina Quinase
LB Linfócitos B LT Linfócitos T
µm Micrômetro
MicroTC Microtomografia Computadorizada
MRONJ Medication-Related Osteonecrosis of the Jaws
OCs Osteoclastos
OMAM Osteonecrose dos Maxilares Associada ao uso de Medicamentos
ONMAB Osteonecrose dos Maxilares Associada ao uso de Bisfosfonatos
OPG Osteoprotegerina
PET-CT Positron Emission Tomography
PRP Plasma Rico em Plaquetas
RANKL Receptor activator of nuclear fator kappa-B ligand
ROI Region of Interest – Região de Interesse
RP Radiografia Panorâmica
SPSS Statistical Package for Social Sciences
TC Tomografia Computadorizada
TCFC Tomografia Computadorizada de Feixes Cônicos
TIFF Tagged Image File Format
VEGF Vascular Endothelial Growth Factor
VO Volume Ósseo
Sumário
1 Introdução ............................................................................................................. 10
2 Revisão de Literatura ........................................................................................... 13
2.1 Medicamentos relacionados a OMAM ............................................................. 13
2.1.1 Bisfosfonatos ............................................................................................. 13
2.1.2 Denosumab ............................................................................................... 14
2.1.3 Drogas antiangiogênicas ........................................................................... 15
2.2 Osteonecrose dos maxilares associada ao uso de medicamentos .......... 15
2.2.1 Etiopatogênese ......................................................................................... 17
2.2.2 Características imaginológicas .................................................................. 20
2.2.3 Características histológicas ....................................................................... 21
2.2.4 Prevenção e Tratamento ........................................................................... 22
2.3 Efeitos dos BFs em diferentes tipos ósseos .................................................... 23
3 Capítulo 1 – Artigo ............................................................................................... 26
4 Considerações finais ........................................................................................... 44
Referências .............................................................................................................. 46
Anexos ..................................................................................................................... 55
9
1 Introdução
10
1 Introdução
Bisfosfonatos (BFs) são drogas antirreabsortivas empregadas na prevenção e
no tratamento de doenças caracterizadas por excessiva reabsorção óssea (ALLEN et
al., 2015; SENEL et al., 2010). Esses fármacos podem ser classificados em
nitrogenados e não nitrogenados, respectivamente, de acordo com a presença ou
ausência de nitrogênio em sua composição (KELLINSALMI et al., 2005; SIGUA-
RODRIGUEZ et al., 2014).
Descrita, inicialmente, em 2003, a osteonecrose dos maxilares associada ao
uso de BFs (ONMAB) é uma condição que se caracteriza por exposição óssea
persistente no complexo maxilomandibular de pacientes expostos ao fármaco, não
submetidos à radioterapia da região de cabeça e pescoço (FLEISHER et al., 2016;
RUGGIERO et al., 2015). A nomenclatura desta enfermidade passou por modificações
em 2014, devido à descoberta de que outras drogas antirreabsortivas e/ou
antiangiogênicas poderiam ocasionar efeito colateral similar, intitulando-se
osteonecrose dos maxilares associada ao uso de medicamentos (OMAM)
(RUGGIERO et al., 2015). O risco do desenvolvimento da OMAM tem sido relacionado
a uma variedade de fatores, dentre os quais se destacam: tipo, dosagem, duração da
terapia, medicamentos associados e realização de procedimentos orais invasivos
(RUGGIERO et al., 2014).
O achado clínico habitual da OMAM consiste em área de mucosa ulcerada com
exposição de osso desvitalizado localizada, mais frequentemente, na região póstero-
lingual da mandíbula (MARX, 2003; PAPAIAKOVOU et al., 2017; RUGGIERO et al.,
2013). Os achados imaginológicos são inespecíficos, mas podem indicar a magnitude
e progressão da osteonecrose (FLEISHER et al., 2016). Ao exame histológico,
podem-se observar áreas de necrose óssea, as quais poderão estar associadas com
a presença de colônias bacterianas e infiltrado celular inflamatório crônico (SIGUA-
RODRIGUEZ et al., 2014). A apresentação clínica da OMAM é semelhante para todas
drogas associadas, sejam antirreabsortivas ou antiangiogênicas, envolvendo as
mesmas áreas afetadas na mandíbula e os mesmos fatores predisponentes
(PAPAIAKOVOU et al., 2017).
Apesar do mecanismo etiopatogênico da OMAM ainda não ser bem elucidado,
hipotetiza-se que seja multifatorial. A literatura aponta uma possível interação entre
11
diversos fatores, como a supressão da remodelação óssea, inibição da angiogênese,
toxicidade local a variados tipos celulares, alteração na imunomodulação,
predisposição genética e associação com microrganismos (ALLEN et al., 2015;
FLEISHER et al., 2016).
O tratamento da OMAM ainda não apresenta um consenso na literatura, devido,
em parte, à precariedade de estudos clínicos randomizados e à provável etiologia
multicausal. De forma geral, existem modalidades de terapia não cirúrgica, que visam
controlar dor, infecção e progressão da doença, e tratamentos cirúrgicos para
intervenção em tecido ósseo necrótico. A avaliação odontológica, prévia ao início da
terapia antirreabsortiva/antiangiogênica, mostra-se essencial para prevenção da
osteonecrose. Também, a interrupção da terapia por tempos determinados pode
auxiliar na prevenção e resolução de lesões (FLEISHER et al., 2016, PAPAIAKOVOU
et al., 2017). O desenvolvimento de uma abordagem eficaz depende de pesquisas
que investiguem os fatores específicos que desencadeiam a lesão, bem como
métodos para o diagnóstico precoce da OMAM.
A OMAM é uma condição exclusiva dos ossos maxilares, e apesar dos avanços
em sua compreensão, ainda não estão completamente elucidados os motivos para o
não acometimento de ossos longos (FLEISHER et al., 2016). Embora ambos tipos
ósseos, maxilares e periféricos, sejam constituídos por populações celulares
semelhantes – osteoblastos, osteócitos e osteoclastos, sabe-se que não apresentam
a mesma origem embrionária. Neste sentido, diferenças biomecânicas e funcionais,
como a taxa de remodelação óssea, podem ser significativas (GONG et al., 2017). Da
mesma forma, as respostas à terapia antirreabsortiva/antiangiogênica podem ser
distintas (OYHANART et al., 2015). Diante do exposto, o presente estudo teve como
objetivo realizar a avaliação comparativa da estrutura óssea de mandíbulas e tíbias
de ratos usuários e não usuários de BFs, por meios imaginológicos e
histomorfométricos.
12
2 Revisão de Literatura
13
2 Revisão de Literatura
2.1 Medicamentos relacionados a OMAM
2.1.1 Bisfosfonatos
2.2.1.1 Estrutura química e classificação
Os BFs são análogos sintéticos do pirofosfato inorgânico, um regulador
fisiológico do metabolismo ósseo, que apresenta alta afinidade pelo cálcio. Esses
compostos são obtidos pela substituição do átomo de oxigênio pelo carbono,
tornando-se mais resistentes à degradação enzimática. A estrutura química dos BFs
é formada por dois grupamentos fosfatos ligados, covalentemente, ao carbono central.
Associados ao átomo de carbono encontram-se os radicais R1 e R2. O grupo R1
fornece a afinidade pelos cristais ósseos e o grupo R2 é responsável pela potência e
atividade farmacológica (RUSSELL, 2007; SIGUA-RODRIGUEZ et al., 2014).
Esses compostos podem ser classificados em três categorias, de acordo com
a presença ou ausência/localização do nitrogênio. Clodronato, etidronato e tiludronato
estão inseridos na primeira geração, na qual o grupamento R2 não apresenta
nitrogênio. Alendronato, pamidronato, ibandronato, neridronato e olpadronato
possuem nitrogênio no grupo R2, e fazem parte da segunda geração. Risedronato e
zoledronato compõem a terceira geração, e são caracterizados pela presença de
nitrogênio em cadeia cíclica, no radical R2 (RUGGIERO et al., 2009; RUSSELL et al.,
2008).
2.2.1.2 Mecanismos de ação
Os BFs possuem alta afinidade pela hidroxiapatita óssea, e assim, são
rapidamente carreados da circulação sistêmica e depositados na superfície óssea
mineral, particularmente nos sítios de atividade osteoclástica. O meio ambiente ácido
associado aos osteoclastos (OCs) em fase de reabsorção, propicia a liberação do BF
da superfície óssea e a subsequente captação pelas células osteoclásticas (BROWN,
2017; MIGLIORATI et al., 2005).
Os BFs nitrogenados e não nitrogenados interferem na função osteoclástica
através de mecanismos distintos. Aqueles que possuem nitrogênio atuam diretamente
na via biossintética do mevalonato inibindo a farnesildifosfato (FDF) e a
geranilgeranildifosfato (GGDF), que são requeridas para a formação de proteínas de
14
sinalização celular. Tais proteínas regulam processos da função osteoclástica, como
o arranjo do citoesqueleto, morfologia celular, transporte vesicular e apoptose
(FLEISHER et al., 2016; RUSSELL et al., 2007). Os não aminados possuem ação
citotóxica por produção de análogos do trifosfato de adenosina (ATP) os quais, por
serem resistentes à hidrólise, acumulam-se nos OCs, o que resulta em apoptose por
inativação de enzimas metabólicas intracelulares (RUSSELL et al., 2007;
VASCONCELOS et al., 2012). Após incorporados ao tecido ósseo, os BFs não são
metabolizados e estima-se que tenham uma meia vida de 11,2 anos (FLEISHER et
al., 2016).
2.2.1.3 Efeitos adversos
Apesar de infrequentes, os BFs produzem efeitos adversos como fadiga,
distúrbios gastrointestinais, anemia, edema, alterações da mucosa esofágica,
alterações da função renal, e aumento do risco de fraturas de fêmur atípicas (BROWN,
2017; RUGGIERO et al., 2009). Em 2003, foram relatadas alterações orais em
pacientes usuários dessa classe de medicamento (MARX, 2003), inicialmente
intitulada como osteonecrose dos maxilares associada ao uso de BFs (ONMAB).
2.1.2 Denosumab
O denosumab é um anticorpo monoclonal humano, do tipo IG2, direcionado
contra o receptor activator of nuclear factor kappa-B ligand (RANKL). Inicialmente
aprovado para o tratamento de osteoporose pela Food and Drug Administration (FDA),
em 2010, tem sido administrado para outras condições associadas à disfunção óssea
(BAGAN et al., 2016; PAPAIAKOVOU et al., 2017). Por mimetizar os efeitos
fisiológicos da citocina osteoprotegerina (OPG), impede a formação do complexo
RANK-RANKL, e bloqueia a ativação de OCs, suprimindo a formação e diferenciação
dessas células – agindo, portanto, como uma droga antirreabsortiva (PAPAIAKOVOU
et al., 2017; RUGGIERO et al., 2014). Contrariamente aos BFs, o denosumab não se
incorpora à estrutura óssea, resultando em uma menor meia-vida (25-32 dias versus
10-12 anos dos BFs), com consequente retorno da remodelação óssea a níveis
fisiológicos após interrupção da terapia. Ainda, a literatura sugere que a potência
farmacológica seja maior em relação aos BFs (BAGAN et al., 2016; PAPAIAKOVOU
et al., 2017). Por não ser metabolizado pela via renal, o denosumab não apresenta
contraindicação para pacientes nefropatas. Desde 2010, estima-se que cerca de 30
15
casos de osteonecrose dos maxilares associado ao denosumab foram descritos em
literatura inglesa (YOSHIMURA et al., 2017).
2.1.3 Drogas antiangiogênicas
Na última década, a evolução dos protocolos terapêuticos oncológicos, deveu-
se à busca pela redução da toxicidade em tecidos não neoplásicos. Desta forma,
drogas capazes de controlar o crescimento e sobrevivência de células malignas por
meio da interferência de rotas moleculares específicas envolvidas na carcinogênese,
como a sanguínea, tem sido desenvolvidas. O bevacizumab constitui um anticorpo
monoclonal figurado para inibir, de forma seletiva, todas as isoformas do vascular
endothelial growth factor (VEGF), e representa a primeira classe de droga inibidora da
angiogênese - aprovado na prática clínica, em 2004, inicialmente para tratamento de
câncer colorretal (ASHRAFI et al., 2017). O VEGF é uma proteína de sinalização que
estimula a formação de vasos sanguíneos, e, portanto, esses fármacos inibem a
angiogênese através da interferência na cascata de sinalização. A evidência de que o
VEGF é essencial para a diferenciação e formação óssea possibilita inferir que as
propriedades antiangiogênicas do bevacizumab podem comprometer a integridade
microvascular, culminando com o comprometimento ósseo e interferindo na
cicatrização de tecidos moles (ASHRAFI et al., 2017).
O sunitinib é um inibidor de tirosina quinase (ITQ) capaz de dificultar a função
dos receptores de superfície celular que iniciam o crescimento, diferenciação e
sobrevivência de células malignas. Adicionalmente, inibe o KIT (CD117) e o receptor
estimulante de colônias do fator 1 (CSF1R) (FANTASIA et al., 2015; FLEISHER et al.,
2016; RUGGIERO et al., 2014). Essas drogas mostram-se eficazes no tratamento de
tumores gastrointestinais, carcinomas renais, pulmonares e tumores neuroendócrinos
(FANTASIA et al., 2015; RUGGIERO et al., 2014).
2.2 Osteonecrose dos maxilares associada ao uso de medicamentos
A OMAM caracteriza-se por: (1) terapia prévia ou atual com drogas
antirreabsortivas e/ou antiangiogênicas; (2) exposição óssea, na região maxilofacial,
persistente por mais de 8 semanas após identificação profissional; (3) ausência de
histórico de radioterapia na região de cabeça e pescoço ou de metástase óssea
evidente (ALLEN, 2015; FLEISHER et al., 2016; KHAN et al., 2015; RUGGIERO et al.,
2015).
16
De acordo com Brown (2017), a prevalência entre pacientes oncológicos que
recebem BFs pela via intravenosa varia de 0% a 12,2%, com índices
consideravelmente menores, oscilando de 0,001% a 0,15%, entre pacientes que
recebem BFs por via oral para tratamento da osteoporose (BROWN, 2017). Para o
denosumab, quando utilizado para o tratamento antineoplásico, estima-se que a
incidência da OMAM varie entre 0,7% e 1,9%, com média de 0,04% naqueles
pacientes que o utilizam como agente antiosteoporótico (BAGAN et al., 2016; KHAN
et al., 2015). Em relação às drogas antiangiogênicas, a incidência de OMAM no
tratamento oncológico com bevacizumab é estimada entre 0,3% e 0,4% (FANTASIA
et al., 2015).
A OMAM poderá ocorrer espontaneamente ou, mais comumente, em local de
lesão prévia, como exodontia, doença periodontal, ou trauma dentoalveolar
(FLEISHER et al, 2016; RIZZOLI et al., 2008; PAZIANAS et al., 2007). Ainda, a
duração da terapia com BFs, tipo e forma de administração do medicamento são
fatores predisponentes ao surgimento dessa condição, especialmente associada ao
uso dos aminobisfosfonatos (FLEISHER et al., 2016; SENEL et al., 2010). Ruggiero
(2011) afirma que os agentes quimioterápicos e os esteroides administrados a
pacientes oncológicos podem interferir na cicatrização óssea e devem ser
considerados como fatores de risco. Pacientes imunocomprometidos também
poderão apresentar atraso no processo de remodelação óssea (CAVANNA et al.,
2007; KHAN et al., 2015).
Clinicamente, a OMAM caracteriza-se por área de mucosa ulcerada com
exposição de osso desvitalizado, de coloração branco-amarelada, localizada, mais
frequentemente, em região póstero-lingual mandibular (MARX et al., 2005,
RUGGIERO et al., 2014). No entanto, o estágio inicial da doença não envolve
exposição óssea (PAPAIAKOVOU et al., 2017). Esse achado clínico pode ser
precedido por dor e/ou desconforto na região envolvida, embora regiões de osso
necrótico exposto possam permanecer assintomáticas. O tamanho pode variar desde
um sítio de exodontia não cicatrizado até o completo envolvimento do maxilar.
Ocasionalmente, pode haver parestesia, dor, edema e fístula intra ou extraoral
(RUGGIERO, 2015). Um sistema de estadiamento clínico foi desenvolvido por
Ruggiero et al. (2006) e atualizado pela última vez em 2014, pela AAOMS, com o
17
objetivo de categorizar os casos para direcionamento terapêutico e prognóstico
(Tabela 1) (FLEISHER et al., 2016; RUGGIERO et al., 2011).
Tabela 1 - Estadiamento da Osteonecrose Associada ao Uso de Medicamentos de acordo com a
American Association of Oral and Maxillofacial Surgeons (2014).
ESTÁGIO CARACTERÍSTICAS CLÍNICAS
Categoria de Risco Ausência de osso exposto ou necrótico em pacientes tratados
com drogas antirreabsortivas ou antiangiogênicas.
Estágio 0 Sintomas e achados clínicos não específicos (como dor nos
ossos maxilares e osteosclerose) estão presentes, mas não há
evidência clínica de osso exposto.
Estágio 1 Osso necrótico e exposto, ou osso sondável através de fístula,
em pacientes assintomáticos. Não há evidência de infecção.
Estágio 2 Osso necrótico e exposto, ou osso sondável através de fístula,
associado à infecção, evidenciada pela dor e pela presença de
eritema na região. Pode conter drenagem purulenta ou não.
Estágio 3 Osso necrótico e exposto, ou osso sondável através de fístula,
associado a dor, infecção, e um ou mais dos seguintes sintomas:
fratura patológica, fistula extraoral, comunicação oral-antral ou
oral-nasal, e osteólise estendendo-se até o assoalho do seio
maxilar ou borda inferior da mandíbula.
2.2.1 Etiopatogênese
Várias teorias têm sido postuladas para explicar a patogênese da OMAM, que
parece ter base multifatorial (ALLEN; BURR, 2009; ALLEN, 2015). Acredita-se que o
impacto dos fármacos, em especial os BFs, nos diferentes tipos celulares seja capaz
de contribuir para a dificuldade da reconstituição tecidual após dano/lesão, o que
resulta nas manifestações clínicas (ALLEN; BURR, 2009). Deste modo, cinco
principais linhas de pesquisa buscam entender o mecanismo de desenvolvimento da
doença: metabolismo ósseo, infecção, angiogênese, genética e tecidos moles.
18
2.2.1.1 Metabolismo Ósseo
A homeostase óssea depende do equilíbrio entre osteoblastos e OCs. Os OCs
são células gigantes multinucleadas especializadas em reabsorção óssea, a qual
depende da citocina RANKL, essencial na sua formação, atividade e sobrevivência,
em condições fisiológicas e patológicas. O RANKL, sintetizado por osteoblastos,
linfócitos T e B (LT e LB), osteócitos, macrófagos e fibroblastos, liga-se ao RANK -
presente na superfície de precursores de OCs, CDs, fibroblastos e LTs - promovendo
a ativação de OCs (LAPÉRINE et al., 2016; VASCONCELOS et al., 2013). A OPG,
produzida em parte por LBs maduros, também medeia a reabsorção óssea, sendo um
antagonista do RANKL - inibindo a interação RANK/RANKL (FLEISHER et al., 2016;
LAPÉRINE et al., 2016).
Os BFs, nitrogenados e não nitrogenados, inibem a atividade dos OCs
causando desorganização do citoesqueleto, perda da borda “em escova” (requerida
para reabsorção óssea), defeitos no sistema de transporte vesicular, interferência na
quimiotaxia, recrutamento, ativação e diferenciação de precursores, além de estímulo
à apoptose (BROWN, 2017; FLEISHER et al., 2016). Quando o remodelamento ósseo
se encontra prejudicado pela inibição da atividade osteoclástica, a matriz sofre
degradação e, consequentemente, há acúmulo de osso não vital (BI et al., 2010,
KELLINSALMI et al., 2005, SENEL et al., 2010, SONIS et al., 2009, VASCONCELOS
et al., 2012, XU et al., 2013).
2.2.1.2 Microrganismos
Vários estudos observam, in vivo, a presença de conglomerados bacterianos e
fúngicos em lesões de OMAM (HANSEN et al., 2006, SEDGHIZADEH et al., 2008,
2009, 2012). Esses estudos revelam, ainda, que em todos os espécimes foram
detectados sítios de reabsorção óssea ativos, contendo grande número de bactérias
de múltiplas espécies. O avanço do biofilme para o interior das lacunas pareceu ser
responsável pelas bordas irregulares encontradas ao longo do tecido reabsorvido.
Células eucarióticas, como OCs, não foram encontradas nas cavidades ou próximas
a elas, sugerindo uma ação direta do biofilme bacteriano na reabsorção óssea.
Apesar disso, não há consenso em relação ao fato de o processo infeccioso ocupar
posição primária ou secundária na fisiopatologia da condição (ALLEN; BURR, 2009).
19
2.2.1.3 Angiogênese
A angiogênese descreve o mecanismo de crescimento de novos vasos
sanguíneos a partir daqueles já existentes (MAAHS et al., 2011). Estudos in vitro e in
vivo apontam a capacidade antiangiogênica dos BFs (BI et al., 2010; NAIDU et al.,
2008; WALTER et al., 2009; YAMADA et al., 2009; WOOD et al., 2002). Tais efeitos
tem sido demonstrados na inibição da proliferação, migração e adesão das células
endoteliais (WALTER et al., 2009; YAMADA et al., 2009). Kobayashi et al. (2010)
afirma que a capacidade do ácido zoledrônico em inibir, in vitro, a proliferação e a
migração de células endoteliais ocorrem de maneira dose-dependente (KOBAYASHI
et al., 2010).
2.2.1.4 Genética
Fatores genéticos estão associados com a imunidade, cicatrização de feridas
e proteção celular. A literatura recente enfatiza que a baixa expressão de genes
específicos pode estar associada com o aumento da susceptibilidade à OMAM
(PAPAIAKOVOU et al., 2017). Estudos de farmacogenômica sugerem que
polimorfismos nos genes da farnesil pirofosfato sintase e/ou do citocromo P450
CYP2C8 possam predispor indivíduos ao desenvolvimento de OMAM. Considerando-
se que nem todos os pacientes com comorbidades e esquemas terapêuticos
semelhantes desenvolvem a doença, reforça-se a suspeita de um envolvimento
genético (ALLEN, 2015).
2.2.1.5 Tecidos Moles
Os efeitos dos BFs sobre as células epiteliais da mucosa oral são pouco
esclarecidos. O estudo de Vasconcelos et al. (2012) mostrou, em modelo animal
tratado com BFs nitrogenados e não nitrogenados, que o envolvimento dos tecidos
moles como iniciadores do desenvolvimento da osteonecrose é menos provável do
que se acreditava. No entanto, pesquisas in vitro, demonstraram que os BFs são
capazes de agir sobre fibroblastos e queratinócitos, reduzindo a proliferação celular,
retardando a migração celular e induzindo a apoptose (ALLEN et al., 2015;
LANDESBERG et al., 2011).
20
2.2.2 Características imaginológicas
Radiografia panorâmica (RP) e periapical, tomografia computadorizada (TC),
tomografia computadorizada de feixes cônicos (TCFC), ressonância magnética e
cintilografia óssea estão entre os exames por imagem que podem ser usados para
avaliar a magnitude e a progressão da OMAM (BEDOGNI et al., 2007). As
características mais frequentes nas imagens incluem esclerose óssea, osteólise,
espessamento da lâmina dura, irregularidade cortical, radiolucências,
fragmentação/formação de sequestro, comunicação buco-sinusal e alvéolos dentários
persistentes pós-extração (TREISTER et al., 2010; TORRES et al., 2011).
O estadiamento da patologia, segundo a AAOMS, inclui características clínicas
e imaginológicas. No entanto, a inspeção visual tende a identificar apenas sinais
superficiais, não refletindo necessariamente a total extensão do acometimento ósseo.
Há relatos de casos de OMAM em que não há evidências clínicas de lesão, porém,
imaginologicamente, pode-se observar áreas compatíveis com necrose óssea
(BEDOGNI et al., 2014).
A RP proporciona uma excelente visualização dos ossos maxilares em sua
totalidade, sendo uma ferramenta imaginológica útil e acessível para avaliação de
pacientes sob terapia com drogas antirreabsortivas e/ou antiangiogênicas. Esse
exame é capaz de revelar lesões osteolíticas, principalmente quando há áreas
compatíveis com sequestro ósseo ou osteólise combinada à osteoesclerose (LEITE
et al., 2014). No entanto, a perda mineral deve alcançar de 30% a 50% para que seja
visível neste tipo de imagem (CANKAYA et al., 2011). Essa modalidade apresenta,
ainda, outras desvantagens como a falta de definição das margens entre áreas
afetadas pela osteonecrose e osso saudável e a visão bidimensional limitada de
estruturas tridimensionais. Além disso, por ser uma radiografia convencional, a
imagem pode sofrer magnificação, distorção e sobreposição de estruturas, exigindo
correto posicionamento do paciente e técnica apropriada (LEITE et al., 2014).
A TC mostra-se útil na visualização de detalhes do osso trabecular e cortical,
além de permitir estimar a real extensão das áreas de osteonecrose. No entanto,
apresenta maior custo e alta exposição à radiação, comparativamente à RP
(CANKAYA et al., 2011). A TCFC consiste em uma técnica de imagem que utiliza
menor quantidade de radiação, comparativamente à técnica convencional, com a
21
capacidade de capturar múltiplas imagens, à semelhança da TC. As informações
obtidas são processadas e reconstruídas em diferentes planos (reconstruções
multiplanares), e caso necessário, tridimensionalmente (CANKAYA et al., 2011). Em
estudo in vivo, Cankaya et al. (2011), concluíram que a TCFC apresenta alta
detectabilidade para a OMAM, sendo possível avaliar a distribuição mineral e a
densidade óssea. Porém, Martelli et al. (2017) concluíram, após estudo in vivo, que a
TCFC não constitui um exame sensível para detectar os primeiros sinais de alteração
óssea em indivíduos sob terapia com BFs nitrogenados e não nitrogenados. Como
desvantagem, as imagens geradas podem conter inconsistências e arbitrariedades
nos valores de cinza, devido a fatores como o tamanho do voxel e da janela (field of
view – FOV) utilizados na aquisição das imagens, ou o tipo de detector do tomógrafo
(CAMPOS et al., 2014; PAUWELS et al., 2013).
A microtomografia computadorizada (microTC) está sendo amplamente
utilizada na análise estrutural de ossos, dentes, cerâmicas, polímeros e bioscaffolds.
Esse exame permite a obtenção de informações longitudinais de alta qualidade em
ossos e dentes, porém sua alta dose de radiação e elevado custo constituem
desvantagens que tem limitado seu uso em pesquisas (ANDERSON et al., 2014).
Hesse et al. (2014) utilizaram microTC para avaliação de lacunas de osteócitos em
amostras de tecido ósseo necrótico de pacientes com OMAM e de tecido ósseo vital
de pacientes saudáveis. A morfologia do sistema lacuno-canalicular, através do qual
ocorre a intercomunicação entre os osteócitos, está indiretamente associada ao
metabolismo dos ossos e, portanto, relacionada à qualidade desse tecido. Assim, os
autores realizaram a quantificação tridimensional do volume das lacunas, além de sua
forma e distribuição espacial. Os volumes lacunares dos espécimes com OMAM
apresentaram a mesma variação dos espécimes saudáveis, concluindo-se que esse
não é um fator chave no desenvolvimento de OMAM.
2.2.3 Características histológicas
Marx et al. (2012) compararam, histopatologicamente, amostras obtidas por
meio de ressecção óssea em pacientes apresentando osteomielite supurativa,
osteorradionecrose e OMAM. Em todos os casos de OMAM foi identificado tecido
ósseo necrótico evidenciado por osteoplastos vazios, ausência de osteoblastos na
periferia, sistemas de Havers e canais de Volkmann vazios, e ainda, todo o espaço
22
medular mostrou-se acelular. Em 76% dos espécimes foi possível observar
microrganismos localizados na superfície óssea (MARX et al., 2012). De acordo com
a revisão sistemática de Hinson et al. (2014), o microrganismo mais comumente
identificado em espécimes de OMAM é Actinomyces sp., seguido por Streptococcus.
Colônias de Candida, Staphylococcus e Klebsiella, embora menos comuns, também
foram identificadas.
2.2.4 Prevenção e tratamento
Embora, na última década, esforços tenham sido realizados no sentido de
entender a etiopatogenia da OMAM, ainda não há consenso sobre o manejo da
condição (FLEISHER et al., 2016; KHAN et al., 2015; SIGUA-RODRIGUEZ et al.,
2014). O principal foco do tratamento deve ser o controle da dor e da infecção,
especialmente naqueles pacientes com estado de saúde mais gravemente
comprometido. Tal estratégia pode ser instituída com o uso de antimicrobianos tópicos
e/ou sistêmicos (FLEISHER et al., 2016; MARX et al., 2005; PAPAIAKOVOU et al.,
2017). Ainda, destaca-se que a avaliação odontológica prévia ao início da terapia é
indispensável (PAPAIAKOVOU et al., 2017).
Nos casos em que procedimentos odontológicos ou cirurgias orais não possam
ser evitados, sugere-se que sejam realizados de forma mais atraumática.
Adicionalmente, como medida preventiva/terapêutica, a literatura sugere que seja feito
um drug holiday, isto é, um período de interrupção da terapia
antirreabsortiva/antiangiogência. Em geral, é recomendado que haja interrupção por
3 meses antes da realização de qualquer procedimento cirúrgico intraoral que envolva
tecido ósseo (PAPAIAKOVOU et al., 2017).
Aplicação de fatores de crescimento tem sido amplamente explorados na
prevenção e tratamento da OMAM. Estudos clínicos apresentam a efetividade do
concentrado plaquetário autólogo na redução do tempo da ferida cirúrgica após
extrações dentárias, prevenindo, desta forma, o desenvolvimento da doença (DEL
FABRO et al., 2015). Administração de leucócitos e plaquetas ricos em fibrina, a
segunda geração do concentrado plaquetário, mostra-se associada ao aumento da
atividade osteoblástica (DINCA et al., 2014; LONGO et al., 2014; TSAI et al., 2016).
Aplicação de proteínas morfogenéticas do tipo 2 tem apresentado resultados
23
satisfatórios em pacientes com doença refratária ao tratamento convencional (KIM et
al., 2016).
2.3 Efeitos dos BFs em diferentes tipos ósseos
Os ossos da região maxilofacial originam-se exclusivamente do
neuroectoderma e formam-se principalmente através de ossificação do tipo
intramembranosa, ao passo que os periféricos tem origem no mesoderma e
desenvolvem-se por ossificação endocondral e intramembranosa. Apesar de ambos
tipos ósseos apresentarem estrutura semelhante e serem constituídos por populações
celulares similares – osteoblastos, osteócitos e osteoclastos, diferenças nas
propriedades mecânicas, bem como na taxa de remodelação óssea, podem ser
observadas (GONG et al., 2017). Da mesma forma, as respostas à terapia
antirreabsortiva e/ou antiangiogênicas podem diferir entre os tipos ósseos. A OMAM
é uma condição exclusiva dos maxilares, e apesar dos avanços em sua compreensão,
ainda não estão bem esclarecidos os motivos para o não acometimento de ossos
longos (FLEISHER et al., 2016).
Oyhanart et al. (2015) demonstraram em ratos em crescimento, através de
análise morfométrica e histomorfométrica, que o alendronato, um BF nitrogenado,
altera o desenvolvimento da mandíbula e de ossos longos, na ausência de
procedimentos cirúrgicos. Tal efeito decorre da alteração do mecanismo de
modelação e remodelação óssea, que afeta o processo de ossificação endocondral
que ocorre em ossos longos e em parte da mandíbula. Os autores observaram
diminuição significativa no comprimento da tíbia, fêmur e mandíbula, e menor
espessura da cartilagem epifisária da tíbia de animais que receberam o fármaco, em
comparação com o grupo controle (OYHANART et al., 2015).
Vermeer et al. (2017) investigaram a diferente resposta de OCs e seus
precursores em mandíbula e ossos longos de ratos adultos, frente à terapia com o
aminobisfosfonato ácido zoledrônico. Os autores observaram, através de
histomorfometria, que a taxa de remodelação óssea na tíbia e fêmur diminuiu
significativamente após 6 meses de terapia com o fármaco, o que não ocorreu em
mandíbula. No osso maxilar, houve redução significativa no número de células com
potencial osteoclastogênico que puderam ser isoladas da medula óssea (VERMEER
et al., 2017).
24
Conforme supracitado, a interação entre as moléculas RANK/RANKL/OPG é
essencial para maturação dos OCs e regulação do metabolismo ósseo (FLEISHER et
al., 2016). Utilizando o método ELISA para determinação da concentração de RANKL
e OPG em mandíbulas e tíbias de ratos tratados com ácido zoledrônico, Cankaya et
al. (2013) observaram aumento da OPG em ambos ossos. No entanto, em relação ao
RANKL, os níveis em mandíbula apresentaram-se diminuídos e na tíbia, aumentados,
em comparação com o grupo controle. Ainda, a menor proporção de RANKL/OPG foi
observada na mandíbula dos animais que receberam a droga. Os resultados apontam
para uma inibição da ativação de OCs, com possível diferença no mecanismo de ação
entre mandíbula e ossos longos (CANKAYA et al., 2013). Tal hipótese é corroborada
pelo estudo de Gong et al. (2017), no qual também foram observadas diferenças,
através de ELISA, entre os ossos maxilares (maxila ou mandíbula) e ossos periféricos
(íleo e tíbia) de ratos. A administração de ácido zoledrônico resultou em uma
diminuição na razão RANKL/OPG nos maxilares, e um aumento desta proporção nos
ossos periféricos. Em adição, nesta mesma pesquisa, mostrou-se que células da
medula óssea dos maxilares são mais suscetíveis aos efeitos do BF do que dos ossos
periféricos, uma vez que apresentaram menor taxa de proliferação em cultura (GONG
et al., 2017).
Em relação às propriedades mecânicas após terapia com ácido zoledrônico,
Camacho et al. (2012) compararam a resistência à fratura do côndilo mandibular e
cabeça do fêmur em ratos, através de teste biomecânico. Os resultados mostraram
que o tratamento aumenta a resistência à fratura em ambos os ossos,
proporcionalmente à dose recebida pelos animais. Ainda, utilizando microscopia
eletrônica de varredura, observou-se ausência de microfraturas no grupo
experimental, em comparação à presença dessas em 30% do grupo controle. Assim,
a diminuição no risco de fratura em usuários de BF ocorre pela manutenção ou
melhora das propriedades biomecânicas em ambos tipos ósseos (CAMACHO et al.,
2012).
25
3 Capítulo 1 Artigo
26
3 Capítulo 1 – Artigo
O seguinte artigo foi formatado de acordo com as normas da revista Oral
Diseases – Qualis A1 e fator de impacto 2.011, e submetido para publicação.
27
Imaginological and histomorphometrical evaluation of mandibles and tibias of
rats submitted to zoledronic acid and clodronate treatment
Short title: Medication-related osteonecrosis of the jaws.
Research Article
Stephanie Joana Roman Martelli¹, Melissa Feres Damian, PhD², André Ribeiro
Schinestsck³, Andreia Morales Cascaes, PhD², Ana Carolina Uchoa Vasconcelos,
PhD²
¹ Master Student, Graduate Program in Dentistry, Federal University of Pelotas, Rua
Gonçalves Chaves 457, 96015-560, Pelotas, RS, Brazil.
² Professor, Graduate Program in Dentistry, Federal University of Pelotas, Rua
Gonçalves Chaves 457, 96015-560, Pelotas, RS, Brazil.
³ Post Graduate Student, Graduate Program in Dentistry, Federal University of Pelotas,
Rua Gonçalves Chaves 457, 96015-560, Pelotas, RS, Brazil.
Corresponding author: Stephanie Joana Roman Martelli, Serviço de Patologia Bucal,
Universidade Federal de Pelotas – UFPel, Rua Gonçalves Chaves, 457, Sala 607,
Pelotas-RS, Brasil. CEP: 96015-560. Tel./fax: +55 53 3225 6741.
E-mail: [email protected].
28
Abstract
Objectives: To evaluate imaginologically and histomorphometrically mandibles and
tibiae of rats treated with bisphosphonates (BPs).
Methods: Thirty-four rat specimens (Rattus novergicus, Wistar strain) were distributed
into 3 groups: (1) 12 rats treated with zoledronic acid; (2) 12 rats treated with
clodronate; and (3) the control group, containing 10 rats that received saline solution.
Mandibles and tibias were exposed to cone beam computed tomography (CBCT). The
images were analised to obtain the bone density (BD), using the software OsiriX 7.0.
Histological slides were obtained from the specimens and, using the software Adobe
Photoshop CC, the proportion of bone volume (BV) was quantified.
Results: There was no statistically significant difference comparing the BD neither
among the drug groups, nor between mandible and tibia. The comparison of BV among
BPs and control group was not significant different. However, comparing the two
bones, the mandibles in control group presented higher BV than tibiae of the same
group.
Conclusions: Based on the results, it is possible to conclude that (1) BD is not altered
by bone type, neither by the kind of drug administered, and (2) the therapy with
zoledronic acid and clodronate did not affect the BV in mandibles and tibias of tests
groups.
Keywords: Bisphosphonate; Cone beam computed tomography; jaws.
29
Introduction
Bisphosphonates (BPs) are antiresorptive drugs, which are the most commonly
prescribed for osteoporosis treatment (Allen, 2015; Oyhanart et al, 2015). They are
also used in other conditions related to altered bone turnover, such as Paget’s disease,
multiple myeloma, and bone metastasis originated from malignant neoplasias (Sigua-
Rodriguez et al, 2014; Fleisher et al, 2016). The nitrogen-containing BPs (N-BPs)
present higher pharmacological potency and exert their mechanism of action inhibiting
the mevalonate biosynthetic pathway, affecting the production of signalling molecules
that modulate osteoclast (OC) function. The non-nitrogen-containing BPs (non-N-
BPs), on the other hand, are metabolized into adenosine triphosphate (ATP) cytotoxic
analogs that accumulate inside the OCs. Thus, BP therapy alters bone metabolism
and reduce bone resorption, due to the interference and/or inhibitory effects over cells
involved in this physiological proccess, specially the OCs (Russell, 2007, 2007; Sigua-
Rodriguez et al, 2014; Oyhanart et al, 2015; Fleisher et al, 2016).
The BP-related osteonecrosis of the jaws (BRONJ) was initially described as a
side effect of BP therapy in 2003 (Marx, 2003). In 2014, the American Association of
Oral and Maxillofacial Surgeons (AAOMS) updated the nomenclature, due to reports
of other antiresorptive agents (as denosumab) and antiangiogenic drugs (as sunitinib
and bevacizumab) associated with the disorder, which was renamed to medication-
related osteonecrosis of the jaws (MRONJ) (Ruggiero et al, 2014; Khan et al, 2015;
Fleisher et al, 2016). The condition’s etiology seems to be multifactorial and, clinically,
most commonly develops after dentoalveolar surgery or another kind of trauma,
although spontaneous occurrence is possible (Sigua-Rodriguez et al, 2014; Khan et
al, 2015).
MRONJ occurs exclusively in the jaws, and despite the advances in its
comprehension, it is not completely clear why it does not develop in long bones. It is
supposed that it may be a consequence of: high bone turnover rate of the jaws;
presence of teeth and gum, facilitating the entrance of microrganisms and predisposing
for infections; and oral structures subjected to a variety of stresses, such as
physiologic, iatrogenic or inflammatory, causing trauma to the mucosa with exposure
of bone (Fleisher et al, 2016). Although both types of bone, jaws and peripheral/long
bones, show similar structure and are composed by the same cell population –
30
osteoblasts, osteocytes and OCs, they exhibit distinct embrionary origin and their
biomechanical and functional differences, as the bone turnover rate, might be
significant (Gong et al, 2017). Moreover, the response to antiresorptive therapy might
be diverse considering different bones (Oyhanart et al, 2015).
The clinical and imaginological examination, associated with the patient’s
medical history, are the most reliable diagnostic approach to MRONJ (Khan et al, 2015;
Ruggiero, 2015). Imaging techniques play an essential role not only to infer the disease
progression, but also to early detect MRONJ and tailor therapeutic management for
each patient (Hamada et al, 2014; Eleutherakis-Papaiakovou and Bamias, 2017).
Advanced imaging procedures like computed tomography (CT), cone beam CT
(CBCT) and positron emission tomography (PET)-CT may be more sensible to detect
lesions, compared to conventional images such as panoramic radiography
(Eleutherakis-Papaiakovou and Bamias, 2017). Furthermore, CBCT has become an
important tool in dentistry, due to its lower radiation dose compared to CT, and studies
have shown that it exhibits high sensibility in measuring mineral distribution, detect
periosteal thickening and bone densiy (BD) alterations (Cankaya et al, 2011;
Guggenberger et al, 2014). The microcomputed tomography (microCT) modality has
been widely employed for high quality structural analysis of bone, teeth and other
materials. However, the high cost and radiation dose limit the use in research
(Anderson et al, 2014).
A number of in vivo studies demonstrate BPs effects in bone tissue following
procedures (Cankaya et al, 2011; Maahs et al, 2011; Vasconcelos et al, 2012; Barba-
Recreo et al, 2014; Gong et al, 2017). Nevertheless, there is a lack of data about the
comparison between different types of bones and between antiresorptive drugs.
Hence, the objective of the present study was to compare by means of
histomorphometrical and imaginological parameters, among mandibles and tibiae, and
N-BP (zoledronic acid) and non-N-BP (clodronate), in order to investigate possible
different effects over the bone structure.
Methods
Ethics
31
The present study was approved by the Ethics Committee for Animal Use of
the Pontifical Catholic University of Rio Grande do Sul, and the procedures were
carried out in accordance with the institutional guidelines for animal care and use.
Animals
The sample was comprised of 34 adult female rats (Rattus norvegicus, Wistar
strain), which had a mean age of 120 days and a mean weight of 230g
(VASCONCELOS et al., 2012). The sample size was based in the previous studies of
Maahs et al. (2011) and Vasconcelos et al. (2012), which used similar methodologies.
The animals were randomly allocated to 3 groups, each according to the BP used: (1)
zoledronic acid (ZA) group: 12 animals were treated with the N-BP ZA (Novartis
Pharma AG, Basel, Switzerland) intraperitoneally (0.6 mg/kg, every 28 days)
(Macerata, 2002; Maahs et al, 2011; Vasconcelos et al, 2012); (2) clodronate (CL)
group: 12 animals treated with the non-N-BP CL (Jenahexal Pharma GmbH, Thuringia,
Germany), intraperitoneally (20 mg/kg, every 28 days) (Vasconcelos et al, 2012); and
(3) control group: 10 animals received saline solution (0.9% sodium chloride). The
intraperitoneal administration is commonly used in laboratory rodents, due to the
difficulty in intravenous access and possibility to administer large amounts of fluids. In
addition, it reduces the risk of adverse effects and provides more comfort to the animal
(Turner et al, 2011).
After 120 days of the drug administration, the animals were euthanized by deep
anesthesia with isoflurane (Cristalia, Porto Alegre, RS, Brazil) in an appropriate
anesthesia chamber. The specimens were then examined using a no.5 clinical probe
(SS White, Duflex, Rio de Janeiro, RJ, Brazil) to determine the presence/absence of
macroscopic bone lesion. Afterwards, the mandibles and left tibiae were dissected and
fixed for 24h in 10% buffered formalin (TopGlass, Porto Alegre, RS, Brazil).
CBCTs
All samples were submitted to CBCT, using a Prexion 3D® tomograph
(Terarecon, San Mateo, CA, USA). The mandibles and tibiae were allocated in plastic
boxes, each composed by three 2,5x6,0cm compartments containing 1 mandible or 1
tibia each, immersed in formalin to attenuate radiation. The boxes were positioned in
the machine with the aid of polystyrene boxes, to acquire ideal height for the
32
tomographic scanning. The following technical parameters were applied: 90 kVp, 4
mA, voxel resolution 0.1 mm, 37s, and a 56x52mm field of view (FOV). The obtained
images were stored in a hard drive in Digital Imaging and Communications in Medicine
(DICOM) format.
Tomographic analysis
The tomographic images were visualized and evaluated using the Osirix 7.0
software (OsiriX, http://www.osirix-viewer.com) by a calibrated and blinded examiner.
The Multiplanar Reconstructions Screen (MPR Screen) visualization was used to
observe the sagittal, coronal and axial planes and to determine the region of interest
(ROI). For determination of mandibular ROI, the images were manipulated so the
sagittal axis was placed in the center of the long axis of the left side of the mandible,
parallel to the cortical bone. The axial axis was positioned next to the incisor tooth root.
For each mandible, an anterior and a posterior ROI were determined, generating a
double sample number for each group. For the anterior ROI, the coronal axis was
placed where the bone tissue started to circle the left incisor tooth root, and the BD
measures were made in the bone superior to the root (Figure 1A). For the posterior
ROI, the coronal axis was moved to the distal region of the last molar tooth on the left
side, measuring the BD posteriorly to the molar tooth root (Figure 1B). For
determination of ROI in tibia, the images were manipulated so the sagittal and coronal
axes were positioned over the center of the long axis, and the axial axis placed next to
the midpoint of the long axis. The BD was measured in the superior and inferior regions
of cortical bone, in the central portion of diaphysis (Figure 1C).
The BD values were obtained from the sagittal plane image for all ROIs, using
the Point tool, in Hounsfield Units (HU). For each region, two consecutive slices were
analyzed, measuring 3 points in each anterior and posterior slice of mandible, and 6
points for each tibia slice (3 for the superior cortical and 3 for the inferior cortical).
At last, the mean BD value for the mandible was obtained combining the anterior
and posterior region of each specimen, and combining the superior and inferior cortical
for each tibia.
Histological processing
33
Following the tomographic exposure, all the mandibles and tibiae were
decalcified in 10% nitric acid solution with urea. After processing, the mandibles were
hemi-sectioned, and the left side was selected for analysis, similar to the tomography
exam. Subsequently, an anteroposterior section was made anterior to the first molar
tooth, resulting in an anterior and a posterior fragment, that were paraffin-embedded
separately. The tibiae were sectioned on the midpoint of the sagittal axis, and each
fragment was paraffin-embedded individually. The paraffin blocks were cut into 4-µm
sections and stained with hematoxylin and eosin (H&E).
Histomorphometry
Sixty-eight histological slides were obtained from the 34 mandibles. For the
tibiae, 34 slides were obtained. The sections were digitized using a Leica DM3000 light
microscope (Leica Microsystems GmbH, Wetzlar, Germany), connected to a Leica
DFC7000 T camera (Leica Microsystems GmbH, Wetzlar, Germany) and a AMD
Phenon II X4 3.4 GHz computer with Leica Application Suite software (Leica
Microsystems GmbH, Wetzlar, Germany). The images were captured using 10x
objective and stored in TIFF (Tagged Image File Format) format. For each slide, as
many fields as necessary were captured to cover all the bone tissue presented in the
interest region. In total, 255 images were obtained for the mandibles, and 710 images
for the tibiae.
A calibrated and blinded examiner, using the software Adobe Photoshop CC
(Adobe Systems, San Jose, California, USA) analyzed the images. The bone tissue
present in each field was selected, using selection tools, and quantified using the pixel
value of the Histogram (Mahl et al, 2009)(MAHL et al., 2009). Blood vessels, medullary
spaces, empty spaces and cartilage were removed from the selection. Then,
considering the mean pixel value for each specimen, a bone volume (BV) proportion
was gathered, corresponding to the proportion of total image area occupied by bone
tissue (Figure 2) (Mahl et al, 2009; Oyhanart et al, 2015).
Statistical analysis
For tomographic analysis, the examiner calibration occurred by the evaluation
of 10 random tomographic images, twice, in different moments. For histological
analysis, the calibration consisted in the evaluation of 20 random histological images,
twice, in different moments. The results of these two distinct calibrations were
34
subjected to a paired t-test and Pearson’s correlation coefficient, showing the absence
of a significant difference (p>0.05) and a strong correlation (r>0.9). The comparison
among the BPs and control group, for each bone, was done by means of ANOVA test,
with significance level of 5%. The Student’s t-test was chosen to compare the different
bones, mandible and tibia, with significance level of 5%. The statistics were processed
by the Stata 15.0 software (StataCorp, College Station, Texas, USA).
Results
Macroscopic evaluation
On macroscopic examination, neither mandibles, nor tibiae exhibited visible bone
lesions.
Tomographic analysis
The results for the tomographic evaluation of BD are shown in Table 1.
Mandible
No significant differences were found in the BD values among ZA, CL and control
groups (p=0.419), with the anterior and posterior regions combined.
Tibia
No significant differences were observed among the ZA, CL and control groups
(p=0.871).
Mandible vs. tibia
By comparison of the two bones in each group, no significant differences could be
observed for ZA (p=0.449), CL (p=0.602) or control (p=0.487) groups.
Histomorphometry
The results of histomorphometric evaluation of BV proportion are shown in Table 2.
Mandible
No significant differences in the BV proportion was observed among ZA, CL and control
groups (p=0.939), with the anterior and posterior regions combined.
Tibia
No significant differences were found among ZA, CL and control groups (p=0.698).
35
Mandible vs. tibia
Although the mandible exhibited a higher proportion of BV in all groups, comparing the
two bones, no significant differences could be observed for ZA (p=0.056) and CL
(p=0.089) groups. For control group, a statistical difference was found (p=0.032), with
higher proportion in the jaw, compared to tibia.
Discussion
In this study, the comparison of mandibular cortical BD values among two
distinct BPs and control, measured by CBCT, did not present statistical difference. It
was shown that cortical bone density in BP-treated patients with MRONJ stages 0 to
3, compared to healthy patients, did not differ significantly when assessed with CT. In
addition, the cortical bone width and cancellous bone density were increased in the
patients with MRONJ (Taniguchi et al, 2016). Individuals with MRONJ presented
decreased intracortical CBCT density, when compared to age-gender matched
controls in the study by Gönen et al (2017), although the cortical bone thickness was
also increased. The cancellous bone density values were not significant. The authors
point that the results indicate that BP alters cortical bone metabolism, possibly by
suppressing intracortical bone remodeling in the mandible (Torres et al, 2012; Gonen
et al, 2018).
The comparative evaluation of a jaw and a long bone BD values did not show
significant difference in this research. Gong et al (2017) performed dental extractions
in the jaws and drill hole defects in ilium and tibia of rats that received ZA. No difference
in the BV fraction of jaw bone between ZA group and controls was observed, using
microCT. However, both BV fraction and cortical bone thickness were increased in the
peripheral bones, comparing to control. Using ELISA, in the same study, the authors
demonstrated a decreased RANKL/OPG ratio in the jaws and an increased ratio in
peripheral bones. Altogether, this indicates that ZA strongly suppresses alveolar bone
remodeling undergoing tooth extraction, in conjunction with more active bone formation
in the peripheral bones following injury (Gong et al, 2017).
Vermeer et al (2017), using microCT to evaluate BV fraction and tissue mineral
density (TMD) in jaws, tibiae and femurs of mice not submitted to any surgical
intervention, in three different times (1, 3 and 6 months). The authors observed that
BV fraction and TMD parameters were significant higher in the jaws when compared
36
with long bones (Vermeer et al, 2017). These results were similar for TMD in all time
points of drug administration. The same authors demonstrated in vivo that jaw bone
marrow cells internalize more BPs comparing to long bones, suggesting bone-site-
specific responses to BPs (Vermeer et al, 2013).
The comparative results for CL in the present study, regarding long bones BD,
are in accordance with the literature. Koivukangas et al (2001, 2003) demonstrated
that the long-term treatment with CL, in both growing rats and adult rats, does not result
in BD value changes in femur and tibia, measured using peripheral quantitative CT
(Koivukangas et al, 2001, 2003). Moreover, the resistance to fracture remained
unchanged for the long bones, which was similar to the observations of another study
(Lepola et al, 1996; Koivukangas et al, 2001, 2003). There is no data in English
language literature about the effects of CL in mandible - which does not allow the
comparison with this study.
Oyhanart et al. (2015) evaluated, histologically, the BV in the interradicular bone
area of mandible and tibia of growing rats that received alendronate, without any
surgical intervention. The authors observed increased BV in the experimental group,
compared to control, in conjunction with a decrease in the total thickness of epiphyseal
cartilage of tibia (Oyhanart et al, 2015). The histomorphometric measurement of
mineral apposition rate in adult rats showed that ZA reduces bone formation in the jaws
after 3 months of treatment, but not after 6 months. In long bones (tibia and femur),
this reduction was observed after 3 and 6 months of treatment (Vermeer et al, 2017).
In the same research, it was shown that the long-term treatment with ZA reduced the
number of jaw bone marrow cells, without affecting the number of long bone marrow
cells. The results show that N-BPs can differently affect long bone and jaw bone
turnover in vivo.
Studies that evaluate the effects of non-N-BP on jaws and long bones show
similar results to this research. The volume of cancellous bone and the mineral
apposition rate of femur and tibia of rats were not altered by the treatment with CL, in
a long-term administration, although the bone growth rate of femur decreased (Lepola
et al, 1996; Koivukangas et al, 2001, 2003). In addition, Vasconcelos et al (2012)
quantified vital bone on maxilla of adult rats that received CL, intraperitoneally (on the
same dose used in the present study), by means of histological analysis - without
37
osseous surgical procedures. The authors concluded that the proportion of vital bone
was significantly greater in the control group compared to the CL group. Curiously, the
present study found a significant statistical difference on control group, with higher
proportion in the jaw, compared to tibia. It is known that BPs promote nearly complete
suppression of bone turnover, but in this study, we could not explain why a greater
effect was observed in control group, when compared with test groups.
Hence, it is possible to infer that bone interventions and age of subject might
alter osseous tissue response under N-BP therapy, which may also occur in a bone-
site-specific manner. The absence of difference among groups and bones in the
present study may be partially explained by (1) BP dose and administration period, (2)
absence of different time-point evaluations, and (3) adult animals. Though our BP
administration regimen was sufficient to induce osteonecrosis (Vasconcelos et al,
2012; Martelli et al, 2017), the cumulative dose of ZA administered by Vermeer et al
(2017) was considerably higher than ours (0.5 mg/kg weekly vs. 0.6 mg/kg monthly).
Additionally, the same study evaluated different time-points, showing the interference
of time-related response. The differences in study design, including the stage of
development of individuals, difficult proper comparison among studies. Additionally,
even though CL is the most prescribed non-N-BP, there are a few reports in literature
about the effects of this class of BF using animal model.
38
Figure 1. BD quantification using OsiriX 7.0. (A) Quantification of mandibular anterior ROI. (B) Quantification of mandibular posterior ROI. (C) Quantification of tibial ROI.
39
Table 1. Tomographic analysis. Bone density quantification, in Hounsfield units (HU).
Group N Mandible
Mean (SD)
Tibia
Mean (SD)
Zoledronic acid (ZA) 12 1619.32 (99.66) 1569.67 (199.56) **p = 0.449
Clodronate (CL) 12 1567.59 (143.54) 1598.81 (145.48) **p = 0.602
Control 10 1550.54 (136.85) 1612.22 (238.65) **p = 0.487
*p = 0.419 *p = 0.871
SD = standard deviation; p = p value.
*ANOVA test, comparison among BP and control groups, for each bone.
**Student’s t-test, comparison between different bones, for each drug group.
Table 2. Histomorphometrical analysis. Proportion (%) of bone volume (BV).
Group N Mandible
% (SD)
Tibia
% (SD)
Zoledronic acid (ZA) 12 66.92 (7.52) 59.41 (10.51) **p = 0.056
Clodronate (CL) 12 66.27 (17.32) 55.93 (10.27) **p = 0.089
Control 10 68.11 (8.98) 55.75 (14.25) **p = 0.032
*p = 0.939 *p = 0.698
SD = standard deviation; p = p value.
*ANOVA test, comparison among BP and control groups, for each bone.
**Student’s t-test, comparison between different bones, for each drug group.
Figure 2. Quantification of bone volume (BV) using Photoshop CC. (A) Mandibular histological section. (B) Tibial histological section.
40
Acknowledgements
We gratefully acknowledge the financial support of National Council for
Scientific and Technological Development (CNPq), the Center of Diagnosis of Oral
Diseases and the Federal University of Pelotas for the use of facilities and equipment.
References
Allen MR (2015). Medication-Related Osteonecrosis of the Jaw: Basic and
Translational Science Updates. Oral Maxillofac Surg Clin North Am 27: 497–508.
Anderson PJ, Yong R, Surman TL, Rajion ZA, Ranjitkar S (2014). Application of three-
dimensional computed tomography in craniofacial clinical practice and research. Aust
Dent J 59 Suppl 1: 174–185.
Barba-Recreo P, Del Castillo Pardo de Vera JL, Garcia-Arranz M, Yebenes L,
Burgueno M (2014). Zoledronic acid - related osteonecrosis of the jaws. Experimental
model with dental extractions in rats. J Craniomaxillofac Surg 42: 744–50.
Cankaya AB, Erdem MA, Isler SC, et al (2011). Use of cone-beam computerized
tomography for evaluation of bisphosphonate-associated osteonecrosis of the jaws in
an experimental rat model. Int J Med Sci 8: 667–72.
Eleutherakis-Papaiakovou E, Bamias A (2017). Antiresorptive treatment-associated
ONJ. Eur J Cancer Care Engl 26.
Fleisher K, Kontio R, Otto S (2016). Antiresorptive drug-related osteonecrosis of the
jaw (ARONJ) – a guide to research. 1st Edition. AOCMF.
Gonen ZB, Yillmaz Asan C, Zararsiz G, Kilic E, Alkan A (2018). Osseous changes in
patients with medication-related osteonecrosis of the jaws. Dentomaxillofac Radiol 47:
20170172.
Gong X, Yu W, Zhao H, Su J, Sheng Q (2017). Skeletal Site-specific Effects of
Zoledronate on in vivo Bone Remodeling and in vitro BMSCs Osteogenic Activity. Sci
Rep 7: 36129.
Guggenberger R, Koral E, Zemann W, Jacobsen C, Andreisek G, Metzler P (2014).
Cone beam computed tomography for diagnosis of bisphosphonate-related
osteonecrosis of the jaw: evaluation of quantitative and qualitative image parameters.
Skelet Radiol 43: 1669–78.
41
Hamada H, Matsuo A, Koizumi T, Satomi T, Chikazu D (2014). A simple evaluation
method for early detection of bisphosphonate-related osteonecrosis of the mandible
using computed tomography. J Craniomaxillofac Surg 42: 924–9.
Khan AA, Morrison A, Hanley DA, et al (2015). Diagnosis and management of
osteonecrosis of the jaw: a systematic review and international consensus. J Bone Min
Res 30: 3–23.
Koivukangas A, Tuukkanen J, Hannuniemi R, Jamsa T, Kippo K, Jalovaara P (2001).
Effects of long-term administration of clodronate on growing rat bone. Calcif Tissue Int
69: 350–5.
Koivukangas A, Tuukkanen J, Kippo K, et al (2003). Long-term administration of
clodronate does not prevent fracture healing in rats. Clin Orthop Relat Res: 268–78.
Lepola VT, Hannuniemi R, Kippo K, Lauren L, Jalovaara P, Vaananen HK (1996).
Long-term effects of clodronate on growing rat bone. Bone 18: 191–6.
Maahs MP, Azambuja AA, Campos MM, Salum FG, Cherubini K (2011). Association
between bisphosphonates and jaw osteonecrosis: a study in Wistar rats. Head Neck
33: 199–207.
Macerata R (2002). Zometa (zoledronic acid)—intravenous formulation. Investigator’s
brochure for consultation.
Mahl C, Fontoura F, Borelli P, Silva I, Fontanella V (2009). Mandible’s Radiographic
and Histomorphometric Assessment in Female Rats Medicated with Glucocorticoid
and Biphosphonate. Rev Facul Odontol Porto Alegre 50: 16–19.
Martelli SJR, Damian MF, Gomes APN, Schinestsck AR, Silva AER, Vasconcelos ACU
(2017). Comparison of effects of zoledronic acid and clodronate on the bone structure:
imaginological and histomorphometrical study in vivo. J Oral Pathol Med 46: 632–636.
Marx RE (2003). Pamidronate (Aredia) and zoledronate (Zometa) induced avascular
necrosis of the jaws: a growing epidemic. J Oral Maxillofac Surg 61: 1115–7.
Oyhanart SR, Escudero ND, Mandalunis PM (2015). Effect of alendronate on the
mandible and long bones: an experimental study in vivo. Pediatr Res 78: 618–25.
Ruggiero SL (2015). Diagnosis and Staging of Medication-Related Osteonecrosis of
the Jaw. Oral Maxillofac Surg Clin North Am 27: 479–87.
42
Ruggiero SL, Dodson TB, Fantasia J, et al (2014). American Association of Oral and
Maxillofacial Surgeons position paper on medication-related osteonecrosis of the jaw-
-2014 update. J Oral Maxillofac Surg 72: 1938–56.
Russell RG (2007). Bisphosphonates: mode of action and pharmacology. Pediatrics
119 Suppl 2: S150-62.
Sigua-Rodriguez EA, da Costa Ribeiro R, de Brito AC, Alvarez-Pinzon N, de
Albergaria-Barbosa JR (2014). Bisphosphonate-related osteonecrosis of the jaw: a
review of the literature. Int J Dent 2014: 192320.
Taniguchi T, Ariji Y, Nozawa M, et al (2016). Computed tomographic assessment of
early changes of the mandible in bisphosphonate-treated patients. Oral Surg Oral Med
Oral Pathol Oral Radiol 122: 362–72.
Torres SR, Chen CS, Leroux BG, et al (2012). Mandibular cortical bone evaluation on
cone beam computed tomography images of patients with bisphosphonate-related
osteonecrosis of the jaw. Oral Surg Oral Med Oral Pathol Oral Radiol 113: 695–703.
Turner PV, Brabb T, Pekow C, Vasbinder MA (2011). Administration of substances to
laboratory animals: routes of administration and factors to consider. J Am Assoc Lab
Anim Sci 50: 600–13.
Vasconcelos AC, Berti-Couto SA, Azambuja AA, et al (2012). Comparison of effects of
clodronate and zoledronic acid on the repair of maxilla surgical wounds -
histomorphometric, receptor activator of nuclear factor-kB ligand, osteoprotegerin, von
Willebrand factor, and caspase-3 evaluation. J Oral Pathol Med 41: 702–12.
Vermeer JA, Jansen ID, Marthi M, et al (2013). Jaw bone marrow-derived osteoclast
precursors internalize more bisphosphonate than long-bone marrow precursors. Bone
57: 242–51.
Vermeer J, Renders G, van Duin MA, et al (2017). Bone-site-specific responses to
zoledronic acid. Oral Dis 23: 126–133.
43
4 Considerações
Finais
44
4 Considerações finais
Frente aos resultados obtidos no presente estudo, conclui-se que a DO não é
influenciada pela localização óssea, nem pelo tipo de terapia administrada aos
indivíduos, quando medida através de TCFC. Ainda, a terapia com BFs nitrogenados
e não nitrogenados não foi capaz de alterar o VO, em mandíbulas e tíbias, nos
grupos que receberam o medicamento.
45
Referências
46
Referências
ALLEN, M. Medication-related osteonecrosis of the jaw: basic and translational
science updates. Oral and Maxillofacial Surgery Clinics of North America, v. 27, p.
497-508, 2015.
ALLEN, M.; BURR, D. B. The pathogenesis of bisphosphonate-related osteonecrosis
of the jaw: so many hypotheses, so few data. Journal of Oral and Maxillofacial
Surgery, v. 67, p. 61-70, 2009.
ANDERSON, P. J. et al. Application of three-dimensional computed tomography in the
craniofacial clinical practice and research. Australian Dental Journal, v. 59, p. 174-
185, 2014.
ASHRAFI, F. et al. Osteonecrosis of the jaws in patient received bisphosphonates and
sunitinib separately: a case report. Journal of Research in Pharmacy Practice, v. 6,
p. 182-185, 2017.
BAGAN, J. et al. Medication-related osteonecrosis of the jaw associated with
bisphosphonates and denosumab in osteoporosis. Oral Diseases, v. 22, p. 324-329,
2016.
BAGAN, J. V. et al. Avascular jaw osteonecrosis in association with cancer
chemotherapy: series of 10 cases. Journal of Oral Pathology & Medicine, v. 34, p.
120-123, 2005.
BASSO, F. G. et al. Zoledronic acid inhibits human osteoblast activities. Gerontology,
v. 59, p. 534-541, 2013.
BEDOGNI, A. et al. Bisphosphonate-associated osteonecrosis can hide jaw
metastases. Bone, v. 41, p. 942-945, 2007.
BEDOGNI, A. et al. Staging of osteonecrosis of the jaw requires computed tomography
for accurate definition of the extent of bony disease. British Journal of Oral and
Maxillofacial Surgery, v.53, p. 603-608, 2014.
BI, Y. et al. Bisphosphonates cause osteonecrosis of the jaw-like disease in mice. The
American Journal of Patology, v. 177, p. 280-290, 2010.
47
BIASOTTO, M. et al. A novel animal model to study non-spontaneous
bisphosphonates osteonecrosis of jaw. Journal of Oral Pathology and Medicine, v.
39, p. 390-396, 2010.
BROOKS, J. K. et al. Osteonecrosis of the jaws associated with use of risedronate:
report of 2 new cases. Oral Surgery, Oral Medicine, Oral Pathology, Oral
Radiology, and Endodontology, v. 103, p. 780-786, 2007.
BROWN, J. P. Antiresorptives: safety concerns-clinical perspective. Toxicologic
Pathology, v. 45, p. 859-863, 2017.
CAMACHO-ALONSO, F. et al. Short-term effect of zoledronic acid upon fracture
resistance of the mandibular condyle and femoral head in an animal model. Medicina
Oral, Patologia Oral, Cirugia Bucal, v. 18, p. 421-426, 2013.
CAMPOS, M. J. S. et al. Bone mineral density in cone beam computed tomography:
Only a few shades of gray. World Journal of Radiology, v. 6, p. 607-612, 2014.
CANKAYA, A. B. et al. Use of cone-beam computerized tomography for evaluation of
bisphosphonate-associated osteonecrosis of the jaws ia an experiment rat model.
International Journal of Medical Sciences, v. 8, p. 667-672, 2011.
CANKAYA, M. et al. The effects of chronic zoledronate usage on the jaw and long
bones evaluated using RANKL and osteoprotegerin levels in an animal model.
International Journal of Oral and Maxillofacial Surgery, v. 42, p. 1134-1139, 2013.
CAVANNA, L. et al. Osteonecrosis of the jaw. A newly emerging site-specific osseous
pathology in patients with cancer treated with bisphosphonates. Report of five cases
and review of the literature. European Journal of Internal Medicine, v. 18, p. 417-
422, 2007.
COXON, F. P.; THOMPSON, K.; ROGERS, M. J. Recent advances in understanding
the mechanism of action of bisphosphonates. Current Opinion in Pharmacology, v.
6, p. 307-312, 2006.
CURI, M. M. et al. Bisphosphonate-related osteonecrosis of the jaws – an initial case
series report of treatment combining partial bone resection and autologous platelet-
rich plasma. Journal of Oral and Maxillofacial Surgery, v. 69, p. 2465-2472, 2011.
48
DIEL, I. J. et al. Pathophysiology, risk factors and management of bisphosphonate-
associated osteonecrosis of the jaw: Is there a diverse relationship of amino- and non-
aminobisphosphonates? Clinical Reviews in Oncology/Hematology, v. 64, p. 198-
207, 2007.
ELEUTHERAKIS-PAPAIAKOVOU, E.; BAMIAS, A. Antiresorptive treatment-
associated ONJ. European Journal of Cancer Care, v. 26, 2017.
FANTASIA, J. E. Bisphosphonates--what the dentist needs to know: practical
considerations. Journal of Oral and Maxillofacial Surgery, v. 67, p. 53-60, 2009.
FANTASIA, J. E. The role of antiangiogenic therapy in the development of
osteonecrosis of the jaw. Oral and Maxillofacial Surgery Clinics of North America,
v. 27, p. 547-553.
FLEISHER, K. E.; KONTIO, R.; OTTO, S. Antiresorptive drug-related
osteonecrosis of the jaw (ARONJ) – a guide to research. AOCMF, 2016.
FREIBERGER, J.J. et al. What is the role of hyperbaric oxygen in the management of
bisphosphonate-related osteonecrosis of the jaw: a randomized controlled trial of
hyperbaric oxygen as an adjunct to surgery and antibiotics. Journal of Oral and
Maxillofacial Surgery, v. 70, p. 1573-1583, 2012.
GONG, X. et al. Skeletal site-specific effects of zoledronate on in vivo bone remodeling
and in vitro BMSCs osteogenic activity. Scientific Reports, v. 7, 2017.
HANSEN, T. et al. Osteonecrosis of the jaws in patients treated with
bisphosphonates: histomorphologic analysis in comparison with infected
osteoradionecrosis. Journal of Oral Pathology & Medicine, v. 35, p. 155-160,
2006.
HESSE, B. et al. Acessing osteocyte lacunar geometrical properties in human jaw
bone on the submicron length scale using synchrotron radiation µCT. Journal of
Microscopy, v. 255, p. 158-168, 2014.
HINSON, A. M. et al. Is bisphosphonate-related osteonecrosis of the jaw an infection?
A histological and microbiological ten-year summary. International Journal of
Dentistry, v. 2014, 2014.
49
KELLINSALMI, M. et al. In vitro comparison of clodronate, pamidronate and zoledronic
acid effects on rat osteoclasts and human stem cell-derived osteoblasts. Basic &
Clinical Pharmacology & Toxicology, v. 97, p. 382-391, 2005.
KHAN, A. et al. Diagnosis and management of osteonecrosis of the jaw: a systematic
review and international consensus. Journal of Bone Mineral Research, v. 30, p. 3-
23, 2015.
KHOSLA, S. et al. Bisphosphonate-associated osteonecrosis of the jaw: report of a
task force of the American Society for Bone and Mineral Research. Journal of Bone
and Mineral Research, v. 22, p. 1479-1491, 2007.
KOBAYASHI, Y. et al. Zoledronic acid delays wound healing of the tooth extraction
socket, inhibits oral epithelial cell migration, and promotes proliferation and adhesion
to hydroxyapatite of oral bacteria, without causing osteonecrosis of the jaw, in mice.
Journal of Bone and Mineral Metabolism, v. 28, p. 165-175, 2010.
LANDESBERG, R. et al. Inhibition of oral mucosal cell wound healing by
bisphosphonates. Journal of Oral and Maxillofacil Surgery, v. 66, p. 839-847, 2008.
LANDESBERG, R. et al. Potential pathophysiological mechanisms in osteonecrosis of
the jaw. Annals of the New York Academy of Sciences, v. 1218, p. 62-79, 2011.
LAPÉRINE, O. et al. Dendritic-cell-derived osteoclasts: a new game changer in bone-
resorption-associated diseases. Drug Discovery Today, v. 21, p. 1345-1354.
LEITE, A. F. et al. Imaging findings of bisphosphonate-related osteonecrosis of the
jaws: a critical review of the quantitative studies. International Journal of Dentistry,
v. 2014, 2014.
MAAHS, M. P. et al. Association between bisphosphonates and jaw osteonecrosis: a
study in Wistar rats. Head and Neck, v. 33, p. 199-207, 2011.
MARTELLI, S. J. M. et al. Comparison of effects of zoledronic acid and clodronate on
the bone structure: imaginological and histomorphometrical study in vivo. Journal of
Oral Pathology and Medicine, v. 46, p. 632-636, 2017.
MARX, R. E. et al. Bisphosphonate-induced exposed bone
(osteonecrosis/osteopetrosis) of the jaws: risk factors, recognition, prevention, and
treatment. Journal of Oral and Maxillofacial Surgery, v. 63, p. 1567-1575, 2005.
50
MARX, R. E., TURSUN, R. Suppurative osteomyelitis, bisphosphonate induced
osteonecrosis, osteoradioecrosis: a blinded histopathologic comparison and its
implications for the mechanism of each disease. International Journal of Oral and
Maxillofacial Surgery, v. 41, p. 283-289, 2012.
MARX, R.E. Pamidronate (Aredia) and zoledronate (Zometa) induced avascular
necrosis of the jaws: a growing epidemic. Journal of Oral and Maxillofacial Surgery,
v. 61, p. 1115-1117, 2003.
MELO, M. D.; OBEID, G. Osteonecrosis of the jaws in patients with a history of
receiving bisphosphonate therapy: strategies for prevention and early recognition.
Journal of the American Dental Association, v. 136, p. 1675-1681, 2005.
MIGLIORATI, C. A. et al. Managing the care of patients with
bisphosphonate associated osteonecrosis: an American Academy of Oral Medicine
position paper. Journal of American Dental Association, v. 136, p. 1658-1668,
2005.
NAIDU, A. et al. The effects of bisphosphonates on osteoblasts in vitro. Oral Surgery,
Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology, v. 106, p. 5-
13, 2008.
OYHANART, S. R. et al. Effect of alendronate on the mandible and long bones: an
experimental study in vivo. Pediatric Research, v. 78, p. 618-625, 2015.
PAUTKE, C. et al. Bisphosphonate related osteonecrosis of the jaw: a minipig large
animal model. Bone, v. 51, p. 592-599, 2012.
PAUWELS, R. et al. Variability of dental cone beam CT grey values for density
estimations. British Journal of Radiology, v. 86, 2013.
PAZIANAS, M. et al. A review of the literature on osteonecrosis of the jaw in patients
with osteoporosis treated with oral bisphosphonates: prevalence, risk factors, and
clinical characteristics. Clinical Therapeutics, v. 29, p. 1548-1558, 2007.
RIZZOLI, R. et al. Osteonecrosis of the jaw and bisphosphonate treatment for
osteoporosis. Bone, v. 42, p. 841-847, 2008.
51
RUGGIERO, S. L. An office-based approach to the diagnosis and management of
osteonecrosis. Oral and Maxillofacial Surgery Clinics of North America, v. 21, p.
167-173, 2015.
RUGGIERO, S. L. Bisphosphonate-related osteonecrosis of the jaw: an overview.
Annals of the New York Academy of Sciences, v. 1218, p. 38-46, 2011.
RUGGIERO, S. L. Diagnosis and staging of medication-related osteonecrosis of the
jaw. Oral and Maxillofacial Surgery Clinics of North America, v. 27, p. 479-487,
2015.
RUGGIERO, S. L. et al. Bisphosphonate-related osteonecrosis of the jaw (BRONJ):
initial discovery and subsequent development. Journal of Oral and Maxillofacial
Surgery, v. 67, p. 13-18, 2009.
RUGGIERO, S. L.; WOO, S.B. Biophosphonate-related osteonecrosis of the jaws.
Dental clinics of North America, v. 52, p. 111-128, 2008.
RUSSELL, R.G. et al. Bisphosphonates: an update on mechanisms of action and how
these relate to clinical efficacy. Annals of the New York Academy of Sciences, v.
1117, p. 209-257, 2007.
SAMBROOK, P.; OLVER, I.; GOSS, A. Bisphosphonates and osteonecrosis of the jaw.
Australian Family Physician, v. 35, p. 801-803, 2006.
SEDGHIZADEH, P. P. et al. Identification of microbial biofilms in osteonecrosis of the
jaws secondary to bisphosphonate therapy. Journal of Oral and Maxillofacial
Surgery, v. 66, p. 767-775, 2008.
SEDGHIZADEH, P. P. et al. Metagenomic investigation of microbes and viruses in
patients with jaw osteonecrosis associated with bisphosphonate therapy. Oral
Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology, v.
114, p. 764-770, 2012.
SEDGHIZADEH, P. P. et al. Microbial biofilms in osteomyelitis of the jaw and
osteonecrosis of the jaw secondary to bisphosphonate therapy. Journal of the
American Dental Association, v. 140, p. 1259-1265, 2009.
52
SENEL, F. C. et al. Jaw bone changes in rats after treatment with zoledronate and
pamidronate. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and
Endodontology, v. 109, p. 385-391, 2010.
SIGUA-RODRIGUEZ, E. A. et al. Bisphosphonate-related osteonecrosis of the jaw: a
review of the literature. International Journal of Dentistry, v. 2014, 2014.
SONIS, S. T. et al. Bony changes in the jaws of rats treated with zoledronic acid and
dexamethasone before dental extractions mimic bisphosphonate-related
osteonecrosis in cancer patients. Oral Oncology, v. 45, p. 164-172, 2009.
TORRES, S. R. et al. Fractal dimension evaluation of cone beam computed
tomography in patients with bisphosphonate-associated osteonecrosis.
Dentomaxillofacial Radiology, v. 40, p. 501-505, 2011.
TREISTER, N. S. et al. Use of cone-beam computerized tomography for evaluation of
bisphosphonate-associated osteonecrosis of the jaws. Oral Surgery, Oral Medicine,
Oral Pathology, Oral Radiology, and Endodontology, v. 105, p. 753-764, 2010.
VASCONCELOS, A. C. et al. Laboratory methods and biomarkers in the evaluation of
bisphosphonate effects on body tissues: a literature review. Journal of Oral
Pathology and Medicine, v. 42, p. 577-586, 2013.
VASCONCELOS, A.C. et al. Comparison of effects of clodronate and zoledronic acid
on the repair of maxilla surgical wounds - histomorphometric, receptor activator of
nuclear factor-kB ligand, osteoprotegerin, von Willebrand factor, and caspase-3
evaluation. Journal of Oral Pathology and Medicine, v. 41, p. 702-712, 2012.
VERMEER, J. et al. Bone-site-specific responses to zoledronic acid. Oral Diseases,
v. 23, p. 126-133, 2017.
WALTER, C.et al. Influence of bisphosphonates on endothelial cells, fibroblasts, and
osteogenic cells. Clinical Oral Investigations, v. 14, p. 35-41, 2010.
WOOD, J. et al. Novel antiangiogenic effects of the bisphosphonate compound
zoledronic acid. The Journal of Pharmacology and Experimental Therapeutics,
Baltimore, v.302, p. 1055-1061, 2002.
53
XU, X. L. et al. Basic research and clinical applications of bisphosphonates in bone
disease: what have we learned over the last 40 years? Journal of Translational
Medicine, v. 11, 2013.
YAMADA, J. et al. Anti-angiogenic property of zoledronic acid by inhibition of
endothelial progenitor cell differentiation. The Journal of Surgical Research, v. 151,
p. 115-120, 2009.
YOSHIMURA, H. et al. Denosumab-related osteonecrosis of the jaw in a patient with
bone metastases of prostate cancer: a case report and literature review. Oncology
Letters, v. 14, p. 127-136, 2017.
54
Anexos
55
Anexos
Anexo A – Parecer do Comitê de Ética
56
Anexo B – Parecer do Comitê de Ética para o Uso de Animais
57
Anexo C – Instruções aos autores para submissão de artigo do periódico Oral
Diseases.
Oral Diseases
© John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Edited By: Bruce Baum, Giovanni Lodi
Impact Factor: 2.011
ISI Journal Citation Reports © Ranking: 2016: 27/90 (Dentistry Oral Surgery & Medicine)
Online ISSN: 1601-0825
Author Guidelines
The median processing time from submission to first decision for manuscripts submitted to Oral
Diseases in the prior 12 months is 22 days.
Content of Author Guidelines: 1. General, 2. Ethical Guidelines, 3. Manuscript Submission Procedure, 4.
Manuscript Types Accepted, 5. Manuscript Format and Structure, 6. After Acceptance.
Relevant Documents: Online Open Order Form, Colour Work Agreement Form, Standard Release Form for
photographic consent
Useful Websites: Submission Site, Articles Published in Oral Diseases, Author Services, Wiley-Blackwell’s Ethical
Guidelines, Guidelines for Figures
1. GENERAL
The editors encourage submissions of original articles, review articles, reports of meetings, book reviews and
correspondence in the form of letters to the editor. Oral Diseases does not accept case reports.
Please read the instructions below carefully for details on the submission of manuscripts, the journal's requirements
and standards as well as information concerning the procedure after a manuscript has been accepted for publication
in Oral Diseases. Authors are encouraged to visit Wiley-Blackwell Author Services for further information on the
preparation and submission of articles and figures.
Avoiding allegations of plagiarism
58
The journal to which you are submitting your manuscript employs text matching software (iThenticate) to ensure
against plagiarism. By submitting your manuscript to this journal you accept that your manuscript may be screened
for plagiarism against previously published work. Authors should consider whether their manuscript may raise
concerns via iThenticate, which will signal whether a paper is likely in any way to be plagiarized in a formal sense.
iThenticate will also, however, signal whether a paper may be plagiarized by repeating work of the submitting
authors and thus be regarded as duplicate or redundant publication. Experience shows that, on occasion, large
sections of submitted manuscripts can be close to verbatim in word choice from that seen in other papers from the
authors’ group. This has nothing to do with simple repetition of names/affiliations, but does involve common (not
necessarily "standard") phrases that are more appropriately referenced instead of repeating. Alternatively, they can
be rephrased differently. Previously published results, including numerical information and figures or images, should
be labeled to make it clear where they were previously reported. Papers that present new analyses of results that
have already been published (for example, subgroup analyses) should identify the primary data source, and include
a full reference to the related primary publications. Oral Diseaseswill review and publish accepted mansucripts that
report data included in conference proceedings in abstract form. In such cases, authors must be clear to readers
that part of all of the manuscript's data have already been published in abstract form by so indicating using a footnote
to the title that states the confrence proceedings in which the relevant abstract was published. For full guidance on
text matching and plagiarism, please refer to Section 3 ('Research Integrity') of Wiley's Ethics Guidelines
at https://authorservices.wiley.com/ethics-guidelines/index.html.
2. ETHICAL GUIDELINES
Oral Diseases adheres to the ethical guidelines given below for publication and research.
2.1. Authorship and Acknowledgements
Authorship: Oral Diseases adheres to the International Standards for Authors published by the Committee on
Publication Ethics (COPE). All authors named on a paper should agree to be named on the paper, and all authors
so named should agree to the submission of the paper to Oral Diseases and approve the submitted and accepted
versions of the publication. Any change to the author list should be approved by all authors, including any author
who has been removed from the list.
Oral Diseases also adheres to the definition of authorship set up by The International Committee of Medical Journal
Editors (ICMJE). According to the ICMJE authorship criteria should be based on 1) substantial contributions to
conception and design of, or acquisition of data or analysis and interpretation of data, 2) drafting the article or
revising it critically for important intellectual content and 3) final approval of the version to be published. Authors
should meet conditions 1, 2 and 3.
It is a requirement that the corresponding author submit a short description of each individual's contribution to the
research and its publication. Upon submission of a manuscript all co-authors should also be registered with a correct
e-mail addresses. If any of the e-mail addresses supplied are incorrect, the corresponding author will be contacted
by the Journal Administrator.
Acknowledgements: Authors must acknowledge individuals who do not qualify as authors but who contributed to
the research. Authors must acknowledge any assistance that they have received (e.g. provision of writing
assistance, literature searching, data analysis, administrative support, supply of materials). If/how this assistance
was funded should be described and included with other funding information. “Acknowledgements” should be brief
and should not include thanks to anonymous referees and editors. Where people are acknowledged, a covering
letter demonstrating their consent must be provided.
59
2.2. Ethical Approvals
Human Subjects: Experimentation involving human subjects will only be published if such research has been
conducted in full accordance with ethical principles, including the World Medical Association Declaration of
Helsinki (version 2002) and the additional requirements, if any, of the country where the research has been carried
out. Manuscripts must be accompanied by a statement that the experiments were undertaken with the
understanding and written consent of each subject and according to the above mentioned principles. A statement
regarding the fact that the study has been independently reviewed and approved by an ethical board should also
be included.
Photographs of People: Oral Diseases follows current HIPAA guidelines for the protection of patient/subject
privacy. If an individual pictured in a digital image or photograph can be identified, his or her permission is required
to publish the image. The corresponding author must either submit a letter signed by the patient authorizing Oral
Diseases to publish the image/photo, or complete the 'Standard Release Form for photographic consent' available
at the top of this page or by clicking the “instructions and Forms” link on the ScholarOne Manuscripts submission
site. The approval must be received by the Editorial Office prior to final acceptance of the manuscript for publication.
Otherwise, the image/photo must be altered such that the individual cannot be identified (black bars over eyes,
tattoos, scars, etc.). Oral Diseases will not publish patient photographs that will in any way allow the patient to be
identified, unless the patient has given their express consent.
Editors reserve the right to reject papers if there are doubts as to whether appropriate procedures have been used.
Animal Study: When experimental animals are used the methods section must clearly indicate that adequate
measures were taken to minimize pain or discomfort. Experiments should be carried out in accordance with the
Guidelines laid down by the National Institute of Health (NIH) in the USA regarding the care and use of animals for
experimental procedures or with the European Communities Council Directive of 24 November 1986 (86/609/EEC)
and in accordance with local laws and regulations.
2.3 Clinical Trials
Clinical Trials should be reported using the CONSORT guidelines available at www.consort-statement.org.
A CONSORT checklist and flowchart should also be included in the submission material. Clinical trials can be
registered in any free, public clinical trials registry such as http://www.clinicaltrials.gov or http://isrctn.org/. A list of
further registries is available at http://www.who.int/ictrp/network/primary/en/. As stated in an editorial published
in Oral Diseases(12:217-218), 2006), all manuscripts reporting results from a clinical trial must indicate that the trial
was fully registered at a readily accessible website. The clinical trial registration number and name of the trial
register will be published with the paper.
2.4 DNA Sequences and Crystallographic Structure Determinations
Papers reporting protein or DNA sequences and crystallographic structure determinations will not be accepted
without a Genbank or Brookhaven accession number, respectively. Other supporting data sets must be made
available on the publication date from the authors directly.
2.5 Conflict of Interest and Source of Funding
All sources of institutional, private and corporate financial support for the work within the manuscript must be fully
acknowledged, and any potential grant holders should be listed. Authors are also required to disclose any possible
conflict of interest. These include financial (for example patent, ownership, stock ownership, consultancies,
60
speaker’s fee). Information on sources of funding and any potential conflict of interest should be disclosed at
submission under the heading “Acknowledgements”.
2.6 Appeal of Decision
The decision on a paper is final and cannot be appealed.
2.7 Permissions
If all or parts of previously published illustrations are used, permission must be obtained from the copyright holder
concerned. It is the author's responsibility to obtain these in writing and provide copies to the Publishers.
2.8 Copyright and OnlineOpen
If your paper is accepted, the author identified as the formal corresponding author for the paper will receive an email
prompting them to login into Author Services; where via the Wiley Author Licensing Service (WALS) they will be
able to complete the license agreement on behalf of all authors on the paper. The corresponding author MUST
submit the CTA as it is a requirement for publication.
For authors signing the copyright transfer agreement
If the OnlineOpen option is not selected the corresponding author will be presented with the copyright transfer
agreement (CTA) to sign. The terms and conditions of the CTA can be previewed in the samples associated with
the Copyright FAQs below:
CTA Terms and Conditions http://exchanges.wiley.com/authors/copyright-and-permissions_333.html.
Online Open
OnlineOpen is available to authors of primary research articles who wish to make their article available to non-
subscribers on publication, or whose funding agency requires grantees to archive the final version of their article.
With OnlineOpen, the author, the author's funding agency, or the author's institution pays a fee to ensure that the
article is made available to non-subscribers upon publication via Wiley InterScience, as well as deposited in the
funding agency's preferred archive. For the full list of terms and conditions,
see http://olabout.wiley.com/WileyCDA/Section/id-406241.html. Any authors wishing to send their paper
OnlineOpen will be required to complete the payment form available from our website
at:https://authorservices.wiley.com/bauthor/onlineopen_order.asp. Prior to acceptance there is no requirement to
inform an Editorial Office that you intend to publish your paper OnlineOpen if you do not wish to. All OnlineOpen
articles are treated in the same way as any other article. They go through the journal's standard peer-review process
and will be accepted or rejected based on their own merit.
For authors choosing OnlineOpen
If the OnlineOpen option is selected the corresponding author will have a choice of the following Creative Commons
License Open Access Agreements (OAA):
Creative Commons Attribution License OAA
Creative Commons Attribution Non-Commercial License OAA
Creative Commons Attribution Non-Commercial -NoDerivs License OAA
To preview the terms and conditions of these open access agreements please visit the Copyright FAQs hosted on
61
Wiley Author Services http://exchanges.wiley.com/authors/copyright-and-permissions_333.html and
visithttp://www.wileyopenaccess.com/details/content/12f25db4c87/Copyright--License.html.
If you select the OnlineOpen option and your research is funded by The Wellcome Trust and members of the
Research Councils UK (RCUK) you will be given the opportunity to publish your article under a CC-BY license
supporting you in complying with Wellcome Trust and Research Councils UK requirements.
For more information on this policy and the Journal’s compliant self-archiving policy please
visit: http://www.wiley.com/go/funderstatement.
Additionally. authors are themselves responsible for obtaining permission to reproduce copyright material from other
sources.
3. MANUSCRIPT SUBMISSION PROCEDURE
Oral Diseases only accepts online submission of manuscripts. Manuscripts should be submitted at the online
submission site: http://mc.manuscriptcentral.com/odi. Complete instructions for submitting a manuscript are
available at the site upon creating an account. Assistance for submitting papers can be sought with the editorial
assistant Lisa Walton at: [email protected]
Upon successful submission, the journal administrator will check that all parts of the submission have been
completed correctly. If any necessary part is missing or if the manuscript does not fulfil the requirements as specified
below, the corresponding author will be asked either to adjust the submission according to specified instructions or
to submit their paper to another journal.
3.1. Getting Started
Launch your web browser (supported browsers include Internet Explorer 5.5 or higher, Safari 1.2.4, or Firefox 1.0.4
or higher) and go to the journal's online Submission Site: http://mc.manuscriptcentral.com/odi
Log-in or, if you are a new user click on 'register here'.
If you are registering as a new user.
- After clicking on 'register here', enter your name and e-mail information and click 'Next'. Your e-mail information is
very important.
- Enter your institution and address information as appropriate, and then click 'Next.'
- Enter a user ID and password of your choice (we recommend using your e-mail address as your user ID), and
then select your areas of expertise. Click 'Finish'.
If you are registered as user, but have forgotten your log in details, enter your e-mail address under 'Password
Help'. The system will send you an automatic user ID and a new temporary password.
Log-in and select 'Corresponding Author Centre'.
3.2. Submitting Your Manuscript
After you have logged into your 'Corresponding Author Centre', submit your manuscript by clicking the submission
link under 'Author Resources'.
Enter data and answer questions as appropriate. You may copy and paste directly from your manuscript and you
may upload your pre-prepared covering letter.
Click the 'Next' button on each screen to save your work and advance to the next screen.
62
You are required to register all of your co-authors with a functioning e-mail address. If the e-mail address is incorrect,
you will be contacted by the journal administrator.
You are required to upload your files: Click on the 'Browse' button and locate the file on your computer. Select the
designation of each file in the drop down next to the Browse button. When you have selected all files you wish to
upload, click the 'Upload Files' button.
Review your submission (in HTML and PDF format) before completing your submission by sending it to the Journal.
Click the 'Submit' button when you are finished reviewing.
3.3. Manuscript Files Accepted
Manuscripts should be uploaded as Word (.doc/.docx) or Rich Text Format (.rft) files (not write-protected) plus
separate figure files. GIF, JPEG, PICT or Bitmap files are acceptable for submission, but only high-resolution TIF
or EPS files are suitable for printing. The files will be automatically converted to HTML and PDF on upload and will
be used for the review process. The text file must contain the entire manuscript including title page, abstract, text,
references, acknowledgements, tables, and figure legends, but no embedded figures. In the text file, please
reference figures as for instance 'Figure 1', 'Figure 2' etc to match the tag name you choose for individual figure
files uploaded. Manuscripts should be formatted as described in the Author Guidelines below.
3.4. Blinded Review
All manuscripts submitted to Oral Diseases will be reviewed by two experts in the field. Oral Diseases uses single
blinded review. The names of the reviewers will thus not be disclosed to the author submitting a paper.
3.5. Suggest a Reviewer
Oral Diseases attempts to keep the review process as short as possible to enable rapid publication of new scientific
data. In order to facilitate this process, you must suggest the names and current e-mail addresses of from 2-4
potential reviewers whom you consider capable of reviewing your manuscript in an unbiased way.
3.6. Suspension of Submission Mid-way in the Submission Process
You may suspend a submission at any phase before clicking the 'Submit' button and save it to submit later. The
manuscript can then be located under 'Unsubmitted Manuscripts' and you can click on 'Continue Submission' to
continue your submission when you choose to.
3.7. E-mail Confirmation of Submission
After submission you will receive an e-mail to confirm receipt of your manuscript. If you do not receive the
confirmation e-mail after 24 hours, please check your e-mail address carefully in the system. If the e-mail address
is correct please contact your IT department. The error may be caused by some sort of spam filtering on your e-
mail server. Also, the e-mails should be received if the IT department adds our e-mail server
(uranus.scholarone.com) to their whitelist.
3.8. Manuscript Status
The average time from submission to first decision for manuscripts submitted to Oral Diseases is 20 days. You can
access ScholarOne Manuscripts (formerly known as Manuscript Central) any time to check your 'Author Centre' for
the status of your manuscript. The Journal will inform you by e-mail once a decision has been made.
3.9. Submission of Revised Manuscripts
63
To upload a revised manuscript, locate your manuscript under 'Manuscripts with Decisions' and click on 'Submit a
Revision'. Please remember to delete any old files uploaded when you upload your revised manuscript.
4. MANUSCRIPT TYPES ACCEPTED
Original Research Articles: Manuscripts reporting laboratory investigations, well-designed and controlled clinical
research, and analytical epidemiology are invited. Studies related to aetiology, pathogenesis, diagnosis, prevention
and treatment are all of interest, but all papers must be based on rigorous hypothesis-driven research. Areas of
interest include autoimmune, endocrine, genetic, infectious, metabolic and mucosal diseases; cancer and pre-
cancerous conditions; chemosensory, developmental, geriatric and motor disorders, pain and wound healing.
Randomised trials must adhere to the CONSORT guidelines, and a CONSORT checklist and flowchart must be
submitted with such papers. Please also refer to the notes under section 2.3 above. Observational studies must
adhere to the STROBE guidelines, and a STROBE checklist must be submitted with such papers. Diagnostic
accuracy studies must adhere to the STARD guidelines, and a STARD checklistmust be submitted with such
papers.
Review Papers: Oral Diseases commissions review papers and also welcomes uninvited reviews. Systematic
reviews with or without meta-analyses must adhere to the PRISMA guidelines, and aPRISMA
checklist and flowchart must be submitted with such papers.
Letters to the Editors: Letters, if of broad interest, are encouraged. They may deal with material in papers
published in Oral Diseases or they may raise new issues, but should have important implications. Only one letter
may be submitted by any single author or group of authors on any one published paper.
Case Reports: Oral Diseases does not accept case reports and instead recommends that authors submit
to Clinical Case Reports an open access journal published by Wiley.
Meeting Reports: Will be considered by the editors for publication only if they are of wide and significant interest.
Invited Concise Reviews: These may be submitted by invitation of the Senior Editors only, and consist of around
2500-2750 words, with a maximum of one table or image and 25 references.
Invited Medical Reviews: These may be submitted by invitation of the Senior Editors only, and consist of around
2500-2750 words, with a maximum of one table or image and 25 references.
Invited Commentaries: These may be submitted by invitation of the Senior Editors only.
Invited Editorials: These may be submitted by invitation of the Senior Editors only.
Invited Book Reviews: These may be submitted by invitation of the Senior Editors only.
5. MANUSCRIPT FORMAT AND STRUCTURE
5.1. Page Charge
Articles exceeding 6 published pages, including title page, abstract, references, table/figure legends and tables and
figures, are subject to a charge of GBP70 per additional page. As a guide, one published page amounts
approximately to 850 words, or two to four small tables/figures. Additional supplementary material (including text
and figures), which does not fit within the page limits, can be published online only as supporting information.
5.2. Format
64
Language: Authors should write their manuscripts in British English using an easily readable style. Authors whose
native language is not English should have a native English speaker read and correct their manuscript. Spelling
and phraseology should conform to standard British usage and should be consistent throughout the paper. A list of
independent suppliers of editing services can be found
at http://authorservices.wiley.com/bauthor/english_language.asp. All services are paid for and arranged by the
author, and use of one of these services does not guarantee acceptance or preference for publication.
Presentation: Authors should pay special attention to the presentation of their findings so that they may be
communicated clearly. The background and hypotheses underlying the study as well as its main conclusions should
be clearly explained. Titles and abstracts especially should be written in language that will be readily intelligible to
any scientist.
Technical jargon: should be avoided as much as possible and clearly explained where its use is unavoidable.
Abbreviations: Oral Diseases adheres to the conventions outlined in Units, Symbols and Abbreviations: A Guide
for Medical and Scientific Editors and Authors. Non-standard abbreviations must be used three or more times and
written out completely in the text when first used.
5.3. Structure: All papers submitted to Oral Diseases should include:
Title Page
Structured Abstract (reviews need not include a structured abstract)
Main text
References
(Figures)
(Figure Legends)
(Tables)
Title Page: should be part of the manuscript uploaded for review and include:
A title of no more than 100 characters including spaces
A running title of no more than 50 characters
3-6 keywords
Complete names and institutions for each author
Corresponding author's name, address, email address and fax number
Date of submission (and revision/resubmission)
Abstract: is limited to 200 words in length and should contain no abbreviations. The abstract should be included in
the manuscript document uploaded for review as well as separately where specified in the submission process. The
abstract should convey the essential purpose and message of the paper in an abbreviated form set out under:
Objective(s),
Subject(s) (or Materials) and Methods,
Results,
65
Conclusions(s).
The Main Text of Original Research Articles should be organised as follows
Introduction: should be focused, outlining the historical or logical origins of the study and not summarize the
results; exhaustive literature reviews are inappropriate. It should close with the explicit statement of the specific
aims of the investigation.
Materials and Methods must contain sufficient detail such that, in combination with the references cited, all clinical
trials and experiments reported can be fully reproduced. As a condition of publication, authors are required to make
materials and methods used freely available to academic researchers for their own use. This includes antibodies
and the constructs used to make transgenic animals, although not the animals themselves. Other supporting data
sets must be made available on the publication date from the authors directly.
(i) Clinical trials: As noted above, these should be reported using the CONSORT guidelines available
at www.consort-statement.org. A CONSORT checklist should also be included in the submission material. Clinical
trials can be registered in any of the following free, public clinical trials
registries: www.clinicaltrials.gov, http://clinicaltrials.ifpma.org/clinicaltrials/, http://isrctn.org/.As stated in an editorial
published in Oral Diseases (12:217-218), 2006), all manuscripts reporting results from a clinical trial must indicate
that the trial was fully registered at a readily accessible website. The clinical trial registration number and name of
the trial register will be published with the paper.
(ii)Experimental subjects: As noted above, experimentation involving human subjects will only be published if
such research has been conducted in full accordance with ethical principles, including the World Medical
Association Declaration of Helsinki (version 2002) and the additional requirements, if any, of the country where the
research has been carried out. Manuscripts must be accompanied by a statement that the experiments were
undertaken with the understanding and written consent of each subject and according to the above mentioned
principles. A statement regarding the fact that the study has been independently reviewed and approved by an
ethical board should also be included.Editors reserve the right to reject papers if there are doubts as to whether
appropriate procedures have been used. When experimental animals are used the methods section must clearly
indicate that adequate measures were taken to minimize pain or discomfort. Experiments should be carried out in
accordance with the Guidelines laid down by the National Institute of Health (NIH) in the USA regarding the care
and use of animals for experimental procedures or with the European Communities Council Directive of 24
November 1986 (86/609/EEC) and in accordance with local laws and regulations.
(iii) Suppliers: Suppliers of materials should be named and their location (town, state/county, country) included.
Results: should present the observations with minimal reference to earlier literature or to possible interpretations.
Discussion: may usually start with a brief summary of the major findings, but repetition of parts of the abstract or
of the results sections should be avoided. The section should end with a brief conclusion and a comment on the
potential clinical relevance of the findings. Statements and interpretation of the data should be appropriately
supported by original references.
Acknowledgements: Should be used to provide information on sources of funding for the research, any potential
conflict of interest and to acknowledge contributors to the study that do not qualify as authors. All sources of
institutional, private and corporate financial support for the work within the manuscript must be fully acknowledged,
and any potential grant holders should be listed. Acknowledgements should be brief and should not include thanks
66
to anonymous referees and editors. Where people are acknowledged, a covering letter demonstrating their consent
must be provided.
5.4. References
References should be prepared according to the Publication Manual of the American Psychological Association (6th
edition). This means in-text citations should follow the author-date method whereby the author's last name and the
year of publication for the source should appear in the text, for example, (Jones, 1998). For references with three
to five authors, all authors should be listed only on the first occurrence of the in-text citation, and in subsequent in-
text occurrences only the first author should be listed followed by 'et al.'. The complete reference list should appear
alphabetically by name at the end of the paper.
A sample of the most common entries in reference lists appears below. Please note that a DOI should be provided
for all references where available. For more information about APA referencing style, please refer to the APA
website. Please note that for journal articles, issue numbers are not included unless each issue in the volume begins
with page one.
Journal article
Example of reference with 2 to 7 authors
Beers, S. R., & De Bellis, M. D. (2002). Neuropsychological function in children with maltreatment-related
posttraumatic stress disorder. The American Journal of Psychiatry, 159, 483–486. doi: 10.1176/appi.ajp.159.3.483
Ramus, F., Rosen, S., Dakin, S. C., Day, B. L., Castellote, J. M., White, S., & Frith, U. (2003). Theories of
developmental dyslexia: Insights from a multiple case study of dyslexic adults. Brain, 126(4), 841–865. doi:
10.1093/brain/awg076
Example of reference with more than 7 authors
Rutter, M., Caspi, A., Fergusson, D., Horwood, L. J., Goodman, R., Maughan, B., … Carroll, J. (2004). Sex
differences in developmental reading disability: New findings from 4 epidemiological studies. Journal of the
American Medical Association, 291(16), 2007–2012. doi: 10.1001/jama.291.16.2007
Book edition
Bradley-Johnson, S. (1994). Psychoeducational assessment of students who are visually impaired or blind: Infancy
through high school (2nd ed.). Austin, TX: Pro-ed.
5.5. Tables, Figures and Figure Legends
Figures: All figures and artwork must be provided in electronic format. Please save vector graphics (e.g. line
artwork) in Encapsulated Postscript Format (EPS) and bitmap files (e.g. half-tones) or clinical or in vitro pictures in
Tagged Image Format (TIFF).
Detailed information on our digital illustration standards can be found
at http://authorservices.wiley.com/bauthor/illustration.asp.
Check your electronic artwork before submitting it: http://authorservices.wiley.com/bauthor/eachecklist.asp.
Unnecessary figures and parts (panels) of figures should be avoided: data presented in small tables or histograms,
for instance, can generally be stated briefly in the text instead. Figures should not contain more than one panel
unless the parts are logically connected.
67
Figures divided into parts should be labelled with a lower-case, boldface, roman letter, a, b, and so on, in the same
type size as used elsewhere in the figure. Lettering in figures should be in lower-case type, with the first letter
capitalized. Units should have a single space between the number and unit, and follow SI nomenclature common
to a particular field. Unusual units and abbreviations should be spelled out in full or defined in the legend. Scale
bars should be used rather than magnification factors, with the length of the bar defined in the legend rather than
on the bar itself. In general visual cues (on the figures themselves) are preferred to verbal explanations in the legend
(e.g. broken line, open red triangles etc).
Guidelines for Cover Submissions
If you would like to send suggestions for artwork related to your manuscript to be considered to appear on the cover
of the journal, please follow these general guidelines.
6. AFTER ACCEPTANCE
Upon acceptance of a paper for publication, the manuscript will be forwarded to the Production Editor who is
responsible for the production of the journal.
Proof Corrections
The corresponding author will receive an e-mail alert containing a link to a website. A working e-mail address must
therefore be provided for the corresponding author. The proof can be downloaded as a PDF (portable document
format) file from this site.
Acrobat Reader will be required in order to read this file. This software can be downloaded (free of charge) from
the following website: www.adobe.com/products/acrobat/readstep2.html . This will enable the file to be opened,
read on screen, and printed out in order for any corrections to be added. Further instructions will be sent with the
proof. Hard copy proofs will be posted if no e-mail address is available; in your absence, please arrange for a
colleague to access your e-mail to retrieve the proofs.
Proofs must be returned to the Production Editor within three days of receipt.
As changes to proofs are costly, we ask that you only correct typesetting errors. Excessive changes made by the
author in the proofs, excluding typesetting errors, will be charged separately. Other than in exceptional
circumstances, all illustrations are retained by the publisher. Please note that the author is responsible for all
statements made in their work, including changes made by the copy editor.
Early View (Publication Prior to Print)
Oral Diseases is covered by Wiley-Blackwell's Early View service. Early View articles are complete full-text articles
published online in advance of their publication in a printed issue. Early View articles are complete and final. They
have been fully reviewed, revised and edited for publication, and the authors' final corrections have been
incorporated. Because they are in final form, no changes can be made after online publication. The nature of Early
View articles means that they do not yet have volume, issue or page numbers, so Early View articles cannot be
cited in the traditional way. They are therefore given a Digital Object Identifier (DOI), which allows the article to be
cited and tracked before it is allocated to an issue. After print publication, the DOI remains valid and can continue
to be used to cite and access the article.
Author Services
Online production tracking is available for your article once it is accepted by registering with Wiley-Blackwell's
Author Services.
68
Video Abstracts
Bring your research to life by creating a video abstract for your article! Wiley partners with Research Square to offer
a service of professionally produced video abstracts. Learn more about video abstracts
at www.wileyauthors.com/videoabstracts and purchase one for your article
at https://www.researchsquare.com/wiley/ or through your Author Services Dashboard. If you have any questions,
please direct them to [email protected].