UNESP - Universidade Estadual Paulista “Júlio de Mesquita Filho” Faculdade de Odontologia de Araraquara RAFAEL SCAF DE MOLON PATOFISIOLOGIA DE OSTEONECROSE INDUZIDA POR BISFOSFONATOS E INIBIDORES DE RANK-L Araraquara 2016 2 UNESP - Universidade Estadual Paulista “Júlio de Mesquita Filho” Faculdade de Odontologia de Araraquara RAFAEL SCAF DE MOLON PATOFISIOLOGIA DE OSTEONECROSE INDUZIDA POR BISFOSFONATOS E INIBIDORES DE RANK-L Araraquara 2016 Tese apresentada ao Programa de Pós-Graduação em Odontologia – Área de Periodontia da Faculdade de Odontologia de Araraquara da Universidade Estadual Paulista para titulo de Doutor em Odontologia Orientador: Prof. Dr. Joni Augusto Cirelli 3 de Molon, Rafael Scaf Patofisiologia de osteonecrose induzida por bisfosfonatos e inibidores de RANK-L / Rafael Scaf de Molon .-- Araraquara: [s.n.], 2016. 123 f. ; 30 cm. Tese (Doutorado) – Universidade Estadual Paulista, Faculdade de Odontologia Orientador: Prof. Dr. Joni Augusto Cirelli 1. Perda óssea alveolar 2. Camundongos 3. Osteoclasto 4. Doenças periodontais 5. Artrite reumatoide I. Título Ficha catalográfica elaborada pela Bibliotecária Marley C. Chiusoli Montagnoli, CRB-8/5646 Serviço Técnico de Biblioteca e Documentação da Faculdade de Odontologia de Araraquara / UNESP 4 RAFAEL SCAF DE MOLON PATOFISIOLOGIA DE OSTEONECROSE INDUZIDA POR BISFOSFONATOS E INIBIDORES DE RANK-L Tese para obtenção do grau de doutor Comissão julgadora Presidente e Orientador: Prof. Dr. Joni Augusto Cirelli 2° Examinador: Prof. Dr. Juliano Milanezi de Almeida 3° Examinador: Profa. Dra. Daniela Bazan Palioto 4° Examinador: Prof. Dr. Pedro Paulo Chaves de Souza 5° Examinador: Prof. Dr. Francisco Humberto Nociti Junior Araraquara, 24 de Março de 2016. 5 DADOS CURRICULARES RAFAEL SCAF DE MOLON NASCIMENTO 06.03.1982 - São Paulo, São Paulo FILIAÇÃO Antonio de Molon Filho e Sandra Scaf de Molon 2000 / 2004 Graduação em Odontologia - Faculdade de Odontologia da Universidade do Sagrado Coração, USC, Bauru, SP. 2005 / 2006 Estágio de Atualização na Disciplina de Cirurgia e Traumatologia Buco-Maxilo-Facial da Faculdade de Odontologia de Araraquara - UNESP 2006 / 2008 Especialização em Implantodontia - Sociedade de Promoção Social do Fissurado Lábio Palatal - PROFIS, Bauru, SP 2009 / 2011 Especialização em Periodontia - Faculdade de Odontologia de Araraquara - UNESP 2010 / 2012 Curso de Pós-Graduação em Odontologia, Área de concentração em Periodontia, nível Mestrado - Faculdade de Odontologia de Araraquara - UNESP 2013 / 2015 Doutorado Sanduíche - University of California at Los Angeles - UCLA School of Dentistry, Los Angeles, CA, USA. 6 2012 / 2016 Curso de Pós-Graduação em Odontologia, Área de concentração em Periodontia, nível Doutorado - Faculdade de Odontologia de Araraquara - UNESP 7 Dedicatória Dedico este trabalho... A Deus, por estar presente em todos os momentos da minha vida, por sempre conduzir meus passos em busca do melhor caminho, pela força em todos os momentos difíceis e também pelos momentos de alegria e por ter sempre colocado na minha vida, pessoas maravilhosas que sempre me ajudaram a seguir em frente em busca de novas conquistas. Aos meus pais Antonio e Sandra, pelo exemplo durante minha criação, pelo incentivo e apoio em todas as minhas escolhas, por todo amor, confiança e dedicação, por estarem sempre ao meu lado, permitindo que eu realize todos os meus sonhos e acima de tudo, pelo exemplo positivo que vocês foram e continuam sendo em minha vida. Aos meus irmãos Gustavo, Melissa e Marina, pelo incentivo, pelo apoio, carinho, compreensão e amizade. 8 A minha esposa, Erica Por estar sempre ao meu lado, compartilhando todos os momentos, pelo incentivo a pesquisa e a pós-graduação e por todo o conhecimento e ensinamentos passados desde o inicio da minha caminhada na pós-graduação. Você é um exemplo de ser humano e de profissional. Obrigado por todos esses anos juntos, por todo amor, pelo imenso respeito, pela lealdade e por todo o incentivo. Agradeço a Deus todos os dias por ter tido você em minha vida; Um grande amor caminhando junto torna a busca pelo sucesso mais real e encorajador. Você e todos os nossos sonhos são minha maior motivação. 9 Agradecimentos Especiais Ao meu orientador Prof. Dr. Joni Augusto Cirelli Muito obrigado. Obrigado pelos ensinamentos, exemplo de conduta profissional, pela confiança. Obrigado por acreditar e confiar no meu trabalho, obrigado por me permitir crescer. O seu apoio, ensinamento, e dedicação foi muito importante para minha formação acadêmica e profissional. Minha admiração, respeito e gratidão. Ao Prof. Dr. Sotirios Tetradis Thank you Dr. Tetradis for all the great opportunities, knowledgement, and for the great life I had in LA. Thank you for receiving me so nicely in your lab. Thank you for all the support and efforts in all the projects we worked on. I am very grateful for the respect, trustful, and for all the happy moments during the 18 months I spent in your lab. I also would like to thank all member from your laboratory especially Olga Bezouglaia, and Prof. Dr. Tara Aghaloo. I am sure we will continue our collaboration. A Profa. Dra. Gulnara Scaf Pelas primeiras orientações no desenvolvimento de trabalhos científicos, pela amizade, convívio, pelo apoio, confiança, e pelo exemplo de honestidade e de conduta profissional. Aos grandes amigos da pós-graduação Aos amigos Jõao Paulo Steffens, Fausto Frizzera, Jonatas Esteves, Mario Verzola, Rubens Moreno, Guilherme Oliveira, Luiz Guilherme Freitas, Joao Antônio Souza, Pedro Souza e 10 Vinicius Ibiapina acima de tudo pela amizade sincera conquistada, e por todos os momentos de alegria, companheirismo e convivência diária durante esses seis últimos anos. Tenho certeza que mesmo após o fim dos nossos cursos, nossa amizade permanecerá. Aos queridos amigos de Araraquara João Paulo, Pablo, Fausto, Fernanda Rocha, Giovana, Marcell, Sabrina, Andressa, Mario, Luana, Lívia, Luizinho, Pedrão, Gui, Piauí, João Antônio. Espero que esta amizade permaneça ao longo de nossas vidas. 11 Agradecimentos À Faculdade de Odontologia de Araraquara, Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP Na pessoa de seu Diretor Profa. Dra. Andreia Affonso Barretto Montandon e a Vice-Diretora, Profa. Dra. Elaine Maria Sgavioli Massucato. À Coordenação do Pós-Graduação em Odontologia, Faculdade de Odontologia de Araraquara – UNESP, representada pelo coordenador Prof. Dr. Carlos Rossa Jr e pelo Vice-coordenador Prof. Dr. Joni Augusto Cirelli. Pela enorme admiração, pela competência, dedicação, responsabilidade e exemplo de conduta e profissionalismo. Aos docentes do curso de Pós-Graduação em Odontologia, Periodontia da Faculdade de Odontologia de Araraquara – UNESP Prof. Dr. Elcio Marcantonio Junior, Prof. Dr. José Eduardo César Sampaio, Profa. Dra. Rosemary Adriana Chiérici Marcantonio, Prof. Dr. Joni Augusto Cirelli, Prof. Dr. Carlos Rossa Junior, Profa. Dra. Silvana Regina Perez Orrico, Profa. Dr. Daniela Zandim pela formação, orientação e exemplo de dedicação e conduta profissional. À Fundação de Amparo a Pesquisa do Estado de São Paulo, FAPESP Pelo apoio financeiro concedido durante o doutorado, indispensável para a realização deste trabalho, por permitir e subsidiar a minha vida em Araraquara (Processo # 2012/09968-5). 12 À Coordenação de Aperfeiçoamento de Pessoal do Nível Superior, CAPES Pelo apoio financeiro concedido durante o doutorado Sanduiche, indispensável para a realização deste trabalho, por permitir e subsidiar a minha vida em Los Angeles, US (Processo #11575-13-1). A todos os funcionários da Faculdade de Odontologia de Araraquara – UNESP Especialmente a Claudinha e ao Leandro, por toda a ajuda e ensinamentos durante o processamento histológicos, Exact e na preparação de soluções para o laboratório. Regina Lúcia, Cláudia, D. Maria do Rosário, Maria José e Esther, pela amizade, carinho e respeito que sempre me dispensaram. Aos funcionários da Seção de Pós-Graduação pela competência, paciência e pela colaboração dedicada. Aos funcionários da biblioteca: Marley, Adriano, Ceres, Eliana e Sílvia pela colaboração. A todas as pessoas que participaram direta ou indiretamente na realização desse trabalho, e que não estão citadas nominalmente, meus sinceros agradecimentos. 13 “A nossa ciência, comparada com o mundo que descreve, é pequena e infantil. No entanto, é a coisa mais preciosa que temos” Albert Einstein 14 de Molon RS. Patofisiologia de osteonecrose induzida por bisfosfonatos e inibidores de RANK-L [Tese de Doutorado]. Araraquara: Faculdade de Odontologia da UNESP; 2016. RESUMO A patogênese de osteonecrose dos maxilares (ONJ) e os fatores de risco ainda não estão totalmente elucidados e continuam sendo foco de contínuos estudos visando estabelecer um correto diagnóstico, prevenção e tratamento. Assim, os objetivos do presente estudo foram: (1) desenvolver em camundongos, um modelo experimental de ONJ induzido por bisfosfonatos (BPs) e inibidores de RANK-L associado a doença periodontal espontânea; (2) investigar neste modelo se a descontinuação desses medicamentos alteraria as características clinicas e radiográficas de osteonecrose e, (3) avaliar a influência da artrite reumatoide (AR) no agravamento de ONJ experimental. As análises em todos os estudos foram estabelecidas por meio de avaliações radiográficas, por microtomógrafo, e histológicas. No primeiro estudo, foi estabelecido um novo modelo experimental animal utilizando-se diferentes medicamentos anti-reabsortivos, resultando em características clinicas semelhantes ao que ocorre em humanos, ou seja, extensas áreas necróticas e com exposição óssea a cavidade oral. Os resultados do estudo 2 demonstraram que a descontinuação de OPG-Fc, um inibidor de RANK-L, mas não de BPs reverteu todas as características clinicas e radiográficas de ONJ. Além disso, os resultados do estudo 3 sugerem que a AR associada ao tratamento com altas doses de BPs exacerba a incidência e a severidade de ONJ em camundongos. Com base nos achados do presente estudo, conclui-se que ONJ induzida por BPs e por inibidores de RANK- L como OPG-Fc e RANK-Fc apresentam grande similaridade nas características radiográficas e histológicas, entre si e com a doença em humanos, mas somente a descontinuação do tratamento com OPG-Fc leva a uma reversão completa das alterações clinicas da doença. Os dados suportam a relevância da AR como um fator de risco para o desenvolvimento de ONJ. Palavras-chave: Perda óssea alveolar. Camundongos. Osteoclasto. Doenças periodontais. Artrite reumatoide. 15 de Molon RS. Pathophysiology of osteonecrosis of the jaws induced by bisphosphonates and RANK-L inhibitors [Tese de Doutorado]. Araraquara: Faculdade de Odontologia da UNESP; 2016. ABSTRACT The pathogenesis of the osteonecrosis of the jaw (ONJ) and its risk factors still remain poorly understudied and continue to be the focus on ongoing studies to establish a proper diagnosis, prevention and treatment. Thus, the aims of this study were: (1) to develop in mice, an experimental model of osteonecrosis induced by bisphosphonates (BPs) and RANK-L inhibitors associated with spontaneously periodontal disease. Then, (2) using the same experimental model in mice, to investigate if the discontinuation of these drugs alters the clinical and radiographic features of ONJ. In addition, (3) the influence of systemic diseases such as rheumatoid arthritis (RA), and ONJ was assessed as a possible risk factor for worsening ONJ characteristics. The analyses in all studies were established through radiographic, by means of micro-computed tomography, and histological assessments. In the first study, a new experimental animal model using different antiresorptive drugs was established with clinical features similar to what occurs in humans, with extensive necrotic areas and exposed bone o the oral cavity. The results of the study 2 showed that discontinuing OPG-Fc, a RANK-L inhibitor, but not BPs reversed all the clinical and radiographic features of ONJ. Furthermore, the results of study 3 suggest that treatment with high doses of BPs associated with the presence of RA exacerbated the incidence and severity of ONJ in mice. These data evidenced that BPs and RANK-L inhibitors possess great similarity in the radiographic and histologic findings, including the incidence of osteonecrosis and bone exposure, but only the OPG-Fc discontinuation completely reverses ONJ features. The data further support the relevance of RA as a risk factor for ONJ development. Keywords: Alveolar bone loss. Mice. Osteoclasts. Periodontal disease. Rheumatoid arthritis. 16 SUMÁRIO 1 INTRODUÇÃO..........................................................................................................17 2 PROPOSIÇÃO...........................................................................................................26 3 PUBLICAÇÕES.........................................................................................................27 3. 1 Publicação 1............................................................................................................27 3.2 Publicação 2.............................................................................................................36 3.3 Publicação 3.............................................................................................................58 4 CONSIDERAÇÕES FINAIS....................................................................................99 5 CONCLUSÃO..........................................................................................................104 REFERÊNCIAS.......................................................................................................105 ANEXO.....................................................................................................................116 17 1 INTRODUÇÃO O desenvolvimento e ativação de osteoclastos é um processo essencial na homeostase metabólica mineral e óssea durante o crescimento e renovação do esqueleto. Estes processos envolvem a reabsorção de osso mineralizado por osteoclastos92, (células multinucleadas derivadas de precursores da linhagem de monócitos/macrófagos, e são responsáveis pela degradação da matriz óssea mineralizada), seguida pela subsequente formação e mineralização de nova matriz óssea pela ação dos osteoblastos24,65. Em várias patologias, tais como osteoporose, metástases tumorais osteolíticas, artrite reumatoide e doença periodontal, a quantidade de osso reabsorvido pelos osteoclastos excede aquela substituída durante a formação óssea, levando a uma perda de massa óssea, aumento da fragilidade óssea e risco de fratura89. O aumento da expressão e produção de RANKL (ligante do receptor de ativação do fator nuclear κB), observado por exemplo, com a privação de estrógeno ou durante o curso da doença periodontal e osteoporose, é um mecanismo central da ativação e diferenciação de osteoclastos e aumento da reabsorção óssea39,105. Neste contexto, duas citocinas surgem como protagonistas essenciais e suficientes para induzir a diferenciação de osteoclastos: o fator estimulante de colônias de macrófagos (M-CSF) e RANKL40,58,114. M-CSF liga-se ao c-Fms, um receptor único de domínio transmembrana da família da tirosina quinase, e RANKL se liga ao RANK, um receptor único transmembrana da família de receptores do fator de necrose tumoral (TNF). O mecanismo de ação envolve a ativação da proliferação e sobrevivência de células da linhagem de monócitos-macrófagos e a expressão de RANK pelo M-CSF, permitindo a ação do RANKL que, em conjunto com M-CSF, constitui uma exigência absoluta para a progressão de precursores iniciais ao longo da linhagem de osteoclasto14,23,70. A diferenciação a partir de precursores de osteoclastos para osteoclastos multinucleados é então induzida por RANKL, novamente por meio da expressão de M-CSF-dependente de RANK na superfície celular destes precursores precoces. Tanto M-CSF como RANKL são secretados pelas células do estroma da medula óssea e osteoblastos, bem como por células T e B. A maioria das células que produzem RANKL também produz um receptor, denominado osteoprotegerina (OPG) que atua como antagonista da sinalização de RANKL e osteoclastogênese55. Em última análise, é a relação entre RANKL e OPG que determina o nível de ativação de RANK e, desse modo, o grau em que é ativado a osteoclastogênese. Quase todos os tratamentos farmacológicos atuais envolvendo doenças metabólicas ósseas, 18 como a osteoporose, utilizam drogas que inibem a reabsorção óssea. Dentre estas drogas, estão incluídas: bisfosfonatos (BPs), raloxifeno e calcitonina. Uma nova e recente abordagem visa atuar no sistema RANK/RANKL de uma maneira específica. O papel indispensável da via RANK/RANKL na remodelação óssea foi demonstrada por doenças monogênicas em humanos, em que a ausência funcional de RANK ou RANKL resultaram em osteopetrose45,99. Estudos clínicos demonstram que a inibição de RANKL foi alcançada por meio de diferentes dosagens de Fc-OPG (0.3, 1 e 3 mg/kg) em mulheres na menopausa levando a uma rápida e sustentada inibição da reabsorção óssea18,31. Recentes investigações têm avaliado amplamente uma nova droga, denominada denosumab (DMAb). Este anticorpo monoclonal humano, se liga e inibe RANKL impedindo o processo de reabsorção óssea. Medicamentos anti-reabsortivos tais como BPs, e o inibidor de RANKL DMAb, impedem a reabsorção óssea osteoclástica e são utilizados na clínica para gerenciar doenças ósseas, como malignidade óssea primária ou metastática e osteoporose. Estes medicamentos, estudados em pacientes com câncer e em mulheres na pós-menopausa, agem reduzindo complicações ósseas, diminuindo a carga tumoral, reduzindo a dor óssea, e diminuindo a incidência de fraturas osteoporóticas, contribuindo, desta forma, para melhorar a qualidade de vida dos pacientes69,83,104,106. A eficácia clínica dos BPs está baseada em duas propriedades chave: (1) na habilidade de se ligar fortemente ao osso mineralizado e (2) no efeito inibitório sobre os osteoclastos maduros90. A forte ligação dos BPs ao osso confere aos bisfosfonatos a propriedade única de captação seletiva pelo órgão-alvo a que se destina. Os BPs ligam-se seletivamente ao osso mineral, e assim inibem osteoclastos maduros nos sítios de reabsorção óssea. Ao mesmo tempo em que os BPs admitem uma estrutura química similar à molécula inorgânica pirofosfato, diferem-se deste por possuírem uma ligação fósforo-carbono-fósforo (P-C-P) ao invés de uma ligação fósforo-oxigênio-fósforo (P-O-P). Esta ligação P-C-P é altamente resistente à hidrólise, tornado os BPs fortemente resistentes a degradação biológica. BPs contendo nitrogênio, tais como: o alendronato, ibandronato, minodronato, pamidronato, risedronato, neridronato e zoledronato, apresentam uma cadeia lateral que contém um átomo de nitrogênio. Uma vez absorvido pelos osteoclastos, estes compostos são capazes de inibir a reabsorção óssea por meio de efeitos intracelulares. BPs contendo nitrogênio atuam principalmente através da inibição da via farnesil-pirofosfato sintase (FPP) - uma enzima da via do mevalonato - impedindo, desta forma, a modificação pós-translacional (prenilação) das proteínas de ligação (guanosina trifosfato - GTP), essenciais para função e sobrevivência dos osteoclastos90. Como resultado, BPs inibem o funcionamento dos osteoclastos ao danificar a 19 diferenciação, perturbar o citoesqueleto, diminuir o transporte intracelular, e induzir apoptose (Figura 1)53,103. Figura 1 - Via de sinalização do mevalonato demonstrando o mecanismo de ação dos bisfosfonatos. Autor: Baron et al.16 Fonte: Bone 2011; 48(4): 677-92 O mecanismo de ação do DMAb difere dos BPs. DMAb impede a ligação do RANKL ao receptor RANK, inibindo, assim, o desenvolvimento, a ativação e a sobrevivência de osteoclastos. Os efeitos da inibição de RANK-L foram inicialmente analisados em estudos em animais e em humanos com proteínas fusionadas Fc (Figura 2)19,72,93,95,108. Fc-OPG (derivado da Escherichia coli), OPG-Fc (expressados nas células mamárias do hospedeiro) e RANK-Fc foram fusionados à porção Fc da imunoglobulina humana G1 (IgG1) e os respectivos efeitos foram avaliados19,72. Fc-OPG foi administrado pela primeira vez em humanos em 199818. Neste estudo clínico de primeira fase, os autores constataram um rápido declínio dos marcadores de remodelação óssea, com um efeito durador de até 6 semanas. Contudo, somente em 2004, os efeitos farmacológicos de Fc-OPG derivados da E. coli foram compreendidos111, revelando mutações genéticas em RANK e OPG associadas com desordens 20 esqueléticas humanas caracterizadas por excessiva atividade osteoclástica. Por este motivo, a continuidade no desenvolvimento do Fc-OPG foi interrompida. Como uma alternativa ao uso do Fc-OPG, OPG-Fc passou a ser produzido a partir de células mamárias de hamsters. As características observadas em relação ao Fc-OPG foram efeito prolongado (de até 10 vezes) e potência de até três vezes comparado com os demais construtos57. Em relação a molécula RANK-Fc, esta foi desenvolvida a partir da fusão com o domínio extracelular de RANK à porção Fc da IgG1 100. Contudo, estudos prévios identificaram risco significativo de hipercalcemia, pela ativação de auto-anticorpos, em primatas, contra o receptor RANK por meio de repetidas doses desta molécula. Evidências de uma resposta imune ao utilizar RANK endógeno em pacientes (observações não publicadas - Amgen), levou a descontinuação da produção de RANK-Fc. Assim, a decisão de interromper os estudos com todos os construtos foi estabelecida em favor do desenvolvimento de um anticorpo, tendo como molécula alvo o RANKL, ao invés de receptores, como nos construtos. Figura 2 - Evolução dos inibidores recombinantes de RANKL. Autor: Lacey et al.57 Fonte: Nature Reviews - Drug Discovery 2012; 11(5): 401-19 DMAb apresenta varias vantagens com relação aos construtos OPG-Fc, Fc-OPG e RANK-Fc68. Dentre estas, (1) DMAb não se liga ao TRAIL (ligante indutor de apoptose relacionado ao TNF) ou a outros membros da superfamília do TNF, tais como CD40, TNF-α e TNF-β; (2) a meia vida é prolongada comparada com os construtos, especialmente pela massa 21 molecular; (3) apesar de ambos DMAb e OPG-Fc admitirem o mesmo alvo, o DMAb é mais específico reconhecendo apenas RANKL humano, em contraste com OPG-Fc, que também se liga ao RANKL de ratos e camundongos, bem como TRAIL41,56. Por conseguinte, o mecanismo de ação do DMAb difere-se dos BPs (Figura 3), os quais se ligam ao osso mineral e inibem a função dos osteoclastos, principalmente, por serem absorvidos por osteoclastos em locais de reabsorção óssea. A principal diferença na forma de ação entre DMAb e BPs em osteoclastos é que o segundo age somente no interior das células, enquanto que o DMAb atua no meio extracelular16. Figura 3 - Ilustração dos mecanismos de inibição de osteoclastos mediada por BPs e DMAb. Autor: Baron et al.16 Fonte: Bone 2011; 48(4): 677-92. O tratamento a longo prazo com BPs tem sido associado com um aumento do número de osteoclastos, incluindo osteoclastos gigantes, hiper-nucleados, não aderidos à superfície óssea, e que passam por apoptose110. A inibição da atividade dos osteoclastos por meio dos BPs poderia desencadear um ciclo de feedback que resultaria em um aumento de RANKL no osso. A superprodução de RANKL poderia, por sua vez, estimular os precursores de 22 osteoclastos a fundirem-se em osteoclastos multinucleados. Esta hipótese é apoiada pela evidência de um aumento no número de osteoclastos sobre as superfícies ósseas em camundongos huRANKL ovariectomizados tratados com alendronato, ao passo que fora constatado um desaparecimento quase completo de osteoclastos neste modelo na presença de DMAb79. Resultados preliminares comparando os efeitos do tratamento de artrite com inibidores de RANKL e BPs, indicaram que o OPG reduziu significativamente a presença de RANKL no osso, enquanto que o ácido zoledrônico causou um aumento de duas vezes na expressão deste ligante101. Apesar de o mecanismo de ação farmacológico ser distintamente diferente, BPs e DMAb estão associados com osteonecrose dos maxilares (ONJ), um infrequente, mas grave efeito adverso, particularmente quando administrado em doses elevadas11. ONJ é definido como osso exposto ou osso que pode ser sondado através de uma fístula intra-oral ou extra- oral na região maxilofacial, que persiste por mais de 8 semanas em pacientes em tratamento atual ou anterior com medicamentos anti-reabsortivos e sem histórico de terapia de radiação nos maxilares ou com doença metastática na maxila e mandíbula50,52,87. Com o aumento no número de casos de ONJ em pacientes que fazem uso de DMAb, a Sociedade Americana de Cirurgiões Oral e Maxilofacial (AAOMS) recomendou o uso do termo osteonecrose dos maxilares relacionada a medicamentos (MRONJ)87. Nesse contexto, pacientes expostos a administração intravenosa de BPs para tratamento de doenças malignas, a incidência de ONJ varia entre 0 a 12.222 para cada 100.000 pacientes / ano12,15,17,22 e a incidência de ONJ em pacientes tratados com DMAb varia de 0 a 2.316 para cada 100.000 pacientes / ano43,62,96,98,102. Por outro lado, a incidência de ONJ em pacientes que fazem uso de BPs por via oral, especialmente para tratamento de osteoporose, varia entre 1.04 a 69 para cada 100.000 pacientes / ano32,51,81 e para os pacientes que fazem uso de DMAb varia de 0 a 30.2 para cada 100.000 pacientes / ano13,30,73,76. Embora a incidência de ONJ seja pequena, o desenvolvimento da doença resulta em serias complicações para estes pacientes. O atual sistema de classificação de ONJ envolve os estágios 0-3 e baseia-se na apresentação clínica do paciente. Apesar de um paciente em estágio 0 não apresentar osso exposto, o que o torna de difícil diagnóstico, os estágios 1 a 3 são definidos em parte pelo osso exposto à cavidade oral. Pacientes em estágio 1 são assintomáticos e não mostram sinais de infecção; por outro lado, pacientes em fase 2 apresentam infecção frequentemente associada com dor, eritema e drenagem purulenta. Em um estado mais avançado, a fase 3 envolve a presença de osso necrosado estendendo-se além do alvéolo dentário, com possível chance de fratura patológica da mandíbula, fístula extraoral, e comunicação nasal85. Na 23 grande maioria dos casos, ONJ ocorre após extração dentária e ao redor de dentes com doença periodontal ou periapical67,86. Assim, o histórico do paciente e o exame clínico são as ferramentas de diagnóstico mais sensíveis para esta condição. Áreas de osso necrosado podem permanecer assintomáticas por semanas, meses ou anos podendo resultar em dor ou osso exposto na maxila ou mandíbula85. Estas lesões tornam-se sintomáticas com a inflamação dos tecidos circundantes e/ou quando houver evidência clínica de exposição óssea. Os sinais e sintomas que antecedem o desenvolvimento da osteonecrose clinicamente detectável incluem dor, mobilidade do dente, inchaço da mucosa, eritema, e ulceração. Estes podem ocorrer espontaneamente ou mais comumente no local da cirurgia dento-alveolar84. Embora os primeiros casos de ONJ tenham sidos descritos há mais de 10 anos, tanto a etiologia quanto a patogênese ainda permanecem desconhecidas. Alguns fatores como inibição de osteoclastos e supressão da remodelação óssea, inflamação e infecção, inibição de angiogênese, função alterada das células imunes do hospedeiro, e baixo turnover ósseo são algumas das hipóteses propostas que fundamentam o desenvolvimento e progressão de ONJ9- 11,49. Fatores que aumentam a incidência e o risco de ONJ incluem: (1) dose e duração do tratamento com medicamentos inibidores de reabsorção óssea; (2) cirurgias dento-alveolares e doenças bucais; (3) doenças sistêmicas e (4) infecção bacteriana9-11,78. Esses fatores de risco podem levar a complicações durante a evolução da doença. Dentre as hipóteses existentes para elucidar a patogênese de ONJ, inflamação e infecção desempenham um papel importante (Figura 4). Estudos recentes apontam para alguns fatores adicionais, além do uso de BPs ou DMAb, que contribuem para o desenvolvimento de necrose, tais como: extrações dentárias, doença periodontal, e doença periapical22,67,87. Modelos de estudo de inflamação e infecção tem sido desenvolvidos em animais na tentativa de reproduzir os achados clínicos de ONJ em pacientes com patologias dentais. Os resultados tem mostrado que a presença de infecção e inflamação são suficientes para o desenvolvimento de necrose óssea3,5,6,27,48,115. Além disso, o papel da infecção bacteriana na patogênese de ONJ é destacada pela redução da incidência de osteonecrose em pacientes com câncer após a melhoria na higiene dental20,71,81. Supressão da imunidade inata ou adquirida causada por corticosteroides ou por quimioterapia também induziu o surgimento de ONJ após a administração de BPs87. Este achado fora suportado por um estudo recente21, em que os autores confirmaram a inflamação estéril nos tecidos moles em torno do maxilar como não suficiente para induzir ONJ. Em um modelo experimental em ratos, o tratamento com antibióticos e fechamento do retalho mucoperiosteal no dia da extração dentária impediu o desenvolvimento de osteonecrose1,63. Não obstante, pode-se concluir que o estado 24 imunocomprometido do paciente em função de doenças, tais como diabetes e artrite reumatoide, e uso de medicamentos, como esteroides e quimioterápicos, podem aumentar a prevalência e a severidade de ONJ quando combinados com infecção / inflamação oral ou trauma e na presença de drogas que inibem a reabsorção óssea26,28,29,115. Figura 4 - Ilustração dos mecanismos propostos para o desenvolvimento de ONJ. Autor: Aghaloo et al.2 Fonte: Oral Maxillofacial Surg Clin N Am 2015; 27(4): 489-96. Assim, o progresso significativo no conhecimento da patogênese de ONJ é dependente do desenvolvimento de modelos animais que reproduzam fielmente as manifestações clínicas da doença6. Perguntas mais básicas sobre a fisiopatologia, tais como causa / efeito dos BPs e DMAb, o papel das doses / durações do tratamento, e quais os outros co-fatores que contribuem para o surgimento e/ou severidade da doença, poderiam ser rapidamente respondidas em uma situação pré-clínica, e possivelmente reduziriam os vieses em pesquisas subsequentes em humanos8. Progressos no desenvolvimento de modelos animais de MRONJ começaram lentamente, e em muitos casos, houve poucos avanços na compreensão da doença. Atualmente, modelos experimentais recapitulando as características de ONJ em roedores e em animais de grande porte, tratados com BPs ou DMAb, tem ampliado o conhecimento sobre a patogênese de ONJ3,5,27,77,107,115. Contudo, modelos animais de osteonecrose induzidos por BPs e inibidores de RANKL associando diferentes modelos de indução de doença periodontal 25 e periapical, permitem o melhor entendimento da patofisiologia da doença, com a vantagem de reduzir os vieses dos estudos clínicos e por fim, auxiliar na promoção de uma melhor atenção médica e periodontal aos pacientes com ONJ. Na literatura científica, estudos focando no desenvolvimento de modelos animais e possíveis investigações mostrando a relação da terapia medicamentosa com possível resolução das características clinicas de necrose, bem como a indução concomitante de ONJ, doença periodontal e doença sistêmica (artrite reumatoide) ainda são escassos. Em uma tentativa de esclarecer alguns dos questionamentos citados ao longo do texto com relação a patogênese de ONJ, nós dividimos este estudo em 3 capítulos nos quais os objetivos específicos de cada um serão explicados a seguir. 26 2 PROPOSIÇÃO O presente estudo teve como objetivo desenvolver um modelo experimental de ONJ em camundongos e avaliar a influência de fatores de risco na incidência e severidade da doença. Para isto o estudo foi subdividido em 3 capítulos com objetivos específicos descritos a seguir: Proposição específica publicação 1 Considerando que a patofisiologia de ONJ ainda não está totalmente estabelecida, o uso de modelos experimentais em animais, com particularidades que se assemelham com as características das condições humanas, se tornam ferramentas indispensáveis para fornecer estratégias de tratamento e prevenção. Assim, o objetivo do primeiro estudo foi desenvolver um novo modelo experimental de ONJ em camundongos, induzidos por diferentes inibidores de reabsorção óssea, associado com doença periodontal espontânea. Proposição específica publicação 2 Considerando que as recomendações para reduzir o risco de incidência de ONJ ainda continuam altamente controversas e indefinidas, o objetivo do segundo estudo foi investigar se a descontinuação de diferentes inibidores de reabsorção óssea (receptor activator of NF- kBligand (RANKL) inhibitor OPG-Fc, e Zoledronic Acid) promoveria alteração nas características clinicas e radiográficas, e na diminuição da incidência de ONJ em camundongos Proposição específica publicação 3 Considerando que existem fatores de risco que aumentariam as chances do desenvolvimento de ONJ, incluindo dose e duração dos medicamentos, doenças sistêmicas, infecção bacteriana, entre outras, o objetivo do terceiro estudo foi avaliar interações entre artrite reumatoide e ONJ em camundongos utilizando-se diferentes modelos de indução de ONJ na maxila e mandíbula por meio de análises tomográfica e histológica. Original Full Length Article Spontaneous osteonecrosis of the jaws in the maxilla of mice on antiresorptive treatment: A novel ONJ mouse model Rafael Scaf deMolon a,b,1, Simon Cheong c,1, Olga Bezouglaia a, SarahM.Dry d, Flavia Pirih e, Joni Augusto Cirelli b, Tara L. Aghaloo a,⁎, Sotirios Tetradis a,f,⁎⁎ a Division of Diagnostic and Surgical Sciences, UCLA School of Dentistry, Los Angeles, CA 90095, USA b Department of Diagnosis and Surgery, School of Dentistry at Araraquara, São Paulo State University, Araraquara 14801–903, Brazil c Ostrow School of Dentistry, University of Southern California, Los Angeles, CA, USA d Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA e Division of Associated Specialties, UCLA School of Dentistry, Los Angeles, CA 90095, USA f Molecular Biology Institute, UCLA, Los Angeles, CA 90095, USA a b s t r a c ta r t i c l e i n f o Article history: Received 26 April 2014 Revised 2 July 2014 Accepted 20 July 2014 Available online 2 August 2014 Edited by: Robert Recker Keywords: Osteonecrosis of the jaw ONJ Antiresorptives Bisphosphonates Alveolar bone Osteoclasts Although osteonecrosis of the jaws (ONJ), a serious complication of antiresorptive medications, was reported a decade ago, the exact mechanisms of disease pathophysiology remain elusive. ONJ-like lesions can be induced in animals after antiresorptive treatment and experimental interventions such as tooth extraction or periapical or periodontal disease. However, experimental induction and manipulation of disease progression does not always reflect clinical reality. Interestingly, naturally occurring maxillofacial abscesses, inducing aggressive inflammation of the peri-radicular mucosa with significant osteolysis and alveolar bone expansion, have been reported in mice. Here, we aimed to explore whether osteonecrotic lesions would develop in areas of maxillary peri-radicular infections, in mice on antiresorptive medications with distinct pharmacologic action, thus establishing a novel ONJ animal model. Mice were treated with RANK-Fc or OPG-Fc that bind to RANKL or with the potent bisphosphonate zoledronic acid (ZA). Maxillae were assessed radiographically and histologically. μCT imaging of vehicle mice revealed several maxillae with altered alveolar bone morphology, significant ridge expansion and large lytic areas. However, in RANK-Fc, OPG-Fc and ZA treated animals the extent of bone loss was significantly less, but exuberant bone deposition was noted at the ridge periphery. BV and BV/TV were increased in the diseased site of antiresorptive vs. veh animals. Histologically, extensive inflammation, bone resorption and marginal gingival epithelium migration were seen in the diseased site of vehicle animals. Rank- Fc, OPG-Fc and ZA reduced alveolar bone loss, increased periosteal bone formation, and induced areas of osteonecrosis, and bone exposure that inmany animals covered significant part of the alveolar bone. Collectively, our data demonstrate ONJ-like lesions at sites of maxillary peri-radicular infection, indistinguishable in mice treated with RAKL inhibitors vs. zoledronate. This novel mouse model of spontaneous ONJ supports a central role of osteoclast inhibition and infection/inflammation in ONJ pathogenesis and validates and complements existing animal models employing experimental interventions. © 2014 Elsevier Inc. All rights reserved. Introduction Osteonecrosis of the jaws (ONJ) is a serious side effect of antiresorptive medications such as bisphosphonates (BPs) and denosumab that ranges in severity from painless, small areas of exposed bone to significant bone exposure associated with severe pain, sequestration, infection, fistula or jaw fracture [1–3]. Although many clinical and animal studies attempt to characterize features of ONJ, the pathogenetic mechanisms of the disease remain elusive [4–6]. Osteoclastic inhibition appears central in the disease process since agents that target osteoclast function, but with distinct pharmacologic properties result in the same clinical outcome [1,4–7]. Bisphosphonates and particularly nitrogen-containing ones, such as zoledronic acid (ZA), pamidronate, and alendronate, inhibit farnesyl diphosphate synthase in the cholesterol biosynthesis pathway, which prevents prenylation of small guanosine triphosphatase (GTPase) signaling proteins [8,9]. As a result, BPs inhibit functioning osteoclasts by impairing differentiation, Bone 68 (2014) 11–19 ⁎ Correspondence to: T.L. Aghaloo, Diagnostic and Surgical Sciences, UCLA School of Dentistry, 10833 Le Conte Ave. CHS Rm. 53–009, Los Angeles, CA 90095–1668, USA. Fax: +1 310 825 7232. ⁎⁎ Correspondence to: S. Tetradis, Diagnostic and Surgical Sciences, UCLA School of Dentistry, 10833 Le Conte Ave. CHS Rm. 53–068, Los Angeles, CA 90095–1668, USA. Fax: +1 310 825 7232. E-mail addresses: taghaloo@dentistry.ucla.edu (T.L. Aghaloo), stetradis@dentistry.ucla.edu (S. Tetradis). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bone.2014.07.027 8756-3282/© 2014 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Bone j ourna l homepage: www.e lsev ie r .com/ locate /bone 3 PUBLICAÇÕES 3.1 Publicação 1 disrupting the cytoskeleton, decreasing intracellular transport, and inducing apoptosis [9,10]. In contrast, denosumab binds directly to the receptor activator of nuclear factor kappa-B (RANK) ligand (RANKL) to prevent its interaction with RANK on osteoclasts. This binding inhibits osteoclast formation, differentiation, and function [11]. Although acting through diverse mechanisms, both BPs and denosumab show similar prevalence of ONJ in patients with multiple myeloma, breast, and prostate cancer [12–14]. ONJ occursmostly after tooth extraction or around teeth with dental disease [15,16]. Teeth in adult patients are extracted mainly as a result of unrestorable caries or periodontal disease [17,18]. Thus, infection/ inflammation due to dental disease appears to be present in most ONJ cases. Paralleling these clinical observations, animal findings demon- strate ONJ-like lesions after experimental interventions such as tooth extraction or periapical or periodontal disease [19–30]. These interven- tions are employed to simulate clinical scenarios that precipitate disease pathogenesis in a controlled and reproducible manner. Interestingly, naturally occurring maxillofacial abscesses have been described in mice [31]. Hair impaction from grooming enters the oral cavity, and inserts into the gingival sulcus where bacterial colonization occurs [31–33]. This natural process leads to the development of repro- ducible bone disease in C57BL/6 mice, which serves as an interesting contrast to the experimentally induced dental disease studies in mice and rats [19,20,23]. Here capitalizing on the natural occurrence of alveolar lesions, we aimed to explore the radiographic and histologic changes in the maxillae of mice treated with agents possessing distinct pharmacologic inhibition of osteoclastic activity. Our data suggest that in this novel ONJ animal model, naturally occurring peri-radicular infection and antiresorptive treatment are sufficient to induce osteonecrotic lesions in the mouse maxilla. Materials and Methods Animal Care Animals were kept and treated according to guidelines of the UCLA Chancellor's Animal Research Committee. Throughout the experimental period, mice were housed in plastic cages, fed a standard laboratory diet, and given water ad libitum. Seventy-six 4-month old C57BL/6J male mice (Jackson Laboratories, Bar Harbor, ME, USA) received intra- peritoneal (IP) injections of veh (endotoxin free saline) or 200 μg/kg zoledronic acid (ZA, Z-5744 LKT Laboratories, St. Paul, MN) three times per week or 10 mg/kg mouse RANK-Fc (composed of the extracellular domain of RANK fused to the Fc portion of IgG [34], kindly provided by Amgen, Inc., Thousand Oaks, CA) three times per week, or 10 mg/kg rat OPG-Fc (composed of the RANKL-binding domains of os- teoprotegerin linked to the Fc portion of IgG [34,35], kindly provided by Amgen, Inc., Thousand Oaks, CA) once per week for a total of 12 weeks. The time and dose of these antiresorptives have been previ- ously shown to induce ONJ-like lesions in mice with periapical disease [19,23]. There were 17 veh, 19 ZA, 20 OPG-Fc and 20 RANK-Fc treated animals. At the end of the experiment, animals were sacrificed via isoflurane overdose, and maxillae were dissected, placed in 4% parafor- maldehyde for 48 hours and stored in 70% ethanol. μCT Scanning Dissected maxillae were imaged by μCT scanning (μCT SkyScan 1172; SkyScan, Kontich, Belgium) utilizing 57 kVp, 184 μA, 0.5 mm aluminum filtration and 10 μm resolution. For linear measurements, volumetric image data were converted to DICOM format and imported in the Dolphin Imaging software (Chatsworth, CA) to generate 3D and multiplanar reconstructed images. The periodontal ligament (PDL) space width was measured at the furcation area of the first molar. The cemento-enamel junction (CEJ) to the alveolar crest (AC) distance was measured at the distal surface of the first molar. Both measurements were performed on a sagittal slice through the middle of the furcation area along the mesio-distal axis of the teeth, as previously described [19]. To assess alveolar bone thickness, axial slices were oriented parallel to the occlusal plane and the bone width was measured at the buccal side of the alveolar ridge at the level of the apical third of the roots. To measure alveolar bone volume (BV) and tissue volume (TV), a region of interest (ROI) was delineated from the mesial root of the first molar to the distal root of the third molar, and from the alveolar crest to the root apices, comprising the entire alveolar bone. The teeth roots were not included in the ROI. BV and TV were measured utilizing the CtAn software (CT Analyzer 1.10.1.0 - Skyscan, Kontich, Belgium). Histology and TRAP staining Maxillae were decalcified in 14.5% ethylenediaminetetraacetic acid (EDTA) solution for 3 weeks. Samples were paraffin embedded and 5 μm-thick cross sections were made perpendicular to the long axis of the alveolar ridge at the area of maximum radiographic changes, as assessed by μCT analysis. If no radiographic evidence of disease was present in either of the sides of the alveolar bone, then sections were made along the distal root of the first molar. Hematoxylin and eosin (H&E) stained slides were digitally scanned utilizing the Aperio AT automated slide scanner and the Aperio ImageScope software (Aperio Technologies, Inc., Vista, CA, USA). The area of the alveolar bone, from the alveolar crest to the superior border of the alveolar bone and the floor of the nasal cavity, was defined as the region of interest (ROI). All subsequent measurements were made in this area. Histologic measurements were made as previously described [19]. The distance between themost apical point of the junctional epithelium to themost coronal point of the alveolar crest (JE-AC)wasmeasured on the palatal side of themaxilla. Periosteal bone thicknesswas assessed by measuring the three greatest areas of palatal or buccal periosteal thick- ness that were then averaged. The total number of osteocytic lacunae, the number of empty lacunae, the surface of total bone area and the sur- face of osteonecrotic area(s) were quantified. An area of osteonecrosis was defined as a loss of more than five contiguous osteocytes with confluent areas of empty lacunae. All histology and digital imaging was performed at the Translational Pathology Core Laboratory (TPCL) at UCLA. For enumeration of osteoclasts, tartrate-resistant acid phosphatase (TRAP) staining was performed from all the groups utilizing the leuko- cyte acid phosphatase kit (387A-IKT Sigma Aldrich, St. Louis, MO, USA). Positive cells were identified as multinucleated (≥3) TRAP-positive cells in contact with or very close proximity to the bone surface, in the ROI. Statistics Raw data were analyzed using the GraphPad Prism Software (GraphPad Software, Inc., La Jolla, CA). Descriptive statistics were used to calculate the mean and the standard error of the mean (SEM). Data among groups were analyzed using one-way analysis of variance (ANOVA) followed by the post-hoc Tukey's test for multiple compari- sons. Data between groups (healthy vs. diseased) were compared using the Student's t test. Categorical data (Table 1) were analyzed using the Fisher's exact test. Results μCT imaging of the maxillae revealed many animals with normal alveolar bone architecture, reflected by a uniform periodontal ligament (PDL) space (Fig. 1, thin arrow) and a continuous thin lamina dura around the roots of the teeth in all treatment groups. However, several veh treated mice presented altered alveolar bone morphology. Large lytic areas were noted around the roots and the furcational area of the 12 R.S. de Molon et al. / Bone 68 (2014) 11–19 * Ver anexo A * maxillarymolars that in select animals extended almost to the root apex and surrounded the entire root (Fig. 1, arrow). Additionally, significant superior, buccal and palatal expansion of the alveolar ridge was observed (Fig. 1, arrowheads). Several RANK-Fc, OPG-Fc and ZA treated animals showed similar lytic lesions to the veh group, although the extent of bone loss was significantly less (Fig. 1, arrows). Substantial periosteal bone apposition was noted at the periphery of the alveolar ridge that resulted in marked bone expansion (Fig. 1, arrowheads). The radiographic appearance of the alveolar bonewas indistinguishable among the RANK-Fc, OPG-Fc or ZA groups. To quantify the amount of bone loss in the diseased sites, the dis- tance from the cemento-enamel junction (CEJ) to the alveolar crest (AC) at the distal surface of the 1st molar was measured. An increased CEJ-AC distance denotes increased bone loss. Diseased vs. healthy sites demonstrated statistically significantly increased CEJ-AC distance for all treatment groups (Fig. 2A). Importantly, statistically significantly larger CEJ-AC distance was seen in the diseased site of veh group compared to the same site in the RANK-Fc, OPG-Fc and ZA groups (Fig. 2A). No difference among the three antiresorptive treated groups was detected. To evaluate effects on PDL space, the PDL space width was assessed at the furcation of the first maxillary molars [19,20,23]. No differences were seen among the healthy sites in any of the treatment groups. For all groups, a statistically significant widening of the PDL space was observed in the diseased vs. healthy sites (Fig. 2B). Diseased sites in veh treated animals showed a significantly higher increase in PDL space width compared to the antiresorptive treatment groups (Fig. 2B). To quantify the alveolar bone expansion, the buccal bone thickness at the level of the apical third of the roots wasmeasured (Fig. 2C). A sig- nificant expansion of the alveolar bone was seen at the diseased vs. healthy site in all groups. However, a statistically significantly greater increase was present in the RANK-Fc, OPG-Fc and ZA vs. veh groups, while no difference was detected among antiresorptive treated animals (Fig. 2C). To evaluate the overall effects of the maxillary infection and antiresorptive treatment in the bone architecture, BV, TV and BV/TV of Table 1 Radiographic and histologic findings in veh and antiresorptive treatment groups. Group Total hemimaxillae Healthy (%) Diseased (%) Bone expansion (%) Osteonecrosis (%) Bone exposure (%) Veh 33 20 (60.6)⁎ 13 (39.4)⁎ 13 (39.4)⁎ (100)# 0⁎⁎ (0)⁎ (0)# 0⁎⁎ (0)⁎ (0)# (0)+ RANK-Fc 40 19 (47.5)⁎ 21 (52.5)⁎ 21 (52.5)⁎ (100)# 10 (25)⁎ (47)# 7 (17.5)⁎ (33.3)# (70)+ OPG-Fc 40 19 (47.5)⁎ 21 (52.5)⁎ 21 (52.5)⁎ (100)# 15 (37.5)⁎ (71)# 11 (27.5)⁎ (52)# (73)+ ZA 38 16 (42)⁎ 22 (58)⁎ 22 (58)⁎ (100)# 10 (26.3)⁎ (45)# 8 (21)⁎ (36.4)# (80)+ ⁎⁎ Statistically significantly different compared to the RANK-Fc, OPG-Fc or ZA groups, p b 0.05. ⁎ % of total hemimaxillae. # % of diseased hemimaxillae. + % of hemimaxillae with osteonecrosis. Fig. 1. Radiographic changes of the maxillary alveolar ridge. Representative axial, coronal and sagittal μCT slices and three-dimensional views of the maxillary molars of healthy (veh) or diseased site from veh, RANK-Fc, OPG-Fc and ZA treated animals are shown. Thin arrows point to the lamina dura around the roots of the veh/healthy animals. Thick arrows point to areas of osteolysis and arrowheads to areas or bone expansion in the diseased site of veh, RANK-Fc, OPG-Fc and ZA treated mice. 13R.S. de Molon et al. / Bone 68 (2014) 11–19 the alveolar ridge were measured (Fig. 2D–F). No difference was seen between the healthy vs. diseased sites in veh animals. Healthy alveolar bone in RANK-Fc, OPG-Fc and ZA showed a similar increase in BV vs. the veh group. Diseased sites in the antiresorptive treated mice showed an even further increase in BV, compared to the healthy site of the same animals or thehealthy or diseased site of the veh group (Fig. 2D). TVwas similarly increased in the diseased site of all groups (Fig. 2E), reflecting the observed alveolar bone expansion. BV/TV was significantly decreased in the diseased site of veh group compared to the healthy site of the same animals, but also compared to the healthy or diseased site of all antiresorptive treatment groups (Fig. 2F). As expected, antiresorptive vs. veh animals showed increased BV/TV in the healthy sites (Fig. 2F). Finally, a small but statistically significant decrease in BV/TV was noted for the diseased vs. healthy site in each of the RANK-Fc, OPG-Fc and ZA groups (Fig. 2F). After μCT assessment, histologic evaluation of the maxillae was performed (Fig. 3). Healthy sites in all groups showed normal alveolar bone and marginal epithelium (Fig. 3A–D, turquoise and red arrows). Fig. 2.Quantification of radiographic findings in the healthy and diseased sites of veh, RANK-Fc, OPG-Fc and ZA treatedmice. To quantify amount of bone loss, the distance from the CEJ to the alveolar bone crest (ABC) was measured at the distal surface of the 1st molar (A) and the width of the PDL space was measured at the furcation area of the 1st molar (B). To assess alveolar bone expansion, the thickness of the buccal bone was measured, at the level of the apical third of the roots (C). To quantify the changes in the bone architecture, BV (D), TV (E) and BV/TV (F) were measured at the area of the alveolar ridge. &, statistically significantly different from healthy veh, diseased RANK-Fc, diseased OPG-Fc or diseased ZA, p b 0.001. *, statistically significantly different from indicated groups, p b 0.05. #, statistically significantly different from indicated groups, p b 0.01. $, statistically different from healthy RANK-Fc, healthy OPG-Fc or healthy ZA, p b 0.01. @, statistically significantly different from diseased RANK-Fc, diseased OPG-Fc or diseased ZA, p b 0.01. Fig. 3.Histologic examination of the periodontal and alveolar bone area. Healthy site of (A) veh, (B) RANK-Fc, (C) OPG-Fc, and (D) ZA treated animals. Diseases site of (E and E′) veh, (F and F′) RANK-Fc, (G and G′) OPG-Fc and (H and H′) ZA treated animals. Red arrows point to marginal gingival epithelium, turquoise arrows to alveolar crest, black arrows to areas of inflam- mation, blue arrows to periosteal bone deposition, yellow arrows to osteonecrotic areas, and green arrows to areas of bone exposure. 14 R.S. de Molon et al. / Bone 68 (2014) 11–19 In vehicle treated animals, an abundant inflammatory infiltrate (Fig. 3E and E′, black arrows) composed of acute (neutrophils) and chronic (lymphocytes) inflammatory cells was noted. Inflammation was present in both the epithelium and underlying mucosa. Migration of the marginal epithelium (Fig. 3E and E′, red arrows) was paralleled by marked bone loss (Fig. 3E and E′, turquoise arrows) such that the marginal epithelium distance appeared increased. No histologic evidence of osteonecrosis was seen in any of the samples. However, new bone formation at the alveolar bone perimeter (Fig. 3E and E′, blue arrows) was present. Comparable histologic features were present at the diseased site among animals treated with all three antiresorptive treatments— RANK-Fc (Fig. 3F and F′), OPG-Fc (Fig. 3G and G′) and ZA (Fig. 3H and H′). Similar to the veh treated animals, abundant mixed (acute and chronic) inflammatory infiltrate was noted in both the epithelium and soft tissue (Fig. 3F, F′, G, G′, H, and H′, black arrows). Bone loss was present compared to healthy sites, but appeared less compared to the diseased site of the veh treated mice (Fig. 3F, F′, G, G′, H, and H′, tur- quoise arrows). However, marked marginal epithelium migration was noted (Fig. 3F, F′, G, G′, H, and H′, red arrows), such that the epithelium to alveolar crest distance was reduced. Areas of osteonecrosis, with empty osteocytic lacunae, were present in many specimens from the diseased site of all antiresorptive treated animals (Fig. 3F, F′, G, G′, H, and H′, yellow arrows). In several specimens, the necrotic bone was not covered by oral mucosa, but was exposed to the oral cavity (Fig. 3F, F′, and G′, green arrows). Three main patterns of histologic appearance were noted in the diseased site of antiresorptive treated animals (Fig. 4) that were indis- tinguishable among the antiresorptive treatment groups (RANK-Fc (Fig. 4A–C), OPG-Fc (Fig. 4D–F) or ZA (Fig. 4G–I). First, abundant inflam- mation and periosteal bone formation were present (Fig. 4A, D, and G, black and blue arrows). However, no areas of osteonecrosis were detected. Second, in addition to the inflammatory bone changes, speci- mens showed areas of osteonecrosis with empty osteocytic lacunae (Fig. 4B, E, and H, black, blue and yellow arrows). Epithelial migration towards the osteonecrotic areaswas noted; however, no bone exposure was observed (Fig. 4B). The third pattern, in addition to inflammation and osteonecrosis, presented with bone exposure (Fig. 4C, F, and I, black, yellow and green arrows). Significant epithelial migration along the surface of the necrotic bone to the level of vital bone resulted in sig- nificant exposure of the osteonecrotic areas to the oral cavity (Fig. 4C, F, and I, red arrows) that included the palatal alveolar ridge and extended nearly to the midline. Table 1 summarizes the radiographic and histologic observations of these studies. The incidence of peri-radicular disease in the various treatment groups ranged from 39.4 to 58%with no statistical significant differences detected among the various groups. The diseased site of all animals showed alveolar bone expansion, irrelevant of the treatment. Osteonecrosis and bone exposure were only observed in animals on antiresorptives. Interestingly, only 45–71% of the diseased hemimaxillae showed areas of osteonecrosis. However, from the sites with osteonecrosis, 70–80% presented with bone exposure. No differ- ence on the incidence of osteonecrosis or bone exposure was detected among RANK-Fc, OPG-Fc or ZA treated groups. We then quantified histologic findings and performed comparisons among treatment groups. A significant increase of the JE-AC distance was noted for the diseased site of the veh treated group, probably reflecting the significant periradicular bone loss (Fig. 5A). In contrast, Fig. 4. Three histologic presentations of the diseased site in mice treated with antiresorptives. (A–C) RANK-Fc treated animals. (D–F) OPG-Fc treated animals. (G–I) ZA treated animals. No osteonecrosis (A, D, and G), presence of osteonecrosis (B, E, and H), and presence osteonecrosis (C, F, and I) with bone exposure were noted. Red arrows point to marginal gingival epithelium, black arrows to areas of inflammation, blue arrows to periosteal bone deposition, yellow arrows to osteonecrotic areas, and green arrows to areas of bone exposure. 15R.S. de Molon et al. / Bone 68 (2014) 11–19 antiresorptive treatment reversed the effect on the JE-AC distance that was shorter in the diseased vs. healthy sites for RANK-Fc, OPG-Fc and ZA treated groups, likely as a result of junctional epitheliummigration, but reduced bone resorption (Fig. 5A). Periosteal thickness significantly increased in the diseased vs. healthy site for all groups. However, this increase was greater for the RANK-Fc, OPG-Fc or ZA vs. veh treated animals (Fig. 5B). To evaluate the effect of antiresorptives on osteocytes and induction of osteonecrosis, the number of empty osteocytic lacunae (Fig. 5C) and the osteonecrotic surface area (Fig. 5D)were evaluated at the area of the maxillary alveolar ridge. No empty osteocytic lacunae or areas of osteonecrosis were detected in the healthy or diseased sites of the veh treated mice. Healthy sites of RANK-Fc, OPG-Fc and ZA treated animals showed a slight increase in the number of empty osteocytic lacunae that did not reach statistical significance (Fig. 5C). In contrast, significant increase of empty osteocytic lacunae (Fig. 5C) and areas of osteonecrosis (Fig. 5D) were present in the diseased site of RANK-Fc, OPG-Fc or ZA treated mice. A small but significantly higher number of empty osteocytic lacunaewas seen in the diseased site of OPG-Fc vs. ZA treated mice (Fig. 5C). However, no difference in osteonecrotic areas was noted among the three antiresorptive treatments (Fig. 5D). The osteonecrotic areas covered 16.9 %, 22.9 %, and 16.0 % of the ROI and measured 135,479 +/− 31,403, 180,368 +/− 25,106, and 139,440 +/− 39,923 μm2 for RANK-Fc, OPG-Fc and ZA groups, respectively. To evaluate the effect of the antiresorptive treatments on osteoclast numbers, TRAP stainingwas performed (Fig. 6). Few TRAP+ cells were observed at the healthy site (Fig. 6A) andwere significantly increased at the diseased site (Fig. 6E) of the veh treated animals. As expected, RANK-Fc and OPG-Fc treatment nearly abolished osteoclast formation in both healthy and diseased sites (Fig. 6B, C, F, and G) Interestingly, TRAP+ cells that significantly increased in the diseased site were noted in ZA treated animals (Fig. 6D and H). However, TRAP+ cells in ZA treated animals showed a round shape with pyknotic nuclear mor- phology and were detached and at times moved from the bone surface (Fig. 6I). Quantification of TRAP staining revealed significant inhibition of osteoclast formation by RANK-Fc and OPG-Fc treatment compared to control and ZA groups (Fig. 6J). No statistical difference of the TRAP+ cell number between the diseased site of veh vs. ZA treated mice was found (Fig. 6I). Discussion ONJ occurs mainly after tooth extraction in patients treated with medications that target osteoclastic function and activity, such as BPs and denosumab, for the treatment of neoplastic or metabolic bone disease [2,36]. Several animal models have utilized tooth extraction and high-dose BPs to reproduce clinical, radiographic and histologic features of the human disease [21,24,37]. Since the great majority of tooth extractions in adults is due to advanced carious lesions or periodontal bone loss [17,18], and given that dental preventive measures reduce the risk for ONJ [38,39] and that a significant number of ONJ cases occurs in the absence of tooth extrac- tion, we have hypothesized that dental disease plays a key role in ONJ pathogenesis [19,20,23]. Indeed, we and others have presented rat and mouse ONJ models in the absence of tooth extraction but in the presence of aggressive periodontitis or periapical disease in animals treated with high dose BPs [20,22,23,26] or RANKL inhibitors [19], thus pointing to a central role of infection/inflammation in ONJ pathophysiology [4–6]. For these ONJ animal models, established models of experimental dental disease were employed. Advantages of such approaches are the well characterized progression of dental disease, the localization of dental disease in a split-mouth design providing internal controls, and the control of onset of dental disease in relation to administration of antiresorptive medication. However, experimental induction and ma- nipulation of disease progression does not always reflect clinical reality. To that effect, several ONJ animal models utilize extraction of healthy teeth [21,24,25,27–30], which are rarely extracted in ONJ patients. Similarly, ONJ animal models utilizing periodontitis [20,22,26] employ experimental procedures that depict some, but not necessarily all the components of the pathological process of human periodontal disease [40]. Finally, in ONJ models of antiresorptives and periapical disease drilling the crown ofmolars to induce pulpal exposure [22,26], although captures the pathophysiologic progression of pulp necrosis and periapical infection does not reflect the natural pulpal infection through deep carious lesions. Naturally occurring maxillofacial abscesses have been reported in mice, including the inbred C57BL/6 strain [31–33]. The mice develop these abscesses from barbering practices including grooming, plucking, Fig. 5. Quantification of the histologic findings in the healthy and diseases sites of veh, RANK-Fc, OPG-Fc and ZA treated animals. (A) The shortest epithelial-crest distance was determined. If epithelium extended below the level of the alveolar crest, a negative value was assigned to the measurements. (B) Periosteal thickness, (C) % empty osteocytic lacunae and (D) % osteonecrotic area. &, statistically significantly different from healthy veh, diseased RANK-Fc, diseased OPG-Fc or diseased ZA, p b 0.001. *, statistically signifi- cantly different from indicated groups, p b 0.05. #, statistically significantly different from indicated groups, p b 0.01. 16 R.S. de Molon et al. / Bone 68 (2014) 11–19 or eating fur or whiskers of cage-mates or oneself [41–43]. C57BL/6 mice are one of the most common rodent strains where barbering, mastication, and fragmentation of hair occur [41,43]. Here, the foreign body gets into the oral cavity, pierces the gingival sulcus, and induces dental disease [31]. After hair impaction, bacterial colonization occurs, and cultures consistently reveal Staphylococcus aureus isolated from the abscesses [31–33]. μCT of these areas demonstrates osteolysis with severe bone loss surrounding the molar roots and expansion of the alveolar bone [31]. Histologically, severe inflammatory infiltrate with presence of neutrophils and mononuclear inflammatory cells is report- ed [31,32]. Intrigued by this spontaneous occurrence of peri-radicular infection and resultant extensive inflammation, we investigated the po- tential development of ONJ-like lesions in themaxillae of mice that had been treated with high-dose antiresorptivemedications, thus establish- ing a novel ONJ animal model. An advantage of this model is the natural occurrence or peri-radicular infection/inflammation, without the need of experimental interventions. We utilized two classes of antiresorptive agents with distinct phar- macologic actions. RANK-Fc andOPG-Fc bind and inhibit RANKL function and were used as a surrogate for denosumab, since denosumab recog- nizes the human but not mouse RANKL [34]. Both RANK-Fc and OPG-Fc bind RANKL and potently inhibit bone resorption and increase bone volume in animal models [34,35]. The second type of antiresorptive was zoledronic acid (ZA), a potent nitrogen-containing bisphosphonate widely used in the management of bone malignancy [44]. Utilizing the employed treatment regimens for these antiresorptives, we have previously reported the successful inhibition of osteoclastic function and development of ONJ-like lesions in the mandible of mice around mandibular molars with experimental periapical disease [19,23]. Similar to published data [31,32], μCT assessment of the alveolar bone revealed significant peri-radicular osteolysis and expansion of the buccal and palatal cortices of the alveolar ridge in several veh treat- ed mice. Antiresorptives attenuated interproximal and furcational bone loss and increased overall alveolar bone volume at the diseased sites. These observations are in agreement with findings around experimen- tal periodontal or periapical disease in rats or mice [19,20,23] and parallel clinical findings that demonstrate diffuse osteosclerosis with increased trabecular density and thickening of cortical outlines in areas of ONJ [45]. Histologically, the presence of intense inflammation, significant osteolysis and epithelial migration was seen in the periodontal tissues of several control (veh treated) mice, similar to previous reports [31, 32]. Presence of inflammation was also noted on antiresorptive- treated groups. However, loss of osteocytes and presence of empty osteocytic lacunae consistent with osteonecrosis were also present in these animals. Presence of osteonecrosis has been described in animals on antiresorptives after tooth extraction or experimental disease, em- phasizing the resemblance of the spontaneous ONJ-like lesions observed in our studies with other ONJ-animal models [19–30]. The similar radiographic and histologic disease presentation between this and other ONJ models [19–30], which parallels findings in ONJ patients, validates such animal models as capturing essential features of disease pathophysiology that lead to alveolar bone osteonecrosis. A noteworthy observation in our studies was the great similarity in the radiographic and histologic findings among the RANK-Fc, OPG-Fc and ZA treated groups, including the incidence of osteonecrosis and bone exposure. Indeed, to the best of our knowledge, this is the second report of antiresorptive treatment, other than bisphosphonates, induc- ing ONJ-like lesions in an animalmodel [19] and the first direct compar- ison of these different antiresorptive categories. Our findings parallel human data from BP vs. denosumab treated patients reporting a similar incidence of ONJ [12–14] and strongly point to a central role of osteoclastic inhibition in ONJ pathogenesis. Utilizing this ONJ mouse model, we observed three histologic pat- terns at sites of disease that were similar for all three antiresorptives (Fig. 4 and Table 1). In the first pattern, significant inflammation throughout the alveolar ridge and varying amount of osteolysis were noted. However, similar to the veh treated group, no osteonecrosis was present. In the clinical setting, these animals could potentially par- allel patients on antiresorptive medications and jaw inflammation but without any clinical or radiographic signs of ONJ. In the second pattern, osteonecrotic areas were present within the alveolar bone but were covered by oral mucosa. This disease appearance appears to parallel Stage 0 ONJ in patients with bone necrosis, but without clinical bone ex- posure [2]. Finally, in the third pattern the necrotic alveolar bone was not covered by oral mucosa but was exposed to the oral environment. This presentation is in line with the classic manifestation of clinical ONJ with varying degree of bone exposure and associated infection/in- flammation (Stage 1–3 ONJ) [2]. Our present studies cannot address whether a transition from less towards higher stage ONJ exists, since we only examined the animals at a single time point. However, clinical data reporting 50% of patients with stage 0 disease developing clinical ONJ within 5 months of initial diagnosis [46] would argue for the progression of the less severe histologic osteonecrosis without bone exposure to the more severe presentation of bone necrosis and clinical exposure. A surprising and interesting finding was the difference on osteonecrosis incidence in this ONJ mouse model with spontaneous maxillary infection vs. studies utilizing experimental periapical disease [19,23], although treatment regimens for various antiresorptives were the same. In the current study, approximately 50% of the animals with peri-radicular disease presented with areas of osteonecrosis. In contrast, 88% of ZA [23] or 100% of RANK-Fc or OPG-Fc [19] treated mice with experimental periapical disease showed osteonecrotic areas histologically. A possible explanation for this finding is that maxilla (in the current study) vs. mandible [19,23] respond differently to bone turnover Fig. 6. TRAP staining on histologic sections from healthy (A–D) and diseased sites (E–H) from veh, RANK-Fc, OPG-Fc and ZA treated animals respectively. Select TRAP+ multinucleated cells from veh (I) and ZA (J) treated mice. Quantification TRAP+ cells in the veh, RANK-Fc, OPG-Fc and ZA treated animals (K). *Statistically significantly different, p b 0.05. 17R.S. de Molon et al. / Bone 68 (2014) 11–19 suppression. In contrast to osteoradionecrosis that almost exclusively localizes to the mandible [47], ONJ appears to affect both jaws with high frequency. However, even in the case of ONJ themandible appears to be more frequently affected, particularly in patients treated with antiresorptives for the treatment of osteoporosis [2,48,49]. The predilection of the mandible for osteonecrosis could be due to the decreased vascularity, thicker cortical borders and smaller percentage of trabecular bone [50,51]. A second possible explanation for this observation is the relation of antiresorptive administration with the disease onset. Maxillary lesions developed spontaneouslywithout any intervention. Thus, someanimals developed maxillary inflammatory lesions prior to, while others after the commencement of antiresorptives. In contrast, experimental periodontitis or periapical disease was induced after antiresorptive medications were administered [19,20,23,26]. Possibly the early stages of infection and resultant inflammatory response could play a crucial role in the alveolar bone response to turnover suppression. Such a premise would be significant from a clinical perspective, since it points to the potential association between antiresorptive administration and time of peri-radicular infection. Interestingly, although osteonecrosis incidence was lower in the current ONJ model, bone exposure was more frequent and extensive compared to published studies [19,20,23]. Here, nearly 75% of animals with osteonecrosis showed bone exposure that at times involved a significant part of the maxillary alveolar bone. In contrast, only 28% of animals with osteonecrosis presented bone exposure that was mostly localized to the marginal periodontium when experimental periapical disease was induced [19,23]. The most likely explanation for these observations is the degree of infection and resultant inflammatory changes that in the current study extended to the whole alveolus and caused significant buccal and palatal bone deposition and marked ridge expansion. Our findings have potentially significant implications for studies on ONJ animal models utilizing maxillary molar extraction, particularly if the C57BL6 strain of mice was used [21,24]. Since in many of these studies animals were pretreated with bisphosphonates prior to tooth extraction, the possible presence of naturally occurring peri-radicular lesions in some sites could complicate socket healing and confound ONJ incidence and severity. In conclusion, our data offer potentially important insights into the mechanisms of ONJ. The nearly identical radiographic and histologic features of alveolar bonemorphology andONJ-like appearance between the RANKL inhibitors OPG-Fc and RANK-Fc, and the potent bisphospho- nate ZA parallel clinical findings and strongly indicate a central role of osteoclasts in ONJ pathophysiology. The reduced incidence of osteonecrosis compared to previously published reports suggests the significance of the onset of infection/inflammation in relation to the commencement of antiresorptive treatment, while the increased fre- quency and extent of alveolar bone exposure points to the link between the severity of inflammation and clinical ONJ severity. From a practical point of view, our observations caution the design and interpretation of experimental findings utilizing interventional approaches, as they might be compounded by the occurrence of spontaneous ONJ lesions around naturally occurring maxillary lesions of animals treated with antiresorptives. Finally, this novel model of spontaneous ONJ validates, compliments and enhances published ONJ models that utilize tooth extraction or experimental inductionof dental disease and strongly sup- ports the central importance of infection/inflammation in combination with inhibition of osteoclastic function in ONJ pathogenesis. Disclosures Dr. Tetradis has served as a paid consultant for and has received grant support from Amgen Inc. All other authors state that they do not have any conflicts of interest. Acknowledgments This work was supported by grant support from Amgen Inc. and by NIH/NIDCR DE019465. de Molon RS was supported by fellowships from the State of Sao Paulo Research Foundation #2012/09968-5 and the Coordination for the Improvement of Higher Level -or Education- Personnel - #11575/13-1. Authors' roles: All authors participated in the design, execution and analyses of the studies. TA, RSM, SC, and ST drafted the manuscript. 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J Oral Maxillofac Surg 2004;62:527–34. [49] Marx R. Oral and intravenous bisphosphonate induced osteonecrosis of the jaws: history, etiology, prevention, and treatment2nd ed. ; 2007. [50] Devlin H, Horner K, Ledgerton D. A comparison of maxillary and mandibular bone mineral densities. J Prosthet Dent 1998;79:323–7. [51] Kingsmill VJ, Gray CM, Moles DR, Boyde A. Cortical vascular canals in human mandible and other bones. J Dent Res 2007;86:368–72. 19R.S. de Molon et al. / Bone 68 (2014) 11–19 OPG-Fc but Not Zoledronic Acid Discontinuation Reverses Osteonecrosis of the Jaws (ONJ) in Mice Rafael Scaf de Molon,1,2 Hiroaki Shimamoto,3 Olga Bezouglaia,1 Flavia Q Pirih,4 Sarah M Dry,5 Paul Kostenuik,6 Rogely W. Boyce,7 Denise Dwyer,7 Tara L Aghaloo,1 and Sotirios Tetradis1,8 1Division of Diagnostic and Surgical Sciences, UCLA School of Dentistry, Los Angeles, CA, USA 2Department of Diagnosis and Surgery, School of Dentistry at Araraquara, S, ~a, o Paulo State University, Araraquara, Brazil 3Department of Oral and Maxillofacial Radiology, Osaka University Graduate School of Dentistry, Osaka, Japan 4Division of Associated Specialties, UCLA School of Dentistry, Los Angeles, CA, USA 5Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA 6Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan, Ann Arbor, MI, USA 7Department of Comparative Biology and Safety Sciences, Amgen Inc., Thousand Oaks, CA, USA 8Molecular Biology Institute, UCLA, Los Angeles, CA, USA ABSTRACT Osteonecrosis of the jaws (ONJ) is a significant complication of antiresorptive medications, such as bisphosphonates and denosumab. Antiresorptive discontinuation to promote healing of ONJ lesions remains highly controversial and understudied. Here, we investigated whether antiresorptive discontinuation alters ONJ features in mice, employing the potent bisphosphonate zoledronic acid (ZA) or the receptor activator of NF-kB ligand (RANKL) inhibitor OPG-Fc, utilizing previously published ONJ animal models. Mice were treated with vehicle (veh), ZA, or OPG-Fc for 11 weeks to induce ONJ, and antiresorptives were discontinued for 6 or 10 weeks. Maxillae and mandibles were examined by mCT imaging and histologically. ONJ features in ZA and OPG-Fc groups included periosteal bone deposition, empty osteocyte lacunae, osteonecrotic areas, and bone exposure, each of which substantially resolved 10 weeks after discontinuing OPG-Fc but not ZA. Full recovery of tartrate-resistant acid phosphatase-positive (TRAPþ) osteoclast numbers occurred after discontinuing OPG-Fc but not ZA. Our data provide the first experimental evidence demonstrating that discontinuation of a RANKL inhibitor, but not a bisphosphonate, reverses features of osteonecrosis in mice. It remains unclear whether antiresorptive discontinuation increases the risk of skeletal-related events in patients with bonemetastases or fracture risk in osteoporosis patients, but these preclinical data may nonetheless help to inform discussions on the rationale for a “drug holiday” in managing the ONJ patient. © 2015 American Society for Bone and Mineral Research. KEY WORDS: OSTEONECROSIS OF THE JAW (ONJ); ANTIRESORPTIVES; BISPHOSPHONATES; ZOLEDRONIC ACID; DENOSUMAB; ALVEOLAR BONE; OSTEOCLASTS Introduction Antiresorptive medications, such as bisphosphonates (BPs) and the receptor activator of NF-kB ligand (RANKL) inhibitor denosumab impede osteoclastic bone resorption and are used in the clinic to manage bone diseases, such as primary or metastatic bone malignancy and osteoporosis. These medica- tions, studied in cancer and postmenopausal women with osteoporosis populations, reduce skeletal-related events (SREs), decrease tumor burden, reduce bone pain, decrease incidence of osteoporotic fractures, and help improve patients’ quality of life.(1–4) Despite their distinctly different pharmacologic mecha- nism of action, BPs and denosumab are each associated with osteonecrosis of the jaw (ONJ), an infrequent but serious adverse effect, particularly when administered at high doses.(5) Anti- resorptive-related ONJ is defined as exposed bone or bone that can be probed through an intraoral or extraoral fistula in the maxillofacial region that has persisted for more than 8 weeks in patients on current or previous treatment with antiresorptive agents and no history of radiation therapy to the jaws or obvious metastatic disease to the jaws.(6–8) The termmedical-related ONJ (MRONJ) was recently introduced to include ONJ cases associated with antiangiogenic therapies.(7) Although ONJ has been described for more than a decade,(9,10) the etiology and pathogenesis of the disease remain largely unknown.(5,11–13) Osteoclast inhibition and bone remodeling suppression, inflammation and/or infection, inhibi- tion of angiogenesis, soft tissue toxicity, and altered immune cell Received in original form January 26, 2015; revised form February 20, 2015; accepted February 24, 2015; accepted manuscript online Month 00, 2015. Address correspondence to: Sotirios Tetradis, DDS, PhD, UCLA School of Dentistry, 10833 Le Conte Avenue, CHS Room 53-068, Los Angeles, CA 90095-1668, USA. E-mail: stetradis@dentistry.ucla.edu. Tara L Aghaloo, DDS, MD, PhD, UCLA School of Dentistry, 10833 Le Conte Avenue, CHS Room 53-009, Los Angeles, CA 90095-1668, USA. E-mail: taghaloo@dentistry.ucla.edu Additional Supporting Information may be found in the online version of this article. ORIGINAL ARTICLE JJBMR Journal of Bone and Mineral Research, Vol. xx, No. xx, Month 2015, pp 1–14 DOI: 10.1002/jbmr.2490 © 2015 American Society for Bone and Mineral Research 1 3.2 Publicação 2 function are among the hypotheses proposed to underlie ONJ development and progression.(5,11–13) Clinical observations and experimental findings provide support for several of these hypotheses, suggesting that ONJ is probably a multifactorial disease influenced by many variables.(7) Because antiresorptive medications with distinct pharmaco- logic function induce similar incidence of ONJ, osteoclastic inhibition and bone turnover suppression appear central in antiresorptive-related ONJ.(5,11–13) To that effect, discontinua- tion of antiresorptive treatment before tooth extraction or other dental surgical intervention or after ONJ development has been proposed as potentially beneficial in improving osteoclast function recovery, which could, in turn, increase bone turnover and improve bone healing.(7,14–16) However, such recommen- dations have only a theoretical basis, and clinical or experimen- tal data supporting the benefit of antiresorptive drug withdrawal in preventing ONJ development or promoting resolution are lacking.(7,17) Furthermore, the consequences of such “drug holidays” on the progression of SREs in cancer patients or fragility fractures in osteoporosis patients remain unclear. As such, an important first step in informing treatment planning decisions is to understand whether discontinuation of antiresorptive treatments mitigates the risk of developing ONJ or fosters the resolution of established ONJ.(7,8) Animal models recapitulating ONJ-like lesions in mice, rats, minipigs, and dogs treated with bisphosphonates have provid- ed important insights into ONJ pathophysiology.(18–29) More- over, utilizing OPG-Fc and RANK-Fc as surrogates for denosumab,(30) we reported for the first time ONJ-like lesions in the presence of periapical disease or spontaneous perira- dicular inflammation in mice treated with the RANKL inhib- itors,(31,32) thus supporting the central role of osteoclastic inhibition in ONJ pathogenesis. Similar ONJ-like changes were reported more recently in mice that underwent tooth extractions and were treated with an antibody against mouse RANKL.(33) In this study, utilizing ONJ animal models, we explored the effects of zoledronic acid (ZA) or OPG-Fc discontinuation on radiographic and histologic features of established ONJ in mice. Our findings indicate that, within the experimental time frame, OPG-Fc but not ZA discontinuation reversed features of osteonecrosis in ONJ animal models. Materials and Methods Animal care Mice and surgical procedures were handled according to the guidelines of the Institutional Animal Care and Use Committee of the University of California, Los Angeles. We followed a randomized, prospective, controlled, animal model design following all the recommendations of the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines for the execution and submission of studies in animals (Kilkenny and colleagues). Mice were kept in the animal facilities with controlled temperature (23°C" 2°C), humidity, and a 12- hour light/dark cycle. Throughout the experimental period, mice were housed in plastic cages, fed a standard laboratory Fig. 1. Serum TRACP-5b levels increase after OPG-Fc but not after ZA discontinuation. TRACP-5b serum levels were measured at the end of treatment (0 weeks) and 2, 4, 6, and 10 weeks after drug discontinuation (13, 15, 17, and 21 weeks after the first injection) (n¼ 16/group per period). þStatistically significantly different from indicated groups, p< 0.0001. **Statistically significantly different from indicated groups, p< 0.001. *Statistically significantly different from indicated groups, p < 0.05. 2 SCAF DE MOLON ET AL. Journal of Bone and Mineral Research diet, and given water ad libitum. A total of 144 10-week- old C57BL/6J wild-type male mice (Jackson Laboratories, Bar Harbor, ME, USA) with average weight of 25 g were randomly divided into three experimental groups of 48 animals that received intraperitoneal (ip) injections of endotoxin-free saline (group veh) two times per week, 10mg/kg rat OPG- Fc (composed of the RANKL-binding domains of osteoprote- gerin linked to the Fc portion of IgG,(32,34,35) kindly provided by Amgen, Inc, Thousand Oaks, CA, USA) twice per week (group OPG-Fc), or 200mg/kg zoledronic acid (Z-5744, LKT Laboratories, St. Paul, MN, USA) twice per week (group ZA). These doses were used to increase the incidence of ONJ-like lesions in the mice, as described previously. Periapical disease induction Experimental periapical disease was performed as de- scribed.(29,32,36,37) Briefly, the crowns of the right mandibular 1st and 2nd molars were drilled utilizing a stainless-steel 1/4 size round bur in a high-speed handpiece, avoiding furcal perfora- tion, and the root canals were left exposed to the oral environment, resulting in pulpal necrosis and subsequent Table 1. Summary of Radiographic and Histologic Findings on Various Groups and Experimental Time Points Group Total no. of hemimandible and hemimaxillae Healthy (%) Diseased (%) Osteonecrosis (%) Bone exposure (%) Veh 11w 64 34 (53)a 30 (47)a 0d (0)a (0)b 0f (0)a (0)b (0)þ Veh 17w 64 31 (48)a 33 (52)a 2e (3.1)a (6)b 0d (0)a (0)b (0)c Veh 21w 64 26 (40)a 38 (60)a 1e (1.5)a (2.6)b 0d (0)a (0)b (0)c OPG-Fc 11w 64 32 (50)a 32 (50)a 24 (37.5)a (75)b 13 (20.3)a (40.6)b (54.2)c OPG-Fc 17w 64 26 (40)a 38 (60)a 16g (25)a (42.1)b 3d (4.7)a (7.9)b (18.7)c OPG-Fc 21w 64 24 (37)a 40 (63)a 13g (20.3)a (32.5)b 0d (0)a (0)b (0)c ZA 11w 64 34 (53)a 30 (47)a 19 (29.7)a (63.4)b 10 (15.6)a (33.3)b (52.6)c ZA 17w 64 27 (42)a 37 (58)a 32 (50)a (86.5)b 19 (29.7)a (51.4)b (59.3)c ZA 21w 64 23 (36)a 41 (64)a 31 (48.4)a (75.6)b 21 (32.8)a (51.2)b (67.7)c aPercent of total hemimaxillae and hemimandible. bPercent of diseased hemimaxillae and hemimandible. cPercent of hemimaxillae and hemimandible with osteonecrosis. dStatistically significantly different, p < 0.05 compared with OPG-Fc and ZA. eStatistically significantly different, p < 0.0001 compared with OPG-Fc and ZA. fStatistically significantly different, p < 0.005 compared with OPG-Fc and ZA. gStatistically significantly different, p < 0.0001 compared with ZA. Fig. 2. Three-dimensionalmCT reconstructed images ofmaxilla. (A–C) Healthy site in veh, OPG-Fc, and ZA, respectively. (D, E) Diseased site after 11weeks of treatment. (G–L) Diseased site at 6 and 10 weeks after drug discontinuation. Blue arrows point to normal alveolar bone crest in the interproximal area between the distal root of the first molar and mesial root of the second molar. Red arrows point to periodontal bone loss and areas of osteolysis in the diseased site of OPG-Fc- and veh-treated mice. Yellow arrows point to increased bone deposition. Journal of Bone and Mineral Research ONJ RESOLUTION AFTER OPG-Fc VS ZA DISCONTINUATION 3 periapical infection. The crowns of the left 1st and 2nd molars were kept intact. Spontaneous naturally occurring maxillofacial abscesses Naturally occurring, spontaneous maxillofacial abscesses have been described in mice.(38) Hair inserts into the gingival sulcus and results in bacterial colonization and reproducible, severe periradicular osteolysis.(38–40) Such diseased maxillary sites, identified by periradicular osteolysis by mCT imaging, were compared with sites with healthy dento- alveolar structures.(31) Antiresorptive administration and serum TRACP-5b measurement Mice were pretreated with veh, OPG-Fc, or ZA for 3 weeks, and periapical disease was induced. Animals continued to receive veh, OPG-Fc, and ZA for 8 additional weeks, after which antiresorptives were discontinued (Fig. 1A). Blood was collected via retro-orbital bleeding after 11 weeks of the experiment (at the end of antiresorptives treatment) and 2, 4, 6, and 10 weeks after antiresorptive withdrawal. Serum tartrate-resistant acid phosphatase 5b (TRACP-5b) was measured by enzyme immu- noassay (RatTRAP; JDS, Gaithersburg, MD, USA). Animal euthaniza