Avaliação da ozonioterapia no tratamento da osteonecrose induzida por zoledronato em ratas senis / Evaluation of ozone therapy in the treatment of zoledronate-induced osteonecrosis in senile rats

A ozonioterapia vem se demonstrando uma ferramenta promissora na prevenção de infecções e no auxílio da reparação tecidual, conciliando com os desafios no tratamento da osteonecrose dos maxilares induzida por medicamentos (ONM-M), este projeto objetiva analisar os efeitos da ozonioterapia, em 55 ratas senis (18 meses), entre 300-350g, induzidas a osteonecrose via medicamentosa (Zoledronato 100µg/kg), após exodontia do primeiro molar inferior. Os animais foram divididos em 4 grupos equitativos (10 ratas por grupo), o primeiro grupo SAL, recebeu aplicações de soro fisiológico por 7 semanas, grupo SAL + OZ recebeu aplicações de soro fisiológico por 7 semanas e o tratamento com a ozonioterapia (0,7mg/kg) a cada 2 dias por 28 dias, o grupo ZOL recebeu aplicações de zoledronato (100µg/kg) por 7 semanas e por último o grupo ZOL + OZ recebeu também aplicações de zoledronato no mesmo protocolo e foi tratado com a ozonioterapia (0,7mg/kg) a cada 2 dias por 28 dias. Todos as ratas receberam a antibioticoterapia (Cristacilina® 0,1ml/kg por dia) iniciando 3 dias antes do procedimento de extração, se estendendo até 4 dias de pós-operatório, passaram pela extração do molar na terceira semana de experimento e foram submetidas a eutanásia na sétima semana de experimento. Após a eutanásia as mandíbulas foram ressecadas, reduzidas e preparadas para as análises microtomográficas (caracterização óssea do osso senil (MCT0) e após terapia com zoledronato (MCT1ZOL) contra seu par controle (MCT1SAL), parâmetros volumétricos (Bv,Bv.Tv,Tb.Th,Tb.N,Tb.Sp,Po.Tot) dos grupos experimentais), histométricas (porcentagem de osso neoformado e porcentagem de osso não vital) e imunoistoquímicas (expressão de TNFa, IL-1b, VEGF, OCN e TRAP). Os resultados da caracterização óssea não apresentaram diferença quando comparado os grupos experimentais (p> 0,05), possivelmente devido ao pouco tempo decorrido na terapia com zoledronato. Os demais resultados comparando os grupos experimentais mostrou com diferenças estatisticamente significativas (p< 0,05) uma característica de osso vítreo, denso, sem vitalidade, pobre em vascularização, com elevados valores para marcadores de inflamação, traduzindo isso em osteonecrose dos maxilares relacionada com a medicação, destoando principalmente do grupo controle SAL, que apresentou melhora na reparação alveolar e características de osso vital e vascularizado. A ozonioterapia (ZOL+OZ, SAL+OZ) apresentou valores significantes estatisticamente quando comparado ao grupo sem tratamento, traduzindo em melhora na vascularização do tecido ósseo, em melhora reparacional do alvéolo, modulação da inflamação local e o aparecimento/manutenção de células osteoblásticas ativas (p< 0,05). Mostrando-se uma terapia viável no controle/tratamento da osteonecrose dos maxilares relacionado com medicamentos(AU)

Ozone therapy has been shown to be a promising tool in the prevention of infections and in the aid of tissue repair, reconciling with the challenges in the treatment of medication-induced jaw osteonecrosis (ONM-M), this project aims to analyze the effects of ozone therapy in 55 rats senile (18 months), between 300-350g, induced to osteonecrosis via medication (Zoledronate 100µg / kg), after extraction of the lower first molar. The animals were divided into 4 equitable groups (ten rats per group), the first SAL group, received saline applications for 7 weeks, SAL + OZ group received saline applications for 7 weeks and ozone therapy (0, 7mg / kg) every 2 days for 28 days, the ZOL group received applications of zoledronate (100µg / kg) for 7 weeks and lastly the ZOL + OZ group also received applications of zoledronate in the same protocol and was treated with ozone therapy (0.7mg / kg) every 2 days for 28 days. All rats received antibiotic therapy (Cristacilina® 0.1ml / kg per day) starting 3 days before the extraction procedure, extending up to 4 days after the operation, underwent molar extraction in the third week of the experiment and were submitted to euthanasia in the seventh week of experiment. After euthanasia, the mandibles were resected, reduced and prepared for microtomographic analysis (bone characterization of senile bone (MCT0) and after therapy with zoledronate (MCT1ZOL) against its control pair (MCT1SAL), volumetric parameters (Bv, Bv.Tv, Tb .Th, Tb.N, Tb.Sp, Po.Tot) of the experimental groups), histometric (percentage of newly formed bone and percentage of non-vital bone) and immunohistochemistry (expression of TNFa, IL-1b, VEGF, OCN and TRAP) . The results of bone characterization did not show any difference when comparing the experimental groups (P> 0.05), possibly due to the short time elapsed in zoledronate therapy. The other results comparing the experimental groups showed with statistically significant differences (P < 0.05) a characteristic of vitreous bone, dense, without vitality, poor in vascularization, with high values for inflammation markers, translating this into a related jaw osteonecrosis with medication, disagreeing mainly with the SAL control group, which showed improvement in alveolar repair and characteristics of a vital and vascularized bone. Ozone therapy (ZOL + OZ, SAL + OZ) showed statistically significant values when compared to the untreated group, translating into an improvement in bone tissue vascularization, a reparational improvement of the alveolus, modulation of local inflammation and the appearance/maintenance of cells active osteoblasts (P < 0.05). Showing to be a viable therapy in the control/treatment of osteonecrosis of the jaws related to drugs(AU)

Delayed alveolar bone repair and osteonecrosis associated with Zoledronic Acid therapy in rats: macroscopic, microscopic and molecular analysis

Abstract Objective This study aims to evaluate bone repair and the development of the medication related osteonecrosis of the jaw (MRONJ) associated with the use of zoledronic acid in Wistar rats. Methodology 48 male Wistar rats were divided into four groups: ZA, treated with intraperitoneal zoledronic acid, 0.6 mg/kg every 28 days, totaling five doses; control (C), treated with 0.9% sodium chloride; ZA-surgical (SZA) and C-surgical (SC), submitted to extraction of the right upper molars 45 days after the first application. Alveolar bone repair was evaluated by macroscopic and histological analysis. Protein expression evaluations were performed by qPCR. Results Macroscopic evaluation showed that 91.66% (11) of the animals in the SZA group and 41.66% (5) from the SC group presented solution of epithelium continuity (P<0.05). All animals in the SZA group and none in the SC group had bone sequestration. The area of osteonecrosis was higher in the SZA group than in the SC group (P<0.05). In molecular evaluation, the SZA group presented changes in the expression of markers for osteoclasts, with increased RANK and RANKL, and a decrease in OPG. Conclusion The results highlighted strong and evident interference of zoledronic acid in bone repair of the socket, causing osteonecrosis and delayed bone remodeling.

The efficacy of sustained-release chitosan microspheres containing recombinant human parathyroid hormone on MRONJ

Abstract Treatment of patients with bisphosphonate usage is a significant concern for oral surgeons because it interferes with jaw bone turnover and regeneration. In case of adverse effects manifesting related to bisphosphonate use, oral surgeons are usually treating and keep the patient's symptoms under control. In this study, we aimed to investigate a new treatment protocol for medication-related osteonecrosis of the jaw (MRONJ). This treatment protocol consisted of administering human parathyroid hormone (hPTH) loaded chitosan microspheres which were prepared by ionotropic gelation method or/and the prepared microspheres were suspended in a poloxamer gel. After in-vitro optimization studies, the efficacy of the chosen formulations was evaluated in-vivo studies. Zoledronic acid was administered daily to forty-eight adult female Sprague-Dawley rats, divided into four experimental groups, at a daily concentration of 0.11 mg/kg over three weeks to induce the MRONJ model. At the end of this period, maxillary left molar teeth were extracted. In the first group, the subjects received no treatment. In the negative control group, poloxamer hydrogel containing empty microspheres were immediately applied to the soft tissues surrounding the extraction socket. The treatment group-1 was treated with local injections of poloxamer hydrogel containing hPTH. The treatment group-2 was treated with a single local injection of poloxamer hydrogel containing hPTH-loaded chitosan microspheres. Both treatment groups received a total of 7 µg of hPTH at the end of the treatment protocol. Our study demonstrates successful attenuation of MRONJ through a local drug delivery system combined with hPTH, as opposed to previously attempted treatment strategies.

Zoledronic acid effect on bone of growing rats / Efecto del ácido zoledrónico en el hueso de ratas en crecimiento

Zoledronic acid (ZA) is an antiresorptive drug used in children with bone diseases like osteogenesis imperfecta, juvenil osteoporosis, fibrous dysplasia and primary bone tumors. The aim of the present study was to evaluate the effects of ZA dose accumulation on growing bone during different periods of treatment in normal rats. Methods: A 4x2 factorial design was used to study the effect of the dose of ZA (D: 0-2.5-12.5-25 µg Z/kg body weight/s.c. weekly) and the length of treatment (T: 15-30 days) in normal female Sprague Dawley rats. Bone morphometric, histomorphometric, densitometric and biomechanical studies were performed. Results: Femoral length and cross-sectional area were affected by both D and T. A significant interaction between D and T was observed in length with a lower value at higher dose and 30 days of treatment. Growth plate of the tibia showed a decrease in total thickness with D and T. Histomorphometric and connectivity parameters of trabecular bone were significantly increased with D and several parameters were also affected by T. Cortical bone strength was increased only with T. Biomechanical parameters of trabecular bone showed significant interaction with greater effect at higher D and T. Conclusion: Even though a mild negative effect of the highest dose of ZA on linear and appositional growth was observed, the other bone parameters evaluated were improved. A careful risk/benefit analysis would lead us to conclude that the mild deleterious effects of ZA during growth are outweighed by the benefit obtained with treatment. (AU)

El ácido zoledrónico (AZ) es un fármaco antirresortivo utilizado en niños con enfermedades óseas como osteogénesis imperfecta, osteoporosis juvenil, displasia fibrosa y tumores óseos primarios. El objetivo del presente estudio fue evaluar los efectos de las dosis acumuladas de AZ en el hueso en crecimiento de ratas hembras normales durante diferentes períodos de tratamiento. Métodos: se utilizó un diseño factorial de 4x2 para estudiar el efecto de la dosis de AZ (D: 0-2,5-12,5-25 µg Z / kg de peso corporal /sc semanalmente) y el período de tratamiento (T: 15-30 días) en ratas Sprague Dawley. Se realizaron estudios óseos morfométricos, histomorfométricos, densitométricos y biomecánicos. Resultados: la longitud y el área de sección transversal del fémur se vieron afectadas tanto por D como por T. Se observó una interacción significativa entre D y T en la longitud obteniéndose un valor más bajo a la dosis más alta y a 30 días de tratamiento. El cartílago de crecimiento de la tibia mostró una disminución en el espesor total con D y T. Los parámetros histomorfométricos y de conectividad del hueso trabecular aumentaron significativamente con D y varios parámetros también se vieron afectados por T. La fortaleza ósea cortical aumentó solo con T. Los parámetros biomecánicos del hueso trabecular mostraron una interacción significativa con un mayor efecto a mayor D y T. Conclusión: a pesar que se observó un leve efecto negativo de la dosis más alta de AZ sobre el crecimiento lineal y aposicional, el resto de los parámetros óseos evaluados mejoraron. Un análisis cuidadoso del riesgo /beneficio permite concluir que los efectos negativos leves del AZ durante el crecimiento son superados por el beneficio obtenido con el tratamiento. (AU)

Desarrollo de un modelo experimental de osteonecrosis de maxilar asociada al tratamiento crónico con aminobisfosfonatos / Experimental model of osteonecrosis of the jaw associated to chronic treatment with aminobisphosphonates

La osteonecrosis de maxilar asociada a aminobisfosfonatos (BRONJ) constituye un efecto secundario del tratamiento crónico con los más potentes. Un modelo experimental permitiría determinar la patogenia de dicha alteración. La oveja presenta características orales y del metabolismo óseo similar al humano y permite realizar manipulaciones bucales. Se evaluaron cambios clínicos, remodelación ósea y masa ósea maxilar en ovejas hembras adultas tratadas con zolendronato (ZOL), durante 22 meses y utilizando dosis equivalente al tratamiento de neoplasias. Seis ovariectomizadas (OVX) recibieron ZOL; 5 OVX y 4 SHAM (control) recibieron solución fisiológica. Al inicio, 4 y 22 meses se evaluó calcemia, fosfatemia, crosslaps (CTX) y fosfatasa alcalina ósea. Al final, se evaluó contenido mineral óseo de la hemimandíbula superior (CMO: mg/cm2). Al final del estudio, CTX disminuyó significativamente en ZOL (p<0,05) sin diferencias entre SHAM y OVX. En maxilar, los contenidos de Ca y P (g/g tejido) y CMO (g/cm2 ) disminuyeron en OVX vs. SHAM (p<0,05) y solo Ca y CMO respecto de ZOL (p<0,05). ZOL incrementó el contenido de Ca y CMO, mientras que el de P permaneció significativamente disminuido respecto de SHAM. La sobrevida en SHAM y OVX fue del 100% y en ZOL 77% (2 muertes); 2 ovejas del grupo ZOL presentaron necrosis de maxilar. Conclusiones: fue posible obtener desarrollo de BRONJ por tratamiento crónico con ZOL, el cual redujo notablemente la resorción y, según la relación Ca/P, posiblemente haya afectado la mineralización ósea. (AU)

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a complication of chronic treatment with the most powerful aminobisphosphonates (BPs). An experimental animal model would allow to determine the pathogenesis of this complication. Ewes exhibit similar oral cavity characteristics and bone metabolism as humans, and they are suitable for oral cavity interventions. We examined herein the clinical manifestations, bone remodeling status, and maxillary bone mass in adult female ewes treated with zoledronate (ZOL) for 22 months. Six ovariectomized (OVX) ewes received ZOL; and 5 OVX and 4 SHAM animals received saline solution. At the start of the experiment, and at the 4 and 22 month-time points serum Ca, P, crosslaps (CTX), and bone alkaline phosphatase were measured. Bone mineral content (BMC) of the superior hemimandible was measured at the end of the experiment. At this time point, CTX was significantly decreased only in the ZOL group (p<0.05). Ca and P content (g/g tissue) and BMC in the mandible were significantly decreased in the OVX group compared to SHAM animals (p<0.05) and only Ca content and BMC were decreased when compared to ZOL (p<0.05). ZOL treatment increased the Ca content and BMC, whereas the P content remained low compared to the SHAM group (p<0.05). All ewes from the SHAM and OVX groups and 77% of the animals from the ZOL group survived until the end of the experiment, whereas two ewes of ZOL group exhibited BRONJ. Conclusion: under our experimental conditions, it was possible to induce BRONJ by the chronic ZOL administration, which in turn induced a high reduction in bone resorption as well as possibly impaired bone mineralization, based on the Ca/P ratio in the mandible. (AU)

Osteonecrose dos Maxilares Associada ao uso de Bisfosfonatos / Osteonecrosis of the Jaws Associated with the use of Bisphosphonates

Os bifosfonatos são uma classe de medicamentos, que têm por função a inibição da atividade dos osteoclastos, interferindo na remodelação e no turnover ósseo. São indicados para retardar a metástase óssea em algumas condições malignas, como em mielomas múltiplos, câncer de mama e próstata, e outras condições benignas, como no tratamento da doença de Paget e da osteoporose. Desde 2003, estudos têm associado a osteonecrose avascular dos ossos maxilares ao uso dos bifosfonatos. Dentre os mecanismos de ação dos bifosfonatos, podemos citar a sua atividade antiosteoclástica e antiangiogênica, que altera o metabolismo ósseo, inibindo a reabsorção óssea e diminuindo o turnover ósseo. As exposições ósseas maxilo-mandibulares ocorrem após procedimentos cirúrgicos odontológicos ou, menos comumente, após o uso de próteses apoiadas na fina mucosa de revestimento ósseo da cavidade bucal. O tratamento da osteonecrose associada aos bifosfonatos (OAB) é bastante variado, controverso e desafiador, visto que nenhum tratamento efetivo tem sido proposto até o momento. O objetivo desse relato de caso é descrever a ocorrência de áreas de exposição óssea mandibular em um paciente que fez uso do ácido zolendrônico (Zometa®) bem como apresentar a abordagem terapêutica realizada que resultou em completo recobrimento mucoso das áreas ósseas expostas... (AU)

Biphosphonates are a class of drugs whose function is the inhibition of osteoclast activity, interfering in remodeling and bone turnover. They are indicated to delay bone metastases in some malignancies such as multiple myeloma, prostate cancer and other benign conditions such as in the treatment of Paget's disease and osteoporosis. Since 2003, studies associate avascular osteonecrosis of the jaws to the use of bisphosphonates, especially intra venous. Among the mechanisms of action of bisphosphonates are their osteoclastic and antiangionenic activity which alters bone metabolism, inhibiting bone resorption and reducing bone turnover. Maxillo-mandibular bone exposures occur after surgical dental procedures or less commonly after the use of prostheses supported on thin bone lining mucosa of the buccal cavity. The treatment of osteonecrosis associated with biphosphonates (OAB) is quite varied, controversial and challenging, since no effective treatment has been proposed until this moment. The purpose of this case report is to describe the occurrence of areas of mandibular bone exposure in a patient with history of use of Zolendronic Acid (Zometa) and present the therapeutic approach undertaken which resulted in full mucous covering of the exposed bony áreas... (AU)

Raloxifene neutralizes bone brittleness induced by anti-remodeling treatment and increases fatigue life through non-cell mediated mechanisms / El raloxifeno invierte fragilidad ósea inducida por el tratamiento anti-remodelación y aumenta la resistencia a la fatiga a través de mecanismos mediados no celulares

Pre-clinical data have shown that tissue level effects stemming from bisphosphonateinduced suppression of bone remodeling can result in bone that is stronger yet more brittle. Raloxifene has been shown to reduce bone brittleness through non-cellular mechanisms. The goal of this work was to test the hypothesis that raloxifene can reverse the bone brittleness resulting from bisphosphonate treatment. Dog and mouse bone from multiple bisphosphonate dosing experiments were soaked in raloxifene and then assessed for mechanical properties. Mice treated with zoledronate in vivo had lower post-yield mechanical properties compared to controls. Raloxifene soaking had significant positive effects on select mechanical properties of bones from both vehicle and zoledronate treated mice. Although the effects were blunted in zoledronate bones relative to vehicle, the soaking was sufficient to normalize properties to control levels. Additional studies showed that raloxifene-soaked bones had a significant positive effect on cycles to failure (+114%) compared to control-soaked mouse bone. Finally, raloxifene soaking significantly improved select properties of ribs from dogs treated for 3 years with alendronate. These data show that ex vivo soaking in raloxifene can act through non-cellular mechanisms to enhance mechanical properties of bone previously treated with bisphosphonate. We also document that the positive effects of raloxifene soaking extend to enhancing fatigue properties of bone. (AU)

Los datos preclínicos han demostrado que los efectos a nivel de tejido que se derivan de la supresión del remodelado óseo inducida por bifosfonatos puede dar como resultado un hueso que es más fuerte pero más frágil. Está comprobado que el raloxifeno reduce la fragilidad ósea a través de mecanismos no celulares. El objetivo de este trabajo fue probar la hipótesis de que el raloxifeno puede revertir la fragilidad ósea resultante del tratamiento con bifosfonatos. Se emplearon huesos de perro y ratón de múltiples experimentos con diferentes dosis de bifosfonatos los cuales fueron sumergidos en raloxifeno y luego se evaluaron sus propiedades mecánicas. Ratones tratados con zoledronato in vivo mostraron propiedades mecánicas post-rendimiento más bajas en comparación con los controles. Luego de sumergirlos en raloxifeno se observaron efectos positivos significativos en algunas propiedades biomecánicas tanto en los huesos de ratones tratados con vehículo como con zoledronato. Aunque los efectos se atenuaron en los huesos tratados con zoledronato en relación con los tratados con vehículo, el raloxifeno fue suficiente para normalizar las propiedades a niveles basales. Estudios adicionales mostraron que los huesos sumergidos en raloxifeno tuvieron un efecto positivo significativo en los ciclos de fractura (+ 114%) en comparación con los huesos de ratón sumergido en vehículo. Finalmente, el raloxifeno mejoró significativamente las propiedades de costillas de perros tratados durante 3 años con alendronato. Estos datos muestran que la inclusión ex vivo en raloxifeno puede actuar a través de mecanismos no celulares para mejorar las propiedades mecánicas de huesos previamente tratado con bifosfonatos. También documentamos que los efectos positivos del raloxifeno mejoran las propiedades de fatiga del hueso. (AU)