Este trabalho apresenta o desenvolvimento de metodologias cromatográficas para a análise de Bisfosfonatos em medicamentos acabados, matérias-prima e em fluidos biológicos utilizando Cromatografia Iônica com detecção Indireta no UV e detecção por Condutividade com Supressão de Eluente. No capítulo 1, descreve-se a pesquisa bibliográfica das propriedades farmacológicas dos BP´s, suas principais rotas sintéticas e sobre os métodos analíticos apresentados na literatura científica. No capítulo 2, foi descrito o desenvolvimento de um método para a determinação dos BP´s etidronato, clodronato, pamidronato e alendronato em matéria-prima e para medicamentos de alendronato, utilizando Cromatografia Iônica e detecção indireta no Ultravioleta. A metodologia foi validada e aplicada na análise de medicamentos contendo alendronato em comprimidos de referência e em comprimidos genéricos. No capítulo 3, descreve-se estudos visando a determinação de etidronado em plasma humano, utilizando Cromatografia Iônica Multidimensional com detecção indireta no Ultravioleta. No capítulo 4, foi desenvolvida outra metodologia para a determinação do BP´s etidronato, clodronato, pamidronato e alendronato em matéria-prima e em medicamentos contendo alendronato, utilizando Cromatografia Iônica e detecção por Condutividade com supressão de eluente. A metodologia foi validada e aplicada na análise de medicamentos contendo alendronato em comprimidos similares e em comprimidos manipulados. No capítulo 5, uma metodologia utilizando Cromatografia Iônica e detecção por Condutividade com supressão de eluente foi desenvolvida e validada para determinar clodronato em urina humana. / This work presents the development of chromatografic methodologies for analysis of Bisphosphonates in drugs, raw material and biological fluids using Ion Chromatography with indirect UV detection and conductivity detection with eluent suppression. In chapter 1, an extensive bibliographical research was accomplished in relationship to the pharmacological properties of bisphosphonates, their more important synthetic routes and about the analytical methods presented in the scientific literature. In chapter 2, the development of a method was described for the determination of BP´s etidronate, clodronate, pamidronate and alendronate in raw material and for alendronate tablets, using Ionic Chromatography with indirect UV detection. The methodology was applied to the analysis of medicines containing alendronate in both forms, generic and reference. In chapter 3, studies conducted to determinate etidronate in human plasma, using Multidimensional ionic chromatography with indirect UV detection are decribed. In chapter 4, a methodology developed for the determination of etidronate, clodronate, pamidronate and alendronate in raw material and for alendronate tablets using ionic chromatography and detection for conductivity with eluente suppression is described. The methodology was applied in the analysis of medicines with alendronate generic and reference. In chapter 5, a methodology using ion chromatography and detection for conductivity with eluente suppression was developed and validated to determine clodronate in human urine.

The isoprenoid biosynthetic pathway (IBP) has been highly implicated in a number of cellular malignancies, including proliferation, invasion, and migration. Epidemiological studies have found clinically relevant inhibitors of the IBP, such as the statin family and nitrogenous bisphosphonates, reduce the risk of prostate cancer advancement. In vitro work has implicated statin's and nitrogenous bisphosphonate's inhibition of GGPP and protein geranylgeranylation as the components responsible for their reduction of prostate cancer progression. However, their depletion of nearly all isoprenoid intermediates as well as their organ specificities make understanding the specific role of protein geranylgeranylation in prostate cancer metastasis impossible. Consequently, we have developed a novel library of seven alkyl bisphosphonate ethers found to potently reduce GGDPS with little to no activity against the related FDPS enzyme. Inhibition of GGDPS in three human prostate cancer cell lines reduced GGPP and protein geranylgeranylation without affecting protein farnesylation, translating into a reduction in cell migration and invasion. Interestingly, the GGDPS inhibitors reduced protein geranylgeranylation at lower concentrations in the highly metastatic PC3 cell line as compared to the less metastatic LNCaP and 22Rv1 cell lines. Additionally, the PC3 cell line was found to have higher levels of endogenous IBP intermediates as compared to the less metastatic cells. Translation in vivo using two murine models of human prostate cancer metastasis found a reduction in soft tissue tumor burden that corresponded to a biochemical reduction in protein geranylgeranylation. In conclusion, selective reduction of GGPP and protein geranylgeranylation was sufficient to reduce the metastatic potential of prostate cancer in vitro and in vivo.

L'ostéoporose ménopausique par une accélération du remodelage osseux, conduit à une masse osseuse basse et à des altérations de la microarchitecture, augmentant ainsi le risque de fracture. Les bisphosphonates diminuent l'activité du remodelage osseux en inhibant la résorption osseuse qui conduit à un maintien de la densité minérale osseuse et à une diminution du risque fracturaire. Ce traitement étant la plupart du temps chronique, l'étude de ses effets sur les différents niveaux d'organisation de l'os, du cristal à sa macrostructure, est primordiale. A travers deux études portant sur l'ibandronate et l'alendronate, nous avons montré que leur utilisation préservait la minéralisation osseuse tissulaire. Cependant, une utilisation prolongée (6,4 ans) de l'alendronate, nous a permis de mettre en évidence un mécanisme d'action impliquant des modifications de la structure des cristaux avec un effet sur les propriétés mécaniques locales. Si l'efficacité anti fracturaire de l'alendronate est démontrée après dix ans de traitement, nos résultats suggèrent des altérations de la qualité osseuse à une échelle plus locale

The work presented in this thesis focuses on the development of strategies and smart bioactive materials for the treatment of osteoporosis. High and low molecular weight soluble hyaluronic acid-bisphosphonate (HA-BP) derivatives were investigated for their ability to inhibit osteoclasts. Low molecular weight HA-BP (L-HA-BP) was most effective in inhibiting active resorption of both murine and human osteoclasts (without affecting osteoblasts) compared to free bisphosphonate (BP). Precursor monocytes were unaffected, suggesting the specificity of HA-BP towards osteoclasts. This new class of functionalized hyaluronic acid could lead to rapid development of tailor-made pro-drugs for targeted treatment of osteoporosis. Polyphosphoesters (PEP) have been widely studied for their pro-osteoblast effects, primarily due to their involvement in cellular energy production pathway leading to the formation of inorganic phosphates that contribute to mineralized bone. Given that the effect of PEP on human osteoclasts is little studied, this work on poly(ethylene sodium phosphate) (PEP.Na) explores the potential to use PEP.Na as an inhibitor of osteoclast activity for the first time. PEP.Na exposure led to a dose-dependent toxicity of osteoclasts with reduction in their capacity to form resorption pits over 24h. Currently, there is a dearth of in vitro cell-culture systems that can study osteoclast-related resorption and osteoblast-related mineralization in a single co-culture system, and to simultaneously quantify the effects of soluble factors on these processes. Described here, is the development of a novel and simple two-sided co-culture system that can overcome these limitations with reliable and quantifiable readouts. In comparison with traditional one-sided co-culture systems, the two-sided co-culture was able to generate similar readouts for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) markers. There is also the advantage of distinctly separate and quantifiable readouts for mineralization and resorption, which has been demonstrated using Pamidronate. Finally, HA-BP was synthesized with pre-determined amounts of BP groups. The BP groups attached to HA allowed the tunable incorporation of BMP-2 in hydrogels. The charge-based affinity of BMP-2 and BP allowed stable incorporation of specific amounts of BMP-2, which could be tuned by the ratio of BP groups. 125I-labelled BMP-2 was loaded into hydrogels and their release was studied. Radioactive measurements revealed the tunable sequestration and controlled release of protein over time. This result was corroborated by ALP measurements of cells exposed to released BMP-2. ALP production was found to be almost 5-fold higher in HA-BP hydrogels loaded with BMP-2 which suggested that the sequestered BMP-2 is not only available to cells but also remains highly potent, even in entrapped form, The release of BMP-2 is dependent upon the rate of diffusion, swelling in hydrogels and degradation pattern of the gels and may assist in the long-term and rapid regeneration of osteoblasts in vitro.

Oral bisphosphonate drug therapy is a common and effective treatment for osteoporosis. Little is known about the long-term effects of bisphosphonates on bone quality. This study examined the structural and mechanical properties of trabecular bone following 0-16 years of bisphosphonate treatment. Fifty-three iliac crest bone samples of Caucasian women diagnosed with low turnover osteoporosis were identified from the Kentucky Bone Registry. Forty-five were treated with oral bisphosphonates for 1 to 16 years while eight were treatment naive. A section of trabecular bone was chosen from a micro-computed tomography (Scanco µCT 40) scan of each sample for a uniaxial linearly elastic compression simulation using finite element analysis (ANSYS 14.0). Morphometric parameters (BV/TV, SMI, Tb.Sp., etc.) were computed using µCT. Apparent modulus, effective modulus and estimated failure stress were calculated. Biomechanical and morphometric parameters improved with treatment duration, peaked around 7 years, and then declined independently of age. The findings suggest a limit to the benefits associated with bisphosphonate treatment and that extended continuous bisphosphonate treatment does not continue to improve bone quality. Bone quality, and subsequently bone strength, may eventually regress to a state poorer than at the onset of treatment. Treatment duration limited to less than 7 years is recommended.

Background: Using a nude rat model of site-specific metastatic bone disease (MBD), we developed a semiquantitative histological score for rapid assessment of lytic lesions in bone. This provides additional information to conventional histological measurement by clarifying the extent and location of metastatic infiltration and the tumor growth pattern. The score can also be used to assess the action of bisphosphonates on bone metastases. Materials and Methods: Male nude rats (n = 12 per group) were inoculated with the human breast cancer cell line MDA-MB-231 via the femoral artery. Following appearance of radiographically visible osteolytic lesions on day 18, the animals received phosphate-buffered saline (PBS; controls) or ibandronate (IBN, 10 µg P/kg) daily until day 30. Whole body radiographs were obtained on days 18 and 30, and osteolytic areas (OA) were determined by radiographic computer-based analysis (CBA). On day 30, MBD was assessed in both tibias using conventional histological CBA and the new scoring system. Results: Metastatic tumor area correlated with the total sum of the new score in both PBS- (r = 0.762) and IBN-treated animals (r = 0.951; p < 0.001). OA correlated well with the total sum in both groups (r = 0.845 and 0.854, respectively; p < 0.001). Conclusion: Significant reduction of bone marrow and cortical infiltration of tumor cells with IBN suggested local control of metastases. / Hintergrund: Mit Hilfe eines etablierten Tiermodells zur Erzeugung lokalisationsspezifischer Knochenmetastasen in der Nacktratte wurde ein semiquantitatives histologisches Graduierungssystem zur schnellen Bewertung osteolytischer Knochenmetastasen entwickelt. Das Graduierungssystem liefert hinsichtlich der Metastasenlokalisation, deren Ausmaß und Infiltrationsmuster wertvolle Zusatzinformationen zu den konventionellen histologischen Untersuchungsmethoden. Damit kann beispielsweise auch die pharmakologische Wirkung von Bisphosphonaten auf die Knochenmetastasierung beurteilt werden. Material und Methoden: Männlichen Nacktratten (n = 12 pro Gruppe) wurden Zellen der humanen Brustkrebszellinie MDA-MB-231 in die Oberschenkelarterie inokuliert. Ab dem Auftreten radiologisch erkennbarer Osteolysen 18 Tage nach Inokulation erhielten die Tiere bis zum Studienende (Tag 30) täglich entweder eine subkutane Applikation einer Phosphat-Puffer-Lösung (Kontrollgruppe) oder Ibandronat (IBN, 10 µg P/kg; Behandlungsgruppe). Konventionelle Röntgenaufnahmen wurden an den Tagen 18 und 30 nach Tumorinokulation angefertigt und die Osteolysenflächen mittels Computerauswertung bestimmt. Nach Studienende wurde der Metastasenbefall in beiden Tibiae sowohl konventionell histologisch als auch mittels des neuen Graduierungssystems ausgewertet. Ergebnisse: Die Metastasenfläche korrelierte mit der kummulativen Punktsumme des Graduierungssystems sowohl in der Kontrollgruppe (r = 0,762; p < 0,001) als auch in der Ibandronat- Gruppe (r = 0,951; p < 0,001). Ebenso war die Osteolysenfläche eng mit der Punktesumme in beiden Gruppen korreliert (r = 0,845 und 0,854; p < 0,001). Schlussfolgerung: Die signifikante Reduktion von Knochenmark- und Kortikalisbefall durch IBN deuten auf eine gute lokale Kontrolle des Metastasenwachstums hin. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

A Osteogênese Imperfeita (OI) é uma doença genética do tecido conjuntivo caracterizada por fragilidade óssea e susceptibilidade à fratura sob mínimo ou nenhum trauma. O objetivo deste trabalho foi estudar características clínicas e moleculares de crianças e adultos com Osteogênese Imperfeita e analisar o efeito do tratamento medicamentoso com bifosfonados em relação aos biomarcadores metabólicos e ósseos em pacientes adultos. Esta tese se dividiu em dois capítulos onde 1) foi realizado um estudo retrospectivo sobre as características clínicas no momento do diagnóstico de OI, com ênfase nas características clínicas, especialmente em relação às fraturas ósseas; 2) avaliação clínica e análise da mutação c.-14C>T no gene IFITM5 foi estuda em uma população com características sugestivas de OI tipo V; e 3) estudo retrospectivo em adultos com OI divididos em 2 grupos tratados com bifosfonados e não tratados. Em relação ao tratamento com bifosfonados foram avaliados os seguintes parâmetros: tipo de droga e duração do tratamento, valores de biomarcadores metabólicos e ósseos por um período de 5 anos, incidência de fraturas num de período de 5 ou 10 anos e densidade mineral óssea da coluna lombar, quadril total e colo femural no início e no final do tratamento. Nossos resultados mostraram que 1) no momento do diagnósico de OI características como escleras azuladas, dentinogênese imperfeita, ossos wormianos e fraturas de membros inferiores e superiores podem ser observadas. Pacientes com formas mais graves de OI foram diagnosticados mais precocemente quando comparados com pacientes com formas leves. Nenhuma criança com OI apresentou fraturas posteromediais das costelas, fratura de escápula ou lesões metafisárias. Essas informações associadas a história da saúde da criança são relevantes para a realização do diagnóstico diferencial. 2) OI tipo V correspondeu a 4% dos casos de OI atendidos no Centro de Referência para OI do HCPA. Indivíduos com OI V associada a mutação c.-14C> T no gene IFITM5 apresentaram características clínicas distintas como formação de calo hiperplásico, calcificação das membranas interósseas, deslocamento da cabeça radial e deformidade de coluna, porém a expressão da doença é variável. 3) Observamos que o tratamento de adultos com OI a longo prazo não foi associado com redução na incidência das fraturas e não se refletiu de forma significativa nos níveis de biomarcadores metabólicos e ósseos, porém houve uma melhora significativa na densidade mineral óssea da coluna lombar associada à terapia. Por ser uma doença rara com prevalência variável e ampla variabilidade fenotípica e genotípica, estudos clínicos e moleculares bem como estudos sobre o efeito do tratamento medicamentoso são imprescindíveis, contribuindo no melhor entendimento da doença, aconselhamento genético acurado e propiciando melhores estratégias de prevenção e tratamento para esta população. / Osteogenesis Imperfecta (OI) is a genetic connective tissue disease characterized by bone fragility and susceptibility to fracture under minimal or no trauma. The aim of this study was to evaluate clinical and molecular features of children and adults with OI and analyze the effect of the drug treatment with bisphosphonates in regarding to metabolic and bone biomarkers in adult patients. This thesis was divided by two chapters: 1) a retrospective study was performed where the clinical characteristic at the moment of diagnosis of OI, the clinical characteristics specially related to bone fractures was evaluated; 2) clinical evaluation and mutation analysis of c.-14C>T in the IFITM5gene was studied in a population with clinical charcteristics suggestive of OI type V; and 3) retrospective study in adults with OI divided in two groups treated with biphosphonates and not treated. Bisphosphonate treatment was evaluated according to the parameters: type of drug and duration of treatment, metabolic and bone biomarkers values for a period of 5 years, incidence of fractures in a period of 5 or 10 years and bone mineral density of the lumbar spine, total hip and femoral neck at baseline and at the end of treatment. Our results showed that 1) at the time of OI diagnosis features such as bluish slerae, dentinogenis imperfecta, wormian bones, and fractures of upper and lower limbs can be observed. Patients with more severe forms of OI were diagnosed earlier when compared with patients with mild forms. No OI children presented posteromedial fractures of the ribs, scapula fracture or metaphyseal lesions. This information associated with the child's health history are relevant for carrying out the differential diagnosis. This information is relevant for carrying out the differential diagnosis. 2) OI type V corresponds to 4% of OI cases at the Reference Center for OI at HCPA. Subjects with OI V associated to the mutation c.-14C> T in the IFITM5 gene presented distinctives clinical features as hyperplastic callus formation, calcification of interosseous membranes, dislocation of the radial head and spinal deformity, but the expression of the disease is variable. 3) We observed that long-term treatment with bisphosphonates (BP) for adults with OI was not associated with reduced incidence of fractures and was not reflected significantly in the levels of metabolic and bone biomarkers, but there was a significant improvement in bone mineral density of the lumbar spine associated to the therapy. Because it is a rare disease with a prevalence variable and wide phenotypic and genotypic variability, clinical and molecular studies and studies of the effect of drug treatment are essential, contributing to the better understanding of the disease, accurate genetic counseling and providing better strategies for prevention and treatment for this population.

A Osteogênese Imperfeita (OI) é uma doença genética do tecido conjuntivo caracterizada por fragilidade óssea e susceptibilidade à fratura sob mínimo ou nenhum trauma. O objetivo deste trabalho foi estudar características clínicas e moleculares de crianças e adultos com Osteogênese Imperfeita e analisar o efeito do tratamento medicamentoso com bifosfonados em relação aos biomarcadores metabólicos e ósseos em pacientes adultos. Esta tese se dividiu em dois capítulos onde 1) foi realizado um estudo retrospectivo sobre as características clínicas no momento do diagnóstico de OI, com ênfase nas características clínicas, especialmente em relação às fraturas ósseas; 2) avaliação clínica e análise da mutação c.-14C>T no gene IFITM5 foi estuda em uma população com características sugestivas de OI tipo V; e 3) estudo retrospectivo em adultos com OI divididos em 2 grupos tratados com bifosfonados e não tratados. Em relação ao tratamento com bifosfonados foram avaliados os seguintes parâmetros: tipo de droga e duração do tratamento, valores de biomarcadores metabólicos e ósseos por um período de 5 anos, incidência de fraturas num de período de 5 ou 10 anos e densidade mineral óssea da coluna lombar, quadril total e colo femural no início e no final do tratamento. Nossos resultados mostraram que 1) no momento do diagnósico de OI características como escleras azuladas, dentinogênese imperfeita, ossos wormianos e fraturas de membros inferiores e superiores podem ser observadas. Pacientes com formas mais graves de OI foram diagnosticados mais precocemente quando comparados com pacientes com formas leves. Nenhuma criança com OI apresentou fraturas posteromediais das costelas, fratura de escápula ou lesões metafisárias. Essas informações associadas a história da saúde da criança são relevantes para a realização do diagnóstico diferencial. 2) OI tipo V correspondeu a 4% dos casos de OI atendidos no Centro de Referência para OI do HCPA. Indivíduos com OI V associada a mutação c.-14C> T no gene IFITM5 apresentaram características clínicas distintas como formação de calo hiperplásico, calcificação das membranas interósseas, deslocamento da cabeça radial e deformidade de coluna, porém a expressão da doença é variável. 3) Observamos que o tratamento de adultos com OI a longo prazo não foi associado com redução na incidência das fraturas e não se refletiu de forma significativa nos níveis de biomarcadores metabólicos e ósseos, porém houve uma melhora significativa na densidade mineral óssea da coluna lombar associada à terapia. Por ser uma doença rara com prevalência variável e ampla variabilidade fenotípica e genotípica, estudos clínicos e moleculares bem como estudos sobre o efeito do tratamento medicamentoso são imprescindíveis, contribuindo no melhor entendimento da doença, aconselhamento genético acurado e propiciando melhores estratégias de prevenção e tratamento para esta população. / Osteogenesis Imperfecta (OI) is a genetic connective tissue disease characterized by bone fragility and susceptibility to fracture under minimal or no trauma. The aim of this study was to evaluate clinical and molecular features of children and adults with OI and analyze the effect of the drug treatment with bisphosphonates in regarding to metabolic and bone biomarkers in adult patients. This thesis was divided by two chapters: 1) a retrospective study was performed where the clinical characteristic at the moment of diagnosis of OI, the clinical characteristics specially related to bone fractures was evaluated; 2) clinical evaluation and mutation analysis of c.-14C>T in the IFITM5gene was studied in a population with clinical charcteristics suggestive of OI type V; and 3) retrospective study in adults with OI divided in two groups treated with biphosphonates and not treated. Bisphosphonate treatment was evaluated according to the parameters: type of drug and duration of treatment, metabolic and bone biomarkers values for a period of 5 years, incidence of fractures in a period of 5 or 10 years and bone mineral density of the lumbar spine, total hip and femoral neck at baseline and at the end of treatment. Our results showed that 1) at the time of OI diagnosis features such as bluish slerae, dentinogenis imperfecta, wormian bones, and fractures of upper and lower limbs can be observed. Patients with more severe forms of OI were diagnosed earlier when compared with patients with mild forms. No OI children presented posteromedial fractures of the ribs, scapula fracture or metaphyseal lesions. This information associated with the child's health history are relevant for carrying out the differential diagnosis. This information is relevant for carrying out the differential diagnosis. 2) OI type V corresponds to 4% of OI cases at the Reference Center for OI at HCPA. Subjects with OI V associated to the mutation c.-14C> T in the IFITM5 gene presented distinctives clinical features as hyperplastic callus formation, calcification of interosseous membranes, dislocation of the radial head and spinal deformity, but the expression of the disease is variable. 3) We observed that long-term treatment with bisphosphonates (BP) for adults with OI was not associated with reduced incidence of fractures and was not reflected significantly in the levels of metabolic and bone biomarkers, but there was a significant improvement in bone mineral density of the lumbar spine associated to the therapy. Because it is a rare disease with a prevalence variable and wide phenotypic and genotypic variability, clinical and molecular studies and studies of the effect of drug treatment are essential, contributing to the better understanding of the disease, accurate genetic counseling and providing better strategies for prevention and treatment for this population.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O objetivo dos autores foi avaliar microscopicamente, a influência de dois regimes de aplicação do clodronato dissódico na movimentação dentária e reabsorção radicular de Rattus novergicus. Foram utilizados 63 ratos, adultos, machos, com dentição permanente completa e peso aproximado de 300g. Os animais foram divididos em três grupos com 21 espécimes cada: Grupo Controle, animais submetidos à movimentação dentária induzida sem aplicação do medicamento; Grupo A, animais submetidos à movimentação com aplicação do Clodronato por via subcutânea em dias alternados; Grupo B, animais submetidos à movimentação e aplicação da droga por via subcutânea apenas no quinto e décimo segundo dias. Para avaliar os eventos celulares que ocorrem durante todo o ciclo de movimentação, os grupos foram subdivididos em três, com sete animais cada um e foi realizada a eutanásia no sétimo, décimo e décimo quarto dias. Este procedimento foi realizado com anestesia por inalação de dietil-éter e administração intra-abdominal de 40mg/Kg de pentobarbital sódico. As peças foram incluídas em parafina e os cortes teciduais (4-6m), corados por Hematoxilina-Eosina, foram usados para observações gerais e avaliação quantitativa em microscópio de luz. A análise dos resultados foi feita utilizando análise de variância (ANOVA) e o teste de Tukey foi utilizado para comparações múltiplas entre as médias. Foi considerado o nível de significância em 5%. A taxa de movimentação foi menor no Grupo A quando comparado ao grupo controle sendo estatisticamente significante (p< 0,01). No Grupo B, a taxa de movimentação foi menor que no grupo controle e maior que no Grupo A, porém sem significância estatística. Para as variáveis lacunas de reabsorção e número de osteoclastos houve diferença estatisticamente significativa quando o grupo controle foi comparado aos dois outros grupos. De acordo com os resultados, o Clodronato reduz a reabsorção radicular e a movimentação dentária, mas este último aspecto pode ser minimizado a depender do regime de aplicação do medicamento. / The purpose of this study was to evaluate microscopically the influence of disodium clodronate in tooth movement and root resorption in Rattus novergicus using two ways of applying the drug. We used 63 male Wistar rats, adults, with permanent dentition and approximate weight of 300g. The animals were divided into three groups with 21 specimens: control group, animals subjected to induced tooth movement without the application of the drug, Group A, animals subjected to induced tooth movement with application of Clodronate by subcutaneously every other day and Group B, animals subjected to induced tooth movement with application of the drug by subcutaneous only in the fifth and tenth-second days. To assess the cellular events that occur throughout the cycle of movement, the groups were subdivided into three, with seven animals each and after the experimental period, euthanasia was performed on the seventh, tenth and tenth to fourth days. This procedure was performed under anesthesia by inhalation of diethyl ether and intra-abdominal administration of 40 mg/kg of sodium pentobarbital. The obtained pieces were fixed and embedded in paraffin, stained with hematoxylin-eosin, then, tissue sections (4-6m) were used for general observations and quantitative assessment by light microscopy. The statistical analysis was performed using analysis of variance (ANOVA) and Tukey's test was used for multiple comparisons between means. The level of significance considered was 5%. The rate of movement was lower in Group A when compared to the control group (p <0.01). In Group B, the rate of turnover was lower than in the control group and higher than in Group A, but without statistical significance. For the variables of resorption lacunae and number of osteoclasts there was no statistical difference when the control group was compared to the other groups. According to the results, Clodronate reduces root resorption and tooth movement, but the latter aspect can be minimized depending on the application rate of the drug.

Orientador: Horácio Faig Leite / Banca: Raquel Guedes Fernandes / Banca: Maria Angélica Gargione Cardoso / Banca: Carlos Eduardo Dias Colombo / Banca: Luiz Eduardo Blumer Rosa / Resumo: Este trabalho avaliou a ação local do alendronato sódico, da hidroxiapatita e da associação alendronato com hidroxiapatita na reparação de defeitos ósseos em fêmures de ratas ovariectomizadas. Noventa e oito animais foram divididos em sete grupos: controle (C), amido (Am), alendronato 1mol (A1), alendronato 2moles (A2), hidroxiapatita 1 mol (HA1), hidroxiapatita 2moles (HA2) e associação alendronato e hidroxiapatita (A+HA). As ratas pesando, aproximadamente, 250g foram ovariectomizadas e, após trinta dias, os defeitos ósseos, medindo 2,5mm de diâmetro, foram confeccionados nos fêmures esquerdos. Os defeitos foram preenchidos com alendronato sódico, hidroxiapatita e/ou com ambos, sendo que o grupo C não recebeu material de preenchimento e o grupo Am apenas o amido. Os animais foram sacrificados sete e 21 dias após a cirurgia. Foram realizadas análise histológica e histomorfométrica da área do defeito ósseo e os resultados submetidos à análise estatística. Histologicamente, as principais diferenças ocorreram após 21 dias. Os grupos C, Am, HA1 e HA2 apresentaram fechamento linear do defeito ósseo em todos espécimes e a maioria dos animais dos grupos A1, A2 e A+HA não exibiu neoformação óssea na região central do defeito, permanecendo este preenchido por tecido conjuntivo fibroso. No período de sete dias não houve diferença estatística significante entre todos os grupos experimentais em relação a neoformação óssea e, após 21 dias, o grupo HA2 apresentou a maior quantidade de osso neoformado. Estatisticamente, não houve diferença entre os grupos A1, A2 e A+HA nos dois períodos de estudo. Concluiu-se que o alendronato sódico, isolado ou associado com a hidroxiapatita, prejudicou a reparação óssea neste modelo experimental e a hidroxiapatita utilizada mostrou-se biocompatível e osteocondutora, com os melhores resultados observados no grupo HA2. / Abstract: This work evaluated the action of sodium alendronate, of hydroxyapatite and the association alendronate with hydroxyapatite in the repair of bone defects in ovariectomized rats femurs. Ninety eight animals were divided into seven groups: control (C), starch (Am), alendronate 1mol (A1), alendronate 2moles (A2), hydroxyapatite 1 mol (HA1), hydroxyapatite 2 moles (HA2) and the association alendronate and hydroxyapatite (A+HA). The rats weighing, approximately, 250g were ovariectomized and, after thirty days, bone defects, measuring 2,5mm, were created in the lefts femurs. The bone defects were filled with alendronate, hydroxyapatite or with both, but the group C not received none material, and the group Am, only starch. The animals were sacrified at seven and 21 days after surgery. Histological and histomorphometric analyses were performed and the results obtained were submitted to statistical analysis. Histologically, the principal differences occurred after 21 days, with the groups C, Am, HA1 and HA2 showing a linear closure of bone defect in every specimen. The most of animals of the groups A1, A2 and A+HA did not show central bone neoformation in bone defects, and there was fibrous connective tissue in this region. After seven days, there was not significance statistical difference among all experimental groups in relation to bone neoformation and, after 21 days, the group HA2 showed the most quantity of new bone formation. Statistically, there were no differences among the groups A1, A2 and A+HA in both studied period. It was concluded that the sodium alendronate, alone or combinated with hydroxyapatite, harmed the bone repair in this experimental model and the hydroxyapatite was biocompatible and osteoconductive, with the best results in group HA2. / Doutor