The use of nanoparticles for disease treatment is an increasingly popular area of research. The potential for multi-functionality allows nanoparticles to be used as transport and delivery vehicles for drugs and as diagnostic aides, among other applications, to address the unmet needs of many disease treatments. One such class of disease is osteoporosis including severe disorders, like Paget’s disease, Osteogenesis Imperfecta and Legg Calve Perthes disease. In this dissertation, we discuss a nanoparticle system consisting of gold nanoparticles surface functionalized with primary amine bisphosphonates, which is a classification of pharmaceuticals that is common in the treatment of osteoporosis. Functionalized nanoparticles allow for greater intracellular concentrations of pharmaceutical, while the properties of the gold nanoparticles provide the ability to track the pharmaceutical and enhance imaging. We have synthesized and characterized bisphosphonate functionalized gold nanoparticles of controlled size of approximately 15 nm, which are suitable for cellular uptake, and functionalized the surface using self-assembly with pamidronate and alendronate. In one major finding of this study, inductively coupled plasma mass spectrometry was used to estimate approximate surface density of the bisphosphonates on the gold nanoparticles. This resulted in concentrations of approximately 0.65 molecules per nm2 (approximately 154 Å2/molecule) for pamidronate functionalized on gold, and approximately 2.6 molecules per nm2 (approximately 39 Å2/molecule) for alendronate functionalized on gold. This allows for more accurate estimates of pharmaceutical concentrations, during in vitro and in vivo studies. Additionally, we investigated the effects of bisphosphonate functionalized gold nanoparticles on the viability and morphology of osteoclast and osteoblast cells in vitro. We found that attaching the bisphosphonates to the surface of the nanoparticles leads to increased apoptotic effects of the bisphosphonates on the osteoclast cells compared to free bisphosphonates. Further, we showed bisphosphonate functionalized gold nanoparticles may have an effect on nuclei morphology that may provide an additional means of modulating bone resorption rather than just through influencing viability. Further we showed that it may be possible to target concentrations that are safe for osteoblasts, which is critical in determining potential treatment concentrations. These viability results bring to light a number of potential considerations into the optimization of potential treatments, such as dosing concentrations. Finally, detailed results are given on effects of bisphosphonate functionalized gold nanoparticles on important behavior and activity of osteoclast and osteoblast cells in vitro. We showed that while using concentrations below the toxicity threshold, some of the normal activity of the cells could be maintained. RANKL and ALP expression in osteoblasts were maintained when removing viability as a variable. Additionally, bone nodule formation was also maintained for osteoblasts and co-cultured in vitro systems. Finally, we showed that the introduction of bone in the in vitro studies adds a new degree of consideration as to the interaction of the bisphosphonates with the hydroxyapatite surface. This strong interaction with bone is an important consideration in further developing potential treatments for osteoporotic disease. This dissertation provides insights into the use of bisphosphonate functionalized gold nanoparticles as a potential treatment and means of study for bone remodeling disorders.

This study examines the nanoindentation (energy inclusive) properties of 0.2 mg/kg alendronate treated ribs at one and three years against a vehicle treated control in a fresh-frozen, non-cold-mounted, condition. This was to verify if the tissue-level properties for 0.2 ALN treated beagles would increase because of an increased level of mineralization despite a microdamage increase. A total of twelve (12) skeletally mature (1–2 years old) female beagle dogs were treated daily for three years and one year with oral doses of vehicle (VEH, 1 mL/kg saline) or alendronate (ALN, 0.2 Merck, Rahway, NJ). The 0.2 mg dose corresponds, on a milligram per kilogram basis, to those used for treatment of postmenopausal osteoporosis. Transversely cut samples were ground and polished to 0.3μm, and were then mounted while nanoindentation was performed. The data obtained were analyzed using two modes of diamond area functions: ideal function and general function. The statistical analysis for the data were carried out using a repeated measured ANOVA (SAS V 9.1, Cary NC.) with the measured and calculated mechanical property (elastic modulus or hardness) or energy property (elastic work or plastic work) as the dependent variable and treatment (control, 1 year or 3 years) modeled as the subject. Either Turkey-Kramer or Bonferroni method was used to compute the pair-wise difference. The results indicate that when compared to one year, the three years of alendronate medication for postmenopausal osteoporosis did not have any effect on the strength of the canine cortical bone, whereas this had effect on the hardness of the subjects. This increase in the medication time resulted in an increase in the elastic work but a decrease in the plastic work. The two methods (modes) of diamond area function analysed showed different mechanical properties (elastic modulus and hardness).

Indiana University-Purdue University Indianapolis (IUPUI) / It has been proposed that the accumulation of microdamage in bone of aging individuals may play a causative and synergistic role in increased fracture incidence. If microdamage production were somehow increased, or reparative remodeling was somehow decreased, the scales may tip towards pathologic fracture. It is known that bisphosphonates increase microdamage accumulation in ribs, lumbar vertebrae, and ilium. The specific aim of this study was to histomorphometrically quantify the microdamage effect of the bisphosphonates alendronate and risedronate therapy on alveolar bone surrounding the first mandibular molar in the dog to determine if this response differs from that in non-bisphosphonate treated dogs. Thirty-four dogs were randomly assigned in two test, and one control groups. Test groups received pharmacologically equivalent doses of either alendronate (11 dogs) or risedronate (11 dogs). The control group (12 dogs) received subcutaneous injections of saline solution. The mandibular right first molar was analyzed for this study. Histomorphometric measurements were made using a x150 Nikon Optiphot-2 fluorescence microscope equipped with brightfield sources (Nikon, Inc.) using the semi-automatic Bioqant digitizing system (R & M Biometrics). There was no significant differences in cortical bone area across treatment groups for any of the regions, nor were any expected. Overall, there was almost twice as much crotical bone found in the Middle (Combined) regions compared with the Coronal (Combined) regions. The precent cortical area was universally high across all treatment groups averaging in the mid-90% range. The Apical region averaged 96.05%, followed by the Coronal region with 95.04% and the Middle region with 93.80%. The number of labeled osteons per cortical area in the alendronate and risedronate groups both tended to be lower relative to the control group (0.92/mm2 and 0.93/mm2 vs. 1.26mm2, respectively), but were not significantly different. On average, the coronal regions had nearly three times the LOn/CtAr as the Middle and Apical regions (1.90/mm2 vs. 0.63/mm2 and 0.57/mm2 respectively). Only in one region was MAR statistically higher in the Coronal (1mm) region, relative to all other regions compared. The Middle region demonstrated a low MAR. The WTh was significantly higher in the risedronate and alendronate groups than that of the control group for the Coronal region. This illustrates that with respect to the bisphosphonates, there is more formation and less resorption. In one region of a significantly lower WTh for the alendronate group relative to the risedronate group was noted. This implies a more potent inhibition in the risedronate treated groups. The WTh for the entire Coronal was statistically lower than every Middle measurement, but was not different than observed in the Apical region. This would tend to signify that in the Coronal, the turnover rate is more of a rapid nature, and therefore the osteons are not as large, while in the Apical, there were so many missing values due to the low rate of turnover, the numbers are skewed to the low end. In the Coronal (Combined) region, the risedronate (108.79 days) group exhibiting a significantly higher FP than the alendronate (62.88 days) and the control (56.13 days) groups. This would imply an increased potency of risedronate over alendronate. Regionally, the FP was significantly lower in the Coronal, relative to Middle or Apical. This is consistent with a more rapid turnover in the Coronal regions observed earlier. The Acf for alendronate (6.41/mm2 per day) and risedronate (5.69/mm2 per day) both tended to be lower by approximately 40% when compared with the control group (10.11/mm2 per day). Overall, the Acf for the Coronal region was 14.15/mm2 per day vs. 2.98/mm2 per day for the Middle and 9.13/mm2 per day for the Apical regions. This shows a significantly increased amount of turnover events taking place not just in the Coronal region, but in the region immediately adjacent to the tooth in the Coronal region. In no region did bone formation differ significantly when treated with bisphosphonates. The Coronal (1mm) region was statistically greater than every region it was measured against, individual and combined. Based on this observation, the second hypothesis that within the first molar alveolar site, bisphosphonate therapy with alendronate, and risedronate would inhibit remodeling more in the coronal region than in the middle and apical region, is rejected. When measuring microdamage accumulation (CrDn), only in the Middle (1mm) region was a significant difference across treatment groups notes. There were no other statistical differences across groups for any other regions. This observation demonstrates that bisphosphonate treatment does not increase the accumulation of microcracks in the dentate alveolar bone. Therefore, the first hypothesis that within the dentate mandible, bisphosphonate therapy with alendronate and risedronate would increase microdamage accumulation around the first molar compound to control, is rejected. When CrDn was compared by region, significant differences were noted. As expected, the Coronal (1mm) region demonstrated a significantly increased CrDN compared with the Apical and Middle regions. Coupled with the information that the BFR is increased in the Coronal and Middle (1-3mm) regions would argue for a reparative function of remodeling in the Coronal and outer Middle regions, which is in response to microdamage accumulation. Significant differences were observed in the Middle (1mm) and Middle (Combined) regions, with the alendronate group demonstrating an increased CrSDn relative to control. There was no statistical difference across treatment groups for any of the regions studied. When compared by regions, the Coronal (1mm) was statistically higher than all regions it was measured against. The Middle regions demonstrated elevated CrSDn relative to the apical region, which displayed the lowest CrSDn values of all regions. One final measure of microdamage is mean crack length. There were no statistically significant differences across any groups for any regions. The only significant differences, when observed across regions, was in reference to the Middle (1mm) region, which was significantly larger than the Coronal (1mm), Coronal (Combined), and the Middle (1-3mm) region. Otherwise, there is no observable trend, and no significant difference between regions. In conclusion, this study found that there was no an increase in microdamage in the dentate mandible of the dogs with bisphosphonate therapy, thereby rejecting the first hypothesis. While there were isolated regions of remodeling inhibition, the hypothesis that bisphosphonate therapy would inhibit remodeling more in the coronal region than in the middle and apical region is rejected. Therefore, based on the findings of this study, we conclude that bisphosphonates do inhibit remodeling in the dentate alveolus generally, but inhibition is not localized to any particular region. Finally, the administration of bisphosphonates do not result in an increase in microdamage accumulation in the dentate alveolus of dogs.

O tratamento de escolha para a avulsão dentária é o reimplante. Quando este é realizado tardiamente o tratamento da superfície radicular deve ser instituído com o objetivo de prevenir a reabsorção radicular. O objetivo deste trabalho foi comparar o efeito tópico das soluções de alendronato de sódio e de fluoreto de sódio no tratamento da superfície radicular em reimplantes tardios de dentes de ratos. Foram utilizados 72 incisivos superiores direitos extraídos e reimplantados. No grupo I, após o período extra-alveolar a seco de 60 minutos, fez-se o preenchimento do canal radicular com pasta de hidróxido de cálcio (CALEN®) e o reimplante. Nos grupos II, III e IV, após o período extra-alveolar a seco de 60 minutos, procedeu-se o tratamento da superfície radicular. No grupo II, os dentes foram imersos em solução de ácido cítrico pH 1,0 por 5 minutos, lavados em soro fisiológico por 5 minutos e mantidos em solução de fluoreto de sódio a 2% pH 5,5 por 20 minutos. No grupo III, os dentes foram imersos em solução de ácido cítrico pH 1,0 por 5 minutos, lavados em soro fisiológico por 5 minutos e mantidos em solução de alendronato de sódio a 3,2 mg/l por 10 minutos. No grupo IV, os dentes foram imersos em solução de hipoclorito de sódio a 1% por 30 minutos, lavados em soro fisiológico por 5 minutos e mantidos em solução de alendronato de sódio a 3,2 mg/l por 10 minutos. Os canais radiculares dos dentes dos grupos II, III e IV foram preenchidos com pasta de hidróxido de cálcio (CALEN®) e reimplantados. Transcorridos os períodos de 30, 90 e 120 dias do reimplante, os animais foram mortos, as peças obtidas e processadas em laboratório para análise microscópica. Os resultados mostraram que nenhum dos tratamentos foi capaz de evitar a anquilose; o tratamento com ácido cítrico e alendronato e ácido cítrico e fluoreto de sódio não evitou a ocorrência de reabsorções inflamatórias intensas; o tratamento com ácido cítrico e alendronato apresentou áreas mais intensas de reabsorção por substituição que o tratamento com hipoclorito de sódio e alendronato e não se observou reabsorções moderadas nem intensas no grupo tratado com hipoclorito de sódio e alendronato. / The treatment of choice for tooth avulsion is replantation. When this is done later, the treatment of root surface should be established with the objective to prevent root resorption. The objective of this research was to compare the topical effect of solutions of sodium alendronate and sodium fluoride in the treatment of root surface in delayed tooth replantations of rats teeth. It was used 72 rat maxillary right central incisors extracted and replanted. In group I, after the dry extra-alveolar period of 60 minutes, it was done the filling of the root canal with calcium hydroxide paste (CALEN®) and replantation. In the groups II, III and IV, after the dry extra-alveolar period of 60 minutes, it was proceeded the treatment of the root surface. In the group II, the teeth were immersed in citric acid solution pH 1.0 for 5 minutes, saline solution for 5 minutes and sodium fluoride at 2% pH 5.5 for 20 minutes. In the group III, the teeth were immersed in citric acid solution pH 1.0 for 5 minutes, saline solution for 5 minutes and sodium alendronate at 3.2 mg/l for 10 minutes. In the group IV, the teeth were immersed in sodium hypochlorite at 1% for 30 minutes, saline solution for 5 minutes and solution of sodium alendronate at 3.2 mg/l for 10 minutes. The root canals in the groups II, III and IV were filled with calcium hydroxide paste (CALEN®) and replantated. Passed the periods of 30, 90 and 120 days of the replantation, the animals were killed, samples obtained and processed in the laboratory for microscopic analysis. The results showed that none of the treatments was able to avoid ankylosis; treatment with citric acid and alendronate and citric acid and sodium fluoride did not prevent the occurrence of intense inflammatory resorption, the treatment with citric acid and alendronate showed more intense areas of replacement resorption than the treatment with sodium hypochlorite and alendronate, and it was not observed moderate or intense resorptions in the group treated with sodium hypochlorite and alendronate.

Os processos de reabsorção radicular externa, geralmente, estão associados aos traumatismos dentários que atingem os tecidos de sustentação e suporte, principalmente a avulsão e a intrusão. A terapia endodôntica nesses casos, deve visar a estabilização ou paralisação deste processo através da utilização de medicação intracanal que possa inibir a atividade osteoclástica, como os bisfosfonatos desde que, esse fármaco seja biocompatível. Neste sentido, o objetivo desse estudo foi analisar a citotoxicidade do alendronato de sódio sobre fibroblastos do ligamento periodontal humano em cultura celular. As células foram cultivadas na densidade de 1 x 10 3 células/placa. Os grupos experimentais foram: G1 (controle) sem alendronato de sódio e G2, G3 e G4 com o alendronato nas concentrações de 10 -5 , 10 -6 e 10 -7 M respectivamente. Nos tempos experimentais de 1, 6, 12 e 24 horas (curto prazo) foi analisada a viabilidade celular e em 2, 4, 6 e 8 dias (longo prazo) a sobrevivência celular. Os resultados em triplicata foram analisados estatisticamente e mostraram que as culturas tratadas com a maior concentração da droga (G2), apresentaram porcentagens de viabilidade celular significantemente menores (p < 0.01), que as dos outros grupos (G1, G3 e G4) nos tempos de 12 e 24 horas. O crescimento celular nos grupos G2 e G3 foram similares. O G2 apresentou crescimento, significantemente menor que dos demais grupos (p < 0.05). Concluiu-se que o alendronato de sódio, em contato direto com fibroblastos de ligamento periodontal humano em cultura, é citotóxico em concentrações mais elevadas (10 -5 e 10 -6 M) / The external root resorption processes are usually associated with dental trauma, mainly avulsion and intrusion. In such cases, the endodontic therapy aims the process stabilization and paralysation, through utilization of medications that can inhibit the osteoclastic activity, like bisphosphonates, since this drug would be biocompatible. The aim of this study was to analyze the sodium alendronate citotoxicity on human periodontal ligament fibroblasts. Cells were plated in a density of 1 X 10 3 cells/dish. The experimental groups were: GI (control) no sodium alendronate, and GII, GIII and GIV with sodium alendronate at the concentrations of 10 -5 , 10 -6 and 10 -7 M, respectively. The experiment times were 1, 6, 12 and 24 hours (short term) for viability and 2, 4, 6 and 8 days (long term) for cell survival. Data in triplicate were statistically analyzed. Cultures treated with the highest alendronate concentration (GII)showed cell viability percentages significantly lower (p < 0.01) than those of the other groups (GI, GIII and GIV), at 12 and 24 hours. Cell growth on GII and GIII groups was similar. GII presented smaller growth than the other groups (p < 0.05). We concluded that sodium alendronate, on direct contact with human periodontal ligament fibroblasts, is citotoxic in concentrations higher than of 10 -6 M

Made available in DSpace on 2015-03-26T13:46:33Z (GMT). No. of bitstreams: 1 texto completo.pdf: 201136 bytes, checksum: d353460c52709971fecf3b8e2ee9ed29 (MD5) Previous issue date: 2007-12-18 / This work consisted of a biological test with the objective to study the influences of the bifosfonates alendronate of sodium and sodic risedronate and of the statins calcic atorvastatine and lovasatatine in the recuperation of fractures of shin-bones of osteoporotics rats induced by the dexamethasone, in rats of the race Wistar, adults, weighing 250 ± 20g. The process of the induction of the osteoporosis consisted of the administration of dexamethasone, of the dosis of 7,5 mg / kg of physical weight, for road IM, once weekly, during four weeks, of six animals of all the groups, with the exception of the animals that there appointed the control group (G1). The animals of the group 2 (G2), called osteoporotics, received only dexamethasone. After the period of induction of the osteoporosis, the animals of all the groups were subjected to the proceeding of shut fracture of the leftt shin-bones, carried out by a veterinary doctor. Immediately after the induction of the fractures, one began the treatments where the group 3 (G3) received alendronate of sodium in the dosis of 0,25mg/kg; the group 4 (G4) received risedronate of sodium in the dosis of 1 mg / kg; the group 5 (G5) that received calcic atorvastatine in the dosis of 1,2 mg / kg and the group 6 (G6) what it received lovastatine in the dosis of 2mg/kg. All the substances were administered by oral road, daily. In the period of 35 days, after the beginning of the treatments, samples of blood were collected for dosages of calcium, phosphorus, albumin and total proteins in the multiparametric equipment of biochemistry Alizé with kits of the mark BioMerieux, and phosphatase alkaline bone in the equipment of quimioluminescence Access Immunoassay System of Beckman Coulter with kits Ostase. After the sacrifice, there was collected the left shin-bone of each animal what, once fixed they were morfometric analysed, X- rayed and when it was prosecuted for getting the blades, which parliament Masson Tricrômic was colored, for hitological study in light microscopie. The right shin-bone was collected to subject to biomechanical tests. All s measures of height, width and of the bone callus of the shin-bones they were carried out with help of a paquimeter. Besides, there were carried out gauging of the density of these bones, which took place through the relation of the values of weighing and dislocation of liquid in graduated container. The left shin- bones were X-rayed then in profile and the obtained image was subjected to an analysis of radiografic density through the program Image Tool he will be Windows 3.0, for tones of ash obtained of the image. The areas of the analyses were delimited in all the radiografic images of the shin-bones and only the selected areas were taken into account for analysis. Tests of flexing, in the right shin-bones they were carried out with the help of the appliance Instrom (Laboratory of Paper and Cellulose of the Department of Engineering Florestal/UFV). One proceeded the percentage calculation of the density trabecular bone, from the image amostral of trabecular bone, contained in the region subcondral, of each histological cut, in optical microscope equipped with digital camera (TCL-984 P), analysed in monitor of microcomputer of 14 inches, making use of the technique of histomorfometric of counting of points. The biological test was carried out according to delineation completely casualizado, with six treatments in six repetitions. The obtained results were subjected to the variance analysis and to the test F (p <0,05). Control the groups (G1 and G2) they were compared between you, through the test F. The treated groups (G3, G4, G5 and G6) were compared between you, through the test of Tukey to 5% of probability. The comparisons also were carried out between the treated groups and the controls G1 and G2, being that, for the same thing, the test of Dunnet was applied to 5 % of probability. The results did not show significant differences between the group control (G1) and the osteoporotic group (G2), as well as, between them and the treated groups (G3, G4, G5 and G6) and, also, between the groups treated between you, in all the periods, as for the values silken of calcium, phophorus and phosphatase alkaline bone. Through the morfometric and histomorfometric was possible the induction of the osteoporose noted with the glucorticoid, as well as the improvement in the bone density, density trabecular bone and thickness of the bone callus in all the treated groups when the group is prevailing treated with alendronate of sodium that presented better results. The results radiografics and biomechanical tests corroborate with the histomorfometric results. The biochemical results, principally phosphatase alkaline bone, what remained without significant differences in all the groups, when valued together with the morfometric, histomorfometri, radiografic and biomechanical test demonstrate what prevailed the properties antirreabsortives in relation the forming ones of bone in all the treated groups. In spite of the predominance of the properties antirreabsortives in all the treatments, the morfometric, histomorfometric, X-ray and biomechanical tests showed comparable results to the bone recuperation of the normal group, which demonstrates what same do not interfere negatively and which positively in the process of recuperation of fractures in osteoporotics animals. Is excepted, however, which subsequent studies of the dosages, interactions, biodisponibility, besides the toxicological effects of these pharmacological substances, make to themselves necessary. / Este trabalho consistiu de um ensaio biológico com o objetivo de estudar as influências do bifosfonatos alendronato de sódio e risedronato sódico e das estatinas atorvastatina cálcica e lovasatatina na recuperação de fraturas de tíbias de ratas osteoporóticas induzida pelo glicocorticóide dexametasona, em ratas da raça Wistar, adultas, pesando 250 ± 20g. O processo da indução da osteoporose consistiu na administração de dexametasona, na dose de 7,5 mg/kg de peso corporal, por via IM, uma vez por semana, durante quatro semanas, nos seis animais de todos os grupos, à exceção dos animais que constituiu o grupo controle (G1). Os animais do grupo 2 (G2), chamado osteoporótico, receberam apenas dexametasona. Após o período de indução da osteoporose, os animais de todos os grupos foram submetidos ao procedimento de fratura fechada das tíbias esquerdas, realizado por um médico veterinário. Imediatamente após a indução das fraturas, iniciou-se os tratamentos onde o grupo 3 (G3) recebeu alendronato de sódio na dose de 0,25mg/kg; o grupo 4 (G4) recebeu risedrnato de sódio na dose de 1 mg/kg ; o grupo 5 (G5) que recebeu atorvastatina cálcica na dose de 1,2 mg/kg e o grupo 6 (G6) que recebeu lovastatina na dose de 2mg/kg. Todas as substâncias foram administradas por via oral, diariamente. No período de 35dias, após o início dos tratamentos, foram coletados amostras de sangue para dosagens de cálcio, fósforo, albumina e proteínas totais no equipamento multiparamétrico de bioquímica Alizé com kits da marca BioMerieux, e fosfatase alcalina óssea no equipamento de quimioluminescência Access Immunoassay System da Beckman Coulter com kits Ostase. Após o sacrifício, foi coletado a tíbia esquerda de cada animal que, uma vez fixados foram analisados morfometricamente, radiografados, submetidas e processado rotineiramente para obtenção das lâminas, cujos cortes foram corados Tricrômico de Masson, para estudo histologico em microscopia de luz. As tíbias direitas foram também coletadas para realização de testes biomecânicos. Medidas de altura, largura e do calo ósseo das tíbias foram realizadas com auxílio de um paquímetro. Além disso, foram realizados aferições da densidade desses ossos, que ocorreram através da relação dos valores de pesagem e deslocamento de líquido em recipiente graduado. As tíbias esquerdas foram então radiografadas em perfil e a imagem obtida foi submetida a uma analise de densidade radiográfica através do programa Image Tool for Windows 3.0, por tons de cinza obtidos da imagem. As áreas das análises foram delimitadas em todas as imagens radiográficas das tíbias e só foram levadas em consideração para análise as áreas selecionadas.Testes de flexão, nas tíbias direitas foram realizados com o auxílio do aparelho Instrom (Laboratório de Papel e Celulose do Departamento de Engenharia Florestal/UFV). Procedeu-se o cálculo percentual da densidade trabecular óssea, à partir da imagem amostral de osso trabecular, contida na região subcondral, de cada corte histológico, em microscópio óptico equipado com câmara digital (TCL-984 P), analisada em monitor de microcomputador de 14 polegadas, utilizando-se da técnica de histomorfometria de contagem de pontos. O ensaio biológico foi realizado segundo delineamento inteiramente casualizado, com seis tratamentos em seis repetições. Os resultados obtidos foram submetidos à análise de variância e ao teste F (p<0,05). Os grupos controles (G1 e G2) foram comparados entre si, por meio do teste F. Os grupos tratados (G3, G4, G5 e G6) foram comparados entre si, através do teste de Tukey à 5% de probabilidade. As comparações também foram realizadas entre os grupos tratados e os controles G1 e G2, sendo que, para o mesmo, foi aplicado o teste de Dunnet à 5% de probabilidade. Os resultados não mostraram diferenças significativas entre o grupo controle (G1) e o grupo osteoporótico (G2), bem como, entre eles e os grupos tratados (G3, G4,G5 e G6) e, também, entre os grupos tratados entre si, em todos os períodos, quanto aos valores sérico de cálcio, fósforo e fosfatase alcalina óssea. Através da morfometria e histomorfometria foi possível constatar a indução da osteoporose com o glicorticóide, assim como a melhoria na densidade óssea, densidade trabecular óssea e espessura do calo ósseo em todos os grupos tratados prevalecendo o grupo tratado com alendronato de sódio que apresentou melhores resultados. Os resultados radiográficos e biomecânicos corroboram com os resultados histomorfometricos. Os resultados bioquímicos, principalmente fosfatase alcalina óssea, que permaneceram sem diferenças significativas em todos os grupos, quando avaliados em conjunto com a morfometria, histomorfometria, radiografia e teste biomecânicos demonstram que prevaleceram as propriedades antirreabsortivas em relação as formadoras de osso em todos os grupos tratados. Apesar da prevalência das propriedades antirreabsortivas em todos os tratamentos, a morfometria, histomorfométria, radiografia e testes biomecânicos mostrou resultados equiparáveis à recuperação óssea do grupo normal, o que demonstra que os mesmos não interferem negativamente e sim positivamente no processo de recuperação de fraturas em animais osteoporóticos. Ressalva- se, no entanto, que estudos posteriores das dosagens, interações, biodisponibilidades, além dos efeitos toxicológicos dessas substâncias farmacológicas, fazem-se necessários.

Resumo: O presente estudo teve como objetivo avaliar a influência da deficiência de estrógeno promovida pela ovariectomia (OVX) sobre o tecido ósseo ao redor de implantes com osseointegração estabelecida. Foram utilizadas ratas Wistar, com idade aproximada de 60 dias e peso variando entre 180g a 220g, submetidas à colocação de um implante na metáfise tibial. Os tratamentos utilizados foram eficientes na prevenção da perda óssea. Os animais que receberam a administração do alendronato apresentaram os melhores resultados para todas as análises realizadas. A deficiência de estrógeno decorrente da OVX acarretou prejuízos para a BMD, densidade radiográfica da região de osso medular e torque de remoção dos implantes. Esse quadro pôde ser revertido com a utilização dos tratamentos instituídos, com o alendronato apresentando os melhores resultados. / Abstract: The aim of the present study was to evaluate the influence of estrogen deficiency due to ovariectomy (OVX) on the bone around osseointegrated titanium implants. Wistar female rats - approximately 60 days old, weighting from 180 to 220 g - were used. Digital radiographs were taken and the implants were submitted to the removal toque. The results showed a negative effect of the OVX on the BMD at the femur and lumbar regions, on the radiographic bone density at the cancellous bone and on the biomechanical characteristics of the bone tissue around the implants. The treatments were efficient preventing bone loss. The animals that received alendronate therapy showed the best results for all the analyzed variables. we can conclude that the estrogen deficiency due to OVX had a negative influence on the BMD, on the radiographic bone density at the cancellous bone and on the removal torque of the implants; however, such negative influence can be reversed with the adopted therapies, among which the alendronate seemed to be the best method for prevention and treatment of bone loss due to OVX. / Orientador: Silvana Regina Perez Orrico / Coorientador: Elcio Marcantonio Junior / Banca: Luiz Antonio Pugliesi Alves de Lima / Banca: Roberto Henrique Barbeiro / Mestre

Os processos de reabsorção radicular externa, geralmente, estão associados aos traumatismos dentários que atingem os tecidos de sustentação e suporte, principalmente a avulsão e a intrusão. A terapia endodôntica nesses casos, deve visar a estabilização ou paralisação deste processo através da utilização de medicação intracanal que possa inibir a atividade osteoclástica, como os bisfosfonatos desde que, esse fármaco seja biocompatível. Neste sentido, o objetivo desse estudo foi analisar a citotoxicidade do alendronato de sódio sobre fibroblastos do ligamento periodontal humano em cultura celular. As células foram cultivadas na densidade de 1 x 10 3 células/placa. Os grupos experimentais foram: G1 (controle) sem alendronato de sódio e G2, G3 e G4 com o alendronato nas concentrações de 10 -5 , 10 -6 e 10 -7 M respectivamente. Nos tempos experimentais de 1, 6, 12 e 24 horas (curto prazo) foi analisada a viabilidade celular e em 2, 4, 6 e 8 dias (longo prazo) a sobrevivência celular. Os resultados em triplicata foram analisados estatisticamente e mostraram que as culturas tratadas com a maior concentração da droga (G2), apresentaram porcentagens de viabilidade celular significantemente menores (p < 0.01), que as dos outros grupos (G1, G3 e G4) nos tempos de 12 e 24 horas. O crescimento celular nos grupos G2 e G3 foram similares. O G2 apresentou crescimento, significantemente menor que dos demais grupos (p < 0.05). Concluiu-se que o alendronato de sódio, em contato direto com fibroblastos de ligamento periodontal humano em cultura, é citotóxico em concentrações mais elevadas (10 -5 e 10 -6 M) / The external root resorption processes are usually associated with dental trauma, mainly avulsion and intrusion. In such cases, the endodontic therapy aims the process stabilization and paralysation, through utilization of medications that can inhibit the osteoclastic activity, like bisphosphonates, since this drug would be biocompatible. The aim of this study was to analyze the sodium alendronate citotoxicity on human periodontal ligament fibroblasts. Cells were plated in a density of 1 X 10 3 cells/dish. The experimental groups were: GI (control) no sodium alendronate, and GII, GIII and GIV with sodium alendronate at the concentrations of 10 -5 , 10 -6 and 10 -7 M, respectively. The experiment times were 1, 6, 12 and 24 hours (short term) for viability and 2, 4, 6 and 8 days (long term) for cell survival. Data in triplicate were statistically analyzed. Cultures treated with the highest alendronate concentration (GII)showed cell viability percentages significantly lower (p < 0.01) than those of the other groups (GI, GIII and GIV), at 12 and 24 hours. Cell growth on GII and GIII groups was similar. GII presented smaller growth than the other groups (p < 0.05). We concluded that sodium alendronate, on direct contact with human periodontal ligament fibroblasts, is citotoxic in concentrations higher than of 10 -6 M

O tratamento de escolha para a avulsão dentária é o reimplante. Quando este é realizado tardiamente o tratamento da superfície radicular deve ser instituído com o objetivo de prevenir a reabsorção radicular. O objetivo deste trabalho foi comparar o efeito tópico das soluções de alendronato de sódio e de fluoreto de sódio no tratamento da superfície radicular em reimplantes tardios de dentes de ratos. Foram utilizados 72 incisivos superiores direitos extraídos e reimplantados. No grupo I, após o período extra-alveolar a seco de 60 minutos, fez-se o preenchimento do canal radicular com pasta de hidróxido de cálcio (CALEN®) e o reimplante. Nos grupos II, III e IV, após o período extra-alveolar a seco de 60 minutos, procedeu-se o tratamento da superfície radicular. No grupo II, os dentes foram imersos em solução de ácido cítrico pH 1,0 por 5 minutos, lavados em soro fisiológico por 5 minutos e mantidos em solução de fluoreto de sódio a 2% pH 5,5 por 20 minutos. No grupo III, os dentes foram imersos em solução de ácido cítrico pH 1,0 por 5 minutos, lavados em soro fisiológico por 5 minutos e mantidos em solução de alendronato de sódio a 3,2 mg/l por 10 minutos. No grupo IV, os dentes foram imersos em solução de hipoclorito de sódio a 1% por 30 minutos, lavados em soro fisiológico por 5 minutos e mantidos em solução de alendronato de sódio a 3,2 mg/l por 10 minutos. Os canais radiculares dos dentes dos grupos II, III e IV foram preenchidos com pasta de hidróxido de cálcio (CALEN®) e reimplantados. Transcorridos os períodos de 30, 90 e 120 dias do reimplante, os animais foram mortos, as peças obtidas e processadas em laboratório para análise microscópica. Os resultados mostraram que nenhum dos tratamentos foi capaz de evitar a anquilose; o tratamento com ácido cítrico e alendronato e ácido cítrico e fluoreto de sódio não evitou a ocorrência de reabsorções inflamatórias intensas; o tratamento com ácido cítrico e alendronato apresentou áreas mais intensas de reabsorção por substituição que o tratamento com hipoclorito de sódio e alendronato e não se observou reabsorções moderadas nem intensas no grupo tratado com hipoclorito de sódio e alendronato. / The treatment of choice for tooth avulsion is replantation. When this is done later, the treatment of root surface should be established with the objective to prevent root resorption. The objective of this research was to compare the topical effect of solutions of sodium alendronate and sodium fluoride in the treatment of root surface in delayed tooth replantations of rats teeth. It was used 72 rat maxillary right central incisors extracted and replanted. In group I, after the dry extra-alveolar period of 60 minutes, it was done the filling of the root canal with calcium hydroxide paste (CALEN®) and replantation. In the groups II, III and IV, after the dry extra-alveolar period of 60 minutes, it was proceeded the treatment of the root surface. In the group II, the teeth were immersed in citric acid solution pH 1.0 for 5 minutes, saline solution for 5 minutes and sodium fluoride at 2% pH 5.5 for 20 minutes. In the group III, the teeth were immersed in citric acid solution pH 1.0 for 5 minutes, saline solution for 5 minutes and sodium alendronate at 3.2 mg/l for 10 minutes. In the group IV, the teeth were immersed in sodium hypochlorite at 1% for 30 minutes, saline solution for 5 minutes and solution of sodium alendronate at 3.2 mg/l for 10 minutes. The root canals in the groups II, III and IV were filled with calcium hydroxide paste (CALEN®) and replantated. Passed the periods of 30, 90 and 120 days of the replantation, the animals were killed, samples obtained and processed in the laboratory for microscopic analysis. The results showed that none of the treatments was able to avoid ankylosis; treatment with citric acid and alendronate and citric acid and sodium fluoride did not prevent the occurrence of intense inflammatory resorption, the treatment with citric acid and alendronate showed more intense areas of replacement resorption than the treatment with sodium hypochlorite and alendronate, and it was not observed moderate or intense resorptions in the group treated with sodium hypochlorite and alendronate.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-04-03T13:20:01Z No. of bitstreams: 1 ledamariliafonsecalucinda.pdf: 1555881 bytes, checksum: 1cf24f935ed553401a8a63db1a26cd5c (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-04-03T18:58:54Z (GMT) No. of bitstreams: 1 ledamariliafonsecalucinda.pdf: 1555881 bytes, checksum: 1cf24f935ed553401a8a63db1a26cd5c (MD5) / Made available in DSpace on 2017-04-03T18:58:54Z (GMT). No. of bitstreams: 1 ledamariliafonsecalucinda.pdf: 1555881 bytes, checksum: 1cf24f935ed553401a8a63db1a26cd5c (MD5) Previous issue date: 2009-04-30 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Introdução: A osteoporose é caracterizada pela diminuição do volume ósseo total e entre as principais causas da doença estão a redução de estrogênio, durante a menopausa e o uso crônico de glicocorticóides. Diversos produtos são usados em seu tratamento e entre estes alguns produtos naturais tais como os fitoestrógenos. Estudos “in vitro” com o extrato de Ginkgo biloba (EGb) demonstraram efeito estrogênico e ação protetora sobre os osteoblastos. Portanto o EGb poderia ser importante no tratamento da osteoporose. Métodos:72 ratas foram distribuídas em 6 grupos: controle, osteoporose, controle positivo (alendronato de sódio - 0,2mg/Kg/dia), EGb1 (14 mg EGb/Kg/dia), EGb2 (28 mg EGb/Kg/dia ) e EGb3 (56 mg EGb/Kg/dia). A osteoporose foi induzida através de injeções intramusculares de dexametasona na dose de 7mg/Kg, uma vez por semana, durante cinco semanas. Após a indução se iniciou o tratamento de 20 e 30 dias, exceto no grupo controle. Após a eutanásia, foram removidas as mandíbulas esquerdas para análise radiográfica digital, seguiu-se posteriormente a avaliação da cortical mandibular e do suporte ósseo periodontal (SOP) na superfície mesial e distal do primeiro molar mandibular. Para a análise histomorfométrica foram removidos as mandíbulas direitas e os fêmures direitos, para avaliação do percentual ósseo trabecular do fêmur (POT) e do percentual ósseo alveolar (POA) do segundo molar mandibular. Os grupos controles foram comparados aos grupos osteoporose (teste “t” Student). Os demais grupos, exceto os controles, foram analisados através do teste de ANOVA seguido do teste post hoc de Tukey ou Dunnett (p<0,05). Resultados: Foi observado através da análise radiográfica mandibular que o glicocorticóide causou diminuição do SOP, o alendronato de sódio apresentou recuperação do percentual do SOP, da mesma forma que o EGb nos tratamentos de 20 e 30 dias nas doses de 28mg/kg/dia e 56mg/kg/dia. O grupo EGb3 apresentou maior espessura cortical mandibular nos tratamentos de 20 e 30 dias. Através da histomorfometria dos animais tratados por 20 e 30 dias foi observado que o glicocorticóide causou diminuição no POT da epífise do fêmur e do POA mandibular (p,0,05). O grupo controle positivo apresentou aumento significativo do POT do fêmur (30 dias) quando comparado ao grupo osteoporose. O POA mandibular do grupo controle positivo apresentou aumento não significativo em relação ao grupo osteoporose (20 e 30 dias). Os grupos EGb 1 e EGb 2 apresentaram um aumento (p<0.05) do POT do fêmur quando comparados aos animais do grupo osteoporose (20 e 30dias). Da mesma forma os grupos EGb 2 e EGb 3 (20 dias) e os grupos EGb1, EGb2 e EGb3 (30 dias) apresentaram aumento significativo do POA mandibular quando comparado ao grupo osteoporose. Conclusão: O EGb recuperou a osteoporose induzida por glicocorticóide nos parâmetros avaliados neste estudo, sugerindo que o EGb possa ser efetivo no tratamento da osteoporose. / Introduction: Osteoporosis is a disease characterized by the reduction of bone volume. Among the main causes of osteoporosis is the reduction of the levels of estrogen and the chronic use of glucocorticoids. Several products are used on the osteoporosis treatment, including natural products, among them some promising alternatives as the phytoestrogens. In vitro studies showed that the extract of Ginkgo biloba (EGb) has a estrogenic effect and a protect action on the osteoblasts cell. Therefore the EGb may be important in the treatment of osteoporosis. Methods: 72 female rats were divided into six groups: control, osteoporosis, positive control (sodium alendronate-0.2mg/Kg/day), EGb1 (14mg/Kg/day), EGb2 (28mg/Kg/day) and (56mg/Kg/day). Osteoporosis induction was done trough intramuscular injection of dexamethasone in a dose of 7mg/Kg, once a week, during five weeks. Treatments were conducted after osteoporosis induction for 20 and 30 days. Following euthanazia, the left mandibles were removed for the digital radiographic analysis. The cortical and the periodontal bone support (PBS) at the mesial and distal surfaces of the first molar were analyzed. For the histomorphometric analysis the right femurs and mandibles were removed to evaluate the percentage of the trabecular bone (PTB) of the femur epiphysis and the percentage of the alveolar bone (PAB) of the interradicular septum of the second molar. The control groups were compared with the osteoporosis group (Student’s t-test). The others groups, except the control groups, were analyzed by ANOVA test followed by Tukey or Dunnett’s post-hoc test (p<0.05). Results: It was observed trough the mandibular radiographic analysis that the glucocorticoid caused the PBS reduction, the sodium alendronate recovered the percentage of PBS in the same way that EGb in the 28mg/kg/day and 56mg/kg/day doses in the treatments of 20 and 30 days. The EGb3 showed increase in the cortical mandibular thickness in the treatments of 20 and 30 days. Trough the histomorphometric analysis of the animals treated for 30 and 20 days, it was observed that glucocorticoids reduced the PTB of the femur and the PAB of the mandible (p<0,05). The positive control group (30 days) showed a statistical significant increase of the PTB of the femur when compared to the osteoporosis group (30 days). The PAB of the mandible in the positive control group showed no significant increase when compared to the osteoporosis group (20 and 30 days). The groups EGb1 and EGb2 groups showed an increase of the PTB of the femur when compared to the osteoporosis group (20 and 30 days), in the same way the EGb2 and EGb3 groups (20 days) and the EGb1, EGb2, and EGB3 groups (30 days) showed a statically significant increase of the PAB when compared to the osteoporosis group. Conclusion: The EGb recovered the effects oft glucocorticoid induced-osteoporosis in the evaluated parameters of this study, it suggests that the extract of Ginkgo biloba may be effective in the treatment of osteoporosis.