Devido à sua peculiar estrutura mineralizada, o tecido ósseo ainda representa um dos desafios para os estudos em bioengenharia regenerativa. Nesse sentido, estratégias realizadas envolvendo o sistema de liberação de drogas através de polímeros bioreabsorvíveis apontam resultados promissores. Esse projeto teve como objetivo empregar PLGA [poli (ácido lático-co-glicólico)] carregado com sinvastatina (SIN) em defeitos ósseos críticos criados em osso parietal de ratos. Foi realizado um defeito de 5.0 mm de diâmetro no osso parietal esquerdo, sendo que no grupo controle (C) foi deixado apenas o coágulo sanguíneo. O grupo experimental foi subdividido como segue: no subgrupo (M), uma membrana de PLGA foi colocada recobrindo o defeito ósseo, de modo que as bordas ultrapassaram a borda do defeito. No outro subgrupo (MSI), microesferas de PLGA 50/50 contendo sinvastatina a 2,5% foram depositadas no interior do defeito, sendo posteriormente recobertas pela membrana de PLGA; no grupo (MSS) foram colocadas microesferas de PLGA sem sinvastatina para avaliar o efeito osteocondutor das microesferas. Os animais foram sacrificados em dois períodos 30 e 60 dias após a cirurgia e as calotas removidas e processadas para análise morfológica em microscopia de luz, microscopia eletrônica de transmissão e varredura, imuno-histoquímica para análise da expressão de OPN (osteopontina), BSP (sialoproteína óssea) e OSAD (osteoaderina) e análise imunocitoquímica da distribuição ultra estrutural da proteína OPN. Os resultados observados mostraram que a formação do novo osso iniciou-se pelas margens do defeito e também pela área central em grupos tratados. A análise da regeneração nos defeitos tratados com SIN mostrou uma disposição ordenada das fibrilas colágenas e uma matriz com aspecto de tecido ósseo em fase mais madura. O estudo mostra que a regeneração do tecido pode ser acelerada e que a matriz neorformada é depositada de forma mais organizada pela liberação gradual da SIN. / Due to its unique mineralized structure, bone regeneration is still a challenge. Numerous strategies had been proposed during the past years, drug delivery system using polymeric scaffolds is a promising strategy. The aim of this study was to employ PLGA [poly (lactide-co-glycolic acid)] loaded with simvastatin(SIN) in bone defects created in rat calvaria. Bone defects with 5.0 mm diameter was created in the left side of the calvarias. In Control group (C) their defects were filled only with blood clot. The experimental group was subdivided. In subgroup (M), a PLGA membrane was covered the defect. In the other subgroup (MSI), PLGA microspheres loaded with 2.5% simvastatin filled the defect over the blood clot inside the defect and subsequently covered with PLGA membrane, another group was criated to evaluet the osteoconductor potencial of microesphers without the (SIN), the MSS group. The animals were sacrificed in 30, 60 after surgery, and the calvarias were removed and processed for light and transmission and scanning electron microscopy analyzes, Osteopontin (OPN), Osteoadherin (OSAD), Bone sialoprotein (BSP) imunnolabeling and immunocytochemical analyzes of the ultrastructural distribution of osteopontin in the neoformed bone. The in vivo experiment revealed that the microspheres containing simvastatin significantly enhanced the disposition of collagen fibrils in immature bone and bone formation in the rabbit calvaria critical size defect.

Wong, Mei Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 207-251). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / PUBLICATIONS BASED ON WORK IN THIS THESIS --- p.vii / ABBREVIATIONS --- p.viii / TABLE OF CONTENTS --- p.x / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Diabetes Mellitus --- p.1 / Chapter 1.2 --- Structure and Functions of the Pancreas --- p.2 / Chapter 1.2.1 --- Size of Pancreatic β-Cells --- p.4 / Chapter 1.2.2 --- Signaling Pathways of Insulin Secretion from Pancreatic β-Cells --- p.4 / Chapter 1.3 --- Classification of Diabetes --- p.6 / Chapter 1.3.1 --- Type 1 Diabetes --- p.6 / Chapter 1.3.2 --- Type 2 Diabetes --- p.8 / Chapter 1.4 --- Pathologies of Type 2 Diabetes --- p.9 / Chapter 1.4.1 --- Hyperglycemia --- p.9 / Chapter 1.4.1.1 --- A dvanced Glycosylation End Products --- p.11 / Chapter 1.4.1.2 --- Protein Kinase C Activation --- p.13 / Chapter 1.4.1.3 --- The Glucosamine Pathway --- p.14 / Chapter 1.4.1.4 --- Oxidative Stress --- p.15 / Chapter 1.4.2 --- Insulin Resistance --- p.15 / Chapter 1.4.3 --- Loss of β-Cell Mass and β-Cell Dysfunction --- p.18 / Chapter 1.5 --- Complications of Diabetes Mellitus --- p.21 / Chapter 1.5.1 --- Cardiovascular Diseases --- p.21 / Chapter 1.5.2 --- Diabetic Retinopathy --- p.22 / Chapter 1.5.3 --- Diabetic Nephropathy --- p.23 / Chapter 1.5.4 --- Neuropathy --- p.24 / Chapter 1.6 --- Anti-Diabetic Drugs for Type 2 Diabetes Mellitus --- p.25 / Chapter 1.6.1 --- Secretagogues --- p.25 / Chapter 1.6.2 --- Sensitizers --- p.26 / Chapter 1.6.3 --- Alpha-Glucosidase Inhibitors --- p.27 / Chapter 1.6.4 --- Peptide Analogs --- p.27 / Chapter 1.6.4.1 --- Incretin Mimetics --- p.27 / Chapter 1.6.4.2 --- Dipeptidyl Peptidase-4 Inhibitors --- p.28 / Chapter 1.7 --- Insights of Porcine Islets in Treatment of Diabetics --- p.28 / Chapter 1.8 3 --- -Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMG CoA Reductase) --- p.31 / Chapter 1.8.1 --- Statins --- p.32 / Chapter 1.8.2 --- Pleiotropic Effects of Statins --- p.36 / Chapter 1.8.2.1 --- Statins and Isoprenylated Proteins --- p.36 / Chapter 1.8.2.2 --- Statins and Endothelial Functions --- p.38 / Chapter 1.8.2.3 --- Statins and Platelet Functions --- p.39 / Chapter 1.8.2.4 --- Statins and Plaque Stability --- p.39 / Chapter 1.8.2.5 --- Statins and Vascular Inflammation --- p.40 / Chapter 1.9 --- Clinical Studies of Statins on Diabetics --- p.41 / Chapter 1.10 --- Possible Factors Involved in Simvastatin-Regulated Insulin Secretion --- p.44 / Chapter 1.10.1 --- AMP-Activated Protein Kinase --- p.44 / Chapter 1.10.2 --- Caveolin-1 --- p.46 / Chapter 1.10.3 --- Sterol-Regulatory Elementary Binding Protein --- p.50 / Chapter 1.10.4 --- Protein Phosphatase 2A --- p.52 / Chapter 1.10.5 --- Calcium Sensing Receptor --- p.55 / Chapter 1.11 --- Objectives of Study --- p.59 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.60 / Chapter 2.1 --- Materials --- p.60 / Chapter 2.1.1 --- Solutions --- p.60 / Chapter 2.1.2 --- Antibodies --- p.63 / Chapter 2.2 --- Methods --- p.64 / Chapter 2.2.1 --- Maintenance of Pancreas Function --- p.64 / Chapter 2.2.2 --- Islet Isolation --- p.65 / Chapter 2.2.3 --- Hematoxylin and Eosin (H&E) Staining --- p.65 / Chapter 2.2.4 --- Simvastatin and Simvastatin-Na+ --- p.66 / Chapter 2.2.5 --- AICAR --- p.67 / Chapter 2.2.6 --- Compound C --- p.67 / Chapter 2.2.7 --- Incubation of Islets --- p.67 / Chapter 2.2.8 --- Western Blot --- p.68 / Chapter 2.2.9 --- Enzyme-Linked Immunosorbent Assay (ELISA) --- p.69 / Chapter 2.2.10 --- Statistical Analysis --- p.71 / Chapter CHAPTER 3 --- HISTOLOGY OF PORCINE PANCREATIC ISLETS OF LANGERHANS --- p.72 / Chapter 3.1 --- Comparison of Sizes of Porcine Pancreatic Islets in Histological Sections of Pancreas --- p.72 / Chapter CHAPTER 4 --- PROTEIN EXPRESSION OF HMG COA REDUCTASE --- p.75 / Chapter 4.1 --- Effect of Incubation Time on HMG CoA Reductase Expression --- p.75 / Chapter 4.2 --- Short-Term Effect of Simvastatin on HMG CoA Reductase Expression --- p.78 / Chapter 4.3 --- Long-Term Effect of Simvastatin on HMG CoA Reductase Expression --- p.81 / Chapter 4.4 --- Effect of Osmolality on HMG CoA Reductase Expression --- p.83 / Chapter 4.5 --- Effect of Simvastatin on Ser871 p-HMG CoA Reductase Expression --- p.87 / Chapter CHAPTER 5 --- EVALUATION OF THE ROLE OF SIMVASTATIN IN INSULIN SECRETION VIA HMG CO A REDUCTASE REGULATION --- p.90 / Chapter 5.1 --- Effect of Simvastatin on Insulin Secretion --- p.90 / Chapter 5.2 --- Effect of Different Concentrations of Simvastatin on Insulin Secretion --- p.94 / Chapter 5.3 --- Effect of Simvastatin on Insulin Content --- p.96 / Chapter CHAPTER 6 --- ROLE OF AMPK EXPRESSION IN INSULIN SECRETION PATHWAY --- p.100 / Chapter 6.1 --- Effect of Simvastatin on Thr172 p-AMPK α and AMPK α1 Expressions --- p.100 / Chapter 6.2 --- Evaluation of the Role of Simvastatin in AMPK Regulation --- p.104 / Chapter 6.3 --- Evaluation of the Role of PP2A in AMPK Regulation --- p.108 / Chapter 6.4 --- Evaluation of the Role of Simvastatin on Insulin Secretion via AMPK Regulation --- p.111 / Chapter 6.4.1 --- AMPK Regulation on Releasable Insulin Secretion --- p.111 / Chapter 6.4.2 --- AMPK Regulation on Non-Releasable Insulin Content and Total Insulin Content --- p.112 / Chapter CHAPTER 7 --- EFFECT OF SIMVASTATIN ON THE EXPRESSION OF REGULATORY PROTEINS INVOLVED IN INSULIN SECRETION --- p.119 / Chapter 7.1 --- Effect of Simvastatin on SREBP-2 Expression --- p.119 / Chapter 7.2 --- Effect of Simvastatin on Caveolin-1 Expression --- p.121 / Chapter 7.3 --- Effect of Simvastatin on Calcium Sensing Receptor Expression --- p.123 / Chapter CHAPTER 8 --- EFFECT OF SIMVASTATIN-NA+ ON INSULIN SECRETION --- p.126 / Chapter 8.1 --- Effect of Simvastatin-Na+ on HMG CoA Reductase Expression --- p.126 / Chapter 8.2 --- Effect of Simvastatin-Na+ on Insulin Secretion --- p.128 / Chapter 8.3 --- Effect of Different Concentrations of Simvastatin-Na+ on Insulin Secretion --- p.130 / Chapter 8.4 --- Effect of Simvastatin-Na+ on Insulin Content --- p.132 / Chapter CHAPTER 9 --- EFFECT OF PRAVASTATIN ON INSULIN SECRETION --- p.136 / Chapter 9.1 --- Effect of Pravastatin on Insulin Secretion --- p.136 / Chapter 9.2 --- Effect of Pravastatin on Insulin Content --- p.138 / Chapter CHAPTER 10 --- EFFECT OF METHYL-B-CYCLODEXTRIN ON INSULIN SECRETION --- p.142 / Chapter 10.1 --- Effect of Methyl-β-cyclodextrin on Insulin Secretion --- p.142 / Chapter 10.2 --- Effect of Methyl-β-cyclodextrin on Insulin Content --- p.144 / Chapter CHAPTER 11 --- DISCUSSION --- p.149 / Chapter 11.1 --- Importance of Studying Porcine Pancreatic Islets and Islet Distribution --- p.150 / Chapter 11.2 --- Screening of Concentration and Incubation Time of Simvastatin on Porcine Pancreatic Islets --- p.152 / Chapter 11.3 --- Glucose-Independent Effect of Simvastatin on Protein Expression of HMG CoA Reductase --- p.154 / Chapter 11.4 --- Role of AMPK in HMG CoA Reductase-Modulated Insulin Secretion --- p.159 / Chapter 11.5 --- Role of SREBP-2 in Simvastatin-Modulated Regulation --- p.174 / Chapter 11.6 --- Role of Calcium Sensing Receptor in Simvastatin-Modulated Regulation --- p.175 / Chapter 11.7 --- Role of Caveolin-1 in Simvastatin-Modulated Regulation --- p.179 / Chapter 11.8 --- "Effects of Simvastatin-Na+， Pravastatin and Methyl-β-cyclodextrin, and Importance of Endoplasmic Reticulum in Insulin Secretion" --- p.183 / Chapter CHAPTER 12 --- CONCLUSIONS AND FURTHER STUDIES --- p.197 / Chapter 12.1 --- Conclusions --- p.197 / Chapter 12.2 --- Further Studies --- p.203 / REFERENCES --- p.207

More than one million people worldwide receive some kind of bone graft each year. Grafts are often needed following bone tumour removal or traumatic fractures to fill voids in the bone and to aid in the healing process. The most common method involves bone transplantation, in which bone tissue is taken from one site to fill the defect in another site. The procedure thus involves two surgeries, which leads to an increased risk of complications. New, synthetic graft materials that can be used to fill defects and minimise the complications associated with bone tissue harvesting are therefore necessary. The synthetic materials available today lack the inherent biological factors of bone that stimulate the bone regeneration process. Much of today’s research concerning synthetic bone graft materials aims to solve this issue and researchers have suggested several different strategies. The purpose of this thesis is to improve the performance of acidic calcium phosphate cements, which are materials used as synthetic bone grafts. By combining these cements with drugs or ion additives, local delivery could be achieved with the potential to stimulate bone formation. Two different combinations were attempted in this thesis: cement in combination with simvastatin, or cement in combination with strontium halide salts. Both simvastatin and strontium are known to positively affect bone formation. The efficacy of the cements with the additives was evaluated using different bone cell cultures. The results regarding simvastatin showed that the cement’s mechanical property was not affected upon drug loading, and that the drug was released by a diffusion-controlled mechanism. Moreover, results showed that simvastatin stimulated the bone-forming cells (osteoblasts) to produce more bone tissue, while it inhibited bone-degrading cells (osteoclasts) from degrading the cement. These findings suggest that simvastatin could aid in the bone regeneration process in the local area surrounding the cement. The main purpose of the study using strontium halide salts was to increase the cement’s X-ray contrast, which is a property used to monitor cement during injection. In addition, strontium is believed to positively affect bone cells. The X-ray contrast did increase after the addition of 10 wt% strontium bromide or strontium iodide, while the cell study results did not indicate any significant effects on the bone-forming cells. In the last section of this thesis, zebrafish were used as a model to evaluate bone formation upon treatment with degradation products from synthetic bone grafts. The zebrafish is a small organism with 70 % gene homology to humans; due to its transparency, fast development and ease of handling, it is an interesting model for high-throughput studies. Silicate, which is an ionic degradation product of many different bone substitute materials, was used as a proof-of-concept to visualise bone formation in these fish. The results showed an increased bone formation upon treatment with 0.625 μM silicate ions. The results suggest that this model could be used as a complement to bone cell culture studies in pre-clinical evaluations of the degradation products of bone substitute materials, thus helping researchers to design materials with degradation products that could stimulate bone formation.

CHAVES, Anderson Valério. Hidroxiapatita e magnetita associadas à scaffolds de quitosana para aplicação em regeneração óssea. 2015. 95 f. Dissertação (Mestrado em química)- Universidade Federal do Ceará, Fortaleza-CE, 2015. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-10-04T15:24:02Z No. of bitstreams: 1 2015_dis_avchaves.pdf: 3670185 bytes, checksum: a8c09405a794892f2eb3e3b598501636 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2016-10-10T19:43:24Z (GMT) No. of bitstreams: 1 2015_dis_avchaves.pdf: 3670185 bytes, checksum: a8c09405a794892f2eb3e3b598501636 (MD5) / Made available in DSpace on 2016-10-10T19:43:24Z (GMT). No. of bitstreams: 1 2015_dis_avchaves.pdf: 3670185 bytes, checksum: a8c09405a794892f2eb3e3b598501636 (MD5) Previous issue date: 2015 / Hydroxyapatite (HA) has been widely used in the biomedical area, especially in orthopedic applications in the treatment of osteoporosis aiming its use as injectable HA nanoparticles can be targeted to various body locations to treat bone defects. The HA is very similar to mineral component of bone material. It is well established that osteoblasts (bone forming cells) grow better on HA-coated metal than for metals isolated. Simvastatin (SIMV) is a drug currently used to reduce cholesterol (oral), but recent studies have shown an interesting ability of this drug to increase the local regeneration process, when combined with hydroxyapatite. In this work, magnetite nanoparticles (Fe3O4) with an iron oxide magnetic properties recently studied as potentiating agent proliferation of bone cells, have been associated with HA with the aim of increasing regenerative processes. This system has undergone the hydrothermal treatment for controlling the crystal properties of the coating. Separately, Fe3O4 and HA are biocompatible and when combined, the nanoparticles improve several important markers of osteoblast differentiation, such as collagen synthesis and deposition of calcium by keeping their magnetic properties. It is recognized that the surface chemistry, surface energy and surface topography have a significant influence on osteoblast responses. In particular, nowadays, the critical size bone defect depends on the engineering "scaffold" which are structural supports 3D, allowing for cell infiltration and subsequent integration to the native tissue. For this reason, the material synthesized and functionalized with SIMV was added in chitosan polymeric matrices. The formed composite showed average particle size of 20 nm (HA-5h), 18 nm (Fe3O4) and 30-70nm (Fe3O4-HA). The XRD patterns showed characteristic peaks of the crystalline phases of the composites according to the literature, as well as the IR bands. / A hidroxiapatita (HA) tem sido amplamente utilizada na área de biomédica, especialmente para aplicações ortopédicas no tratamento da osteoporose, objetivando seu uso como nanopartículas injetáveis que podem ser dirigidas para vários locais do corpo para tratamento de defeitos ósseos. A HA é um material muito semelhante ao mineral componente do osso. Está bem estabelecido que os osteoblastos (células formadoras do osso) crescem melhor em metais revestidos por HA do que em metais isolados. A sinvastatina (SINV) é um medicamento utilizado atualmente para redução de colesterol (via oral), porém, estudos recentes mostraram uma interessante capacidade desse fármaco no aumento do processo de regeneração local quando associado com hidroxiapatita. Neste trabalho, nanopartículas de magnetita (Fe3O4), um óxido de ferro com propriedades magnéticas, recentemente estudado como agente potencializador de proliferação de células ósseas, foram associadas com HA com o objetivo de aumentar processos regenerativos. Este sistema sofreu tratamento hidrotérmico para controlar as propriedades cristalinas do revestimento. Separadamente, Fe3O4 e HA são biocompatíveis e, quando combinados, as nanopartículas melhoram vários indicadores de diferenciação de osteoblastos importantes, tais como a síntese de colágeno e a deposição de cálcio, mantendo as suas propriedades magnéticas. Reconhece-se que a química de superfície, a energia de superfície e a topografia da superfície têm uma influência significativa sobre as respostas de osteoblastos. Em particular, nos dias de hoje, os defeitos ósseos de tamanho crítico dependem da engenharia de “scaffolds” que são suportes estruturais em 3D, permitindo a infiltração celular e posterior integração com o tecido nativo. Por esse motivo, o material sintetizado e funcionalizado com SINV foi introduzido em matrizes poliméricas de quitosana. Os compósitos formados apresentaram tamanho médio de partícula de 20 nm (HA-5h) e 18 nm (Fe3O4) e 40-100 nm (Fe3O4-HA). Os difratogramas mostraram os picos característicos das fases cristalinas dos compósitos de acordo com a literatura, bem como as bandas de infravermelho.

Orientador: Leonardo Marchini / Banca: Luciane Dias de Oliveira / Banca: Márcia Sampaio Campos / Resumo: Devido ao aumento na taxa de óbitos relacionados à aterosclerose e AVC a sinvastatina tornou-se o medicamento eleito pelo SUS, por sua eficácia no combate às altas taxas de colesterol plasmático e diabete melitos. Enfermidades estas, decorrentes tanto de histórico familiar de hipercolesterolemia, quanto devido a hábitos de vida sedentária e de alimentação desregrada, que vem acometendo adultos jovens e mulheres em idade fértil; que já fazem uso de um ou mais medicamentos. Associado às vezes a uma gravidez não planejada, afetando também o feto. Inúmeros trabalhos têm sido feitos com o intuito de conhecer melhor sobre a atuação desse fármaco utilizado durante o período gestacional, tanto que, diversos medicamentos em princípio são contra indicados, principalmente nos primeiros três meses. Entretanto a sinvastatina, embora controversa a sua contra indicação, é o fármaco de escolha nos postos de saúde e essas jóvens mulheres grávidas estão fazendo uso dos mesmos. O objetivo desse trabalho foi o de fornecer maior embasamento a essa contra indicação. Foram utilizadas 24 ratas em período gestacional, onde desde o primeiro dia da prenhez confirmada as mesmas receberam por gavagem o fármaco manipulado pela farmácia de manipulação "A Especialista". Os animais foram divididos em 4 grupos: as ratas controle, que receberam óleo de amêndoas doce e as ratas que receberam 10, 20 e 40 mg/kg de sinvastatina diariamente. Os resultados foram plotados em planilhas do Excel e submetidos aos testes estatísticos não paramétricos e serviram para corroborar a contra indicação do fármaco pois houve aumento significativo do número de reabsorções fetais nos cornos uterinos comprometendo a prenhez, reduzindo o número de fetos gerados nas ratas que receberam 40 mg/kg, além de diminuir o peso e comprimento dos fetos em geral / Abstract: Simvastatin is currently the medicine chosen actually due to hight atherosclerosis death index and its effectiveness in combating high rates of plasma cholesterol and diabetes mellitus. Diseases caused both by family histories of hypercholesterolemia as well, due to sedentary lifestyles and eating disorders, which are affecting young adults and women of childbearing age who, due to this family history, had used one or more drugs which interact intensifying their effect, .associated some times to, an unplanned pregnancy, and drug interaction, affecting both. Several studies have been done in order to learn and get better foundation about the actions of these drugs during pregnancy, so that several medications, in principle, are contraindicated. However simvastatin, controversial contraindication, is the choosen drug in health care, and those pregnant women with a family history of hypercholesterolemia are making use of them. Therefore, the purpose of this study is to confirm this contraindication. The sample consisted of 24 female rats with confirmed pregnancy from day one. The entire sample received by gavage the drug - manipulated by the Specialist pharmacy. The animals were divided into four groups: 1. control (SC), where the rats received sweet almond oil and three other groups (S10, S20 and S40) where the rats received simvastatin 10, 20 and 40 mg / kg daily, respectively. The results were plotted in an excel spreadsheet and submitted to a nonparametric statistical test. They served to confirm our initial goal confirming the simvastatin contraindication drug due to uterin reabsortion increased number reducing little rats number, wheight and size. Thus compromising pregnancy / Mestre

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Nos Ãltimos vinte anos, o campo de biomateriais à base de fosfato de cÃlcio tem crescido rapidamente, desempenhando um papel-chave como substituinte Ãsseo. A hidroxiapatita (HA) apresenta semelhanÃa quÃmica e estrutural com a fase mineral dos ossos e dos dentes, à biocompatÃvel e osteocondutiva, tem excelente afinidade quÃmica e biolÃgica com os tecidos Ãsseos e tem aplicaÃÃes biomÃdicas importantes como preenchimento de defeitos Ãsseos e em âscaffoldsâ na engenharia de tecidos. Nanocristais de HA foram sintetizados com sucesso por co-precipitaÃÃo e via hidrotÃrmica. Para a caracterizaÃÃo estrutural, tamanho e morfologia das fases obtidas foram utilizadas tÃcnicas de DifraÃÃo de Raios-x (DRX), Espectroscopia na RegiÃo do Infravermelho com Transformada de Fourier (FTIR), AdsorÃÃo/dessorÃÃo de N2, Potencial Zeta (PZ) e Microscopia EletrÃnica de TransmissÃo (TEM). Estruturas mesoporosas foram obtidas com diÃmetros de poros variando entre 12 e 31 nm. Os nanocristais de HA apresentaram uma morfologia definida, em forma de bastÃo, e foram incorporadas a um polÃmero para formaÃÃo de um âscaffoldâ. A droga sinvastatina (SINV), que influencia a regeneraÃÃo Ãssea quando aplicada localmente, foi associada aos biocompÃsitos obtidos. A caracterizaÃÃo dos âscaffoldsâ foi realizada por Microscopia EletrÃnica de Varredura (MEV). / In the last twenty years, the field of biomaterials based on calcium phosphates has grown rapidly, playing a key role as a bone substituent. Hydroxyapatite has chemical and structural similarity to the mineral phase of bone and teeth, is biocompatible and osteoconductive. It has excellent chemical and biological affinity with the bone tissue and important biomedical applications such as filling bone defects and scaffolds in tissues engineering. Hydroxyapatite nanocrystals were successfully synthesized by co-precipitation and hydrothermal route. X-ray diffraction, Fourier-transform infrared spectroscopy, N2 adsorption/desorption, Zeta Potential and Transmission Electron Microscopy were used to structural characterization, size and morphology of the nanoparticles. Mesoporous structures were obtained with pore diameters ranging from 12 to 31 nm. The nanoparticles showed a rod-shaped mophorlogy and were incorporated into a polymer to form a scaffold. The drug simvastatin, which influences bone regeneration when locally applied, was associated with the biocomposite. The characterization of the scaffolds was performed by Scanning Electron Microscopy.

Devido à sua peculiar estrutura mineralizada, o tecido ósseo ainda representa um dos desafios para os estudos em bioengenharia regenerativa. Nesse sentido, estratégias realizadas envolvendo o sistema de liberação de drogas através de polímeros bioreabsorvíveis apontam resultados promissores. Esse projeto teve como objetivo empregar PLGA [poli (ácido lático-co-glicólico)] carregado com sinvastatina (SIN) em defeitos ósseos críticos criados em osso parietal de ratos. Foi realizado um defeito de 5.0 mm de diâmetro no osso parietal esquerdo, sendo que no grupo controle (C) foi deixado apenas o coágulo sanguíneo. O grupo experimental foi subdividido como segue: no subgrupo (M), uma membrana de PLGA foi colocada recobrindo o defeito ósseo, de modo que as bordas ultrapassaram a borda do defeito. No outro subgrupo (MSI), microesferas de PLGA 50/50 contendo sinvastatina a 2,5% foram depositadas no interior do defeito, sendo posteriormente recobertas pela membrana de PLGA; no grupo (MSS) foram colocadas microesferas de PLGA sem sinvastatina para avaliar o efeito osteocondutor das microesferas. Os animais foram sacrificados em dois períodos 30 e 60 dias após a cirurgia e as calotas removidas e processadas para análise morfológica em microscopia de luz, microscopia eletrônica de transmissão e varredura, imuno-histoquímica para análise da expressão de OPN (osteopontina), BSP (sialoproteína óssea) e OSAD (osteoaderina) e análise imunocitoquímica da distribuição ultra estrutural da proteína OPN. Os resultados observados mostraram que a formação do novo osso iniciou-se pelas margens do defeito e também pela área central em grupos tratados. A análise da regeneração nos defeitos tratados com SIN mostrou uma disposição ordenada das fibrilas colágenas e uma matriz com aspecto de tecido ósseo em fase mais madura. O estudo mostra que a regeneração do tecido pode ser acelerada e que a matriz neorformada é depositada de forma mais organizada pela liberação gradual da SIN. / Due to its unique mineralized structure, bone regeneration is still a challenge. Numerous strategies had been proposed during the past years, drug delivery system using polymeric scaffolds is a promising strategy. The aim of this study was to employ PLGA [poly (lactide-co-glycolic acid)] loaded with simvastatin(SIN) in bone defects created in rat calvaria. Bone defects with 5.0 mm diameter was created in the left side of the calvarias. In Control group (C) their defects were filled only with blood clot. The experimental group was subdivided. In subgroup (M), a PLGA membrane was covered the defect. In the other subgroup (MSI), PLGA microspheres loaded with 2.5% simvastatin filled the defect over the blood clot inside the defect and subsequently covered with PLGA membrane, another group was criated to evaluet the osteoconductor potencial of microesphers without the (SIN), the MSS group. The animals were sacrificed in 30, 60 after surgery, and the calvarias were removed and processed for light and transmission and scanning electron microscopy analyzes, Osteopontin (OPN), Osteoadherin (OSAD), Bone sialoprotein (BSP) imunnolabeling and immunocytochemical analyzes of the ultrastructural distribution of osteopontin in the neoformed bone. The in vivo experiment revealed that the microspheres containing simvastatin significantly enhanced the disposition of collagen fibrils in immature bone and bone formation in the rabbit calvaria critical size defect.

Orientador: Edson Rosa Pimentel / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T20:28:03Z (GMT). No. of bitstreams: 1 Oliveira_LeticiaPradode_M.pdf: 4812379 bytes, checksum: ed278304758adcd22ac4272f33a145ac (MD5) Previous issue date: 2013 / Resumo: As estatinas, inibidores da 3-hidroxi-3-metilglutaril-coenzima A redutase, são fármacos utilizados para o tratamento de hipercolesterolemia - níveis elevados de colesterol no sangue. Embora sejam considerados medicamentos seguros e eficazes, possuem inúmeros efeitos adversos como, por exemplo, miopatias e hepatopatias. Além disso, há relatos de lesões em tendões associadas ao uso destes medicamentos e os mecanismos responsáveis por este efeito adverso não estão bem esclarecidos. Portanto, este estudo teve como objetivo avaliar as possíveis alterações morfológicas, bioquímicas e funcionais no tendão calcanear de ratos após tratamento crônico com estatinas. Os ratos foram divididos nos seguintes grupos: tratados com a atorvastatina (A-20 e A-80), sinvastatina (S-20 e S-80), carboximetilcelulose utilizado como o veículo (V) e um grupo que não recebeu tratamento (ST). As doses de atorvastatina e sinvastatina foram calculadas utilizando extrapolação alométrica com base nas doses de 20mg/dia e 80 mg/dia, recomendadas para os seres humanos. Os ratos foram sacrificados após dois meses de tratamento e os tendões foram coletados para análises morfológicas, bioquímicas e funcionais. Para as análises morfológicas em microscopia de luz os cortes de tendões foram corados com Hematoxilina-eosina e Azul de Toluidina. Foram realizadas medidas de birrefringência através da microscopia de polarização. Para análises bioquímicas, os tendões foram processados e analisados de acordo com as seguintes técnicas: eletroforese em gel de agarose para análise de glicosaminoglicanos sulfatados; zimografia para detecção de metaloproteinases (MMPs) dos tipos -2 e -9; Western Blotting para colágeno I, dosagem de proteínas não-colagênicas, glicosaminoglicanos e hidroxiprolina. Os tendões também foram submetidos a testes biomecânicos. Todos os resultados foram analisados utilizando ANOVA-one-way, seguida pelo teste de Tukey, com exceção dos dados de birrefringência, que foram analizados pelo teste de Mann-Whitney. A concentração de proteínas não-colagênicas em todos os grupos tratados com estatinas foi menor em relação ao grupo V. Houve um aumento significativo de pro-MMP-2 no grupo A-80 e MMP-2 ativa em S-20 comparado ao grupo ST. Foi observado também um aumento significativo de pro-MMP-9 nos grupos A-80 e S-20 e um aumento de MMP-9 ativa no grupo A-20 em relação ao grupo ST. O grupo A-20 apresentou menor quantidade de colágeno I quando comparado ao grupo ST e no grupo S-20 foi observada uma maior concentração de hidroxiprolina em relação aos grupos ST e V. A dosagem de glicosaminoglicanos mostrou um aumento significativo no grupo A-20 e o grupo S-80 apresentou menor concentração deste componente em relação ao grupo ST e V respectivamente. As medidas de birrefringência dos grupos A-20, A-80 e S-80 revelaram um menor grau de organização das fibras de colágeno nestes grupos em relação ao grupo V. Durante os ensaios biomecânicos os tendões dos grupos A-20, A-80 e S-20 foram menos resistentes à ruptura quando comparados com o grupo ST. Nossos resultados sugerem claramente que o tratamento com estatinas provoca notáveis alterações no tendão calcanear de ratos. Além de promover o desequilíbrio entre a síntese e degradação de componentes da matriz extracelular e a desorganização das fibras de colágeno, as estatinas também trouxeram prejuízos às propriedades biomecânicas dos tendões, fatores que associados, podem tornar os tendões mais predispostos a rupturas e possivelmente induzir micro lesões / Abstract: Statins are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. These drugs are used to treat hypercholesterolemia - high cholesterol levels in the blood. Although considered safe and effective drugs, they have numerous adverse effects such as myopathy and liver toxicity. In addition, there are reports of tendon lesions associated with statins and the mechanisms responsible for this adverse effect are not clear. Therefore, this study aimed to evaluate possible morphological, biochemical and functional alterations in Achilles tendon of rats after chronic treatment with statins. The rats were divided into the following groups: treated with simvastatin (S-20 and S-80), atorvastatin (A-20 and A-80), carboxymethylcellulose used as the vehicle (V) and a group that received no treatment (NT). Doses of statins were calculated using allometric scaling with reference to 80mg/day and 20mg/day doses recommended for humans. The rats were euthanized after two months of treatment and the tendons were collected for morphological, biochemical and functional analysis. For morphological analysis by light microscopy, sections of tendons were stained with hematoxylin-eosin and toluidine blue. Birefringence measurements were performed by polarizing microscopy. For biochemical analysis, the tendons were processed and analyzed according to the following techniques: Agarose gel electrophoresis for the analysis of sulphated glycosaminoglycans; zymography for detecting metalloproteinases (MMPs) -2 and -9; Western Blotting for collagen I, dosage of non-collagenous proteins, glycosaminoglycans and hydroxyproline. The tendons were also submitted to biomechanical tests. All of the results were analyzed by ANOVA-one-way followed by Tukey test, except the birefringence data that were analyzed by Mann-Whitney test. The concentration of non-collagenous proteins in all groups treated with statins was lower than in V group. There was a significant increase in pro-MMP-2 in A-80 group and active MMP-2 in S-20 compared to the ST group. We observed a significant increase in pro-MMP-9 in A-80 and S-20 groups. The A-20 group showed a significant increase in the levels of active MMP-9 in relation to the ST group. The A-20 group showed less collagen I when compared to the ST group and in the S-20 group was observed a higher concentration of hydroxyproline in relation to the both ST and V groups. The dosage of glycosaminoglycans showed a significant increase in A-20 group and S-80 group showed lower concentration of this component in relation to the ST and V groups respectively. The birefringence measurements of the A-20, A-80 and S-80 groups showed a lower degree of organization of the collagen fibers in these groups than in the V group. During the biomechanical tests the tendons of the A-20, A-80 and S-20 groups were less resistant to rupture when compared to the ST group. Our results clearly suggest that treatment with statins causes remarkable changes in the Achilles tendon. Besides promoting the imbalance between the synthesis and degradation of extracellular matrix components and disorganization of collagen fibers, statins also brought losses to the biomechanical properties of tendons. These factors associated can make the tendons more prone to ruptures and possibly induce micro injuries / Mestrado / Histologia / Mestra em Biologia Celular e Estrutural

Orientador: Gilbert Bannach / Banca: Éder Tadeu Gomes Cavalheiro / Banca: Roni Antônio Mendes / Banca: Flavio Junior Caires / Banca: Aroldo Geraldo Magdalena / Resumo: Neste trabalho são estudadas novas formas sólidas dos fármacos genfibrozila(GEM) e sinvastatina(SIM) e suas misturas binárias com os coformadores isonicotinamida (INCT), picolinamida (PA), nicotinamida (NA),benzamida (BZ), niacina (NCN) e pirazinamida (PZN) com uso do método mecanoquímico. Os fármacos são agentes antilipêmicos pertencentes à classe dos fibratos (GEM) e estatinas (SIM). A ênfase do trabalho é na formação de cocristais, no entanto, a existência de polimorfismo também foi estudada. A caracterização dos polimorfos e misturas binárias foram realizadas com uso de técnicas termoanalíticas: termogravimetria - análise térmica diferencial (TG-DTA), calorimetria exploratória diferencial (DSC), termomicroscopia de luz polarizada (PLTM) e também por técnicas espectroscópicas: espectroscopia vibracional na região do infravermelho médio (FTIR) e difratometria de raios X pelo método do pó (PXRD) e de monocristal (SCXRD). Com isso, primeiramente, foram investigadas evidências da existência de polimorfismo dos fármacos quando submetidos a um tratamento térmico e também de moagem. A GEM exibiu polimorfismo mediante cristalização a partir do fundido, com relação monotrópica com a forma de partida, a qual acredita-se possuir estrutura semelhante à forma partida. Já a SIM não apresentou indícios de polimorfismo. Com relação ao método mecanoquímico de moagem, os fármacos e coformadores não apresentaram polimorfismo nas condições selecionadas. Em seguida, foram estudados os doze sis... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In this work, new solid forms of the drugs gemfibrozil (GEM) and simvastatin (SIM) and their binary mixtures with coformers isonicotinamide (INCT), picolinamide (PA), nicotinamide (NA), benzamide (BZ), niacin (NCN) and pyrazinamide (PZN), through mechanochemistry method are studied. The drugs are antilipemic agents belonging to the fibrates (GEM) and statins (SIM) classes. The focus of this work is on the formation of cocrystals, however, the existence of polymorphism was also studied. The characterization of the polymorphs and binary mixtures were performed using thermoanalytical techniques: thermogravimetry - differential thermal analysis (TG-DTA), differential scanning calorimetry (DSC), polarized light thermomicroscopy (PLTM) and also by spectroscopic techniques: medium infrared (FTIR), powder X-ray diffraction (PXRD) and single crystal X-ray diffraction (SCXRD). Thereby, first of all, it was investigated evidence of polymorphism of the drugs studied when they were submitted to thermal treatment and also mechanochemistry grinding. GEM presented polymorphism by crystallization from the melt, exhibiting a monotropic relationship with the starting form, which is believed to have a similar structure with the starting form. The SIM did not exhibit evidence of polymorphism. About the mechanochemistry grinding method, the drugs and coformers did not show polymorphism in chosen conditions. Afterwards, the twelve system proposed were studied. It was confirmed the formation of cocr... (Complete abstract click electronic access below) / Doutor

The mevalonate pathway plays an important role in cancer biology and has been targeted with farnesyl transferase inhibitors, although their efficacy is limited due to significant adverse effects. Statins and bisphosphonates inhibit the mevalonate pathway at different steps, thus having negative effects at various levels on cancer cells. A combination of these drugs may result in an amplified cytotoxic effect and allow for use of significantly lower doses of the drugs involved. Statins inhibit the mevalonate pathway at 3-hydroxy-3-methylglutaryl coenzyme A reductase and bisphosphonates at farnesyl pyrophosphate synthase. Our results show that low-dose combinations of simvastatin and alendronate have a synergistic cytotoxic effect on androgen-independent prostate cancer PC-3 cells, but not on androgen-dependent LNCaP or DU 145 prostate cancer cells. These two drugs cause a sequential blockade of the mevalonate pathway and significantly affect survival and apoptotic pathways by down-regulating phospho-AKT and activating c-JUN and ERK.