Orientador: Carlos Rossa Junior / Banca: Raquel Mantuaneli Scarel Caminaga / Banca: Eliete Neves da Silva Guerra / Resumo: Curcumin é um polifenol amarelo extraído do rizoma de uma planta tropical, do tipo herbácea. Possui ações anti-inflamatória, antioxidante, antiangiogênica, imunomodulatória, citotóxica, antimicrobiana e antiapoptótica. A aplicação terapêutica do curcumin vem sendo avaliada em modelos pré-clínicos e estudos de várias doenças. As limitações da eficácia do curcumin in vivo são atribuídas à sua má solubilidade em veículos aquosos e baixa taxa de absorção no trato gastrointestinal. O objetivo desse trabalho foi avaliar o efeito dose-resposta do composto sintético análogo ao curcumin (CMC2.24) em diferentes doses (1 mg, 3 mg, 10 mg, 30 mg) sobre o processo inflamatório e osteoclastogênese em modelo de doença periodontal in vivo e in vitro, para analisar qual é a dose mínima necessária para que o composto tenha o efeito biológico. A doença periodontal foi induzida em ratos por meio de injeção de 30 µg de LPS de Escherichia coli, realizadas 3x/semana durante 4 semanas. Os controles receberam injeções do mesmo volume do veículo de diluição do LPS (PBS). A administração de CMC2.24 (1, 3, 10 e 30mg/kg) foi feita por via intragástrica (gavagem oral) diariamente, durante 29 dias (um dia antes da primeira injeção de LPS/PBS). A expressão de fosfatase ácido tartarato resistente (TRAP), indicativa de diferenciação osteoclástica, foi avaliada por meio de imunohistoquímica, a proporção de células inflamatórias, em cortes corados com H/E, por estereometria, a presença de ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Curcumin is a yellowish polyphenol extracted from the rizome of a herbaceous tropical plant. It has multiple biological activities described, including anti-inflammatory, antioxidant, antiangiogenic, immunomodulatory, cytotoxic, antimicrobial and pro- and antiapoptotic activities. Its therapeutic application is actively evaluated in preclinical models and clinical studies of various diseases. In vivo use of curcumin is limited by its pharmacodynamic propereties, such as poor solubility in aqueous vehicles, low absorption rate in the gastrointestinal tract and short half-life in the peripheral circulation. The aim of this study was to evaluate the dose-response effect of a synthetic compound that is structurally to natural curcumin (CMC2.24) in a model of experimental periodontal disease that mimics the host-microbial interaction and the resultant inflammation and bone resorption that are hallmarks of this conditions in humans. Periodontal disease was induced in mice by injection of 30 ug LPS of Escherichia coli, carried out 3x / week for 4 weeks. Controls received injections of the same volume of vehicle (PBS). Administration of CMC2.24 (1, 3, 10 and 30mg / kg) was performed daily (starting the day before the first LPS/PBS injection) by oral gavage (1, 3, 10 and 30 mg/Kg) during the whole experimental period. Immunohistochemical detection of tartrate-resistant acid phosphatase (TRAP) was used to identify osteoclasts in the area of interest; the inflammatory... (Complete abstract click electronic access below) / Mestre

Dysregulated microRNAs in osteoclasts could cause many skeletal diseases. The therapeutic manipulation of these pathogenic microRNAs necessitates novel, efficient delivery systems to facilitate microRNAs modulators targeting osteoclasts with minimal off-target effects. Bone resorption surfaces characterized by highly crystallized hydroxyapatite are dominantly occupied by osteoclasts. Considering that the eight repeating sequences of aspartate (D-Asp8) could preferably bind to highly crystallized hydroxyapatite, we developed a targeting system by conjugating D-Asp8 peptide with liposome for delivering microRNA modulators specifically to bone resorption surfaces and subsequently encapsulated antagomir-148a (a microRNA modulator suppressing the osteoclastogenic miR-148a), i.e. (D-Asp8)-liposome-antagomir-148a. Our results demonstrated that D-Asp8 could facilitate the enrichment of antagomir-148a and the subsequent down-regulation of miR-148a in osteoclasts in vivo, resulting in reduced bone resorption and attenuated deterioration of trabecular architecture in osteoporotic mice. Mechanistically, the osteoclast-targeting delivery depended on the interaction between bone resorption surfaces and D-Asp8. No detectable liver and kidney toxicity was found in mice after single/multiple dose(s) treatment of (D-Asp8)-liposome-antagomir-148a. These results indicated that (D-Asp8)-liposome as a promising osteoclast-targeting delivery system could facilitate clinical translation of microRNA modulators in treating those osteoclast-dysfunction-induced skeletal diseases.

Indiana University-Purdue University Indianapolis (IUPUI) / Fluoride has been used in the treatment of osteoporosis because of its apparent ability to directly initiate bone formation. However, fluoride's therapeutic efficacy is controversial. Clinical trials in the range of 50 to 75 mg/day demonstrated severe side effects and a lack of consistent therapeutic benefits. Animal studies have not fully proven a positive effect of fluoride on bone strength. The objective of this study was to determine the histomorphometric changes in the cortical bone of rabbits caused by high doses of fluoride. The hypothesis was that high-dose fluoride intake enhances bone modeling and inhibits bone remodeling. Twenty-four young adult (four months old) female, Dutch Belted rabbits were randomly divided in two groups. The control group received no fluoride in their drinking water, while the experimental group received 100-ppm fluoride. Both groups received approximately 12-ppm fluoride in their food. A pair of tetracycline labels was given two weeks apart before initiation of the experiment. Fluoride treatment was given for six months. A terminal pair of calcein green labels was given before the animals were euthanized. Histomorphometric measurements were made using stereological point-hit and linear-intercept methods. The histomorphometric findings were correlated with fluoride serum and bone levels and also with strength tests. The study demonstrated that fluoride increases bone modeling by increasing periosteal bone apposition and endosteal bone resorption. The net effect of fluoride was an enlargement of the cortical bone and bone marrow and, therefore, the total tissue cross-section. However, the observed increase in bone mass produced by fluoride did not have a positive effect on the mechanical properties of bone. Fluoride did not produce a change in the primary histomorphometric parameters of osteoid surface (OS/BS%) or mineralizing surface (MS/BS%). Fluoride treatment produced an increase in the cortical periosteal modified mineral apposition rate (CPMAR). The remaining dynamic indices (i.e. endosteal MAR, remodeling MAR, cortical endosteal BFR and total BFR, activation frequency and formation period) were not affected by fluoride. The study failed to show an inhibitory effect of fluoride on bone remodeling.

Includes bibliographical references (leaves 355-403) / 403 leaves : plates (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dental School, 2002

Thesis (M.Sc.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science, Pharmacy and Pharmaceutical Sciences. Title from pdf file main screen (viewed on July 31, 2009). Includes bibliographical references.

published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Objetivos: este estudo se propôs avaliar a correlação entre atividade do músculo masseter e alteração óssea mandibular, posterior ao último implante, um ano após a reabilitação com próteses implantossuportadas, em pacientes com fissura labiopalatina já reparada. Material e Método: As alterações dimensionais ósseas foram medidas em radiografias panorâmicas digitalizadas realizadas antes e um ano após a reabilitação de dezessete pacientes. A eletromiografia dos músculos masseteres foi realizada durante a contração voluntária isométrica máxima (CVIM) por 5s, mastigação habitual de cenoura (MH), mastigação unilateral direita (MUD) e esquerda (MUE) de látex. Resultados: ocorreu aumento ósseo significativo na mandíbula de 1,98mm (p<0,001) para o tipo de prótese inferior overdenture implantossuportada. O lado direito é maior que o esquerdo em 0,9mm. Um significante aumento da amplitude para a MH (9V) e para a MUE (19V) foi observado, independente do tipo de prótese. Um aumento significante para a MUE (19V) foi observado para a prótese overdenture. Ocorreu um aumento estatisticamente significante para o ato mastigatório durante os três tipos de mastigação (MH=0,13s, MUD=0,06s, MUE=0,12s), independente do tipo de prótese. Para o ciclo mastigatório também foi observado aumento estatisticamente significante, independente do tipo de prótese durante a MH (0,12s) e MUE (0,11s). Conclusão: Ocorreu aposição óssea na região posterior da mandíbula para a prótese overdenture e apesar do aumento da atividade muscular, não foi encontrada correlação estatisticamente significante com a atividade muscular do masseter. / Purpose: The purpose of the present study was to evaluate the correlation between masseter muscle activity and changes occurred in mandibular bone height, posterior to the last implant, one year after implant-supported prosthesis rehabilitation in repared cleft lip and palate patients. Materials and Methods: changes in mandibular bone height were measured in digitalized images of panoramic radiographies taken before and one year after oral rehabilitation of seventeen repared cleft lip and palate patients (Radiocef 2 Radiomemory). Parameters for electromyography of masseter muscle were: maximum voluntary isometric clench (MVC) for 5s, habitual chewing of carrots (HC) and unilateral right (URL) and left (ULL) chewing of latex. Results: a mean increase in mandibular bone height of 1,98mm (p<0,001) for the implant retained overdenture was observed. Bone apposition was higher in the right side. Significant increase in the amplitude was observed during HC (9V) and during ULL (19 V), regardless prosthesis type and side. A significant increase was observed during URL (19 V) for the implant retained overdenture. A significant statistical increase for the masticatory act was observed during the three types of mastication (HC=0,13s, URL=0,06s, ULL=0,12s), regardless prosthesis types. A significant statistical increase was observed for the masticatory cycle during HC (0,12s) and ULL (0,11s), regardless prosthesis type. Conclusion: Mandibular bone apposition in the posterior region to the last implant, in patients using implant retained overdentures and improvement in muscular activity in both types of prosthesis were observed, although no significant correlations were encountered between them.

O tecido ósseo possui alto grau de rigidez e resistência à pressão, característica relacionada às suas funções de proteção, sustentação e locomoção. Patologias como periodontite, artrite reumatóide e osteoporose decorrem do desequilíbrio da dinâmica óssea, que ocorre quando os osteoclastos estão em maior atividade em relação aos osteoblastos. O fluoreto (F-) é incorporado nos tecidos mineralizados após ingestão e a sua administração aumenta a formação óssea in vivo, de maneira dependente da dose e da linhagem dos animais. As linhagens A/J e 129P3/J possibilitam a análise dos fenômenos moleculares envolvidos na suscetibilidade e resistência de células ósseas aos efeitos do F- no osso. Neste estudo, avaliou-se a administração in vivo de F- na osteoclastogênese, através da análise da atividade da enzima fosfatase ácida resistente ao tartarato (TRAP), contagem de células TRAP- positivas e quantificação da atividade das metaloproteinases MMP-2 e -9. Para tal, células hematopoiéticas provenientes da medula óssea de camundongos das linhagens 129P3/J e A/J foram cultivadas com M-CSF e RANKL em associação ou não com F-, nas concentrações de 10- 9, 10-7, 10-5, 5x10-5, 5x10-5, 10-4 e 10-3 M. Os dados mostraram que a atividade da TRAP foi semelhante entre as células das duas linhagens de animais, independente do F-. Concomitante à diferenciação osteoclástica, o tratamento das células com F-, independente da concentração, aumentou significativamente a atividade da TRAP em células da linhagem A/J. O aumento da atividade de TRAP foi associada com o aumento do número de osteoclastos formados, na concentração de 10-3 M F-. Já em células dos animais 129P3/J, o F- não alterou a atividade da TRAP bem como a osteoclastogênese. A atividade de MMP-9 foi aumentada em relação à da MMP-2, porém ambas não foram moduladas pelo F-, independente da linhagem. Assim, concluiu-se que as células de ambas as linhagens não diferem com relação à diferenciação de osteoclastos, mas respondem diferentemente ao F-, sendo as células dos animais A/J e 129P3/J sensíveis e resistentes à osteoclastogênese induzida pelo F-, respectivamente. / The bone tissue has a high degree of rigidity and pressure resistance, characteristic related to its protection, support and locomotion functions. Diseases such as periodontitis, osteoporosis and rheumatoid arthritis arise from dynamic bone imbalance that occurs when osteoclasts present increased activity compared to osteoblasts. Fluoride (F-) is incorporated in mineralized tissues after ingestion and its administration increases in vivo bone formation in dose and strain-dependent manner. The strains A/J and 129P3/J make possible the analysis of molecular phenomena involved in susceptibility and resistance of bone cells to the effect of F- in bone. In this study, we evaluated the effect of F- on the in vitro osteoclastogenesis, by analyzing the fosfatase tartrate-resistant acid (TRAP) activity, TRAP positive cell counts and the activity of MMP-2 and -9 measurements in A/J and 129P3/J differentiated hematopoietic stem cells. Hematopoietic cells were differentiated with M-CSF and RANKL and treated or not with F- in the concentrations of 10-9, 10-7, 10-5, 5x10-5, 5x10-5, 10-4 and 10-3 M.The data show that TRAP activity, an osteoclast marker, was similar between cells from both strains of mice, regardless of F-. In addition, the treatment of A/J cells with F- significantly increased TRAP activity, in a dose-independent manner. This was accompained by the increased TRAP+-osteoclast counts, at 10-3 M F- dose. However, while the MMP-9 activity was aumented when compared to MMP-2, it was not altered by F-, regardless of the strain. Thus, we concluded that cells from both strains do not differ regarding the osteoclast differentiation potential, but they respond differently to F-, whereas A/J and 129P3/J cells are sensitive and resistant to F--induced osteoclastogenesis, respectively.

The skeleton is a dynamic organ that undergoes a continual process known as bone remodeling. Bone remodeling is necessary to maintain structural integrity, heal micro-fractures caused by from daily wear and tear, and to store and release essential ions and minerals. Remodeling is a highly regulated process, with bone resorption precisely balanced by bone formation under homeostatic conditions. In the setting of rheumatoid arthritis (RA), an inflammatory condition affecting joints, this balance is lost and bone around inflamed joints is eroded. These so-called "bone erosions" compromise joint function, causing disability. Osteoclasts, multinucleated cells of hematopoietic origin, are the only cells known to resorb bone. Osteoclasts are found at erosion sites in human joints, and data from mouse models of inflammatory arthritis suggest that osteoclasts are required for erosions to form in bone. The canonical pathway of osteoclast differentiation requires stimulation of myeloid precursors by the cytokine Receptor Activator of NF-Kappa B ligand (RANKL) through its receptor, RANK. In the inflamed joint, RANKL expression can be induced on mesenchymal lineage cells by inflammatory cytokines such as tumor necrosis factor alpha (TNF alpha). Surprisingly, our lab observed bone erosions and osteoclast formation in a mouse model of RA in the absence of RANK. Thus we hypothesized that in addition to RANKL expression, the cytokine milieu in RA may directly stimulate osteoclast formation. It was recently reported that the inflammatory cytokines TNF alpha and interleukin-6 (IL-6) in combination stimulate osteoclast differentiation, independent of exogenous RANKL. We have reproduced these results and shown that these osteoclast-like cells form entirely independently of RANK signaling. However, TNF alpha/IL-6 induced osteoclast formation still requires the transcription factor Nuclear Factor of Activated T cells (NFATc1), a master regulator of RANK-mediated osteoclast differentiation, as well as co-stimulatory signaling provided by the immunoreceptor tyrosine based activation motif (ITAM)-containing DNAX-activating protein (DAP12) molecules. We also showed that TNF alpha/IL-6 induced osteoclast formation requires activity of IL-6 receptor (IL-6R), as osteoclast formation can be inhibited through co-culture with an IL-6R blocking antibody (MR16-1). Finally, using an in vivo mouse model of RA in RANK-deficient mice, we tested whether blocking IL-6R with MR16-1 antibody protects against the formation of periarticular bone erosions. Our results suggest that a RANK-independent pathway of osteoclast formation contributes to inflammatory bone erosions. Targeting this pathway may improve outcomes for RA patients.

Includes bibliographical references (leaves 355-403)