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O'Brien, Elizabeth Ann. "Regulation of osteoclast activity : differential adhesion of osteoclasts to the bone surface." Thesis, University of Liverpool, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343930.
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Rowlands, Marit-Naomi. "In vitro production of osteoclasts." Thesis, University of Liverpool, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.250270.
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Nesbitt, Stephen Anthony. "Collagen binding proteins in osteoclasts." Thesis, Open University, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265344.
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Herrera, Bruno Schneider. "Óxido nítrico e periodontite experimental: caracterização de mediadores intracelulares da atividade osteoclastogênica, conseqüências locais e sistêmicas." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/42/42136/tde-11092008-154717/.
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A doença periodontal é a doença crônica mais prevalente nas doenças orais. Dentre os mediadores desse processo contam-se a Resolvina E1 (RvE1), um mediador pró-resolução da inflamação capaz de diminuir a perda óssea secundária á doença periodontal em coelhos, e o oxido nítrico (NO), o qual pode ser produzido em grandes quantidades pela ação de citocinas e estimular a diferenciação e atividade osteoclástica. O objetivo deste estudo foi investigar os efeitos da RvE1 em osteoclastos (OCs) em cultura e os mecanismos envolvidos, bem como o papel do NO na progressão da periodontite experimental em ratos e as alterações sistémicas resultantes de danos oxidativos. A diferenciação de OCs foi induzida em células de medula óssea de camundongos C57BL/6 em cultura (7 dias) e tratadas com diferentes doses de RvE1. NFkB e Akt fosforilada foram analisadas por Western blotting e a expressão gênica de NO sintase (NOS) induzível (iNOS) por \"real-time\" PCR. A participação dos receptores ChemR23 e BLT-1 na resposta à RvE1 foi estudada em membranas isoladas de OCs empregando radioligantes. A perda óssea alveolar e danos em órgãos periféricos foram analisados em ratos com periodontite induzida por ligadura (P) e sob tratamento de longo prazo com o inibidor não-seletivo de NOS, L-NAME. Os animais receberam L-NAME durante as 2 semanas prévias à indução da periodontite e até o momento do sacrifício (3, 7 ou 14 após a ligadura). A perda óssea alveolar foi avaliada radiograficamente, e análises do conteúdo de proteínas contendo nitrotirosina (NT), espécies reativas do ácido tiobarbitúrico (TBARs) e atividade da mieloperoxidase (MPO) foram realisadas em amostras de coração, baço, rim, fígado e pulmão. RvE1 (3 ng/mL) através da ativação do receptor para BLT-1 (mas não ChemR23) inibiu a diferenciação e atividade de OCs (p<0,05) após 5 ou 7 dias de cultura, assim como a fosforilação dos dois sítios da Akt e a traslocação do NF-kB para o núcleo, um evento chave tanto na diferenciação de OCs (p<0,05) como na diminuição da expressão de iNOS. In vivo, ratos P (dia 7) mostraram um aumento na expressão de NT cardíaca e MPO renal em comparação ao grupo Sham (S; p<0,05). L-NAME resultou em aumento de NT hepática no grupo P no dia 3 (p<0,05), mas diminuição da NT cardíaca no dia 7 (p<0,01). Em comparação ao grupo P, ratos P+LN mostraram um aumento significativo na MPO hepática, cardíaca e renal no dia 3 (p<0,05), mas diminuição de MPO (dia 7) e TBARs esplênico (dia 3, p<0,05). Em resumo, mostramos que a RvE1 ligando-se ao receptor para BLT-1 inibe a diferenciação e atividade de OCs interferindo com a sinalização de Akt e NF-kB, e consequentemente inibindo a expressão da iNOS, e que o NO tem um papel central na periodontite, não só relacionado a consequências locais na perda óssea alveolar, como também em órgãos periféricos distantes.The periodontal disease is the most prevalent chronic disease in oral diseases. Among the mediators of this process, is the Resolvin E1 (RvE1), a novel mediator pro-resolving of inflammation that is capable to decrease alveolar bone loss secondary to periodontal disease in rabbits; and the nitric oxide (NO), that can be produced in large amount, induced by cytokines and it can stimulate the osteoclast differentiation and activity. The aim of this study is to investigate the effects of RvE1 on osteoclasts (OCs) culture and the pathway involved, also the role of NO in the progression of experimental periodontitis in rats and systemic alterations due to oxidative damage. The OCs differentiation was induced in bone marrow cell culture from C57BL/6 mice (7 days) and treated with various doses of RvE1. NFkB and Akt phosphorylation were analyzed with Western blotting and the genic expression of NO synthase (NOS) inducible (iNOS) with \"Real Time\" PCR. The role of receptors ChemR23 and BLT-1 was accessed in OCs isolated membranes performing radioligants. The alveolar bone loss and peripheral organ damage was assessed in rats with ligature-induced periodontitis (P) under a long-term treatment of a NOS inhibitor, L-NAME. The animals received L-NAME from two weeks prior to periodontitis induction and until their sacrifice (3, 7 and 14 days after ligature). The alveolar bone loss was evaluated radiographically, and the protein nitrotyrosine (NT) content, reactive species of thiobarbituric acid (TBARs) and myeloperoxidase activity (MPO) were analyzed in samples of heart, spleen, kidney, lungs and kidneys. RvE1 (3 ng/mL) trough BLT-1 receptor activation (but not ChemR23) inhibits the OCs differentiation and activity (p<0.05) after 5 or 7 days of the culture, as well as the Akt phosphorylation and NF-kB translocation to the nucleus, a key event both in OCs differentiation (p<0.05) and iNOS expression decreases. In vivo, P rats (day 7) show an increase of heart NT and renal MPO, but lower lung MPO activity in comparison to the Sham group (S; p<0.05). L-NAME leads to an increase the liver NT expression in P rats on day 3 (p<0.05), but decreases the cardiac NT on day 7 (p<0.01). In comparison with the P group, P+LN rats showed significantly increased liver, heart and kidney MPO content on day 3 (p<0.05), but lower lung MPO (day 7) and spleen TBARs (day 3) content (p<0.05). In summary we have shown that RvE1 binding on BLT-1 receptor inhibits OCs differentiation and activity by interfering with Akt and NF-kB signaling and consequently iNOS inhibition, and NO has a central role on periodontitis, not only related to the local consequences on alveolar bone resorption, but also on distant peripheral organs.
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Franco, Gilson Cesar Nobre. "Analise da farmacocinetica e dos indices PK/PD da doxiciclina no plasma, fluido gengival e saliva e avaliação de seu efeito sobre a osteoclastogenese mediada por RANKL." [s.n.], 2007. http://repositorio.unicamp.br/jspui/handle/REPOSIP/288516.
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Orientadores: Pedro Luiz Rosalen, Francisco Carlos Groppo, Toshihisa KawaiTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Doxiciclina (Dox) é um antimicrobiano pertencente à família das tetraciclinas com um amplo espectro de ação contra bactérias Gram-positivas e Gram-negativas. Além de suas propriedades antimicrobianas, Dox é atualmente empregada na periodontia como um modulador da resposta do hospedeiro (MRH), ao inibir a atividade da enzima metaloproteinase de matriz (MMP), a qual está relacionada ao processo de destruição tecidual. Neste contexto, este trabalho teve os seguintes objetivos: 1-determinar os parâmetros farmacocinéticos e integrar os índices PK/PD da Dox para o plasma, fluido gengival (FG) e saliva; 2-analisar os efeitos in vitro e in vivo da Dox sobre a osteoclastogênese com a finalidade de elucidar possíveis propriedades biológicas adicionais deste fármaco como MRH. Para a análise farmacocinética, 12 voluntários receberam dose oral única de 100 mg de Dox. Sangue, FG e saliva foram coletados em tempos pré-determinados e a concentração da Dox nestes fluidos foi determinada por bioensaio. A análise dos principais índices PK/PD da Dox foi realizada considerando o CIM para P. gingivalis. Para o segundo objetivo, o efeito da Dox sobre os processos de diferenciação e ativação osteoclástica foi verificado, respectivamente, pela contagem de células TRAP+ multinucleadas geradas a partir de células precursoras estimuladas com sRANKL na presença ou ausência de Dox e pela análise das lacunas de reabsorção formadas por estas células, quando cultivadas sobre discos de dentina. In vivo, o efeito da Dox sobre a osteoclastogênese foi determinado através da indução deste processo em calvária de camundongo. Solução de sRANKL/LPS foi injetada na região da calvária e os animais receberam, por gavagem, Dox ou placebo diariamente. Após 10 dias, a calvária foi removida para análise histoquímica. Em acréscimo, a atividade da Dox sobre a expressão de genes responsáveis pelos processos de diferenciação e ativação osteoclástica foi analisada por RT-PCR. Durante os experimentos in vitro e in vivo, a produção e atividade da MMP foram verificadas através de Western-blot e Zimograma respectivamente. Os resultados demonstraram que as maiores concentrações de Dox foram observadas no plasma, seguido pelo FG e saliva. A análise dos índices PK/PD da Dox indicou que a dose de 100 mg foi insuficiente para se obter os valores ideais antimicrobianos preconizados na /CIM. Os experimentos in vitro e in vivo sobre o efeito da Dox como MRH demonstraram que este fármaco inibiu os processos de diferenciação e ativação dos osteoclastos. Dox também modulou a expressão de proteínas diretamente relacionadas a osteoclastogênese, incluindo TRAP, Catepsina K e c-Myc. Finalmente, embora a síntese da MMP não tenha sido afetada, a atividade da MMP foi reduzida na presença de Dox. Portanto, os resultados do presente estudo sugerem que uma dose inicial maior do que 100 mg é necessária para alcançar o valor preconizado para ASC/CIM e Cmax/CIM, com a finalidade de se obter os melhores resultados clínicos antimicrobianos. A análise da Dox como MRH indicou que este fármaco pode atuar neste processo não somente pela sua capacidade de inativar a MMP, e sim, por apresentar a propriedade de inibir a diferenciação e ativação osteoclástica, incluindo a modulação de sua expressão gênica. literatura para os parâmetros ASC/CIM e Cmax
Abstract: Doxycycline (Dox), a member of the tetracycline family, is an antimicrobial agent with a broad-spectrum of activity against Gram-positive and Gram-negative bacteria. In addition to its antimicrobial properties, Dox is used in the treatment of periodontal diseases as a host response modulator by inhibiting the activity of an important enzyme, matrix metalloproteinase (MMP), which is related to the process of tissue destruction. In this context, this study had the following aims: 1-to determine the pharmacokinetic parameters of Dox and to integrate the PK/PD indices for plasma, gingival crevicular fluid (GCF) and saliva; 2-to analyze the effects in vitro and in vivo of Dox on the osteoclastogenesis and on the osteoclast activation in order to elucidate additional biological properties of Dox on the host response modulation (HRM). Twelve volunteers received single oral administration of Dox (100 mg). Blood, GCF and saliva were collected and the concentrations were measured by bioassay technique. The PK/PD analyses were carried out using the MIC for P. gingivalis. For the second objective, the effect of Dox on the osteoclast differentiation and activation processes was determined, respectively, by the counting of TRAP+ multinuclear cells derived from osteoclast precursory cells sRANKL-stimulated in the presence or absence of Dox and by the analysis of the resorption areas formed by these cells when cultured on dentin discs. In vivo, Dox¿s effect on the osteoclastogenesis was verified using the model of osteoclastogenesis induction in mouse calvaria. sRANKL/LPS was injected in the supra-calvaria area and the animals received Dox or placebo daily by gavage. After the experimental period of 10 days, the calvariae were removed for histochemistry analyses. In addition, the effect of Dox on the expression of genes related to the osteoclast differentiation and activation processes was carried out using RT-PCR technique. MMP production and activity were ensured during in vitro and in vivo experiments by Western-blot and Zymography, respectively. The results demonstrated that Dox achieved the highest concentration in the plasma, following by GCF and saliva. PK/PD analyses showed that the dose of 100 mg was insufficient to get the antimicrobial levels indicated in the literature for AUC/MIC and Cmax/MIC indices. In vitro and in vivo studies of Dox¿s effects on the HRM demonstrated that this drug could inhibit the osteoclast differentiation and activation process. Dox also showed an important property of down-regulation in the expression of proteins directly related to osteoclastogenesis, including TRAP, Cathepsin K and c-Myc. Finally, although Dox did not affect the expression of MMP protein, MMP activity was remarkably decreased by Dox. Therefore, the present study suggests that higher doses than 100 mg would be necessary to obtain effective antimicrobial levels and the effect of DOX on the HRM can be due to not only by MMP inhibition but also by the direct effect on RANKL-mediated osteoclast differentiation and activation, including its gene regulation
Doutorado
Farmacologia, Anestesiologia e Terapeutica
Doutor em Odontologia
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Cayana, Ezymar Gomes. "Efeito da administração intermitente do PTH (1-34) na periodontite experimental em ratas expostas à fumaça de cigarros." [s.n.], 2010. http://repositorio.unicamp.br/jspui/handle/REPOSIP/290845.
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Orientadores: Enilson Antônio Sallum, João Baptista César NetoTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: O objetivo deste estudo foi investigar histológica e histoquimicamente a influência da inalação da fumaça de cigarros (IFC) e da administração intermitente de PTH 1-34 sobre a perda óssea alveolar na região de furca em ratas submetidas a periodontite experimental induzida por meio de ligaduras. Animais foram aleatoriamente distribuídos nos grupos: 1 - placebo (veículo) controle (n=11); 2- IFC + placebo (n=15); 3- PTH 1-34 (n=10); 4 - IFC + PTH 1-34 (n=15). Dentes controlaterais, não receberam ligaduras e serviram como controle. Após 60 dias com ligaduras, os animais foram sacrificados. A avaliação histométrica foi realizada quantificando a área de perda óssea na região da bifurcação e a análise histoquímica por meio de reação de fosfatase ácida tártarato resistente (TRAP). Os dados coletados foram analisados estatisticamente utilizando a análise de variância ANOVA e o teste Tukey (=5%). Nos dentes com ligaduras, uma análise intergrupo revelou aumento estatisticamente significante da perda óssea como resultado do modelo de periodontite induzida quando o grupo 2 foi comparado com os grupos 1, 3 e 4 respectivamente (p<0,05). O número de células marcadas positivamente pelo TRAP na superfície linear da crista óssea demonstrou um aumento significativo no número de osteoclastos para o grupo 2 quando comparado com os grupos 1, 3 e 4, respectivamente (P<0,05). Dentro dos limites do presente estudo, pode-se concluir que o PTH 1-34 na ausência ou presença de IFC pode reduzir significativamente a perda óssea resultante da periodontite experimental induzida por ligaduras
Abstract: The aim of the present investigation was to histologically and histoquimically evaluate, in an animal model (rats), the influence of cigarette smoke inhalation (CSI) and intermittent administration of PTH in rodents would block the alveolar bone loss when a ligature-induced periodontitis is used. Animals were randomly assigned in groups: 1 - placebo (vehicle) Control-ligated (n=11); 2 - CSI + placebo- ligated (n=15); 3 - PTH-treated ligated (n=10); 4 - CSI + PTH-treated ligated (n=15). Contralateral teeth were unligated to serve as controls. After 60 days with ligatures, the animals were killed. The histometric avaluete determined the area between the bone crest and cementum surface in the furcation regions of teeth and the number of cells positive for tartrate-resistant acid phosphatase (TRAP). The date were statistically analysed using ANOVA and Tukey's test (alpha=5%). At the ligated sites, intergroup analysis revealed significantly increased the bone loss resulting from ligature-induced periodontitis when group 2 compared with group 1, 3 and 4, respectively (P<0,05). The number of TRAP-Positive cell in the linear surface of the bone crest showed an increase for the group 2 compared with the group 1, 3 and 4, respectively (P<0,05). Whithin the limits of the present study, it can be concluded that PTH in the absence or presence of CSI may be minimize significantly the bone resorption associated with periodontitis
Doutorado
Periodontia
Doutor em Clínica Odontológica
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Taylor, Adam. "The role of Rab GTPases in osteoclasts." Thesis, Available from the University of Aberdeen Library and Historic Collections Digital Resources, 2009. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?application=DIGITOOL-3&owner=resourcediscovery&custom_att_2=simple_viewer&pid=59017.
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Hussein, Osama. "Interaction of breast cancer cells with osteoclasts." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103695.
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Breast cancer is a major health problem. Metastatic disease is generally incurable. In the majority of patients, the skeleton bears the major metastatic burden. Bony lesions of metastatic breast cancer are usually osteolytic. Osteolytic metastases are formed by the pathological activation of osteoclasts. Anti-osteoclastic drugs are standard of care for patients suffering from breast cancer metastases. We conducted this project to decipher the signalling mechanisms responsible for osteoclasts activation in response to exposure to mediators released from mammary carcinoma cells. We evaluated the apoptotic profiles of osteoclasts in vitro in the presence of soluble factors derived from mammary carcinoma cells cultures. We observed a significant inhibition of osteoclasts apoptosis secondary to exposure to breast cancer cells-derived factors. This effect was not reversed with bisphosphonates. The pro-apoptotic protein BIM in osteoclasts was a target of modulation by breast cancer cells-derived factors. We proceeded to characterize the osteoclasts intracellular signalling pathways modulated by mammary carcinoma cells-derived factors. We identified phospholipase C γ (PLC γ) and the mammalian target of rapamycin (mTOR) as mechanistic meditors of the anti-apoptotic effect of cancer cells on osteoclasts. We tested the therapeutic benefit of rapamycin administration in a mouse model of experimental bone metastases of mammary carcinoma. In this model, rapamycin therapy inhibited metastasis-associated osteolysis, prolonged animal survival and reversed some tumour-induced immune changes in the host. Taken together, these studies provide new insights into the pathophysiology of breast cancer metastasis to bone, and demonstrate that targeting osteoclast signalling mediators, such as mTOR provides therapeutic benefits in experimental bone metastasis model.Le cancer du sein est un problème de santé important. La maladie métastatique est généralement incurable. Dans la majorité de patients, le squelette soutient le fardeau métastatique principal. Les lésions osseuses du cancer du sein métastatique sont habituellement ostéolytique. Des métastases ostéolytique sont constituées par l'activation pathologique des osteoclasts. Les drogues anti-osteoclastiques sont mesures de soin essentiales pour des patients souffrant des métastases de cancer du sein. Nous avons conduit ce projet pour déchiffrer les mécanismes de signalisation responsables de l'activation d'osteoclasts en réponse à l'exposition aux médiateurs libérés des cellules mammaires de carcinome. Nous avons évalué les profils apoptotiques des osteoclasts in vitro en présence des facteurs solubles dérivés des cultures mammaires de cellules de carcinome. Nous avons observé une inhibition significative d'apoptosis d'osteoclasts secondaire à l'exposition aux facteurs cellule-dérivés par cancer du sein. Cet effet n'a pas été renversé avec des bisphosphonates. La pro-apoptotic protéine BIM dans les osteoclasts était une cible de la modulation par des facteurs cellule-dérivés par cancer du sein. Nous avons procédé caractériser les voies de signalisation intracellulaires d'osteoclasts modulées par des facteurs cellule-dérivés par carcinome mammaire. Nous avons identifié la phospholipase C (PLC γ) et la cible mammifère du rapamycin (mTOR) en tant que meditors mécanistes de l'effet anti-apoptotic des cellules de cancer sur des osteoclasts. Nous avons examiné l'avantage thérapeutique de l'administration de rapamycin dans un modèle de souris des métastases expérimentales d'os du carcinome mammaire. Dans ce modèle, la thérapie de rapamycin a empêché l'osteolysis métastase-associé, a affecté des survies animales prolongée et renversé quelques changements immunisés induits par la tumeur.
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Gray, A. "Isolation, generation and characterization of equine osteoclasts." Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599624.
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a) Equine osteoclasts (OCs) were identified in ex vivo samples of developing long bone. They were particularly numerous in areas of active bone formation and remodelling such as at the longitudinal septum between cartilage and subchondral bone and in the spongiosum. Equine OCs were identified as being typically multinuclear and expressing very high levels of tartrate resistant acid phosphatase (TRAP) and cathepsin K. Only low levels of cathepsin B were detected in equine OCs. b) Techniques were developed to isolate equine OCs for the first time in vitro. The phenotype of these cells was clearly defined; they were multinuclear, expressed TRAP, the vitronectin receptor (VNR) and were observed in direct association with areas of resorption when cultured on ivory slices. However, equine OCs could only be isolated in small numbers in vitro and were often of poor viability. c) Techniques were developed to generate equine osteoclast-like cells (OCLs) for the first time in vitro from bone marrow cells (BMCs) with or without osteoblastic support cells. d) Equine OCLs demonstrated high levels of cathepsin K activity but only low levels of cathepsin B activity as determined by the use of fluorogenic substrates for these enzymes. e) The bisphosphonate pamidronate (APD) caused a dose-dependent inhibition of resorption by equine OCLs. APD also dose-dependently reduced the number of OCLs present in MNCEP cultures after 7d incubation but paradoxically increased the resorption undertaken by similar cultures at certain concentrations. f) Calcitonin inhibited resorption by equine OCLs apparently by inducing cytoplasmic retraction. g) Resorption by OCLs was also inhibited by insulin and the proton-pump inhibitor, bafilomycin.
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Ford, Lorna. "An investigation into the effects of endocannabinoids and the COX-2 metabolite of 2-Arachidonyl glycerol on bone cells." Thesis, Available from the University of Aberdeen Library and Historic Collections Digital Resources, 2009. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?application=DIGITOOL-3&owner=resourcediscovery&custom_att_2=simple_viewer&pid=33596.
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Tan, Jamie We-Yin. "The investigation of RANKL TNF-like core domain by truncation mutation." University of Western Australia. School of Surgery and Pathology, 2003. http://theses.library.uwa.edu.au/adt-WU2004.0032.
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Osteoclasts are multinucleated cells found exclusively in bone and are derived from the haematopoietic cells of monocytes/macrophage lineage. The cell-to-cell interaction between osteoblastic/stromal cells and osteoclast precursor cells is necessary for osteoclastogenesis. Receptor Activator of NF-κB ligand (RANKL) was identified as a membrane-bound TNF ligand family member that is the ‘master’ cytokine expressed on osteoblastic/stromal cells, which stimulate osteoclastogenesis through cell-to-cell contact with osteoclast precursors. RANKL is considered to be a factor that is necessary and sufficient for the induction of osteoclastogenesis (Lacey, et al., 1998). RANKL is a type II transmembrane cytokine of the TNF ligand superfamily and has an active TNF-like core domain at the extracellular domain. This active TNF-like core domain is thought to be the region through which it binds to it’s active receptor, RANK, for the activation of signal transduction pathways for the initiation of processes leading to osteoclastogenesis (Lacey, et al., 1998; Li, et al., 1999). It was hypothesized that any change in the active TNF-like core domain might affect the ability of RANKL binding to RANK and consequently affect the activation of signal transduction pathways and osteoclastogenesis. Hence, this thesis sought to investigate the effects of changes in the active TNF-like core domain by truncation mutation on the ability of RANKL binding to RANK and consequently affect the activation of signal transduction pathways and osteoclastogenesis. A cDNA fragment encoding the full-length TNF-like core domain of rat RANKL (rRANKL) (aa160-318) was cloned into the bacterial expression pGEX vectors and stably expressed in Eschechia coli as a fusion protein with the C-terminus of glutathione S-transferase (GST). Four mutants (aa160-302, aa160-268, aa239-318 and aa246-318) were also generated by truncation mutation in the TNF-like core domain, and cloned into the pGEX vector to produce GST-rRANKL mutants. The proteins were over-expressed and affinity purified to 95% in purity. GST-rRANKL (160-318) containing the full length TNF-like core domain was able to induced osteoclastogenesis in spleen cells in the presence of M-CSF and in RAW264.7 cells in the absence of M-CSF. It was also found to activate mature osteoclast activity in vitro, ex vivo and in vivo. It has the highest binding affinity to RANK and the greatest potency for NF-κB activation as well as the induction of osteoclastogenesis compared to the truncated mutants. Mutants generated by truncation of the TNF-like core domain revealed that the TNF-like core domain is important for the interaction with the RANK, for high binding affinity, NF-κB activation and induction of osteoclastogenesis. In general, the truncated mutants not only displayed a reduction in the binding affinity to RANK, but also a reduction in NF-κB activation, and significantly reduced potency in the induction of osteoclastogenesis. Interestingly, mutant GST-rRANKL (160-268) showed a higher affectivity than the other mutants did, in that it had greater binding affinity to RANK, and in NF-κB activation than the rest of the truncated mutants. Mutants GST-rRANKL (239-318) and GST-rRANKL (246-318) on the other hand, showed little potency in the induction of osteoclast formation, however, might have an inhibitory effect through competition with full length GST-rRANKL (160-318) as well as inducing a response in vivo resulting in an increase in the serum calcium level. In conclusion, this thesis demonstrated that the TNF-like core domain of RANKL is active, and imperative in the binding to RANK, activating signal transduction pathways and induction of osteoclastogenesis. Changes in the active TNF-like core domain affected the ability, affinity and efficiency of RANKL binding to the receptor, RANK and consequently affected the activation of signal transduction pathways and osteoclastogenesis.
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Pitombo, Jonleno Coutinho Paiva [UNESP]. "Impacto de andrógenos na diferenciação e atividade de osteoclastos em cultura celular." Universidade Estadual Paulista (UNESP), 2016. http://hdl.handle.net/11449/138870.
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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Os mecanismos de ação dos andrógenos sobre homeostase e regulação das células que participam do turnover ósseo em fêmeas ainda são pouco compreendidos. Este trabalho teve como principal objetivo avaliar a participação de andrógenos na diferenciação e atividade de osteoclastos in vitro. Células totais de medula óssea de fêmur e tíbia de camundongos fêmeas foram utilizadas como fonte de células precursoras de osteoclastos, sendo cultivadas utilizando-se α-MEM suplementado e em presença de RANK-L (30ng/mL) e M-CSF (50ng/mL). As células foram tratadas com testosterona (T) diidrotestosterona (DHT) e antagonistas de receptores de hormônios sexuais, como flutamida (FLU) e fulvestranto (FUL). O anastrozol (ANA) foi usado para inibição da enzima aromatase e o etanol (0,01%) foi utilizado como controle. Após cinco dias, as células foram fixadas, coradas com TRAP e contadas, considerando-se células TRAP-positivas com 3 ou mais núcleos. Para o ensaio de atividade, foram utilizadas placas revestidas com fosfato de cálcio inorgânico e a área de reabsorção foi calculada com o auxílio de software. O estágio de diferenciação osteoclástica foi avaliado por RT-qPCR e a modulação da expressão de receptores para hormônios sexuais foi avaliada por Western Blot. Os andrógenos (T e DHT) não exerceram efeitos sobre a diferenciação e atividade de osteoclastos (ANOVA; p>0,05). Por outro lado, os tratamentos com ANA, FLU e FUL, associados ou não a T, regularam positivamente a diferenciação e atividade de osteoclastos. A expressão gênica de RANK, Catepsina K, NFATc1 e β3 integrina não foi alterada pelos tratamentos propostos (ANOVA; p>0,05). Além disso, os tratamentos com T, DHT, FLU e FUL modularam a expressão proteica do receptor de andrógeno (AR) e dos receptores de estrógeno (ERα e ERβ) por Western Blot. Tomados em conjunto, nossos resultados indicam que os andrógenos exercem limitada participação na diferenciação e atividade de osteoclastos de camundongos fêmeas e que este processo é mediado, ao menos em parte, por ações indiretas da T e pela modulação de receptores de hormônios sexuais.
The action mechanisms of androgens on homeostasis and the regulation of cells that participate in bone turnover in females are still poorly understood. This study had as main objective to evaluate the participation of androgens in the differentiation and activity of in vitro osteoclasts. Total bone marrow cells from femur and tibia of female mice were used as a source of precursor cells of osteoclasts, they were cultivated using supplemented α-MEM and in the presence of RANK-L (30ng/mL) and M-CSF (50ng/mL). The cells were treated with testosterone (T), dihydrotestosterone (DHT) and antagonists of sexual hormone receptors such as flutamide (FLU) and fulvestrant (FUL). Anastrozole (ANA) was used for inhibiting the aromatase enzyme and ethanol (0.01%) was used as a control. After five days, the cells were fixed, colored with TRAP and counted, considering TRAP-positive cells those ones containing 3 or more nuclei. For the activity assay, were used plaques covered with inorganic calcium phosphate and the area of reabsorption was calculated with the assistance of a software. The osteoclast differentiation stage was evaluated by RT-qPCR and the modulation of the expression of receptors for sexual hormones was assessed by Western Blotting. The androgens (T and DHT) did not exert effects in differentiation and activity of osteoclasts (ANOVA, p> 0.05). On the other hand, the treatments with ANA, FLU and FUL, associated or not to T, positively regulated the differentiation and activity of osteoclasts. The genic expression of RANK, Cathepsin K, NFATc1 and β3 integrin was not altered by the proposed treatments (ANOVA, p> 0.05). Moreover, the treatments with T, DHT, FLU and FUL modulated the protein expression of the androgen receptor (AR) and of the estrogen receptors (ERα and ERβ) by Western Blotting. Taken together, our results indicate that the androgens exert limited participation in differentiation and activity of osteoclasts of female mice and that this process is mediated, at least in part, by indirect actions of T and by the modulation of sexual hormone receptors.
CNPq: 133815/2014-5
FAPESP: 2013/12014-6
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Wang, Cathy Ting-Peng. "Molecular dissection of RANKL signaling pathways in osteoclasts." University of Western Australia. School of Surgery and Pathology, 2007. http://theses.library.uwa.edu.au/adt-WU2008.0037.
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[Truncated abstract] Bone remodeling is intricately regulated by osteoclast-mediated bone resorption and osteoblast-mediated bone formation. The elevation in osteoclast number and/or activity is a major hallmark of several common pathological bone disorders including post-menopausal osteoporosis, osteoarthritis, Paget's disease, and tumour-mediated osteolysis. Receptor activator of nuclear factor kappa B ligand (RANKL) is a key cytokine for osteoclast differentiation and activation. The association of RANKL to its cognate receptor, RANK, which is expressed on osteoclast precursors and mature osteoclasts, is essential for osteoclast formation and activation. The intimate interaction between RANKL and RANK triggers the activation of a cascade of signal transduction pathways including NF-κB, NFAT, MAPK and PI3 kinase. Although osteoclast signaling pathways have been intensively studied, the precise molecules and signaling events which underlie osteoclast differentiation and function remain unclear. In order to dissect the molecular mechanism(s) regulating osteoclast differentiation and activity, this thesis herein explores the key RANKL/RANK-mediated signaling pathways. Four truncation mutants within the TNF-like domain of RANKL [(aa160-302), (aa160-268), (aa239-318) and (aa246-318)] were generated to investigate their potential binding to RANK and the activation to RANK-signal transduction pathways. All were found to differentially impair osteoclastogenesis and bone resorption as compared to the wild-type RANKL. The impaired function of the truncation mutants of RANKL on osteoclast formation and function correlates with their reduced ability to activate crucial RANK signaling including NF-κB, IκBα, ERK and JNK. Further analysis revealed that the truncation mutants of RANKL exhibited differentially affinity to RANK as observed by in vitro pull-down assays. ... It is possible that Bryostatin 1 acts via upregulation of a fusion mechanism as the RANKL-induced OCLs are morphologically enlarged, exhibiting increased nuclei number expressing high level of DC-Stamp. Furthermore, Rottlerin was shown to inhibit NF-κB activity, whereas Bryostatin 1 showed the opposing effects. Both inhibitor and activator were also found to modulate other key osteoclastic signaling pathways including NFAT and total c-SRC. These findings implicate, for the first time, Protein Kinase C delta signaling pathways in the modulation of RANKL-induced osteoclast differentiation and activity. Taken together, the studies presented in this thesis provide compelling molecular, biochemical and morphological evidence to suggest that: (1) RANKL mutants might potentially serve as peptide mimic to inhibit RANKL-induced signaling, osteoclastogenesis and bone resorption. (2) A cross talk mechanism between extracellular Ca2+ and RANKL exist to regulate on osteoclast survival. (3) TPA suppressed RANKL-induced osteoclastogenesis predominantly during the early stage of osteoclast differentiation via modulation of NF-κB. (4) Selective inhibition of Protein Kinase C signaling pathways involved in osteoclastogenesis might be a potential treatment method for osteoclast-related bone diseases. (5) Protein Kinase C delta signaling pathways play a key role in regulating osteoclast formation and function.
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Luukkonen, J. (Jani). "Osteopontin and osteoclasts in rheumatoid arthritis and osteoarthritis." Doctoral thesis, Oulun yliopisto, 2019. http://urn.fi/urn:isbn:9789526223643.
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Abstract Rheumatoid arthritis and osteoarthritis are two chronic joint diseases, which cause two of the largest socioeconomical burdens among all joint diseases according to the World Health Organization. Both diseases are associated with changes in bone structure and bone cell, especially osteoclast, function. The etiology or pathogenesis of these diseases are not completely understood. Traditionally, osteoarthritis is seen as a disease resulting from mechanical wear of cartilage and bone, and rheumatoid arthritis as an autoinflammatory disease of synovial tissue. However, also in osteoarthritis chronic inflammation is present in synovial tissue, and in rheumatoid arthritis large changes in bone structure are seen. The field of study focusing on this connection between inflammation and bone is called osteoimmunology and it can explain many features of these chronic diseases linking joint health to disturbances in bone homeostasis. Here, the study focused on the function of osteoclasts in normal and pathological environments, and on the factors that have an effect on bone resorption, with a special emphasis on the protein osteopontin. Samples of synovial fluid and serum from rheumatoid arthritis and osteoarthritis patients were analyzed for factors affecting osteoclasts, and in vitro cell cultures of human derived osteoclasts were used to analyze osteoclast function in normal and pathological environment. The phosphorylation of osteopontin was found to be increased in rheumatoid arthritis, along with multiple other inflammatory factors that also affect osteoclasts, such as IL-6, IL-8 and VEGF. Osteoclast cell cultures showed how the use of different patient samples significantly affected osteoclastogenesis, due to so-called inflammatory osteoclastogenesis. Additionally, we show that osteoclasts deposit osteopontin into the resorption lacunae during bone resorption. Based on the results, the inflammatory component present in both osteoarthritis and rheumatoid arthritis significantly affects osteoclast function, and its further study in the future may reveal new therapeutic possibilities. Especially the new discoveries of osteopontin’s role in normal osteoclast function and its changes seen between osteoarthritis and rheumatoid arthritis may prove to have therapeutic potentialTiivistelmä Nivelreuma ja nivelrikko ovat kroonisia nivelsairauksia, jotka Maailman terveysjärjestön (WHO) mukaan aiheuttavat eniten sosioekonomista haittaa. Molemmissa sairauksissa luiden rakenteessa ja luusolujen, erityisesti osteoklastien, toiminnassa tapahtuu muutoksia. Kummankaan taudin etiologiaa tai patogeneesiä ei täysin tunneta. Perinteisesti ajatellaan, että nivelrikko johtuu rusto- ja luukudoksen mekaanisesta kulumisesta ja nivelreuma nivelkalvon autoinflammatoorisesta tulehduksesta. Kuitenkin nivelrikossa nähdään myös selkeä nivelkalvon krooninen tulehdus ja nivelreumassa suuria luun rakenteen muutoksia. Tutkimusala, joka tutkii tulehduksen ja luun yhteyttä, on nimeltään osteoimmunologia. Tässä väitöskirjassa tutkitaan osteoklastien toimintaa ja niihin vaikuttavia tekijöitä, erityisesti proteiini osteopontiinia, normaalissa ja tautiympäristössä. Analysoin osteoklasteihin vaikuttavia tekijöitä nivelrikko- ja nivelreumapotilaiden näytteistä sekä osteoklastien toimintaa soluviljelmissä. Soluviljelmissä käytettiin nivelreuma- ja nivelrikkopotilaiden näytteitä mahdollisimman totuudenmukaisen ympäristön luomiseksi osteoklasteille. Tutkimuksessa osoitettiin, kuinka osteopontiinin fosforylaatio on lisääntynyt nivelreumapotilaiden nivelnesteessä. Myös useiden muiden osteoklasteihin vaikuttavien tekijöiden, kuten IL-6:n, IL-8:n ja VEGF:n, havaittiin lisääntyneen nivelreumassa. Osteoklastien soluviljelmissä havaittiin selkeät erot siinä, miten eri potilasnäytteet vaikuttavat osteoklasteihin ja erityisesti tulehduksen aiheuttamaan osteoklastien syntyyn. Osoitan myös, miten osteoklastit erittävät osteopontiinia luunhajotuskuoppaan luun hajotuksen aikana. Tutkimustulosten mukaan krooninen tulehdustila nivelrikossa ja nivelreumassa vaikuttaa huomattavasti osteoklastien toimintaan. Uskon, että lisätutkimukset tällä saralla voivat paljastaa uusia hoidollisia mahdollisuuksia. Erityisesti uudet löydökset osteopontiinin roolista osteoklastien toiminnassa sekä muutoksista nivelrikossa ja nivelreumassa vaativat jatkotutkimuksia, jotta proteiinin kliininen merkittävyys saadaan selvitettyä
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Cleaton-Roberts, Melanie. "Development and characterisation of polyclonal and monoclonal antibodies raised against cathepsin K." Thesis, University of Liverpool, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.250227.
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Das, Subhajit. "Study of critical pathways important for the pathophysiology and pharmacology of osteoclasts." Thesis, University of Aberdeen, 2013. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=204054.
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Bone and skeletal joint disorders affect millions of people with significant morbidity and mortality. Over the last few decades it has become apparent that the physiological structure of bone is maintained by the balanced functioning of bone-forming osteoblast and bone-resorbing osteoclast cells. Thus study of the regulation and function of osteoclasts became the focus of scientific research to find therapeutic targets for bone related disorders. The aim of this study was to elucidate the roles of two pathways, namely Receptor Activator of NFB (RANK) signaling pathway and mevalonate pathway in relation to the pathology and pharmacology of osteoclasts. RANK mutations associated with osteopetrosis were studied to elucidate the molecular mechanism of activation of the RANK signaling pathway. The data demonstrate that, unlike other TNF receptors, the C-terminal PreLigand Assembly Domain (PLAD or ‘IVVY' motif) is not essential for ligand-induced activation of RANK signaling. The study of the extracellular RANK mutations provided the opportunity to examine role of three cysteine rich domains (CRD) within extracellular RANK in its interaction with RANK ligand. The binding affinities of RANK ligand to wild type and six extracellular mutant RANK proteins were studied by surface plasmon resonance. It showed that CRD 1, 3 and 4 played crucial roles, despite previous crystallography studies predicting roles for only CRD 2 and CRD 3 in RANK ligand binding. In the second part of the thesis the mevalonate pathway was studied in relation to mevalonate kinase deficiency and osteoporosis. Mevalonate kinase deficiency is a hereditary disorder caused by mutations within the mevalonate kinase gene and manifests as autoimmune disorder due to deficiency of end products in mevalonate pathway. Due to rarity of the disorder, access to patient samples is extremely limited and we aimed to develop an in vitro model of mevalonate kinase deficiency to develop a better understanding of regulation of the mevalonate pathway. In osteoporosis, nitrogen-containing bisphosphonate drugs target farnesyl pyrophosphate synthase (FPPS) within the mevalonate pathway. A small number of patients develop resistance to bisphosphonate therapy, but the molecular mechanism is unknown. It was hypothesized that upregulation of FPPS would confer resistance to bisphosphonates and this study showed that upregulated endogenous FPPS introduced bisphosphonate resistance at therapeutic concentrations in vitro. Furthermore, it was observed that mitochondrial isoforms of FPPS were unlikely to play any role in bisphosphonate resistance. In conclusion, these data suggest that therapeutic targeting of the PLAD motif in RANK may not be as effective as previously proposed, but the extracellular domain of RANK may be a potential target for the development of novel therapies in the prevention and treatment of osteoporosis. In addition, prior screening for expression levels of FPPS patients with osteoporosis for may identify those at risk of resistance to nitrogen-containing bisphosphonate therapy.
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Hocking, Lynne J. "Genetics of Paget's disease of bone." Thesis, University of Aberdeen, 2002. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU160239.
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In Chapter 4, I investigated the roles of the RANK signalling partners RANK ligand (RANKL) and osteoprotegerin (OPG) in the pathogenesis of sporadic and familial PDB. One polymorphism in the RANK gene and five polymorphisms in the OPG gene were examined in sporadic PDB cases and in sex- and age-matched controls. No allele-disease or genotype-disease association was observed for the RANKL polymorphism, suggesting RANKL is not directly involved in susceptibility to sporadic PDB. Genotypes at two OPG polymorphisms did significantly predict disease status in individuals affected with sporadic PDB, suggesting a role for OPG in the pathogenesis of sporadic PDB. The five OPG polymorphisms were also examined in families affected with PDB. No evidence was found to either suggest or exclude the involvement of any of the OPG polymorphisms in familial PDB. In Chapter 5, I performed a genome-wide search for PDB susceptibility loci in families with inherited PDB. Three regions of potential linkage were identified at 2q36, 5q35 and 10p11. Fine mapping was performed for the candidate region on chromosome 5q35, and eight families with a high probability of linkage to 5q35 were identified. In seven of the families, a shared haplotype transmitted only with affected family members was present. The shared haplotype varied between families and no common allele existed in the seven families for any of the nine markers studied. However, one area of shared haplotype occurred in all seven families across three of the markers, supporting evidence for a susceptibility gene for PDB on 5q35 in these families and narrowing the candidate region. In summary, this study has further highlighted the importance of genetic heterogeneity in the pathogenesis of PDB, excluding the previously identified PDB2 susceptibility locus and identifying three novel regions potentially harbouring susceptibility loci in the families studied. This study has also further defined the role of members of the RANK signalling pathway in the pathogenesis of familial and sporadic PDB.
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Buitrago, Liseth Yamile Wilches. "Papel da frutose 1,6-bisfosfato na osteoclastogênese e reabsorção óssea in vitro." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/17/17133/tde-23042018-120322/.
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O remodelamento ósseo é um processo metabólico, dentro do qual os osteoblastos e os osteoclastos, participam ativamente. Portanto, qualquer alteração neste equilíbrio, pode provocar uma modificação na densidade mineral do osso, situação observada em certas doenças osteolíticas como osteoporose, artrite reumatóide e periodontite. Nos últimos anos, há um crescente interesse em avaliar o papel da glicólise na proliferação, sobrevivência e diferenciação dos diferentes tipos celulares. Em particular, tem sido evidenciado o efeito regulador da frutose 1,6-bisfosfato (FBP), um intermediário da via glicolítica de alta energia. Considerando que ainda não existem dados na literatura que correlacionem a FBP com o funcionamento dos osteoclastos, este trabalho tem por finalidade avaliar seu papel na osteoclastogênese e reabsorção óssea in vitro. Para isso, pré-osteoclastos murinos derivados da medula óssea foram diferenciados em osteoclastos na presença de M-CSF, RANKL e duas concentrações da FBP (100 e 300 ?M). Os resultados obtidos amostram que a FBP inibe a diferenciação osteoclástica em uma relação dose-dependente, sem afetar a viabilidade celular. Observa-se também, que o tratamento com FBP diminui a expressão de genes marcadores como, Nfatc1, Trap e Catepsina K (p < 0.01) e das proteínas NFATc1 e catepsina K. Como também, promove uma redução na atividade reabsortiva dos osteoclastos depois de 96 h de cultura. O efeito inibidor da FBP não depende da atividade da piruvato quinase M2 (PKM2). Em conjunto, estes dados sugerem que a FBP é um metabolito regulador importante da osteoclastogênese, demonstrando ser um agente potencial para o tratamento de doenças osteolíticas.Bone remodeling is a coordinated metabolic process, where the osteoblasts and osteoclasts participate actively. Therefore, any alteration in this balance may cause a change in the bone mineral density, a condition observed in certain bone loss-associated diseases such as osteoporosis, rheumatoid arthritis and periodontitis. Recently, there has been a growing interest in assessing the role of the glycolysis on the proliferation, survival, and differentiation of the different cell types. In particular, it has been demonstrated the protective effect of the Fructose 1,6-bisphosphate (FBP), a high-energy glycolytic intermediate. Considering that there is no evidence in the literature that associate FBP with the function of osteoclasts, this work aims to evaluate its role in osteoclastogenesis and bone resorption in vitro. To this end, murine bone marrow derived pre-osteoclasts were differentiated into osteoclasts in the presence of M-CSF, RANKL and two concentrations of FBP (100 and 300 ?M). The results showed that FBP inhibits the differentiation of osteoclasts in a dose dependent manner, without affecting the cell viability. It was also observed that the treatment with the FBP decreases the expression of marker genes such as Nfatc1, Trap and Cathepsin K (p < 0.01) and the NFATc1 and cathepsin K protein levels. As well, the treatment with FBP resulted in markedly fewer osteoclast activity after 96 h of culture. FBP osteoclast inhibitory effect does not involve Pyruvate Kinase M2 (PKM2) activity. Together, these data denote the important regulatory role of the FBP on osteoclastogenesis, proving to be a potential agent for the treatment of bone loss-associated diseases.
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Magalhães, Fernando Augusto Cintra. "Efeito protetor da Interleucina-4 na reabsorção óssea periodontal induzida por agonista de TLR2 (Pam2CSK4) /." Araraquara, 2018. http://hdl.handle.net/11449/154085.
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Orientador: Pedro Paulo Chaves de SouzaResumo: A periodontite é resultado do desequilíbrio entre o biofilme bacteriano e a resposta imune do hospedeiro. Componentes bacterianos, como o lipopolissacarídeo (LPS) e as lipoproteínas, são reconhecidos pelo sistema imune e desencadeiam a produção de citocinas que auxiliam no combate à infecção, mas também induzem a destruição tecidual. A participação do LPS na destruição óssea já é bem estabelecida, porém o papel das lipoproteínas na periodontite permanece carente de investigação. Na periodontite, citocinas pró-inflamatórias participam do processo de destruição do tecido ósseo. Neste processo, são secretadas também citocinas osteoprotetoras. Dentre elas, a interleucina 4 (IL-4) é reconhecida pela propriedade de inibir a produção citocinas pró inflamatórias como IL-1, IL-6 e TNF-α. O papel protetor de IL-4 na osteoclastogênese e na doença periodontal induzida por lipoproteína ainda não foi investigado. Nosso estudo foi divido em dois capítulos. No capítulo 1, hipotetizamos que a lipoproteína sintética Pam2CSK4 (PAM2) poderia induzir a reabsorção óssea periodontal. Para isso, foram utilizados camundongos C57bl/6, que receberam injeções a cada 2 dias, por 24 dias, do veículo, LPS de Escherichia. coli ou PAM2, entre o primeiro e segundo molar superior. Após o período experimental, os animais foram eutanasiados e destinados à análise por microCT, análise histológica e imunohistoquímica para marcação dos osteoclastos. A PAM2 apresentou a capacidade de induzir a perda óssea alveolar, ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The pathogenesis of periodontitis is a result of imbalance between the bacterial biofilm and the host immune response. Bacterial components such as lipopolysaccharide (LPS) and lipoproteins, activate the immune system leading to periodontal distruction. The participation of LPS in periodontal bone destruction is well established, but there is a lack of information about the role of lipoproteins in periodontitis. In the pathogenesis of periodontitis, these molecular patterns are recognized by host immune system and trigger the production of cytokines that participate of antimicrobial response, but also induce tissue destruction. On the other hand, antinflamatory cytokines produced by Th2 cells, such as IL-4, have an osteoprotective phenotype. The role of IL-4 in lipoprotein-induced periodontitis was not yet investigated. Thus, this thesis was divided in two chapters. In chapter 1, we investigated the role of lipoproteins in the pathogenesis of periodontitis in mice. In this study, we hypothesized that the synthetic lipoprotein Pam2CSK4 (PAM2) can induce periodontal bone resorption. C57bl / 6 mice received bilateral injections every other day for 24 days of: vehicle, Escherichia coli LPS or PAM2, between the first and second upper molars. Twenty-four hours after the last injection, the mice were euthanized and the jaw bones were scanned for micro computed tomography, decalcified and processed for histological analysis and stained for tartrate-resistant acid phosphatase, phenoty... (Complete abstract click electronic access below)
Doutor
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Wilkinson, Debbie Isabelle. "Visualisation of osteoclast membrane domains." Thesis, University of Aberdeen, 2010. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=158808.
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Osteoclasts polarise upon activation and form four distinct membrane domains; the basolateral domain, the sealing zone, the functional secretory domain and the ruffled border. The ruffled border is the resorptive organelle of the cell and provides a large surface area for the release of protons and enzymes into the space beneath the osteoclast. Defects in osteoclast formation or function can lead to diseases such as osteopetrosis. Ruffled border formation is a critical event in osteoclast function but the process by which it and other membrane domains form is only partially understood. Vesicular trafficking is essential for the tight regulation of the osteoclast membrane domains and it has been shown previously that treatment with pharmacological inhibitors causes disruption of trafficking. The aims of this PhD were to increase our understanding of vesicular trafficking in osteoclasts and to optimise ways of visualising osteoclast membrane domains. My studies of patients with osteoclast-poor osteopetrosis identified defects in RANKL as a cause of the defect. This in turn has identified a potential therapy of recombinant RANKL for patients with this form of the disease. Although purification of wild type or mutant RANKL was not completely successful, it did suggest that the mutant forms of RANKL were not functional. I have used pharmacological inhibitors to study osteoclast membrane domains, and found that transmission electron microscopy is an essential tool for studying membrane changes following pharmacological inhibition at the ultrastructural level. I also established that the study of vesicular trafficking to analyse formation of membrane domains can make excellent use of immuno-electron methods. Furthermore, genetic diseases associated with defective ruffled border formation such as XLA and osteopetrosis provide useful tools to further analyse the dynamics involved in the formation and maintenance of the ruffled border, as well as revealing more about the diseases themselves.
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Mancini, Lucia. "Nitric oxide regulation of bone metabolism." Thesis, Queen Mary, University of London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343902.
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Mellis, David. "The study of RANK mutations associated with the diseases of osteoclast dysfunction." Thesis, University of Aberdeen, 2010. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=166647.
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Osteoclasts are the cells that resorb bone to maintain a healthy skeleton. Receptor activator of NFkB (RANK) is a receptor that is critical for the formation, activity and survival of osteoclasts. A number of mutations have been identified within RANK that cause bone diseases with opposite osteoclast phenotypes. The aim of this thesis was to study the downstream consequences of these disease-associated mutations for RANK protein processing and activation of the RANK signalling pathway. Early onset Paget’s disease of bone (ePDB), Familial Expansile Osteolysis (FEO) and Expansile Skeletal Hyperphosphatasia (ESH) are conditions featuring focal areas of increased bone resorption driven by overactive osteoclasts. These conditions are caused by heterozygous insertion mutations in the signal peptide region of the RANK gene, but the mechanisms underlying the development of overactive osteoclasts are not known. In this thesis, in vitro study of the mutant RANK proteins demonstrated that homozygous overexpression caused inactivation of RANK signalling due to intracellular accumulation of RANK within an extended form of the endoplasmic reticulum. By contrast, when expressed in a heterozygous manner, the mutant proteins were found at the plasma membrane and caused prolonged ligand-dependent signalling. Taken together and as predicted by the clinical situation, these data strongly suggest that heterozygous expression of the mutant RANK proteins hold the key to the hyperactive osteoclast phenotype associated with these diseases. Osteoclast-poor osteopetrosis is a disease in which osteoclasts do not form leading to a high bone mass phenotype. Single base pair mutations within RANK have been identified in some patients with this condition. These mutant RANK proteins were studied and the findings related to regions within RANK in which the mutations occur that have been shown to be critical for its function. In addition, osteoclast formation was assessed in cultures of peripheral blood mononuclear cells isolated from patients with osteopetrosis with unidentified genetic background in order to further characterise the osteoclast phenotype for each patient. In summary, the findings presented in this thesis begin to elucidate the molecular mechanisms leading to diseases of bone with opposite osteoclast phenotypes that, paradoxically, are all caused by inactivating mutations in the RANK gene.
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Gu, Yuchun. "Investigation of ion channels on bone cells." Thesis, University of Birmingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.369360.
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Wang, Wen. "Investigating the role of CCN1, CCN2, and CCN6 in osteoclast and osteoblast physiology." Thesis, University of Aberdeen, 2012. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=204059.
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CCN protein family members (CYR61, CTGF, Nov, Wisp-1, Wisp-2 and Wisp-3) have important roles in many different processes including angiogenesis, inflammation, remodelling of extracellular matrix and tumorigenesis. In bone, CCN1 increases osteoblastogenesis via Wnt3A signalling and activation of -catenin which, in turn, upregulates CCN1 expression. The exact role of CCN1, CCN2, and CCN6 in osteoclast physiology are not known but we have recently shown that recombinant human (rh)CCN1 inhibits osteoclastogenesis in vitro. The aim of this study was to determine: 1) the expressions of all six members of the CCN protein family in osteoblasts and osteoclasts; 2) the functions of recombinant human CCN2, CCN6 in osteoclastogenesis; 3) whether osteoblast-derived CCN1 may mediate the effect of CCN1 on osteoclast formation and the roles of osteoblast-derived CCN1 and/or osteoclast-derived CCN1 in osteoblast and/or osteoclast differentiation; 4) which signalling pathways are involved in the function of CCN1 in osteoclasts and osteoblasts. We found CCN1-5 but not CCN6 expressed in murine osteoclasts and osteoblasts. All six members were expressed in human OA osteoblasts but only CCN1-3 were detected in human osteoclasts using quantitative RT-PCR. rhCCN1 (in agreement with our previous observations), and also 2 and 6 inhibited human and mouse osteoclast formation in a concentration-dependent manner. We generated and validated an expression construct to specifically overexpress CCN1 in osteoblasts. Incorporation of CCN1-specific siRNA reduced CCN1 expression to between 12.5% and 50% of control osteoblast cultures. In both co-cultures with direct contact between osteoblasts and osteoclast co-cultures as well as Transwell cultures, overexpression of CCN1 in osteoblasts decreased the formation of TRAP positive multinucleated osteoclast-like cells, while siRNA mediated knockdown of CCN1 in the osteoblasts resulted in increased osteoclast-like cell formation. These data suggest that osteoblast-derived CCN1 is a secreted negative regulator of osteoclastogenesis. Moreover, overexpression or knockdown of CCN1 in osteoclast precursors inhibited or increased osteoclast differentiation whilst overexpression or knockdown CCN1 in osteoblasts increased or inhibited osteoblast mineralization respectively. Further investigation found that CCN1 increased Wnt and MAPK signalling in osteoblasts cultured in mineralization medium and inhibited Wnt and IGF-1 signalling during osteoclast differentiation. In conclusion, paracrine and autocrine effects of CCN1 have been demonstrated in osteoclasts and osteoblasts in this study and Wnt, MAPK, amd IGF-1 signalling pathways, may be involved in these effects.
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Tran, Anh Nhi. "Examining the role of autophagy in osteoclast function." Thesis, University of Aberdeen, 2018. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=238273.
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Osteoclasts are cells that degrade bone, by forming a ruffled border (RB) membrane, contained within an actin-rich attachment site (the sealing zone; SZ). Lysosomal vesicles fuse to the RB, and release their contents into the extracellular space to degrade bone matrix. LC3, a marker of autophagosomes, localises to the RB, implying that either canonical autophagy (i.e. autophagosomes) or non-canonical autophagy (a process where LC3 localises to non-autophagic membrane) is involved in the resorptive function of osteoclasts. To examine this in detail, this study used a model with reduced canonical autophagy (FIP200 conditional knockout mouse), and two non-canonical autophagy deficient models (Rubicon knockdown in RAW 264.7-cell derived osteoclasts and an Atg16L1 WD40 domain knockout mouse). Using advanced imaging and molecular techniques I examined whether impairing either process affected LC3 RB localisation and resorption. Reducing canonical autophagy through FIP200 deficiency did not significantly affect GFP-LC3 RB localisation or bone homeostasis. However, impairing non-canonical autophagy resulted in a trend towards increased resorption in vitro. In Atg16L1 WD40 domain-deficient osteoclasts, this may be due to the significantly larger SZs formed in the mutants, which were often stable and contained LysoTracker-positive acidic vesicles within them, putatively signalling increased resorption. As LC3 was frequently observed at the RB, I then examined the LC3-interacting lysosomal adaptor protein, PLEKHM1. I showed that in the PLEKHM1 functional knockout mouse model (R714 STOP), osteoclasts still form RBs but have impaired resorption in vitro. Detailed analysis of multiple aspects of resorption in PLEKHM1 deficient osteoclasts, was required to uncover these defects which may underlie the osteopetrotic phenotype observed in PLEKHM1 deficient mice. Overall this work reveals the potential role of non-canonical autophagy in osteoclast function. Additional dissection of this pathway in osteoclasts may uncover further new insights regarding the regulation of bone resorption and defects underlying bone disorders.
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Ek-Rylander, Barbro. "Studies on the structure and function of the purple acid phosphatase of rat osteoclasts." Stockholm : Dept. of Immunology, Microbiology, Pathology and Infectious Diseases, Karolinska Institute, 1995. http://catalog.hathitrust.org/api/volumes/oclc/33858972.html.
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Pitombo, Jonleno Coutinho Paiva. "Impacto de andrógenos na diferenciação e atividade de osteoclastos em cultura celular /." Araraquara, 2016. http://hdl.handle.net/11449/138870.
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Orientador: Luis Carlos SpolidorioBanca: Ticiana Sidorenko de Oliveira Capone
Banca: Thallita Pereira Queiroz
Resumo: Os mecanismos de ação dos andrógenos sobre homeostase e regulação das células que participam do turnover ósseo em fêmeas ainda são pouco compreendidos. Este trabalho teve como principal objetivo avaliar a participação de andrógenos na diferenciação e atividade de osteoclastos in vitro. Células totais de medula óssea de fêmur e tíbia de camundongos fêmeas foram utilizadas como fonte de células precursoras de osteoclastos, sendo cultivadas utilizando-se α-MEM suplementado e em presença de RANK-L (30ng/mL) e M-CSF (50ng/mL). As células foram tratadas com testosterona (T) diidrotestosterona (DHT) e antagonistas de receptores de hormônios sexuais, como flutamida (FLU) e fulvestranto (FUL). O anastrozol (ANA) foi usado para inibição da enzima aromatase e o etanol (0,01%) foi utilizado como controle. Após cinco dias, as células foram fixadas, coradas com TRAP e contadas, considerando-se células TRAP-positivas com 3 ou mais núcleos. Para o ensaio de atividade, foram utilizadas placas revestidas com fosfato de cálcio inorgânico e a área de reabsorção foi calculada com o auxílio de software. O estágio de diferenciação osteoclástica foi avaliado por RT-qPCR e a modulação da expressão de receptores para hormônios sexuais foi avaliada por Western Blot. Os andrógenos (T e DHT) não exerceram efeitos sobre a diferenciação e atividade de osteoclastos (ANOVA; p>0,05). Por outro lado, os tratamentos com ANA, FLU e FUL, associados ou não a T, regularam positivamente a diferenciação e atividade d... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The action mechanisms of androgens on homeostasis and the regulation of cells that participate in bone turnover in females are still poorly understood. This study had as main objective to evaluate the participation of androgens in the differentiation and activity of in vitro osteoclasts. Total bone marrow cells from femur and tibia of female mice were used as a source of precursor cells of osteoclasts, they were cultivated using supplemented α-MEM and in the presence of RANK-L (30ng/mL) and M-CSF (50ng/mL). The cells were treated with testosterone (T), dihydrotestosterone (DHT) and antagonists of sexual hormone receptors such as flutamide (FLU) and fulvestrant (FUL). Anastrozole (ANA) was used for inhibiting the aromatase enzyme and ethanol (0.01%) was used as a control. After five days, the cells were fixed, colored with TRAP and counted, considering TRAP-positive cells those ones containing 3 or more nuclei. For the activity assay, were used plaques covered with inorganic calcium phosphate and the area of reabsorption was calculated with the assistance of a software. The osteoclast differentiation stage was evaluated by RT-qPCR and the modulation of the expression of receptors for sexual hormones was assessed by Western Blotting. The androgens (T and DHT) did not exert effects in differentiation and activity of osteoclasts (ANOVA, p> 0.05). On the other hand, the treatments with ANA, FLU and FUL, associated or not to T, positively regulated the differentiation and activity ... (Complete abstract click electronic access below)
Mestre
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Hu, Rong. "Regulation of osteoclast differentiation by transcription factors MITF, PU.1 and EOS." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1166644761.
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Napimoga, Marcelo Henrique. "Indução de osteoclastogene independente de RANK por uma fração lipidica isolada de Porphyromonas gingivalis." [s.n.], 2005. http://repositorio.unicamp.br/jspui/handle/REPOSIP/288635.
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Orientador: Reginaldo Bruno GonçalvesTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-08-05T08:24:42Z (GMT). No. of bitstreams: 1 Napimoga_MarceloHenrique_D.pdf: 980827 bytes, checksum: d6e89bf1b359918d8c502f0a7367d2c1 (MD5) Previous issue date: 2005
Resumo: Porphyromonas gingivalis (Pg) sintetiza diversas classes de fosfolipídeos. Entretanto, pouco se sabe sobre os efeitos biológicos que estes lipídeos exercem sobre a reabsorção óssea. Células precursoras de osteoclastos de camundongos, incluindo RAW264.7 e células da medula óssea, foram estimuladas com um lipídeo recém caracterizado isolado de Pg (F24), LPS de Pg, RANKL e um isômero estrutural isolado de mamíferos (DMPE), seguida de coloração das células por fosfatase ácida (TRAP) para detecção de osteoclastos. A formação de lacunas de reabsorção em discos de dentina foi realizados para identificar a presença de osteoclastos maduros. Proteínas de células precursoras RAW264.7 com afinidade a F24 foram identificadas como sendo nucleolin através de espectometria de massa, e confirmada sua presença através de ensaio em microscopia confocal. Utilizando Western-blot e RNAi, a sinalização intracelular desencadeada pela F24 durante a osteoclastogênese foi analisada. A F24 mas não o LPS ou DMPE induziu números significantemente maiores de células multinucleadas TRAP+ quando comparado ao RANKL, assim como a formação de lacunas de desmineralização em discos de dentina, o qual não foi inibida pelo repressor do RANKL, a OPG. LPS de Pg, mas não a F24, induziu a produção de TNF-a, IL-1ß e IL-6 pelas células RAW264.7. Microscopia confocal mostrou que a F24 está co-localizada com este receptor na superfície de células RAW264.7. A F24 induziu a fosforilação de maior intensidade do p38 e JNK comparado ao RANKL. Este novo fosfolipídeo isolado de P. gingivalis, distinto do LPS, mostrou-se capaz de induzir osteoclastogênese utilizando nucleolin como a molécula receptora através de uma via independente do RANK
Abstract: Porphyromonas gingivalis (Pg) synthesizes several classes of complex phospholipids in addition to LPS. However, little is known about the biological effects of these phospholipids on bone resorption. Mouse osteoclast precursors cells including, RAW264.7 and bone marrow cells, were stimulated with phospholipids isolated from Pg (F24), Pg LPS, RANKL and a mammalian structural isomer (DMPE). Tartrate-resistant acid phosphatase (TRAP) staining and a dentine-pit formation assay were used for the identification of activated mature osteoclasts. F24¿s counter-ligand expressed on RAW264.7 cells were identified by affinity purification and mass-spectrometry based proteomics, and counter-examined by a confocal microscopy. Using Western-blot, signaling pathways triggered by F24 during osteoclastogenesis were examined with RNAi technology. F24, but not LPS or DMPE, induced significantly higher number of TRAP+ multinuclear cells from osteoclast precursors than RANKL, along with dentine-pit formation, which was not inhibited by RANKL decoy receptor OPG. Pg LPS, but not F24, induced TNF-a, IL-1ß and IL-6 by RAW264.7 cells. Proteomics analyses identified nucleolin as a ligand for F24. Confocal microscopy revealed the co-localization of F24 and its ligand on the surface of RAW264.7 cells. F24 induced stronger phosphorylation of p38 MAP kinase than RANKL. A novel P. gingivalis phospholipid that is distinct from LPS represents a new class of RANK-independent osteoclast differentiation factor
Doutorado
Microbiologia e Imunologia
Doutor em Biologia Buco-Dental
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Marques, Marcelo Rocha 1976. "Avaliação do efeito do PTH nas citocinas IL-1beta, IL-6, TNF-alfa, nas metaloproteinases da matriz 2 e 9, e na atividade osteoclastica em ratos com periodontite induzida." [s.n.], 2007. http://repositorio.unicamp.br/jspui/handle/REPOSIP/290043.
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Orientador: Silvana Pereira BarrosTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-08-11T01:50:55Z (GMT). No. of bitstreams: 1 Marques_MarceloRocha_D.pdf: 1159258 bytes, checksum: b480791f2296297d8bf75042e1af33c1 (MD5) Previous issue date: 2007
Resumo: O hormônio paratireóideo (PTH), um mediador da remodelação óssea, é o principal regulador da homeostasia do cálcio, sendo capaz de promover reabsorção e aposição ósseas. Em trabalhos realizados recentemente, foi verificado que o tratamento com PTH, administrado de maneira intermitente, diminuiu a perda óssea e também células inflamatórias no tecido gengival, em ratos com periodontite induzida. No intuito de melhor explicar os resultados obtidos nesses trabalhos, doença periodontal foi induzida em primeiros molares de ratos e após 15 e 30 dias de tratamento com PTH os animais foram sacrificados com o objetivo investigar o periodonto para: 1) avaliar a expressão gênica, por meio de RT-PCR, e localizar, por meio de reação imunohistoquímica, algumas citocinas inflamatórias (IL-1b, IL-6, TNF-alfa) e certas metaloproteinases da matriz (MMP-2 e MMP-9), 2) verificar, por meio de histoquímica, a atividade de fosfatase ácida tatarato resistente (TRAP) na superfície óssea alveolar, e 4) medir a atividade gelatinolítica das MMPs 2 e 9. Como resultados, observou-se que o PTH diminuiu a expressão de mRNA para MMP-2 (15 e 30 dias experimentais) e IL-6 (30 dias experimentais) nas gengivas dos animais estudados; que a localização de IL-1beta, MMP-2 e MMP-9 se deu basicamente no tecido conjuntivo da gengiva, e que a IL-6 foi detectada principalmente na superfície do osso alveolar. O número de células TRAPpositivas foi diminuído nos animais tratados 30 dias com PTH, e atividade gelatinolítica de MMP-9 foi diminuída após 15 dias de tratamento com o hormônio. Dentro dos limites deste estudo, pode-se concluir que a administração intermitente de PTH pôde modular moléculas (MMP-2, MMP-9, IL-6 e número de células TRAP-positivas) que têm relação com degradação tecidual na doença periodontal em ratos
Abstract: Intermittent Parathyroid hormone (PTH) administration has been used as an anabolic agent, and when it is administrated in rats with induced periodontitis, it is able to decrease alveolar bone loss, and reduce the inflammatory cells on the marginal gingival tissue. A possible action of the PTH in cytokines and metalloproteinase (MMPs), related with breakdown tissue in periodontitis, was hypothesized. After inducing periodontitis with cotton ligature in rats and PTH intermittent treatment during 15 and 30 days, inflamed gingival tissue was removed, and a decrease of IL-6 and MMP-2 mRNA expression was observed by semi-quantitative RT-PCR analysis. Zymography assay demonstrated that PTH treatment decreased MMP-9 activity after 15 experimental days. After jaw decalcification, TRAP positive cells were found in higher number on the alveolar bone surface in the group without PTH treatment, after 30 experimental days. Using immunohistochemistry, MMP-2, MMP-9, IL-beta and TNF-alpha were identified in all groups and they were found on the gingival tissue and IL-6 on alveolar bone surface. Within the limits of this study, it can be concluded that intermittent PTH administration may modulate IL-6, MMP-2, MMP-9 and TRAP-positive cells in experimental periodontitis
Doutorado
Histologia e Embriologia
Doutor em Biologia Buco-Dental
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Bighetti, Bruna Barros. "Avaliação das citocinas TNF-α, RANKL e OPG e do número de osteoclastos no reparo de defeito ósseo em calvária de ratos diabéticos tratados com matriz óssea desmineralizada." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/25/25149/tde-25112016-114638/.
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Neste trabalho, foi avaliado a participação dos osteoclastos bem como a ação das citocinas RANKL, OPG e TNF-α durante a formação e remodelação óssea em defeitos ósseos de tamanho crítico em ratos normoglicêmicos e diabéticos tratados ou não com a MAOD. Para isso, foram utilizados 250 ratos machos Wistar. Trinta ratos foram utilizados para coleta dos fêmures e tíbias, os quais foram processados para obtenção da MAOD. Os demais 220 ratos foram divididos em Grupo Não Diabétido (CTL, n=110) e Grupo Diabético (DIAB, n= 110) induzido pela aplicação de uma dose única de 47 mg/Kg de massa corporal de estreptozotocina. Um defeito transósseo de 8 mm de diâmetro foi realizado nos ossos parietais dos ratos, sendo que, nos subgrupos CTL MAOD e DIAB MAOD, os defeitos foram preenchidos com MAOD e nos grupos CTL COAG e DIAB COAG apenas com coágulo sanguíneo. Após 0, 7, 14, 21 e 42 dias, as calotas cranianas foram coletadas para determinação da densidade de volume, número de osteoclastos/mm2 na área do defeito, quantificação por imunoistoquimica e expressão do RNAm para as proteínas RANKL, OPG e TNF-α. Os resultados para volume do tecido ósseo neoformado foi maior nos grupos CTL COAG e CTL MAOD, bem como no grupo DIAB MAOD quando comparado com DIAB COAG (CTL MAOD > CTL COAG e DIAB MAOD > DIAB COAG). O número de osteoclastos nos grupos CTL aumentaram significantemente (3,69 osteoclasto/mm2), enquanto que nos grupos MAOD aumentaram gradualmente até os 42 dias (2,8 osteoclasto/mm2). Os resultados para imunomarcação mostraram que a MAOD promove 1,28 vezes maior expressão de OPG, bem como de TNF-α tanto no grupo CTL (1,59 vezes) como no DIAB (1,76 vezes). Os resultados para expressão do RNAm para OPG mostrou que a média dos valores do grupo COAG comparado com a do grupo MAOD foi 1,91 vezes maior no grupo COAG. Já os valores para expressão de RANKL permaneceram constantes no grupo DIAB MAOD, com aumento significativo de 2,57 vezes aos 42 dias, sendo 4,3 vezes maior, quando comparado com a média dos outros grupos no mesmo período. Conclui-se que nos animais normoglicemicos, o tratamento com a MAOD aumenta a expressão de OPG, RANKL e TNF-α, assim como a atividade osteoclástica, promovendo reabsorção da MAOD e formação de tecido ósseo, enquanto que nos animais diabéticos, a atividade osteoclástica foi reduzida, sem alteração nos níveis de OPG e RANKL, reduzindo a reabsorção da MAOD e consequentemente da formação óssea.Participation of osteoclasts was evaluated in reabsorption process of demineralized allogenic bone matrix (DABM) as well as the activity of cytokines RANKL, OPG and TNF- α during formation and bone remodeling in critial size defect of normoglycemic and diabetic rats treated or not with DABM. Therefore, 250 male Wistar rats were used. Thirty rats had femurs and tibias collected and processed to obtain DABM. 220 rats were divided into control group (CTL, n=110) and diabetic group (DIAB, n= 110) injected by a single dose of 47 mg/Kg of body weight streptozotocin. Were made 8mm bone defect on skulls of rats, in subgroups CTL DABM and DIAB DABM, defects were filled with DABM and subgroups CTL CLOT and DIAB CLOT were filled with blood clot. After 0, 7, 14, 21 and 42 days, the skulls were collected to determine the volume density, number of osteoclasts/mm2 into defects area, quantification by immunohistochemistry and RNAm expression of RANKL, OPG and TNF-α cytokines. The results of volume density of newly formed bone was higher in CTL CLOT and CTL DABM, as well as in DIAB DABM compared to DIAB CLOT (CTL DABM > CTL CLOT and DIAB DABM > DIAB CLOT). The number of osteoclasts in CTL groups increased to 3,69 osteoclasts/mm2, while in subgroups treated with DABM gradually increased up until 42 days (2,8 osteoclasts/mm2). Immunohistochemistry showed that DABM promotes an increase of 1.28-fold of OPG expression, as well as TNF-a expression in CTL group (1.59-fold) and DIAB group (1.76-fold). The results of RNAm expression of OPG showed that the average values of the CLOT subgroup compared to the average values of DABM subgroup was 1.91- fold higher in CLOT subgroup. The values of RANKL RNAm expression increase 2.57-fold at 42 days, being 4.3-fold higher than the average os the other groups in the same period. In conclusion, in the normoglicemic animals (CTL group), the treatment with DABM increase the expression of OPG, RANKL and TNF-α as the activity of osteoclasts, leading to DABM resorption and bone tissue formation, while in diabetic animals, the osteoclast activity was reduced, without changes in the leves of OPG and RANKL, decreasing DABM resorption and bone formation.
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Yamamoto, Fernanda Paula. "Estudo da presença de osteonecrose na madíbula após exodontia de molares em ratos tratados com alendronato de sódio." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/23/23141/tde-09112010-111814/.
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A osteonecrose dos ossos maxilares relacionada ao uso prolongado de bisfosfonatos (OMRB), associada a procedimentos cirúrgicos, constitui uma entidade com menos de dez anos de ocorrência na clínica estomatológica. Os bisfosfonatos (BFs) são medicamentos antireabsortivos altamente efetivos no tratamento de diversas doenças ósseas, além de metástases. Embora a maioria dos casos de OMRB tenha sido relatada após o uso de potentes BFs da terceira geração, o alendronato de sódio (ALN), um bisfosfonato de segunda geração, é amplamente utilizado no tratamento e prevenção de doenças ósseas. O presente estudo experimental in vivo visou analisar a possível presença de OMRB no processo alveolar de ratos tratados com ALN após exodontia do segundo molar inferior. Para tanto, utilizou-se 30 ratos Wistar, machos, com 7 semanas de vida, divididos em dois grupos, ALN (tratado com alendronato) e CTL (controle, tratado com solução salina). O ALN foi administrado diariamente por injeção subcutânea na dose de 2,5 mg/kg peso por 14 dias. Nesse momento, foi realizada a exodontia, havendo sido eutanasiados cinco animais de cada grupo, 7, 14 e 21 dias após a exodontia. Após a eutanásia, a região da mandíbula foi fixada em 2,5% formaldeído e 2% glutaraldeído, em tampão cacodilato 0,1M - pH 7,4 e descalcificada em EDTA a 4,13% por 30 dias. Os espécimes foram analisados em cortes corados em HE, e foi realizada histomorfometria para análise da reabsorção da crista alveolar mesial e distal, além do tecido ósseo neoformado no interior do alvéolo, que também foi analisado através de imuno-histoquímica para as proteínas não colágenas OPN e BSP e para evidenciação de vasos sanguíneos neoformados com o anticorpo anti-CD105. Os osteoclastos foram evidenciados através de histoquímica para fosfatase ácida resistente ao tartarato (TRAP). Além disso, as células e eventos da reparação foram examinados por microscopia eletrônica de transmissão. Os resultados mostraram que os animais tratados com ALN exibiram OMRB de grau zero em todos os períodos estudados, além da presença de osteoclastos latentes TRAP-positivos e histomorfometria, onde a não reabsorção das cristas alveolares ocasionaram a permanência do osso alveolar, em contato com bactérias. Observou-se ainda, um claro atraso na formação óssea, quando comparado ao controle, além da diminuição dos vasos sanguíneos nos estágios iniciais. A localização e o padrão de marcação para as proteínas OPN e BSP foram considerados normais, observados evidente marcação em áreas ósseas imaturas e em linhas cimentantes. Assim, concluiu-se que o ALN administrado neste protocolo provocou OMRB leve, provavelmente causada pela diminuição da angiogênese, presença de osso alveolar não remodelado expostos a colônias bacterianas, além da diminuição da atividade osteoblástica.The bisphosphonate-related osteonecrosis of the jaw (BRONJ) associated with surgery procedures, is an entity with less than 10 years of occurrence in dental practice. The bisphosphonates (BFs) are anti-resorbing drugs highly effective in the treatment of several bone diseases, including metastasis. Although the majority of cases of BRONJ had been reported with the potent third generation of BFs, sodium alendronate (ALN), a second generation bisphosphonate, is widely used for treatment or prevent bone diseases as osteoporosis. The present experimental study aimed to analyze the possible presence of BRONJ in the alveolar process of rats treated with ALN following extraction of the mandibular second molar. For this, we used thirty 7-week-old male Wistar rats, divided into two groups, ALN (treated with alendronate) and CTL (control, treated with saline solution). The administration of ALN received daily subcutaneous injection at a dose of 2.5 mg/kg for 14 days. At that moment, the extraction was performed. After 7, 14 e 21 days of surgery, five animals of each group were euthanized and the mandibular region fixed in 2.5% formaldehyde + 2% glutaraldehyde in cacodylate buffer 0.1M - pH 7.4 and decalcified in 4.13% EDTA for 30 days. The specimens were morphologically analyzed in HE stained sections, and, histomorphometry was used to analyze the resorption of mesial and distal alveolar crests, as well as the bone formed into the alveolus. Immunohistochemistry for the noncollagenous proteins OPN and BSP and for CD105 to revel neoformed blood vessels was also performed. The osteoclasts were revealed through tartrate-resistant acid phosphatase (TRAP) histochemistry. Moreover, the cells and events of alveolar healing were examined by transmission electron microscopy. The results showed light degree of BRONJ in the ALN treated animals at all the studied periods, and the presence of latent osteoclasts near the non-resorbing crest alveolar. Bacteria were observed in contact with the non-resorbed alveolar bone. An evident delay in bone formation when compared to control group was also observed, as well as the decrease of blood vessels in early stages. The distribution of immature bone and in cement lines of OPN and BSP was considered normal. Thus, we concluded that the present protocol of ALN yielded light BRONJ areas, probably by reduced angiogenesis, lock of remodeling of alveolar, presence of bacterial colonies and evident decreased osteoblastic ativitity.
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Shaikh, Atik Badshah. "Role of CKIP-1 in suppression of osteoblast mediated bone repair in a collagen induced non-human primate arthritis model." HKBU Institutional Repository, 2017. https://repository.hkbu.edu.hk/etd_oa/478.
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Rheumatoid arthritis (RA) is a systemic, inflammatory disease, which predominantly affects multiple joints. RA is characterized by swollen joints and peri-articular bone erosion. Conventional RA treatments have shown to reduce inflammation and slow down bone erosion, but repair of bone erosion is limited. Additionally, failure to repair for bone erosion in rheumatoid arthritis (RA) is caused by inadequate osteoblast-mediated bone formation resulting from focal inflammatory environment. Hence, there is immediate need to facilitate greater insight and develop a new therapeutic strategy to aid osteoblast -mediated repair of bone loss in RA. CKIP-1 is an intracellular inhibitor, that can negatively regulate osteoblast lineage cells differentiation and activity. CKIP-1 levels were found to be aberrantly over expressed in bone specimens from RA patients and arthritis mice, which was associated with reduced bone formation and increased disease severity. By genetic approach, overexpressed CKIP-1 in osteoblast exacerbated bone erosion and articular inflammation in CIA mice, whereas deficiency of CKIP-1 in osteoblast alleviates bone erosion in CIA mice. By pharmacological approach, RNA interference-based silencing of osteoblastic CKIP-1 led to bone formation-mediated reparative process at erosive site and reduced articular inflammation in arthritis induced mice. To extend above findings to a more relevant species that more closely resemble humans, we aimed to investigate the role of osteoblastic Casein kinase-2 interacting protein-1 (CKIP-1) in failure to repair bone erosion in non-human primate (NHP) arthritis model in this study. We found that CKIP-1 mRNA expression in osteoblasts of arthritic NHP was significantly suppressed by CKIP-1 siRNA treatment. Moreover, silencing of CKIP-1 in osteoblast of arthritis monkey improved clinical signs such as reduction in arthritis score, joint swelling and increase in body weight. Similarly, suppression of osteoblastic CKIP-1 resulted in better organized bony and articular structure with, fewer bone erosion sites as observed in x ray and micro CT images. Moreover, we found increase in bone mass, bone formation parameters such as BFR/BS and MAR and histological examination revealed attenuation of peri articular bone erosion and intraarticular inflammation in CKIP-1 siRNA treated arthritis monkeys. Taken together, these data strongly suggest that highly expressed osteoblastic CKIP-1 plays an important role in failure to repair articular bone erosion by inhibiting bone formation in RA. Targeting osteoblastic CKIP-1 could serve as a new therapeutic strategy by bone repair augmentation in RA.
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Buckley, Katherine Anne. "The effects of extracellular nucleotides and parathyroid hormone on bone remodelling." Thesis, University of Liverpool, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366663.
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Osteoclasts are multinucleated, terminally differentiated cells found in bone tissue, which have the unique ability to resorb calcified substrates. The study of human osteoclasts has been restricted in the past due to difficulties in obtaining these cells. Recently, however, cell culture techniques have been devised that allow human osteoclasts to be grown in culture from their mononuclear precursors found in peripheral blood, therefore providing a constant supply of these cells. These cultures are based on the discovery of RANKL (receptor activator for NFJ(B ligand), which has recently been shown to playa central role in osteoclast formation and activation. This thesis has initially characterised cells grown in such cultures to confirm that they are authentic human osteoclasts, possessing osteoclast markers and with the ability to resorb calcified substrates. Once these cultures were established, and the authenticity of human osteoclasts grown in these cultures was confirmed, these cells were used to study the effects of extracellular nucleotides on human osteoclast acti vity. Adenosine-5' -triphosphate (ATP) is well known as an intracellular energy source. This and other nucleotides, however, also exist extracellularly, where they are agonists at a group of receptors termed P2 receptors. This receptor family is subdivided into P2X ligand gated ion channels, and P2Y G-protein coupled receptors. Bone-forming osteoblast cells, and boneresorbing osteoclast cells both express multiple SUbtypes of these receptors. Studies examining the effects of extracellular nucleotides on osteoclasts have been largely limited to non-human cells in the past due to the difficulty in obtaining human osteoclasts. This thesis, therefore, has examined the effects of extracellular nucleotides on human osteoclast activity. Human osteoclasts and their precursors were shown to express mRNA for nearly all of the P2Y and P2X receptor subtypes. ATP was found to both activate human osteoclast formation, by acting directly on P2X receptors expressed by osteoclast precursors, and to stimulate osteoclast resorption indirectly by acting at osteoblast-expressed P2Y) receptors to induce elevated RANKL expression by these cells. Activation of P2Y receptors expressed by osteoclasts was shown to result in downstream activation of MAPKinase pathways. Parathyroid hormone (PTH) is considered to be one of, if not the most important systemic factor in the regulation of bone. Co-stimulation of UMR-I 06 osteoblast-like cells with this hormone and P2Y) agonists resulted in potentiation of P2Y) agonist-induced [Ca2+Ji release by PTH, while PTH alone produced no [Ca2+]j elevation at all. The mechanism of this potentiation was attributed to Gs activation following PTH receptor stimulation, Gq having no involvement. Co-stimulation of these cells by PTH and P2Y) agonists also resulted in synergistic immediate early gene expression. These findings suggested that extracellular nucleotides are able to sensitize osteoblasts to the actions of PTH, providing a mechanism for localizing the response to this systemic hormone in bone, consistent with the discrete pattern of remodelling observed in the skeleton. Finally, the involvement of PTH on osteoclast formation was investigated. PTH was found to inhibit this process via activation of contaminating T lymphocytes present in osteoclast cultures. In conclusion this thesis presents evidence to suggest that extracellular nucleotides are important local signaling molecules in bone, affecting both osteoclast and osteoblast activity, alone and in combination with systemic factors such as PTH. Additionally, a novel action of PTH acting via lymphocytes to affect osteoclast formation is described.
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Bord, Sharyn. "The role of matrix metalloproteinases in human bone modelling and remodelling." Thesis, Anglia Ruskin University, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243631.
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Midha, Swati. "Osteogenesis in porous biomaterials for bone regeneration." Thesis, Ulster University, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.674920.
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Gartland, Alison. "The role of the P2X7 receptor in bone cell formation." Thesis, University of Liverpool, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343968.
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Neale, Susan Dorothy. "The role of macrophages in pathological bone resorption /." Title page, table of contents and abstract only, 1998. http://web4.library.adelaide.edu.au/theses/09MSM/09msmn348.pdf.
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Waring-Green, Victoria. "Characterisation of a novel antigen found in osteoclasts and tumour cells." Thesis, University of Liverpool, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.526832.
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Williams, Allan James. "Thyroid hormone and thyroid stimulating hormone actions in osteoblasts and osteoclasts." Thesis, Imperial College London, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.497252.
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Hoflack, Bernard, Pierre Jurdic, Thilo Riedl, Anne Gallois, and Maria Arantzazu Sanchez-Fernandez. "Osteoclasts control osteoblast chemotaxis via PDGF-BB/PDGF receptor beta signaling." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-184120.
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BACKGROUND:
Bone remodeling relies on the tightly regulated interplay between bone forming osteoblasts and bone digesting osteoclasts. Several studies have now described the molecular mechanisms by which osteoblasts control osteoclastogenesis and bone degradation. It is currently unclear whether osteoclasts can influence bone rebuilding.
METHODOLOGY/PRINCIPAL FINDINGS:
Using in vitro cell systems, we show here that mature osteoclasts, but not their precursors, secrete chemotactic factors recognized by both mature osteoblasts and their precursors. Several growth factors whose expression is upregulated during osteoclastogenesis were identified by DNA microarrays as candidates mediating osteoblast chemotaxis. Our subsequent functional analyses demonstrate that mature osteoclasts, whose platelet-derived growth factor bb (PDGF-bb) expression is reduced by siRNAs, exhibit a reduced capability of attracting osteoblasts. Conversely, osteoblasts whose platelet-derived growth factor receptor beta (PDGFR-beta) expression is reduced by siRNAs exhibit a lower capability of responding to chemotactic factors secreted by osteoclasts.
CONCLUSIONS/SIGNIFICANCE:
We conclude that, in vitro mature osteoclasts control osteoblast chemotaxis via PDGF-bb/PDGFR-beta signaling. This may provide one key mechanism by which osteoclasts control bone formation in vivo.
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Hoflack, Bernard, Pierre Jurdic, Thilo Riedl, Anne Gallois, and Maria Arantzazu Sanchez-Fernandez. "Osteoclasts control osteoblast chemotaxis via PDGF-BB/PDGF receptor beta signaling." PLOS one, 2008. https://tud.qucosa.de/id/qucosa%3A28994.
Full textAbstract:
BACKGROUND:
Bone remodeling relies on the tightly regulated interplay between bone forming osteoblasts and bone digesting osteoclasts. Several studies have now described the molecular mechanisms by which osteoblasts control osteoclastogenesis and bone degradation. It is currently unclear whether osteoclasts can influence bone rebuilding.
METHODOLOGY/PRINCIPAL FINDINGS:
Using in vitro cell systems, we show here that mature osteoclasts, but not their precursors, secrete chemotactic factors recognized by both mature osteoblasts and their precursors. Several growth factors whose expression is upregulated during osteoclastogenesis were identified by DNA microarrays as candidates mediating osteoblast chemotaxis. Our subsequent functional analyses demonstrate that mature osteoclasts, whose platelet-derived growth factor bb (PDGF-bb) expression is reduced by siRNAs, exhibit a reduced capability of attracting osteoblasts. Conversely, osteoblasts whose platelet-derived growth factor receptor beta (PDGFR-beta) expression is reduced by siRNAs exhibit a lower capability of responding to chemotactic factors secreted by osteoclasts.
CONCLUSIONS/SIGNIFICANCE:
We conclude that, in vitro mature osteoclasts control osteoblast chemotaxis via PDGF-bb/PDGFR-beta signaling. This may provide one key mechanism by which osteoclasts control bone formation in vivo.
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Hu, Shiqiong. "Mechanics and Dynamics of Cell Adhesion : Experimental Study of the Osteoclasts." Lyon, Ecole normale supérieure, 2010. http://www.theses.fr/2010ENSL0594.
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Osteoclasts are large, multinucleated cells, which resorb mineralized bone. When an osteoclast encounters a substrate, dot-like actin-rich structures, the podosomes, appear and assemble into clusters, rings or a belt. We experimentally investigate, from a cell population to a single podosome, their function and dynamics. Over a cell population, kinetic measurements show that the cell surface area A scales as A ~ K2, where K is the number of nuclei, indicating a flat morphology. By defining quantities that account for the spatial distribution of the actin within the cell, we demonstrate that the podosomes organization only depends on the time after differentiation, and not on K. In a single osteoclast, the observation of a strong coupling between cell spreading and podosomes formation lead us to propose that podosomes play an important role in osteoclast motility. Analysis of osteoclast migration, and the forces it applies on the substrate, demonstrates that the internal dynamics of the actin within the cell does not only correlate with cell migration, but drives it. Finally, in order to understand the internal dynamics of a single podosome, we improved the model of Biben et al. (2005) by considering on the one hand, actin polymerization, and on the other hand, diffusion and attachment kinetics of the gelsolin, an actin severing protein. We find that podosomes are mainly governed by the actin dynamics, regardless of gelsolin concentrationLes ostéoclastes sont des cellules multinucléées, responsables de la résorption osseuse. Quand ils sont déposés sur un substrat, des structures ponctuelles riches en actine, les podosomes, apparaissent et s'assemblent en clusters, anneaux ou ceinture. Nous avons étudié expérimentalement leur fonction et leur dynamique, depuis une population entière jusqu'à l'échelle d'un unique podosome. Sur une population de cellules, des mesures cinétiques montrent que la surface de la cellule A varie comme A ~ K2, où K est le nombre de noyaux ; ce résultat indique une forme aplatie. Par ailleurs, la mesure de quantités qui prennent en compte l'organisation spatiale de l'actine dans la cellule montre que l'organisation des podosomes ne dépend que du temps écoulé après différentiation, et non de K. Dans un seul ostéoclaste, l'observation d'un fort couplage entre l'étalement d'une cellule et la formation des podosomes nous a conduit a suggérer que les podosomes jouent un rôle important dans la mobilité des ostéoclastes. L'analyse de la migration d'ostéoclastes, ainsi que des forces appliquées sur le substrat, montre que la dynamique interne de l'actine dans la cellule est non seulement corrélée avec la migration cellulaire, mais la gouverne. Enfin, afin de comprendre la dynamique interne d'un podosome, nous avons amélioré le modèle de Biben et al. (2005), en prenant en compte d'une part, la polymérisation de l'actine, et d'autre part, la diffusion et la cinétique d'attachement de la gelsoline, une protéine responsable de la coupe des filaments d'actine. Nous montrons que les podosomes sont principalement gouvernés par la dynamique de l'actine, indépendamment de la concentration en gelsoline
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Hollberg, Karin. "Studies on the phenotype and function of osteoclasts using osteopetrotic and rachitic animal models /." Stockholm : Karolinska institutet, 2007. http://diss.kib.ki.se/2007/978-91-7357-313-9/.
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Fernandes, Leandro Araújo [UNESP]. "Ação da terapia fotodinâmica no tratamento da doença periodontal experimentalmente induzida em ratos tratados ou não com nicotina: estudo histológico, histométrico e imunoistoquímico." Universidade Estadual Paulista (UNESP), 2010. http://hdl.handle.net/11449/104707.
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fernandes_la_dr_araca.pdf: 1070170 bytes, checksum: 5b88fec778e5c29fd6d418138e92a16f (MD5)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O objetivo do presente estudo foi avaliar a influência da terapia fotodinâmica (PDT) como coadjuvante ao tratamento não cirúrgico da doença periodontal (DP) experimentalmente induzida em ratos tratados ou não com nicotina. Cento e oitenta ratos foram divididos em 2 grupos de 90 animais cada. Os do Grupo Controle (C) receberam aplicações subcutâneas de soro fisiológico; os do Grupo Nicotina (N) receberam aplicações subcutâneas de nicotina. As injeções foram realizadas duas vezes ao dia e iniciadas 30 dias antes da indução da DP, continuando pós tratamento periodontal até os respectivos períodos de sacrifício. A DP foi induzida através da colocação de ligadura com fio de algodão na região dento-gengival dos primeiros molares inferiores esquerdos. Após 7 dias da indução, a ligadura foi removida e, os animais foram divididos em subgrupos de acordo com os seguintes tratamentos locais: Tratamento I - raspagem e alisamento radicular (RAR) e irrigação com soro fisiológico; Tratamento II - RAR e irrigação com azul de toluidina O (TBO) e Tratamento III – RAR, irrigação com TBO e, após 1 minuto, aplicação do laser em baixa intensidade (LLLT) (GaAsAl, 660 nm, 4J), realizando a PDT. Dez animais de cada subgrupo foram sacrificados aos 7, 15 e 30 dias. Os espécimes foram processados laboratorialmente e analisados histológica, histométrica e imunoistoquímicamente. Aos 7, 15 e 30 dias, em ambos os grupos, o tratamento RAR apresentou um tecido conjuntivo desorganizado, com elevado número de neutrófilos e discreto número de fibroblastos. O tecido ósseo apresentou áreas de necrose e trabéculas ósseas finas. Nos períodos de 7 e 15 dias os animais dos grupos C e N, tratados pela PDT, apresentaram um tecido conjuntivo bem desenvolvido com moderado número de fibroblastos, discreto infiltrado inflamatório crônico e tecido ósseo moderadamente...
The aim of this study was to evaluate the influence of photodynamic therapy (PDT) as an adjunct to non-surgical treatment of periodontal disease (PD) experimental induced in rats treated with or without nicotine. One hundred and eighty rats were divided into 2 groups of 90 animals each. Control group (C) received subcutaneous infusions of saline, group of Nicotine (N) received subcutaneous infusions of nicotine. The injections were performed twice a day and started 30 days before induction of PD, continuing after periodontal treatment until their period of sacrifice. PD was induced by placing cotton ligature in the dento-gingival region of the left lower first molars. After 7 days of induction, the ligature was removed and the animals were divided into subgroups according to the following local treatments: treatment I - scaling and root planing (SRP) and irrigation with saline; Treatment II - SRP and irrigation with O toluidine blue (TBO) and Treatment III - SRP, irrigation with TBO and 1 minute after application of low intensity laser therapy (LLLT) (GaAlAs, 660 nm, 4J), performing the PDT. Ten animals of each subgroup were sacrificed at 7, 15 and 30 days. The laboratory specimens were processed and analyzed histologically, histometrically and immunohistochemically. At 7, 15 and 30 days in both groups, treatment SRP showed a disorganized tissue with high numbers of neutrophils and a small number of fibroblasts. The bone tissue showed areas of necrosis and trabecular bone thin. At 7 and 15 days the animals in groups C and N, treated by PDT, showed a well developed connective tissue with a moderate number of fibroblasts, mild chronic inflammatory infiltrate and bone moderately developed. At 30 days, they presented an intact periodontal ligament, and organized collagen fibers in parallel. The connective tissue was well-developed, healthy and with no inflammatory... (Complete abstract click electronic access below)
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Muguruma, Yukari. "The origin and differentiation of the osteoclast /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/5681.
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Alkindi, Mohammed. "Effects of soluble factors released by oral squamos cell carcinoma on osteoclasts." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103726.
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Objective: Bone invasion represent significant problem in managing head and neck cancers, however the mechanisms of interactions between oral squamous cell carcinoma (OSCC) and bone cells are poorly understood. We hypothesized that tumor cells can directly stimulate osteoclastogenesis. Methods: OSCC cell lines, bone-invasive BHY and metastatic but not bone-invasive HN were cultured and conditioned medium (CM) was collected. Osteoclast formation from RAW 264.7 mouse monocytic cell-line was assessed. Results: When RAW 264.7 were primed with receptor activator of nuclear factor κB ligand (RANKL) and then treated with BHY-CM, marked 2-6 fold induction of osteoclastogenesis was observed. In contrast, HN-CM did not significantly affect osteoclastogenesis. In addition, BHY-CM, but not HN-CM promoted survival of mature osteoclasts. Using pharmacological inhibitors, we found that Protein kinase C (PKC)/Extracellular signal-regulated kinase (ERK)1/2/Mitogen activated protein kinase (MAPK) p38 as well as Phosphatidyl-inositol 3-kinases (PI3K)/Serine/threonine protein kinase Akt/Mammalian target of rapamycin (mTOR) pathways mediate BHY-CM induced osteoclastogenesis. Conclusion: OSCC-cells produce soluble factors that stimulate osteoclastogenesis from RANKL-primed precursors. Tumor-derived factors act by stimulating ERK1/2 and p38 MAPK pathways in osteoclast precursors.Objectif: L'invasion du tissu osseux est un problème majeur dans le traitement des cancers de la tête et du cou, cependant les mécanismes d'interactions entre le carcinome de cellules de squamous oral (OSCC) et les cellules du tissu osseux sont mal compris. Nous avons posé comme hypothèse que les cellules tumorales peuvent stimuler directement le phénomène d'ostéoclastogenèse. Méthodes: Deux différentes populations cellulaires de la lignée OSCC furent utilisées: les cellules BHY ayant un potentiel de colonisation du tissu osseux et les cellules HN ayant un potentiel métastatique mais non colonisant. Ces deux lignées cellulaires ont été cultivées et le milieu de culture conditionné (CM) a été collecté. La formation de cellules ostéoclastiques à partir de cellules de la lignée monocytaire de souris RAW 264.7 a été évaluée. Résultats: Une augmentation significative du phénomène d'ostéoclastogenèse d'un facteur 2 à 6 fut observée lors d'une activation des cellules RAW 264.7 avec RANKL suivit d'un traitement avec BHY-CM. De plus, la survie des cellules ostéoclastiques matures était favorisée en présence de BHY-CM uniquement. L'utilisation d'inhibiteurs pharmacologiques nous a permis de mettre en évidence que la stimulation du phénomène d'ostéoclastogenèse induite par BHY-CM est médiée par les voies de signalisations PKC/ERK/p38 et PI3K/AKT/mTOR. Conclusion : Les cellules OSCC produisent des facteurs solubles stimulant la formation d'ostéoclastes à partir de précurseurs activées par RANKL. Les facteurs dérivés de tumeurs agissent en stimulant les voies de signalisation ERK1/2 et p38 dans les précurseurs ostéoclastiques.
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Lymperi, Stefania. "The role of osteoblasts and osteoclasts in the haemopoietic stem cell niche." Thesis, Imperial College London, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.501195.
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Quang, Jonathan Q. "Bone marrow stromal cell proliferation in mice lacking NFATc1 in mature osteoclasts." Thesis, Boston University, 2013. https://hdl.handle.net/2144/12195.
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Thesis (M.A.)--Boston UniversityThe transcription factor Nuclear factor of activated T cells c1 (NFATc1) is a master regulator of osteoclastogenesis. Previous knockout studies have shown that global NFATc1 deletion in younger mice leads to poor osteoclastogenesis and osteopetrosis. Here we show that the cathepsin k-cre mediated deletion of NFATc1 in mature osteoclasts leads to a phenotype with notable differences including the presence of abnormally large, multinucleate, TRAP-positive osteoclasts and the effacement of the bone marrow by stromal cells resembling a fibrotic reaction. We characterize this phenotype in a multitude of ways. We show that the fibrosis phenotype: (1) presents between age E15.5 and P5, (2) is dependent on the presence of osteoclasts and is downstream of RANK, the cell surface receptor that triggers osteoclast differentiation (3) and is not likely an osteoblast lineage-intrinsic phenotype. We hypothesized that NFATc1 in osteoclasts negatively regulates a secreted stromal lineage proliferative factor that functions in coupling. Our in-vitro assays using media conditioned by cultured wild-type or knockout osteoclasts were unable to show differences in neither BMSC proliferation nor differentiation. Last, we identify genes that are highly upregulated in NFATc1 knock-out osteoclasts and describe those that may function in regulation of stromal-lineage differentiation or proliferation.
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McDermott, Emma. "Characterisation of the osteoclast ruffled border using advanced imaging techniques." Thesis, University of Aberdeen, 2018. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=236980.
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The osteoclast ruffled border is a highly convoluted, complex membrane that is necessary for bone resorption. It is thought to form following mass lysosomal fusion with the boneapposing plasma membrane and vesicular trafficking is vital for its formation and function. The aim of this PhD was to better understand the ultrastructure, formation and function of the ruffled border using TEM and advanced imaging techniques. Ruffled border reformation following calcitonin treatment was visualised and the stages of ruffled border formation were described. Ruffled borders in healthy and osteopetrotic osteoclasts were also imaged by TEM and characterised using a morphological grading system. The key findings of this thesis are as follows: (1) vacuoles, not lysosomes, are the primary contributors of membrane to the ruffled border and the membrane projections of the ruffled border form passively as a consequence of channel formation, not actively by membrane folding, (2) extracellular vesicles are located, and appear to be released, at the ruffled border. Various functional aspects of the ruffled border were also investigated. Vesicles near the ruffled border were identified and characterised by immunoelectron microscopy based on their content and morphology. We found no morphological defects in ruffled borders in mice deficient in Plekhm1. In osteoclasts derived from patients with a SNX10 mutation, we found that while the cells retained the capacity to form well-developed ruffled borders, they did so less often than healthy control osteoclasts. Importantly, we observed that even in a population of healthy osteoclasts, ruffled border morphology is highly heterogeneous because they are at different stages in the resorption cycle. In conclusion, the data in this thesis provide novel findings, previously unseen details regarding how resorbing osteoclasts interact with the bone surface, and have revealed unique insights into ruffled border morphology, formation and the vesicles with which it interacts.