Orientador: Joni Augusto Cirelli / Resumo: Com o alto índice de sucesso dos implantes, o desenvolvimento de novas ligas e tratamentos de superfície tem sido o grande enfoque das pesquisas da área. O objetivo desse trabalho foi caracterizar a liga binária composta de Ti-15Mo que recebeu um tratamento químico a base de ácido fosfórico, seguido ou não por tratamento de hidróxido de sódio e também tratamento de laser com deposição de apatita para em seguida avaliar a resposta in vitro de osteoblastos de ratos. A metodologia aplicada avaliou a liga através dos testes de microscopia eletrônica de varredura associada à espectroscopia por energia dispersiva de raios X, medidas de rugosidade e área efetiva com microscópio confocal a laser e ângulo de contato. Os ensaios biológicos verificaram a resposta dos osteoblastos a essas superfícies observando a morfologia, adesão e proliferação celular, além da quantificação de nódulos de mineralização, níveis de atividade de fosfatase alcalina e expressão gênica de marcadores da osteogênese. Os resultados demonstram que os tratamentos de superfície realizados atingiram os objetivos de modificar as características físico-químicas do Ti-cp e Ti-15Mo, de forma que os eventos intermediários observados na RBMSC sugerem favorecer a osseointegração. As células osteoblásticas se comportaram de forma semelhante entre as superfícies tratadas, sem diferenças significativas, sugerindo que a liga Ti-15Mo é uma alternativa para o uso na Implantodontia pelas vantagens de sua propriedade mecânica. Es... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: With the high success rate of implants, the development of new alloys and surface treatments has been the major focus of research in the area. The aim of this study was to characterize the binary composite alloy of Ti-15Mo that received a chemical treatment based on phosphoric acid, followed or not by sodium hydroxide and also laser treatment with deposition of apatite to next evaluate the in vitro response of rat osteoblasts. The applied methodology evaluated the alloy through scanning electron microscopy associated to X-ray dispersive energy spectroscopy, roughness measurements and effective area with laser confocal microscope and contact angle. The biological tests verified the response of osteoblasts to these surfaces by observing cell morphology, adhesion and proliferation, as well as the quantification of mineralization nodules, levels of alkaline phosphatase activity and gene expression of osteogenesis markers. The results demonstrate that the surface treatments performed reached the goals of modifying the physicochemical characteristics of the Ti-cp and Ti-15Mo alloy, so that the intermediate events observed in the RBMSC suggest that they favor osseointegration. The osteoblastic cells behaved similarly in both alloys and surface treatments, without significant differences, suggesting that the Ti-15Mo alloy is an alternative for the use in the Implantology for the advantages of mecanical property. These results are expected to determine benefits for possible biological... (Complete abstract click electronic access below) / Doutor

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22143号 / 医博第4534号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 森本 尚樹, 教授 別所 和久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Orientador: Leonardo Perez Faverani / Resumo: Objetivo: O presente estudo teve como objetivo comparar o efeito da Atorvastatina, aplicada de forma local e sistêmica, em defeitos críticos de calotas de ratos. Material e médodos: 36 (trinta e seis) ratos (Rattus norvegicus, albinus, Wistar), adultos foram divididos aleatoriamente em 3 grupos, o grupo aplicação de membrana de colágeno com água destilada (GAD) com defeitos de tamanho crítico contendo água destilada; o grupo aplicação Sistêmica de Atorvastatina (GAS) foram realizado defeitos de tamanho crítico e os animais foram tratados com atorvastatina (3,6mg/kg/dia) por gavagem; e o grupo de aplicação local de Atorvastatina (GAL) com defeitos de tamanho crítico contendo Atorvastatina. Cada grupo foi avaliado através da histometria, mensuração do defeito residual, área de osso néo formado (AON), área de membrana e tecido mole, contagem de células (osteócitos, osteoblastos, células inflamatórias) e imunoistoquímica (OSTEOCALCINA e CD31), nos períodos de 14 e 28 dias. Resultados: Os dados mostraram diminuição do defeito residual para GAS quando comparada ao GAL (p=0,024) e ao GAD (p=0,033), o GAS revelou diminuição de número de osteócitos em comparação ao GAD (p=0,026), e em comparação com GAL (p=0,020). Os osteoblastos não apresentaram diferença entre os grupos (p>0,05), a quantidade de fibroblastos mostrou maiores somente para o GAL de 14 para 28 dias (p=0,019). Aos 28 dias, tanto para GAL quanto GAS, a quantidade de células inflamatórias foram maiores comparadas ao GAD (p... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Objective: This study aimed to compare the effect of Atorvastatin, applied locally and systemically, on critical defects of rat caps. Material and medicaments: 36 (thirty-six) rats (Rattus norvegicus, albinus, Wistar), adults were randomly divided into 3 groups, the collagen membrane application group with distilled water (GAD) with critical size defects containing distilled water; the systemic application group of Atorvastatin (GAS) was performed with critical size defects and the animals were treated with atorvastatin (3.6mg/kg/day) per gavage; and the local application group of Atorvastatin (GAL) with critical size defects containing Atorvastatin. Each group was evaluated by histometry, measurement of the residual defect, area of neo formed bone (AON), area of membrane and soft tissue, cell count (osteocytes, osteoblasts, inflammatory cells) and immunohistochemistry (OSTEOCALCINE and CD31), in periods of 14 and 28 days. Results: The data showed a decrease of residual defect for GAS when compared to GAL (p=0.024) and GAD (p=0.033), GAS showed a decrease of osteocytes in comparison to GAD (p=0.026), and in comparison to GAL (p=0.020). The osteoblasts did not present difference between the groups (p>0.05), the amount of fibroblasts showed higher only for the GAL from 14 to 28 days (p=0.019). At 28 days, both for GAL and GAS, the amount of inflammatory cells were higher compared to GAD (p<0.05). The immunohistochemistry marking for CD31 showed no change, and CSF in osteoblasts... (Complete abstract click electronic access below) / Doutor

Parathyroid hormone (PTH) analogs are the main anabolic pharmacological agent for osteoporosis. PTH is an endogenous hormone, of which amino acids 1-34 bind the parathyroid hormone receptor (PTH1r), a G-coupled protein receptor expressed in kidney, fat, and bone. PTH increases trabecular bone mass by promoting the differentiation of the mesenchymal stem cell (MSC) into the osteogenic lineage. The Zinc Finger Protein 467 (Zfp467) is a potential downstream target of PTH1r and an important mediator of the MSC into the adipogenic lineage. Taken together, we ask whether Zfp467 knockout cells will show greater osteogenic potential and increased sensitivity to PTH treatment. We also seek to investigate a mechanistic signaling pathway of PTH1r involving Zfp467. Calvarial osteoblast (COB) and bone marrow stromal cells (BMSCs) from Zfp467 wild type (WT) and knockout (KO) mice were osteogenically differentiated and treated with either continuous (48h) or intermittent (6h/42h) PTH for 7-14 days. At 7 and 14 days, alkaline phosphatase (ALP) and von Kossa staining were conducted, respectively. At 7 days after differentiation, qPCR was used to analyze genes involved in osteogenesis, adipogenesis, WNT signaling, and mitochondrial respiration. ELISA was used to measure cAMP levels. Seahorse XF96 assays were used to measure oxygen consumption rates (OCRs) and extracellular acidification rates (ECARs). Western blot was used to measure PTH1r. Additionally, adipogenic differentiation and Oil Red O staining were performed on BMSCs. ALP and von Kossa results showed that Zfp467 KO cells exhibited increased osteogenesis and an increased response to PTH treatment (continuous and intermittent) as compared to WT controls. qPCR analysis of Alp, Rankl, and Sp7 further supported an increased osteogenic potential of the KO. Also, Oil Red O staining revealed suppressed adipogenesis in KO BMSCs and qPCR analysis showed suppressed Adiponectin and Ppary in KO COBs. Additionally, Pth1r and PTH1r expressions were significantly higher in KO and short PTH treatments (~10m) induced a remarkable reduction in Zfp467 of WT cells. Furthermore, the KO showed suppressed Pgc1a, similar OCR, and increased ECAR as compared to WT. The KO also exhibited higher cAMP levels and was more responsive to PTH-induced increases of cAMP at 10 minutes of PTH exposure. However, qPCR analysis of Lef1 and Sost showed no difference regarding the WNT pathway. Our data support an anti-osteogenic and pro-adipogenic role for Zfp467. The KO displays less adipogenesis, more osteogenesis, and is consistently more sensitive to the osteogenic effects of PTH. The upregulation of Pth1r and PTH1r in KO cells offers an explanation for this increased sensitivity. We propose a mechanism where the suppression of Zfp467 upregulates Pth1r and PTH1r activation suppresses Zfp467, resulting in a constitutively active positive feedback loop. Further still, the KO shows potentially suppressed mitochondrial biogenesis (through Pgc1a analysis), similar oxidative phosphorylation (through OCRs), increased glycolysis (through ECARs), and increased PKA signaling (through cAMP assays), yet their exact connections to the PTH1r-Zfp467 signaling pathway have yet to be investigated.

No description available.

BACKGROUND: In applications on dental/orthopedic implants and bone regeneration, biomaterials contained magnesium have been widely used. However, the mechanism underlying the biologic effects is still largely unknown. In addition, previous reports of osteogenic effect of magnesium mainly relied on studies using ATCC osteosarcoma cell lines but not normal human osteoblasts. OBJECTIVE: This study was designed to test the effect of magnesium on osteogenic phenotypic behaviors of normal human osteoblasts. METHODS: Normal human osteoblasts derived from human alveolar bone were cultured in triplicate in growth media with varies concentrations of supplemental magnesium: 0.5mM, 1mM, 2mM, 4mM, 8mM and 16mM as the study groups and 0mM as a control group for the time intervals of 7 days, 10 days, 14 days and 21days. Cell proliferation was measured by crystal violet dye staining. Expression of osteocalcin was measured by Quantikine Elisa and mineralization of cultures was measured by Alizarin Red staining. The data were normalized per cell basis. Statistical analysis was done using ANOVA and Student’s t test. Results: Osteocalcin expression was upregulated in groups with supplemented magnesium at 0.5mM (1.16folds, p<0.01 ), 1.0mM (1.22folds, p<0.01 ), 2.0mM (1.37folds, p<0.01 ) at day 21 compared to control, while at 4mM ( p<0.01 ) and above showed down-regulation. Alizarin Red stained cultures showed higher degree of mineralization at 1mM ( p=0.0228 ) and 2mM ( p=0.0142) compared to control. Groups with 4mM and above showed less calcium deposition. Similar results have been gained also on day 10 and day 14 for both assays. CONCLUSION: Osteogenesis of normal human osteoblasts could be significantly upregulated by 2mM supplemental magnesium. These data are important for manufacturing magnesium-containing biomaterials for bone tissue regeneration and implants.

No / Little has been written about the functional outcome of patients treated with bone transport to reconstruct a distal tibial defect. The aim of this study was to investigate the functional capabilities of patients who had undergone reconstruction with distraction osteogenesis for the treatment of a distal tibial defect in one lower limb. At least eighteen months after completion of treatment, eight patients who had no pain and were able to walk and climb stairs without difficulty performed isometric ankle plantar flexion maximum voluntary contractions while the electromyographic activity of the tibialis anterior and triceps surae muscles was simultaneously recorded. Seven of the patients also underwent gait analysis. Data for the involved limb were compared with those collected for the contralateral limb. During gait, stance time (p = 0.01), the plantar flexion angular displacement and peak moment developed during the second half of stance (p < 0.046), and the amount of ankle power generated (p = 0.02) were significantly decreased in the involved limb compared with the contralateral limb. Similar decreases were observed in the plantar flexion (p = 0.01) and dorsiflexion (p = 0.01) maximum voluntary contractions and the corresponding electromyographic activity (p = 0.01). These results suggest that adaptive changes had occurred at the level of the transported muscles, which affected both routine and maximal effort capabilities. These findings contribute to our understanding of the functional limitations of patients who have undergone bone transport with its obligatory shortening of muscle length.

The use of nanowires, nano and micro needles in biomedical applications has markedly increased in the past years, mainly due to attractive properties such as biocompatibility and simple fabrication. Specifically, these structures have shown promise in applications including cell separation, tumor cell capture, intracellular delivery, cell therapy, cancer treatment and as cell growth scaffolds. The work proposed here aims to study two platforms for different applications: a vertical magnetic nanowire array for mesenchymal stem cell differentiation and a micro needle platform for intracellular delivery. First, a thorough evaluation of the cytotoxicity of nanowires was done in order to understand how a biological system interacts with high aspect ratio structures. Nanowires were fabricated through pulsed electrodeposition and characterized by electron microscopy, vibrating sample magnetometry and energy dispersive X-ray spectroscopy. Studies of biocompatibility, cell death, cell membrane integrity, nanowire internalization and intracellular dissolution were all performed in order to characterize the cell response. Results showed a variable biocompatibility depending on nanowire concentration and incubation time, with cell death resulting from an apoptotic pathway arising after internalization. A vertical array of nanowires was then used as a scaffold for the differentiation of human mesenchymal stem cells. Using fluorescence and electron microscopy, the interactions between the dense array of nanowires and the cells were analyzed, as well as the biocompatibility of the array and its effects on cell differentiation. A magnetic field was additionally applied on the substrate to observe a possible differentiation. Stem cells grown on this scaffold showed a cytoskeleton and focal adhesion reorganization, and later expressed the osteogenic marker osteopontin. The application of a magnetic field counteracted this outcome. Lastly, a micro needle platform was fabricated through lithography and electrodeposition, characterized using the previously mentioned techniques and then evaluated as a vector for intracellular delivery. Fluorescence and electron microscopy imaging were first performed to assess the biocompatibility, cell spreading and the interface of the cells and the needles. Intracellular delivery of a fluorescent dye was achieved via inductive heating of the needles, with the results showing a dependency of delivery and cell survivability on the exposure time.

Biodegradable polyesters, such as poly(DL-lactic-co-glycolic acid) (PLGA), have been used to fabricate porous bone scaffolds to support bone tissue development. These scaffolds allow for cell seeding, attachment, growth and extracellular matrix production in vitro and are replaced by new bone tissue when implanted into bone sites in vivo. Hydroxyapatite (HAP) and μ-tricalcium phosphate (μ-TCP) ceramics have been incorporated into PLGA bone scaffolds and have been shown to increase their osteoconductivity (support cell attachment). Although HAP, μ-TCP, and biodegradable polyesters are osteoconductive, there is no evidence that these scaffold materials are osteoinductive (support cell differentiation). Calcium and phosphate ions, in contrast, have been postulated to be osteogenic factors that enhance osteoblast differentiation and mineralization. Recently, a zirconia-hybridized pyrophosphate stabilized amorphous calcium phosphate (Zr-ACP) has been synthesized which permits controlled release of calcium and phosphate ions and thus is hypothesized to be osteoinductive. Incorporation of Zr-ACP into a highly porous poly(DL lactic-co-glycolic acid) (PLGA) scaffold could potentially increase the osteoinductivity of the scaffold and therefore promote osteogenesis when implanted in vivo. To determine the osteoinductivity of Zr-ACP, a MC3T3-E1 mouse calvarial-derived osteoprogenitor cell line was used to measure cell response to Zr-ACP. To accomplish this objective, Zr-ACP was added to cell culture at different stages in cell maturation (days 0, 4 and 11). DNA synthesis, alkaline phosphatase (ALP) activity, osteopontin synthesis and collagen synthesis were determined. Results indicate that culture in the presence of Zr-ACP significantly increased cell proliferation, ALP activity and osteopontin synthesis but not collagen synthesis. To determine the feasibility of incorporating Zr-ACP into a PLGA scaffold, PLGA/Zr-ACP composite foams (5% or 10% (w/v) polymer:solvent with 25 wt% or 50 wt% Zr-ACP) were fabricated using a thermal phase inversion technique. Scanning electron microscopy revealed a highly porous structure with pores ranging in size from a few microns to about 100 μm. The amorphous structure of the Zr-ACP was maintained during composite fabrication as confirmed by X-ray diffraction measurements. Composite scaffolds also showed significantly greater compressive yield strengths and moduli as compared to pure polymer scaffolds. The results of this study indicate that Zr-ACP enhances the osteoblastic phenotype of MC3T3-E1 cells in vitro and can be incorporated into a porous PLGA scaffold. Porous PLGA/Zr-ACP composites are promising for use as bone scaffolds to heal bone defects. / Master of Science

Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / Title from title screen of research.pdf file (viewed on December 22, 2006). The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2006" Includes bibliographical references.