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Evaluation of bioactivity of alkali- and heat-treated titanium using fluorescent mouse osteoblasts / 蛍光タンパク導入マウス由来骨芽細胞を用いたアルカリ加熱処理チタンの生体活性能の評価Tsukanaka, Masako 24 March 2014 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18146号 / 医博第3866号 / 新制||医||1002(附属図書館) / 31004 / 京都大学大学院医学研究科医学専攻 / (主査)教授 鈴木 茂彦, 教授 妻木 範行, 教授 戸口田 淳也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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INHIBITED MINERALIZATION IN OSTEOBLASTS CULTURED UNDER VARIED SIMULATED PARTIAL GRAVITY CONDITIONS AND THE USE OF PHYTONUTRIENTS FOR MITIGATING THE EFFECTS OF REDUCED GRAVITYBraveboy-Wagner, Justin, 0000-0002-6301-1394 January 2022 (has links)
The multifaceted adverse effects of reduced gravity on the skeletal system pose a significant challenge to human spaceflight. There is an interest in investigating any hypothetical differences between partial gravity and microgravity, and in the unmet need to identify countermeasures to both. A hypothesis to be tested is that reduced gravity impairs a variety of osteogenic cell functions, such as proliferation and differentiation, and that these inhibitory effects can be mitigated by nutritional countermeasures or by interrupting signaling pathways that drive undesired osteogenic remodeling. Utilizing the Random Positioning Machine, it is possible to simulate a variety of reduced gravity levels relevant to future manned space missions: Mars, Moon, and Microgravity of the Low Earth Orbit (LEO) environment.
In this study, the effects of altered gravity on the physiology and morphology of cultured osteoblasts were investigated, specifically on their proliferation, osteogenic differentiation, and matrix mineralization. In assessing the role of mechanotransduction in microgravity-induced cytoskeletal dysfunction, this thesis also explored whether selective inhibition of specific signaling steps within the Rho-ROCK pathway can be used to modulate the effects of microgravity on osteoblast differentiation and function. Finally, in developing new countermeasures, an investigation was made into the effectiveness of curcumin and carnosic acid, two nutritional antioxidants with pro-osteogenic properties, contrasted with the trace element zinc, as potential alimentary supplements that may mitigate or alleviate the deleterious effects of microgravity. Results showed that short-term (6 days) culture yielded a dose-dependent reduction in proliferation and the enzymatic activity of alkaline phosphatase (ALP), while long-term studies (21 days) showed a distinct dose-dependent inhibition of mineralization. By contrast, expression levels of key osteogenic genes (Alkaline phosphatase, Runt-related Transcription Factor 2, Sparc/osteonectin) exhibited a threshold behavior: gene expression was significantly inhibited when the cells were exposed to Mars-simulating partial gravity, and this was not reduced further when the cells were cultured under simulated Moon or microgravity conditions. My data suggests that impairment of cell function with decreasing simulated gravity levels is graded and that the threshold profile observed for reduced gene expression is distinct from the dose dependence observed for cell proliferation, ALP activity, and mineral deposition. My studies into the gravity-induced re-organization of the cytoskeleton indicate that selective interruption of the Rho-ROCK pathway at ROCK can prevent morphological changes that result in impaired differentiation and mineralization. Further, I found that nutraceuticals partially reversed the inhibitory effects of SMG on ALP activity and promoted osteoblast proliferation and differentiation in the absence of traditional osteogenic media. I further observed a synergistic effect of the intermix of the phytonutrients on ALP activity. Intermixes of phytonutrients may serve as convenient and effective nutritional countermeasures against bone loss in space. / Bioengineering
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IMPACT OF PETROLEUM RELATED COMPOUNDS ON MESENCHYMAL STEM CELL DERIVED PROGENITOR CELLSGutgesell, Robert Michael January 2022 (has links)
There is concern over the impact that petroleum related compounds (PRCs) associated with mining activity in the Athabasca Oil Sands Region (AOSR) are having on local wildlife. With the increase in oil sands mining activity in the AOSR there has been a corresponding decline in the fertility of indicator species in the AOSR. One of the primary sources of PRCs in the environment is oil sands process affected water (OSPW), which is stored in tailings ponds. Several PRCs, including naphthenic acid fraction components (NAFC), have endocrine disrupting effects, which may, in part, explain reduced fertility in indicator species. For example, male North American river otters (Lontra canadensis) living in areas impacted by mining activity have lower baculum strength those unaffected by mining activity. Weaker baculums are associated with increases in fracture rates and reduced fertility. Baculum strength is maintained throughout life by bone remodeling, a process that requires the differentiation of osteoblasts. NAFCs can impact several pathways integral to the development and path selection of mesenchymal stem cells into osteoblasts or adipocytes. Therefore, the objective of this thesis was to test the hypothesis that NAFCs inhibit osteoblast differentiation and induce adipocyte differentiation from progenitor cells. We exposed osteoblast progenitor cells and adipocyte progenitor cells to NAFCs. We demonstrated that NAFCs inhibit osteoblast differentiation and activate the glucocorticoid receptor pathway. We also found that NAFCs do not induce adipogenesis in adipocyte precursor cells. Lastly, we showed that NAFCs are PPARγ ligands that inhibit the expression of PPARγ associated genes. These insights into the effects of NAFCs on osteoblast and adipocyte progenitor cells suggest NAFCs may contribute to lower baculum strength and impaired adipose tissue function of animals living in the AOSR. These effects my reduce the fertility and population of wildlife in the AOSR. / Thesis / Master of Science (MSc) / There is concern that chemicals from oil sands mining in the Athabasca oil sands region are hurting the reproductive health of animals in the wild. Some of these animals, including bears, wolves, and river otters, need a bone in their penis called a baculum to reproduce. Studies have shown that some chemicals, including those from mining activity can make the baculum bone weaker. For bone to stay strong, bone cells always need to be developing to fix the bone tissue. The goal of our study was to find how chemicals from mining activity can affect the development of bone cells. We found that a group of chemicals that come from oil sands mining called naphthenic acid fraction components (NAFCs) stop bone cells from developing and making new bone. We also know that having more fat cells in bone is associated with weaker bones. We also looked at whether NAFCs could increase the development of fat cells. However, NAFCs did not increase the development of fat cells. Together, this research shows that NAFCs can make bones like the baculum weaker by slowing the development of new bone, but not by increasing fat cells. Our research suggests that exposure to NAFCs may make baculums weaker which may be bad for the reproductive health of animals living near oil sands mining activity.
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OSTEOACTIVIN IN SKELETON: CHARACTERIZATION OF OSTEOACTIVIN KNOCKOUT MICE & THERAPEUTIC IMPLICATIONSStinnett, Hilary M. 30 April 2015 (has links)
No description available.
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GENETICALLY MANIPULATED MOUSE MODELS FOR THE STUDY OF INSULIN-LIKE GROWTH FACTOR I IN BONEZHANG, MEI 11 March 2002 (has links)
No description available.
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Design, Fabrication, and Analysis of Polymer Scaffolds for Use in Bonce Tissue EngineeringMinton, Joshua A. 20 August 2013 (has links)
No description available.
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TRANSCRIPTIONAL REGULATION OF OSTEOACTIVIN EXPRESSION BY BMP-2 IN OSTEOBLASTSSingh, Maneet January 2011 (has links)
Osteoactivin (OA) is a glycoprotein required for the differentiation of osteoblasts. In osteoblasts, Bone Morphogenetic Protein-2 (BMP-2) activated Smad1 signaling enhances OA expression. However, the transcriptional regulation of OA gene expression by BMP-2 is still unknown. The aim of this study was to characterize BMP-2-induced transcription factors that regulate OA gene expression during osteoblast differentiation. The stimulatory effects of BMP-2 on OA transcription were established by cloning the proximal 0.96kb of rat OA promoter region in a luciferase reporter vector in various osteogenic cell types. Further, by deletion and mutagenesis analyses of the cloned OA promoter, key binding sites for osteogenic transcription factors namely, Runx2, Smad1, Smad4 and homeodomain proteins (Dlx3, Dlx5 and Msx2) were identified and characterized. Utilizing specific siRNAs to knock down Runx2, Smad1, Smad4, Dlx3, Dlx5 or Msx2 proteins in osteoblasts, we found that Runx2, Smad1, Smad4, Dlx3 and Dlx5 proteins up-regulate OA transcription, whereas, Msx2 down-regulated OA gene expression. These specific effects of transcription factors on OA promoter regulation were confirmed by forced expression of transcription factors. Most notably, BMP-2-stimulated cooperative and synergistic interactions between Runx2-Smad1 proteins and Dlx3-Dlx5 proteins that up-regulate OA promoter activity. Electrophoretic mobility shift and supershift assays demonstrated that BMP-2 stimulates interactions between Runx2, Smad1 and Smad4 and homeodomain transcription factors with the OA promoter regions flanking the -585 Runx2 binding site, the -248 Smad binding site and the region between the -852 and the -843 homeodomain binding sites relative to transcription start site. The OA promoter region was occupied by Runx2 and also Dlx3 transcription factors during proliferation stages of osteoblast differentiation. As the osteoblasts progress from proliferation to matrix maturation stages of differentiation, the OA promoter was predominantly occupied by Runx2 and to a lesser extent Dlx5 in response to BMP-2. Finally, during matrix mineralization stages of osteoblast differentiation, BMP-2-induced a robust recruitment of Dlx5, Smad1, Dlx3 and Msx2 proteins with simultaneous dissociation of Runx2 from the OA promoter region. In conclusion, the BMP-2-induced osteogenic transcription factors Runx2, Smad1, Smad4, Dlx3, Dlx5 and Msx2 provide key molecular switches that regulate OA transcription during osteoblast differentiation. / Cell Biology
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The Interaction Between Connective Tissue Growth Factor and Bone Morphogenetic Protein-2 During Osteoblast Differentiation and FunctionMundy, Christina Maria January 2014 (has links)
Connective tissue growth factor (CTGF/CCN2) and bone morphogenetic protein (BMP)-2 are both produced and secreted by osteoblasts. Both proteins have been shown to have independent effects in regulating osteoblast proliferation, maturation and mineralization. However, how these two proteins interact during osteoblast differentiation remains unknown. In Chapters 2 and 3, we utilized two cell culture model systems, osteoblasts derived from CTGF knockout (KO) mice and osteoblasts infected with an adenovirus, which over-expresses CTGF (Ad-CTGF), to investigate the effects of CTGF and BMP-2 on osteoblast development and function in vitro. To observe differences in osteoblast maturation and mineralization, we performed alkaline phosphatase (ALP) staining and activity and alizarin red staining, respectively. Contrary to a previously published report, osteoblast maturation and mineralization were similar in osteogenic cultures derived from KO and wild type (WT) calvaria in the absence of BMP-2 stimulation. Interestingly, in KO and WT osteoblast cultures stimulated with BMP-2, the KO osteoblast cultures exhibited increased alkaline phosphatase staining and activity and had larger, fused nodules stained with alizarin red than WT osteoblast cultures. This increase in osteoblast differentiation was accompanied by increased protein levels of phosphorylated Smad 1/5/8 and mRNA expression levels of bone morphogenetic protein receptor Ib. These data confirm enhanced osteoblast maturation and mineralization in BMP-2 induced KO osteoblast cultures. We also examined osteoblast differentiation in cultures that were infected with Ad-CTGF and in control cultures. Continuous over-expression of CTGF resulted in decreased ALP staining and activity, alizarin red staining, and mRNA expression of osteoblast markers in both unstimulated and BMP-2 stimulated cultures. Impaired osteoblast differentiation in cultures over-expressing CTGF was accompanied by decreased protein levels of phosphorylated Smad 1/5/8. In addition to the functional assays that we performed on WT and KO osteoblast cultures, we performed ChIP assays to investigate differences in binding occupancy of transcription factors on the Runx2 and Osteocalcin promoters in BMP-2 induced WT and KO osteoblast cultures. We demonstrate that in BMP-2 induced WT and KO osteoblast cultures, there was greater Smad 1 and JunB occupancy on the Runx2 promoter and Runx2 occupancy on the Osteocalcin promoter in BMP-2 induced KO osteoblast cultures compared to WT cultures. Collectively, the data demonstrate that CTGF acts to negatively regulate BMP-2 induced signaling and osteoblast differentiation. In Chapter 4, we synthesized an active His-tagged BMP-2 recombinant protein to track surface binding of BMP-2 in CTGF WT and KO osteoblasts. We amplified mature BMP-2 in genomic DNA, which was inserted correctly into a pET-28b(+) vector. We ran a SDS-PAGE gel and stained with Coomassie blue to show that we successfully induced BMP-2 in bacteria cells, extracted the protein using urea, and purified and eluted the protein using Nickel charged agarose beads and imidazole elution buffer. Furthermore, by Western blot analysis using anti-His antibody, we confirmed the presence of the His-tag on the BMP-2 protein. Lastly, ALP staining on osteoblast cultures stimulated with our synthesized BMP-2 exhibited increased staining compared to the unstimulated osteoblast cultures, which confirmed the activity of our His-tagged BMP-2 protein. Future studies utilizing this protein will demonstrate that CTGF acts as an extracellular antagonist by limiting the amount of BMP-2 available for receptor binding. / Cell Biology
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Epigenetic regulation of osteoblast differentiationNajafova, Zeynab 09 August 2016 (has links)
No description available.
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Glucose diffusivity in tissue engineering membranes and scaffolds : implications for hollow fibre membrane bioreactorSuhaimi, Hazwani January 2015 (has links)
Unlike thin tissues (e.g., skin) which has been successfully grown, growing thick tissues (e.g., bone and muscle) still exhibit certain limitations due to lack of nutrients (e.g., glucose and oxygen) feeding on cells in extracapillary space (ECS) region, or also known as scaffold in an in vitro static culture. The transport of glucose and oxygen into the cells is depended solely on diffusion process which results in a condition where the cells are deprived of adequate glucose and oxygen supply. This condition is termed as hypoxia and leads to premature cell death. Hollow fibre membrane bioreactors (HFMBs) which operate under perfusive cell culture conditions, have been attempted to reduce the diffusion limitation problem. However, direct sampling of glucose and oxygen is almost impossible; hence noninvasive methods (e.g., mathematical models) have been developed in the past. These models have defined that the glucose diffusivity in cell culture medium (CCM) is similar to the diffusivity in water; thus, they do not represent precisely the nutrient transport processes occurring inside the HFMB. In this research, we define glucose as our nutrient specie due to its limited published information with regard to its diffusivity values, especially one that corresponds to cell/tissue engineering (TE) experiments. A series of well-defined diffusion experiments are carried out with TE materials of varying pore size and shapes imbibed in water and CCM, namely, cellulose nitrate (CN) membrane, polyvinylidene fluoride (PVDF) membrane, poly(L-lactide) (PLLA) scaffold, poly(caprolactone) (PCL) scaffold and collagen scaffold. A diffusion cell is constructed to study the diffusion of glucose across these materials. The glucose diffusion across cell-free membranes and scaffolds is investigated first where pore size distribution, porosity and tortuosity are determined and correlated to the effective diffusivity. As expected, the effective diffusivity increases correspondingly with the pore size of the materials. We also observe that the effective glucose diffusivity through the pores of these materials in CCM is smaller than in water. Next, we seeded human osteoblast cells (HOSTE85) on the scaffolds for a culture period of up to 3 weeks. Similar to the first series of the diffusion experiments, we have attempted to determine the effective glucose diffusivity through the pores of the scaffolds where cells have grown at 37°C. The results show that cell growth changes the morphological structure of the scaffolds, reducing the effective pore space which leads to reduced effective diffusivity. In addition, the self-diffusion of glucose in CCM and water has also been determined using a diaphragm cell method (DCM). The results have shown that the glucose diffusivity in CCM has significantly reduced in comparison to the water diffusivity which is due to the larger dynamic viscosity of CCM. The presence of other components and difference in fluid properties of CCM may also contribute to the decrease. We finally employ our experimentally deduced effective diffusivity and self-diffusivity values into a mathematical model based on the Krogh cylinder assumption. The glucose concentration is predicted to be the lowest near the bioreactor outlet, or in the scaffold region, hence this region becomes a location of interest. The governing transport equations are non-dimensionalised and solved numerically. The results shown offer an insight into pointing out the important parameters that should be considered when one wishes to develop and optimise the HFMB design.
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