Theses and Dissertations from UMD
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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM
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Item The Influence of Vimentin Intermediate Filaments on Human Mesenchymal Stem Cell Response to Physical Stimuli(2017) Sharma, Poonam; Hsieh, Adam H; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mesenchymal stem cells (MSCs) are increasingly being investigated as a therapeutic cell population for a variety of diseases. However, these therapies are limited by an imperfect understanding of how MSCs interact with and respond to their physical environment. Cell response to external stimuli is mediated by the cytoskeleton. Of the cytoskeletal proteins, understanding of vimentin intermediate filaments’ influence on MSC behavior is still lacking, despite increasing evidence that they are involved in many cellular processes. In this work, we investigated the influence of vimentin intermediate filaments in modulating MSC characteristics and behavior by using lentiviral shRNA transduction to decrease vimentin levels in MSCs through RNA interference. First, the contribution of vimentin intermediate filaments to the deformability of MSCs within agarose hydrogels was examined. Vimentin-deficient MSCs were found to be less deformable than control cell populations and this resistance to deformation may be due to the compensatory role of actin microfilaments. Next, to determine how vimentin affects the ability of MSCs to interact with various microenvironments, we examined cell spreading on different extracellular matrix proteins, multiple substrate stiffness’, and in response to fluid shear stress. An intact vimentin network was found to be necessary for unimpaired spreading on fibronectin, but only on stiffer substrates. Further, vimentin appears to be involved in resisting cell area changes in response to low fluid shear stress. Vimentin’s physical interaction with focal adhesions, rather than an impact at the transcriptional or translational level, may contribute to the cell spreading response observed. Finally, in the third part of this work, we examined the influence of vimentin on chondrogenic differentiation of MSC populations. Unexpectedly, we found that vimentin may not be involved in chondrogenic differentiation in late stage chondrogenic cultures. Instead, the culture condition-dependent microenvironment may have a greater impact, particularly in gene expression of matrix degrading enzymes and the αV integrin subunit. Altogether, these studies indicate a role for vimentin in the MSC response to physical stimuli. Moreover, this work furthers the dialogue surrounding MSCs’ interaction with different environments, the understanding of which will be critical for the development and evaluation of cell-based therapies.Item Engineering Zonal Cartilage Through Utilization of a Mesenchymal Stem Cell Population(2012) Coates, Emily Elizabeth; Fisher, John; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Articular cartilage has a limited ability to repair itself after damage due to injury or disease. Regenerative therapies using chondrocytes, the primary cartilage cell population, result in poor quality repair tissue and often cause further damage at the donor site. Furthermore, there are no current therapies which aim to regenerate the zonal organization and function of the tissue. In an effort to address both cell source limitations and zonal tissue regeneration the goal of the presented work was to utilize a mesenchymal stem cell (MSC) population to generate abundant numbers of chondrocytes with zonal phenotypes. To this end, zonal subpopulations of articular chondrocytes were isolated, characterized for differences in gene and protein expression, and exposed to scaffold environments designed to aid in phenotype retention. From these results, and reports in the literature, it was clear a major functional difference between zones was the production of a lubricating protein, proteoglycan 4 (PRG4), in the superficial zone only. Middle and deep zone cells were found to be phenotypically similar and distinct from superficial zone cells. It was further found that gene expression of PRG4 by superficial zone cells in alginate culture can be significantly enhanced by incorporation of matrix molecules hyaluronic acid (HA) and chondroitin sulfate (CS) to the scaffold environment. HA and CS also had favorable effects on MSC chondrogenesis by upregulating chondrogenic transcription factor Sox9 gene expression, and downregulating type I collagen (fibroblastic marker) gene expression. The potential of soluble signals derived from zonal (superficial or middle/deep) cartilage explants to drive MSC chondrogenesis was also investigated. Results show that signals derived from cartilage explants can induce chondrogenesis to varying degrees, with superficial zone explants inducing robust and sustained differentiation. This differentiation was found to be dependent on the proximity of the MSCs and tissue explants, implying that communication between MSCs and chondrocytes is necessary for chondrogenic induction. Coculture with superficial zone explants also upregulated MSC gene expression of PRG4. This research highlights the important functional differences between zonal chondrocyte populations and identifies MSCs as a progenitor population capable of differentiating into zone-specific chondrocytes.