Fischell Department of Bioengineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/6628
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Item Quantitative Phenotyping of Brain Endothelial Cell-Cell Junctions for Physiological and Pathophysiological Applications(2019) Gray, Kelsey M; Stroka, Kimberly M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The integrity of endothelial cell-cell junctions is required for the maintenance of normal physiological processes. The expression of junctional proteins is particularly important in the endothelial cells of the blood-brain barrier (BBB), the cellular unit that protects the brain via regulated transport between the peripheral blood and the central nervous system. Dysfunction of the BBB is linked with decreased junctional protein localization and is implicated in several diseases including Alzheimer’s disease and multiple sclerosis. On the other hand, the tight junctions of the BBB impede the delivery of medications targeting the brain. Therefore, understanding the key players driving junction stability could hold significant promise for therapeutic discovery and drug delivery applications. Despite this, the mechanisms underlying junction disruption aren’t fully understood. While several studies have linked different junction protein patterns with altered barrier function, the quantification of this parameter remains limited due to the lack of efficient measurement techniques. Here, we aimed to investigate the influence of junction phenotype on brain endothelial barrier properties. To accomplish this, we developed the Junction Analyzer Program (JAnaP) to semi-automatically calculate edge-localization protein phenotypes. Application of the JAnaP to measure the junctional proteins VE-cadherin and ZO-1 in different physiological and pathophysiological conditions revealed that discontinuous junctions contribute more to barrier permeability compared to continuous, linear junctions. Continuous junctions were also increased in endothelial cells with decreased contractility, mediated biochemically or by lowered subendothelial matrix stiffness. Finally, breast cancer cell secreted factors increased immature adherens junctions, likely through VEGF signaling, but minimally affected tight junction presentation. Thus far, the development and application of the JAnaP has revealed insights into the effects of junction patterns on barrier function, the mechanobiology of endothelial cells, and the response of brain endothelial cells to biochemical cues involved in breast cancer metastasis. Understanding the conditions driving altered junction presentation, and the resultant effects on barrier integrity, could lead to the development of therapeutics capable of traversing the BBB for delivery to the brain or for diseases associated with BBB dysfunction. Future use of this program holds significant potential for physiological and pathophysiological study in various endothelial and epithelial cell systems.Item EFFECTS OF 3D PRINTED VASCULAR NETWORKS ON HUMAN MESENCHYMAL STEM CELL VIABILITY IN LARGE BONE TISSUE CONSTRUCTS(2015) Ball, Owen Matthew; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)There is a significant clinical need for engineered bone graft substitutes that can quickly, effectively, and safely repair segmental bone defects. One emerging field of interest involves the growth of engineered bone tissue in vitro within bioreactors, the most promising of which, are perfusion bioreactors. Utilizing a tubular perfusion system bioreactor, which allows media to perfuse freely around alginate scaffolds laden with human mesenchymal stem cells, large-scale bone constructs can be created by simply aggregating these beads together in the desired shape. However, these engineered constructs lack inherent vasculature and quickly develop a necrotic core, where no nutrient exchange occurs. Through the use of 3D printed vascular structures, used in conjunction with a TPS bioreactor, cell viability after just one day of aggregation was found to increase by as much as 50 percent in the core of these constructs, with in silico modeling predicting construct viability at steady state.