Browsing by Subject "Extracellular matrix"

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    Identification of extracellular matrix components essential for a conductive Geobacter sulfurreducens biofilm
    (2011-11) Rollefson, Janet Beth
    Electron transfer from Geobacter sulfurreducens cells to electrodes or metal oxides requires proper expression and localization of redox-active proteins as well as attachment mechanisms that interface bacteria with surfaces and other cells. Type IV pili and c-type cytochromes have long been considered important components of this conductive network. In this work, a large-scale mutagenesis of G. sulfurreducens was performed and mutants were screened for extracellular electron transfer and attachment phenotypes, identifying new genes essential for a conductive Geobacter network. A mutant defective in polysaccharide export to the extracellular matrix (Δ1501, ΔxapD::kan) was identified based on its altered surface attachment. Characterization of this mutant revealed the importance of extracellular polysaccharides for proper attachment and anchoring of the external c-type cytochromes necessary for a conductive biofilm network. Furthermore, decreased polysaccharide content was found in commonly studied cytochrome and type IV pili mutants, with defects in cell to cell and cell to surface attachment correlating with levels of extracellular polysaccharides. The extracellular matrix of G. sulfurreducens is therefore a complex network of polysaccharides, type IV pili, and c-type cytochromes. Disruption of any one of these extracellular components alters overall matrix properties and impedes extracellular electron transfer and attachment.
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    Modulation of mesenchymal stem cell invasion into decellularized engineered tissues
    (2013-06) Weidenhamer, Nathan Kyle
    Mechanical forces play an important role in shaping the organization and alignment of the extracellular matrix (ECM) in developing and mature tissues, where the organization gives the tissue its unique functional properties. This dissertation will discuss research that is fundamental to the understanding of the interaction of cells with the extracellular matrix (ECM), a concept that is vital to creating functional engineered tissues, such as arteries and heart valves. The work presented is divided into two main focus areas; the influence of mechanical forces on the development of cell and ECM alignment and the influence of ECM alignment and culture conditions on cell invasion. The first focus area developed a novel method to create and stretch tubular cell sheets by seeding neonatal dermal fibroblasts (nHDF) onto a rotating silicone tube. Fibroblasts proliferated to create a confluent monolayer around the tube and a collagenous, isotropic tubular tissue over 4 weeks of static culture. These silicone tubes with overlying tubular tissue constructs were mounted into a cyclic distension bioreactor and subjected to cyclic circumferential stretch at 5% strain, 0.5 Hz for 3 weeks. Tissue subjected to cyclic stretch compacted axially over the silicone tube in comparison to static controls, leading to a circumferentially-aligned tissue with higher membrane stiffness and maximum tension. In a subsequent study, the tissue constructs were constrained against axial compaction during cyclic stretching. The resulting alignment of fibroblasts and collagen was perpendicular (axial) to the stretch direction (circumferential). When the cells were devitalized with sodium azide before stretching, similarly constrained tissue did not develop strong axial alignment. This work suggests that both mechanical stretching and mechanical constraints are important in determining tissue organization, and that this organization is dependent on an intact cytoskeleton. The second focus area explored the influence of ECM alignment and culture conditions on human mesenchymal stem cell (hMSC) invasion into decellularized tissues. These studies showed that the soluble factors insulin and ascorbic acid promote the invasion of hMSCs into decellularized engineered tissues. We speculate that this is due to an increase in motility and proliferation of hMSCs when exposed to these factors. Furthermore, hMSC invasion into aligned and non-aligned matrices was not different, although there was a difference in cell orientation between aligned and non-aligned matrices. Finally, we show that, regardless of culture conditions or ECM alignment, hMSCs appear to be differentiating toward a myofibroblast-like phenotype.