| Popis: |
Mechanical cues have been shown to influence cell gene expression, cell protein expression, and cell behaviors critical for homeostasis and disease progression. Cells experience the mechanical cue of confinement in vivo, such as within the extracellular matrix, and in vitro, such as within tissue engineered scaffolds. Despite its prevalence, the impact of mechanical confinement on cell fate is poorly understood. Cues from the mechanical microenvironment are primarily sensed and responded to by the cytoskeleton, which transmits forces to the nucleus and can thereby alter gene expression. The nucleus itself is also a mechanosensor, sensing external forces and again altering gene expression. Mesenchymal stem cells (MSCs) and lung fibroblasts are known to be sensitive to mechanical forces, yet the effect of mechanical confinement on these cells is unclear. In this dissertation, we investigated how mechanical confinement induced by engineered microchannels influences MSC morphology and migration. Notably, we show that confinement alters the relative contributions of cytoskeletal and contractile machinery in MSC migration in unconfined and confined spaces. We next investigated how mechanical confinement induced by microchannels influences MSC and fibroblast nucleus volume. When certain cytoskeletal machinery was inhibited, nucleus volume was altered only in MSCs in wide channels, suggesting diverging roles of the cytoskeleton in regulating nuclear deformation and migration in different degrees of confinement and in different cell types. While performing this work, we observed a lack of assays that provide precise control over the degree of confinement induced on cells, yield a large sample size, enable long-term culture, and enable easy visualization of cells over time. Therefore, we designed, created, and validated a confining micropillar assay that achieves these requirements. Using these confining micropillars, we investigated the effect of confinement on lung fibroblast to myofibroblast transition (FMT), a hallmark of idiopathic pulmonary fibrosis. Cell density was more predictive of FMT than the degree of confinement induced by micropillar arrays. These results improve our understanding of how MSCs and lung fibroblasts respond to confinement, which will aid in the rational design of MSC-based therapies and FMT-targeting therapies. |
