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The periodontium is a dynamic tissue that undergoes constant remodeling to maintain its structure and function. There is much evidence in the literature that autophagy can be activated by both mechanical stretching and inflammation, however the mechanisms involved are highly context dependent. Autophagy may also be important for bone remodeling in the oral cavity. Two scenarios where bone remodeling is key to the physiological outcome are orthodontic tooth movement and inflammatory-induced periodontal disease. This thesis aims to investigate the hypothesis that autophagy is involved in bone remodeling during orthodontic tooth movement and periodontal disease. While the stem cells concerned in both osteogenesis and osteoclastogenesis are different, they both originate from the bone marrow, therefore the same primary human bone marrow cells containing the specific stem cell populations required for osteogenesis or osteoclastogenesis were used to establish in vitro models for mechanical strain and chronic inflammation, and the activation and differentiation of these bone marrow derived cells were analysed in response to the conditions created in these model systems. Orthodontic tooth movement invokes constant mechanical force onto the periodontium, with tensile stretching force initiating osteogenesis. Mechanical strain is also linked to an increase in autophagy, a cellular degradation pathway that may be protective or can result in cell death. The hypothesis was that cells exposed to tensile strain undergo an altered state of autophagic flux which contributes to osteogenesis. In vivo and in vitro models of tooth movement were used to analyse osteogenic and autophagy marker expression. Initially tensile strain was found to increase autophagy but decrease expression of osteogenic markers. However, by 24 hours there was a decrease in autophagy and osteogenesis marker expression was increased. Artificially activating autophagy in cells undergoing mechanical stretching resulted in enhanced Runt-related transcription factor 2 (RUNX2) expression, a marker of osteogenic differentiation. Autophagy activation is perhaps a protective mechanism as an initial response to mechanical strain, after which osteogenesis may occur. Patients undergoing orthodontic treatment are at risk of developing periodontitis, a chronic inflammatory disease caused by the presence of Gram-negative bacteria leading to inflammation of the periodontium. It is also clear that disease susceptibility and progression are affected by host factors, however the mechanisms behind this are unknown. Lipopolysaccharide (LPS) tolerance has been demonstrated in many chronic inflammatory diseases and may alleviate or exacerbate disease, but its role in periodontitis is not fully established. The hypothesis was that periodontitis results in LPS tolerance which affects osteoclastogenesis. Hyaluronan (HA) signalling was explored as a potential mechanism, as HA synthesis is upregulated in chronic inflammation. This thesis demonstrates that inflammatory monocytes increase HA synthesis in response to LPS, and that HA enhances differentiation of osteoclast precursors. However, LPS tolerance of inflammatory monocytes resulted in a rapid reduction in HA synthesis, which may be autophagy-dependent. This reduction in HA synthesis resulted in a reduction in osteoclast differentiation, which may explain why periodontal disease associated alveolar bone loss does not always occur to the same severity in different patients. In conclusion, autophagy was found to have roles in the regulation of osteogenesis and osteoclastogenesis under tensile strain and chronic inflammation, respectively, and therefore may be an appropriate future target for pharmaceutical intervention.

This Thesis investigates the damage sensing capabilities of the electrical potential measurement technique in carbon fibre reinforced polymer composites. Impact damage was introduced in multidirectional laminates and its effect on potential distribution studied. It was found that delaminations and fibre breakages within the laminate can be detected and located by measuring potential changes on the external composite surface. The extent and size of potential changes were significantly affected by the position of the current electrodes in relation to the potential measurement probes. A numerical model was developed investigating the effect of different size delaminations, located in various positions within the lamina, on electrical potential distributions on the external ply, and a quantitative analysis of the numerical results is presented. The numerical simulations demonstrated that the measured potential changes on the external ply were in proportion to the delamination size. The numerical and experimental results were compared and the optimum configuration of current electrodes and potential probes for damage detection selected. The response of electrical potential to mechanical strain, in unidirectional and multidirectional samples was also investigated. It was found that the conductive medium, used for introducing the current, defines the piezo-resistance performance of the composite. A finite element model was developed able to predict the effect of inhomogeneous current introduction in unidirectional specimens on electrical potential and piezo-resistance. The effects of temperature and water absorption on potential measurements were also presented.

Metastasis is the ultimate cause of most cancer-related deaths. As such, it is critical to understand the processes that control the spread of cancer through the body. To metastasize, cancer cells must leave the primary tumor, access the circulatory system, adhere to the endothelium of the metastatic site, and migrate out into the surrounding tissue. A major limitation in the discovery and development of drugs that prevent the spread of cancer is a lack of high throughput in vitro assays that accurately recreate the cell-cell interactions and mechanical forces that occur during cancer cell adhesion and exit from the vasculature at the secondary site. Here we study cancer cell adhesion in the presence of physiologic shear stress and the effects of mechanical strain on cancer progression and metastasis. We developed an adhesion assay to investigate cancer cell adhesion in the presence of shear stress. Adhesion assays performed under flow yield markedly different results from static adhesion assays. Treatment of breast cancer cells with integrin inhibitors demonstrated that these compounds had minimal effect on cancer cell adhesion to endothelial cells under static conditions, whereas under shear many of these compounds significantly reduced adhesion of cancer cells. A static adhesion assay of breast cancer cells to various types of ECM showed higher adhesion of the less aggressive MCF-7 cell line in comparison to the more aggressive MDA-MB-231 cell line. In contrast, flow incorporating assays showed increased adhesion of the MDA-MB-231 cell line. Finally, a high throughput screen using a kinase inhibitor library of 80 compounds identified seven hits, several of which were multiple hits for the same target, in the shear assay, whereas no hits were found in the same assay performed under static conditions. This assay was then modified to read cancer cell adhesion in real time, enabling the investigation of cancer cell adhesion kinetics to several ECM with various drug treatments. Cancer cell adhesion had a complex relationship with shear stress, cancer cell line, and ECM. Application of mechanical strain effects cancer cell signaling and function. Cyclic mechanical strain reduced proliferation and increased drug resistance in MDA-MB-231 breast cancer cells. Mechanical strain increased Yap/Taz nuclear localization, while decreasing nuclear phosphorylated Smad 2/3. Mechanical strain increased adhesion to endothelial cells while decreasing transmigration through an endothelial monolayer. These findings were supported by RNA sequencing data. The addition of shear stress to adhesion assays in a high throughput device enabled rapid investigation of inhibitors of cancer cell adhesion while more accurately recapitulating conditions within the body. Application of mechanical strain altered cancer cell proliferation, drug resistance, adhesion and invasion. Biophysical forces are present throughput the tumor microenvironment and play an important role in regulating cancer metastasis and progression

The extracellular matrix (ECM) plays an important role in cell regulation, development, and homeostasis. Not only does it provide a structural scaffold for cell migration and interaction, but also acts as a deposit for cytokines and chemokines that regulate cellular function. Cells can modify the structure, composition, and biochemical characteristics of the ECM, and are able to apply mechanical forces that can alter the ECM and it components. One essential component of the ECM is fibronectin (FN). A protein that forms fibers in the ECM, and has various binding sites that allow intermolecular interactions among other FN molecules, with other ECM constituent proteins, carbohydrates, cell-ECM binding proteins such as integrins, and various cytokines and signaling molecules. FN has several mechanosensitive cryptic binding sites that open upon protein unfolding due to mechanical tension. During inflammation and other physiological processes, swelling of tissue and ECM remodeling can induce mechanical stretching or relaxing of the ECM, which can in turn induce a change a in the conformation of Fn. This thesis contributes to the understanding of how force-induced conformational changes in FN can have an impact on cytokine binding, by focusing on how mechanically stretching FN fibers affect the binding of interleukin-7 (IL-7) to FN. IL-7 is a cytokine belonging to the g-c interleukin family that plays an essential role in the survival, proliferation and differentiation of naive and memory T-cells in lymphatic tissues, and the development of B and T-cells in the thymus. It is produced by stromal cells in the lymph nodes and thymus, and has been previously shown to bind to fibronectin (FN). In this thesis, a Förster Resonance Energy Transfer (FRET) based method to correlate ligand-binding to changes in FN conformation is presented. This method provides a quantitative tool to assess how changes in FN conformation result in modified cytokine binding. The developed method was used to assess whether FN’s conformational changes due to mechanical force application result in changes in binding of IL-7. We found that stretching of FN fibers increased IL-7 binding, and localized the FN binding site on the CD-loop region of IL-7 using a synthetic CD-loop peptide which also showed increased binding to stretched fibrillar FN. Upon structural analysis of IL-7 binding to its receptors IL7RA and IL2RG, we propose that the CD-loop is available for simultaneous interaction with FN while IL-7 is bound to its cognate cell receptors. We also propose a negative-feedback mechanism reciprocally regulating the tensional state of FN fibers and IL-7 signaling. Our data show then, for the first time, a mechano-regulated mechanism of IL-7 binding to the ECM, where stretching FN fibers might locally concentrate IL-7 in an extracellular matrix bound state. Finally, first steps towards determining what impact these findings may have on cellular function are presented, to motivate the refinement and/or development of adequate assays to test the physiological implications of our findings. Since IL-7 is produced by lymph node stromal cells (LNSCs), and stromal cells from the thymus, and these cells in turn respond to IL-7, we aimed to characterize mechano-biological behaviour of these cells, their ECM, and make the first steps towards elucidating whether these cells can bring about changes in the ECM that could result in differential binding of IL-7 to fibronectin in the ECM, and in turn, whether changes in IL-7 binding levels can have an effect on their function. The work presented here could be of relevance for the further understanding of how mechanically induced changes of the ECM affect chemokine and cytokine binding and the effects of this on cell function. These aspects should be considered in the future for the development of current and/or new IL-7 based immunotherapies that take into consideration possible changes in ECM due to disease, inflammation or ageing.

Abstract: Strain gauges have been powerful tools in experimental stress analysis. This importance is expected to continue, even though other means of strain measurement are continually introduced to the market. The strain gauge is a simple device that can be easily installed to measure mechanical strain. By far, the one-dimensional single-filament has been the most common strain gauge. Conventional strain gauges are typically made from thin-foil metal; thus the resistivity change under stress is insignificant. On the other hand, semiconductor material demonstrated considerable resistivity change as a result of the applied mechanical strain or stress. In this work, the piezoresistive characteristics of silicon crystal are investigated to realize a robust Micro Electro Mechanical (MEMS) strain sensor. This sensor has been developed to withstand harsh environmental conditions, such as those in Structural Health Monitoring (SHM) applications. Silicon strain gauges have demonstrated higher gauge factor, sensitivity, and accuracy compared to conventional thin-foil strain gauges. Unfortunately, silicon strain gauges suffer from large temperature effect, which influences their performance dramatically. This temperature effect puts various challenges on the development and application of semiconductor sensors. On top of these challenges are the temperature compensation of the output signal, packaging, and fabrication. In addition, transferring strain through different structural layers causes substantial loss in the sensed strain values. Piezoresistivity theory is presented and applied to develop a new MEMS strain sensor. Taking into account all geometric and material characteristics, various tools and techniques, such as indicial equations, Finite Element Modeling (FEM) (ANSYS10.0®), and experimental evaluation, were employed to go through the development cycle of the piezoresistive strain sensor. In addition, alignment errors during microfabrication have been investigated. The proper microfabrication parameters and piezoresistors configuration were selected by investigating the silicon crystal material properties based on the crystallographic directions. A microfabrication process flow has been developed exploring a group of fabrication processes available at the University of Alberta micromachining and nanofabrication facility (NanoFab). In order to minimize the loss in the transferred strain, geometric features were created in the silicon substrate. These geometric features have resulted in stress discontinuity in their vicinity, which introduced the concept of geometrical gauge factor. The geometrical gauge factor is a new concept that can ‘virtually’ improve the performance of any piezoresistive sensor by utilizing the silicon carrier to increase the differential stress around the piezoresistive sensing elements. The main limitation to use this concept is whether or not material properties will accommodate the resulted stress concentration without failing the sensor. The developed sensor was evaluated, tested, and characterized at the University of Alberta and Syncrude Canada Edmonton Development Research Center. Uniaxial tension was utilized to calibrate a number of chip designs. The temperature coefficient of resistance (TCR) was also evaluated. Preliminary packaging procedure was proposed and applied. Comparing the performance characteristics of the developed MEMS strain sensor to a 350Ω thin-foil strain gauge showed that the piezoresistive MEMS strain sensor had better performance characteristics: sensitivity and resolution, with room for significant improvements. In addition, the MEMS strain sensor can be successfully applied under varying temperature conditions. The solution of the above challenges and the small size of MEMS sensors have resulted in a novel MEMS-based strain sensor with low power consumption, compared to conventional strain gauges. This low power consumption promotes this sensor in wireless SHM systems as the sensing unit, which can extend such technology to wider range of applications. The MEMS strain sensor has potential to provide a valuable tool to improve the current SHM systems as well as allowing higher number of sensors to be economically deployed. As a result, the reliability of both the SHM system and equipment will be enhanced, which will reflect positively on the economic performance of the equipment.

The field of tissue engineering holds great promise for the treatment of cardiovascular disease. Cardiovascular tissues reside in a mechanically dynamic environment and it is therefore important to understand how vascular cells respond to mechanical signals from the extracellular matrix (ECM) to which they are adhered. The goal of this thesis is to provide a better understanding of the effects of mechanical strain on cardiovascular cell gene expression, particularly in terms of genes associated with the calcification process in the vasculature. Tissues were engineered by seeding smooth muscle cells (SMCs) into three-dimensional scaffolds. Mechanical strain was applied using a custom-made device to exert a uniaxial strain of 7% at a frequency of 1 Hz. SMCs utilized distinct integrin receptors to adhere to various substrates and cellular gene expression was regulated by the scaffold chemistry. These cells were found to express a variety of bone-associated genes, such as osteopontin, matrix gla protein (MGP), alkaline phosphatase, and the transcription factor CBFA-1. Strikingly, however, expression of these genes was downregulated in tissues exposed to cyclic strain at all time points ranging from 5 to 150 days. Further, long-term strain played a protective role against calcification, as unstrained tissues exhibited increased calcium deposition compared to strained tissues. The mechanism of strain-induced osteopontin and MGP gene expression by SMCs was also studied by investigating the involvement of the ECM and matrix-associated protein kinase (MAPK) signaling pathways. The MAPK signal transduction pathways ERK and p38 were studied and neither pathway regulated the expression of either osteopontin or MGP with strain application. However, blocking ERK signaling inhibited the expression of MGP, implicating the involvement of that pathway in the regulation of MGP expression. Overall, the results in this thesis suggest that without an appropriate mechanical environment, SMCs in three-dimensional culture undergo a phenotypic conversion to an osteoblast-like pattern of gene expression. These studies shed light onto how mechanical strain is transduced through the cell and regulates the gene expression of known mediators in the vascular calcification process. Understanding the role of strain in the vasculature can provide insight into causes of disease and lead to better treatment options.

The fields of regenerative medicine and cellular engineering hold tremendous promise in the treatment of a large number of medical conditions. The key to these efforts lies in understanding how cells sense and respond to stimuli in their environment. One critically important stimulus that guides the development and function of a variety of tissues is mechanical force. The goal of this thesis is to improve the current understanding of how cells receive and process mechanical signals. Previous work suggests that cell adhesion structures called focal contacts may play a central role in the transduction of mechanical signals. The hypothesis guiding the studies in this thesis is that mechanical force in the form of strain, applied via the extracellular matrix, will cause changes in the composition and signaling activity of focal contacts. To address this hypothesis, and the mechanisms involved in the cell response, an experimental system was developed to apply strain to smooth muscle cells cultured on flexible silicone rubber substrates coated with adhesion proteins. Following strain application, the distribution of the focal contact components vinculin, alpha-actinin, and paxillin between the detergent-insoluble cytoskeletal pool and the soluble cytoplasmic pool were evaluated by immunofluorescence microscopy and western blotting. Cyclic strain, but not single step changes in strain, were found to increase insoluble vinculin and paxillin, as well as the tyrosine phosphorylation state of paxillin. In order to determine whether a soluble signaling step was involved in this process, incorporation of fluorescent vinculin into the focal contacts of permeabilized cells was observed during strain. Again, cyclic strain, but not step changes in strain, caused enrichment of vinculin in cell adhesions. These results suggest that a threshold number of mechanical perturbations must be exceeded to alter net focal contact composition, and that one part of the mechanism is mediated by a direct effect of force on adhesions. A better understanding of force-induced changes in cell adhesions may provide insight into the role of mechanical signals in health and disease, and may allow the rational application of these signals for therapeutic use in guiding cell function in cell and tissue engineering.