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Tendon and ligament injuries are common orthopedic problems that have an enormous impact on the quality of life of affected patients. Despite the frequency at which these injuries occur, current treatments are unable to restore native function to the damaged tissue. Because of this, reinjury is common. It is well known that mechanical stimulation is beneficial for promoting tendon and ligament development and tissue homeostasis; however, the specific mechanisms remain unclear. The transcription factor scleraxis (Scx) is an interesting candidate for mediating the tendon and ligament mechanoresponse, as it has been shown that Scx expression is induced by cyclic mechanical strain in tenocytes and is required for mechanically-induced stem cell tenogenesis. Moreover, Scx expression is increased in adult tendons following exercise. The studies described in this dissertation therefore focus on the combined role of Scx and mechanical stimulation in two contexts: 1) influencing ligament cell differentiation and 2) regulating adult tenocyte behavior. In the first study, transient Scx overexpression combined with mechanical strain in a 3D collagen hydrogel model was investigated as a means of deriving mature ligament cells from stem cells for use in ligament tissue engineering. Scx overexpression in C3H10T1/2 cells cultured in collagen hydrogels under static strain resulted in increased construct contraction and cell elongation, but no concurrent increase in the expression of ligament-related genes or production of glycosaminoglycans (GAG). When combined with low levels of cyclic strain, Scx overexpression resulted in increased mechanical properties of the tissue constructs, increased GAG production, and increased expression of ligament-related genes compared to cyclic strain alone. Together, these results demonstrate that Scx overexpression combined with cyclic strain can induce ligament cell differentiation and suggest that Scx does so by improving the mechanosensitivity of cells to cyclic strain. In the second study, the role of Scx in adult tenocyte mechanotransduction was explored using RNA-sequencing (RNA-seq) and small interfering RNA (siRNA) technologies. Equine tenocytes were exposed to siRNA targeting Scx or a control siRNA and maintained under cyclic mechanical strain prior to being submitted for RNA-seq. Comparison of the resulting transcriptomes revealed that Scx knockdown decreased the expression of several genes encoding important focal adhesion adaptor proteins. Correspondingly, Scx-depleted tenocytes showed abnormally long focal adhesions, decreased cytoskeletal stiffness, and an impaired ability to migrate on soft surfaces. This suggests that Scx regulates the tenocyte mechanoresponse by promoting the expression of focal adhesion-related genes. Combined, the results of these studies support a role for Scx in tendon and ligament cell mechanotransduction and identify the regulation of genes related to maintaining the cell-extracellular matrix connection and cytoskeletal dynamics as a potential mechanism. These findings enhance our understanding of how mechanical stimulation influences cell behavior and provide new research directions and methodologies for future studies of tendon and ligament mechanobiology. / Ph. D.

Pressure ulcers (PU) are localized damage to skin and underlying tissue that forms in response to ischemia and subsequent hypoxia from external applied mechanical loads such as pressure. We demonstrate how a multiscale and multiphysics finite element model can capture the process of pressure ulcer formation.

Mechanobiology is an emerging field that aims to understand how physical forces regulate cell function, morphology, and development. Cells interpret forces, such as the deformation of the membrane to encapsulate a particle, or the rigidity of the extracellular matrix (ECM), and make decisions about cell adhesion, motility, and differentiation. These cell-ECM interactions are important to maintaining homeostasis, and the disruption of this interface has pathological consequences. Common diseases, such as Alzheimer’s disease, cancer, and atherosclerosis each arise, in part, from an abnormality in the mechanotransduction pathway. Hence, understanding the contribution of this pathway and the role of the ECM in cell function, proves to be a useful tool in improving drug targeting and understanding disease progression. While size, shape and surface chemistry of nanoparticle uptake has been extensively studied, varying the particle mechanics can also be a useful design strategy to manipulate particles and improve uptake and targeting. Using model polystyrene-co-N-isopropylacrylamide (pS-co-NIPAM) particles, with varying elastic moduli, it was observed that as the particles became stiffer, there was a subsequent decrease in bound/internalized particles for phagocytic RAW264.7 macrophage and non-phagocytic HepG2 hepatoma carcinoma cells, showing that both of these cell types are sensitive to particle mechanics, even in a higher stiffness regime (MPa). ECM mechanics have recently been implicated in tissue stiffness changes that precede and drive disease development. Recent research has started looking into these effects in the progression of neurodegenerative diseases. This research found that the elasticity of the brain becomes softer with aging, and even softer in patients with AD. Analogous to the pS-co-NIPAM studies, this tissue softening could have implications on amyloid-beta endocytosis as well as neuron dystrophy in response to the peptide. Understanding the role of the ECM in the progression of AD in vitro could provide a better approach to determine an in vivo mechanism behind Alzheimer’s disease pathology. In order to mimic a softer ECM substrate, SH-SY5Y neuroblastoma and human primary neurons were plated on 2-D polyacrylamide and 3-D collagen gels with varying stiffness ranging from 0.15-25kPa. Both cell types grown using these substrates show a sensitivity to their ECM environment, and display an increase in cell spreading and the number of F-actin stress fibers with an increase in substrate rigidity. Moreover, the extent of Aβ internalization and aggregate production increased with ECM stiffness for SH-SY5Y neuroblastoma. Intracellular Aβ processing remains a central question to understanding the early-stage events in AD pathogenesis. As the ECM can modify Aβ endocytosis and aggregation, the ECM is likely influencing downstream neurotoxic effects of AD. Despite an increase in the plaque production on the soft substrates, both SH-SY5Y neuroblastoma and primary neurons showed a decreased toxicity to Aβ with decreasing substrate stiffness. This decrease in toxicity is associated with cytoskeletal actin remodeling, as cells plated on plastic, but pretreated with cytochalasin D displayed a recovery in viability in response to the oligomeric species. The softening of the ECM initiates actin cytoskeletal depolymerization, as a protective mechanism against neuronal loss and AD progression. This work demonstrates that the ECM impacts Aβ endocytosis and aggregation, and the ECM prompts neuroprotective actin reorganization against the neurotoxic effects of AD. Further, it is demonstrated the biophysical role of ECM stiffness in modifying Aβ internalization, plaque production, and toxicity offers an improved in vitro model of critical AD components. By better understanding the cytoskeletal reorganization triggered by a softening ECM, potential novel avenues of therapeutic intervention could later be determined to stop the progression of the disease.

Cells in the body respond to mechanical loads in ways that are crucial to normal and disease physiology. Understanding these processes is difficult due to the complex mechanical environment in vivo. In this research, we have developed several cell-stretching devices capable of subjecting cell cultures to non-uniform stretch distributions in order to investigate pathological responses of vascular smooth muscle cells to physiologic stretches. 10T1/2 cells were cyclically stretched with these devices for 24 hours upon silicone membranes, PDMS tubes, and within 3D PEGDA hydrogels. After stretching, altered cell behaviors were measured, including orientation, proliferation (quantified by BrdU incorporation), and gene expression (quantified by real-time, RT-PCR). Cells demonstrated marked changes in orientation, proliferation, and mRNA expression, which all varied with cellular location in the non-uniform environment. More specifically, increased orientation, increased proliferation, and more dramatically altered mRNA expression were found in regions of high, uniaxial stretch, relative to regions of low, near-equibiaxial stretch. These findings demonstrate the capabilities of graded stretch distributions to produce graded cell responses, indicating potentially localized smooth muscle cell behavior in a diseased artery. The novel devices employed herein will hopefully improve our understanding of these complicated cellular pathways, ultimately allowing for improved treatment or prevention of vascular disease.

Cell mechanics are directly related to the biological functionality of a cell, and therefore have been extensively studied. Current understanding of the unique relationships associated with mechanical loading conditions and the biological outcomes of a cell is far from complete [1]. The main objective of this thesis work was the design of a device capable of determining mechanical properties including stiffness and Young’s modulus of a biological cell. The device was implemented using micro-electro mechanical systems technology (MEMS), and the cell testing was limited to yeast cells for the purpose of this research. The design consisted of a micro-gripper which performed controlled cell squeezing with a spring of known stiffness. Differential displacements were obtained allowing for the calculation of cell mechanical properties. The incorporation of spatially periodic structures on the moving components of the gripper enabled measurements with 10 nm precision based on discrete Fourier transformation and phase [2].

In silico models of intracranial aneurysm (IA) evolution aim to reliably represent the mechanical blood flow environment, the biology of the arterial wall and, crucially, the complex link between the two, namely the mechanobiology of healthy and diseased arteries. The ultimate goal is to create diagnostic tools for personalized management and treatment of aneurysm disease. Towards that target, the work presented in this thesis aims to establish a directly interactive link between experimental (in vivo and in vitro) and computational work for biologically and clinically relevant research on aneurysm disease. Mechanobiological hypotheses were firstly investigated in a novel 1D mathematical conceptual model of aneurysm evolution: for the first time these included representations of endothelial heterogeneity and smooth muscle cell (SMC) active stress response and apoptosis. The 1D investigations analysed and assessed the role of wall shear stress (WSS) homeostasis in elastin degradation, and the evolving role of the adventitia as a protective sheath in health and primary load-bearer in disease. The 1D framework was applied to a specific patient's aneurysm using both imaging and histological data to parameterise the model, calculating a material parameter for the adventitital collagen. The predicted evolution captured aspects of tissue changes measured with time focusing on the remodelled tissue wall thickness consistent with the experimental measurements, and physiological cyclic deformation in order to propose an approach to modelling adventitia's adaptive role to load bearing. Furthermore, an existing Fluid-Solid-Growth (FSG) computational framework was adapted and calibrated for the same patient-specific case with the help from the experimental data and the analysis from the 1D framework. This FSG model quantifies the arterial mechanical environment and captures the mechanical response of the fibrous arterial constituents. Comparing 1D and 3D investigations to establish consistency for our models, the 3Dmodel tested the hypothesis of WSS homeostasis, additionally introducing the element of spatial heterogeneity in the definition, and a new hypothesis linking cyclic deformation with collagen growth that ensures a physiological mechanical environment in stabilised aneurysms. Moreover, the FSG framework was applied in a specific rabbit aneurysm case and extended to link growth and remodeling to the detailed representation of the pulsatile blood flow mechanical environment. This research illustrates the power of computational modelling when coupled with rich data sets on the physiology, histology and geometry of healthy and diseased vascular tissue. In particular, the integrative modelling framework provides the foundation for establishing mechanobiological links crucial to aneurysm progression, and a basis for further research towards creating reliable aneurysm clinical tools.

The composition and conformational state of biological molecules have a profound influence on cell behavior and large-scale processes including development and disease progression. Fibronectin fibers are a prevalent component of the extracellular matrix that are believed to adopt a wide array conformations with different functions. Two factors that are hypothesized to regulate fibronectin conformation, and hence fibronectin biological function, are allosteric regulators, such as heparan sulfates, and mechanical strain. However, the relative influence of allosteric regulators and mechanical forces on fibronectin conformation has not been determined. This conformational regulation is especially important in the context of the heparin 2 binding domain (modules III12 to III14), which is known to bind and present numerous growth factors, such as vascular endothelial growth factor, to cells. This thesis will highlight three contributions to this field. First, a new, and remarkably simple technique was developed that permits the detection of the non-equilibrium fibronectin conformations. This technique is founded on the identification of monoclonal antibodies that have altered affinities for fibronectin based on heparin treatment or mechanical strain dependence, or that bind fibronectin equally well in all conditions. Second, the impact of both heparin and mechanical strain on the binding of VEGF to the hep2 region of fibronectin was investigated. It was discovered that both strain and heparin co-regulate VEGF binding. Finally, studies of cell attachment and migration on single fibers of fibronectin with controlled strain states provided the first direct evidence that mechanical strain regulates cell attachment, spreading, and migration on a fibronectin matrix. This body of work demonstrating that the conformational changes in fibronectin lead to altered biological activity has broad impact in a number of fields due to the ubiquitous presence and requirement of fibronectin in cell and tissue function.

The Intervertebral disc (IVD), comprised of two distinct regions, namely the fibrous annulus fibrosus (AF) and the gelatinous nucleus pulposus (NP), is a fibrocartilage pad located between adjoining vertebrae of the spine. The function of the IVD is to provide stability to the spine, while maintaining movement. IVD degeneration, also known as degenerative disc disease (DDD), is the process whereby the IVD tissue degrades, resulting in loss of function to the disc. Low back pain (LBP) is associated with the degeneration of the IVD, making it important to investigate the pathogenesis of DDD, as this could lead to novel therapies for the prevention and/or treatment of LBP. Mechanical stimuli (MS) are known to be important for IVD cell matrix homeostasis, with cells of the AF and NP responding to physiological forces with a trend towards increased matrix anabolism, while non-physiological forces lead to matrix catabolism. Furthermore, recent evidence suggests that IVD cells derived from degenerate tissue may have lost their ability to respond to physiological MS in the 'normal' anabolic manner, potentially leading to the progression of DDD. It is therefore important to investigate the response of IVD cells derived from both non-degenerate and degenerate tissue to MS, to ascertain whether there is a difference with degeneration. If the response is found to be altered with degeneration, then elucidation of the potentially altered mechanotransduction pathway utilised by degenerate cells could lead to the discovery of novel therapeutic targets for the treatment of DDD. To date, the majority of IVD MS studies have concentrated on the response of NP cells to hydrostatic pressure, with only a limited number of AF studies available. Thus, the first aim of this PhD was to investigate the response of human AF cells derived from non-degenerate and degenerate IVDs to the physiologically relevant mechanical stimulus of cyclic tensile strain (CTS), to ascertain whether the response (regulation of matrix protein and matrix degrading enzyme gene expression) was frequency-dependent or altered with IVD degeneration. Using an in vitro mechanical loading system (Flexcell® Tension Plus™ system, Flexcell International) capable of delivering a CTS of 10% strain, 0.33Hz or 1.0Hz for 20 minutes, the response of AF cells derived from non-degenerate IVDs was found to be frequency-dependent (reduced catabolism at 1.0Hz, with decreased MMP -3 and ADAM-TS -4 gene expression; and catabolic at 0.33Hz, with decreased types I and II collagen and increased MMP -9 gene expression). Furthermore, the response of AF cells to 1.0Hz CTS was shown to be altered with IVD degeneration, depicted by a switch from reduced catabolism (decreased MMP -3 and ADAM-TS -4) in non-degenerate AF cells, to reduced anabolism (decreased aggrecan and type I collagen gene expression) in degenerate AF cells. Subsequently, the second aim of the PhD was to attempt to elucidate the mechanotransduction pathways operating in human AF cells derived from non-degenerate and degenerate IVDs, to ascertain whether the mechanotransduction pathway was altered with IVD degeneration. An identical mechanical stimulation regime was used (1.0Hz CTS) in parallel with functional inhibitors against the cytokines interleukin (IL) -1 and -4, and the cell surface receptors, RGD-recognising integrins. Additionally, the involvement of the cytokine associated transcription factors, nuclear factor kappa beta (NFκB) and signal transducer and activator of transcription (STAT) -6, as well as the integrin-associated kinase, focal adhesion kinase (FAK) was investigated in 1.0Hz CTS-treated non-degenerate AF cells. The response to 1.0Hz CTS (reduced catabolism) of AF cells derived from non-degenerate IVDs occurred in an IL -1, IL -4 and RGD-recognising integrin-dependent manner, with FAK being phosphorylated. Of significant interest, the altered response of AF cells derived from degenerate IVDs to 1.0Hz CTS (reduced anabolism) occurred independently of either cytokine and independently of RGD-recognising integrins, suggesting an altered mechanotransduction pathway in operation and warranting further investigation.

It has been well established that the fundamental behaviors of mammalian cells are influenced by the physical cues that they experience from their surrounding environment. With respect to cells in our bodies, mechanically-driven morphological and phenotypic changes to our cells have been linked to responses critical to both normal development and disease progression, including lung, heart, muscle and bone disorders, and cancer. Although significant advancements to our understanding of cell behavior have been made using 2D cell culture methods, questions regarding how physical stretch guides cell behavior in more complex 3D biological systems remain unanswered. To address these questions, we used microfabrication techniques to develop vacuum-actuated stretchers for high throughput stretching and dynamic mechanical screening of 3D microtissue cultures. This thesis contains five research chapters that have utilized these devices to advance our understanding of how cells feel stretch and how it influences their behavior in a 3D matrix. In the first research chapter (chapter 2), we characterized how stretch is transferred from the tissue-level to the single-cell level and we investigated the cytoskeletal reinforcement response to long-term mechanical conditioning. In the second research chapter (chapter 3), we examined the effects of an acute dynamic stretch and found that 3D cultures soften through actin depolymerization to homeostatically maintain a mean tension. This softening response to stretch may lengthen tissues in our body, and thus may be an important mechanism by which airway resistance and arterial blood pressure are controlled. In the third and forth research chapters (chapter 4-5), we investigated the time dependencies of microtissues cultures and we found that their behavior differed from our knowledge of the rheological behavior of cells in 2D culture. Microtissues instead followed a stretched exponential model that seemed to be set by a dynamic equilibrium between cytoskeletal assembly and disassembly rates. The difference in the behavior from cells in 2D may reflect the profound changes to the structure and distribution of the cytoskeleton that occur when cells are grown on flat surfaces vs. within a 3D environment. In the fifth and final research chapter (chapter 6), we examined how mechanical forces may contribute to the progression of tissue fibrosis through activating latent TGF-β1. Our results suggest that mechanical stretch contributes to a feed forward loop that preserves a myofibroblastic phenotype. Together these investigations further our understanding of how cells respond to mechanical stimuli within 3D environments, and thus, mark a significant contribution to the fields of mechanobiology and cell mechanics.