Cell-level and tissue-level mechanical properties are key to healthy biological functions, and many diseases and disorder arise or progress due to altered cell and tissue mechanics. Pulse electric field (PEFs), which employ intense external electric fields to cause electroporation, a phenomenon characterized by increased cell membrane permeability, also can cause significant changes to cell and tissue mechanics. Here, we investigate the mechanics of brain and brain cancer cells, specifically focusing on how PEFs impact cell mechanics and PEF-induced blood-brain barrier disruption. In our first study, we investigate single-cell mechanical disruption of glioblastoma cells after reversible electroporation using Nanonet Force Microscopy (NFM). A precise network of extracellular-matrix mimicking nanofibers enabled cell attachment and contraction, resulting in measurable fiber deflections. Cell contractile forces were shown to be temporarily disrupted after reversible electroporation, in an orientation and field-dependent manner. Furthermore, we found that cell response is often a multi-stage process involving a cell-rounding stage, biphasic stage, and a cell re-spreading stage. Additionally, cell viability post-PEFs was orientation-dependent. In another study, we investigated the mechanical properties of brain cancer for various-grade glioma cells (healthy astrocytes, grade II, grade III, and grade IV (glioblastoma) cells). A microfluidic constriction channel caused cell deformation as cells, driven by hydrostatic pressure, entered a narrow constriction. Finite element models of cell deformation and a neural network were used to convert experimental results (cell entry time and cell elongation within the channel) into elastic modulus values (kPa). We found that the that low-grade glioma cells showed higher stiffnesses compared to healthy and grade IV glioma cells, which both showed similar values. These results warrant future studies to investigate these trends further. PEFs can induce Blood-brain barrier (BBB) disruption, an effect we studied using a multiplexed, PDMS microdevice. A monolayer of human cerebral endothelial cells on a semi-permeable membrane was used to model the BBB, and permeability was assessed by the diffusion of a fluorescent dye from an upper to lower channel. A custom tapered channel and branching channel design created a linear gradient in the electric field within the device that enabled six electric field strengths to be tested at once against two unexposed (control) channels. Normalization of permeability by the control channels significantly removed experimental noise. We found that after high-frequency bipolar irreversible electroporation (HFIRE) electric pulses, permeability transiently increased within the first hour after electroporation, in a voltage- and pulse-number dependent manner. However, we found significant electrofusion events after pulsing at high voltages, which reduced monolayer permeability below baseline values. This device enables efficient exploration of a wide range of electroporation parameters to identify the optimal conditions for blood-brain barrier disruption. In another blood-brain barrier study, we incorporate dense, polystyrene nanofiber networks to create ultra-thin, ultra-porous basement-membrane-mimics for In vitro blood-brain barrier models. Fiber networks are fabricated using the non-electrospinning Spinneret-based Tunable Engineered Parameters (STEP) technique. Endothelial cells cultured on one side of the fiber network are in close contact with supporting cell types (pericytes) cultured on the backside of the fibers. Contact-orientation co-cultures have been shown to increase blood-brain barrier integrity, and our nanofiber networks increase the physiological realism of basement-membrane mimics for improve modeling. Finally, we investigate how cell viability post-electroporation is impacted by cell morphology. The impact of cell morphology (shape and cytoskeletal structure) on cell survival after electroporation is not well understood. Linking specific morphological characteristics with cell susceptibility to electroporation will enhance fundamental knowledge and will be widely useful for improving electroporation techniques where cell viability is desirable (gene transfection, electrofusion, electrochemotherapy) or where cell viability is undesirable (tumor ablation, cardiac ablation). Precise control of cell shape and orientation enabled by nanofiber scaffolds provides a convenient and expedient platform for investigating a wide variety of factors (morphological and experimental) on cell viability. Altogether, these investigations shed new light on cell mechanical changes due to disease and pulsed electric fields, and suggest opportunities for improving brain cancer therapies. / Doctor of Philosophy / In biology, structure and function are interrelated. Cells and tissue have structures that enable them to perform their proper function. In the case of disease, cell and tissue properties are altered, leading to dysfunction. Alternatively, healthy structures sometime hinder effective treatments, and therefore can be therapeutically disrupted to improve treatments. In this study, we investigate single-cell and multi-cellular mechanical change due to disease or after pulsed electric fields (PEFs), with a specific focus on the brain. Pulsed electric fields (PEFs) use electrodes to deliver short, intense pulses of electrical energy to disrupt cell membranes and change cell mechanics. We studied as single-cell contractility, cancer cell stiffness, and blood-brain barrier (BBB) disruption by PEFs. We found that PEFs cause significant change to cell shape and mechanics, and can disrupt the BBB. By studying several grades of brain cancers, we found that low-grade brain cancer (gliomas) showed increased stiffness compared to healthy and highly diseased (grade IV) cells. To mimic the BBB, we used microfluidic devices to grow specialized brain cells (endothelial cells) on permeable membranes and nanofibers networks and showed that these devices can mimic structures found in animals/humans. Finally, we studied how cell properties (such as shape) determine whether cells will survive PEFs. Taken together, our investigations improve the understanding of brain mechanics during disease and after PEFs, and suggest the usefulness of PEFs for improved brain cancer therapies.

<p>To elucidate the mechanisms of cellular mechanotransduction, it is necessary to employ biomaterials that effectively merge biofunctionality with appropriate mechanical characteristics. Agarose is a standard biopolymer used in cartilage mechanobiology but lacks necessary adhesion motifs for cell-matrix interactions to complete mechanostransduction studies. Collagen type I is a natural biomaterial used in cartilage mechanotransduction studies but creates an environment much softer than native cartilage tissue.  In these studies, agarose was blended at two final concentrations (2% w/v and 4% w/v) with collagen type I (2 mg/mL). The overarching goal was to determine whether a composite hydrogel of agarose and collagen can create a mechanically and biologically suitable matrix for chondrocyte studies. First, hydrogels were characterized by rheologic and compressive properties, contraction, and structural homogeneity. Following baseline characterization, primary murine chondrocytes were embedded (1 × 106 cells/mL) within the hydrogels to assess the longer-term <em>in vitro</em> impact on matrix mechanics, cell proliferation, sulfated glycosaminoglycan (sGAG) content, and cellular morphology. To begin probing questions about physiologic loading conditions that chondrocytes experience <em>in</em> <em>vivo</em>, a custom compression loading system was validated using cell-laden hydrogels. Briefly, the 4% agarose – 2 mg/mL collagen I hydrogel composites were able to retain chondrocyte morphology over 21 days in culture, resulted in continual sGAG production, and had bulk mechanics similar to that of the stiffest hydrogel material tested, indicating this hydrogel class may be promising towards developing an effective hydrogel for chondrocyte mechanotransduction and mechanobiology studies, a critical step towards a fuller understanding of cell-matrix interactions. </p>

<p>Breast cancer is the most commonly diagnosed cancer type worldwide. Given high survivorship, increased focus has been placed on long-term treatment outcomes and patient quality of life. While breast-conserving surgery (BCS) is the preferred treatment strategy for early-stage breast cancer, anticipated healing and breast deformation (cosmetic) outcomes weigh heavily on surgeon and patient selection between BCS and more aggressive mastectomy procedures. Unfortunately, surgical outcomes following BCS are difficult to predict, owing to the complexity of the tissue repair process and significant patient-to-patient variability. To overcome this challenge, we developed a predictive computational mechanobiological model that simulates breast healing and deformation following BCS. The coupled biochemical-biomechanical model incorporates multi-scale cell and tissue mechanics, including collagen deposition and remodeling, collagen-dependent cell migration and contractility, and tissue plastic deformation. Available human clinical data evaluating cavity contraction and histopathological data from an experimental porcine lumpectomy study were used for model calibration. The computational model was successfully fit to data by optimizing biochemical and mechanobiological parameters through the Gaussian Process. The calibrated model was then applied to define key mechanobiological parameters and relationships influencing healing and breast deformation outcomes. Variability in patient characteristics including cavity-to-breast volume percentage and breast composition were further evaluated to determine effects on cavity contraction and breast cosmetic outcomes, with simulation outcomes aligning well with previously reported human studies. The proposed model has the potential to assist surgeons and their patients in developing and discussing individualized treatment plans that lead to more satisfying post-surgical outcomes and improved quality of life.</p>

<p dir="ltr">During many biological processes, cells move through their surrounding environment by exerting mechanical forces. The mechanical forces are mainly generated by molecular interactions between actin filaments (F-actins) and myosin motors within the actin cytoskeleton. Forces are transmitted to the surrounding extracellular matrix via adhesions. In this thesis, we employed agent-based computational models to study the interactions between F-actins and myosin in the motility assay and the cell migration process. In the first project, the myosin motility assay was employed to study the collective behaviors of F-actins. Unlike most of the previous computational models, we explicitly represent myosin motors. By running simulations under various conditions, we showed how the length, bending stiffness, and concentration affect the collective behavior of F-actins. We found that four distinct structures formed: homogeneous networks, flocks, bands, and rings. In addition, we showed that mobile motors lead to the formation of distinct F-actin clusters that were not observed with immobile motors. In the second project, we developed a 3D migration model to define how cells mechanically interact with their 3D environment during migration. Unlike cells migrating on a surface, cells within 3D extracellular matrix (ECM) must remodel the ECM and/or squeeze their body through the ECM, which causes 3D cell migration to be significantly more challenging than 2D migration. Our model describes realistic structural and rheological properties of ECM, cell protrusion, and focal adhesions between cells and the ECM.</p>

In vitro mechanotransduction studies, uncovering the basic science of the response of cells to mechanical forces, are essential for progress in tissue engineering and its clinical application. Many varying investigations have described a multitude of cell responses, however as the precise nature and magnitude of the stresses applied are infrequently reported and rarely validated, the experiments are often not comparable, limiting research progress. This thesis provides physical and biological validation of a widely available fluid stimulation device, a see-saw rocker, as an In vitro model for cyclic fluid shear stress mechanotransduction. This allows linkage between precisely characterised stimuli and cell monolayer response in a convenient six-well plate format. Computational fluid dynamic models of one well were analysed extensively to generate convergent, stable and consistent predictions of the cyclic fluid velocity vectors at a rocking frequency of 0.5 Hz, accounting for the free surface. Validation was provided by comparison with flow velocities measured experimentally using particle image velocimetry. Qualitative flow behaviour was matched and quantitative analysis showed good agreement at representative locations and time points. A maximum shear stress of 0.22Pa was estimated near the well edge, and time-average shear stress ranged between 0.029 and 0.068Pa, within the envelope of previous musculoskeletal In vitro fluid flow investigations. The CFD model was extended to explore changes in culture medium viscosity, rocking frequency and the robustness to position on the rocking platform. Shear stress magnitude was shown to increase almost linearly with an increase in the viscosity of culture medium. Compared with 0.5 Hz, models at 0.083 and 1:167 Hz, the operational limits of the see-saw rocker, indicated a change in shear stress patterns at the cell layer, and a reduction and increase in mean shear stress respectively. At the platform edge at 0.5 Hz, a 1.67-fold increase in time-average shear stress was identified. Extensive biological validations using human tenocytes underlined the versatility of the simple In vitro device. The application of fluid-induced shear stress at 0.5 Hz under varying regimes up to 0.714Pa caused a significant increase in secreted collagen (p < 0.05) compared to static controls. Tenocytes stimulated at a shear stress magnitude of 1.023Pa secreted significantly less collagen compared to static controls. The potential for a local maximum in the relationship between collagen secretion rate and shear stress was identified, indicating a change from anabolic to catabolic behaviour. Collagen biochemical assay results were echoed with antibody stains for proteins, where a co-localisation of connexin-32 with collagen type-I was also identified. A custom algorithm showed that four hours of fluid-induced shear stress of 0:033Pa intermittently applied to tenocytes encouraged alignment and elongation over an eight day period in comparison to static controls. Primary cilia were identified in human tenocyte cultures and bovine flexor tendon tissue; however primary cilium abrogation In vitro using chloral hydrate proved detrimental to cell viability. Collaborative investigations identified that ERK signalling and c-Fos transcription factor expression peaked after the application of 0.012Pa at 0.083 Hz for 20 minutes and anabolic collagen gene expression relative quantities increased after 48 hours of rocking at 0.083 Hz. In conclusion, validated shear stresses within a six-well plate, induced by cyclic flow from a see-saw rocker, provides an exceptional model for the In vitro study of dynamic fluid shear stress mechanobiology. Biological investigations have been linked to precise applied shear stress, creating a foundation for understanding the complex relationship between tenocytes and fluid-induced shear stress In vitro. Using this model, research is repeatable, comparable and accurately attributed to shear stress, accelerating the scientific advancement of musculoskeletal mechanobiology.

Biological tissue differs from other materials in many ways. Perhaps the most crucial difference is its ability to grow. Growth processes may give rise to stresses that exist in a body in the absence of applied loads and these are known as residual stresses. Residual stress is present in many biological systems and can have important consequences on the mechanical response of a body. Mathematical models of biological structures must therefore be able to capture accurately the effects of differential growth and residual stress, since greater understanding of the roles of these phenomena may have applications in many fields. In addition to residual stresses, biological structures often have a complex morphology. The theory of 3-D elasticity is analytically tractable in modelling mechanical properties in simple geometries such as a cylinder. On the other hand, rod theory is well-suited for geometrically-complex deformations, but is unable to account for residual stress. In this thesis, we aim to develop a map between the two frameworks. Firstly, we use 3-D elasticity to determine effective mechanical properties of a growing cylinder and map them into an effective rod. Secondly, we consider a growing filament embedded in an elastic foundation. Here, we estimate the degree of transverse reinforcement the foundation confers on the filament in terms of its material properties. Finally, to gain a greater understanding of the role of residual stress in biological structures, we consider a case study: the chameleon's tongue. In particular we consider the role of residual stress and anisotropy in aiding the rapid projection of the tongue during prey capture. We construct a mechanical model of the tongue and use it to investigate a proposed mechanism of projection by means of an energy balance argument.

Ovarian cancer is the deadliest of all the gynecologic cancers and is known for its clinically occult and asymptomatic dissemination. Most ovarian malignancies are diagnosed in the late stages of the disease and the high rate of morbidity is thought to be due, in part, to the highly metastatic nature of ovarian carcinomas. Cancer metastasis relies on the ability of cells to migrate away from primary tumors and invade into target tissues. Though the processes are distinct, cancer cell invasion relies on the underlying migration machinery to invade target tissues. Cell migration requires the coordinated effort of numerous spatially-regulated signaling pathways to extend protrusions, create new adhesion to the extracellular matrix (ECM), translocate the cell body, and retract the cell rear. Our lab established that the cyclic-AMP dependent protein kinase (PKA) subunits and enzymatic activity are localized to the leading edge of migrating cells and are required for cell movement. Despite the importance for localized PKA activity during migration, neither its role in regulating ovarian cancer cell migration and invasion nor the mechanism regulating leading edge PKA activity have been determined. Therefore, the objective of the enclosed work is to establish the importance of PKA for ovarian cancer cell migration and invasion and elucidate the molecular mechanism governing leading edge PKA. We demonstrate, for the first time, that PKA activity and spatial distribution through A-Kinase Anchoring Proteins (AKAPs) is required for efficient ovarian cancer cell migration and invasion. Additionally, we establish a link between leading edge PKA activity in migrating cells, ECM stiffness sensing, and the regulation of both PKA activity and ovarian cancer cell migration by the mechanical properties of the ECM. Finally, we delineate the hierarchy of cell signaling events that regulate leading edge PKA activity and, ultimately, the migration of ovarian cancer cells. Specifically, we elucidate a mechanism where leading edge protrusions elicit leading edge calcium currents through the stretch-activated calcium channel (SACC) of the transient receptor potential family melastatin 7 (TrpM7) to activate actomyosin contractility. ECM substrate stiffness is sensed by the actin cytoskeleton and actomyosin contractility, which, in turn, regulates the activity of leading edge PKA activity. These studies have provided important insights into the regulation of cell migration and have established the mechanistic details governing leading edge PKA activity during cell migration.

<p>Osteoporosis is a common metabolic bone disorder characterized by low bone mass and microstructural degradation of bone tissue due to derailed bone remodeling process. A deeper understanding of mechanobiological phenomenon modulating bone remodeling response to mechanical load in a healthy bone is crucial to develop treatments for this bone remodeling disease by restoring bone integrity, and preventing further bone loss and fracture. Rodent models have been provided invaluable insight into the mechanobiological mechanisms regulating the bone adaptation response to dynamic mechanic stimuli. However, use of avian models may suggest novel insight into the mechanisms managing bone adaptation to dynamic load since the bird bones have some distinctive features to the mammal bones. </p><p> This dissertation sheds light on these aspects by means of assessing mechanical environment of cortical and cancellous tissue to in vivo dynamic compressive loading within the mouse tibia and chukar partridge tibiotarsus using microCT-based finite element model in combination with diaphyseal strain gauge measures. While the mouse tibial loading model showed that cancellous strains were lower than those in the midshaft cortical bone, cancellous strains were greater than those in the midshaft cortical bone for the bird tibiotarsal loading model. Sensitivity analyses for both the mouse model and the bird model demonstrated that the material property of cortical bone was the most significant model parameter. Despite the correlations between the computationally-modeled strains and strain gradients, and histologically-measured bone formation thickness at the mid-diaphyseal cross-section of the mouse tibia, no correlation existed between the modeled strains and bone formation measures at the mid-diaphyseal cross-sections of the bird tibiotarsus. A weak correlation found between the mid-diaphyseal strain gradients and bone formation thickness for birds. Further studies in this direction will enhance the interpretation of how the bone adaptation mechanism in a healthy bone is modulated to maintain bone integrity. </p>

Blood vessels are dynamic structures whose properties are continuously adapted by resident vascular cells. Existing mechanobiological models tend to ignore regulatory signalling and cell population dynamics, both key determinants of arterial growth and remodelling (G&R). In this D.Phil., a combined theoretical, experimental and computational approach is used to formulate, refine and implement a novel model of the arterial wall that includes vascular mechanics, microstructure, biochemical metabolism and signalling, and cell phenotype and population dynamics. A mathematical chemo-mechano-biological (CMB) model is formulated by coupling a biomechanical model of the arterial wall as a cylindrical nonlinear elastic membrane to a system of biologically-informed evolution laws governing fibroblast cell-mediated, transforming growth factor (TGF)-&beta;-regulated collagen metabolism. Model simulation of inflammatory aneurysm development suggests that increasing TGF-&beta; levels promotes a cell-driven profibrotic response leading to aneurysm stabilisation, illustrating the model's ability to couple chemo-biological processes to tissue-level mechanical evolution. To inform the theoretical framework experimentally, a recent mouse model of post-developmental disruption of medial smooth muscle TGF-&beta; signalling is for the first time subjected to hypertension, and characterised by biaxial mechanical testing and (immuno)histological staining. Increased adventitial TGF-&beta; levels following perturbation are associated with strong profibrotic responses (increased cellularity, collagen deposition, thicker walls) altering tissue mechanics (lower biaxial stress, higher structural stiffness). Simulation of realistic arterial geometries is enabled by coupling the 1D CMB model to a three-dimensional structural solver. Heterogeneous spatial distributions of mechanical, microstructural and chemo-biological variables determining the evolution of complex saccular aneurysm geometries can be simulated with this 3D implementation. A novel chemo-mechano-biological model of vascular cell dynamics and regulatory signalling governing arterial G&R is formulated, informed by specifically-generated experimental data, and implemented in an advanced 3D computational framework. This will allow for virtual investigation of therapies acting on chemo-biological agents of arterial G&R, with potential benefits for vascular disease patients.

Small heat-shock proteins (sHsps) are part of a broad cellular sys- tem that functions to maintain a stable proteome under stress. They also perform a variety of regulatory roles at physiological conditions. Despite the multitude of sHsp targets, their interactions with partners are not well understood due to highly dynamical structures. In this thesis, I apply a variety of biophysical and structural approaches to examine distinct interactions made by the abundant human sHsps αβ-crystallin and Hsp27. First, I find that αβ-crystallin binds a cardiac-specific domain of the muscle sarcomere protein titin. A cardiomyopathy-causative variant of αβ-crystallin is shown to disrupt this interaction, with demonstrated implications for tissue biomechanics. Next, I investigate the conformation and unfolding behaviour of another sarcomere-associated protein, filamin C, finding support for the hypothesis that it is mechanosensitive. This leads into an interrogation of the interaction between filamin C and Hsp27, which we find is modulated by phosphorylation of Hsp27. This modulation only manifests during filamin C unfolding, pointing toward a protective chaperoning mode against over-extension during mechanical stress. This finding is bolstered by up-regulation and interaction of both proteins in a mouse model of heart failure. I establish a system for similar studies of a third sHsp, cvHsp, which is muscle-specific and implicated in various myopathies but scantly understood at the molecular level compared to αβ-crystallin and Hsp27. Finally, I probe the stoichiometries and kinetics of complexes formed between αβ-crystallin and Hsp27 themselves, which co-assemble into a highly polydisperse ensemble. This involved the development of a high-resolution native mass spectrometry method for disentangling heterogeneous systems. Together these findings add to our understanding of the roles and mechanisms of ATP-independent molecular chaperones.