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The overall goal of this research is to elucidate the effects of stiffness on the activation of pulmonary endothelial cells by inflammatory cytokines. The hypothesis tested is that increasing matrix stiffness in the (patho) physiological range will exacerbate the response of cultured endothelial cells to inflammatory stimuli. To test this hypothesis, we are culturing control and TNF-a stimulated rat lung microvascular endothelial cells (RLMVECs) on hydrogels with tunable stiffnesses of 5, 20, and 45 kPa (measured using compression testing), modeling the stiffness of healthy, intermediate and fibrotic lung tissue respectively. The cellular readout was assessed through RT-qPCR, microscopy, and monocyte adhesion for basal expression and upregulation of vascular cell adhesion molecule-1 (VCAM-1) in quiescent and TNF-a stimulated cultured endothelial cell. This model of microvascular pulmonary inflammation, mimicking a normal, intermediate, and fibrotic lung, is aimed at establishing a correlation between substrate stiffness and inflammation. This research demonstrates the significant increase of basal VCAM-1 gene expression as well as monocyte adhesion as substrate stiffness increases. When using inhibition, it was also found that VCAM-1 is partially activated through the Rho/ROCK, YAP/TAZ, and NF-kB pathway. Our results contribute to a mechanistic understanding of disease pathologies such as idiopathic pulmonary fibrosis, in which treatment is just about limited to a full lung transplant and facilitate testing of new drug therapies. / Bioengineering

Cardiovascular disease is the world’s number 1 killer. The cardiovascular system helps to pump blood throughout the human body and maintain a systemic balance. However, medial vascular calcification results when this system becomes off balance, such as in cases of high blood pressure leading to hypertension. Many factors are involved in this process, but the most important is the vascular smooth muscle cell phenotypic switch to osteoblast-like cells. When vascular smooth muscle cells are subject to mechanical stimuli, mechanotransduction occurs, causing an intracellular signaling cascade leading to a phenotypic switch associated with the Wnt signaling pathway and osteogenic markers. There is a lack of understanding of the defined linkages of pathways that lead to the development of the osteoblast-like cell type. Therefore, examining human aortic smooth muscle cells under hypertensive conditions could decrease the prevalence of cardiovascular disease worldwide.

Organisms are constantly subjected to mechanical stimuli originating from their environment or internal activities. To sense and properly handle such mechanical cues, the conversion of mechanical signals into electrochemical signals occurs continuously within the organisms. Mechanosensitive ion channels (MSCs) are pivotal participants in this process, being ready to open to allow the movement of ions across cellular membranes in response to mechanical stimulation. Piezo1 and Piezo2 mechanosensitive cation channels are evolutionarily conserved MSCs, taking part in various physiological processes, including bone formation and touch sensation. Mutations in Piezo1 or Piezo2 lead to pathological conditions and overexpression of the channels is associated with several diseases. Piezo modulators can be used not only as useful tools for targeted interventions of such pathological outcomes but also as important contributors to the characterization and study of Piezo channels. This dissertation is a part of such endeavors to identify Piezo modulators. Proteinogenic sulfur-containing amino acids (PSCAAs), which is a term to call methionine (Met) and cysteine (Cys) collectively, and propofol were chosen as candidates for modulating Piezo channel activities, given their potential to modify membranes as gating of Piezo channels is known to be greatly influenced by mechanical properties of cellular membranes.The effects of PSCAAs and propofol on Piezo channels were evaluated through electrophysiological recording and calcium imaging, which were performed using Pieoz1-deficient (P1KO) HEK293T cells transfected with either human Piezo1 or human Piezo2 and HEK293T cells that stably overexpress Piezo1, respectively. It was observed that perfusion of 100 µM Met on Piezo1-transfected P1KO HEK293T enhanced Piezo1 currents and, interestingly, delayed Piezo1 inactivation, in the whole-cell and outside-out configurations. However, 100 µM D-Met was incapable of inducing calcium influx into the Piezo1-overexpressing cell line in calcium imaging. Double-mutant Piezo1, which is unable to inactivate due to a substitution of arginine for Met (M2225R) and a substitution of lysine for arginine (R2456K), was unaffected by 100 µM D-Met in the outside-out configuration. These results indicate that Met augments Piezo1 currents by slowing down Piezo1 inactivation instead of contributing to activation of more Piezo1 channels. Piezo2 currents, on the other hand, were not altered by 100 µM D-Met in the cell-attached and whole-cell configurations. 100 µM Cys induced responses similar to those of Met, increasing Piezo1 currents while delaying inactivation. 50 µM propofol inhibited Piezo1 currents in outside-out, whole-cell, and cell-attached recording and counteracted Yoda1-induced calcium influx into Piezo1-overexpressing HEK293T cells. Piezo2 currents were also suppressed by 50 µM propofol. This study reveals that PSCAAs enhance Piezo1 current while delaying its inactivation and propofol inhibits both Piezo1 and Piezo2 channels. These findings offer potential strategies for targeted therapeutic modulation of Piezo channels, advancing the treatment options for Piezo-related conditions and expanding our understanding of mechanosensory signal regulation.

Au cours de la ventilation liquide totale (VLT), les cellules pulmonaires sont exposées à des perfluorocarbones (PFC) dont les propriétés physiques diffèrent fortement du milieu standard de culture cellulaire (DMEM) et encore plus des propriétés de l'air. Dans cette thèse nous étudions les effets d’une exposition au PFC sur la réponse des cellules épithéliales pulmonaires, en effectuant une étude approfondie des propriétés structurales, mécaniques et fonctionnelles. La réponse des cellules A549 (alvéolaire), HBE (bronchique) et AM (Macrophage alvéolaire) exposées au PFC est étudiée par comparaison au DMEM. Les variations de la structure de F-actine, de la densité d'adhésion focale et de la distribution du glycocalyx sont évaluées par fluorescence. Les changements de propriétés mécaniques et de paramètres d’adhésion sont mesurés par la Magnétocymétrie (MTC) étendue à l’analyse multiéchelle. La mécanique cellulaire est caractérisée par deux modèles microrhéologiques reflétant deux types de comportement possibles du cytosquelette (CSK). L'adhésion à la matrice cellulaire est analysée par un modèle stochastique de dé-adhésion, décrivant la composante non-réversible de la réponse cellulaire. Les rôles fondamentaux de la structure de F-actine et de la couche de glycocalyx sont respectivement évalués par dépolymérisation de F-actine et en dégradant le glycocalyx. Les résultats montrent que l'exposition au PFC induit un remodelage de la structure de F-actine, un affaiblissement du CSK et une diminution de l'adhésion. Ces résultats démontrent que le PFC déclenche une réponse particulière des cellules épithéliales caractérisée par une diminution de la tension intracellulaire, l'affaiblissement de l'adhésion et la redistribution du glycocalyx. L’origine de cette adaptation cellulaire est physique et très probablement reliée à l’augmentation de l'énergie interfaciale associée à la basse tension de surface d’un PFC chimiquement apolaire. La faible tension de surface du PFC est également responsable d'une augmentation de la compliance pulmonaire pendant VLT et a des impacts profonds dans les paramètres respiratoires, parallèlement à la modification de la réponse cellulaire. / During Total Liquid Ventilation (TLV), lung cells are exposed to perfluorocarbon (PFC) whose physical properties highly differ from aqueous medium (DMEM) standardly used for cell culture and farther air properties. In this thesis, we study the effects of PFC exposure on the response of pulmonary epithelial cell by performing a thorough assessment of their structural, mechanical and functional properties. The response of A549 cells (alveolar), HBE (bronchial), and AM (alveolar macrophages) exposed to PFC is studied by comparison to DMEM. Changes in F-actin structure, focal adhesion size and density and glycocalyx expression are evaluated by fluorescence. Changes in cell mechanics and adhesion parameters are measured by a multiscale Magnetic Twisting Cytometry (MTC) method. Cell mechanics is analyzed by two microrheological models reflecting two possible cytoskeleton features. Cell-matrix adhesion is analyzed by a stochastic multibond de-adhesion model describing the non-reversible component of the cell response by MTC. The key roles of F-actin structure and glycocalyx layer are established by respectively depolymerising F-actin and degrading glycocalyx. Results show that PFC exposure induces F-actin remodelling, cytoskeleton softening and adhesion weakening. They demonstrate that PFC triggers an epithelial cell response which is characterized by decay in intracellular tension, adhesion weakening and glycocalyx redistribution. The origin of this cellular adaptations is physical and most likely related to the increase in interfacial energy, associated to the low surface tension of the non polar perflurorocarbon, The low surface tension of PFC is also responsible for an increase in lung compliance during TLV and has deep impacts during ventilation parallel to the modification of cell response.

This study explored hydrostatic pressure as a mechanobiological parameter to control in vitro endothelial cell tubulogenesis in 3-D hydrogels as a model microvascular tissue engineering approach. For this purpose, the present investigation used an endothelial spheroid model, which we believe is an adaptable microvascularization strategy for many tissue engineering construct designs. We also aimed to identify the operating magnitudes and exposure times for hydrostatic pressure-sensitive sprout formation as well as verify the involvement of VEGFR-3 signaling. For this purpose, we used a custom-designed pressure system and a 3-D endothelial cell spheroid model of sprouting tubulogenesis. We report that an exposure time of 3 days is the minimum duration required to increase endothelial sprout formation in response to 20 mmHg. Notably, exposure to 5 mmHg for 3 days was inhibitory for endothelial spheroid lengths without affecting sprout numbers. Moreover, endothelial spheroids exposed to 40 mmHg also inhibited sprouting activity by reducing sprout numbers without affecting sprout lengths. Finally, blockade of VEGFR-3 signaling abolished the effects of the 20-mmHg stimuli on sprout formation. Based on these results, VEGFR-3 dependent endothelial sprouting appears to exhibit a complex pressure dependence that one may exploit to control microvessel formation.

Fibrosis of vital organs remains one of the leading causes of death in the developed world, where it occurs predominantly in soft tissues (liver, lung, kidney, heart) through fibroblast proliferation and deposition of extracellular matrix (ECM). In the process of fibrosis, remodeling and deposition of ECM results in stiffening of cellular microenvironment; cells also respond to these changes in the stiffness through engagement of their cytoskeleton and signaling via cell-ECM contacts. Thus, understanding to what extent the stiffness of the cellular microenvironment changes as a consequence of fibrotic progression, and how cells respond to this change, is critical. In this thesis, we quantitatively measured stiffness of the lung parenchyma and its changes during fibrosis. We find that the average stiffness increases by approximately 10-fold. We then investigated how changes in ECM rigidity affect the cytoskeletal phenotype of lung fibroblasts. We find a complex relation between expression of the glycoprotein Thy-1 (CD90) and ECM rigidity-dependent cytoskeletal phenotype (i.e. “mechanotransduction”). Finally, we investigate a mechanism for the regulation of rigidity sensing by Thy-1 and its involvement in intracellular signaling through cell-ECM contacts. Taken together, this work helps define in vivo parameters critical to the fibrogenesis program and to define unique cellular phenotypes that may respond or contribute to mechanical homeostasis in fibrotic diseases.

Hypercholesterolemia is a dominant risk factor for a variety of cardiovascular diseases and involves a chronic inflammatory component in which neutrophil activity plays a critical role. Recently, fluid shear stress mechanotransduction has been established as a control mechanism that regulates the activity of neutrophils by reducing the formation of pseudopods and the surface expression of CD18 integrins, thereby rendering these cells rounded, deformable, and non-adhesive. This is critical for maintaining a healthy circulation, because chronically activated neutrophils not only release excess cytotoxic and degradative agents but also exhibit a reduced efficiency to pass through the small vessels of the microcirculation leading to increased microvascular resistance. We hypothesized that aberrant neutrophil mechanosensitivity to fluid shear stress due to the altered blood environment (i.e., excess plasma cholesterol) is a contributing factor for elevated hemodynamic resistance in the microcirculation associated with hypercholesterolemia. For this purpose, the present work firstly showed that the sensitivity of neutrophils to fluid shear stress depends on the cholesterol-dependent fluidity of the cell membrane, and that, in the face of hypercholesterolemia, the neutrophil mechanosensitivity highly correlated with the plasma levels of free cholesterol. The second part of this project demonstrated that, when subjected to shear stress fields, leukocyte suspensions exhibited transient (within 10 min of flow onset) time-dependent reductions in their apparent viscosity. Moreover, shear-induced changes in viscosity of cell suspensions were influenced by disturbances of membrane cholesterol and fluidity in a fashion similar to that for shear-induced pseudopod retraction. Finally, the third part of this work provided evidence that neutrophils played a role in hypercholesterolemia-related impairment of flow recovery response to transient ischemia. In conclusion, results of the current work provided the first evidence that cholesterol is an important component of the neutrophil mechanotransducing capacity and impaired neutrophil shear mechanotransduction may disturb the blood flow rheology, leading to elevations in the apparent viscosity as well as in the resistance. This cholesterol-linked perturbation may be a contributing factor for the pathologic microcirculation associated with hypercholesterolemia.

Tissue engineering and regenerative medicine have emerged as viable approaches to repairing osteochondral tissue damage, especially with the implementation of biomaterials and mesenchymal stem cells (MSCs). Poly(vinyl alcohol) (PVA) is a synthetic and non-biodegradable polymer that has received attention as a tissue engineering scaffold and cartilage replacement due to its inherent viscoelasticity and biocompatibility. This work investigated the use of mechanical cues to trigger mechanotransduction pathways and thereby guide human MSCs towards a desired differentiation lineage. PVA scaffolds with a range of compressive moduli (1 - 600 kPa) were fabricated by varying molecular weight, solution concentration, and freeze-thaw cycles. Mass loss rates and changes in stiffness were not significantly different after 7 days of dynamic compression or static culture in standard MSC culture medium. Short-term dynamic loading of human MSC-seeded PVA scaffolds resulted in an increase in cell viability and collagen production for loaded versus static samples over 7 days of culture. Through a simple dynamic compressive loading sequence MSC viability and matrix protein production may increase on synthetic, bioinert PVA scaffolds. Lastly upstream processing of polymer fabrication and cell culture was conducted in preparation for studies on a custom designed dynamic compressive loading machine for cell-seeded scaffolds.

La pose d’une Prothèse Totale de Hanche est l’une des chirurgies orthopédiques les plus pratiquées, et représente un enjeu économique et de santé publique majeur. Ainsi, il est essentiel de comprendre le comportement mécanique de l’os et sa réaction à la suite d’une telle chirurgie. La simulation numérique joue un rôle intéressant dans cette perspective, permettant la reproduction et l’analyse de la réponse osseuse aux stimulus externes. L’os est un matériau complexe présentant une structure hiérarchique et poreuse, et une capacité naturelle d’adaptation structurelle grâce à des cellules spécifiques sensibles aux mouvements de fluide. Basé sur ces caractéristiques, un modèle multi-échelle a été développé au cours de cette thèse dans le but de modéliser la réponse de l’os soumis à des sollicitations mécaniques externes. Le modèle développé repose sur la méthode d’homogénéisation pour les structures périodiques basé sur un développement asymptotique. Il simule l’os cortical comme une structure homogène, composé d’une microstructure périodique, d’une porosité de 5%, saturé de fluide interstitiel qui suit dans ce cas la loi de Darcy. La première application du modèle développé est un cas d’étude, consistant en un volume d’os chargé en compression, permettant la détermination d’une raideur poroélastique équivalente. En considérant principalement deux cas extrêmes de conditions aux limites en fluide, l’analyse de la réponse structurelle correspondante permet d’avoir un aperçu de la contribution du fluide dans le comportement mécanique d’un tel matériau, et en particulier de sa raideur équivalente. Ce paramètre est soit réduit (lorsque le fluide peut sortir de la structure), soit augmenté (lorsque le fluide est confiné dans la structure). Pour valider ce modèle, une étude numérique et expérimentale sont proposées. La validation numérique permet l’estimation de la pertinence du modèle en faisant varier certains paramètres d’entrée comme les propriétés matériaux ou les conditions aux limites. Puis, une validation expérimentale est mise en place. En comparaison, des données issues d’un échantillon d’os trabéculaire de hanche mis en compression sont utilisées. La raideur équivalente de l’échantillon est calculée et comparée à celle obtenue expérimentalement. Les courbes obtenues présentent des résultats similaires et permettent d’attester de la validité du modèle compte tenu des circonstances d’essais. Ainsi, le modèle numérique développé, s’inscrit dans l’objectif de fournir un modèle bio-fidèle de l’os, afin de déterminer les paramètres critiques permettant d’avoir une influence sur le remodelage osseux. En prévision de l’élaboration et de la production de nouvelles générations de prothèses, ce modèle numérique d’os présente à la fois le compromis intéressant de la pertinence scientifique sans requérir des ressources numériques excessives, nécessaires à son application en tant qu’outil de prévision pré-opératoire. / Total Hip Arthroplasty is nowadays one of the most performed orthopedic surgery and is representing a major health and economic issue. Thus, it is essential to provide a better understanding of bone mechanical behavior and its reaction to the implantation of a device such as a hip prosthesis. Numerical simulation plays a key role on this challenge, allowing for the reproduction and analysis of the bone response to the external stimuli. Bone is a complex material showing a hierarchical and porous structure, and natural ability to remodel itself thanks to specific cells, which are sensitive to fluid flows. Based on these characteristics, a multiscale numerical model has been developed in order to simulate the bone response under external mechanical solicitations. The developed model relies on the homogenization technique for periodic structures based on an asymptotic expansion. It simulates cortical bone as a homogeneous structure. It is constituted of a porous microstructure with a 5% saturated with bone fluid, which, in the considered conditions, follows the Darcy’s law. The first application of the developed model is a case study, consisting in the loading of a finite volume of bone, allowing for the determination of an equivalent poroelastic stiffness. Focusing on two extreme fluid boundary conditions, the analysis of the corresponding structural response provides an overview of the fluid contribution to the poroelastic behavior, impacting the equivalent stiffness of the considered material. This parameter is either reduced (when the fluid can flow out of the structure) or increased (when the fluid is confined the structure). To validate the developed model, both numerical and experimental validation are proposed. The numerical validation consists in the estimation of the model accuracy when varying parameters such as material properties or boundary conditions. Then, an experimental validation is set up. As a reference case, a previous work on a cubic trabecular bone sample, extracted from a human hip and put under a compressive load, has been used. Increasing the load applied on the top of the bone specimen, the displacement is extracted, allowing the computation of the equivalent strain-stress curve. The equivalent stiffness of the bone specimen, calculated numerically by the developed numerical tool, is then compared with the one from the experiments. A good agreement between the curves attests the validity of the developed numerical model, accounting for both the solid matrix and fluid contributions. The presented poroelastic numerical, is here developed in the perspective of providing a bio-reliable model of bones, to determine the critical parameters that might impact bone remodeling. Towards the design and manufacturing of new generation of prosthesis, this bone model shows both accuracy and ease of computation, which will be required for its application as a preoperative or design tool.