Nano-FET biosensors are an emerging nanoelectronic technology capable of real-time and label-free quantification of soluble biological molecules. This technology promises to enable novel in vitro experimental approaches for investigating complex biological systems. In this study, we first explored osteocyte mechanosensitivity under different mechanical stimuli and found that osteocytes are exquisitely sensitive to different oscillatory fluid flow conditions. We therefore aimed to characterize protein-mediated intercellular communication between mechanically-stimulated osteocytes and other bone cell populations in vitro to elucidate the underlying mechanisms of load-induced bone remodeling. To this end, we devised a novel nano-manipulation based fabrication method for manufacturing nano-FET biosensors with precisely controlled device parameters, and further investigated the effect of these parameters on sensor performance.

Integrins are heterodimers that mediate cell adhesion in many physiological processes. Binding of integrins to ligands provides anchorage and signals for the cell. However, how force regulates integrin/ligand dissociation is unclear. Atomic force microscopy was used to measure the force dependence of lifetimes of single bonds between a FN fragment and integrin α₅β₁. First, lifetime-force relationships demonstrated that force prolonged bond lifetimes in the 10-30 pN range, a behavior called catch bonds. Changing divalent cations from Ca²⁺/Mg²⁺ to Mg²⁺/EGTA and to Mn²⁺ caused more pronounced catch bonds. A truncated α₅β₁ construct containing the headpiece but not the legs (trα₅β₁-Fc) formed much longer-lived catch bonds in the same force range. Bindings of two activating mAbs, 12G10 and TS2/16, left shift the catch bond and converted catch bonds to slip bonds, respectively. Catch bonds may provide a mechanical mechanism for the cell to regulate adhesion by applying different forces. Second, FNIII₇₋₁₀/α₅β₁-Fc/GG-7 bond was stretched to ~ 30 pN and then relaxed to ~ 7 pN at which the bond's lifetime was measured. The strong bond state induced by the 30 pN stretching stayed stable even after the force was reduced to 7 pN. In other words, lower the force would not weaken FNIII₇₋₁₀/α₅β₁-Fc bond once it had been stretched. Similar behaviors were observed for FNIII₇₋₁₀/trα₅β₁-Fc and FNIII₇₋₁₀/mα₅β₁interactions. In addition, the efficiency of the force to induce such a strong bond state for FNIII₇₋₁₀/α₅β₁-Fc interaction in 2 mM Mg²⁺/EGTA condition was characterized. The probability of force to induce the strong bond state increased as force increased and when the force reached 26 pN, all bonds were transit to the strong state. Moreover, reversible unbending of α₅β₁binding with FNIII₇₋₁₀ under mechanical force were observed, which proved that integrin bending and unbending was dynamic. Importantly, integrin could restore bent conformation even when engaged with its ligand, providing a mechanism for mechanotransduction. Third, structural changes of α₅β₁under force were observed. The structural changes did not change the trend of lifetime-force relationships of FNIII₇₋₁₀/α₅β₁/GG-7 bond. Moreover, the lifetime for the structural changes to occur and molecular length changes caused by them were characterized.

Atherosclerosis is a multifactoral inflammatory disease that occurs in predisposed locations in the vasculature where blood flow is disturbed. In vitro studies have implicated reactive oxygen species as mediators of mechanotransduction leading to inflammatory protein expression and ultimately atherogenesis. While these cell culture-based studies have provided enormous insight into the effects of WSS on endothelial biology, the applicability to the in vivo setting is questionable. We hypothesized that low magnitude oscillatory WSS acts through reactive oxygen species (ROS) to increase expression of inflammatory cell adhesion molecules leading to the development of atherosclerotic lesions. The overall objective for this thesis was to develop an in vivo flow model that produces low magnitude oscillatory WSS which could be used to investigate the in vivo molecular mechanisms of mechanotransduction. We created a novel aortic coarctation model using a shape memory nitinol clip. The clip reproducibly constricts the aorta creating a narrowing of the lumen resulting in a stenosis. This mechanical constraint produces a region of flow separation downstream from the coarctation. We have characterized the coarctation in terms of the efficacy, pressure loss, and fluid dynamics. We then measured the endothelial response of shear sensitive redox and inflammatory markers. Lastly, we utilized genetically modified mice and mice treated with pharmacological inhibitors to investigate the mechanisms involved in the expression of WSS induced inflammatory and redox markers. We found that inducing a coarctation of the aorta using a nitinol clip uniquely created a hemodynamic environment of low magnitude oscillatory WSS without a significant change in blood pressure. Using this model we found that the in vivo endothelial phenotype associated with acutely disturbed flow was characterized by increased production of superoxide and increased expression of select inflammatory proteins. In comparison, the phenotype associated with chronically disturbed flow was characterized by a more modest increase in superoxide and increased levels of multiple inflammatory proteins. We determined that in regions of acutely disturbed flow in vivo, VCAM-1 expression was not modulated by reactive oxygen species. Additionally, p47 phox-dependent NADPH Oxidase activity does not have a functional role in WSS induced superoxide generation in the endothelium. In summary, we have created a novel murine model of low magnitude oscillatory WSS that can be used to investigate the in vivo molecular mechanisms associated with atherogenesis. While previous data obtained in vitro indicated that depletion of an individual ROS was sufficient to inhibit flow-induced inflammatory protein expression, our findings, to the contrary, showed that antioxidant treatment in vivo does not inhibit shear-dependent inflammatory protein expression. Our results suggest that atherogenesis in the in vivo environment is significantly more complicated than the in vitro environment and that parallel pathways and compensatory mechanisms are likely activated in vivo in response to WSS. These results could have significant implications in the efficacy of antioxidant treatment of atherosclerosis and could explain the complexity of results observed in clinical trials.

Force production by a muscle is critical to maintaining proper function and overall health of a human or animal. Muscle adapts to increased loading with hypertrophy by activating a number of intracellular signaling cascades that regulate protein synthesis. The overall hypothesis is that force-dependent processes acutely activate growth-related signaling during active force generation. This project took two approaches. The first employed a general survey of muscles in which age-dependent changes in muscle activity differed. No conclusive activity-dependent signaling emerged however coordinated signaling among kinases broke down with age. The second approach utilized an in situ muscle preparation in which force production or metabolic costs were specifically controlled. Similar sub-maximal force levels generated by different methods found that force, per se, is not a primary modulator of growth-related signaling but that ERK phosphorylation is dependent on fiber-activation. Prolonging the duration of electrical stimulation applied to the nerve or increasing the frequency at which stimulations are applied was expected to increase the metabolic stress associated with contraction. Several growth-related kinases correlated with markers of metabolic stress, i.e. increased AMPK activity and decreased glycogen content, which were decoupled from force decline. This suggests energy depletion, specific to stimulation pattern, strongly influences the immediate response to high force contraction signaling. The overall conclusion is that signaling molecules previously implicated in force-dependent signaling lie much too downstream to relay strict force-dependent signaling.

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.

Bone mechanotransduction is a fundamental process underlying the remarkable ability of bones to perceive surrounding physical cues and adapt their mass, structure and overall strength to their mechanical environment. Therefore, it is central to many aspects of bone biology and disease. The key to a mechanistic understanding of this process lies in better knowledge of critical signaling molecules that relay the mechanical information inside bone cells. In this thesis, we investigate the role of polycystin-1 (PC1), a proposed fluid flow sensor in kidney epithelial cells, in transducing mechanical signals in bone cells. Loss of PC1 in osteoblast lineage cells using osterix-Cre (Osx-Cre) causes mild osteopenia in mice with reduced calvarial and trabecular bone formation, and markedly attenuated anabolic bone formation responses to in vivo mechanical loading of long bones. Loss of PC1 in limb bud mesenchymal cells at an early stage causes mildly increased bone formation and a tendency to exhibit enhanced anabolic responses to in vivo mechanical loading of long bones. These findings suggest that PC1 has a complex role in different bone cell populations both during development and in bone mechanotransduction. PC1 has been shown to mediate tensile force-induced proliferation in osteoprogenitor cells (OPCs) in craniofacial sutures. To investigate the role of PC1 in periosteal osteoprogenitor mechanotransduction, we establish a shockwave-induced periosteum mechanical stress model. Shockwave treatment triggers dramatically increased cell proliferation, potent osteogenic activity, and intramembranous new bone formation in the periosteum. We show that loss of PC1 in periosteal cells (Prx1-Cre) does not affect periosteal mechanoresponsiveness to shockwave mechanical stress. These findings suggest that the role of PC1 in OPCs is likely tissue or force dependent. Fluid shear stress (FSS) in the lacunar-canalicular network is a major force element that osteocytes experience and respond to in vivo. To study the role of PC1 in FSS-mediated osteocyte/osteoblast mechanotransduction, we establish a laminar FSS system with custom-made flow chambers and a PC1-deficient osteoblast cell line. Our data show that PC1 is essential for regulation of FSS-induced initial \(Ca^{2+}\) influx in osteoblasts and mediates osteoblast FSS responses in a COX-2 and AP-1 independent manner.

The assembly, form, and function of the heart is regulated by complex mechanical signals originating from intrinsic and extrinsic sources, such as the cytoskeleton and the extracellular matrix. During development, mechanical forces influence the self-assembly of highly organized ventricular myocardium. However, mechanical overload induces maladaptive remodeling of tissue structure and eventual failure. Thus, mechanical forces potentiate physiological or pathological remodeling, depending on factors such as frequency and magnitude. We hypothesized that mechanical stimuli in the form of microenvironmental stiffness, cytoskeletal architecture, or cyclic stretch regulate cell-cell junction formation and cytoskeletal remodeling during development and disease. To test this, we engineered cardiac tissues in vitro and quantified structural and functional remodeling over multiple spatial scales in response to diverse mechanical perturbations mimicking development and disease. We first asked if the mechanical microenvironment impacts tissue assembly. To investigate this, we cultured two-cell cardiac µtissues on flexible substrates with tunable stiffness and monitored cell-cell junction formation over time. As myocytes transitioned from isolated cells to interconnected µtissues, focal adhesions disassembled near cell-cell interfaces and mechanical forces were transmitted almost completely through cell-cell junctions. However, µtissues cultured on stiff substrates mimicking fibrotic microenvironments retained focal adhesions near the cell-cell interface, potentially to reinforce the cell-cell junction in response to excessive forces generated by myofibrils in stiff microenvironments. Intercellular electrical conductance between myocytes was measured as a function of connexin 43 immunosignal and the length-to-width ratio of cell pairs. We observed that conductance was correlated to connexin 43 immunosignal and cell pair length-to-width ratio, indicating that tissue architecture can affect electrical coupling. The impact of mechanical overload was also determined by applying chronic cyclic stretch to engineered cardiac tissues. Stretch activated gene expression patterns characteristic of pathological remodeling, including up-regulation of focal adhesion genes, and impacted sarcomere alignment and myocyte shape. Furthermore, chronic cyclic stretch altered intracellular calcium cycling in a manner similar to heart failure and decreased contractile stress generation, suggestive of maladaptive remodeling. In summary, we show that the assembly, form, and function of cardiac tissue is sensitive to a wide range of mechanical cues that emerge during physiological and pathological growth. / Engineering and Applied Sciences

Cell migration is a fundamental biological process involved in tissue development, wound repair, and diseases such as cancer metastasis. It is a biomechanical process involving the adhesion of a cell to a substratum, usually an elastic extracellular matrix, as well as the physical contraction of the cell driven by intracellular actomyosin network. In the migration of cells as a group, known as collective migration, the cells are also physically linked to one another through cell-cell adhesions. How mechanical interactions with cell substratum and with neighboring cells regulate movements during collective migration, nevertheless, is poorly understood. To address this question, the effects of substrate stiffness on sheet migration of MCF10A epithelial cells were systematically analyzed. Speed, persistence, directionality and coordination of individual cells within the migrating sheet were all found to increase with substrate stiffening. Substrate stiffening also enhanced the propagation of coordinated movement from the sheet edge into the monolayer, which correlated with an upregulation of myosin-II activity in sheet edge cells. This mechano-response was dependent on cadherin-mediated cell-cell adhesions, which are required for the transmission of directional cue. Importantly, myosin-II contractility modulated cadherin- dependent cell-cell coordination, suggesting that contractile forces at cadherin adhesions regulate collective migration. To measure forces transmitted through cell-cell adhesions, a quantitative approach was developed in which cell-cell forces were deduced from cell-substrate traction forces, based on force balance principles and simple cell mechanics modeling. This method enabled the analysis of cell-cell mechanical interactions in small cell clusters of complex topology. The dynamic fluctuations of cell-cell forces over time revealed that force transmission between non-adjacent cells is typically limited, but is enhanced when the cell across which forces are being transmitted has reduced myosin-IIA or talin-1. This suggests that cells in a group may differentially regulate their levels of myosin-II contractility and cell-matrix mechanotransduction to promote longer-range force transmission during collective migration. Together, the results in this dissertation led to a working model of collective cell migration as regulated by cell-matrix mechanical properties and cell-cell mechanical interactions. This model, as well as the quantitative techniques developed here, will drive future studies on the mechanisms underlying collective migration.

Hypercholesterolemia is a risk factor for osteoporosis but the underlying mechanism is unknown. Previous evidence suggests that osteoporosis results from an impaired regulation of osteoblasts by fluid pressure fluctuations in the bone matrix. Recently, our laboratory showed that enhanced cholesterol in the cell membrane, due to hypercholesterolemia, alters leukocyte mechanosensitivity. We predict a similar link between osteoblasts and hypercholesterolemia leading to osteoporosis. Specifically, we hypothesize that extracellular cholesterol modifies the osteoblast sensitivity to pressure. MC3T3-E1 cells were exposed to hydrodynamic pressures regimes (mean=40mmHg, amplitude=0-20mmHg, frequency=1Hz) for 1-12 hours. To assess the impact of membrane cholesterol enrichment, cells were pre-treated with 0-50 µg/mL cyclodextran:cholesterol conjugates. We assessed the pressure effects on mitosis and F-actin stress fiber formation (SFF) of cells. Exposure of cells to 50/30 mmHg pressure transiently increased the number of cells in the S- and G2M-phases of mitosis after 6 and 12 hours, respectively. Relative to controls, osteoblast-like cells exposed to all pressures exhibited significantly (p

Living organisms are made up of a multitude of individual cells that are surrounded by biomolecules and fluids. It is well known that cells are highly regulated by biochemical signals; however it is now becoming clear that cells are also influenced by the mechanical forces and mechanical properties of the local microenvironment. Extracellular forces causing cellular deformation can originate from many sources, such as fluid shear stresses arising from interstitial or blood flow, mechanical stretching during breathing or compression during muscle contraction. Cells are able to sense variations in the mechanical properties (elasticity) of their microenvironment by actively probing their surroundings by utilizing specialized proteins that are involved in sensing and transmitting mechanical information. The actin cytoskeleton and myosin-II motor proteins form a contractile (actomyosin) network inside the cell that is connected to the extracellular microenvironment through focal adhesion and integrin sites. The transmission of internal actomyosin strain to the microenvironment via focal adhesion sites generates mechanical traction forces. Importantly, cells generate traction forces in response to extracellular forces and also to actively probe the elasticity of the microenvironment. Many studies have demonstrated that extracellular forces can lead to rapid cytoskeletal remodeling, focal adhesion regulation, and intracellular signalling which can alter traction force dynamics. As well, cell migration, proliferation and stem cell fate are regulated by the ability of cells to sense the elasticity of their microenvironment through the generation of traction forces. In vitro studies have largely explored the influence of substrate elasticity and extracellular forces in isolation, however, in vivo cells are exposed to both mechanical cues simultaneously and their combined effect remains largely unexplored. Therefore, a series of experiments were performed in which cells were subjected to controlled extracellular forces as on substrates of increasing elasticity. The cellular response was quantified by measuring the resulting traction force magnitude dynamics. Two cell types were shown to increase their traction forces in response to extracellular forces only on substrates of specific elasticities. Therefore, cellular traction forces are regulated by an ability to sense and integrate at least two pieces of mechanical information - elasticity and deformation. Finally, this ability is shown to be dependent on the microtubule network and regulators of myosin-II activity.