Investigating Mechanical Interactions of Cells with Their Environment

Recent studies have shown that cells not only respond to chemical signals such as growth factors or chemoattractants, but they are also capable of detecting mechanical stimuli and responding to them. The process during which these mechanical stimuli are detected and transferred to chemical signals, that cells can process, is called mechanotransduction. The mechanical stimuli that can affect cells can be either an external stimulus applied to cells, such as shear flow or cyclic compression and tension, or they can be linked to the mechanical properties of their substrates. One of the mechanical properties of a substrate that can affect cellular behavior is known to be stiffness, mostly measured by elastic modulus. Stiffness influences a wide variety of cellular behaviour such as cell shape, adhesion to substrate, proliferation, and differentiation. Anchorage dependent cells are in direct contact with their environment, which then leads to complicated interactions. These interactions can be both biological and mechanical. In the current research, the mechanical interactions are often called the “mechanical responses” of cells. For anchorage-dependent migrating cells, mechanical responses can be the substrate deformations induced by the forces generated by cells also called cell traction forces. These mechanical responses can be studied in three levels of complexity. The first level is when cells are cultured on a 2D matrix and responses are also studied in 2D. The second level of complexity is when cells are cultured on a 2D matrix and the biological behaviour of cells, such as growth or migration, is studied in 2D, however, the mechanical responses of cells are studied in 3D, meaning that not only in plane deformation and forces are studied, but out of plane ones are also assessed. The third level of complexity is when cells are cultured inside a 3D matrix and both biological responses and mechanical responses are studied in 3D. In the current research, the second level of complexity is chosen. After testing different types of materials, polyacrylamide (PAAm) was chosen as the model biomaterial. Following mechanical characterization of PAAm samples, substrates were prepared with three different elastic moduli. Both biological responses and mechanical responses of human corneal epithelial cells (HCECs) were studied. For biological responses, cell viability, activation, adhesion molecules, apoptosis and migration behaviour were studied. For mechanical responses, confocal microscopy in junction with image processing technique, digital volume correlation (DVC), was used to measure cell induced deformations. It was found that elastic modulus, as a mechanical stimulus, affects not only biological behaviour of cells, but also their mechanical behaviour. Decreasing elastic modulus led to significantly lower migration speed of HCECs, slightly higher number of apoptotic cells as well as significantly higher number of necrotic cells. Furthermore, while no significant changes in adhesion molecules occurred, dramatic changes in cytoskeleton structure was seen on cells cultured on compliant matrices. Also, the DVC code was capable of detecting both in plane and out of plane deformations from confocal images. It was found that substrate elastic modulus can change the pattern of displacements on compliant substrate compared to stiff ones. Results of the present study suggest that the deformation pattern and magnitude does not change over the body of cells and that they are rather similar in the leading edge and trailing edge. Deformation under the nucleus was also assessed and for compliant and stiff substrates were present while no deformation was found under the cells cultured on medium stiffness substrates. It was also speculated that mechanical interaction of HCECs with their substrates can be more complicated than currently known and cells seem to be able to exert moments on their substrate as well as forces. Results presented in this thesis demonstrate that HCECs are sensitive to substrate stiffness and elastic modulus can affect their behaviour. Furthermore, considering the complexity of HCECs mechanical interaction with their substrates, it is critical to study both biology and mechanics for full comprehension of cellular interaction with the ocular environment

A new constitutive model for the time-dependent behavior of rocks with consideration of damage parameter

Deformation and time-dependent behavior of rocks are closely related to the stability and safety of underground structures and mines. In this paper, a numerical-analytical model is presented to investigate time-dependent damage and deformation of rocks under creep. The proposed model is obtained by combining the elastic-visco-plastic model based on the theory of over-stress and stress hardening law with the sub-critical crack growth model. The advantage of this model is that it is in incremental form and therefore can be implemented numerically. First, the governing equations of the model and its numerical computational algorithm are described. The proposed constitutive model is then implemented in the FLAC code using the FISH function. Determination of model parameters and calibration is done by various laboratory tests performed on a type of gypsum. The creep test was performed on gypsum under a stress of 13 MPa, which is equal to 70% of its compressive strength. After determining the parameters, by fitting the creep curve of the presented analyticalnumerical model, a good agreement is observed with the creep curve obtained from the laboratory data. It is also observed that during creep, the damage parameter and wing crack length increase

Corneal epithelial cells exposed to shear stress show altered cytoskeleton and migratory behaviour

<div><p>Cells that form the corneal epithelium, the outermost layer of the cornea, are exposed to shear stress through blinking during waking hours. In this <i>in vitro</i> study, the effect of fluid shear stress on human corneal epithelial cells (HCECs) was investigated. Following exposure to shear stresses of 4 and 8 dyn/cm², HCECs showed cytoskeletal rearrangement with more prominent, organized and elongated filamentous actin. Cytoskeletal changes were time-dependent, and were most significant after 24 hours of shear stress. Higher rates of migration and proliferation, as evaluated by a scratch assay, were also observed following 24 hours of low shear stress exposure (4 dyn/cm²). This result contrasted the poor migration observed in samples scratched before shear exposure, indicating that shear-induced cytoskeletal changes played a key role in improved wound healing and must therefore precede any damage to the cell layer. HCEC cytoskeletal changes were accompanied by an upregulation in integrin β₁ and downregulation of ICAM-1. These results demonstrate that HCECs respond favourably to flow-induced shear stress, impacting their proliferation and migration properties as well as phenotype.</p></div

Diagram showing experiments and the sequence in which they are performed in this study.

<p>Diagram showing experiments and the sequence in which they are performed in this study.</p

Optical micrograph of the scratch pattern used in this study.

<p>Optical micrograph of the scratch pattern used in this study.</p

HCEC cytoskeleton organization.

<p>Actin filaments were stained with fluorescently labeled phalloidin. a) Control cells (i.e., not exposed to shear stress). b) Cells exposed to low shear stress (4 dyn/cm²) for 14 hours (b-1) and 24 hours (b-2). Organization of the cytoskeleton with actin filament bundles are visible in cells exposed to low levels of shear stress for 24 hours. c) Cells exposed to high shear stress (8 dyn/cm²) for 24 hours with less visible (c-1) and more visible (c-2) filamentous actin cytoskeleton structure. White arrows indicate stretched actin filaments. Images captured using a Zeiss laser scanning confocal microscope.</p

Experimental conditions of <i>in vitro</i> studies of corneal epithelial cells exposed to flow-induced shear stress.

<p>Experimental conditions of <i>in vitro</i> studies of corneal epithelial cells exposed to flow-induced shear stress.</p

Effect of shear stress on migration of HCECs in a scratch wound <i>in vitro</i> model.

<p>Optical micrographs of changes of wound width over time. a) Study #1, confluent monolayers were scratched and then immediately exposed to 4 (low) or 8 (high) dyn/cm² for 24 hours. b) Study #2, confluent monolayers were exposed to 4 (low) or 8 (high) dyn/cm² for 24 hours and then scratched. Wound width was assessed for up to 3 days after wounding. Optical micrographs were taken using a Nikon inverted optical microscope.</p

The effect of shear stress on apoptosis and necrosis.

<p>a) Total apoptotic and necrotic cells; mean ± SD. b) Distribution of cell population (mean values only; standard deviation has been omitted for clarity). HCECs were exposed to 0 (control), 4 (low) and 8 (high) dyn/cm² shear stress for 24 hours. Caspase-mediated apoptosis was measured with FAM-VAD-FMK and PI to detect necrotic cells. * <i>p</i> < <i>0</i>.<i>04</i> compared to control, n = 5 to 6.</p