Session Z27: Mechanics of Cells and Tissues IV

Abnormal vein geometry and pathologic blood flowrates in dialysis patients create complex hemodynamics and loss of dialysis access

Over 750,000 people in the United States receive hemodialysis to treat end-stage renal disease annually, which requires an arteriovenous fistula (AVF). However, AVF maturation and use can result in dysfunctional vein remodeling, stenosis and thrombosis in the cephalic arch (CA) downstream to the AVF, causing access loss. We present a novel method for device fabrication and imaging that recreate complex hemodynamics in large blood vessels at high flowrates in vitro. Briefly, we reconstructed patient-specific 3D models of the CA from venogram, ultrasound and Doppler imaging and fabricated fluidic models of each patient’s CA using 3D printing and soft lithography. We then perfused these models with a fluid that match the density and viscosity of blood at flowrates measured in the patients. Fluorescent tracers in the fluid were imaged in the models at different flowrates, ranging from physiologic to pathologic. We noted laminar flow at physiologic flowrates in all models and complex flow behaviors at pathologic values. We identified distinct flow patterns in the patient models at pathologic flowrates: flow perpendicular to the vessel wall; vortex formation along indentations in the wall; disordered, turbulent flow.   

Bilayered tubes display internal undulations as arteries do under pressure

Endothelial tissues in arteries are widely known to undergo a wrinkling process when their internal pressure drops below a critical value. This mechanism occurs naturally in living systems due to stiffness mismatch in the tube layers when the driving pressure cycles between systolic and diastolic. Our work shows that a synthetic inner-lined elastic tube with a stiffness mismatch between the lining and the support displays similar behavior. We investigate the wrinkles' geometry, amplitude, and wavelength as a function of the pressure changes, comparing experimental results with a nonlinear physical model we developed recently.

We also experimentally studied the wrinkled patterns on single-layer membranes buckled under geometric constraints in a circular sector. The wavelength selection mechanism completely differs from the material mismatch of synthetic inner-lined tubes but remarkably reproduces the geometry of the observed wrinkling patterns.   

Modeling the mechanics of cell-cell junction formation and dynamics in vascular networks

Abstract: The dynamics and stability of cell-cell junctions plays an important role in various physiological processes, ranging from morphogenesis to cancer. In developing vascular networks, in particular, endothelial junction formation and dynamics govern the overall structure and thereby function of the vasculature.  Therefore, it is crucial to understand how the formation and subsequent evolution of cell-cell adhesion junctions are controlled by single-cell behaviors such as cell migration, cell protrusion, polarization, and cell-cell interactions mediated by mechanics or chemical sensing . Starting with a simple case of a two-body problem of cells, we study the mechanics of junction formation and how it is controlled by protrusive activity and cell-cell interactions. We further show how the feedback between cadherin bond kinetics at the cell-cell junction and actomyosin contractile forces determine the long-term junction stability. We then generalize our model to larger numbers of cells to study network formation and remodeling. Using such mechano-chemical models, one can gain insights into how cell-cell junctions affect the mechanical integrity and network topology of tissues.   

Neuronal growth investigated by traction force and atomic force microscopy

During brain development, neurons actively grow axons that steer over distances ranging from tens to hundreds of cell diameters in length to locate target dendrites from other neurons and form neuronal networks. Axonal growth is largely controlled by a complex interplay between intrinsic and extrinsic factors, including genetic programming, biochemical signaling, cytoskeletal dynamics, and extracellular cues. Here I will present experimental results obtained by measuring traction forces and stresses exerted by neurons on the growth substrate during axonal extension. We also investigate how the traction forces and the elastic modulus of neurons change upon disruption of cell adhesion and of the actin intracellular dynamics . Our results highlight the interplay between neuron biomechanical properties, internal cytoskeletal dynamics, and external cues in generating traction forces, and open up new directions for future investigations of axonal growth and the formation of neuronal networks.   

Probing effects of vimentin on cell cytoskeleton dynamics through Differential Dynamic Microscopy (DDM)

The cell cytoskeleton is a complex network of actin, microtubules, and intermediate filaments, which play a fundamental role in various cellular processes, such as cell polarization, intracellular transport, and cell migration. While microtubules are central to cell-polarization and have been studied in great detail, vimentin intermediate filaments also seem to play a role, as disrupting vimentin impairs polarized cell motion and the directional mobility of vesicles. In our investigation, we employed Differential Dynamic Microscopy (DDM) to explore the dynamic behavior of the microtubules and vesicles in wild-type and vimentin-null mouse embryonic fibroblasts. Here, we  measured the dynamics and spatial organization of microtubule network using DDM in presence and absence of vimentin.  Our research reveals that cells lacking vimentin show 1.5 times higher decay times compared to cells with vimentin, which suggest that vimentin enhances the dynamics of microtubule filaments in the cell. Furthermore, Fluorescence Recovery after Photobleaching studies indicate that, despite an equivalent tubulin concentration in both wild-type (WT) and vimentin-null (VN) cells, the rate of fluorescence recovery is higher in cells with vimentin. Our results indicate a role of vimentin in mediating microtubule dynamics, which has important implications for how cells organize and coordinate polarized motion.

Osmotic Pressure Microscopy: New method to quantify cellular mechanical properties in walled cells.

Plant cells, similar to yeast and bacteria, are surrounded by a cell wall, a stiff extracellular matrix that limits their deformation. Plant organs can grow and even move owing to the high cellular hydrostatic pressure (5 to 20 atm) deforming the rigid walls. Quantifying mechanical properties at the cellular scale is therefore essential to reveal how plant regulate their own shape and movements. However, the most common methods to extract these properties, Pressure Probes and Atomic Force Microscopy, suffer from severe limitations. We are developing a new method, Osmotic Pressure Microscopy (OPM) to measure in living cells the parameters most relevant to understand plant movements and growth. The method involves the combination of 3D microscopy, image analysis, osmotic treatments, and modelling, to quantify physical parameters such as cellular 3D geometry, turgor pressure, bulk modulus and cell wall elasticity. OPM does not require dedicated or complex experimental setups, making it accessible to most biology labs. Preliminary results on different plant organs such as leaf and anther demonstrate the potential and versatility of our new method.   

Reverse-engineering plant cell mechanical properties

Unlike animal systems, plant cell mechanics depend mainly on their stiff extra-cellular matrix - the cell wall - which is stretched due to high hydrostatic turgor pressure (~10 atm) imposed by the cell. Plants evolved strategies to manipulate cell wall properties, controlling organ growth and movement. Measuring the micro-scale mechanical properties of individual plant cells is challenging. Indentation methods are used for the measurement but sensitivity of indenter size and geometry, hinder the separation of the cell wall elasticity and pressure. We developed a novel method to reverse engineer the cellular mechanical properties using osmotic treatments and Finite Element Method(FEM) simulations of pressurized cells. Osmotic experiments reveal the turgor pressure which can be used as an input for mechanical modeling along with the deformed 3D cell wall geometry extracted from experiments. FEM simulations are used to reverse engineer the remaining mechanical properties like Young’s modulus of the cell wall(E) and boundary conditions on the cells. Applying this method to the epidermis of Arabidopsis thaliana early leaf, our results unveil tissue-level stretching, the cellular distribution of E, and the significant influence of surrounding cells on individual cell’s expansion.   

The Mechanical Behavior of Arabidopsis Cotyledons with Genetic Mutations Affecting Cell Structure and Cell Adhesion

The growth of plant cells and their integration into tissues relies on the control of local mechanical properties in the middle lamella, the cell wall, and the cytoskeleton. While mutations affecting constituents of these regions, including actin or pectin, have been shown to influence cotyledon development, the direct influence of these biomolecular components to cell/tissue mechanical properties and cell adhesion remain unclear. To address the current gap, we perform mechanical testing on the cotyledons of Arabidopsis mutants. The combination of a miniature load frame and in situ microscopy highlights the contributions of molecular components to the strength and mechanical failure of plant materials. This connection between cellular biochemistry and mechanical behavior in the leaves of Arabidopsis informs potential modifications to economically valuable plant species.    

Investigation of Adhesion Forces on the Surface of Borrelia burgdorferi Using AFM

The Lyme disease spirochete, Borrelia burgdorferi (Bb), is the leading cause of arthropod-borne disease in the USA. Adhesion is important to the establishment of infection and contributes to dissemination, persistence, and immune evasion. Therefore, understanding bacterial adhesion at the molecular level is crucial. Atomic Force Microscopy (AFM) can provide both the topography and mechanical properties of biological samples. With AFM, we measured adhesive forces between non-functionalized AFM tips and adhesins on the surface of live Bb. Using contact-mode force spectroscopy we extracted the tip-bacteria adhesion characteristics of three different B. burgdorferi cell lines (Wild,  AD3BLKO, HPJ) with progressively higher mutations. From over 6000 total measurements, probability density functions for each mutant line were extracted. Results of the experiment show significant adhesion forces between non-functionalized AFM cantilevers and B. burgdorferi, exhibiting a linear decrease in maximum adhesion with an increase in genes removed. Similarly, binding probability from successful vs unsuccessful adhesion events exhibits a linear decrease as a function of increased mutation. Current work is being done to characterize the adhesion force between fibronectin functionalized cantilevers and proteins isolated from B. burgdorferi.   

A general solution of a regular 2D spring network to model the E. coli peptidoglycan shell.

The cell wall of gram-negative E. coli bacteria derives its mechanical resilience from a 2D network of glycan strands covalently cross-linked by short peptides. The network is constantly growing by the insertion of new material while holding up to extreme stresses. It is only incompletely understood how the astonishing capabilities of this network derive from the molecular properties of its constituents and network geometry. We use a 2D triangular spring network to provide a bridge between molecular and continuum-elastic modeling. We derive general constitutive laws, valid for large deformations, non-linear responses, and arbitrary anisotropies. We then find the minimal model compatible with the available data on morphology and mechanics of E. coli cell walls. We show that, in the large-deformation regime, the stiffness of the network is always proportional to the magnitude of the stress, and therefore, to the turgor pressure of the cell.   

IPF ECM-Derived Hydrogels Promote the Inflammatory Cytokine Expression of Cultured IPF Fibroblasts and Trigger an Inflammatory and Proliferative Immune Cell Profile

Idiopathic pulmonary fibrosis (IPF) is a chronic disease that causes scarring of lung tissue, leading to a reduction in oxygen transport. At the cellular level, IPF is characterized by overproduction and accumulation of extracellular matrix (ECM) secreted by fibroblasts. Previous work has shown that culturing fibroblasts on stiff 2D surfaces can alter gene expression in both IPF fibroblasts (IPF-F) and normal human lung fibroblasts (NHLF), but fail to recapitulate key signatures of the fibrotic microenvironment in vivo. This compromises their usefulness as model systems in which to interrogate IPF biology and test candidate therapeutics.

To better understand fibroblast biology in IPF, we developed a 3D hydrogel model derived from human fibrotic lung tissue that allows us to study the effects of fibroblast activation and cytokine expression without the confounding influence of mechanosignaling from stiff plastic substrates. Hydrogels were generated from lung tissue via decellularization, lyophilization, pulverization, and digestion of scaffold prior to forming gels. 2D culture served as control. Fibroblast 3D hydrogel cultures were assayed for contractility, imaged via optical microscopy, and analyzed with ImageJ. Conditioned media was collected from fibroblast cultures and used to treat monocytes and macrophages. Gene expression was assayed via quantitative PCR, and protein expression was assayed via Western Blot. The mechanical properties of hydrogels were characterized with rheometry and atomic force microscopy and correlated with contractility of the IPF-F when cultured in our ECM hydrogels, as compared to NHLF. Our results broadly suggest that hydrogels recapitulate the increased inflammatory immune cells as a result of the fibrotic ECM in vitro model and altered mechanical properties in the IPF disease state.

In conclusion, our hydrogels drive expression of inflammatory cytokines that are lost in standard 2D culture.  These observations reinforce the need for improved in vitro model systems that retain environmental cues in the IPF lung. Our hydrogel platform will serve as a robust in vitro platform for further investigations of fundamental issues underlying the progression of IPF.   

Oral: A mathematical and computational model to study the role of the cardiac jelly during morphogenesis

One of the key components of the cardiac morphogenesis of vertebrates is the dynamics of the extracellular matrix, also known as cardiac jelly, located between the myocardium and endocardium layers during embryonic development. Besides its viscoelastic properties, the thickness inhomogeneities in the cardiac jelly during these stages are crucial for correct development. Experimental data have shown a strong correlation between this and the process of delamination that forms the structure of the heart. 

In this study, we introduce a detailed mathematical model to describe the dynamics of the cardiac jelly. Our model captures the intricate interplay between its elasticity, viscosity, and time-dependent deformation. This approach allows us to look deeper into the physical properties that govern the cardiac jelly's behavior during embryonic development.

The main application of our model is to investigate fracture generation within the cardiac jelly. Using our mathematical framework and a finite element computational implementation, we simulate a 3D model of the cardiac jelly. This enables us to study the generation of fracture patterns depending on the geometrical and material properties of the system and to compare our results with experimental observations in zebrafish embryos.    

Reproducing Experimentally Observed Alternans in Cardiac Tissue Using Fractional Diffusion

Heart disease is the leading cause of mortality in the US, despite the existence of many treatments to aid cardiovascular problems. Alternans of the heart's action potential has been shown to be a dangerous marker for mortality with over 80% of people identified with it dying within 2 years if not treated. Experimental studies have shown how alternans in space can lead to the initiation of complex arrhythmias because of the spatiotemporal dispersion in refractoriness they produce. Computational studies have shown mechanisms behind the dynamics of alternans, however to date they fail to reproduce the dynamics quantitatively, requiring much larger tissue domains compared with experiments. In this talk we argue that the voltage propagation in cardiac tissue in the mesoscopic scale can be described by fractional diffusion and this allows for models to be fitted in tissue sizes as in experiments.   

A Modified Fitzhugh-Nagumo Model that Reproduces the Action Potential and Dynamics of the Ten Tusscher et. al. Cardiac Model in Tissue

The two-variable Fitzhugh-Nagumo (FHN) model is a widely used cardiac and neural action potential simulating model due to its simplicity; however, it lacks many of the dynamics observed in cardiac experiments that can be reproduced by complex ionic cell models, such as the 19-variable Ten Tusscher et. al. (TNNP) model. We have parameterized a modified version of the FHN model that reproduces the dynamics in space of more complex cardiac cell models. We combined a series of modifications that previously were applied to the FHN model – mainly, the addition of a nullcline at zero voltage for the fast variable, that eliminates the hyperpolarization of the traditional FHN model and the modification of the slow nullcline from linear to quadratic, which allows for alternan behavior and a better fit to experiments and other models. This new model is fitted using particle swarm optimization (PSO) to fit the action potential for a large number of pacing periods so that the restitution of the action potential is matched between the two models. We created a modified FHN model that matches most of the AP shape of the TNNP model for a large range of periods and dynamics in space. This model allows for faster proof of concept investigations that can then help guide the more time-consuming simulations with complex ionic models.