Kenneth A. Barbee

Mathematical model for the effects of adhesion and mechanics on cell migration speed

Migration of mammalian blood and tissue cells over adhesive surfaces is apparently mediated by specific reversible reactions between cell membrane adhesion receptors and complementary ligands attached to the substratum. Although in a number of systems these receptors and ligand molecules have been isolated and identified, a theory capable of predicting the effects of their properties on cell migration behavior currently does not exist. We present a simple mathematical model for elucidating the dependence of cell speed on adhesion-receptor/ligand binding and cell mechanical properties. Our model can be applied to propose answers to questions such as: does an optimal adhesiveness exist for cell movement? How might changes in receptor and ligand density and/or affinity affect the rate of migration? Can cell rheological properties influence movement speed? This model incorporates cytoskeletal force generation, cell polarization, and dynamic adhesion as requirements for persistent cell movement. A critical feature is the proposed existence of an asymmetry in some cell adhesion-receptor property, correlated with cell polarity. We consider two major alternative mechanisms underlying this asymmetry: (a) a spatial distribution of adhesion-receptor number due to polarized endocytic trafficking and (b) a spatial variation in adhesion-receptor/ligand bond strength. Applying a viscoelastic-solid model for cell mechanics allows us to represent one-dimensional locomotion with a system of differential equations describing cell deformation and displacement along with adhesion-receptor dynamics. In this paper, we solve these equations under the simplifying assumption that receptor dynamics are at a quasi-steady state relative to cell locomotion. Thus, our results are strictly valid for sufficiently slow cell movement, as typically observed for tissue cells such as fibroblasts. Numerical examples relevant to experimental systems are provided. Our results predict how cell speed might vary with intracellular contractile force, cell rheology, receptor/ligand kinetics, and receptor/ligand number densities. A biphasic dependence is shown to be possible with respect to some of the system parameters, with position of the maxima essentially governed by a balance between transmitted contractile force and adhesiveness. We demonstrate that predictions for the two alternative asymmetry mechanisms can be distinguished and could be experimentally tested using cell populations possessing different adhesion-receptor numbers.

Shear stress-induced reorganization of the surface topography of living endothelial cells imaged by atomic force microscopy.

We report the first topographical data of the surface of living endothelial cells at sub-light-microscopic resolution, measurements essential for a detailed understanding of force distribution in the endothelium subjected to flow. Atomic force microscopy was used to observe the surface topography of living endothelial cells in confluent monolayers maintained in static culture or subjected to unidirectional shear stress in laminar flow (12 dyne/cm2 for 24 hours). The surface of polygonal unsheared cells was smooth, with mean excursion of surface undulation between peak height (over the nucleus) and minima (at intercellular junctions) of 3.4 +/- 0.7 microns (mean +/- SD); the mean height to length ratio was 0.11 +/- 0.02. In cells that were aligned in the direction of flow after a 24-hour exposure to laminar shear stress, height differentials were significantly reduced (mean, 1.8 +/- 0.5 micron), and the mean height to length ratio was 0.045 +/- 0.009. Calculation of maximum shear stress and maximum gradient of shear stress (delta tau/delta x, where tau is shear stress at the cell surface) at constant flow velocity revealed substantial streamling of aligned cells that reduced delta tau/delta x by more than 50% at a nominal shear stress of 10 dyne/cm2. Aligned cells exhibited ridges extending in the direction of flow that represented imprints of submembranous F-actin stress-fiber bundles mechanically coupled to the cell membrane. The surface ridges, approximately 50 nm in height and 200 to 1000 nm in width, were particularly prominent in the periphery of the aligned cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of cell viability during bioprinting processes

Bioprinting is an emerging technology in the field of tissue engineering and regenerative medicine. The process consists of simultaneous deposition of cells, biomaterial and/or growth factors under pressure through a micro-scale nozzle. Cell viability can be controlled by varying the parameters like pressure and nozzle diameter. The process itself can be a very useful tool for evaluating an in vitro cell injury model. It is essential to understand the cell responses to process-induced mechanical disturbances because they alter cell morphology and function. We carried out analysis and quantification of the degree of cell injury induced by bioprinting process. A parametric study with different process parameters was conducted to analyze and quantify cell injury as well as to optimize the parameters for printing viable cells. A phenomenological model was developed correlating the percentage of live, apoptotic and necrotic cells to the process parameters. This study incorporates an analytical formulation to predict the cell viability through the system as a function of the maximum shear stress in the system. The study shows that dispensing pressure has a more significant effect on cell viability than the nozzle diameter. The percentage of live cells is reduced significantly (by 38.75%) when constructs are printed at 40 psi compared to those printed at 5 psi.

A mechanism for heterogeneous endothelial responses to flow in vivo and in vitro

Exposure of endothelium to a nominally uniform flow field in vivo and in vitro frequently results in a heterogeneous distribution of individual cell responses. Extremes in response levels are often noted in neighboring cells. Such variations are important for the spatial interpretation of vascular responses to flow and for an understanding of mechanotransduction mechanisms at the level of single cells. We propose that variations of local forces defined by the cell surface geometry contribute to these differences. Atomic force microscopy measurements of cell surface topography in living endothelium both in vitro and in situ combined with computational fluid dynamics demonstrated large cell-to-cell variations in the distribution of flow-generated shear stresses at the endothelial luminal surface. The distribution of forces throughout the surface of individual cells of the monolayer was also found to vary considerably and to be defined by the surface geometry. We conclude that the endothelial three-dimensional surface geometry defines the detailed distribution of shear stresses and gradients at the single cell level, and that there are large variations in force magnitude and distribution between neighboring cells. The measurements support a topographic basis for differential endothelial responses to flow observed in vivo and in vitro. Included in these studies are the first preliminary measurements of the living endothelial cell surface in an intact artery.

Ceramic-based multisite electrode arrays for chronic single-neuron recording

A method is described for the manufacture of a microelectrode array for chronic, multichannel, single neuron recording. The ceramic-based, multisite electrode array has four recording sites patterned onto a ceramic shaft the size of a single typical microwire electrode. The sites and connecting wires are applied to the ceramic substrate using a reverse photolithographic procedure. Recording sites (22/spl times/80 /spl mu/m) are separated by 200 /spl mu/m along the shaft. A layer of alumina insulation is applied over the whole array (exclusive of recording sites) by ion-beam assisted deposition. These arrays were capable of recording single neuron activity from each of their recording sites for at least three weeks during chronic implantation in the somatosensory cortex of rats, and several sites had recordings that lasted for more than 8 weeks. The vertical arrangement of the recording sites on these electrodes is ideal for simultaneously recording across the different layers of brain areas such as the cerebral cortex and hippocampus in chronic preparations.

An approach to targeted drug delivery based on uniform magnetic fields

The capability to deliver high effective dosages to specific sites in the human body has become the holy grail of drug delivery research. Drugs with proven effectiveness under in vitro investigation often reach a major roadblock under in vivo testing due to a lack of an effective delivery strategy. In addition, many clinical scenarios require delivery of agents that are therapeutic at the desired delivery point, but otherwise systemically toxic. We propose a method for targeted drug delivery by applying uniform magnetic fields to an injected superparamagnetic colloidal fluid carrying a drug. The experimental and theoretical models presented give insight into the use of magnetic microspheres for site-specific delivery of therapeutic agents and blood flow occlusion for embolotherapy.

In Vitro Cell Shearing Device to Investigate the Dynamic Response of Cells in a Controlled Hydrodynamic Environment

AbstractMechanical stresses and strains play important roles in the normal growth and development of biological tissues, yet the cellular mechanisms of mechanotransduction have not been identified. A variety of in vitro systems for applying mechanical loads to cell populations have been developed to gain insight into these mechanisms. However, limitations in the ability to control precisely relevant aspects of the mechanical stimuli have obscured the physical relationships between mechanical loading and the biochemical signals that mediate the cellular response. We present a novel in vitro cell shearing device based on the principles of a cone and plate viscometer that utilizes microstepper motor technology to control independently the dynamic and steady components of a hydrodynamic shear-stress environment. Physical measurements of the cone velocity demonstrated faithful reproduction of user-defined input wave forms. Computational modeling of the fluid environment for the unsteady startup confirmed small inertial contributions and negligible secondary flows. Finally, we present experimental results demonstrating the onset rate dependence of functional and structural responses of endothelial cell cultures to dynamically applied shear stress. The controlled cell shearing device is a novel tool for elucidating mechanisms by which mechanical forces give rise to the biological signals that modulate cellular morphology and metabolism.

Strain measurements in cultured vascular smooth muscle cells subjected to mechanical deformation

Early work in the field of biomechanics employed rigorous application of the principles of mechanics to the study of the macroscopic structural response of tissues to applied loads. Interest in the functional response of tissues to mechanical stimulation has lead researchers to study the biochemical responses of cells to mechanical loading. Characterization of the experimental system (i.e., specimen geometry and boundary conditions) is no less important on the microscopic scale of the cell than it is for macroscopic tissue testing. We outline a method for appropriate characterization of cell deformation in a cell culture model; describe a system for applying a uniform, isotropic strain field to cells in culture; and demonstrate a dependence of cell deformation on morphology and distribution of adhesion sites. Cultured vascular smooth-muscle cells were mechanically deformed by applying an isotropic strain to the compliant substrate to which they were adhered. The state of strain in the cells was determined by measurement of the displacements of fluorescent microspheres attached to the cell surface. The magnitude and orientation of principal strains were found to vary spatially and temporally and to depend on cell morphology. These results show that cell strain can be highly variable and emphasize the need to characterize both the loading conditions and the actual cellular deformation in this type of experimental model.

Mechanisms of cell death and neuroprotection by poloxamer 188 after mechanical trauma

The mechanisms of cell death and the progressive degeneration of neural tissue following traumatic brain injury (TBI) have come under intense investigation. However, the complex interactions among the evolving pathologies in multiple cell types obscure the causal relationships between the initial effects of the mechanical trauma at the cellular level and the long‐term dysfunction and neuronal death. We used an in vitro model of neuronal injury to study the mechanisms of cell death in response to a well‐defined mechanical insult and found that the majority of dead cells were apoptotic. We have previously reported that promotion of membrane repair acutely with the non‐ionic surfactant poloxamer 188 (P188) restored cell viability to control values at 24 h postinjury. Here, we showed that P188 significantly inhibits apoptosis and prevents necrosis. We also examined the role of mitogen‐activated protein kinases (MAPKs) in cell death. There was a rapid, transient activation of extracellular signal‐regulated kinases, c‐Jun N‐terminal kinase, and p38s after mechanical insult. Of these, activation of the proapoptotic p38 was the greatest. Treatment with P188 inhibited p38 activation; however, direct inhibition of p38 by SB203580, which selectively inhibits the activity of the p38 MAPK, provided only partial inhibition of apoptosis and had no effect on necrosis. These data suggest that multiple signaling pathways may be involved in the long‐term response of neurons to mechanical injury. Furthermore, that the membrane resealing action of P188 provides such significant protection from both necrosis and apoptosis suggests that acute membrane damage due to trauma is a critical precipitating event that is upstream of the many signaling cascades contributing to the subsequent pathology.

Mechanical membrane injury induces axonal beading through localized activation of calpain

Diffuse axonal injury (DAI), a major component of traumatic brain injury, is characterized by a sequence of neurochemical reactions initiated at the time of trauma and resulting in axonal degeneration and cell death. Calcium influx through mechanically induced axolemmal pores and subsequent activation of calpains are thought to be responsible for the cytoskeletal damage leading to impaired axonal transport. Focal disruption of cytoskeleton accompanied by the accumulation of transported membranous cargo leads to axonal beading which is the characteristic morphology of DAI. By applying fluid shear stress injury on cultured primary neurons, acute calcium (Ca(2+)) and calpain responses of axons to mechanical trauma were investigated. Intracellular Ca(2+) concentration ([Ca(2+)](i)) shows a steady increase following injury that can be blocked by sealing membrane pores with Poloxamer 188 and by chelating intra- or extracellular Ca(2+). Calpain activity increases in response to mechanical injury and this increase depends on Ca(2+) availability and on axolemmal permeability. Both the [Ca(2+)](i) increase and calpain activity exhibit focal peaks along the axons which co-localize with mitochondria and predict future axonal bead locations. These findings suggest that mechanoporation may be the initiating mechanism resulting in ensuing calcium fluxes and subsequent calpain activity and that post-injury membrane repair may be a valid therapeutic approach for acute intervention in DAI.