A disk-type apparatus for applying fluid shear stress on cultured endothelial cell

To study the effect of fluid shear stress on cultured endothelial cells, we have developed an apparatus for the stress creation, which consists of a stainless steel disk driven by an electric DC motor and a stage to place a culture dish and to adjust the distance between the disk and the dish. When the disk is rotated, a concentric fluid movement occurs in the culture medium in the dish and exerts the shear stress on the endothelial cells cultured on the bottom of the dish. A theoretical analyses concerning the induced concentric flow velocity predicted that when the angular velocity of the disk rotation (omega) is slow enough to maintain a Reynolds' number of the order of 10, the exerted wall shear stress tau w on the endothelial cell monolayer is given for a constant as tau w = mu r omega/d where mu is the viscosity of the medium, d the distance from the plate to the monolayer and r the radial distance from the center of the dish. When omega is varied in a sinusoidal mode tau w also becomes sinusoidal, thus allowing to apply a pulsatile stress. In vitro experiments carried out to examine the validity of the theoretical results, using a suspension of polystyrene as a tracer with the ordinary culture medium and 99% ethanol, revealed excellent agreement of the measured velocity profiles with the predicted ones. The results demonstrated that the present apparatus can create both the steady and pulsatile wall shear stress on the culture cell layer as expected, unless Reynolds' number greatly exceeds the level of 10.     

Correspondence of low mean shear and high harmonic content in the porcine iliac arteries

BACKGROUND: Temporal variations in shear stress have been suggested to affect endothelial cell biology. To better quantify the range of dynamic shear forces that occur in vivo, the frequency content of shear variations that occur naturally over a cardiac cycle in the iliac arteries was determined. METHOD OF APPROACH: Computational fluid dynamic calculations were performed in six iliac arteries from three juvenile swine. Fourier analysis of the time-varying shear stress computed at the arterial wall was performed to determine the prevalence of shear forces occurring at higher frequencies in these arteries. RESULTS: While most of each artery experienced shear forces predominantly at the frequency of the heart rate, the frequency spectra at certain regions were dominated by shear forces at higher frequencies. Regions whose frequency spectra were dominated by higher harmonics generally experienced lower mean shear stress. The negative correlation between shear and dominant harmonic was significant (p=0.002). CONCLUSIONS: Since lesion development typically occurs in regions experiencing low time-average shear stress, this result suggests that the frequency content of the shear exposure may also be a contributing factor in lesion development. A better understanding of the vascular response to shear components of different frequencies might help rationalize the notion of "disturbed flow" as a hemodynamic entity.     

Design optimization of a helical endothelial cell culture device

The specific roles of mass transfer and fluid dynamic stresses on endothelial function, important in atherogenesis, are not known. Further, the effects of mass transfer and fluid dynamic stresses are difficult to separate because areas of “abnormal” mass transfer and “abnormal” wall shear stress tend to co-localize (where abnormal is defined as any deviation from the mass transfer rate or wall shear stress present in a long straight artery with the same flow rate and diameter). Our goal was to design a cell culture device which gives maximum separation between areas of abnormal shear stress and areas of abnormal mass transfer. We used design optimization principles to design a helical cell culture device. Using shear stress and mass transfer fields predicted by solving the governing equations, the area of the device which was exposed to low rates of mass transfer and normal levels of wall shear stress was determined. The design optimization method then maximized this area by varying the design variables, resulting in the optimum design. The optimum design had Reynolds number = 50, helical radius = 3.23 and helical pitch = 3.82. The area of the device which was exposed to low rates of mass transfer and regular levels of wall shear stress was about 4.5 times the inlet cross-sectional area of the device or about 5% of the device total internal surface area. An optimum design was successfully determined and the methodology used was shown to be robust. The area of the device which was exposed to low rates of mass transfer and regular levels of wall shear stress occurred in a defined region which should aid further experimental work.     

Wall shear stress during pulsatile flow distal to a normal porcine aortic valve

The shear stress at the wall has been of interest as one of the possible fluid dynamic factors that may be damaging in the region of prosthetic valves. The purpose of this study was to measure the axial wall shear stresses in the region of a 29 mm tissue annulus diameter porcine stent mounted prosthetic aortic valve (Hancock, Model 242). Studies were performed in an in vitro pulse duplicating system. The axial wall shear stress was calculated from velocities obtained near the wall with a laser Doppler anemometer. The largest axial wall shear stress was 29 dyn cm-2 and it occurred at the highest stroke volume used (80 ml). At a stroke volume of 50 ml, the largest axial wall shear stress was 17 dyn cm-2 and at a stroke volume of 35 ml, it was 15 dyn cm-2. Stresses of these magnitudes are far below those reported to be damaging to the endothelial surface. These stresses may be high enough, however, to affect platelet function.     

Leukocytes rolling and recruitment by endothelial cells: hemorheological experiments and numerical simulations

The recruitment of leukocytes from the blood stream and their subsequent adhesion to endothelial walls are essential stages to the immune response system during inflammation. The precise dynamic mechanisms by which molecular mediators facilitate leukocyte arrests are still unknown. In this study combined experimental results and computer simulations are used to investigate localized hydrodynamics of individual and collective behavior of clusters of leukocytes. Leukocyte-endothelial cell interactions in post-capillary venules of Wistar rats cremaster muscle were monitored by intravital microscopy. From these experiments the hemorheologic and hemodynamical measured parameters were used in time dependent three-dimensional computer simulations, using a mesoscopic lattice Boltzmann flow solver for shear thinning fluids. The dynamics of leukocyte clusters under generalized Newtonian blood flow with shear thinning viscosity was computed and discussed. In this paper we present quantified distributions of velocity and shear stress on the surface of leukocytes and near vessel wall attachment points. We have observed one region of maximum shear stress and two regions of minimum shear stress on the surface of leukocytes close to the endothelial wall. We verified that the collective hydrodynamic behavior of the cluster of recruited leukocytes establishes a strong motive for additional leukocyte recruitment. It was found that the lattice Boltzmann solver used here is fully adaptive to the measured experimental parameters. This study suggests that the influence of the leukocytes rolling on the increase of the endothelial wall shear stress may support the activation of more signalling mediators during inflammation.     

Shear stress increases the residence time of adhesion of Pseudomonas aeruginosa

Although ubiquitous, the processes by which bacteria colonize surfaces remain poorly understood. Here we report results for the influence of the wall shear stress on the early-stage adhesion of Pseudomonas aeruginosa PA14 on glass and polydimethylsiloxane surfaces. We use image analysis to measure the residence time of each adhering bacterium under flow. Our main finding is that, on either surface, the characteristic residence time of bacteria increases approximately linearly as the shear stress increases (∼0–3.5 Pa). To investigate this phenomenon, we used mutant strains defective in surface organelles (type I pili, type IV pili, or the flagellum) or extracellular matrix production. Our results show that, although these bacterial surface features influence the frequency of adhesion events and the early-stage detachment probability, none of them is responsible for the trend in the shear-enhanced adhesion time. These observations bring what we believe are new insights into the mechanism of bacterial attachment in shear flows, and suggest a role for other intrinsic features of the cell surface, or a dynamic cell response to shear stress.     

To what extent does a minimal atherosclerotic plaque alter the arterial wall shear stress distribution? A model study by an electrochemical method

An electrochemical surface shear stress measurement was applied to a model of very thin unilateral arterial stenosis (height of 1/8 of the model pipe diameter with very smooth surface). Three dimensional wall shear stress distribution was measured under steady flow field from a relatively low Reynolds number, Re = 270, to a high Reynolds number, Re = 1200. There was a characteristic high and low wall shear distribution pattern around the stenosis. There were also remarkable high shear stress areas on the opposite wall and both side walls of the stenosis. It was clearly shown that three dimensional structure of the flow field, hence, the wall shear stress distribution, is affected by a minimal change on the arterial wall.     

Influence of cardiac flow rate on turbulent shear stress from a prosthetic heart valve

Elevated turbulent shear stresses associated with sufficient exposure times are potentially damaging to blood constituents. Since these conditions can be induced by mechanical heart valves, the objectives of this study were to locate the maximum turbulent shear stress in both space and time and to determine how the maximum turbulent shear stress depends on the cardiac flow rate in a pulsatile flow downstream of a tilting disk valve. Two-component, simultaneous, correlated laser velocimeter measurements were recorded at four different axial locations and three different flow rates in a straight tube model of the aorta. All velocity data were ensemble averaged within a 15 ms time window located at approximately peak systolic flow over more than 300 cycles. Shear stresses as high as 992 dynes/cm2 were found 0.92 tube diameters downstream of the monostrut, disk valve. The maximum turbulent shear stress was found to scale with flow rate to the 0.72 power. A repeatable starting vortex was shed from the disk at the beginning of each cycle.     

Microfluidic accumulation assay probes attachment of biofilm forming diatom cells

Testing of fouling release (FR) technologies is of great relevance for discovery of the next generation of protective marine coatings. In this paper, an accumulation assay to test diatom interaction under laminar flow with the model organism Navicula perminuta is introduced. Using time lapse microscopy with large area sampling allows determination of the accumulation kinetics of the diatom on three model surfaces with different surface properties at different wall shear stresses. The hydrodynamic conditions within the flow cell are described and a suitable shear stress range to perform accumulation experiments is identified at which statistically significant discrimination of surfaces is possible. The observed trends compare well to published adhesion preferences of N. perminuta. Also, previously determined trends of critical wall shear stresses required for cell removal from the same set of functionalized interfaces shows consistent trends. Initial attachment mediated by extracellular polymeric substances (EPS) present outside the diatoms leads to the conclusion that the FR potential of the tested coating candidates can be deducted from dynamic accumulation experiments under well-defined hydrodynamic conditions. As well as testing new coating candidates for their FR properties, monitoring of the adhesion process under flow provides additional information on the mechanism and geometry of attachment and the population kinetics.     

The impact of process stress on suspended anchorage-dependent mammalian cells as an indicator of likely challenges for regenerative medicines

To quantify the engineering shear constraint on processing, the effect of capillary shear stress (pipe flow) on suspended anchorage-dependent mammalian cells has been investigated. Exposure of cultured rat aortic smooth muscle cells to repeated capillary shear stress (2-120 N m(-2)) causes a decrease in total number of cells, number of intact cells and number of cells able to grow. The optimum wall shear stress for cell survival was found to be 10-50 N m(-2) (flowrate 4-20 mL/min, I.D. 0.45 mm). Cell populations which are able to grow after exposure to shear stress do not exhibit reduced growth rate or altered metabolism.     

CFD-aided design of a dynamic filter for mammalian cell separation

In the present work, a rotating disk filter was designed for mammalian cell separation with the aim of avoiding both cell damage and membrane fouling. Different geometric and operational variables of the rotating disk filter were studied using computational fluid dynamics (CFD) by varying rotor radius, rotor angle, membrane-rotor distance, and angular velocity. The combinations of these variables followed a statistical design, so that an analysis of the CFD results provided correlations describing the average shear stress on the membrane surface and the maximum shear stress in the whole module as a function of the variables studied. Based on these correlations, and on the shear resistance levels of Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cell lines, which were investigated using a cone-and-plate viscosimeter, it was possible to determine the geometry and angular velocity that would minimize both cell damage and membrane fouling. After construction, the filter was tested in filtration experiments at increasing permeate fluxes. Cell viability remained >90% for the duration of the experiments (2.5 h), and no indication of fouling was observed. It was shown that the designed dynamic filter is able to effectively avoid both cell damage and membrane fouling, and thus can be used for mammalian cell harvesting and perfusion.     

Numerical 3D-simulation of pulsatile wall shear stress in an arterial T-bifurcation model

The structure of pulsatile blood flow and wall shear stress in a 90° T-bifurcation model is analysed numerically. The nonlinear Navier-Stokes equations for time-dependent incompressible Newtonian fluid flow are approximated using a newly developed pressure correction, finite element method. The wall shear stress is calculated from the finite element velocity field. The investigation shows viscous flow phenomena such as flow separation and stagnation and the distribution of high and low wall shear stress during the pulse cycle. Furthermore, the effect of a sharp corner the bifurcation edge on the wall shear stress is analysed. Detailed local flow investigation is required to examine fluid dynamic contribution to the development of arterial diseases such as atherosclerosis and thrombosis.     

A numerical study of flow in curved tubes simulating coronary arteries

Numerical simulations of pulsatile flow in coronary arteries which take into account the curvature associated with the bending of arteries over the surface of the heart are presented for resting, excited and drug induced states. The study was motivated by reported observations of atherosclerotic plaque localization on the inner curvature of coronary arteries. The simulated flow field appears quasi-steady under resting conditions with wall shear stress always highest on the outside wall and only a single secondary flow vortex in the half tube. However, reversal of wall shear stress direction at the inside wall does occur under resting flow conditions and this is not a quasi-steady characteristic. The flow field is markedly unsteady under excited conditions with wall shear stress sometimes peaking on the inside wall and an increase in the magnitude of wall shear stress reversal on the inside wall. However, only a single secondary flow vortex in the half tube is observed. Implications of the simulations for the role of fluid mechanics in coronary artery atherosclerosis are also discussed.     

Fluid dynamics of a textured blood-contacting surface

This study examined the fluid dynamics of a textured blood-contacting surface using a computational fluid-dynamic modeling technique. The texture consisted of a regular array of microfibers of length 50 or 100 microm, spaced 100 microm apart, projecting perpendicularly to the surface. The results showed that the surface texture served as a flow-retarding solid boundary for a laminar viscous flow, resulting in a lowered wall shear stress on the hase-plane surface. However, the maximum wall shear stress on the fibers was much higher than the shear stress on the nontextured phase plane. At all fractions of fiber height down past 10 microm, the permeability of the textured region greatly exceeded the analytically predictable permeability of an equivalent array of infinite-height fihers. The lowered suiface shear stress appears to explain in part the enhanced deposition of formed blood elements on the textured surface.     

Dynamic curvature strongly affects wall shear rates in a coronary artery bifurcation model

This study was motivated by the need for a better understanding of coronary artery blood flow patterns and their possible role in atherosclerosis formation. Of particular interest in this study was the effects of the dynamic deformation due to myocardial contraction on wall shear rate patterns in the coronary arteries. A better understanding of these effects on wall shear rate in a bifurcation geometry and an evaluation of the importance of these effects was desired. A three-dimensional computer model of a bifurcation lying on the surface of a sphere with time-varying radius of curvature was employed to simulate the motion and deformation of the arteries. The results indicated low mean shear rates along the myocardial wall and very high shear rate variations (over 100% of the static mean shear rate) along the outer wall. The results obtained using a quasi-static analysis were found to underestimate the dynamic wall shear rate variation along the myocardial and outer walls. It was concluded that dynamic geometry effects are important in determining sites of low mean and oscillating wall shear that have been associated with atherogenesis in curved, bifurcating arteries.     

Platelet lysis and aggregation in shear fields.

A rotational viscometer was used to study the effects of shear stress on platelets in human platelet-rich plasma (PRP). For 5-min exposure times, shear stresses above 160 dynes/cm2 induced platelet lysis (as determined by release of platelet lactic dehydrogenase). For 30-s exposure times, shear stresses greater than 600 dynes/cm2 were required to induce platelet lysis. The platelet counts of sheared PRP were decreased to as low as one-fifth the original count due largely to shear-induced aggregation. The count is a minimum at intermediate stress levels (200-400 dynes/cm2). Higher stresses induce disaggregation as well as lysis. The diminution in the counts was partially reversed in 2 h incubation after cessation of shearing. Experiments were carried out with three different viscometer configurations so that the shear stress and the solid surface area access could be varied independently. Surface access was not a significant variable in the conditions of the experiments. Thus aggregation and lysis may be induced by stress effects alone as well as by solid surface effects. The results also show that the response of platelets to shear stress is strongly dependent on exposure time. Platelets are much less resistant to shear stress than red cells for relatively long exposure times. However, the converse is true for very short exposure times.     

Hydrodynamic thickening of lubricating fluid layer beneath sliding mesothelial tissues

The delicate mesothelial surfaces of the pleural space and other serosal cavities slide relative to each, lubricated by pleural fluid. In the absence of breathing motion, differences between lung and chest wall shape could eventually cause the lungs and chest wall to come into contact. Whether sliding motion keeps lungs and chest wall separated by a continuous liquid layer is not known. To explore the effects of hydrodynamic pressures generated by mesothelial sliding, we measured the thickness of the liquid layer beneath the peritoneal surface of a 3-cm disk of rat abdominal wall under a normal stress of 2 cm H2O sliding against a glass plate rotating at 0-1 rev/s. Thickness of the lubricating layer was determined microscopically from the appearance of fluorescent microspheres adherent to the tissue and glass. Usually, fluid thickness near the center of the tissue disk increased with the onset of glass rotation, increasing to 50-200 microm at higher rotation rates, suggesting hydrodynamic pumping. However, thickness changes often differed substantially among tissue samples and between clockwise and counter-clockwise rotation, and sometimes thickness decreased with rotation, suggesting that topographic features of the tissue are important in determining global hydrodynamic effects. We conclude that mesothelial sliding induces local hydrodynamic pressure gradients and global hydrodynamic pumping that typically increases the thickness of the lubricating fluid layer, moving fluid against the global pressure gradient. A similar phenomenon could maintain fluid continuity in the pleural space, reducing frictional force and shear stress during breathing.     

Dynamic shear stress in parallel-plate flow chambers

An in vitro model using a parallel-plate fluid flow chamber is supposed to simulate in vivo fluid shear stresses on various cell types exposed to dynamic fluid flow in their physiological environment. The metabolic response of cells in vitro is associated with the wall shear stress. However, parallel-plate flow chambers have not been characterized for dynamic fluid flow experiments. We use a dimensionless ratio h / lambda(v), in determining the exact magnitude of the dynamic wall shear stress, with its oscillating components scaled by a shear factor T. It is shown that, in order to expose cells to predictable levels of dynamic fluid shear stress, two conditions have to be met: (1) h / lambda(v) < 2, where h is the distance between the plates and lambda(v) is the viscous penetration depth; and (2) f(0) < f(c) / m, where the critical frequency f(c) is the upper threshold for this flow regime, m is the highest harmonic mode of the flow, and f(0) is the fundamental frequency of fluid flow.     

Radial artery wall shear stress evaluation in patients with arteriovenous fistula for hemodialysis access

It has been extensively documented that changes in blood flow induce vascular remodeling and this phenomenon seems to be correlated to the shear forces imposed on the vessel wall by motion of blood. Wall shear stress, the tractive force that acts on the endothelium, has been shown to influence endothelial cell function. To study changes in wall shear stress that develop on the vessel wall upon changes of blood flow, we set up a technique that allows estimation of shear stress in the radial artery of patients on chronic hemodialysis therapy. The technique is based on color-flow Doppler examination of the radial artery before and after surgical creation of radiocephalic fistula for hemodialysis. Calculation of time function wall shear stress and blood flow rate in the radial artery is performed on the basis of arterial diameter, center-line velocity waveform and blood viscosity, using a numerical method developed according to Womersley's theory for pulsatile flow in tubes. The results presented confirm that the model developed is suitable for calculation of the wall shear stress that develops in the radial artery of patients before and after surgical creation of an arteriovenous fistula for hemodialysis. This methodology was developed for characterization of wall shear stress in the radial artery but may be well applied to other vessels that can be examined by echo-Doppler technique.     

Measured wall shear stress distribution pattern upstream and downstream of a unilateral stenosis by an electrochemical method

An electrochemical surface shear stress measurement was applied to a model of unilateral arterial stenosis. The unilateral stenosis model was made up of a removable stenosis plug, in an electrochemical shear stress measurement test section with 100 cathodes. Three dimensional wall shear stress distribution was measured under steady flow field. At a relatively low Reynolds number, Re = 270, there was a characteristic high and low wall shear distribution pattern downstream of the unilateral stenosis. There were also remarkable high shear stress areas on the opposite wall up- and downstream, and both side walls of the stenosis upstream. It was clearly shown that detailed three dimensional structure of the flow field must be studied in order to correlate it to pathological findings.