Haemodynamic flow conditions at the initiation of high-shear platelet aggregation: a combined in vitro and cellular in silico study.

The influence of the flow environment on platelet aggregation is not fully understood in high-shear thrombosis. The objective of this study is to investigate the role of a high shear rate in initial platelet aggregation. The haemodynamic conditions in a microfluidic device are studied using cell-based blood flow simulations. The results are compared with in vitro platelet aggregation experiments performed with porcine whole blood (WB) and platelet-rich-plasma (PRP). We studied whether the cell-depleted layer in combination with high shear and high platelet flux can account for the distribution of platelet aggregates. High platelet fluxes at the wall were found in silico. In WB, the platelet flux was about twice as high as in PRP. Additionally, initial platelet aggregation and occlusion were observed in vitro in the stenotic region. In PRP, the position of the occlusive thrombus was located more downstream than in WB. Furthermore, the shear rates and stresses in cell-based and continuum simulations were studied. We found that a continuum simulation is a good approximation for PRP. For WB, it cannot predict the correct values near the wall.

A study on the effects of covered stents on tissue prolapse.

Polyvinyl alcohol (PVA) cryogel covered stents may reduce complications from thrombosis and restenosis by decreasing tissue prolapse. Finite element analysis was employed to evaluate the effects of PVA cryogel layers of varying thickness on tissue prolapse and artery wall stress for two common stent geometries and two vessel diameters. Additionally, several PVA cryogel covered stents were fabricated and imaged with an environmental scanning electron microscope. Finite element results showed that covered stents reduced tissue prolapse up to 13% and artery wall stress up to 29% with the size of the reduction depending on the stent geometry, vessel diameter, and PVA cryogel layer thickness. Environmental scanning electron microscope images of expanded covered stents showed the PVA cryogel to completely cover the area between struts without gaps or tears. Overall, this work provides both computational and experimental evidence for the use of PVA cryogels in covered stents.

Augmentation of bone tunnel healing in anterior cruciate ligament grafts: application of calcium phosphates and other materials.

Bone tunnel healing is an important consideration after anterior cruciate ligament (ACL) replacement surgery. Recently, a variety of materials have been proposed for improving this healing process, including autologous bone tissue, cells, artificial proteins, and calcium salts. Amongst these materials are calcium phosphates (CaPs), which are known for their biocompatibility and are widely commercially available. As with the majority of the materials investigated, CaPs have been shown to advance the healing of bone tunnel tissue in animal studies. Mechanical testing shows fixation strengths to be improved, particularly by the application of CaP-based cement in the bone tunnel. Significantly, CaP-based cements have been shown to produce improvements comparable to those induced by potentially more complex treatments such as biologics (including fibronectin and chitin) and cultured cells. Further investigation of CaP-based treatment in the bone tunnels during ACL replacement is therefore warranted in order to establish what improvements in healing and resulting clinical benefits may be achieved through its application.

Quantitative prediction of improvement in cardiac function after revascularization with MR imaging and modeling: initial results.

PURPOSE: To evaluate a model that can be used quantitatively to predict changes in postrevascularization left ventricular function based on classification of myocardial tissue as hibernating, scarred, or normal with cine magnetic resonance (MR) imaging. MATERIALS AND METHODS: Eleven patients with chronic left ventricular dysfunction were studied before and after revascularization with cine MR imaging. Regional myocardial contractility and wall thickness were used in the model to predict postrevascularization ejection fraction (EF). The actual EF from the postrevascularization MR images was compared with the EF from the prerevascularization images predicted with the model by using regression analysis and Bland-Altman analysis. RESULTS: Correlation between the actual EF after revascularization and the EF predicted by using the model yielded an R value of 0.98, with a standard error of 1.3 EF percentage points. Predicting changes in function in a myocardial segment was less successful because only 55% of segments classified as hibernating actually improved resting function after revascularization. In nonimproved segments, 78% were either adjacent to infarcted segments or had nontransmural wall thinning. CONCLUSION: A simple mathematical model combined with functional information provided by MR imaging was used to predict improvements in global EF resulting from revascularization.

Contractile responses in arteries subjected to hypertensive pressure in seven-day organ culture.

Early stage changes in hypertensive arteries have a significant effect on the long-term adaptation of the arteries. Compared to the long-term adaptation, little is known about the early dimensional and functional changes in hypertensive arteries in the first few days of hypertension. To study the early stage changes in hypertensive arteries, porcine common carotid arteries were cultured for seven days in a simplified ex vivo artery organ culture system with pulsatile flow under hypertensive (200+/-30 mm Hg) or normotensive (100+/-20 mm Hg) pressure conditions while maintaining a physiological mean wall shear stress of 15 dyn/cm2. Vessel viability was demonstrated by contractile diameter responses to norepinephrine (NE), carbachol (CCh), and sodium nitroprusside (SNP) as well as staining for mitochondrial activity and cell apoptosis/necrosis. The results show that arteries demonstrated strong contractile responses to NE, CCh, and SNP, basal tone, and viable mitochondria in the organ culture system for seven days. Hypertensive arteries demonstrated a stronger contractile response than normotensive arteries (p<0.05). Diameter enlargement was observed in hypertensive arteries as compared to arteries cultured under normotensive conditions. In conclusion, the pulsatile culture system can maintain arteries viable with active vasomotion tone for up to seven days. Hypertensive pressure causes arterial adaptation by significantly increasing arterial diameter and contractile response within the first seven days.

A mechanistic model of acute platelet accumulation in thrombogenic stenoses.

Thrombosis on an atherosclerotic lesion can cause heart attack or stroke. Thrombosis may be triggered by plaque rupture or erosion, creating a thrombogenic stenosis. To measure and model this situation, collagen-coated stenoses have been exposed to nonanticoagulated blood in a baboon ex vivo shunt. The maximum rate of platelet accumulation, measured using a gamma camera, was highest in the throat region of moderate and severe stenoses, and increased with increasing stenosis severity. A species transport model of platelet accumulation was developed, which included mechanisms of convection, shear-enhanced diffusion, near-wall platelet concentration, and a kinetic model of platelet activation and aggregation. The model accurately reproduced the average spatial pattern and time rate of platelet accumulation in the upstream and throat regions of the stenosis, where shear-enhanced diffusivity increased platelet transport in the stenosis throat. Downstream of the throat where flow is complicated by recirculation, the model computed a transport-limited region with lower than measured platelet accumulation, suggesting that fluid-phase platelet activation may significantly affect both transport and adhesion rates in the poststenotic region. This model may provide an initial quantitative estimate of the likelihood of occlusive thrombus in individual patients due to plaque erosion, artery spasm, incomplete angioplasty, or plaque rupture.

Mechanical properties of a novel PVA hydrogel in shear and unconfined compression.

Poly(vinyl alcohol) (PVA) hydrogels have been proposed as promising biomaterials to replace diseased or damaged articular cartilage. A critical barrier to their use as load-bearing tissue replacements is a lack of sufficient mechanical properties. The purpose of this study was to characterize the functional compressive and shear mechanical properties of a novel PVA hydrogel. Two formulations of the biomaterial were tested, one with a lower water content (75% water), and the other with higher water content (80% water). The compressive tangent modulus varied with biomaterial formulation and was found to be statistically strain magnitude and rate dependent. Over a strain range of 10-60%, the compressive modulus increased from approximately 1-18 MPa, which is within the range of the modulus of articular cartilage. The shear tangent modulus (0.1-0.4 MPa) was also found to be strain magnitude dependent and within the range of normal human articular cartilage, but it was not statistically dependent on strain rate, This behavior was attributed to the dominance of fluid flow and related frictional drag on the viscoelastic behavior. Compressive failure of the hydrogels was found to occur between 45 and 60% strain, depending on water content.

Steady flow and wall compression in stenotic arteries: a three-dimensional thick-wall model with fluid-wall interactions.

Severe stenosis may cause critical flow and wall mechanical conditions related to artery fatigue, artery compression, and plaque rupture, which leads directly to heart attack and stroke. The exact mechanism involved is not well understood. In this paper a nonlinear three-dimensional thick-wall model with fluid-wall interactions is introduced to simulate blood flow in carotid arteries with stenosis and to quantify physiological conditions under which wall compression or even collapse may occur. The mechanical properties of the tube wall were selected to match a thick-wall stenosis model made of PVA hydrogel. The experimentally measured nonlinear stress-strain relationship is implemented in the computational model using an incremental linear elasticity approach. The Navier-Stokes equations are used for the fluid model. An incremental boundary iteration method is used to handle the fluid-wall interactions. Our results indicate that severe stenosis causes considerable compressive stress in the tube wall and critical flow conditions such as negative pressure, high shear stress, and flow separation which may be related to artery compression, plaque cap rupture, platelet activation, and thrombus formation. The stress distribution has a very localized pattern and both maximum tensile stress (five times higher than normal average stress) and maximum compressive stress occur inside the stenotic section. Wall deformation, flow rates, and true severities of the stenosis under different pressure conditions are calculated and compared with experimental measurements and reasonable agreement is found.

Early effects of arterial hemodynamic conditions on human saphenous veins perfused ex vivo.

Exposure to the arterial hemodynamic environment is thought to be a potential trigger for the pathological remodeling of saphenous vein grafts. Using matched pairs of freshly isolated human saphenous vein, we analyzed the early effects of ex vivo hemodynamic conditions mimicking the venous (native) compared with arterial (graft) environment on the key components of vascular remodeling, ie, matrix metalloproteinase (MMP)-9 and MMP-2 and cell proliferation. Interestingly, we found that arterial conditions halved latent MMP-9 (50+/-11%, P=0.01) and MMP-2 (44+/-6%, P=0.005) levels relative to matched vein pairs maintained ex vivo under venous perfusion for up to 3 days. Immunostaining supported decreased MMP levels in the innermost area of arterially perfused veins. Either decreased synthesis or increased posttranslational processing may decrease MMP zymogen levels. Biosynthetic radiolabeling showed that arterial perfusion actually increased MMP-9 and MMP-2 production. When we then examined potential pathways for MMP zymogen processing, we found that arterial conditions did not affect the expression of MT-MMP-1, a cell-associated MMP activator, but that they significantly increased the levels of superoxide, another MMP activator, suggesting redox-dependent MMP processing. Additional experiments indicated that increased superoxide under arterial conditions was due to diminished scavenging by decreased extracellular superoxide dismutase. Arterial perfusion also stimulated cell proliferation (by 220% to 750%) in the majority of vein segments investigated. Our observations support the hypothesis that arterial hemodynamic conditions stimulate early vein graft remodeling. Furthermore, physiological arterial flow may work to prevent pathological remodeling, particularly the formation of intimal hyperplasia, through rapid inactivation of secreted MMPs and, possibly, through preferential stimulation of cell proliferation in the outer layers of the vein wall.

Transmural pressure induces matrix-degrading activity in porcine arteries ex vivo.

Extracellular matrix components must be degraded and resynthesized for vascular remodeling to occur. We hypothesized that the hemodynamic environment regulates activity of matrix metalloproteinases (MMPs), the primary agents for in vivo matrix degradation, during vascular remodeling in response to changes in transmural pressure and shear stress. Pathological hemodynamic conditions were reproduced in an ex vivo system in which we maintained porcine carotid arteries for 24 and 48 h. Total levels of MMP-2 and MMP-9 extracted from tissue homogenates and analyzed by SDS-PAGE zymography were stimulated by transmural pressure and were unaffected by shear stress changes. Degradation of two specific gelatinase substrates, gelatin and elastin, increased with increasing pressure, but the degradation was not affected by shear stress changes in tissue specimens analyzed using in situ zymography (gelatin) and fluorescent measurement of endogenous elastin degradation (elastin). Our results suggest that transmural pressure activates at least two members of the MMP family and that activity of these enzymes is accompanied by degradation of matrix components, effects that may be implicated in hypertensive vascular remodeling.

A nonlinear axisymmetric model with fluid-wall interactions for steady viscous flow in stenotic elastic tubes.

Arteries with high-grade stenoses may compress under physiologic conditions due to negative transmural pressure caused by high-velocity flow passing through the stenoses. To quantify the compressive conditions near the stenosis, a nonlinear axisymmetric model with fluid-wall interactions is introduced to simulate the viscous flow in a compliant stenotic tube. The nonlinear elastic properties of the tube (tube law) are measured experimentally and used in the model. The model is solved using ADINA (Automatic Dynamic Incremental Nonlinear Analysis), which is a finite element package capable of solving problems with fluid-structure interactions. Our results indicate that severe stenoses cause critical flow conditions such as negative pressure and high and low shear stresses, which may be related to artery compression, plaque cap rupture, platelet activation, and thrombus formation. The pressure filed near a stenosis has a complex pattern not seen in one-dimensional models. Negative transmural pressure as low as -24 mmHg for a 78 percent stenosis by diameter is observed at the throat of the stenosis for a downstream pressure of 30 mmHg. Maximum shear stress as a high as 1860 dyn/cm2 occurs at the throat of the stenoses, while low shear stress with reversed direction is observed right distal to the stenosis. Compressive stresses are observed inside the tube wall. The maximal principal stress and hoop stress in the 78 percent stenosis are 80 percent higher than that from the 50 percent stenosis used in our simulation. Flow rates under different pressure drop conditions are calculated and compared with experimental measurements and reasonable agreement is found for the prebuckling stage.

Fluid mechanics of vascular systems, diseases, and thrombosis.

The cardiovascular system is an internal flow loop with multiple branches circulating a complex liquid. The hallmarks of blood flow in arteries are pulsatility and branches, which cause wall stresses to be cyclical and nonuniform. Normal arterial flow is laminar, with secondary flows generated at curves and branches. Arteries can adapt to and modify hemodynamic conditions, and unusual hemodynamic conditions may cause an abnormal biological response. Velocity profile skewing can create pockets in which the wall shear stress is low and oscillates in direction. Atherosclerosis tends to localize to these sites and creates a narrowing of the artery lumen--a stenosis. Plaque rupture or endothelial injury can stimulate thrombosis, which can block blood flow to heart or brain tissues, causing a heart attack or stroke. This small lumen and elevated shear rate in a stenosis create conditions that accelerate platelet accumulation and occlusion. The relationship between thrombosis and fluid mechanics is complex, especially in the post-stenotic flow field. New convection models have been developed to predict clinical from platelet thrombosis in diseased arteries. Future hemodynamic studies should address the complex mechanics of flow-induced, large-scale wall motion and convection of semisolid particles and cells in flowing blood.

A novel method for efficient drug delivery.

Local delivery of anti-thrombotic and anti-restenotic drugs is desired to achieve high concentrations of agents which may be rapidly degraded systemically or which exhibit very short half-lives in vivo. In this article, the operating characteristics of a novel local drug delivery method are described and its effectiveness demonstrated computationally and experimentally. Computational models used a finite volume method to determine the concentration field. Optical dye density measurements of Evans blue in saline were performed in an in vitro steady flow system. Modeling parameters were kept in the physiologic range. Experimental flow visualization studies demonstrated high concentrations of infusate near the vessel wall. Computational studies predicted high, clinically significant drug concentrations along the wall downstream of the infusion device. When the radial infusion velocity is large (infusion flow rate, Qinf>0.5% of the main flow rate, Q), the wall concentration of the infused drug remains high, e.g., levels are greater than 80% of the infusate concentration 5 cm downstream of the infusion device. At lower infusion rates (Qinf<0.001Q), the drug concentration at the wall decreases exponentially with axial distance to less than 25% of the infusate concentration 5 cm downstream of the infusion device, although therapeutic drug levels are still readily maintained. The near wall drug concentration is a function of flow conditions, infusion rate, and the drug diffusivity. Good agreement was obtained between computational and experimental concentration measurements. Flow simulation and experimental results indicate that the technique can effectively sustain high local drug concentrations for inhibition of thrombosis and vascular lesion formation.

Effects of frictional losses and pulsatile flow on the collapse of stenotic arteries.

High-grade stenosis can produce conditions in which the artery may collapse. A one-dimensional numerical model of a compliant stenosis was developed from the collapsible tube theory of Shapiro. The model extends an earlier model by including the effects of frictional losses and unsteadiness. The model was used to investigate the relative importance of several physical parameters present in the in vivo environment. The results indicated that collapse can occur within the stenosis. Frictional loss was influential in reducing the magnitude of collapse. Large separation losses could prevent collapse outright even with low downstream resistances. However, the degree of stenosis was still the primary parameter governing the onset of collapse. Pulsatile solutions demonstrated conditions that produce cyclic collapse within the stenosis. This study predicts certain physiologic conditions in which collapse of arteries may occur for high-grade stenoses.

Convective mass transfer at the carotid bifurcation.

The convective conditions in regions of hemodynamic separation may produce uneven local mass transfer at the arterial wall which may lead to an atherogenic response. This study estimates the potential variation in local mass transfer of oxygen at the human carotid bifurcation under steady flow conditions. The three-dimensional separated flow at the bifurcation was studied using a computational analysis of the basic conservation equations of mass, momentum, and species. Mass transfer between the blood and the wall was estimated throughout the sinus region for a condition where the concentration at the wall was constant. Flow separation at the carotid bifurcation created a complex concentration field. The mass transfer was five times lower along the outer wall of the carotid sinus than the artery wall immediately upstream or downstream of the sinus. The region of low mass transfer was similar to the region of low shear stress but not identical. This distribution of low mass transfer correlated strongly with intimal thickening as measured previously from human specimens. Quantitative differences in mass transfer at the arterial wall should not be discarded as an important mechanism by which hemodynamics influences atherogenesis at this site of clinical disease.

Computational simulation of turbulent signal loss in 2D time-of-flight magnetic resonance angiograms.

Time-of-flight magnetic resonance (MR) angiography is currently limited in the evaluation of arterial stenoses by flow-induced signal loss. This signal loss has been attributed to phase dispersion and to phase misregistration. We have developed a fluid mechanics model of 2D time-of-flight MR angiograms to study the amount of signal loss caused by random turbulence. The simulations were created by stochastic analysis of particle pathlines determined by computational fluid dynamics for turbulent flow. The images obtained by the model compare well to actual MR images of flow in stenoses. By selectively removing the random turbulent motion in the simulation, it can be seen that random phase dispersion is the dominant mechanism of signal loss. Phase misregistration and mean flow phase dispersion act as secondary effects. The MR simulation model recreates accurately the variation of signal loss over a range of echo times. The model can be used further to explore and design new pulse sequences. For example, the current study showed that high slew rate gradient waveforms can significantly reduce poststenotic signal loss. In conclusion, computational modeling of MR angiography can be a useful approach for the analysis of MRA signal loss and the design of improved pulse sequences.

Reduced blood flow accelerates intimal hyperplasia in endarterectomized canine arteries.

The purpose of this study was to evaluate a technique that accelerates intimal hyperplasia by reduction of blood flow. Bilateral endarterectomies were performed in both femoral and carotid arteries in six dogs. One week later, all animals underwent banding of an artery distal to the injured region to reduce the blood flow by 50%. The contralateral injured arteries served as controls. At 11 weeks, the specimens were harvested and analyzed. Five of 12 (42%) of the flow-restricted arteries and nine of 12 (75%) of the non-flow-restricted arteries were patent at 11 weeks (P<0.05). Marked stenotic intimal hyperplastic lesions developed in the flow-restricted arteries (69% stenosis) as compared with the non-flow-restricted arteries (37% stenosis). Mean(s.d.) intimal thickness, intimal areas, and intimal/medial area ratio were 0.52(0.19) mm, 3.17(1.11) mm2, and 1.12(0.33)%, respectively, in the flow-restricted arteries. Their counterparts in the non-flow-restricted arteries were 0.21(0.09) mm, 1.70(1.09) mm2, and 0.58(0.14)%, respectively (P<0.05). Extracellular matrix comprised 48% of total intimal volumes in the flow-restricted arteries. Cell proliferation and occluded arteries were also characterized. These data demonstrate that reduction of blood flow significantly accelerated intimal hyperplasia and occlusion rates in endarterectomized arteries. Advanced intimal hyperplastic lesions (>50% stenosis) possess a high extracellular matrix content. This new animal model is a reliable generator of advanced stenotic lesions in a relatively short time period and can be used to study biologic mechanisms of stenosis and evaluate therapeutic interventions.

Boundary layer infusion of nitric oxide reduces early smooth muscle cell proliferation in the endarterectomized canine artery.

To evaluate the direct effect of nitric oxide (NO) on vascular smooth muscle cell (SMC) proliferation in vivo, we used an expanded polytetrafluoroethylene (ePTFE)-based local infusion device to deliver an NO donor, proline/NO (PROLI/NO), to the luminal boundary layer of endarterectomized artery and the distal anastomosis of the graft in a canine model. Once delivered to the blood, PROLI/NO releases NO by a mechanism involving pH-dependent decomposition. Six dogs underwent bilateral femoral artery endarterectomies. ePTFE infusion devices, blindly primed with PROLI/NO to one artery or proline to the contralateral vessel, were anastomosed proximal to the injured segments so that each animal served as its own control. PROLI/NO or proline was continuously delivered for 7 days from an osmotic reservoir, through the wall of the graft infusion device. Euthanasia was carried out at 7 days, and the processed specimens were blindly analyzed for SMC proliferation at both graft anastomoses and endarterectomized segments by a bromodeoxyuridine index assay. All dogs survived with no clinical side effects. In comparing the treated and control vessels, NO released from PROLI/NO significantly reduced SMC proliferation by 43% (13.24 +/- 1.24% versus 23.24 +/- 1.01%, P = 0.004) at the distal anastomoses and by 68% (10.58 +/- 1.63% versus 25.17 +/- 3.39%, P = 0.007) at endarterectomized segments. However, there was no significant difference in blood flow measurements between treated and control arteries (56.25 +/- 6.50 ml/min versus 46.50 +/- 3.20 ml/min, P = 0.094). These data demonstrate that local boundary layer infusion of NO released from PROLI/NO significantly reduces SMC proliferation in injured arteries with no effect on regional blood flow. This study suggests a new strategy to inhibit early SMC proliferation in injured arteries and probably to control intimal hyperplastic lesion formation in the manipulated vessels.

Time-course study of intimal hyperplasia in the endarterectomized canine artery.

In an effort to characterize intimal hyperplastic lesions, we have undertaken a time-course study in a canine endarterectomy model of intimal hyperplasia. Twenty dogs underwent surgical endarterectomies of the carotid arteries. A total of 23 of 27 (85%) injured arteries were patent, which consisted of 6, 8, 5, and 4 arteries found to be patent at 1, 2, 5, and 11 weeks, respectively. Measurable intimal thickening developed at 1 week (0.08 +/- 0.01 mm) and at 2 weeks (0.13 +/- 0.02 mm), maximized at 5 weeks (0.29 +/- 0.03 mm), and subsided at 11 weeks (0.21 +/- 0.01 mm) after injury. Endothelial cells covering intimal hyperplastic tissues were seen only at 11 weeks. The intimal cell proliferation rate reached a maximum of 24% at 1 week, decreased dramatically at 2 weeks, and remained at low levels but higher than baseline levels at 5 and 11 weeks. Extracellular matrix (ECM) content accounted for 29% of total intimal volume at 1 week after endarterectomy and increased to 37, 40, and 47% at 2, 5, and 11 weeks, respectively. These data demonstrate that maximum intimal cell proliferation occurs at 1 week and maximum intimal hyperplasia at 5 weeks after arterial injury. Intimal ECM content increased with time after injury throughout the duration of this study. The uniform and consistent intimal lesion that was established in this large animal model is clinically relevant and can be used to study cellular and molecular mechanisms of restenosis and to evaluate therapeutic interventions.

The accuracy of magnetic resonance phase velocity measurements in stenotic flow.

Nuclear magnetic resonance (MR) can be used to measure velocities in fluid flow using the technique of phase velocity mapping. Advantages of MR velocimetry include the simultaneous mapping of the entire flow field through a non-contacting, magnetic window. The phase velocity mapping technique assumes that velocity is constant over the measurement time (typically around 10 ms). For many fluid flows, this assumption is not valid. The current study showed that MR phase velocity measurements of velocity through stenotic flow can be in error by over 100% immediately upstream and downstream of the stenosis throat and by 20% far downstream of the throat in comparison with laser Doppler anemometer measurements taken at the same location. Highly turbulent flow also led to significant errors in velocity measurement. These errors can be attributed to several sources including low signal-to-noise ratio, additional phase shifts due to non-constant velocities, and non-stationary transit-time effects. Velocity measurement errors could be reduced to under 30% at all measurement locations through the use of MR sequences with high signal-to-noise ratios, low echo times, and thick slices.