Shear stimulated red blood cell microparticles: Effect on clot structure, flow and fibrinolysis.

BACKGROUND: Microparticles (MPs) have activity in thrombus promotion and generation. Erythrocyte microparticles (ErMPs) have been reported to accelerate fibrinolysis in the absence of permeation. We hypothesized that shear induced ErMPs would affect fibrin structure of clots and change flow with implications for fibrinolysis. OBJECTIVE: To determine the effect of ErMPs on clot structure and fibrinolysis. METHODS: Plasma with elevated ErMPs was isolated from whole blood or from washed red blood cells (RBCs) resuspended in platelet free plasma (PFP) after high shear. Dynamic light scattering (DLS) provided size distribution of ErMPs from sheared samples and unsheared PFP controls. Clots were formed by recalcification for flow/lysis experiments and examined by confocal microscopy and SEM. Flow rates through clots and time-to-lysis were recorded. A cellular automata model showed the effect of ErMPs on fibrin polymerization and clot structure. RESULTS: Coverage of fibrin increased by 41% in clots formed from plasma of sheared RBCs in PFP over controls. Flow rate decreased by 46.7% under a pressure gradient of 10 mmHg/cm with reduction in time to lysis from 5.7 ± 0.7 min to 12.2 ± 1.1 min (p < 0.01). Particle size of ErMPs from sheared samples (200 nm) was comparable to endogenous microparticles. CONCLUSIONS: ErMPs alter the fibrin network in a thrombus and affect hydraulic permeability resulting in decelerated delivery of fibrinolytic drugs.

Mechanical Strength and Conductivity of Cementitious Composites with Multiwalled Carbon Nanotubes: To Functionalize or Not?

The incorporation of carbon nanotubes into cementitious composites increases their compressive and flexural strength, as well as their electrical and thermal conductivity. Multiwalled carbon nanotubes (MWCNTs) covalently functionalized with hydroxyl and carboxyl moieties are thought to offer superior performance over bare nanotubes, based on the chemistry of cement binder and nanotubes. Anionic carboxylate can bind to cationic calcium in the hydration products, while hydroxyl groups participate in hydrogen bonding to anionic and nonionic oxygen atoms. Results in the literature for mechanical properties vary widely for both bare and modified filler, so any added benefits with functionalization are not clearly evident. This mini-review seeks to resolve the issue using an analysis of reports where direct comparisons of cementitious composites with plain and functionalized nanotubes were made at the same concentrations, with the same methods of preparation and under the same conditions of testing. A focus on observations related to the mechanisms underlying the enhancement of mechanical strength and conductivity helps to clarify the benefits of using functionalized MWCNTs.

Elongational Stresses and Cells.

Fluid forces and their effects on cells have been researched for quite some time, especially in the realm of biology and medicine. Shear forces have been the primary emphasis, often attributed as being the main source of cell deformation/damage in devices like prosthetic heart valves and artificial organs. Less well understood and studied are extensional stresses which are often found in such devices, in bioreactors, and in normal blood circulation. Several microfluidic channels utilizing hyperbolic, abrupt, or tapered constrictions and cross-flow geometries, have been used to isolate the effects of extensional flow. Under such flow cell deformations, erythrocytes, leukocytes, and a variety of other cell types have been examined. Results suggest that extensional stresses cause larger deformation than shear stresses of the same magnitude. This has further implications in assessing cell injury from mechanical forces in artificial organs and bioreactors. The cells' greater sensitivity to extensional stress has found utility in mechanophenotyping devices, which have been successfully used to identify pathologies that affect cell deformability. Further application outside of biology includes disrupting cells for increased food product stability and harvesting macromolecules for biofuel. The effects of extensional stresses on cells remains an area meriting further study.

Hemolysis estimation in turbulent flow for the FDA critical path initiative centrifugal blood pump.

Hemolysis in medical devices and implants has been a primary concern over the past fifty years. Turbulent flow in particular can cause cell trauma and hemolysis in such devices. In this work, the effects of turbulence on red blood cell (RBC) damage are examined by simulating the flow field through a centrifugal blood pump that has been identified as a case study through the critical path initiative of the US Food and Drug Administration (FDA). In this study, a new model was employed to predict hemolysis in the turbulent flow environment in the pump selected for the FDA critical path initiative. The operating conditions for a centrifugal blood pump were specified by the FDA for rotational speeds of 2500 and 3500 rpm. The model is based on the analysis of the smaller eddies within the turbulent flow field, since it is assumed that turbulent flow eddies with sizes comparable to the dimensions of the RBCs lead to cell trauma. The Kolmogorov length scale of the velocity field is used to identify such small eddies. Using model parameters obtained in prior work through comparisons to capillary and jet flow, it is found that hemolysis for the 2500-rpm pump was predicted well, while hemolysis for the 3500-rpm pump was overpredicted. Results indicate refinement of the model and empirical constants with better experimental data is needed.

Effect of Morphologically Controlled Hematite Nanoparticles on the Properties of Fly Ash Blended Cement.

Several types of hematite nanoparticles (α-Fe2O3) have been investigated for their effects on the structure and properties of fly ash (FA) blended cement. All synthesized nanoparticles were found to be of spherical shape, but of different particle sizes ranging from 10 to 195 nm depending on the surfactant used in their preparation. The cement hydration with time showed 1.0% α-Fe2O3 nanoparticles are effective accelerators for FA blended cement. Moreover, adding α-Fe2O3 nanoparticles in FA blended cement enhanced the compressive strength and workability of cement. Nanoparticle size and size distribution were important for optimal filling of various size of pores within the cement structure.

Production of erythrocyte microparticles in a sub-hemolytic environment.

Microparticles are produced by various cells due to a number of different stimuli in the circulatory system. Shear stress has been shown to injure red blood cells resulting in hemolysis or non-reversible sub-hemolytic damage. We hypothesized that, in the sub-hemolytic shear range, there exist sufficient mechanical stimuli for red blood cells to respond with production of microparticles. Red blood cells isolated from blood of healthy volunteers were exposed to high shear stress in a microfluidic channel to mimic mechanical trauma similar to that occurring in ventricular assist devices. Utilizing flow cytometry techniques, both an increase of shear rate and exposure time showed higher concentrations of red blood cell microparticles. Controlled shear rate exposure shows that red blood cell microparticle concentration may be indicative of sub-hemolytic damage to red blood cells. In addition, properties of these red blood cell microparticles produced by shear suggest that mechanical trauma may underlie some complications for cardiovascular patients.

Rheology of Virgin Asphalt Binder Combined with High Percentages of RAP Binder Rejuvenated with Waste Vegetable Oil.

Waste cooking oils (WCOs) show promise as a rejuvenator for reclaimed asphalt pavement (RAP) binders. Their use helps to make RAP a renewable resource and to address environmental concerns related to WCO disposal. While studies suggest that 100% RAP for pavement is feasible, RAP will likely be combined with a virgin binder and aggregate incrementally in the field. In this study, the rheological properties of the virgin binder blended with a simulated RAP binder and 10% waste vegetable oil (WVO) as a rejuvenator were examined. Viscosities below that of a PG 64-22 virgin binder were observed with WVO in blends of 40 or 60% RAP and the virgin binder. The virgin-60% RAP/WVO binder blend retained a Superpave grade of the virgin binder and was found to satisfy requirements for both rutting parameter and fatigue resistance. Results indicated that WVO significantly reduced the effects of long-term ageing, making the blend containing RAP durable. The effect of RAP content on WVO/virgin binder blends was most pronounced on the rutting parameter. A "molecular lubrication" model is suggested as a mechanism for the viscosity reduction with WVO.

The Applicability of a Drop Penetration Method to Measure Contact Angles on TiO2 and ZnO Nanoparticles.

In this study, six solvents (water, diiodomethane, bromonaphthalene, formamide, ethanol and ethylene glycol) were examined for three nanoparticle substrates, zinc oxide and titanium dioxide (21 nm and 100 nm), with the goal of assessing the suitability of a modified drop penetration method (DPM) for orders of magnitude smaller particles. Nanoparticles were compressed into flat discs and the solvent dropped on the surface while the image with time was recorded. Contact angles were in reasonable agreement with literature over the range of 20-80°, but failed to provide acceptable results for surface energy components. It was necessary to eliminate certain solvents and substrates not meeting the selection criteria.

A Flow Induced Autoimmune Response and Accelerated Senescence of Red Blood Cells in Cardiovascular Devices.

Red blood cells (RBCs) passing through heart pumps, prosthetic heart valves and other cardiovascular devices undergo early senescence attributed to non-physiologic forces. We hypothesized that mechanical trauma accelerates aging by deformation of membrane proteins to cause binding of naturally occurring IgG. RBCs isolated from blood of healthy volunteers were exposed to high shear stress in a viscometer or microfluidics channel to mimic mechanical trauma and then incubated with autologous plasma. Increased binding of IgG was observed indicating forces caused conformational changes in a membrane protein exposing an epitope(s), probably the senescent cell antigen of band 3. The binding of immunoglobulin suggests it plays a role in the premature sequestration and phagocytosis of RBCs in the spleen. Measurement of IgG holds promise as a marker foreshadowing complications in cardiovascular patients and as a means to improve the design of medical devices in which RBCs are susceptible to sublethal trauma.

Possible erythrocyte contributions to and exacerbation of the post-thrombolytic no-reflow phenomenon.

BACKGROUND: Reperfusion injury often occurs with therapeutic intervention addressing the arterial occlusions causing acute myocardial infarction and stroke. The no-reflow phenomenon has been ascribed to leukocyte plugging and blood vessel constriction in the microcirculation. OBJECTIVE: To assess possible red cell contributions to post-thrombolytic no-reflow phenomenon. METHODS: Blood clots were formed by recalcifying 1 ml of citrated fresh human venous blood and then lysed by adding 1,000 units of streptokinase (SK) at several intervals within 1 hour. Red cell deformability was tested by both a microscopic photometric and a filtration technique, viscosity by a cone and plate viscometer, and erythrocyte aggregation by an optical aggregometer. RESULTS: Two sampling methods were devised for the microscopic photometric test, both of which indicated increases of erythrocyte stiffness after being lysed from the clot by SK. In accompanying experiments, the viscosity, aggregation and filterability of the post-lytic erythrocytes were assessed. Results indicated increased viscosity in Ringer's, decreased aggregation index and filterability through a 5 µm pore size Nuclepore membrane. CONCLUSION: Findings demonstrated that post-lytic changes in red cell deformability do occur which could contribute to the no-reflow phenomenon.

Modified dextran, heparin-based triggered release microspheres for cardiovascular delivery of therapeutic drugs using protamine as a stimulus.

In this study, we describe the synthesis of an amine-modified acetalated dextran polymer, which is combined with heparin (HP) as the basis for a novel controlled release system. Dextran-amine (DEXAM) conjugates, synthesised using reductive amination, were incorporated into DEXAM/HP microspheres. HP binds to positively charged ammonium ions of the DEXAM conjugates, contributing to the structural integrity of the microspheres. Crystal violet (CV) was encapsulated inside DEXAM/HP microspheres as a model drug to test the system. Protamine with a high affinity for HP functioned as a trigger to release CV. DEXAM/HP microspheres were characterised by particle size, encapsulation efficiency, scanning electron microscope images, and in vitro release profile. Release of CV from microspheres varied with primary amine content of DEXAM conjugates, amount of HP, and concentration of protamine added. The system is considered for controlled delivery of agents without the necessity of chemical modification.

Flow-Field Simulations and Hemolysis Estimates for the Food and Drug Administration Critical Path Initiative Centrifugal Blood Pump.

The design of blood pumps for use in ventricular assist devices, which provide life-saving circulatory support in patients with heart failure, require remarkable precision and attention to detail to replicate the functionality of the native heart. The United States Food and Drug Administration (FDA) initiated a Critical Path Initiative to standardize and facilitate the use of computational fluid dynamics in the study and development of these devices. As a part of the study, a simplified centrifugal blood pump model generated by computer-aided design was released to universities and laboratories nationwide. The effects of changes in fluid rheology due to temperature, hematocrit, and turbulent flow on key metrics of the FDA pump were examined in depth using results from a finite volume-based commercial computational fluid dynamics code. Differences in blood damage indices obtained using Eulerian and Lagrangian formulations were considered. These results are presented and discussed awaiting future validation using experimental results, which will be released by the FDA at a future date.

An Approach for Assessing Turbulent Flow Damage to Blood in Medical Devices.

In this work, contributing factors for red blood cell (RBC) damage in turbulence are investigated by simulating jet flow experiments. Results show that dissipative eddies comparable or smaller in size to the red blood cells cause hemolysis and that hemolysis corresponds to the number and, more importantly, the surface area of eddies that are associated with Kolmogorov length scale (KLS) smaller than about 10 µm. The size distribution of Kolmogorov scale eddies is used to define a turbulent flow extensive property with eddies serving as a means to assess the turbulence effectiveness in damaging cells, and a new hemolysis model is proposed. This empirical model is in agreement with hemolysis results for well-defined systems that exhibit different exposure times and flow conditions, in Couette flow viscometer, capillary tube, and jet flow experiments.

Heterogeneous phase fibrinolysis rates by damped oscillation rheometry.

BACKGROUND: Devices gauging viscoelastic properties of blood during coagulation like the thromboelastograph support fundamental research as well as point of care needs. Associated fibrinolysis data are based on endogenous species or plasminogen activator added to a homogeneous sample prior to clot formation. Digestion in a monolithic structure differs from the physical situation of thrombolytic therapy where surface reactions dominate. OBJECTIVE: This study aims to develop rheological testing for heterogeneous phase fibrinolysis. METHOD: Fibrinolysis rates were determined by phase change of a solid clot induced by autologous plasma/streptokinase (SK) in a rheometer sensitive to viscous damping. RESULTS: Initial slope or overall change in the logarithmic damping factor indicated fibrinolytic rates. Rates depended on clot geometry, phase volumes, clot composition and SK concentration. CONCLUSION: The damped oscillation rheometer can be adapted to determine relative rates of heterogeneous fibrinolysis in vitro.

An In Vitro Thrombolysis Study Using a Mixture of Fast-Acting and Slower Release Microspheres.

PURPOSE: To test the hypothesis that a mixture combining fast and slower release rate microspheres can restore blood flow rapidly and prevent formation of another blockage in thrombolysis. METHODS: We used polyethylene glycol (PEG) microspheres which provide the release of the encapsulated streptokinase (SK) on the scale of minutes, and Eudragit FS30D (Eud), a polymethacrylate polymer, for development of delayed release microspheres which were desirable to prevent a putative second thrombus. Eud microspheres were coated with chitosan (CS) to further extend half-life. Experiments included the development, characterization of Eud/SK and CS-Eud/SK microspheres, and in vitro thrombolytic studies of the mixtures of PEG/SK and Eud /SK microspheres and of PEG/SK and CS-Eud/SK microspheres. RESULTS: CS-Eud/SK microspheres have slightly lower encapsulation efficiency, reduced activity of SK, and a much slower release of SK when compared with microspheres of Eud/SK microspheres. Counter-intuitively, slower release leads to faster thrombolysis after reocclusion as a result of greater retention of agent and the mechanism of distributed intraclot thrombolysis. CONCLUSIONS: A mixture of PEG/SK and CS-Eud/SK microspheres could break up the blood clot rapidly while providing clot-lytic efficacy in prevention of a second blockage up to 4 h.

Hemodynamics of the renal artery ostia with implications for their structural development and efficiency of flow.

BACKGROUND: Energy losses at tube or blood vessel orifices depend on the extent of flare as measured by the dimensionless ratio of the fillet radius of curvature to diameter (r/D). OBJECTIVE: The goal of this study was to assess the effect of ostial fillet radii on energy losses at the aorta-renal artery junctions since as much as a quarter of cardiac output passes through the kidneys. METHOD: Pressure loss coefficients K for the renal artery ostia as a function of r/D have been determined for representative anatomical variants using finite volume simulations. Estimates of fillet radii in humans from image analysis were employed in simulations for comparison of loss coefficients. RESULTS: Values for K drop 45% as r/D increases over the range 0-1.3. Image analysis indicates that the ostia are not symmetric in humans with (r/D)superior much larger than (r/D)inferior. Simulations show the loss coefficient depends almost entirely on the superior fillet radius. CONCLUSIONS: Superior fillet radii for both renal arteries are similar to the optimal value to reduce energy losses while the inferior radii are not. Ostial asymmetry may have been induced by higher levels of shear stress present on the superior portion of a developing symmetric ostium of small r/D.

Hemolysis Related to Turbulent Eddy Size Distributions Using Comparisons of Experiments to Computations.

Turbulent blood flow in medical devices contributes to blood trauma, yet the exact mechanism(s) have not been fully elucidated. Local turbulent stresses, viscous stresses, and the rate of dissipation of the turbulent kinetic energy have been proffered as hypotheses to describe and predict blood damage. In this work, simulations of experiments in a Couette flow viscometer and a capillary tube were used to examine extensive properties of the turbulent flow field and to investigate contributing factors for red blood cell hemoglobin release in turbulence by eddy analysis. It was found that hemolysis occurred when dissipative eddies were comparable in size to the red blood cells. The Kolmogorov length scale was used to quantify the size of smaller turbulent eddies, indicating correspondence of hemolysis with number and surface area of eddies smaller than about 10 µm when a k-ε turbulence model is adopted.

Aqueous dual-tailed surfactants simulated on the alumina surface.

Atomistic molecular dynamics (MD) simulations were used to compare the morphology of aqueous surfactant self-assembled aggregates on a flat alumina substrate. The substrate was modeled using the CLAYFF force field, and it was considered fully protonated. Three ionic surfactants were considered, all with a sulfate headgroup. The first surfactant was the single-tailed, widely studied sodium dodecyl sulfate (SDS), for which previous simulation results are available on several substrates. The results obtained for this surfactant were used for benchmarking the behavior of two dual-tailed surfactants. These latter surfactants have equal structure, except that in one case both linear tails are composed by seven fully protonated carbon atoms [CH3(CH2)6CHOSO3(CH2)6CH3(-), 2H7], whereas in the other, one tail is composed by seven fully protonated carbon atoms and the other tail is composed by seven fully fluorinated carbon atoms [CF3(CF2)6CHOSO3(CH2)6CH3(-), H7F7]. Our results suggest that preferential interactions lead to surfactant aggregates for H7F7 that differ compared to both those obtained for SDS and 2H7. Although molecular-level geometric structural differences can be invoked to explain differences between H7F7 and SDS aggregates, those between H7F7 and 2H7 aggregates can only be ascribed to atomic-scale interactions. Because as the adsorbed amount of surfactant increases, the self-assembled surfactant aggregates change, suggesting that the substrate on which adsorption occurs effectively evolves as adsorption progresses, compared to bare alumina. The morphological differences observed in our simulations coupled with molecular-level microphase separation might explain, in part, the unusual retrograde adsorption isotherm that has been observed experimentally for H7F7 surfactants on alumina.

Arterial deformation with renal artery aneurysm as a basis for secondary hypertension.

The goal of this work is to better understand the association between renal artery aneurysms and secondary hypertension through modeling the blood flow and the mechanics of diseased arteries. A large number of patients with renal artery aneurysms exhibit hypertension. Following surgical intervention, some patients experience improvement in their hypertension while others do not. This indicates that for some patients, the aneurysm is directly related to their hypertension, possibly through a hemodynamic effect on hormonal blood pressure control. In previous work, we proposed a possible mechanism for this--that high pressure inside an aneurysm may cause the arterial wall to deform and constrict the artery, creating a "pseudostenosis". Here, we use fluid structure interaction simulations to investigate the deformation of the renal artery in the presence of an aneurysm. Aneurysms on the superior surface and at the main bifurcation of the artery have been modeled, and the effect of changes in mechanical properties and geometry were investigated. For some cases, it was found that an aneurysm could cause flow distortions that led to higher than normal pressure losses without significant vessel wall deformation. For other cases, conditions were determined that induced movement of the arterial wall with constriction of the underlying artery. The maximum occlusion observed was 54% for a symmetric aneurysm at the main bifurcation with a severely weakened wall with a Young's modulus of 1 × 10(3) Pa.

Biphasic release of protein from polyethylene glycol and polyethylene glycol/modified dextran microspheres.

Dextrans show great promise for delivery of therapeutic agents. Dextran acetates (DAs) were synthesized with increasing degrees of substitution (DA1 < DA2 < DA3) by the reaction of the polysaccharide dextran (70 kDa) with acetic anhydride. A series of polyethylene glycol (PEG)/DA microspheres were prepared and tested with bovine serum albumin (BSA) functioning as a model protein. Particle size (0.74-0.85 µm) and encapsulation efficiency (56-70%) increased with the degree of substitution along with a slower release rate of protein from PEG/DA microspheres. Time to release 90% of protein rose from 31 to 118 min. Percentage of BSA released from PEG and PEG/DA3 microspheres with time (min) was modeled mathematically [Y(PEG) = 100(1 - e(-0.12t)); Y(PEG/DA3) = 100(1 - e(-0.024t))] to predict cumulative delivery from mixtures in vitro over a period of hours when constrained to a target level at 30 min. The system is examined for potential application in thrombolytic therapy.