Syed F. Mohammad

An In Vitro Test Model to Study the Performance and Thrombogenecity of Cardiovascular Devices

During the development of cardiovascular devices, it is necessary to evaluate their performance in vitro under experimental conditions that closely resemble their use in vivo. Therefore, an in vitro test model was developed to evaluate the performance of cardiovascular devices with blood. The in vitro test model consists of a pneumatically driven 50 cc polyurethane ventricle with either mechanical or experimental polyurethane tricuspid semilunar valves, connected to a horseshoe-shaped blood reservoir. A sampling port permits frequent removal of blood for hematologic evaluation. All the couplings are of the quick connect type to facilitate assembly and exchange of parts. The total volume of blood in the test model is approximately 1.4 L, and experiments are conducted in the absence of an air-blood interface. Except for mechanical valves and the chosen test interface, the entire blood contacting surface consists of polyurethane. Fresh bovine heparinized blood is used for the experiments. After filling the reservoir and the ventricle with blood, the concentration of heparin is adjusted with protamine to an APTT level of three times normal (80-100 sec). Once this level of anticoagulation is achieved, the blood is circulated for the next 2-3 hr at the desired test parameters (beats/min, cardiac output, etc.). A number of hematologic parameters are monitored during the test, including APTT, PT, plasma free hemoglobin and ADP-induced platelet aggregation. With the help of this model a number of devices, including experimental polyurethane tricuspid semilunar valves, Silastic tube valves, valves with heparin coated leaflets, and small (50 cc) experimental ventricles have been studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Magnetic field enhancement of antibiotic activity in biofilm forming Pseudomonas aeruginosa

Device related infection initiated by biofilm bacteria are often difficult to resolve with antimicrobial therapy. Study results indicate that application of static magnetic fields may enhance the activity of gentamicin against biofilm forming Pseudomonas aeruginosa adherent to a polymer substrate. Results indicate a maximal reduction of 86.5 +/- 7.2% (n = 6) in the number of adherent viable bacteria compared with a control for samples exposed to a 5 gauss (G) magnetic field and gentamicin. The effect appears to be limited to magnetic fields between 5 and 20 G. Experiments using glass, Chronoflex (Polymedica, Golden, CO), Biomer (Ethicon, Somerville, NJ), and polystyrene substrate showed that the effect was independent of substrate surface. Autoradiograms from In111 uptake experiments showed that bacteria colonizing the substrate surface were significantly reduced in samples subjected to a magnetic field and gentamicin.

In vitro Pharmacological Inhibition of Human Vascular Smooth Muscle Cell Proliferation for the Prevention of Hemodialysis Vascular Access Stenosis

Background: Vascular access for chronic hemodialysis often fails as a result of stenosis caused primarily by the proliferation of vascular smooth muscle cells (VSMC). Various drugs have been shown to inhibit the proliferation of VSMC under different conditions. Methods: In this study, we compared the inhibitory effect of ten drugs on the proliferation of human aortic smooth muscle cells (SMC) in culture. Quiescent cells were cultured in the presence of growth factors, fetal bovine serum and incremental concentrations of the test drug. Cell proliferation was assessed by the MTT reduction assay. Results: Aspirin, enalaprilat, heparin, hydroxyurea, indomethacin and tirofiban were ineffective. While dipyridamole, paclitaxel, tranilast and verapamil inhibited cell proliferation, the concentrations required were significantly higher than the clinical plasma levels achieved after systemic administration. Conclusion: Local delivery of these drugs to the target site may therefore be a more effective and appropriate strategy for the prevention of hemodialysis vascular access stenosis.

Heparin binding on poly(L-lysine) immobilized surface

Abstract A poly(ethylene-vinyl alcohol) (PEVAL) copolymer surface with immobilized poly( L -lysine) · HBr (PLL · HBr) has been used as a model surface to study the interaction of heparin with polycationic surfaces. The amount of heparin bound from PBS was 0.52 ± 0.19 μg/cm 2 on a smooth PLL immobilized PEVAL surface and 1.69 ± 0.26 μg/cm 2 on a porous PLL-PEVAL surface. Heparin adsorption kinetic studies indicated that heparin adsorption from plasma or blood exhibited a “two step” profile, which may be related to the effects of competitive binding between heparin and proteins, membrane porosity, and solution viscosity. The time needed to reach heparin binding saturation was 10 min in PBS and 30 min in plasma or blood at flow rate of 100 ml/min. However, under similar experimental conditions, heparin binding in PBS did not reach saturation for 2 h at flow rate of 3 ml/min. The difference in time required to reach saturation for two different flow rates (3 and 100 ml/min) was attributed to the heparin concentration gradient between bulk and surface. Bound heparin was eluted with a basic solution. The recovery of heparin bound from PBS, plasma, and blood was 85 ± 3%, which implied that most of the heparin was tightly bound to protonated amino groups on the side chain of PLL. The data suggest that electrostatic interactions between heparin and PLL may be the driving force for heparin binding. This study offers information for understanding heparin binding onto polycationic surfaces, especially in biological systems.

Enhanced risk of infection with device-associated thrombi.

Thrombosis and infection are two well-recognized risks with prosthetic devices that contact blood. Many of the currently used biomaterials may present an attractive surface for thrombus development as well as bacterial adhesion and colonization. Clinical experience with vegetative endocarditis patients has suggested that thrombosis may lead to enhanced risk of infection, and the possibility that adherent bacteria may enhance the risk of thrombosis has been noted by several investigators. To investigate the correlation between thrombosis and infection, a series of tests were conducted to assess the affinity of pathogen with surfaces in the absence and presence of blood components. Coronary stents were used as a model device to attract thrombi in a recirculating loop in vitro. Fresh heparinized blood was used to investigate thrombus development and bacterial interaction. 111Indium-labeled Staphylococcus epidermidis and 111Indium-labeled platelets were utilized to quantify bacterial interaction with thrombi under various test conditions. Anticoagulants, antiplatelet agents, and antibiotics were utilized in attempts to selectively influence bacteria, platelets, or thrombosis. The results suggest that under appropriate conditions, bacteria may preferentially adhere to platelet rich thrombus. These observations also suggest that by reducing the risk of thrombosis, the risk of device-associated infection may also be reduced.

Prostacyclin (PGI2) inhibits the enhancement of granulocyte adhesion to Cuprophane induced by immunoglobulin G

Abstract Several purified plasma proteins have been found to influence leukocyte adhesion to artificial surfaces such as Cuprophane. This study demonstrates that albumin, fibrinogen, and hemoglobin inhibit leukocyte adhesion to Cuprophane while IgG, IgM, and thrombin enhance this reaction. The inhibitory effect of fibrinogen and enhancing effect of IgG on leukocyte adhesion to Cuprophane are likely due to modification of the Cuprophane surface by adsorbed proteins. Prostacylin (PGI2) was found to inhibit leukocyte adhesion to Cuprophane promoted by IgG. The inhibition of adhesion produced by PGI2 is concentration dependent and saturable. Other prostaglandins such as PGE1, 6-keto-PGF1 α, PGA2, and PGB2 at high concentration (100 μM) did not exert significant inhibiton of the same reaction.

Thromboembolization associated with sudden increases in flow in a coronary stent ex vivo shunt model.

To observe the dynamics of thromboembolism (TE) in an animal model, a carotid-carotid arterial ex vivo shunt was developed. A coronary stent deployed in a 3.5 mm polyvinylchloride (PVC) tubing segment was used as a model device in the shunt. A light-scattering microemboli detector monitored the embolic content of the blood flowing through the shunt at 50-150 ml/min as determined by a clamp-on ultrasound flow probe. The stent was found to actively develop thrombi and release emboli for 1-3 hours when the activated clotting time (ACT) was maintained between 125 and 150 seconds. The shunt flow rate fluctuated considerably (from 50 to 150 ml/min) depending on the animals activity. When the time profile of this fluctuating flow rate was super imposed on the time profile of embolization, it was noted that sudden increases in flow rate were associated with incidents of embolization. Statistically, sudden flow rate increases of 100% or more were accompanied by embolic events 95% of the time (p < 0.01). Based on the results of this study, it was postulated that the increased embolization may be due to the fluid forces associated with accelerating flow. To explore this postulate, in vitro studies were conducted to compare the effects of pulsatile flow with steady flow on stent induced TE. Results of this study suggested a significant increase (100%) in both stent thrombosis and embolism during pulsatile flow compared with steady flow.

Device induced thromboembolism in a bovine in vitro coronary stent model.

The potential of a new bovine in vitro model to evaluate various aspects of device induced thromboembolism was studied using two test modes. First, the effect of an antithrombotic drug on stent induced thromboembolism was assessed. The antithrombotic potential of an antiplatelet agent was compared with that of the other conventional antithrombotic agents (aspirin, dipyridamole) used in the past with this in vitro model. Stent associated thrombus was assessed gravimetrically at the end of the experiment. Emboli were assessed continuously using a light scattering microemboli detection system. Second, the sensitivity of the model to flow induced thromboembolism was studied using a combination of surface roughness and stenosis. Thrombus was assessed visually, and emboli were assessed as described earlier. The results show that 1) this in vitro model is sensitive to the action of antithrombotic drugs, and to the effect of hemodynamics on thromboembolism; 2) the antiplatelet drug used in this study was effective in attenuating thromboembolism; 3) a stenosis in combination with roughness produced more emboli than roughness alone; and 4) the model was useful for the study of physical and biochemical aspects of thromboembolism.

A comparison of the antiplatelet effect of aspirin on human and bovine platelets

The risk of thromboembolism in human patients or animal models with blood contacting prosthetic devices is well documented. Aspirin is used frequently as an antiplatelet agent to minimize this risk. Although the inhibitory effect of aspirin on human platelets has been clearly established, preliminary studies to examine this effect on bovine platelets revealed a minimal inhibition of platelet function in vivo as well as in vitro. Because a considerable amount of implant research is carried out in bovine models, it was considered important to evaluate carefully the antiplatelet effect of aspirin on bovine platelets. To evaluate the effect of aspirin, experiments were conducted on human and bovine blood in vitro as well as after the administration of aspirin up to 4,000 mg/day (p.o.) in calves and 2,500 mg/day in humans. Appropriate amounts of buffered aspirin were added to heparinized or citrated blood incubated for 25 min and centrifuged to obtain platelet rich plasma (PRP). An aliquot of PRP was then challenged by a predetermined concentration of ADP (an aggregating agent) or collagen to evaluate platelet aggregation and release reaction. After aspirin was administered in vivo, blood was drawn from the animal at predetermined intervals to evaluate platelet function. In human blood, the inhibitory effect of aspirin was discernable at 80 mg/L in vitro and 2,500 mg/day when ingested orally. Under identical experimental conditions, no inhibition of bovine platelet aggregation was observed using dosages of aspirin up to 1,000 mg/L in vitro or 4,000 mg/day in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of plasma proteins in formation of obstructive protamine complexes

Formation of complexes between heparin and protamine (in saline), or heparin, plasma proteins, and protamine (in plasma) was assessed by measurements of light transmission through different test solutions. To examine the formation of these complexes, 125I-labeled protamine was used. Addition of 125I-protamine to plasma or blood resulted in the sedimentation of 125I-protamine in the form of insoluble complexes. This complex formation was not affected by the presence of heparin, suggesting that protamine-plasma protein interaction may be primarily responsible for precipitation of 125I-protamine. To assess the capability of these complexes to obstruct the pulmonary circulation, an in vitro experimental model was developed. Citrated serum, plasma, blood, or saline were allowed to flow through a glass bead column with the help of a peristaltic pump. A pressure transducer positioned before the column allowed pressure measurements at a constant flow rate during the experiment. Mixing of protamine with plasma or blood prior to their passage through the glass bead column resulted in a significant increase in pressure suggesting that the column was being clogged with insoluble complexes. The increase in pressure occurred both in the presence and absence of heparin in plasma or blood. Under identical experimental conditions, the increase in pressure was insignificant when protamine was added to saline or serum regardless of whether heparin was present or absent. This was further confirmed by the use of 125I-protamine. These observations suggest that protamine forms insoluble complexes with certain plasma proteins. Based on these observations, it is hypothesized that following intravenous administration, protamine immediately forms complexes in circulating blood.(ABSTRACT TRUNCATED AT 250 WORDS)