Shingo Yamane

Protein adsorption onto ceramic surfaces

Ceramics seldom have been used as blood-contacting materials. However, alumina ceramic (Al2O3) and polyethylene are incorporated into the pivot bearings of the Gyro centrifugal blood pump. This material combination was chosen based on the high durability of the materials. Due to the stagnant flow that often occurs in a continuous flow condition inside a centrifugal pump, pivot bearing system is extremely critical. To evaluate the thombogenicity of pivot bearings in the Gyro pump, this study sought to investigate protein adsorption, particularly albumin, IgG, fibrinogen, and fibronectin onto ceramic surfaces. Al2O3 and silicon carbide ceramic (SiC) were compared with polyethylene (PE) and polyvinylchloride (PVC). Bicinchoninic acid (BCA) protein assay revealed that the amount of adsorbed proteins onto Al2O3 and SiC was significantly less than that on PVC. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that numerous proteins adsorbed onto PVC compared to PE, Al2O3, and SiC. Identification of adsorbed proteins by Western immunoblotting revealed that the adsorption of albumin was similar on all four materials tested. Western immunoblotting also indicated lesser amounts of IgG, fibrinogen, and fibronectin on Al2O3 and SiC than on PE and PVC. In conclusion, ceramics (Al2O3 and SiC) are expected to be thromboresistant from the viewpoint of protein adsorption.

The effects of heparin coating of oxygenator fibers on platelet adhesion and protein adsorption.

UNLABELLED Platelet adhesion on the cardiopulmonary bypass oxygenator membrane is associated with impaired hemostasis. We investigated the effects of heparin coating of the oxygenator membrane on protein adsorption and platelet adhesion on the surface. Noncoated and heparin-coated polypropylene membranes were incubated in whole blood with small- (1 U/mL) or large-dose (5 U/mL) heparin as an anticoagulant for 3 h at 37 degrees C. The amount of platelets adhering on each fiber was assessed by using enzyme immunoassays using monoclonal antibodies directed against CD42b (GP Ib) and CD61 (GP IIb/IIIa). Platelet activation was assessed by measuring plasma guanosine monophosphate 140 levels. The amount and composition of the adsorbed proteins on the surface were analyzed by using a bicinchoninic acid protein assay and by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting technique. The heparin coating of the fibers significantly reduced platelet adhesion on the surface. However, platelet activation was reduced by heparin coating only with small-dose heparinization. The adsorption of platelet adhesive proteins such as fibrinogen and von Willebrand factor was not altered, whereas that of fibronectin was increased by heparin coating. We conclude that heparin coating of the oxygenator fibers can decrease platelet adhesion without affecting adsorption of major adhesive proteins. Surface heparin coating is associated with an increased fibronectin adsorption on the fibers. IMPLICATIONS Heparin coating can reduce platelet adhesion and activation in the presence of small-dose heparinization, potentially reducing the inflammatory response and activation of thrombosis and fibrinolysis.

LONG-TERM IN VIVO LEFT VENTRICULAR ASSIST DEVICE STUDY WITH A TITANIUM CENTRIFUGAL PUMP

A totally implantable centrifugal artificial heart has been developed. The plastic prototype, Gyro PI 601, passed 2 day hemodynamic tests as a functional total artificial heart, 2 week screening tests for antithrombogenicity, and 1 month system feasibility. Based on these results, a metallic prototype, Gyro PI 702, was subjected to in vivo left ventricular assist device (LVAD) studies. The pump system employed the Gyro PI 702, which has the same inner dimensions and the same characteristics as the Gyro PI 601, including an eccentric inlet port, a double pivot bearing system, and a magnet coupling system. The PI 702 is driven with the Vienna DC brushless motor actuator. For the in vivo LVAD study, the pump actuator package was implanted in the preperitoneal space in two calves, from the left ventricular apex to the descending aorta. Case 1 achieved greater than 9 month survival without any complications, at an average flow rate of 6.6 L/min with 10.2 W input power. Case 2 was killed early due to the excessive growth of the calf, which caused functional obstruction of the inlet port. There was no blood clot inside the pump. During these periods, neither case exhibited any physiologic abnormalities. The PI 702 pump gives excellent results as a long-term implantable LVAD.

Development of a silicone hollow fiber membrane oxygenator for ECMO application

A new silicone hollow fiber membrane oxygenator for extracorporeal membrane oxygenation (ECMO) was developed using an ultrathin silicone hollow fiber, with a 300 microm outer diameter and a wall thickness of 50 microm. The hollow fibers were mechanically cross-wound on the flow distributor to achieve equal distribution of blood flow without changing the fiber shape. The housing, made of silicone coated acryl, was 236 mm long with an inner diameter of 60 mm. The surface area was 1.0 m2 for prototype 211, and 1.1 m2 for prototype 209. The silicone fiber length was 150 mm, and the silicone membrane packing density was 43% for prototype 211 and 36% for prototype 209. Prototype 211 has a priming volume of 208 ml, and prototype 209 has a priming volume of 228 ml. The prototype 211 oxygenator demonstrates a gas transfer rate of 120 +/- 5 ml/min (mean +/- SD) for O2 and 67 +/- 12 ml/min for CO2 under 2 L of blood flow and 4 L of O2 gas flow. Prototype 209 produced the same values. The blood side pressure drop was low compared with the silicone sheet oxygenator (Avecor, 1500ECMO). These results showed that this new oxygenator for ECMO had efficiency similar to the silicone sheet oxygenator that has a 50% larger surface area. These results suggest that the new generation oxygenator using an ultrathin silicone hollow fiber possesses sufficient gas transfer performance for long-term extracorporeal lung support.

Protein adsorption and platelet adhesion on the surface of an oxygenator membrane.

Platelet adhesion on an oxygenator membrane is associated with thrombocytopenia or thrombus formation during extra-corporeal circulation. The authors evaluated protein adsorption and platelet adhesion on three oxygenator hollow fiber membranes fabricated with polypropylene, silicone, and double layer polyolefin. Adsorbed proteins were analyzed by bicinchoninic acid protein assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and Western blot. Platelet adhesion was assessed with enzyme immunoassays using monoclonal antibodies directed against CD42b and CD61. After 3 hr of incubation at 37°C in whole blood, the amount of adsorbed protein was the least on silicone and increased from silicone < double polyolefin < polypropylene. The adsorbed protein pattern was similar; however, silicone showed less adsorption for all protein bands, and the γ; chain of fibrinogen was not detected. In contrast, double polyolefin showed the highest fibrinogen adsorption. The optical density at a wavelength of 450 nm for CD42b was 1.47 ± 0.35 in polypropylene, 1.16 ± 0.38 in silicone, and 1.85 ± 0.19 in double polyolefin (p < 0.01 vs silicone) and for CD61 0.98 ± 0.39 in polypropylene, 0.91 ± 0.22 in silicone, and 1.69 ± 0.25 in double polyolefin (p < 0.01 vs silicone and polypropylene). These data suggest that silicone is advantageous for long term extracorporeal respiratory support in terms of less platelet adhesion and no plasma leakage through the pores.

Platelet adhesion to heparin coated oxygenator fibers under in vitro static conditions: impact of temperature.

Heparin coating of cardiopulmonary bypass (CPB) circuitry may attenuate the platelet consumption associated with CPB. We investigated the effect of temperature on the interaction between platelet and heparin coated surfaces under in vitro static conditions. Heparin coated and non coated oxygenator fibers were incubated with heparinized whole blood at 37°C and 22°C. The incubation time was set at 30, 60, 180, and 300 minutes. The number of platelets adhering to each fiber was assessed with enzyme immunoassay using monoclonal antibody against platelet receptor protein CD 61(GPIIbIIIa). As an index of platelet activation, plasma soluble(s) P-selectin levels were measured by enzyme-linked immunosorbent assay. Under normothermia, the number of adherent platelets on the non coated surface increased significantly after 300 min of incubation. Platelet adhesion was reduced significantly by heparin coating of the surface and was kept constant after 300 min. Under hypothermia, heparin coating was also associated with significant reduction of platelet adhesion. The levels of sP-selectin did not correlate with the extent of platelet adhesion. Our results suggest that heparin coating is effective in decreasing platelet adhesion to the synthetic surface tested regardless of the temperature under static conditions. Inhibition of platelet activation on the heparin coated surface may be masked by standard dose heparinization.

The Effect of Apheresis on Adhesion Molecules

Abstract:  The adhesion molecules on the leukocytes and the endothelial cells mediate interaction between their cells. The plasma levels of soluble adhesion molecules increase in patients with ischemic heart disease, atherosclerotic aortic disease. Hypercholesterolemia is one of risk factors for atherosclerosis, and it is considered that the expression of adhesion molecules in endothelial cells is related to the development of atherosclerosis. Low‐density lipoprotein (LDL) apheresis has been applied to patients with hypercholesterolemia. LDL apheresis may have an effect on adhesion molecules in patients with hypercholesterolemia.

Newly Developed Hollow Fiber Membrane for Extracorporeal Membrane Oxgenator (ECMO)

Membrane oxygenators consisting of microporous polypropylene membranes are routinely used in open heart surgeries and they provide a high performance of 0 2 and CO2 transfer. However, during long term perfusion cases including ECMO and PCPS procedures the microporous membrane may have plasma leakage through the membrane pores due to the protein adsorption on the membrane. This plasma leakage drastically . diminishes the gas transfer. Presently the only available membrane that is adequate for long term perfusion in the US is a silicone oxygenator consisting of flat silicone sheet membrane that was developed over 30 years ago and manufactured by Avecor Inc., MN. .

Cytokine production and protein adsorption in a stainless steel filter used for leukocyte reduction.

A new metallic filter made from a stainless steel fiber has been under development. To evaluate biocompatibility of this filter, the authors compared cytokine production with that of stainless steel fibers and polyester fibers by using a mononuclear cell culture. Furthermore, adsorbed proteins on each fiber were identified by using sodium-dodecyl sulfate (SDS)-polyacrlyamide gel electrophoresis (PAGE). The levels of tumor necrosis factor (TNF)-alpha in the cultured supernatant without fibers as the control, with polyester fibers, and with stainless steel fibers were 28.1 +/- 8.1, 39.3 +/- 2.6, and 29.1 +/- 6.7 pg/ml, respectively. The levels of interleukin (IL)-1 beta were 7.6 +/- 3.2, 8.9 +/- 1.5, and 8.9 +/- 2.1 pg/ml, respectively. The IL-4 levels were less than 0.25 pg/ml, and the interferon-tau levels were less than 7.8 pg/ml in all three conditions. The amount of adsorbed proteins was 3.39 +/- 0.27 microgram/cm2 for the polyester fibers and 2.72 +/- 0.23 microgram/ cm2 for the stainless steel fibers. The protein bands adsorbed to the polyester fibers by SDS-PAGE analysis were observed at approximately 180, 120, 90, 76, 67, 59, 56, and 28 kd molecular weight. In contrast, the protein bands adsorbed to the stainless steel fibers were observed at 90, 76, 67, 62, 56, 28, and 12 kd molecular weight. Thus, the proteins adsorbed to the stainless steel fibers differed from those on the polyester fibers. By western blot analysis, the amounts of albumin, IgG tau chain, and fibronectin adsorbed on the stainless steel fibers were smaller than those on the polyester fibers. The results of this study suggest that the stainless steel fibers do not stimulate monocytes, Th1, and Th2 cells. In addition, lesser adsorption of IgG tau chain and fibronectin may indicate that the stainless steel is a superior material for anti thrombogenicity compared to polyester.