Attenuation of hypothermia-induced platelet activation and platelet adhesion to artificial surfaces in vitro by modification of hemoglobin to carry S-nitric oxide and polyethylene glycol.

Hypothermic cardiopulmonary bypass alters platelet function and hypothermia is associated with postoperative myocardial ischemia. Thrombogenic surfaces such as extracorporeal circuits, vascular graft materials, and components of atherosclerotic plaque induce activation of platelets. The effects of human hemoglobin (Hb) covalently modified to carry S-nitric oxide (NO) functional groups (SNO-Hb), polyethylene glycol (PEG-Hb), and SNO-PEG-Hb on platelet activation were studied. Platelet activation was assessed by cytometric analysis of GPIIb-IIIa activation and P-selectin expression at hypothermic condition (22 degrees C) after stimulation with Hb derivatives. Platelet adhesion and aggregation were measured in a parallel glass plate chamber coated with unmodified Hb, SNO-Hb, PEG-Hb, SNO-PEG-Hb, and collagen. Platelet binding of antibodies to GPIIb-IIIa and P-selectin was significantly enhanced by hypothermic condition and by unmodified Hb. There was significantly less platelet binding of antibodies to GPIIb-IIIa and P-selectin with SNO-Hb, PEG-Hb, and SNO-PEG-Hb compared with unmodified Hb. There was significantly less platelet attachment, adhesion, and aggregation on the SNO-Hb, PEG-Hb and SNO-PEG-Hb coated surfaces compared with unmodified Hb-coated and -uncoated surfaces. SNO-Hb, PEG-Hb, and SNO-PEG-Hb induced less platelet activation at hypothermic temperature, and induced less platelet adhesion and aggregation on thrombogenic surfaces compared with unmodified Hb. The inhibitory effect may be derived from antiadhesive properties of Hb, antiplatelet actions of NO, and molecular barrier action of PEG.

Development of a membrane oxygenator for ECMO using a novel fine silicone hollow fiber.

One of the limitations of conventional silicone hollow fiber oxygenators compared with microporous membrane oxygenators is poor gas permeability. However, the silicone hollow fiber is free from plasma leakage, which is the major life limiting factor of the microporous membrane oxygenator. It has been difficult to fabricate a fine, thin hollow fiber for reduction of resistance to gas permeability because of the poor mechanical strength of conventional silicone materials. The authors developed a novel silicone material with sufficient mechanical strength, and a fine silicone hollow fiber with a diameter of 30 microns and wall thickness of 50 microns, which is approximately half that of a conventional silicone hollow fiber. Using this newly developed silicone hollow fiber, the authors developed a compact extracapillary flow membrane oxygenator. The oxygenator consists of fine silicone hollow fibers inserted in a housing made of polycarbonate. The housing is a cylindrical case, 20 cm long and 55 mm in inside diameter. The hollow fibers are cross-wound. The surface area of the membrane is 2.0 m2, and priming volume is 230 ml. Gas transfer performance of the newly developed oxygenator was evaluated by in vitro experiments. Oxygen and carbon dioxide transfer rates were 195 ml/min and 165 ml/min, at a blood flow rate 3 L/min. The novel silicone membrane oxygenator developed in this study can be used for extended duration in such applications as extracorporeal membrane oxygenation.

Preparation and characterization of SNO-PEG-hemoglobin as a candidate for oxygen transporting material.

Acellular hemoglobin (Hb) derivates developed as oxygen carriers are known to cause hypertensive reactions due to their nitric oxide (NO) scavenging action. To modulate this undesired activity, we have developed a new Hb derivative, s-nitrosylated polyethylene glycol (PEG)-modified hemoglobin (SNO-PEG-Hb), which can deliver oxygen and NO. After human Hb was modified with PEG to increase its molecular weight, the free sulfhydryl groups of Hb were s-nitrosylated with s-nitrosoglutathione. Administration of unmodified Hb into anesthetized rats caused a hypertensive reaction, while s-nitrosylated Hb derivatives such as SNO-Hb and SNO-PEG-Hb did not raise blood pressure. The plasma half-lives of heme and NO bound to SNO-PEG-Hb were 11.5 and 2.4 hours respectively, indicating that the s-nitrosylated Hb derivative may act as a slow-releasing agent for NO. Based on these findings, SNO-PEG-Hb is a useful candidate for a blood substitute and tool for oxygen therapeutics.

Development of the needle insertion robot for percutaneous vertebroplasty.

Percutaneous Vertebroplasty (PVP) is an effective and less invasive medical treatment for vertebral osteoporotic compression fractures. However, this operative procedure is quite difficult because an arcus vertebra, which is narrow, is needled with accuracy, and an operator's hand is exposed to X-ray continuously. We have developed a needle insertion robot for Percutaneous Vertebroplasty. Its experimental evaluation on the basic performance of the system and needle insertion accuracy are presented. A needle insertion robot is developed for PVP. This robot can puncture with accuracy and an operator does not need to be exposed to X-ray. The mechanism of the robot is compact in size (350 mm x D 400 mm x H270 mm, weight: 15 kg) so that the robot system can be inserted in the space between C-arm and the patient on the operating table. The robot system is controlled by the surgical navigation system where the appropriate needle trajectory is planned based on pre-operative three-dimensional CT images. The needle holding part of the robot is X-ray lucent so that the needle insertion process can be monitored by fluoroscopy. The position of the needle during insertion process can be continuously monitored. In vitro evaluation of the system showed that average position and orientation errors were less than 1.0 mm and 1.0 degree respectively. Experimental results showed that the safety mechanism called mechanical fuse released the needle holding disk properly when excessive force was applied to the needle. These experimental results demonstrated that the developed system has the satisfactory basic performance as needle insertion robot for PVP.

Studies of the development of optical fiber sensors for biochemical analysis.

An optical fiber sensor utilizing Thymol blue and an ion-exchange resin complex in a cellulose acetate membrane was developed. By monitoring several different chromophores of Thymol blue, the sensor could measure the pH of the solution from 1.0 to 12.0 with good reproducibility. An optical fiber glucose sensor utilizing a cellulose acetate membrane containing glucose oxidase, 2,7-diaminofluorene dihydrochloride, and sodium N-(3-sulfopropyl)-3,3',5,5'-tetramethylbenzidine was developed. Reflectance changes at 580 nm were large enough to trace changes in glucose concentration in physiological saline solution.

Experimental study on hemolysis in centrifugal blood pumps: improvement of flow visualization method.

To evaluate rotary blood pumps, flow visualization is commonly applied to determine the flow patterns in a centrifugal blood pump, which have a relationship to its hemolytic performance. However, it is very troublesome to visualize the flow near the vanes due to the high rotational speed of the impeller. The rotational speed of the impeller in a centrifugal blood pump is usually several hundred revolutions per minute. In this study, we combined a high-speed video camera based imaging method and an optical system in which the image of the rotating impeller was kept stationary. In the optical system, a prism rotating at half the speed of the impeller reflected the image of the impeller. The resultant reflected image was observed by a high-speed video camera through a half mirror. With this optical setup, the image through the half mirror became stationary, and the path of a specific tracer particle could be traced for a longer duration. A longer duration of measurements and better quality of the obtained images were realized through this improvement. Movement of a specific tracer from the inlet portion to the outlet portion of the impeller could be examined using the developed method.

A biolized, compact, low noise, high performance implantable electromechanical ventricular assist system.

An implantable electromechanical ventricular assist system (VAS) intended for permanent human use was developed. It consisted of a conically shaped pumping chamber, a polyolefin (Hexsyn) rubber diaphragm attached to a pusher-plate, and a compact actuator with a direct current brushless motor and a planetary rollerscrew. The outer diameter was 97 mm, and the total thickness was 70 mm. This design was chosen to give a stroke volume of 63 ml. The device weighs 620 g, with a total volume of 360 ml. The pump can provide 8 L/min flow against 120 mmHg afterload with a preload of 10 mmHg. The inner surface of the device, including the pumping chamber and diaphragm, was made biocompatible with a dry gelatin coating. To date, two subacute (2 and 6 day) calf studies have been conducted. The pump showed reasonable anatomic fit inside the left thorax, and the entire system functioned satisfactorily in both the fill-empty mode using the Hall effect sensor signals and the conventional fixed rate mode. There were no thromboembolic complications despite no anticoagulation therapy. The system now is being endurance tested greater than 10 weeks (9 million cycles). This VAS is compact, low noise, easy to control, and has excellent biocompatibility.

Permeability characteristics of hemoglobin derivatives across cultured endothelial cell monolayers.

To better understand the vascular activity of hemoglobin-based (Hb-based) oxygen carriers, the endothelial permeability characteristics of Hb derivatives having various molecular masses were defined by using monolayers of bovine endothelial cells cultured on microporous membranes. The endothelial permeability of unmodified bovine Hb was almost twice that of bovine serum albumin. Intramolecularly cross-linked human Hb showed slightly but significantly reduced permeability as compared with unmodified bovine Hb. Polyethyleneglycol modification or haptoglobin binding to Hb further reduced the permeability. These properties were intensified in conditions in which the endothelial barrier function was reduced by pretreatment with either interleukin-6 (100 ng/mL, 21 hours) or lipopolysaccharide (1 microg/mL, 10 hours). In contrast, there was little permeability of liposome-encapsulated Hb, and it was almost unaffected by the pretreatments. These data provide the first information that Hb derivatives with smaller molecular masses show larger transendothelial flux. Because Hb is a potent scavenger of endothelium-derived relaxing factor (EDRF), our observations support the idea that smaller Hb-based acellular oxygen carriers are potent vasoconstrictors as a result of abluminal EDRF scavenging.

A compact centrifugal pump for cardiopulmonary bypass.

A majority of the cardiopulmonary bypass (CPB) systems still utilize bulky roller pumps. A direct-drive small centrifugal pump intended for second-generation CPB pump has been developed. The pump has a 50 mm diameter impeller and provides a 6 L/min flow at 3,000 rpm against 300 mm Hg. A flexible drive shaft allows us to separate the pump head from the console resulting in easier manipulation. An in vitro study showed that the pump generated less hemolysis (index of hemolysis = 0.0011, comparable to the value for Bio-medicus BP-80). To improve blood flow around the shaft-seal region and to reduce thrombus formation around the shaft, six holes were drilled through the impeller. In biventricular bypass experiments using calves, our pump demonstrated excellent antithrombogenicity and durability for 48 h. And the compact and atraumatic centrifugal pump system showed excellent performance and easy manipulation under actual CPB conditions in animal.