Hiroaki Harasaki

Biodegradable Pericardial Implants for Bladder Augmentation: A 2.5-Year Study in Dogs

Bladder augmentation using biodegradable pericardial tissue was evaluated in canine bladders. Acetic acid and acetic anhydride treated pericardial tissue grafts were stored in 75% ethanol for 18 to 27 months before implant. Ten dogs weighing 20 to 25 kg. were subjected to a 50% partial cystectomy. After careful separation of the mucosa, bladder muscle and adventitial layers a pericardial graft volumetrically equivalent to the portion of the bladder removed was sutured to the bladder remnant in 2 layers. In 1 control dog the bladder was opened, 50% of the bladder was removed and the bladder was closed primarily. In another control dog the excised bladder was replaced with fresh chemically treated patch material that was never subjected to ethanol storage. Excretory urography and cystography were performed on all dogs. Urodynamics with filling pressures and bladder volumes measured before and after the operation at intervals of up to 36 months confirmed that adequate bladder capacity was achieved. There were no operative complications. Postmortem histological evaluations revealed a smooth epithelialized inner surface with no traces of any surface irregularities or suture lines. The bladder apex showed an intact epithelium and the absence of a smooth muscle layer. The biodegradable acetylated tissue provides an intact structural reservoir for urine and serves as a template for epithelial regeneration. This permits volumetric bladder enlargement while the graft is progressively reabsorbed with time.

Cardiovascular implant calcification: a survey and update.

Calcification of cardiovascular prosthetic implants is a common and important problem. This review provides an update based upon the Conference on Cardiovascular Implant Calcification held as part of the 13th World Congress of the International Society for Heart Research, 1989. A variety of cardiovascular prostheses are affected clinically by calcification, including bioprosthetic heart valves, aortic homografts and trileaflet polymeric valve prostheses. In addition, experimental studies have demonstrated calcification of artificial heart devices in ventricular assist systems in long-term calf studies. The pathophysiology of this disease process is incompletely understood. A common element between the various types of cardiovascular implant calcification is the localization of calcific deposits to devitalized cells and membranous debris. Prevention of cardiovascular implant calcification by either biomaterial modifications or regional drug therapy (controlled release) is being investigated.

eNOS-overexpressing endothelial cells inhibit platelet aggregation and smooth muscle cell proliferation in vitro.

Endothelial cell seeding of synthetic small diameter vascular grafts (SSDVG) has been shown to diminish thrombosis and intimal hyperplasia, resulting in improved graft patency. However, endothelial cell retention on seeded grafts when exposed to physiological shearing conditions remains poor. We report that the genetic engineering of endothelial cells to overexpress endothelial nitric oxide synthase (eNOS), may create improved anti-thrombotic and anti-hyperplastic endothelial cell phenotypes for SSDVG seeding. eNOS-overexpressing endothelial cells may potentially overcome the biochemical loss due to shear induced reduction in endothelial cell coverage on SSDVG. Bovine aortic endothelial cells (BAEC) were transfected with the human eNOS gene, and co-incubated with either human whole blood or bovine aortic smooth muscle cells (BASMC) in vitro. eNOS-transfected BAEC significantly overexpressed eNOS compared to control beta-Gal-transfected and untransfected BAEC up to 120 h post transfection. In co-incubation and co-culture assays, human platelet aggregation decreased by 46% and BASMC proliferation decreased by 67.2% when compared to incubation with untransfected BAEC.

Biocompatibility of heparin-coated extracorporeal bypass circuits: A randomized, masked clinical trial

Cardiopulmonary bypass circuits cause morbidity during cardiac operations. Plasma proteins and cellular components are stimulated by contact with the cardiopulmonary bypass circuit and can cause bleeding and postperfusion syndrome. This is especially true in patients undergoing reoperative cardiac procedures, which carries a higher risk of postoperative bleeding and prolonged ventilation compared with primary cardiac surgical procedures. Recently, cardiopulmonary bypass circuit surfaces have been coated with antithrombotic agents to improve their biocompatibility. This study evaluated the effect of a heparin-coated cardiopulmonary bypass system (Duraflo II, Baxter Bentley Healthcare Systems, Irvine, Calif.) on thrombin formation, platelet stimulation, and leukocyte activation in patients undergoing reoperative coronary artery bypass grafting or valve operation. Fifty patients were selected and randomly assigned to a standard noncoated control system (n = 26) or the Duraflo heparin-coated system (n = 24). Similar heparin doses were used in both groups (3 mg/kg). The heparin-coated group used a completely heparin-coated bypass circuit including the cardiotomy reservoir; arterial filters were heparin-coated in both groups. Samples were obtained before cardiopulmonary bypass, 30 minutes into cardiopulmonary bypass, 5 minutes after crossclamp removal, and 5 minutes after protamine administration. Thrombin formation (thrombin-antithrombin III by enzyme-linked immunosorbent assays) and platelet activation (beta-thromboglobulin by enzyme-linked immunosorbent assays; P-selectin expression by flow cytometry) were assayed. Leukocyte activation was determined by quantitative and qualitative analysis of arterial filters by scanning electron microscopy in six patients from each group. In both circuits, thrombin values increased markedly 30 minutes into cardiopulmonary bypass compared with baseline values (p < 0.001) (heparin-coated, 7 +/- 5 to 96 +/- 115 ng/ml; noncoated, 10 +/- 9 to 115 +/- 125 ng/ml). Platelet activation as measured by beta-thromboglobulin (heparin-coated, 104 +/- 100 to 284 +/- 166 IU/ml; noncoated, 81 +/- 74 to 288 +/- 277 IU/ml) and P-selectin expression (heparin-coated, 1.5% +/- 1.5% to 6.4% +/- 6.1%; noncoated, 1.4% +/- 1.1% to 6.2% +/- 4.3%) also significantly increased 30 minutes into cardiopulmonary bypass compared with baseline values (p < 0.001). Platelet activation and thrombin generation did not differ between the two circuits at any time. Granulocyte activation and platelet deposition did not differ between the two circuits when arterial filters were evaluated. Both groups had similar heparin and protamine administration, blood transfusions, postoperative alveolar-arterial oxygen gradient, time to extubation, length of intensive care unit stay, and overall morbidity and mortality. Clinical outcome and blood loss did not differ between the groups. We conclude that heparin-coated cardiopulmonary bypass circuits did not improve biochemical or clinical markers of biocompatibility in a reoperative patient population.

Chronic nonpusatile blood flow: I. Cerebral autoregulation in chronic nonpulsatile biventricular bypass: Carotid blood flow response to hypercapnia

To investigate the response of the carotid blood flow and general circulation to hypercapnia in chronic nonpulsatile blood flow, we performed 18 carbon dioxide gas inhalation studies on three calves undergoing a centrifugal biventricular bypass with ventricular fibrillation. An ultrasonic flow probe was put on the carotid artery during biventricular bypass pump implantation, and pump flows were maintained at 90, 100, and 120 ml/kg per minute for 1 week each. The carbon dioxide inhalation studies were performed twice a week. Hypercapnia was induced by administering pure carbon dioxide gas through a nasal tube at flow rates of 0, 5, 7.5, 10, 12.5, and 15 L/min for 5 minutes each at three different nominal pump flow rates, and the resultant arterial blood gas and hemodynamic changes were recorded. No significant correlation existed between the carotid blood flow and mean aortic pressure, which varied from 70 to 140 mm Hg, but the carotid blood flow correlated significantly (p < 0.01) with the systemic pump flow rate. A significant (p < 0.01) linear relationship was found between the carotid blood flow and arterial carbon dioxide tension. For each 1 mm Hg change in arterial carbon dioxide tension, there was a 2.8 % change in the carotid blood flow. The percent changes in the carotid blood flow in response to arterial carbon dioxide tension were calculated as 2.9%, 3.7%, and 2.5% for each 1 mm Hg change in arterial carbon dioxide tension at pump flows of 90, 100 and 120 ml/kg per minute. No significant differences in the carotid blood flow response to hypercapnia were detected among the three systemic pump flow rates. These results thus suggested that chronic nonpulsatile blood flow had no detrimental effects on cerebral autoregulation.

Evaluation of platelet and coagulation function in different animal species using the xylum clot signature analyzer.

Platelet and coagulation function were evaluated in four different animal species with a newly developed clot signature analyzer (CSA). CSA is unique in that it simultaneously measures global platelet and coagulation function under flow using whole blood. No anticoagulant, chemical, or immunologic agent is required. Three CSA parameters are measurable: platelet mediated hemostasis time (PHT), collagen induced thrombus formation time (CITF), and clotting time (CT). Bovine, ovine, and canine species were chosen because these are the animal models most frequently used in in vivo testing of cardiovascular implants. These parameters, as well as data from whole blood platelet aggregometry (commonly used for platelet function studies because of the response to exogenous agonists), and platelet counts from these animals, were measured and compared with those in humans. In all three parameters, the canine species showed distinctively shorter time values than other species, including humans, suggesting that the dog is not an ideal animal model for the evaluation of blood-surface interactions. Ovine and human blood showed similar PHT and CT values, but CITF time values were significantly shorter in sheep than in humans. With bovine blood, PHT was most prolonged among the four species compared. CT and CITF times in calves were shorter than those in humans, although the difference in CITF time was not statistically significant. Adenosine diphosphate induced platelet aggregation showed the same order of responsiveness in four species as did CITF. It was noted that the intermeasurement variation was rather high for CSA parameters, especially in PHT, warranting caution when this parameter is used to study time-dependent changes after device implantation.

Effects of stent design and serum cholesterol level on the restenosis rate in atherosclerotic rabbits

We investigated the effect of serum cholesterol level and stent design on the restenosis rate within the stent after balloon angioplasty and stent implantation using atherosclerotic rabbits. Two types of nickel/titanium stents with gaps (open stent) and without gaps (closed stent) between the wire coils were implanted into the aorta of the rabbits 10 weeks after atherosclerosis had been induced using a standard high cholesterol diet and balloon abrasion. Each rabbit had an open stent and a closed stent implanted into the infrarenal abdominal aorta. Between these two stents a control segment of the aorta was treated with angioplasty alone. The animals were divided into two groups according to the diet protocol as follows: in group I (n = 9) a high cholesterol diet was stopped after stent implantation; in group II (n = 10) a high cholesterol diet was maintained after stent implantation. Digital subtraction angiograms were obtained every 4 weeks for up to 24 weeks and the narrowest diameter of the arterial segments within each stent and in the segment between stents was measured. The diameter narrowing within the closed stent was greater in the high cholesterol group compared with the low cholesterol group: 12 weeks (2.57 +/- 0.09 mm in group I vs 2.14 +/- 0.15 mm in group II, mean +/- S.E., p < 0.05); 16 weeks (2.55 +/- 0.09 mm vs 2.14 +/- 0.12 mm, p < 0.05); 20 weeks (2.59 +/- 0.06 mm vs 1.98 +/- 0.12 mm, p < 0.01); and 24 weeks (2.45 +/- 0.11 mm vs 2.01 +/- 0.11 mm, p < 0.05). No significant differences in the narrowest diameter of the arterial segments were observed between high and low cholesterol groups in the angioplasty alone areas or within the open stents. There was a significant difference in the narrowest diameter between stents with versus those without gaps (at 12, 16, and 20 weeks poststenting in group I and at 4, 8, 12, 16, 20, and 24 weeks in group II). Thus the stent with the least metal is correlated with less stenosis and intimal hyperplasia. From these data we conclude that both stent design and serum cholesterol are important factors for restenosis after stent implantation.

Vascular stenting in normal and atherosclerotic rabbits. Studies of the intravascular endoprosthesis of titanium-nickel-alloy.

Percutaneous transluminal balloon angioplasty would be more effective if the rate of recurrent stenosis were reduced. To evaluate the prevention of restenosis after percutaneous transluminal angioplasty, intravascular endoprosthetic stents of titanium-nickel-alloy were implanted transluminally in seven normal and 21 atherosclerotic rabbits. In normal rabbits, a 3.5-mm diameter stent was implanted in the aorta and a 2.5-mm diameter stent in the right iliac artery, which were followed with serial angiograms from 6 weeks (n = 7) to 8 months (n = 4). There was a mean stenosis of 13.1% in the 2.5-mm and 13.6% in the 3.5-mm stent. There was no significant narrowing compared with the adjacent control segments of artery; histopathology showed a thin, fibrous neointima with smooth muscle cells. Each atherosclerotic rabbit was balloon dilated at two separate stenotic sites; each site was 2.0 cm in length. The aortic site (with 28.8 +/- 13.8% mean stenosis [+/- SD]) was dilated with a 3.5-mm balloon, and the iliac site (with 36.5 +/- 14.2% stenosis) was dilated with a 2.5-mm balloon. In each site, an intravascular stent of corresponding diameter and 7-mm length was implanted in one half of the dilated segment, assigned randomly, and the other half served as the angioplasty control. Angiographically observed restenosis rates and the corresponding histopathology were similar in the atherosclerotic segments that had angioplasty alone versus the atherosclerotic segments that had angioplasty plus stenting. The mean neointimal thickness in the aortas and iliac arteries, respectively, measured 247 +/- 181 microns (+/- SD) and 218 +/- 77 microns after 6 weeks (n = 8) versus 321 +/- 168 and 308 +/- 189 microns after 20 weeks (n = 5, p = NS). At 20 weeks follow-up, there was 29.1 +/- 29.8% (median, 16.4%) stenosis in the aortic stent versus 38.9 +/- 24.1% (median, 34.0%) stenosis in the percutaneous transluminal angioplasty control segment of aorta (n = 5, p = NS) and 81.4 +/- 25.5% stenosis in the iliac artery stent versus 89.3 +/- 15.3% stenosis in the PTA control segment of the right iliac artery (n = 5, p = NS). Comparing stenotic arterial segments treated with angioplasty alone with angioplasty plus intravascular stenting in the atherosclerotic rabbits showed that there was no significant difference in either the histopathologic changes or the restenosis rates.

Laparoscopic Inferior Vena Cava and Right Atrial Thrombectomy Utilizing Deep Hypothermic Circulatory Arrest

BACKGROUND AND PURPOSE Surgery for renal cancer associated with a level III or IV tumor thrombus often involves cardiopulmonary bypass, deep hypothermia, and exploration of the right atrium and inferior vena cava (IVC). This major open operation necessitates a large median sternotomy incision and a midline abdominal or chevron incision. Herein, we investigate the feasibility of purely laparoscopic IVC and right atrial thrombectomy utilizing deep hypothermic circulatory arrest. MATERIALS AND METHODS In six male calves weighing 70 to 80 kg, the right common carotid artery and right internal jugular vein were cannulated for subsequent cardiopulmonary bypass. One laparoscopic team performed right radical nephrectomy and complete mobilization of the intra-abdominal IVC by a four-port approach. Simultaneously, a second laparoscopic team obtained three-port thoracoscopic access to incise the pericardium and expose the right atrium. In sequence, cardiopulmonary bypass, complete exsanguination, cardiac arrest, and core hypothermia of 18 degrees C were achieved. A coagulum thrombus was created by needle injection into the IVC. Combined laparoscopic and thoracoscopic incision, exploration, and thrombectomy of the IVC and the right atrium were then performed in a bloodless field. An angioscope was inserted inside the heart and the IVC to confirm complete thrombus clearance visually. The IVC and right atrium were then laparoscopically suture repaired, cardiopulmonary bypass was reestablished, and the animal was gradually rewarmed. Once sinus rhythm was reestablished at normal body temperature, the animal was weaned off the pump. RESULTS The mean total operative time was 494.5 minutes (range 355-705 minutes). The mean time needed to lower the core temperature was 63.5 minutes (range 50-120 minutes), and the mean time required to rewarm the animal was 101.8 minutes (range 70-130 minutes). The mean blood volume drained into the pump was 2633.3 mL (range 1400-3200 mL), and the mean estimated blood loss was 350 mL (range 200-750 mL). Reestablishment of sinus cardiac rhythm and weaning off the pump was successful in all animals prior to acute euthanasia. CONCLUSIONS Laparoscopic radical nephrectomy with thrombectomy for level III or IV tumor thrombi utilizing deep hypothermic circulatory arrest is feasible in the calf model using minimally invasive techniques exclusively. The procedure is technically complex and requires the combined efforts of expert urologic and cardiac operative teams. Survival studies are planned.

Conjugated human hemoglobin as a physiological oxygen carrier--pyridoxalated hemoglobin polyoxyethylene conjugate (PHP).

Problems associated with specific physiological properties of Hb-based blood substitutes, such as a low P50, short plasma half-life and nephrotoxicity are still major issues to be addressed. Extensive investigations aimed at overcoming these problems have resulted in the preparation of pyridoxalated-hemoglobin-polyoxyethylene conjugate (PHP). PHP was developed from human hemoglobin by two major chemical modifications; pyridoxylation for the purpose of lowering the oxygen affinity (P50 of 19.5 ± 1.2 mmHg), and coupling with polyoxyethylene (POE) to increase its molecular weight (to approximately 90 kdaltons). The circulating half-life of PHP is about 40 hours in dogs. Toxicologicai and physiological studies including renal function assessments have demonstrated that PHP does not have untoward effects on major organ functions. Its efficacy in transporting oxygen has been shown in ET and intracoronary perfusion, and in in vitro studies with sickle cells. Studies to date suggest that PHP is a promising candidate as a physiological oxygen carrier. In this paper the properties of PHP, its safety and efficacy aspects, and its potential as a clinical oxygen carrier are reviewed based on studies conducted in the Authors laboratory.