Temperature and perfusion responses of muscle and lung tissue during chronic heating in vivo.

For the first time, both temperature and perfusion responses have been obtained from in vivo studies of chronically heated lung and muscle tissue of calves. In each tissue, the spatial temperature distribution was measured by thermistors placed in needles at several distances from an implanted heated disc. A perfusion parameter was defined for a bioheat transfer model that describes temperature dynamics with distance from the heated disc. Estimates of perfusion were obtained by a least-squares fit of the model output to a step change in heat flux. Except for short transient experiments several times a week, a constant heat flux of 0.04, 0.06 or 0.08 Wcm(-2) was maintained at the disc surface for up to seven weeks. At the higher heat fluxes, the steady-state tissue temperature decreased with heating duration. Also, the characteristic time constants of the tissues decreased with heating duration. Muscle perfusion showed a statistically significant increase during chronic heating. Tissue adapts to chronic heating above 42 degrees C by allowing more capillary blood flow that increases heat loss to reduce tissue temperature.

Particle image velocimetry investigation of intravalvular flow fields of a bileaflet mechanical heart valve in a pulsatile flow.

BACKGROUND AND AIM OF THE STUDY: Our previous studies of bileaflet mechanical heart valves (MHV) explanted from sheep revealed patterns of localized platelet aggregation on valve surfaces, which may have clinical relevance. Since flow phenomena may promote localized platelet aggregation, an evaluation of flow within a valve lumen was conducted. METHODS: Phase-locked particle image velocimetry (PIV) measurements were obtained within the lumen of a 'mitral' model bileaflet MHV with transparent acrylic leaflets and housing, in a pulsatile flow loop. Instantaneous, two-dimensional flow maps of a central plane, perpendicular to the flow and leaflet pivot axes, were obtained at discrete times during the simulated cardiac cycle. Flow conditions were cardiac output, 3.5 l/min; rate, 72 beats/min; and systolic duration, 300 ms, using blood analog fluid refractive index-matched to acrylic. Leaflet closing velocities and angles were found using double-exposure imagery, and maximum leaflet closing velocity was extrapolated from regression analysis. RESULTS: During full opening, flow within the three lumenal orifices formed a three-peak axial velocity profile. Vorticity was concentrated in shear layers adjacent to downstream leaflet surfaces and in downstream wakes. Forward flow peak velocity was 90 cm/s, with a steep velocity gradient in the central orifice. During closing, the central-gap regurgitant flow formed a jet (peak velocity, 144 cm/s). High vorticity occurred near leaflet leading and trailing edges. During full closure, first a transient (<3 ms) 'stopping vortex' developed near the leaflet trailing edge, followed by a wall jet which formed at the leaflet-housing junction. Maximum leaflet closing velocity was 1.4 m/s. CONCLUSION: Localized jets, steep velocity gradients, high vorticity and vortex recirculation have been observed in vitro near model MHV surfaces. In vivo, each of these flow phenomena, when occurring near valve surfaces, may promote localized platelet aggregation. For the acrylic leaflets, maximum velocity was comparable with results reported for pyrolytic carbon leaflets. PIV of fully transparent models is a promising method for evaluating lumenal flows.

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.

Whole blood platelet aggregation in humans and animals: a comparative study.

BACKGROUND: Many animal species are used to evaluate the performance and blood compatibility of cardiovascular devices, but interspecies differences in platelet activity have not been well characterized. This study measures platelet response to six agonists in human, dog, and calf blood. MATERIALS AND METHODS: We used whole blood impedance lumi-aggregometry to measure platelet aggregation and ATP release in blood samples from adult humans (n = 19), mongrel dogs (n = 19), and Holstein calves (n = 7). The agonists were collagen, ristocetin, arachidonic acid, thrombin, and three concentrations of both ADP and epinephrine. RESULTS: Only collagen (1 microg/ml) and ADP (5, 10, and 20 microM) caused aggregation and ATP release in all samples. Canine platelets responded to all six agonists at all doses. Human platelets responded to everything except epinephrine at 2 and 100 microM. Bovine platelets responded only to collagen, ADP, and thrombin. In bovine platelets, aggregation from collagen and ATP release from thrombin were significantly lower than the corresponding responses in human and canine blood. The aggregation induced by 10 microM ADP was significantly higher in canine than in human platelets. CONCLUSION: Human, canine, and bovine platelets have very different responses to agonists. In these models, collagen (1 microg/ml) and ADP (10 microM) are the agonists of choice for investigating whole blood platelet aggregation because they provide the most consistent results between species. For ATP release, 1 U/ml thrombin is the recommended agonist and the dose for all three species.

Characterization of tissue morphology, angiogenesis, and temperature in the adaptive response of muscle tissue to chronic heating.

Previous investigations on the in vivo effects of chronic heat on tissue suggest a response whereby heated tissue temperatures decrease over time. This response occurred in conjunction with localized angiogenesis, which possibly contributed to the temperature decreases by increasing local perfusion and enhancing tissue heat transfer. Our own studies were the first to use a chronic heat source to heat tissue at initial interfacial temperatures between 40 degrees C and 46 degrees C. Initial temperatures above 45.3+/-2.2 degrees C caused necrosis of adjacent tissue. Through an adaptive response, the necrosis was removed by 7 weeks and replaced by a highly vascularized tissue capsule at 41.8+/-0.5 degrees C. The present study sought to characterize the spatial distribution, number of capillaries, and temperatures associated with this adaptive response. Heated and control muscle tissue sections were removed after 2, 4, and 7 weeks of heating at 0.08 W/cm2. Tissue layer thicknesses and capillary densities were measured and correlated with corresponding tissue temperatures. Necrosis was present adjacent to the heat source at 2 and 4 weeks; however by 7 weeks, a highly vascularized fibrous tissue capsule had replaced nearly all necrosis. Capillary densities, particularly near the heat source, were significantly greater at 7 weeks than at either 2 or 4 weeks. Capillary densities in heated tissue capillary fronts tripled from 2 to 7 weeks (106.4+/-14.3 caps/mm2 versus 39.1+/-18.5 caps/mm2). Furthermore, a mean temperature of 41.7+/-0.9 degrees C was measured in heated tissue capillary fronts at all durations, suggesting that this may be a threshold temperature for heat-induced angiogenesis or endothelial cell survival. These findings more completely characterize the perfusion component of the current mathematical model for heat transfer in tissue and will help to establish guidelines for the functional heat loss that an implantable, heat-producing device may allow.

Analysis of subcutaneous fluid in rats.

Subcutaneous implantation in rats is a commonly used model for biomaterial calcification studies. Although this model is frequently used, its components have not been characterized with respect to calcification. Exudate from the subcutaneous spaces of 18 young rats was collected using diffusion chambers. These chambers consisted of polymethylmethacrylate tubes with 0.22 micron pore filters covering each end allowing fluid, but not cells, to enter the chambers. Glutaraldehyde treated bovine pericardial strips were implanted subcutaneously, inside the chambers and outside the chambers, to test the calcification inducing abilities of the various environments. The animals were killed on postoperative day 10, and the exudate and materials were collected. The exudate was analyzed for ionic calcium, total calcium, inorganic phosphorus, and albumin, and for cells by a differentiated cell smear. The materials were analyzed for calcification by radiography, histology, and atomic absorption. Calcification was present in the materials inside the chambers where no cells were present and in the materials that were not in chambers. The distinct features of the exudate were elevated ionic calcium, a high Ca x P product, and elevated phosphorus.

Heat from an implanted power source is mainly dissipated by blood perfusion.

Heat dissipation and its effects on tissue and blood interfaces are common problems associated with the development and increased use of artificial hearts, because all of the implantable actuators for artificial hearts generate waste heat due to inefficiencies of energy conversion. To determine the mechanisms of heat dissipation from artificial hearts, heated disks producing constant heat fluxes of 0.08 watts/cm2 were implanted adjacent to the left lung and the latissimus dorsi muscle in calves for 2 weeks, 4 weeks, and 7 weeks. At the end of each experiment, a series of acute studies was performed in which blood perfusion to the heated tissue was decreased or stopped to observe the contribution of blood perfusion to heat dissipation. The cooling effect of ventilation was also examined to determine its relative contribution to heat dissipation in lung tissue by decreasing the minute ventilation volume. The importance of blood perfusion for heat dissipation was demonstrated by the temperature rise after cessation of blood perfusion to the heated tissue. The contribution of ventilation to heat dissipation in the heated lung tissue was minimal. Contribution of total blood perfusion to heat dissipation was increased with time in the muscle tissue, which has relatively low resting blood perfusion, but not in the lung tissue, which has relatively high blood perfusion. In the heated muscle tissue, the in vivo adaptive response to chronic heat was functionally shown by the increased perfusion. In conclusion, blood perfusion was the main mechanism of heat dissipation from tissues that were adjacent to an implanted power source.

Sensitivity analysis of one-dimensional heat transfer in tissue with temperature-dependent perfusion.

Design criteria for implantable heat-generating devices such as the total artificial heart require the determination of safe thresholds for chronic heating. This involves in-vivo experiments in which tissue temperature distributions are obtained in response to known heat sources. Prior to experimental studies, simulation using a mathematical model can help optimize the design of experiments. In this paper, a theoretical analysis of heat transfer is presented that describes the dynamic, one-dimensional distribution of temperature from a heated surface. Loss of heat by perfusion is represented by temperature-independent and temperature-dependent terms that can reflect changes in local control of blood flow. Model simulations using physiologically appropriate parameter values indicate that the temperature elevation profile caused by a heated surface adjacent to tissue may extend several centimeters into the tissue. Furthermore, sensitivity analysis indicates the conditions under which temperature profiles are sensitive to changes in thermal diffusivity and perfusion parameters. This information provides the basis for estimation of model parameters in different tissues and for prediction of the thermal responses of these tissues.

Anatomic fitting studies of a total artificial heart in heart transplant recipients. Critical dimensions and prediction of fit.

Anatomic fitting studies of the Cleveland Clinic-Nimbus total artificial heart were performed in 33 patients undergoing heart transplantation. The pump fit in the pericardial space in 20 men (80%) and 4 women (50%). There was no significant difference between the Fit and Non-Fit groups in external chest dimensions. Among 42 intrathoracic dimensions, the distance from the center of the mitral valve to the diaphragm (Fit: 5.6 +/- 2.2 cm, Non-Fit: 3.6 +/- 0.4 cm, p < 0.00001) and the distance from the caudal end of the pulmonary valve to the diaphragm (Fit: 9.4 +/- 1.6 cm, Non-Fit: 6.3 +/- 0.8 cm, p < 0.0001) were the most critical. To predict anatomic fit, an index (A x B x C) was obtained from chest X-ray measurements (A, the craniocaudal distance from the dorsal region of the 8th left rib to the left diaphragm; B, the maximum left chest width; and C, the maximum anteroposterior sternum-vertebrae dimension). The pump fit in 88.5% of the patients with an index above 1200 cm3, whereas it fit in only 14.3% of the patients with an index below 1200 cm3 (p < 0.001). This index was an easily obtainable, good predictor of anatomic fit.

In vitro evaluation of automatic control performance of a total artificial heart with changes in pump orientation.

Unlike in animal experiments, the pump orientation of a total artificial heart (TAH) can change remarkably in humans with the recipient's posture (upright, supine, or prone), thus affecting its filling characteristics. The left master alternate control mode of the Cleveland Clinic-Nimbus (CC-N) TAH adjusts beat rate by maintaining the left pump at 90% filling, producing a Frank-Starling like preload sensitivity. In order to verify that the CC-N TAH functions properly regardless of the gravity effects on pump filling, the preload sensitivity curves of the CC-N TAH were evaluated on a mock circulatory loop with the simulated supine (right pump up) and prone (left pump up) positions in humans. The right preload sensitivity was slightly higher when the right pump was up versus down, and likewise the left preload sensitivity was higher when the left pump was up versus down. Despite these gravity effects on pump filling, right and left preload sensitivity remained within physiologic range and the automatic control of the CC-N TAH functioned properly without significant postural effects.

Microencapsulated vaccines to provide prolonged immunity with a single administration.

Vaccines that provide lasting immunity with a single administration of the antigen can reduce the cost of routine immunization programs while increasing their efficacy by lessening the need for patient compliance. The authors have been developing methods for using biodegradable polymer microspheres to encapsulate vaccines. These microcapsules are designed to provide timed release of the antigen on a schedule that mimics conventional booster shots. The microspheres are made from poly-DL-lactide-co-glycolide. The rate of biodegradation of this polymer is controllable by varying the molar ratio of the monomers. High performance liquid chromatography was used to measure release kinetics in vitro, and a process was developed for the encapsulation of water soluble protein antigens. This process then was used to prepare a microencapsulated vaccine for type A botulism made using a recombinant C fragment antigen. A series of 27 adult C57BL/6J mice were used to study the efficacy of this vaccine. Six mice injected with saline filled microspheres served as a control group. Plasma samples were taken weekly to measure antibody levels using enzyme linked immunosorbent assay. At 14 weeks, 21 immunized mice and six control subjects were used for an aerosol challenge test with botulinum toxin. All control subjects died within 72 hrs. Fifteen (71%) of the immunized mice survived.

Platelets are deposited early post-operatively on the leaflet of a mechanical heart valve in sheep without post-operative anticoagulants or antiplatelet agents. A scanning electron microscopic observation of the pyrolytic carbon surface in a mechanical heart valve.

Pyrolytic carbon has been used for mechanical heart valves as a thromboresistant, wear resistant, and fatigue resistant material. Thrombosis and thromboembolism, however, remain major mechanical heart valve associated complications and may frequently occur during the early post-operative period. In depth morphologic studies on blood-pyrolytic carbon surface interactions are limited. The purpose of this study was to evaluate the blood compatibility of the pyrolytic carbon surface of St. Jude Medical mechanical heart valves that were implanted in the mitral position of sheep without the administration of post-operative anticoagulants or antiplatelet agents for 2, 4, and 6 weeks. Almost the entire leaflet and orifice ring surfaces were observed by scanning electron microscopy. Although the surfaces appeared clean macroscopically, when observed by electron microscopy, the surface were mottled, mainly by solitary platelets and aggregations. There were only a few leukocytes or red blood cells observed. No fibrin clots were observed on the leaflets. The density of platelet deposition was higher in the vicinity of the pivots and near the edges of the leaflets. The sizes of the platelet aggregations decreased with longer duration. The outer surfaces of the pivot guards were covered by various amounts of deposition composed of platelet aggregations and thrombi. Thus, the administration of antiplatelet agents is recommended during the early post-operative period after mechanical heart valve implantation.

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 nonpulsatile blood flow. II. Hemodynamic responses to progressive exercise in calves with chronic nonpulsatile biventricular bypass.

We investigated the effects of stepwise treadmill exercise on animal (calf) hemodynamic variables during chronic nonpulsatile biventricular bypass with ventricular fibrillation. Seven days was allowed for recovery from the effects of anesthesia and surgery; each animal's natural heart was then fibrillated. The pump flows were maintained at nominal rates of 90, 100, and 120 ml.kg-1.min-1 for 1 week each, with the order varying from experiment to experiment. A total of 30 incremental exercise tests were performed on five animals. No significant changes in mean aortic pressure were observed during nonpulsatile perfusion at the three nominal flow rates of nonpulsatile flow either before or during exercise. The systemic vascular resistance decreased significantly during exercise (from 705 +/- 22 to 547 +/- 81 dyne.sec.cm-5, p < 0.01, and from 604 +/- 25 to 510 +/- 15 dyne.sec.cm-5, p < 0.05, at nominal flow rates of 100 and 120 ml.kg-1.min-1, respectively). There were also significant (analysis of variance, Scheffe test, p < 0.05) differences in systemic vascular resistance among three nominal flow rates both before and during exercise. These results suggest that the autonomic nerve reflex control of the cardiovascular system in physical exercise was functioning normally in animals with chronic nonpulsatile blood flow.

Chronic nonpulsatile blood flow. III. Effects of pump flow rate on oxygen transport and utilization in chronic nonpulsatile biventricular bypass.

The relationship between blood flow and oxygen transport was studied in five calves with chronic nonpulsatile biventricular bypass. Seven days was allowed for recovery from the effects of anesthesia and operation; the natural heart was then fibrillated. Pump flows were maintained at nominal rates of 90, 100, or 120 ml.kg-1.min for 1 week each, with the sequence varied from experiment to experiment. Venous and arterial blood samples were taken at rest for blood gas analysis. Serum lactate analysis was done twice a week, on the third and seventh days after each pump flow change. Serum catecholamine levels were assayed on the seventh day of each flow rate. Progressive exercise tests were also conducted during each test segment. Basal oxygen consumption of a 4-month-old calf was 6.3 +/- 0.3 ml.kg-1.min-1. The mixed venous oxygen tension decreased when pump flow rate was reduced (29.6 +/- 1.0, 28.3 +/- 1.2, and 23.8 +/- 0.9 mm Hg at 120, 100, and 90 ml.kg-1.min-1 of pump flow, respectively), and oxygen extraction increased linearly when pump flow rate was reduced. Hemoglobin concentration significantly affected oxygen extraction rate. Serum lactate concentration increased significantly at a 90 ml.kg-1.min-1 perfusion compared with concentrations at other pump flow rates (7.81 +/- 2.42 mEq/L at 90 ml.kg-1.min-1 vs 0.71 +/- 0.19 and 0.73 +/- 0.81 mEq/L at 100 and 120 ml.kg-1.min-1, respectively; p < 0.01, analysis of variance, Scheffe F test). Maximum oxygen extraction during exercise was 78%. These results suggest that a critical flow level between 90 and 100 ml.kg-1.min-1 maintains oxidative metabolism in the calf with chronic nonpulsatile flow. The resulting oxygen delivery was slightly higher than that indicated in the literature. Maximal oxygen extraction was normal.

Left ventricular echocardiographic and histologic changes: impact of chronic unloading by an implantable ventricular assist device.

OBJECTIVES: We studied the effects of chronic left ventricular unloading by a ventricular assist device and assessed left ventricular morphologic and histologic changes. BACKGROUND: The implantable left ventricular assist device has been effective as a "bridge" to cardiac transplantation. Although there are reports documenting its circulatory support, little is known about the effects of chronic left ventricular unloading on the heart itself. METHODS: We performed intraoperative transesophageal echocardiography at the insertion and explanation of a HeartMate left ventricular assist device in 19 patients with end-stage heart failure. They were supported by the assist device for 3 to 153 days (mean [+/-SD] 68 +/- 33). Measurements were taken retrospectively to obtain left atrial and ventricular diameters and interventricular septal and posterior wall thicknesses. Histologic examinations were made from the left ventricular myocardial specimens of 15 patients at the times of insertion and explanation for heart transplantation. Insertion and explanation specimens were compared qualitatively (0 to 3 scale) for wavy fibers, contraction band necrosis and fibrosis, with quantitative measurement of minimal myocyte diameter across the nucleus. RESULTS: Left atrial and left ventricular diastolic and systolic diameters decreased immediately after insertion of the left ventricular assist device (from 46 to 35, 63 to 41 and 59 to 36 mm, respectively, all p < 0.001). Left ventricular wall thickness increased from 10 to 14 mm (p < 0.001) for the interventricular septum and from 10 to 13 mm for the posterior wall (p<0.001). No echocardiographic measurements showed significant subsequent changes at the chronic stage. Myocardial histologic findings demonstrated a reduction in myocyte damage (from 1.9 to 0.5, p<0.001, for wavy fiber and from 1.3 to 0.2, p<0.01, for contraction band necrosis) and an increase in fibrosis (from 1.3 to 1.9, p<0.05), but without significant change in myocyte diameter (from 15.6 to 16.8 micrometer, p=0.065). CONCLUSIONS: Left ventricular unloading with the implantable assist device induces an immediate increase in wall thickness, consistent with the reduction in chamber size, thereby decreasing wall stress. Chronic unloading allows myocardial healing and fibrosis without evidence for ongoing myocyte damage or atrophy. Left ventricular assist device insertion may have a role in "resting" the ventricle for selected patients with heart failure.

Reduced expression of platelet surface glycoprotein receptor IIb/IIIa at hyperthermic temperatures.

BACKGROUND: Hyperthermic temperatures exist from the heat dissipation of the implantable energy source of an artificial heart. This procedure as well as therapies for cancer and thermal injuries pose a new medical problem. Among many reported effects of heat on biologic systems, platelet functions such as maximal aggregation and adhesion are known to be reduced. Using flow cytometry, we have studied platelet dysfunction at elevated temperatures and have gained a mechanistic comprehension of the loss of platelet function. EXPERIMENTAL DESIGN: Platelet rich plasma was incubated at differing temperatures for 1 hour. Immediately after, the platelets were stained using mAb against glycoprotein IIb/IIIa (GPIIb-IIIa) (CD41a) and other platelet surface glycoproteins (GP) involved in aggregation and adhesion. Relative fluorescence intensity was measured using single-labeled, laser flow cytometry to determine changes in GP surface expression. In addition, scanning electron microscopy was used to evaluate morphologic changes. RESULTS: Hyperthermic temperatures between 40 and 44 degrees C significantly lowered the mAb cell surface binding in vitro of GP that participate in aggregation and adhesion. The most dramatic temperature-dependent loss of mAb binding was demonstrated by anti-GPIIb-IIIa, the mAb against the fibrinogen receptor. mAb binding to this receptor at 44 degrees C was decreased to 6.2% of a base-line fluorescence intensity of 654 (arbitrary units). The ADP-induced aggregation of platelets incubated at the same temperature also decreased to 2.1% of maximum aggregation. Other mAb, such as those against the von Willebrand factor receptor (GPIb) (CD42b), the thrombospondin receptor (GPIV) (CD36), and GPIIIa (CD61), also showed statistically significant reduction of mAb binding but to a lesser degree. Finally, scanning electron microscopy as well as side-scatter density plots from flow cytometry revealed that platelets became more spherical after incubation at 44 degrees C. CONCLUSIONS: The significant reduction in mAb binding correlates with functional impairment exhibited during hyperthermic incubation. Our results support the loss of binding ability of surface GP that are involved in aggregation and adhesion as a mechanism of platelet dysfunction upon heating. GPIIb-IIIa appeared the most susceptible to heat and the principal agent in thermal induced loss of platelet function. Significant morphologic changes at 44 degrees C, the critical temperature at which ADP-induced aggregation ceases, may contribute as well.

A comprehensive hematologic study in calves with total artificial hearts.

Thromboembolism and infection remain potential threats for long-term circulatory assist and replacement devices. The alteration of the hemostatic system and of blood cell functions caused by device implantation may predispose the recipient to these complications. Many sensitive coagulation assays and the technology of flow cytometry would be powerful tools for this investigation. The availability of such immunologic technologies for animal species other than humans has yet to be established. In a series of in vitro tests we found that the following assays, among others, are usable in calves: TAT, TxB2, platelet surface glycoprotein IIbIIIa, and membrane aminophospholipid. F1.2, D-dimer, beta TG, PF-4, and platelet surface expression of GMP-140 and receptors for fibronectin, thrombospondin, and vWF were not measurable. A sustained mild decrease in hematocrit levels in six calves with the Cleveland Clinic-Nimbus total artificial heart for 11-120 days was attributed to an increase in circulating blood volume, but not to red blood cell damage. Whole blood platelet aggregation was suppressed only for the first 3 post operative days, with decreased GPIIbIIIa expression. Polymorphonuclear phagocytosis, chemotaxis, and superoxide anion production were not altered. Device infection and thromboembolism occurred in one of 13 cases overall.

Assessment of circulating blood volume in calves with a total artificial heart.

Measurement of the circulating blood volume (CBV) is essential to a proper understanding of the hemodynamic performance of total artificial hearts (TAHs). Recently, the authors employed CBV measurements using indocyanine green dye in calves with a TAH. The advantages of this method over previous methods using radionuclides include simplicity, low cost, and the capability of repeated and frequent measurements. Reproducibility of the measurements was demonstrated in three normal calves with a relative standard deviation of 3.9 +/- 2.4%. CBV was measured in eight calves with the Cleveland Clinic-Nimbus TAH and compared with that of seven calves that underwent mitral valve replacement. Small standard deviations in pre operative values in both TAH and mitral valve replacement groups demonstrated the precision of CBV measurements. Although there was no change in CBV in the mitral valve replacement group, CBV in the TAH group increased to more than twice the pre operative value after 2 weeks. Although the right atrial pressure increased similarly after TAH implantation, there was no correlation (r = 0.08) between the right atrial pressure and CBV, which suggested a possible inaccuracy in estimating CBV from the right atrial pressure. A negative correlation between the hematocrit value and CBV suggested that hemodilution might be one of the causes of anemia observed in our TAH animals.

Accelerated healing of cardiovascular textiles promoted by an RGD peptide.

Polytetrafluoroethylene (PTFE) and polyethylene terephthalate (Dacron polyester) fabrics are used extensively in cardiovascular devices, e.g. heart valve sewing cuffs and vascular prostheses. While devices containing these fabrics are generally successful, it is recognized that fabrics cause complications prior to tissue ingrowth due to their thrombogenic nature. A surface active synthetic peptide, called PepTite Coating (PepTite), which was modeled after the cell attachment domain of human fibronectin has been marketed as a biocompatible coating. This peptide stimulates cell attachment through the arginine-glycine-aspartic acid (RGD) sequence. Modification of medical implants with PepTite has been shown to promote ingrowth of surrounding cells into the material leading to better tissue integration, reduced inflammation and reduced fibrotic encapsulation. In this study, polyester and PTFE textiles were modified with PepTite. The effectiveness of this coating in enhancing wound healing was investigated in a simple vascular and cardiac valve model. Our results indicate that the RGD-containing peptide, PepTite, promoted the formation of an endothelial-like cell layer on both polyester and PTFE vascular patches in the dog model. PepTite was also found to promote the formation of a significantly thinner neointima (pannus) on polyester as compared to that on its uncoated control. These results were corroborated in the cardiac valve model in which a greater amount of thin pannus and less thrombus were seen on coated polyester sewing cuffs than on control uncoated cuffs. This research shows the promising tissue response to RGD coated textiles and the potential role of this peptide in material passivation via accelerated healing.