William E. Burkel

Sequential studies of healing in endothelial seeded vascular prostheses histologic and ultrastructure characteristics of graft incorporation

Abstract Chronological events leading to incorporation of endothelial cell seeded prosthetic vascular grafts were documented in this investigation. Forty-one adult dogs underwent thoracoabdominal bypass using double-velour Dacron grafts. Experimental grafts were preclotted with blood containing enzymatically derived endothelium immediately after derivation, or after 14 days of cultivation. Control grafts were preclotted without addition of endothelial cells. Grafts were studied grossly as well as by light, scanning electron, transmission electron, and fluorescence microscopy, 1 to 28 days postimplantation. Control graft healing proceeded from pannus and perigraft ingrowth. Experimental grafts healed from seeded cells as well. Platelets covered all grafts by Days 1 and 2. Thrombus accumulations on control grafts, first evident on Day 4, became maximal by Day 14. Seeded grafts appeared relatively thrombus free with patches of endothelial cells noted by 4 days. These cells were initially separated by gaps, often containing leukocytes. Endothelium became densely packed with cellular migration and proliferation. Subendothelial tissues were composed of fibrin and smooth muscle. Control and experimental grafts were approximately 10 and 80% endothelialized, respectively, by Day 28. Smooth muscle dominated subintimal tissue in experimental grafts. These cells initially appeared fibroblastoid. Endothelial seeding enhances both pseudointimal development and rapid graft incorporation.

Isolation of adult canine venous endothelium for tissue culture

SummaryIn order to provide autologous adult endothelial cells for the production of cell-lined artificial vascular prostheses, we have developed a method for harvesting large numbers of cells with minimal contamination by other cellular types. In this technique, the vein to be stripped is isolated, removed, and everted over a stainless steel rod. After washing, the vein is incubated in trypsin-EDTA solution followed by collagenase and the endothelial cells flushed off with a stream of culture medium. With care and appropriate timing, the endothelium can be selectively removed leaving the underlying basal lamina intact.

Endothelial cell seeding of expanded polytetrafluoroethylene vena cava conduits: Effects on luminal production of prostacyclin, platelet adherence, and fibrinogen accumulation

The blood-surface interface of 12 mm ID x 5 cm ePTFE vena cava conduits, unseeded (n = 8) and seeded (n = 8) with enzymatically derived autologous endothelial cells, was studied in a canine model at 4 and 12 weeks after graft implantation. Acetylsalicylic acid (325 mg each day) and Coumadin (prothrombin time 1.4 to 1.7 times the control value) were administered preoperatively and continued 4 weeks postoperatively. Platelets labeled with 111In and 125I-labeled fibrinogen were administered 24 hours before graft removal. Luminal platelet adherence, expressed as 10(6) platelets/cm2 of graft surface, was 8.9 +/- 5.6 vs. 56.4 +/- 8.0 (p less than 0.008) and 4.0 +/- 0.9 vs. 12.4 +/- 2.3 (p less than 0.005) in seeded vs. unseeded grafts at 4 and 12 weeks, respectively. Luminal fibrinogen deposition, expressed in micrograms per square centimeter of graft surface, was 11.8 +/- 2.2 vs. 32.0 +/- 2.0 (p less than 0.06) and 6.1 +/- 2.4 vs. 12.4 +/- 6.3 (p less than 0.005) in seeded vs. unseeded grafts at 4 and 12 weeks, respectively. Cumulative 4- and 12-week luminal production of 6-keto-PGF1 alpha from seeded and unseeded grafts represented 11% and 5%, respectively, of that produced from the native iliac vein. Luminal endothelial cell coverage was 71% +/- 22% vs. 33% +/- 9% and 79% +/- 8% vs. 55% +/- 8% (p less than 0.05) in seeded and unseeded grafts at 4 and 12 weeks, respectively. Although endothelialization was not complete in seeded vena cava grafts, it is clear that seeded prostheses exhibited improved thromboresistance compared with unseeded conduits.

Comparison of Static Incubation versus Physiologic Perfusion Techniques for Quantitation of Luminal Release of Prostacyclin and Thromboxane in Canine Arteries and Veins

Intraluminal release of 6-keto-PGF1 alpha and TxB2 in ex vivo canine arteries and veins was assessed during five consecutive 15-min periods using static incubation and physiologic perfusion techniques. Arterial segments were perfused with 90 ml/min pulsatile flow at 100 mm Hg and vein segments with 90 ml/min nonpulsatile flow at 7 mm Hg. During the final 15-min period vessels were stimulated with arachidonic acid (AAS). Perfusion of vein segments caused a higher release of 6-keto-PGF1 alpha during the first 30 min (P less than 0.05) and following AAS (P less than 0.05) than did static incubation. Perfused arterial segments exhibited a higher release than did incubated segments of 6-keto-PGF1 alpha for 45 min (P less than 0.01) as well as following AAS (P less than 0.01). TxB2 release was higher during the entire observation period in perfused arteries and veins compared to incubated vessels (P less than 0.01 and less than 0.05, respectively). There was no correlation between the amounts of 6-keto-PGF1 alpha or TxB2 released when comparing values obtained by one technique to values obtained by the other (P greater than 0.1). These data suggest that flow related shear stress alters vascular prostanoid production, and that such should be accounted for when interpreting results of studies on prostacyclin and thromboxane release from intact vessels.

Arterial 6-keto-PGF1α and TxB2 release in ex vivo perfused canine vessels: Effects of pulserate, pulsatility, altered pressure and flow rate

Certain experimental conditions are known to influence the release of prostacyclin and thromboxane from the vessel wall. The specific effects of altered pulsatility, pressure, and flow rate on intraluminal release of 6-keto-PGF1 alpha and thromboxane B2 were assessed in canine arteries perfused ex vivo for five 15 min periods with arachidonic acid (AA) added during the last period. Control arteries were perfused at 100 mmHg with pulsatile flow of 90 ml/min. Experimental arteries were perfused at 7, 50 and 200 mmHg with pulsatile flow of 90 ml/min, and at 100 mmHg pressure with pulsatile flow of 20, 60, 130 and 180 ml/min, as well as at 100 mmHg with 90 ml/min nonpulsatile flow. Perfusion pump rates of 44 and 96 beats/min were also assessed. The lowest perfusion pressure, 7 mmHg, resulted in a lesser initial release of prostacyclin compared to higher pressures, and there was a tendency to a higher release of prostacyclin with increasing pressures. There was also a tendency for a lesser response to AA in arteries perfused at 200 mmHg, perhaps due to endothelial cell damage. Nonpulsatile flow was associated with a decreased initial release of prostacyclin, and diminished release following addition of AA when compared to pulsatile flow. Altered flow rate elicited no difference in prostacyclin release, although there was a tendency towards a lesser release when perfused at 20 ml/min compared to 130 ml/min or 180 ml/min. Thromboxane release was decreased by nonpulsatile flow but was otherwise unaffected by the experimental conditions tested. It is concluded that pulsatility enhances release of prostacyclin from arteries.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelial Linings in Prosthetic Vascular Grafts

Of all the animals so far studied, man is the only one known to be incapable of healing prosthetic vascular grafts with its own natural endothelium. Consequently there has been considerable effort in the 30 or so years since the first prosthetic grafts became available either to find truly nonthrombogenic artificial materials or to enhance the body’s own natural healing processes in order to provide blood-compatible devices. It has been known from the days of the earliest prosthetic grafts that the fibrin-platelet coagulum that typically lines them is an adequate surface only for grafts of 8 mm i.d. or greater. Unfortunately, the need for smaller prostheses is equal to or greater than that for the large. In the early days, emphasis in graft development was focused on different materials, such as nylon, Dacron, Teflon, tanned bovine carotid arteries, or umbilical veins as conduits. Paralleling the work with prosthetic vessels was the development of the artificial heart and the desire to provide blood-compatible linings for these devices. The idea of cell or tissue fragment seeding of prostheses in order to provide pseudointimal linings originated because of its applicability to artificial hearts (see ref. 2 for a review of this early work). Our own work in this area began with artificial hearts as well, under the auspices of the National Heart, Lung, and Blood Institute and the Artificial Heart Program. At that time the emphasis was on developing microfiber substrates upon which to grow pseudointimal linings and on finding a cell or tissue type that would provide a blood-compatible surface. In order to develop these materials and methods the prosthetic devices were fabricated into nonporous urethane tubes lined with a microfiber scaffold and of a size that could be tested by interposition in the abdominal aorta of a dog (FIG. 1). The cells were introduced into the prostheses and cultivated in vitro until a pseudointima developed.” The progress was slow; materials were not very cell compatible, and the cells that were easily grown in vitro were thrombogenic. However, in the late 1970s several developments occurred that made cell-lined vascular grafts a practical reality. First, we and other groups of investigators finally learned how to isolate and grow adult endothelial cells. Second, we switched from the troublesome microfiber grafts to commercially available vascular prostheses (FIG. 2). Third, a new group of investigators4 discovered that scrapings from the intima of cutaneous veins, seeded into grafts in clotted blood and then immediately implanted into dogs, would produce endothelial linings in these vessels. With these developments research on endothelial cell seeding greatly increased. In the last few years endothelial cell seeding techniques have developed rapidly to the point where clinical reports of success with the method are beginning to appear’ (for a complete review, see refs. 6 and 7). In the following pages we will briefly describe the method and the current status of research in this area. This presentation is not meant to be exhaustive, but to highlight some of the more important observations as well as questions still to be answered.

Nuclide imaging of vascular graft-platelet interactions: Comparison of indium excess and technetium subtraction techniques

Indium-111-labeled platelet adherence to ePTFE thoracoabdominal vascular prostheses in a canine model (n = 10) was quantitated by (1) an indium-111 excess technique, contrasting graft radioactivity to that in a reference region, and (2) a technetium-99m subtraction technique, with radioactivity of circulating platelets eliminated by discounting background blood activity. Variation in graft thrombogenicity was provided by seeding six prostheses with enzymatically derived autologous endothelial cells, and implanting four prostheses without seeding. Grafts were imaged at 1, 4, and 6 weeks postimplantation, with platelet labeling using indium-111-oxine and red blood cell labeling using technetium-99m. At 7 weeks grafts were excised and gamma activity was measured in proximal, middle, and distal segments. Luminal generation of TxB2 and 6-keto-PGF1 alpha from midportions of grafts was assayed. Indium-111 excess ratios at 6 weeks correlated with actual gamma activity of excised grafts (proximal r = 0.80, P less than 0.01; middle r = 0.73, P less than 0.05; distal r = 0.48, ns) but such a correlation did not exist for the technetium-99m subtraction technique (r = -0.05, -0.25, and 0.16, in the three segments, respectively, all ns). The ratio of graft to aortic TxB2 production revealed a positive correlation with graft gamma activity (r = 0.87, P less than 0.01), and the ratio of graft 6-keto-PGF1 alpha to TxB2 production also correlated with gamma counts (r = -0.64, P = 0.05). In this experimental setting technetium-99m subtraction analysis was an imprecise method of detecting graft platelet accumulation, whereas indium-111 excess ratios proved to be a more accurate method of quantitating vascular prosthetic thrombogenicity.

Effects of Thromboxane Synthetase Inhibition on Patency and Anastomotic Hyperplasia of Vascular Grafts

The efficacy of a thromboxane synthetase inhibitor (U-63,557A, Upjohn) in promoting early patency and inhibiting anastomotic intimal hyperplasia in ePTFE grafts was compared to that of acetylsalicylic acid (ASA) in a canine model. Animals were started on ASA 5 gr po qd (Group I, n = 12) or U-63,557A 10 mg/kg po bid (Group II, n = 12) 1 day before placement of bilateral 5-mm-i.d., 13- to 16.5-cm-long ePTFE aortoiliac grafts and continued on the medication for the 16-week study. Six dogs in each group received autologous endothelial cell-seeded grafts, while the other six received unseeded grafts. Patency was determined weekly by assessment of femoral pulses. At the conclusion of the study anastomotic intimal hyperplasia was measured on serial sections through the distal anastomosis using a computer-linked digitizer. In Group I the patencies of seeded and unseeded grafts were not significantly different, being 100 and 83%, respectively. Furthermore, luminal narrowing due to intimal hyperplasia was not significantly different being 9.1 +/- 7.6% (chi +/- SD) in seeded grafts and 8.8 +/- 8.1% in unseeded grafts. On the other hand, in Group II the seeded grafts had significantly improved patency when compared to the unseeded grafts (83% vs 33%, P less than 0.05) and less luminal narrowing (11.4 +/- 11.1% vs 21.9 +/- 19.5%, P less than 0.01). Although U-63,557A administration promoted patency of unseeded grafts compared to no antiplatelet medication (0% patency), it was significantly less effective than ASA in improving patency (P less than 0.05) and inhibiting luminal narrowing (P less than 0.01).

EFFECT OF TESTOSTERONE ON LONG-TERM ORGAN CULTURES OF CANINE PROSTATE

SummaryOrgan cultures of rodent and human prostate glands have shown marked differences in their morphological response to testosterone. In this study, explants from 19 canine prostate glands were cultivated for a minimum of 9 days in Trowell’s T-8 medium. Groups of explants were exposed to media containing from 0.05 to 100 µm testosterone. While the higher testosterone levels (50 and 100 µm) markedly decreased explant viability, explants cultivated at lower levels (0.05 to 5 µm) appeared similar to control explants in testosterone-free Trowell’s T-8 medium. Atmospheric mixtures containing either 95% or 50% oxygen were equally effective.Shortly after the cultures were initiated, large amounts of secretory product were liberated into the lumen. After 9 or more days in vitro, glandular epithelium appeared cuboidal and never revealed the acid phosphatase-rich secretory granules seen in the preculture control. However, the epithelium exhibited an increase in alkaline phosphatase and lipid content following cultivation.

Effects of duration of acetylsalicylic acid administration on patency and anastomotic hyperplasia of ePTFE grafts

Effects of duration of acetylsalicylic acid (ASA) administration on the patency and development of distal anastomotic intimal hyperplasia (DAIH) of endothelial cell (EC) seeded and unseeded prosthetic aortoiliac grafts were studied in a canine model. ASA, 5 gr po qd, was administered to dogs 1 day prior to placement of bilateral, 12 to 17 cm long, 5 mm inside diameter expanded polytetrafluoroethylene (ePTFE) aortoiliac grafts and continued for 2 wk (Group 1, n = 12 dogs) or 16 wk (Group 2, n = 12 dogs). Six dogs in each group received autologous EC seeded grafts, while the others received unseeded grafts. Prosthesis patency was assessed weekly. At the conclusion of the study, DAIH was measured on serial sections using a computer-linked digitizer. The 16 wk patency for Group 1 grafts was 67%, while that for Group 2 grafts was 88% (p less than 0.09). Luminal narrowing due to DAIH was not significantly different between Groups 1 and 2 (7.7 +/- 8.3% [means +/- SD] and 9.0 +/- 7.8% respectively). EC seeding improved the 16 wk combined patency from 62 to 92% (p less than 0.02). A more complete luminal endothelial cell lining was correlated with reduced DAIH (r = -0.4, p less than 0.05). Chronic ASA administration prevented graft thrombosis between 2 and 4 wk postimplantation in this study but was not associated with decreased DAIH.