Endothelial cell seeding reduces thrombogenicity of Dacron grafts in humans Per Ortenwall, M D , PhD, ~ H a n s Wadenvik, M D , PhD, ~ Jack Kutfi, MD, Phi), ~ and Bo Risberg, M D , PhD, ~ G#teborg, Sweden Vascular prostheses in humans do not endothelialize spontaneously. In the present study we explored the feasibility of seeding autologous endothelial cells into prostheses implanted in patients undergoing reconstruction of the infrarenal aorta. In 22 patients one limb of an aortic Dacron bifurcation prosthesis was seeded with autologous endothelial cells harvested from the distal portion of the saphenous vein, The other limb Was shamseeded with culture medium only. The effect of seeding was studied by use of indium 111 radiolabeled platelets and external gamma camera scanning at 1, 4, and 12 months after surgery. No complications ascribable to the seeding procedure were seen. During the first year after surgery a gradual decrease in platelet accumulation occurred over the whole vascular prosthesis. At all time points studied the seeded graft limbs exhibited significantly less deposition of radiolabeled platelets than did control limbs. The observed difference in platelet accttmulation on autologous endothelial seeding-treated graft segments merits further investigation of this technique in humans. (J VASC SURG 1990;11:403-10.) Vascular grafts, made of Dacron or expanded polytetrafluoroethylene (ePTFE), perform satisfactorily in high-flow, large-diameter regions/ However, the patency of tiny-caliber grafts manufactured of these polymers is poor compared to autologous vein grafts. 2 One difference between the vein graft and the synthetic graft is the presence of an endothelial cell (EC) lining; Dacron or microporous ePTFE vascular prostheses do not spontaneously acquire such a lining after implantation in h u m a n s Y Vascular endothelial cells (VECs) exert a number of anticoagulant fimctions, but they can also express pcrocoagulant activities, for example, after stimulation ~ l t h bacterial endotoxin, interleuldn-1, or tumor necrosis factor. 5 Vascular wall thrombogenicity in vivo is a function of the balance between these activities. During normal conditions, the anticoagulant mechanisms
From the Departmentsof Surgery~and InternalMedicine,~ University of G6teborg. Supported by grants from the MedicalFaculty,Universityof G6teborg, The Swedish Societyfor Medical Research, Salus InsuranceCompany~The SwedishSocietyof Medicine,The Medical Societyof G6teborg, The Swedish National Association against Heart and Chest Diseases, and The Swedish Medical Research Council (Project 00660). Reprint requests: Per Ortenwall, MD, Department of Surgery, University of G6teborg, Ostra sjukhuset, CK, S-416 85 G6teborg, Sweden. 24/1/17241
willpredominate. The lack of VECs in synthetic vascular prostheses make these grafts more likely to fail from thrombotic occlusion, especially when the other components in Virchow's triad (blood flow and composition) are altered in an unfavorable way. The concept of autologous endothelial seeding (AES), as a mean of reducing thrombogenicity of synthetic grafts, was introduced by Herring et al.6 in 1978. In experiments carried out on dogs and baboons intraoperative AES produced a complete luminal VEC lining of the vascular prosthesis within 4 to 8 weeks after graft implantation. 7'8 Platelet accumulation was substantially reduced and mean platelet life-span normalized, as measured several weeks after surgery. 9 Furthermore, both patency rates and resistance to thrombotic occlusion, induced by flow reduction, were enhanced, t°q2 In humans only one report is available, demonstrating a VEC-like lining (by scamling electron microscopy) in an AES-treated human vascular prosthesis. 13 Obviously this method cannot be used for evaluation of AES, except in rare cases. Furthermore a biologic probe demonstrating the presence of fimctionally intact endothelium is not yet available. So far the effect of AES has to be assessed by indirect methods, such as the accumulation of platelets labeled with indium 111 (rain) on grafts. The purpose of the present study was to evaluate whether AES could reduce the thrombogenicity o f vascular prostheses in humans. We report a signifi403
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cant reduction in the accumulation of labeled platelets on AES-treated segments of aortic bifurcation grafts, even 1 year after surgery.
MATERIAL AND METHODS Twenty-two patients (19 men and three women) scheduled for abdominal aortic reconstruction, by use of a bifurcated vascular prosthesis were studied, Their mean age was 66.7 years __+5.6 years (range 52 to 74 years). The ankle/brachial pressure index was calculated for each limb before and after operation. Eleven of the patients were operated on because of occlusive disease and 11 because of aneurysms. Three patients had previous vascular surgery (one carotid, two iliac thromboendarterectomies), and one patient had an iliac artery balloon angioplasty. Minimum time lapse between previous vascular intervention was 2 years. All patients had a smoking history, and one had diabetes mellitus. Nine patients were on antihypertensive drugs. All studies were reviewed and approved by the Ethical Committee of the University of G6teborg. Informed consent was obtained from all patients. All techniques used in the present work have been described in detail previously14and are therefore only summarized herein: Surgical procedure Prophylactic antibiotic treatment was given as cefuroxime 1.5 gm intravenously × 3 during the day of surgery. Routine anticoagulation wag 500 ml of dextran 40 preoperatively, 500 ml during the first 8 postoperative hours, followed by 500 ml daily over 4 days, All patients were operated on by the same two vascular surgeons. The cell-harvesting procedure (see herein) was performed in the operating room and took between 45 and 60 minutes. During this time the graft was preclotted with autologous blood, and the proximal anastomosis was performed. Noncrushing vascular clamps were then placed across the graft limbs just distal to the bifurcation. Which graft limb to be seeded, and the order in which to construct the distal anastomoses, was decided randomly. The EC suspension was divided into two 2 ml aliquots. These aliquots were sequentially mixed with equal volumes of autologous whole blood and were injected 5 minutes apart into the graft limb to be seeded. The cells were allowed to settle in the graft limb during completion of the distal anastomosis. Just before completion of the anastomosis, clamps were released and blood was allowed to flush through the graft limb. The nonseeded (con-
Journal of VASCULAR SURGERY
trol) graft limb was treated identically, except that blood was mixed with culture medium only, omitting the ECs. Twenty-one Bionit (USCI Surgical Products, C.R. Bard Inc, Billerica, Mass.) and one Vascutek VP 1200K (Vascutek Ltd, Ayr, Scotland) were implanted. The approach to the abdominal aorta was transabdominal in 18 patients and extraperitoneal in four. Two femoropopliteal reversed-vein bypass graftings and one renal artery reconstruction were performed in three patients at the time of the aortic reconstruction. Thirty-three of the distal anastomoses were constructed at the common or external iliac artery, and 11 were constructed at the common or deep femoral artery. Cell harvesting and culture procedure Five to ten centimeters of distal calf saphenous vein was excised. The vein was cannulated with bi .;at tipped arterial catmulas and flushed with chilled Hank's buffered saline solution. Prewarmed collagenase solution (0.1%, type I) in phosphate-buffered saline was injected into the vein, which was incubated in sterile phosphate buffered saline solution at 37 ° C for 10 to 15 minutes. The effluent was flushed into a sterile centrifuge tube by use of 10 ml of chilled complete medium. All fluids were sterilized by passage through 0.22 ~m filters. After cell harvesting the vein segment was cut open, and the luminal area of the vein was measured. The effluent was flushed through the vein two additional times and centrifuged at 180 g for 10 minutes. The resulting cell pellet was resuspended in 5 ml of complete medium. Four milliliters of this suspension was used for see.,ding of the vascular prosthesis. The remaining 1 r~ll was taken to the cell culture laboratory. Two hundred cubic millimeters were taken for cell counting in a hemocytometer. The other portion of the cell suspension was inoculated onto plastic tissue culture wells, precoated with human fibronectin (10 ~g/cm 2 dish area). The cultures were held at 37 ° C in a humidified 5% carbon dioxide environment, and the culture medium was changed every 3 days. T M Cultures were studied with an inverted phase contrast microscope, and the identity of the cells was checked with a peroxidase-antiperoxidase-staining test for factor VIII-related antigen. 17 Cell viability was checked in the last 16 patients by calculation of plating efficiency, is Radioisotopic procedures These procedures were performed 1, 4, and 3.-", months after surgery.
Volume 11 Number 3 March 1990
Platelet separation and labeling procedure. All patients were forbidden to ingest any aspirin or other nonsteroidal antiinflammatory drugs 10 days before the study. Seventeen milliliters of venous blood was obtained with a 19-gauge butterfly needle into a disposable syringe, containing 3 ml of acid citrate dextrose N I H formula A (ACD-A) as anticoagulant. The anticoagulated blood was transferred into a 30 ml screwcap polycarbonate tube. Platelet-rich plasma (PRP) was obtained by centrifugation of the blood at 260 g for 15 minutes. Platelet rich plasma was then transferred into another 30 ml polycarbonate tube, and the pH was adjusted to 6.3 to 6.5, by adding I ml of ACD-A per 10 ml of PRP. The platelets were centrifuged at 800 g for 10 minutes. The supernatant platelet poor plasma (PPP) was removed for later Use. The platelet pellet was washed with 5 ml modified Tyrode's solution, at 37 ° C. The platelets were resuspended in 1 ml modified Tyrode's solff~ ,on, and the required amount of nlIn oxine was added to the suspension. After incubation for 2 minutes with the isotope at 37 ° C, 2 ml of PPP were added, and the mixture was centrifuged at 8 0 0 g for 10 minutes. The supematant was removed, and the platelet pellet was finally resuspended in 5 ml of PPP. For calculation of labeling efficiency, the plateletbound radioactivity and the radioactivity in the supernatant was measured in a dose calibrator (Radioisotope Calibrator CRC-5RB, Capintec, Montvale, N.J.). 200 txCi was injected into the patients. All procedures were carried out aseptically at room temperature, if not otherwise stated. Graft-platelet interaction. Anterior scanning of the abdomen was performed at 24, 48, 72, and 96 hours after injection of the labeled platelets, with a s~mtillation camera (General Electric; MaxiCamera II [General Electric Co., Medical Systems Div., Milwaukee, Wis.]) interfaced with a computer (Digital Equipment; [Digital Equipment International Ltd., Galway, Ireland]PDP 11-34). Both photo peaks emitted by rain (173 and 247 keV) were collected through a medium energy (max 370 keV) parallelhole collimator, by use of a 15% window, during six consecutive 5 minute periods each time. For visual assessment, images and depicted regions of interest (ROI) were recorded on Polaroid film. The images were stored on computer disc for data processing, by means of a 64 X 64 matrix system, dividing the image into 4096 pixels (Software: Gamma 11 [Digital Equipment Corp., Marlboro, Mass.). These computer-generated gamma images were assessed by means of a color-coded scale for platelet deposition S~2ensity. To correct the image for contributing radiation from the pelvic and vertebral bone marrow the
Seeding of aortobifemoralgrafts 405 background activity from this area was subtracted. 18 Thc matrix system enabled calculation of the accumulated number of counts from any chosen ROI in the image. By dividing with the actual number of pixels necessary to cover the ROI, an average count number per square unit of the ROI was obtained. Division of this average count number by the number of counts obtained from 0. l ml of whole blood, collected at the same time and counted for 200 seconds in a crystal scintillation counter, gave a graft/blood ratio. This ratio, corrected for both isotope decay and platelet senescence, we have designated the thrombogenicity index (TI) using the formula: TI = 100 x Counts in ROI - Background Counts (Number of pixels in ROI) x (Whole blood counts) By keeping the ROIs identical between different imaging times, TI could be compared as a function of time. Scans were viewed in a blinded fashion regarding the seeded or unseeded status of graft limbs. To correct for variations in distance between the scintillation camera and the graft limbs, all calculations of TI were performed on equal segments of limbs to be compared.
Follow-up In association with vascular prosthesis scanning, the paticnts were assessed by clinical and femoral Doppler examination. If these results indicated any postoperative complication, additional angiograms were performed. Statistical analysis Statistical analysis was performcd with a paired t test to compare intraindividual differcnccs in TI at the same imaging time, and an unpaired t test to compare intraindividual changes of TI between differcnt imaging timcs. Coefficient of variation was used for testing of intraobserver reliability. All values are given as mean _+ SD, unless otherwise stated. RESULTS Cell harvesting and culture An average of 142 + 96 × 103 cells were harvested from donor veins with a mean area of 5.9 _+ 1.5 cm. g The mean vascular prosthesis area seeded was 41 + 10 cm2; resulting in a mean veinto-vascular prosthesis area ratio of 0.15 + 0.05~ The mean number of viable cells (cells capable of attaching to culture dishes during the first 24 houis after surgery) seeded/cm 2 graft was 2.2 -+ 1.5 x 10. 3 This estimate of viability was based on 16 cultures: Cell cultures morphologically resembled ECs and
406
Journal of VASCULAR SURGERY
Ortenwall et al.
1 xont~,
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~
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48
mont~hs
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Fig. 1. Thrombogenicity index of the bifurcation area (mean __ SEM), as assessed at 24, 48, 72, and 96 hours after injection of radiolabeled platelets. A reduction occurred during the first year after implantation. The differences are depicted as: ~p < 0.05, ~ p < 0.01.
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Fig. 2. Thrombogenicity index of abdominal aortic remnant (mean _+ SEM), as assessed at 24, 48, 72, and 96 hours after injection of radiolabeled platelets. Platelet deposition on aortic remnant was higher as compared to the vascular prosthesis or the bifurcation area, possibly reflecting platelet deposition on a diseased vessel wall. There was no significant change during the year. stained positive for factor VIII-related antigen. The admixture of other cell types in cultures was judged to be below 5% from visual assessment and grid counting.
Radioisotopic studies Platelet data. Platelet-bound radioactivity compared to plasma always exceeded 95%. Labeling efficacy exceeded 82%. Graft-platelet interactions. Regions of interest studied were (a) the bifurcation area of the graft, (b) the remnant of the abdominal aorta, and (c) equal portions of the two graft limbs. These ROIs were kept identical between different imaging times (Polaroid films), and TIs were calculated for each R O I at each imaging time. Duplicate estimates of TI for all R O I s were performed with a time interval of at least 3 weeks. The coefficient of variation was 8%.
Scans o f the bifurcation area. An increase in platelet accumulation occurred during the 4 consecutive days. Comparison of scans performed after 1 and 12 months, showed a statistically significant reduction of TI around 30% (p < 0.04) (Fig. 1). Scans o f abdominal aortic remnant. This ROI was defined as the area with the same width as, and located immediately above the bifurcation and below the liver. Thrombogenicity index for the abdominal aortic remnants was higher than for the vascular prosthesis. There were no significant changes with time in values obtained during the first year after surgcry (Fig. 2). Scans o f graft limbs. The AES-treated graft limbs had a lower TI compared to control limbs at all imaging times. Ratios of TI for seeded/control graft limbs o~ days 3 and 4 for the total material, and separately for
Volume 11 Number 3 March 1990
Seedingof aortobifemoralgraBs 407 seeded
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Figs. 3, A-C. Thrombogenicity index of seeded and unseeded graft limbs (mean + SEM), as assessed at 1, 2, 3, and 4 days after injection of radiolabeled platelets, (Fig. 3, A - 1 month, Fig. 3, B - 4 months and Fig. 3, C - 12 months after operation). Seeded graft limbs had a lower TI compared to control at all imaging times. These differences are depicted as: ~-p < 0.05, ~ p < 0.01 and ~ p < 0.001. These differences remained despite a significant reduction in TI for both graft limbs.
Table I. Ratios of TI (seeded limb)/TI (control limb) of aortic bifurcation grafts Total material No.
Mean
SD
19 14
0.73 0.67
0.25 0.24
Day 3
21
0.78
Day 4 12 mo Day 3 Day 4
21
0.82
20 19
0.71 0.71
Patients with ratio 1 year. Early postoperative complications ( < 3 0 days). There were no deaths. Distal perioperative embolization necessitated embolectomy in one patient. One patient had a reversible renal failure in the immediate postoperative period, necessitating temporary hemo-
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filtration. One patient was operated on for bowel obstruction caused by adhesions 3 weeks after graft implantation. Pneumonia developed in two patients, and one had a myocardial infarction in the immediate postoperative period. Seromas in the groin developed in three patients, but in no case any graft infection occurred. Late postoperative complications (30 to 365 days). Three patients have experienced midline incisional hernias. One patient had reoperation because of a false aneurysm, which developed at the distal anastomosis of a concomitantly implanted femoropopliteal reversed vein graft. One patient had a myocardial infarction with cardiac arrest in his home 6 months after surgery. He was successfully resuscitated and was discharged from the hospital 3 weeks later. One patient died 3 months after vascular reconstruction from small bowel obstruction, complicated by bowel perforation, septicemia, and adult respiratory distress syndrome. All graft limbs were patent.
DISCUSSION Autologous vein is the graft of choice in smallcaliber vascular reconstructions. 19 The more favorable patency rates obtained with vein grafts, compared to synthetic vascular prostheses, have been ascribed to the lack Of a VEC lining in the latter, since presently used Dacron or cPTFE grafts do not endothelializc in humans. The growing knowledge of the complex biologic mechanisms involving the EC, especially as regards its crucial importance in thrombosis and hemostasis s has led tO efforts in promoting E C growth on vascular prostheses. Since the introduction of AES in 1978, 6 a number of research groups have demonstrated that this technique applied to Dacron and ePTFE synthetic vascular prostheses in experimental animals resulted in complete endothelialization of graft flow surface within 4 to 8 weeks after implantation. 7,8 Studies of graft-platelet interactions revealed an inverse relation between platelet accumulation/thrombus formation and degree of endothelial coverage of the vascular prosthesis. 2° Apart from the decrease in platelet accumulation on graft flow surface, AES resulted in higher patency rate and improved ability to withstand flow reduction. 9-12 However, existing differences between species regarding factors such as platelet behaviors, blood coagulation, fibrinolytic capacity, and capability of VEC to replicate, make data obtained from animal studies hazardous to extrapolate to the clinical situation? ~
Journal of VASCULAR SURGERY
To address this problem human clinical trials arc needed. So far a limited number of studies, with enzymatic EC harvesting, have been published. However, data are difficult to interpret, because of differences with respect to pretreatment of grafts and end points studied. Herring et al. 22 found a significant difference, in favor of AES, as to patency of 39 seeded ePTFE femoropopliteal grafts compared to i5 control grafts. In a similar study by Walker et al. 23 these results could not be reproduced. However, since patency is affected by a wide variety of factors, efforts to relate occlusion rate to one single variable, for example, seeding, will require very large series of patients. In another study by Zilla et al.24 nine seeded ePTFE grafts implanted in the femoropopliteal position were compared to nine controls. No differences regarding patency or deposition of labeled platelets were noticed, but platelet factors indicating platelet activation (platelet factor 4, betathromboglobulin) were significantly lower in the seeded ,as compared to the control group. However, since patients operated on because of arteriosclerotic occlusions are fikely to have a widespread disease, it is difficult to ascertain to which degree this might affect their platelets. That AES as a concept, is feasible in humans, was proved by biopsy from an ePTFE-graft, where VEC could be demonstrated in the midportion of a graft by scanning electron microscopy. 13 For obvious reasons, biopsies of functioning grafts can only rarely be obtained, and microscopic examinations of failed or failing grafts will be of limited value. An ideal diagnostic tool would not only demonstrate the presence of a VEC lining, but also determine the functional state of the ECs. Since such a probe is not available at the present, we decided to study the eft( 7 of AES with respect to graft-platelet interactions. External gamma imaging studies of platelet accumulation on graft flow surfaces are of clinical interest, since platelet deposition is considered to be the initiator of graft thrombosis. 2s A significant reduction of this phenomenon is possibly beneficial in a critical flow situation in a small-caliber graft. The principal advantage of using a large-caliber bifurcated graft as an experimental model, is its ability to serve as its own control, under the assumption of equal blood flows through both graft limbs. The overall high patency rates for aortoiliac/femoral reconstructions makes it tmlikely to expect any differences regarding this parameter. In the present study patency was 100% for both seeded and control limbs during the first year after surgery.
Voliame 11 Number 3 March 1990
Studies of deposition of radiolabeled platelets on a vascular segment by use of a gamma camera interfaced with a computer, allows the investigator to measure the accumulation of platelets in a defined ROI. Thereby, a ratio between the activity in any ROI mad a reference point can be calculated. In blood, the fall in count rate over time is a resuk of physical decay of the isotope and removal of radiolabeled platelets from the circulation. In case no deposition of platelets takes place on the vascular prosthesis, the recorded activity over the graft area will strictly follow the decline of radioactivity in whole blood, and the ratio between vascular prosthesis/blood will be constant. Studies ofiliac artery segments on healthy volunteers have confirmed this hypothesis? 4 On the other hand, an excessive accumulation of ptatelets will result in an increase of the calculated ratio, which will be an estimate of the deposition of platelets. We have therefore chosen to ey'~ress the ptatelet deposition as a graft/blood ratio. Other investigators have used different vascular segments as reference points 26 and used the daily rise in platelet accumulation as an estimate of graft thrombogenicity. 27 Since the actual patient population often suffers from widespread atherosclerotic vascular disease, the use of such a reference may not be valid. However, dravcbacks of the graft/blood ratio include interindividual differences in platelet mean lifespan and attenuation of isotope, which make the obtained values specific for the individual. Thus the ratios obtained cannot be compared between different individuals. Ptatdet activity registered over a vessel is dependent on flow. z8 In the present study it is unlikely that t h e observed differences in ptatelet deposition were affected by blood flow, since postop~' ative ankde-brachial pressure indexes for seeded and unseeded limbs were similar 40.85 _+ 0.18 vs 0.89 _ 0.17), indicating equal mn-offconditions on both sides. Nor were the results affected by Variations in surface area or volume, since both graft limbs were of equal size: Polaroid pictures were obtained every time a graft scan was analyzed, depicting the ROIs. Thus identical ROIs could be used during analysis of the following scans. Since grafts were clearly visible on each rain scan, we did not use flow scans with technetium to outline the vascular conduit. Our data demonstrate that a significant reduction in measurable platelet-vascular prosthesis interaction occurs during the first year after surgery in aortobifemoral knitted Dacron grafts treated with AES. This difference was constant over time. Also, it is unlikely that the observed difference could be as-
Seeding of aortobifemoralgrafts 409 cribed to the effects of mixing culture medium with autologous blood, since the control limb was shamseeded with culture medium only, The single difference in treatment between the two graft limbs was the inoculation of ECs in the seeded limb. In humans a decrease in platelet deposition occurs on implanted Dacron grafts over time. 29 In man the exact ultrastructural correlation to this reduction of thrombogenicity is not known. Studies on graft healing, carried out on animals, have demonstrated an early inflammatory reaction, followed by a gradual ingrowth of fibroblasts and capillaries into the graft interstices. This fibrous tissue anchors the inner lining, which is called pseudointime or neointima? ° This is composed of collagen, fibrin, fibroblasts, scattered macrophages, monocytes, and leukocytes. In the present work, the bifurcation area was used to study the healing of Dacron grafts implanted according to standard surgical routine. The observed reduction of thrombogenicity for this part of the graft during the first postoperative year was 33%, as compared to 67% reported by Goldman et al.~6However, the first measurement in that study was performed on the second postoperative week; furthermore, the aortic arch was used as reference area, and TI was calculated differently. These considerations might well explain the differences between results obtained. The reduction of graft-platelet interaction in AES-treated Dacron graft segments, observed in the present study, were constant over time and consistent with the hypothesis that AES in humans is feasible. However, these data do not allow us to state that we actually succeeded to cover the graft surface with ECs. A complete endothelialization of graft flow surface is unlikely to occur at our EC seeding densities, since these were lower than those usually required to EC coverage in canine studies ofAES. Howeveri the actual number of ECs required to seed a graft in humans is not known. Second, it is unknown which percentage of EC coverage is required to ~educe platelet accumulation on a graft and curtail events of graft thrombosis in low flow situations: Obviously several other features of EC behavior, unrelated to platelet-endothelial interactions, will affect results of AES. For example, the increased turnover rate of ECs reported to occur even after complete endothelialization of graft surface in baboons, could indicate that AES might even increase production of plateletderived growth factor capable of inducing intimal hyperplasia in treated grafts? t The reduced platelet accumulation reported in this study and the lack of detectable negative side
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effects make us advocate further careful clinical evaluation o f AES, especially for use in distal reconstructions. REFERENCES 1. Szilagyi DE, Elliot JP, Smith RF, Reddy DJ, McPharlin M. A thirty-year survey of the reconstructive surgical treatment of aortoiliac occlusive disease. J VASE SURG 1986;3:421-36. 2. Bergan JJ, Veith FJ, Bernhard VM, et al. Randomization of autogenous vein and polytetrafluoroethylene grafts in femoro-distal i:econstruction. Surgery 1982;92:921-30. 3. Berger K, Sanvage LR, Rao AM, Wood SJ. Healing of arterial prostheses in man: its incompleteness. Ann Surg 1972;175: 118-27. 4. Sauvage LR, Berger K, Beilin LB, Smith JC, Wood SJ, Mansfield PB. Presence of endothelium in an axillary femoral graft of knitted Dacron with an external velour surface. Ann Surg 1975;182:749-53. 5. Gimbrone MA. Vascular endothelium: nature's blood container. In: Gimbrone MA, ed. Vascular endothelium in hemostasis and thrombosis. (Contemporary issues in haemostasis and thrombosis. Vol 2). London: Churchill Livingstone, 1986:1-13. 6. Herring M, Gardner A, Glover J. A single-staged technique for seeding vascular grafts with autogenous endothelium. Surgery 1978;84:498-504. 7. Herring M, Dilley R, Jersild RA Jr, Boxer L, Gardner A, Glover J. Seeding arterial prostheses with vascular endothelium. The nature of the lining. Ann Surg 1979;190:84-90. 8. Shepard AD, Eldrup-Jorgensen J, Keough EM, et al. Endothelial cell seeding of small-caliber synthetic grafts in the baboon. Surgery 1986;99:318-26. 9. Clagett GP, Burkel WE, Sharefldn JB, et al. Platelet reactivity in vivo in dogs with arterial prostheses seeded with endothelial cells. Lab Invest 1984;69:632-6. 10. Schmidt SP, Hunter TJ, Sharp WV, Malindzak GS, Evancho MM. Endothelial-seeded four-millimeter Dacron vascular grafts: Effects on blood flow manipulation through the grafts. J VAsc SURG 1984;1:434-41. 11. Allen BT, Long JA, Clark RE, Sicard GA, Hopkins KT, Welch MJ. Influence of endothelial cell seeding on platelet deposition and patency in small diameter Dacron arterial grafts: J VASE SUWG1984;1:224-33. 12. Stanley JC, Burkel WE, Ford JW, et al. Enhanced patency of small-diameter, externally supported Dacron iliofemoral grafts seeded with endothelial cells. Surgery 1982;92:9941005. 13. Herring M, Baughman S, Glovcr J. Endothelium develops on seeded human arterial prosthesis: A brief clinical note. J VASE SURG 1985;2:727-30. 14. Ortcnwall P, Wadenvik H, Kutti J, R.isberg B. Reduction in deposition of indium Ill-labeled platelets after autologous endothelial seeding of Dacron aortic bifurcation gratis in humans: a preliminary report. J VASE SURG 1987;6:17-25. 15. Watldns MT, Sharefldn JB, Zajtchuk R, ct al. Adult human saphenous vein endothelial cells: Assessment of their repro-
16. 17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
ductive capacity for use in endothelial seeding of vascula~ prostheses. J Surg Res 1984;36:588-96. larrel B, Lcvine E, Shapiro S, et al. Human adult endothelial cell growth in culture. J VASE SURG 1984;1:757-64. Baughman S, Herring M, Glover l, Dilley R. The peroxidase antiperoxidase staining of factor VIII-related antigen on cultured endothelial cells. J Biomed Mater Res 1984;18:561-6. Stratton JR, Thiele BL, Ritchie JL. Platelet deposition on Dacron aortic bifurcation grafts in man: quantitation with Indium 111 platelet imaging. Circ 1982;66:1287-93. Veith FJ, Gupta SK, Ascer E, et al. Six-year prospective multicenter randomized comparison of autologous saphenous vein and expanded polytetrafluoroethylene grafts in infrainguinal arterial reconstructions. J VASE SURG1986;3:104-14. Belden TA, Schmidt SP, Falkow LJ, Sharp WV. Endothelial cell seeding of small-diameter vascular grafts. Trans Am Soc Artif Intern Organs 1982;28:173-7. (3rtenwall P, Bylock A, Kje/lstr6m BT, Risberg B. Seeding of ePTFE carotid interposition grafts in sheep and dogs: species-dependent results. Surgery 1988; 103:199-205. Herring MB, Compton RS, Garner AL, LeGrand DR. Clinical experiences with endothelial seeding in Indianapolis. In: Zilla PP, Fasol RD, Deutsch M, eds. Endothelialization of vascular grafts. Basel: Karger, 1987:218-24. Walker MG, Thomson GJL, Shaw j'W. Endothelial cell seeded versus non-seeded ePTFE grafts in patients with severe peripheral vasaalar disease. In: Zilla PP, Fasot RD, Deutsch M, eds. Endothelialization of vascular grafts. Basel: Karger, 1987:245-48. Zilla P, Fasol R, Deutsch M, et al. Endothelial cell seeding of polytetrafluoroethylene grafts in humans: a preliminary report. J VASE SURG 1987;6:535-41. j'osa M, Lie )'T, Bianco RL, Kaye MP. Reduction of thrombosis in canine coronaty bypass vein grafts with dipyridamote and aspirin. Am J Cardiol 1981;47:1248-54. Goldman M. Norcott HC, Hawker RJ, Drolc Z, McCollum CN. Platelet accumulation on mature Dacron grafts in man. Br J Surg 1982;69[suppl]:S38-40. Goldman MD, Simpson D, Hawker RJ, Norcott, McColltun CN. Aspirin and dipyridamole reduce platelet deposition on prosthetic femoro-popliteal grafts in man. Ann Surg 1983;198:713-6. Eldrup-Jorgensen J, Mackey WC, Connolly RJ, et al. Ev,~ ) nation of arterial prostheses in a baboon ex vivo shunt: the effect of graft material and flow on platelet deposition. Am J Surg 1985;150;185-90. McCollum CN, Kester RC, Rajah SM, Learoyd P, Pepper M. Arterial graft maturation: the duration of thrombotic activity in Dacron aortobifemoral grafts mesured by platelet and fibrinogen kinetics. Br J Surg 1981;68:61-4. Burkel WE, Vinter DW, Ford JW, Kahn Rift, Graham LM, Stanley JC. Sequential studies of healing in endothelial seeded vascular prostheses: histologic and ultrastructure characteristics of graft incorporation. J Surg Res 1981;30:305-24. Zacharias RK, Kirkman TR, Clowes AW. Mechanisms of healing in synthetic grafts. ]" VASC SURG 1987;6:429-36.
From the Departmentsof Surgery~and InternalMedicine,~ University of G6teborg. Supported by grants from the MedicalFaculty,Universityof G6teborg, The Swedish Societyfor Medical Research, Salus InsuranceCompany~The SwedishSocietyof Medicine,The Medical Societyof G6teborg, The Swedish National Association against Heart and Chest Diseases, and The Swedish Medical Research Council (Project 00660). Reprint requests: Per Ortenwall, MD, Department of Surgery, University of G6teborg, Ostra sjukhuset, CK, S-416 85 G6teborg, Sweden. 24/1/17241
willpredominate. The lack of VECs in synthetic vascular prostheses make these grafts more likely to fail from thrombotic occlusion, especially when the other components in Virchow's triad (blood flow and composition) are altered in an unfavorable way. The concept of autologous endothelial seeding (AES), as a mean of reducing thrombogenicity of synthetic grafts, was introduced by Herring et al.6 in 1978. In experiments carried out on dogs and baboons intraoperative AES produced a complete luminal VEC lining of the vascular prosthesis within 4 to 8 weeks after graft implantation. 7'8 Platelet accumulation was substantially reduced and mean platelet life-span normalized, as measured several weeks after surgery. 9 Furthermore, both patency rates and resistance to thrombotic occlusion, induced by flow reduction, were enhanced, t°q2 In humans only one report is available, demonstrating a VEC-like lining (by scamling electron microscopy) in an AES-treated human vascular prosthesis. 13 Obviously this method cannot be used for evaluation of AES, except in rare cases. Furthermore a biologic probe demonstrating the presence of fimctionally intact endothelium is not yet available. So far the effect of AES has to be assessed by indirect methods, such as the accumulation of platelets labeled with indium 111 (rain) on grafts. The purpose of the present study was to evaluate whether AES could reduce the thrombogenicity o f vascular prostheses in humans. We report a signifi403
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cant reduction in the accumulation of labeled platelets on AES-treated segments of aortic bifurcation grafts, even 1 year after surgery.
MATERIAL AND METHODS Twenty-two patients (19 men and three women) scheduled for abdominal aortic reconstruction, by use of a bifurcated vascular prosthesis were studied, Their mean age was 66.7 years __+5.6 years (range 52 to 74 years). The ankle/brachial pressure index was calculated for each limb before and after operation. Eleven of the patients were operated on because of occlusive disease and 11 because of aneurysms. Three patients had previous vascular surgery (one carotid, two iliac thromboendarterectomies), and one patient had an iliac artery balloon angioplasty. Minimum time lapse between previous vascular intervention was 2 years. All patients had a smoking history, and one had diabetes mellitus. Nine patients were on antihypertensive drugs. All studies were reviewed and approved by the Ethical Committee of the University of G6teborg. Informed consent was obtained from all patients. All techniques used in the present work have been described in detail previously14and are therefore only summarized herein: Surgical procedure Prophylactic antibiotic treatment was given as cefuroxime 1.5 gm intravenously × 3 during the day of surgery. Routine anticoagulation wag 500 ml of dextran 40 preoperatively, 500 ml during the first 8 postoperative hours, followed by 500 ml daily over 4 days, All patients were operated on by the same two vascular surgeons. The cell-harvesting procedure (see herein) was performed in the operating room and took between 45 and 60 minutes. During this time the graft was preclotted with autologous blood, and the proximal anastomosis was performed. Noncrushing vascular clamps were then placed across the graft limbs just distal to the bifurcation. Which graft limb to be seeded, and the order in which to construct the distal anastomoses, was decided randomly. The EC suspension was divided into two 2 ml aliquots. These aliquots were sequentially mixed with equal volumes of autologous whole blood and were injected 5 minutes apart into the graft limb to be seeded. The cells were allowed to settle in the graft limb during completion of the distal anastomosis. Just before completion of the anastomosis, clamps were released and blood was allowed to flush through the graft limb. The nonseeded (con-
Journal of VASCULAR SURGERY
trol) graft limb was treated identically, except that blood was mixed with culture medium only, omitting the ECs. Twenty-one Bionit (USCI Surgical Products, C.R. Bard Inc, Billerica, Mass.) and one Vascutek VP 1200K (Vascutek Ltd, Ayr, Scotland) were implanted. The approach to the abdominal aorta was transabdominal in 18 patients and extraperitoneal in four. Two femoropopliteal reversed-vein bypass graftings and one renal artery reconstruction were performed in three patients at the time of the aortic reconstruction. Thirty-three of the distal anastomoses were constructed at the common or external iliac artery, and 11 were constructed at the common or deep femoral artery. Cell harvesting and culture procedure Five to ten centimeters of distal calf saphenous vein was excised. The vein was cannulated with bi .;at tipped arterial catmulas and flushed with chilled Hank's buffered saline solution. Prewarmed collagenase solution (0.1%, type I) in phosphate-buffered saline was injected into the vein, which was incubated in sterile phosphate buffered saline solution at 37 ° C for 10 to 15 minutes. The effluent was flushed into a sterile centrifuge tube by use of 10 ml of chilled complete medium. All fluids were sterilized by passage through 0.22 ~m filters. After cell harvesting the vein segment was cut open, and the luminal area of the vein was measured. The effluent was flushed through the vein two additional times and centrifuged at 180 g for 10 minutes. The resulting cell pellet was resuspended in 5 ml of complete medium. Four milliliters of this suspension was used for see.,ding of the vascular prosthesis. The remaining 1 r~ll was taken to the cell culture laboratory. Two hundred cubic millimeters were taken for cell counting in a hemocytometer. The other portion of the cell suspension was inoculated onto plastic tissue culture wells, precoated with human fibronectin (10 ~g/cm 2 dish area). The cultures were held at 37 ° C in a humidified 5% carbon dioxide environment, and the culture medium was changed every 3 days. T M Cultures were studied with an inverted phase contrast microscope, and the identity of the cells was checked with a peroxidase-antiperoxidase-staining test for factor VIII-related antigen. 17 Cell viability was checked in the last 16 patients by calculation of plating efficiency, is Radioisotopic procedures These procedures were performed 1, 4, and 3.-", months after surgery.
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Platelet separation and labeling procedure. All patients were forbidden to ingest any aspirin or other nonsteroidal antiinflammatory drugs 10 days before the study. Seventeen milliliters of venous blood was obtained with a 19-gauge butterfly needle into a disposable syringe, containing 3 ml of acid citrate dextrose N I H formula A (ACD-A) as anticoagulant. The anticoagulated blood was transferred into a 30 ml screwcap polycarbonate tube. Platelet-rich plasma (PRP) was obtained by centrifugation of the blood at 260 g for 15 minutes. Platelet rich plasma was then transferred into another 30 ml polycarbonate tube, and the pH was adjusted to 6.3 to 6.5, by adding I ml of ACD-A per 10 ml of PRP. The platelets were centrifuged at 800 g for 10 minutes. The supernatant platelet poor plasma (PPP) was removed for later Use. The platelet pellet was washed with 5 ml modified Tyrode's solution, at 37 ° C. The platelets were resuspended in 1 ml modified Tyrode's solff~ ,on, and the required amount of nlIn oxine was added to the suspension. After incubation for 2 minutes with the isotope at 37 ° C, 2 ml of PPP were added, and the mixture was centrifuged at 8 0 0 g for 10 minutes. The supematant was removed, and the platelet pellet was finally resuspended in 5 ml of PPP. For calculation of labeling efficiency, the plateletbound radioactivity and the radioactivity in the supernatant was measured in a dose calibrator (Radioisotope Calibrator CRC-5RB, Capintec, Montvale, N.J.). 200 txCi was injected into the patients. All procedures were carried out aseptically at room temperature, if not otherwise stated. Graft-platelet interaction. Anterior scanning of the abdomen was performed at 24, 48, 72, and 96 hours after injection of the labeled platelets, with a s~mtillation camera (General Electric; MaxiCamera II [General Electric Co., Medical Systems Div., Milwaukee, Wis.]) interfaced with a computer (Digital Equipment; [Digital Equipment International Ltd., Galway, Ireland]PDP 11-34). Both photo peaks emitted by rain (173 and 247 keV) were collected through a medium energy (max 370 keV) parallelhole collimator, by use of a 15% window, during six consecutive 5 minute periods each time. For visual assessment, images and depicted regions of interest (ROI) were recorded on Polaroid film. The images were stored on computer disc for data processing, by means of a 64 X 64 matrix system, dividing the image into 4096 pixels (Software: Gamma 11 [Digital Equipment Corp., Marlboro, Mass.). These computer-generated gamma images were assessed by means of a color-coded scale for platelet deposition S~2ensity. To correct the image for contributing radiation from the pelvic and vertebral bone marrow the
Seeding of aortobifemoralgrafts 405 background activity from this area was subtracted. 18 Thc matrix system enabled calculation of the accumulated number of counts from any chosen ROI in the image. By dividing with the actual number of pixels necessary to cover the ROI, an average count number per square unit of the ROI was obtained. Division of this average count number by the number of counts obtained from 0. l ml of whole blood, collected at the same time and counted for 200 seconds in a crystal scintillation counter, gave a graft/blood ratio. This ratio, corrected for both isotope decay and platelet senescence, we have designated the thrombogenicity index (TI) using the formula: TI = 100 x Counts in ROI - Background Counts (Number of pixels in ROI) x (Whole blood counts) By keeping the ROIs identical between different imaging times, TI could be compared as a function of time. Scans were viewed in a blinded fashion regarding the seeded or unseeded status of graft limbs. To correct for variations in distance between the scintillation camera and the graft limbs, all calculations of TI were performed on equal segments of limbs to be compared.
Follow-up In association with vascular prosthesis scanning, the paticnts were assessed by clinical and femoral Doppler examination. If these results indicated any postoperative complication, additional angiograms were performed. Statistical analysis Statistical analysis was performcd with a paired t test to compare intraindividual differcnccs in TI at the same imaging time, and an unpaired t test to compare intraindividual changes of TI between differcnt imaging timcs. Coefficient of variation was used for testing of intraobserver reliability. All values are given as mean _+ SD, unless otherwise stated. RESULTS Cell harvesting and culture An average of 142 + 96 × 103 cells were harvested from donor veins with a mean area of 5.9 _+ 1.5 cm. g The mean vascular prosthesis area seeded was 41 + 10 cm2; resulting in a mean veinto-vascular prosthesis area ratio of 0.15 + 0.05~ The mean number of viable cells (cells capable of attaching to culture dishes during the first 24 houis after surgery) seeded/cm 2 graft was 2.2 -+ 1.5 x 10. 3 This estimate of viability was based on 16 cultures: Cell cultures morphologically resembled ECs and
406
Journal of VASCULAR SURGERY
Ortenwall et al.
1 xont~,
84
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4 Nontl~s
~
12
48
mont~hs
72
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Fig. 1. Thrombogenicity index of the bifurcation area (mean __ SEM), as assessed at 24, 48, 72, and 96 hours after injection of radiolabeled platelets. A reduction occurred during the first year after implantation. The differences are depicted as: ~p < 0.05, ~ p < 0.01.
1 ~onth
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Fig. 2. Thrombogenicity index of abdominal aortic remnant (mean _+ SEM), as assessed at 24, 48, 72, and 96 hours after injection of radiolabeled platelets. Platelet deposition on aortic remnant was higher as compared to the vascular prosthesis or the bifurcation area, possibly reflecting platelet deposition on a diseased vessel wall. There was no significant change during the year. stained positive for factor VIII-related antigen. The admixture of other cell types in cultures was judged to be below 5% from visual assessment and grid counting.
Radioisotopic studies Platelet data. Platelet-bound radioactivity compared to plasma always exceeded 95%. Labeling efficacy exceeded 82%. Graft-platelet interactions. Regions of interest studied were (a) the bifurcation area of the graft, (b) the remnant of the abdominal aorta, and (c) equal portions of the two graft limbs. These ROIs were kept identical between different imaging times (Polaroid films), and TIs were calculated for each R O I at each imaging time. Duplicate estimates of TI for all R O I s were performed with a time interval of at least 3 weeks. The coefficient of variation was 8%.
Scans o f the bifurcation area. An increase in platelet accumulation occurred during the 4 consecutive days. Comparison of scans performed after 1 and 12 months, showed a statistically significant reduction of TI around 30% (p < 0.04) (Fig. 1). Scans o f abdominal aortic remnant. This ROI was defined as the area with the same width as, and located immediately above the bifurcation and below the liver. Thrombogenicity index for the abdominal aortic remnants was higher than for the vascular prosthesis. There were no significant changes with time in values obtained during the first year after surgcry (Fig. 2). Scans o f graft limbs. The AES-treated graft limbs had a lower TI compared to control limbs at all imaging times. Ratios of TI for seeded/control graft limbs o~ days 3 and 4 for the total material, and separately for
Volume 11 Number 3 March 1990
Seedingof aortobifemoralgraBs 407 seeded
L'q~O o o n ~ s , o l
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seeded
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Figs. 3, A-C. Thrombogenicity index of seeded and unseeded graft limbs (mean + SEM), as assessed at 1, 2, 3, and 4 days after injection of radiolabeled platelets, (Fig. 3, A - 1 month, Fig. 3, B - 4 months and Fig. 3, C - 12 months after operation). Seeded graft limbs had a lower TI compared to control at all imaging times. These differences are depicted as: ~-p < 0.05, ~ p < 0.01 and ~ p < 0.001. These differences remained despite a significant reduction in TI for both graft limbs.
Table I. Ratios of TI (seeded limb)/TI (control limb) of aortic bifurcation grafts Total material No.
Mean
SD
19 14
0.73 0.67
0.25 0.24
Day 3
21
0.78
Day 4 12 mo Day 3 Day 4
21
0.82
20 19
0.71 0.71
Patients with ratio 1 year. Early postoperative complications ( < 3 0 days). There were no deaths. Distal perioperative embolization necessitated embolectomy in one patient. One patient had a reversible renal failure in the immediate postoperative period, necessitating temporary hemo-
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filtration. One patient was operated on for bowel obstruction caused by adhesions 3 weeks after graft implantation. Pneumonia developed in two patients, and one had a myocardial infarction in the immediate postoperative period. Seromas in the groin developed in three patients, but in no case any graft infection occurred. Late postoperative complications (30 to 365 days). Three patients have experienced midline incisional hernias. One patient had reoperation because of a false aneurysm, which developed at the distal anastomosis of a concomitantly implanted femoropopliteal reversed vein graft. One patient had a myocardial infarction with cardiac arrest in his home 6 months after surgery. He was successfully resuscitated and was discharged from the hospital 3 weeks later. One patient died 3 months after vascular reconstruction from small bowel obstruction, complicated by bowel perforation, septicemia, and adult respiratory distress syndrome. All graft limbs were patent.
DISCUSSION Autologous vein is the graft of choice in smallcaliber vascular reconstructions. 19 The more favorable patency rates obtained with vein grafts, compared to synthetic vascular prostheses, have been ascribed to the lack Of a VEC lining in the latter, since presently used Dacron or cPTFE grafts do not endothelializc in humans. The growing knowledge of the complex biologic mechanisms involving the EC, especially as regards its crucial importance in thrombosis and hemostasis s has led tO efforts in promoting E C growth on vascular prostheses. Since the introduction of AES in 1978, 6 a number of research groups have demonstrated that this technique applied to Dacron and ePTFE synthetic vascular prostheses in experimental animals resulted in complete endothelialization of graft flow surface within 4 to 8 weeks after implantation. 7,8 Studies of graft-platelet interactions revealed an inverse relation between platelet accumulation/thrombus formation and degree of endothelial coverage of the vascular prosthesis. 2° Apart from the decrease in platelet accumulation on graft flow surface, AES resulted in higher patency rate and improved ability to withstand flow reduction. 9-12 However, existing differences between species regarding factors such as platelet behaviors, blood coagulation, fibrinolytic capacity, and capability of VEC to replicate, make data obtained from animal studies hazardous to extrapolate to the clinical situation? ~
Journal of VASCULAR SURGERY
To address this problem human clinical trials arc needed. So far a limited number of studies, with enzymatic EC harvesting, have been published. However, data are difficult to interpret, because of differences with respect to pretreatment of grafts and end points studied. Herring et al. 22 found a significant difference, in favor of AES, as to patency of 39 seeded ePTFE femoropopliteal grafts compared to i5 control grafts. In a similar study by Walker et al. 23 these results could not be reproduced. However, since patency is affected by a wide variety of factors, efforts to relate occlusion rate to one single variable, for example, seeding, will require very large series of patients. In another study by Zilla et al.24 nine seeded ePTFE grafts implanted in the femoropopliteal position were compared to nine controls. No differences regarding patency or deposition of labeled platelets were noticed, but platelet factors indicating platelet activation (platelet factor 4, betathromboglobulin) were significantly lower in the seeded ,as compared to the control group. However, since patients operated on because of arteriosclerotic occlusions are fikely to have a widespread disease, it is difficult to ascertain to which degree this might affect their platelets. That AES as a concept, is feasible in humans, was proved by biopsy from an ePTFE-graft, where VEC could be demonstrated in the midportion of a graft by scanning electron microscopy. 13 For obvious reasons, biopsies of functioning grafts can only rarely be obtained, and microscopic examinations of failed or failing grafts will be of limited value. An ideal diagnostic tool would not only demonstrate the presence of a VEC lining, but also determine the functional state of the ECs. Since such a probe is not available at the present, we decided to study the eft( 7 of AES with respect to graft-platelet interactions. External gamma imaging studies of platelet accumulation on graft flow surfaces are of clinical interest, since platelet deposition is considered to be the initiator of graft thrombosis. 2s A significant reduction of this phenomenon is possibly beneficial in a critical flow situation in a small-caliber graft. The principal advantage of using a large-caliber bifurcated graft as an experimental model, is its ability to serve as its own control, under the assumption of equal blood flows through both graft limbs. The overall high patency rates for aortoiliac/femoral reconstructions makes it tmlikely to expect any differences regarding this parameter. In the present study patency was 100% for both seeded and control limbs during the first year after surgery.
Voliame 11 Number 3 March 1990
Studies of deposition of radiolabeled platelets on a vascular segment by use of a gamma camera interfaced with a computer, allows the investigator to measure the accumulation of platelets in a defined ROI. Thereby, a ratio between the activity in any ROI mad a reference point can be calculated. In blood, the fall in count rate over time is a resuk of physical decay of the isotope and removal of radiolabeled platelets from the circulation. In case no deposition of platelets takes place on the vascular prosthesis, the recorded activity over the graft area will strictly follow the decline of radioactivity in whole blood, and the ratio between vascular prosthesis/blood will be constant. Studies ofiliac artery segments on healthy volunteers have confirmed this hypothesis? 4 On the other hand, an excessive accumulation of ptatelets will result in an increase of the calculated ratio, which will be an estimate of the deposition of platelets. We have therefore chosen to ey'~ress the ptatelet deposition as a graft/blood ratio. Other investigators have used different vascular segments as reference points 26 and used the daily rise in platelet accumulation as an estimate of graft thrombogenicity. 27 Since the actual patient population often suffers from widespread atherosclerotic vascular disease, the use of such a reference may not be valid. However, dravcbacks of the graft/blood ratio include interindividual differences in platelet mean lifespan and attenuation of isotope, which make the obtained values specific for the individual. Thus the ratios obtained cannot be compared between different individuals. Ptatdet activity registered over a vessel is dependent on flow. z8 In the present study it is unlikely that t h e observed differences in ptatelet deposition were affected by blood flow, since postop~' ative ankde-brachial pressure indexes for seeded and unseeded limbs were similar 40.85 _+ 0.18 vs 0.89 _ 0.17), indicating equal mn-offconditions on both sides. Nor were the results affected by Variations in surface area or volume, since both graft limbs were of equal size: Polaroid pictures were obtained every time a graft scan was analyzed, depicting the ROIs. Thus identical ROIs could be used during analysis of the following scans. Since grafts were clearly visible on each rain scan, we did not use flow scans with technetium to outline the vascular conduit. Our data demonstrate that a significant reduction in measurable platelet-vascular prosthesis interaction occurs during the first year after surgery in aortobifemoral knitted Dacron grafts treated with AES. This difference was constant over time. Also, it is unlikely that the observed difference could be as-
Seeding of aortobifemoralgrafts 409 cribed to the effects of mixing culture medium with autologous blood, since the control limb was shamseeded with culture medium only, The single difference in treatment between the two graft limbs was the inoculation of ECs in the seeded limb. In humans a decrease in platelet deposition occurs on implanted Dacron grafts over time. 29 In man the exact ultrastructural correlation to this reduction of thrombogenicity is not known. Studies on graft healing, carried out on animals, have demonstrated an early inflammatory reaction, followed by a gradual ingrowth of fibroblasts and capillaries into the graft interstices. This fibrous tissue anchors the inner lining, which is called pseudointime or neointima? ° This is composed of collagen, fibrin, fibroblasts, scattered macrophages, monocytes, and leukocytes. In the present work, the bifurcation area was used to study the healing of Dacron grafts implanted according to standard surgical routine. The observed reduction of thrombogenicity for this part of the graft during the first postoperative year was 33%, as compared to 67% reported by Goldman et al.~6However, the first measurement in that study was performed on the second postoperative week; furthermore, the aortic arch was used as reference area, and TI was calculated differently. These considerations might well explain the differences between results obtained. The reduction of graft-platelet interaction in AES-treated Dacron graft segments, observed in the present study, were constant over time and consistent with the hypothesis that AES in humans is feasible. However, these data do not allow us to state that we actually succeeded to cover the graft surface with ECs. A complete endothelialization of graft flow surface is unlikely to occur at our EC seeding densities, since these were lower than those usually required to EC coverage in canine studies ofAES. Howeveri the actual number of ECs required to seed a graft in humans is not known. Second, it is unknown which percentage of EC coverage is required to ~educe platelet accumulation on a graft and curtail events of graft thrombosis in low flow situations: Obviously several other features of EC behavior, unrelated to platelet-endothelial interactions, will affect results of AES. For example, the increased turnover rate of ECs reported to occur even after complete endothelialization of graft surface in baboons, could indicate that AES might even increase production of plateletderived growth factor capable of inducing intimal hyperplasia in treated grafts? t The reduced platelet accumulation reported in this study and the lack of detectable negative side
410
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effects make us advocate further careful clinical evaluation o f AES, especially for use in distal reconstructions. REFERENCES 1. Szilagyi DE, Elliot JP, Smith RF, Reddy DJ, McPharlin M. A thirty-year survey of the reconstructive surgical treatment of aortoiliac occlusive disease. J VASE SURG 1986;3:421-36. 2. Bergan JJ, Veith FJ, Bernhard VM, et al. Randomization of autogenous vein and polytetrafluoroethylene grafts in femoro-distal i:econstruction. Surgery 1982;92:921-30. 3. Berger K, Sanvage LR, Rao AM, Wood SJ. Healing of arterial prostheses in man: its incompleteness. Ann Surg 1972;175: 118-27. 4. Sauvage LR, Berger K, Beilin LB, Smith JC, Wood SJ, Mansfield PB. Presence of endothelium in an axillary femoral graft of knitted Dacron with an external velour surface. Ann Surg 1975;182:749-53. 5. Gimbrone MA. Vascular endothelium: nature's blood container. In: Gimbrone MA, ed. Vascular endothelium in hemostasis and thrombosis. (Contemporary issues in haemostasis and thrombosis. Vol 2). London: Churchill Livingstone, 1986:1-13. 6. Herring M, Gardner A, Glover J. A single-staged technique for seeding vascular grafts with autogenous endothelium. Surgery 1978;84:498-504. 7. Herring M, Dilley R, Jersild RA Jr, Boxer L, Gardner A, Glover J. Seeding arterial prostheses with vascular endothelium. The nature of the lining. Ann Surg 1979;190:84-90. 8. Shepard AD, Eldrup-Jorgensen J, Keough EM, et al. Endothelial cell seeding of small-caliber synthetic grafts in the baboon. Surgery 1986;99:318-26. 9. Clagett GP, Burkel WE, Sharefldn JB, et al. Platelet reactivity in vivo in dogs with arterial prostheses seeded with endothelial cells. Lab Invest 1984;69:632-6. 10. Schmidt SP, Hunter TJ, Sharp WV, Malindzak GS, Evancho MM. Endothelial-seeded four-millimeter Dacron vascular grafts: Effects on blood flow manipulation through the grafts. J VAsc SURG 1984;1:434-41. 11. Allen BT, Long JA, Clark RE, Sicard GA, Hopkins KT, Welch MJ. Influence of endothelial cell seeding on platelet deposition and patency in small diameter Dacron arterial grafts: J VASE SUWG1984;1:224-33. 12. Stanley JC, Burkel WE, Ford JW, et al. Enhanced patency of small-diameter, externally supported Dacron iliofemoral grafts seeded with endothelial cells. Surgery 1982;92:9941005. 13. Herring M, Baughman S, Glovcr J. Endothelium develops on seeded human arterial prosthesis: A brief clinical note. J VASE SURG 1985;2:727-30. 14. Ortcnwall P, Wadenvik H, Kutti J, R.isberg B. Reduction in deposition of indium Ill-labeled platelets after autologous endothelial seeding of Dacron aortic bifurcation gratis in humans: a preliminary report. J VASE SURG 1987;6:17-25. 15. Watldns MT, Sharefldn JB, Zajtchuk R, ct al. Adult human saphenous vein endothelial cells: Assessment of their repro-
16. 17.
18.
19.
20.
21.
22.
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24.
25.
26.
27.
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ductive capacity for use in endothelial seeding of vascula~ prostheses. J Surg Res 1984;36:588-96. larrel B, Lcvine E, Shapiro S, et al. Human adult endothelial cell growth in culture. J VASE SURG 1984;1:757-64. Baughman S, Herring M, Glover l, Dilley R. The peroxidase antiperoxidase staining of factor VIII-related antigen on cultured endothelial cells. J Biomed Mater Res 1984;18:561-6. Stratton JR, Thiele BL, Ritchie JL. Platelet deposition on Dacron aortic bifurcation grafts in man: quantitation with Indium 111 platelet imaging. Circ 1982;66:1287-93. Veith FJ, Gupta SK, Ascer E, et al. Six-year prospective multicenter randomized comparison of autologous saphenous vein and expanded polytetrafluoroethylene grafts in infrainguinal arterial reconstructions. J VASE SURG1986;3:104-14. Belden TA, Schmidt SP, Falkow LJ, Sharp WV. Endothelial cell seeding of small-diameter vascular grafts. Trans Am Soc Artif Intern Organs 1982;28:173-7. (3rtenwall P, Bylock A, Kje/lstr6m BT, Risberg B. Seeding of ePTFE carotid interposition grafts in sheep and dogs: species-dependent results. Surgery 1988; 103:199-205. Herring MB, Compton RS, Garner AL, LeGrand DR. Clinical experiences with endothelial seeding in Indianapolis. In: Zilla PP, Fasol RD, Deutsch M, eds. Endothelialization of vascular grafts. Basel: Karger, 1987:218-24. Walker MG, Thomson GJL, Shaw j'W. Endothelial cell seeded versus non-seeded ePTFE grafts in patients with severe peripheral vasaalar disease. In: Zilla PP, Fasot RD, Deutsch M, eds. Endothelialization of vascular grafts. Basel: Karger, 1987:245-48. Zilla P, Fasol R, Deutsch M, et al. Endothelial cell seeding of polytetrafluoroethylene grafts in humans: a preliminary report. J VASE SURG 1987;6:535-41. j'osa M, Lie )'T, Bianco RL, Kaye MP. Reduction of thrombosis in canine coronaty bypass vein grafts with dipyridamote and aspirin. Am J Cardiol 1981;47:1248-54. Goldman M. Norcott HC, Hawker RJ, Drolc Z, McCollum CN. Platelet accumulation on mature Dacron grafts in man. Br J Surg 1982;69[suppl]:S38-40. Goldman MD, Simpson D, Hawker RJ, Norcott, McColltun CN. Aspirin and dipyridamole reduce platelet deposition on prosthetic femoro-popliteal grafts in man. Ann Surg 1983;198:713-6. Eldrup-Jorgensen J, Mackey WC, Connolly RJ, et al. Ev,~ ) nation of arterial prostheses in a baboon ex vivo shunt: the effect of graft material and flow on platelet deposition. Am J Surg 1985;150;185-90. McCollum CN, Kester RC, Rajah SM, Learoyd P, Pepper M. Arterial graft maturation: the duration of thrombotic activity in Dacron aortobifemoral grafts mesured by platelet and fibrinogen kinetics. Br J Surg 1981;68:61-4. Burkel WE, Vinter DW, Ford JW, Kahn Rift, Graham LM, Stanley JC. Sequential studies of healing in endothelial seeded vascular prostheses: histologic and ultrastructure characteristics of graft incorporation. J Surg Res 1981;30:305-24. Zacharias RK, Kirkman TR, Clowes AW. Mechanisms of healing in synthetic grafts. ]" VASC SURG 1987;6:429-36.
