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The influence of endothelial seeding and platelet inhibition on the patency of ePTFE grafts used to replace small arteries—an experimental study
EurJ VascSurg 2,365-370 (1988)
The Influence of Endothelial Seeding and Platelet Inhibition on the Patency of ePTFE Grafts Used to Replace Small Arteries-an Experimental Study d. B. C a m p b e l l , 1 J. L. G I o v e r 2 a n d B. H e r r i n g 3
1Assistant Professor of Surgery, Louisiana State University Medical Center, Shreveport, Louisiana, U.S.A., 2Chief Surgical Services, William Beaumont Hospital, Royal Oak, Michigan 4 8 0 7 2 , U.S.A., 3Associate Clinical Professor of Surgery, Indiana University Medical Center, Indianapolis, Indiana, U.S.A. Previous studies on the influence of endothelial seeding on graft patency have shown that significant improvement has only been achieved with Dacron and an experimental, porous PTFE graft. Methods of assessing patency or showing statistical significance could be questioned in some of these studies. To determine if the combination of endothelial cell seeding and antiplatelet agents would improve patency in small-diameter, commercially available expanded polytetrafluorethylene (ePTFE) grafts, we placed ePTFE grafts into the left carotid position in two groups of mongrel dogs. All grafts were 4 mm internal diameter and 60 mm long, and were interposed in an end-to-end fashion. Both groups received aspirin (80 mg daily) and dipyridamole (25 mg daily) for 14 days, beginning immediately prior to surgery. In Group I (n = 12), the grafts were seeded with enzymatically harvested autogenous endothelium just prior to implanatation; in Group II (n = 10) the grafts were not seeded. AH grafts were removed at 30 days. Seven of 12 (58%) seeded grafts, but only one control graft (10%) remained patent (P=O.03). Six of the seven seeded grafts exhibited surface endothelium, but the single patent control graft did not. The inner capsule of the seeded grafts consisted of a monolayer of endothelium and a thin acellular subendothelial matrix with an average thickness of 8 p. We conclude that a 14-day course of anti-platelet agents combined with endothelial seeding of ePTFE resulted in significantly improved patency compared to controls, with most patent, seeded grafts developing an endothelial lining in 30 days. Key Words: Endothelial seeding; PTFE; platelet inhibitions.
Introduction
Endothelium repairs itselL inhibits platelet aggregation and adherence by the elaboration of prostacyclin,1 and initiates clot lysis by the elaboration of plasminogen activator.2 Assuming that these properties confer resistance to thrombosis, considerable effort has been directed toward the development of endothelialised vascular prostheses by "seeding" grafts with endothelial cells at the time of implantation. Synthetic conduits implanted without endothelial seeding develop a surface consisting of a fibrin-platelet Reprint requests should be sent to: John L. Glover, Department of Surgery, William BeaumontHospital, 3601 West 13 MileRoad, RoyalOak, Michigan 48072, U.S.A. 0950-821X/88/060365 +06 $03,00/0 © 1988 Grune &Stratton Ltd
meshwork; and platelets deposit onto these surfaces for up to nine months, as measured by decreased platelet survival time. s The development of an endothelial lining by these grafts has been shown to decrease platelet consumption in proportion to the amount of surface endothelialised. 4 Consequently, endothelium-lined conduits might be expected to have a higher patency rate than unlined conduits of the same dimensions. Studies of production of such linings show that inoculation of Dacron and ePTFE grafts with autologous endothelium harvested at the time of implantation results in the development of an endothelial lining, usually by four weeks, s s In small-diameter Dacron grafts, however, seeding alone does not improve patency 9 because occlusion occurs before the seeded cells evolve into a functioning lining. The addition of a two-week course of
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antiplatelet agents prevents thrombosis during this interval and results in improved patency in a canine model. TM There was no study, however, which showed that endothelial seeding impro~/es the patency of commercially available PTFE grafts; and this experiment was done for that reason.
M a t e r i a l s and M e t h o d s Twenty-two mongrel dogs, 1 8 - 2 2 kg were anaesthetised with sodium pentothal, intubated, and maintained on mechanical ventilation. Using an aseptic technique, both external jugular veins were removed, everted on stainless steel rods, and manually spun for 1-2 s in a tube of phosphate buffered saline (PBS) to remove loose debris. The veins were immersed in a solution of 0.2% collagenase (Type II, Worthington) for 20 rain at 37°C. The collagenase-cell suspension was decanted into a 5Oral centrifuge tube containing 5 ml of calf serum. The veins were spun again in PBS and washed with a gentle stream of PBS. These solutions were added to the centrifuge tube which was spun at 2000 g for 15 min to form an endothelial cell pellet which was then suspended in 2 ml of serum-free M-199. E-PTFE grafts (W. L. Gore & Assoc., Flagstaff, Arizona) with an average internodal distance of 30~t, an internal diameter of 4 m m and a length of 60 m m were pre-clotted using 5 ml of autologous venous blood. After the blood was forced through the interstices and allowed to clot, the grafts were manually stripped to remove excess coagulum from the lumen. In the seeded group, the grafts were filled with the endothelial cell solution and both ends occluded. After incubation at room temperature for 5 rain, the grafts were rotated 180 ° and incubated for another 5 min. The animals were anticoagulated with intravenous sodium heparin (1 O0 units/kg), and the left carotid arteries were exposed. Four cm of carotid artery was excised and the grafts interposed, end-to-end, with interrupted 6 - 0 Prolene, after which the wounds were closed. Dipyridamole (Boehringer Ingelheim, Elmsford, New York) 25 mg and aspirin (Lilly, Indianapolis, Indiana) 8 0 m g , were given preoperatively and continued daily for 14 days. On the thirtieth postoperative day, the dogs were anaesthetised; the grafts were mobilised; and a catheter was inserted into the carotid artery I cm proximal to the graft. Through the catheter, the graft was perfused with a solution of 100% glutaraldehyde at a pressure of 1 2 0 m m H g until the perfusate leaving the graft was clear. If no flow through the graft occurred, it was designated as occluded. The patency data was analysed by the two-tailed Fisher's Exact Test. Eur] Vasc Surg Vol 2, December 1988
Transverse sections from the mid-graft and a longitudinal section across each anastomosis were randomly selected and examined by light microscopy. The inner capsule was defined as the tissue that formed between the polymer and the blood flow surface. An independent observer, without knowledge of the kind of graft (seeded or not), analysed the inner capsule for thickness and predominant cell type. For those grafts that demonstrated lining cells that looked like endothelium with light microscopy, representative samples were examined by scanning electron microscopy to confirm the identify of the cells by standard morphologic criteria. To obtain thrombus-free surface area, 35 m m transparencies were taken of the inner graft surfaces and projected onto quadrille paper. The percentage of clot-free graft surface was computed by dividing the total number of squares subtended by the clot-free surface by the total n u m b e r of squares subtended by the graft.
Results Seven of the 12 (58%) seeded grafts but only one control graft (10%) was patent at 30 days (P = 0.03). Histological examination of the randomly selected sections of midgraft from each specimen showed that six of the seven patent seeded grafts had a surface endothelium and that the singie patent control graft did not. The inner capsule of the seeded grafts consisted of a monolayer of endothelium and a thin acellular subendothelial matrix with an average thickness of 8 ~ (Fig. 1). Scanning electron microscopy showed multiple areas of confluent endothelium (Fig. 2); there were occasional scattered surface clots overlying gaps in the endothelial surface (Fig. 3). Examination of longitudinal sections across the proximal and distal anastomoses showed that pannus ingrowth extended onto the grafts for 2 - 5 m m in both seeded and unseeded groups. No subintimal hyperplasia was observed in either group. The one patent unseeded graft failed to show any evidence of midgraft endothelial cells. The flow surface appeared to be compacted fibrin. Occluded grafts in both seeded and unseeded groups demonstrated organising thrombus, and no endothelial cells were seen. Thrombus-free surface area was 56.0 + 10.9 in the seeded group and 15.2 + 7.1 ( m e a n + SEM) in the unseeded group; this difference was significant ( P = O.01). In one seeded graft which was occluded by red thrombus for a portion of its length, the clot-free segment had an endothelial lining, but the portion with thrombus did not.
E n d o t h e l i a l Cell Seeded G r a f t s
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Fig. 1. Typical appearance of a patent, seeded graft. Note confluent monolayer of endothelium overlying a thin subendothelial layer (arrow) and the graft wall (W). ( x 301 ).
Fig. 2. Luminal surface of seeded graft. Note confluent endothelial surface. Several red blood cells are present due to fixation artifact (E = endothelial cell, r = red blood cell). (SEM, × 1995). EurJ Vasc Surg Vol 2, December 1988
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Fig. 3. Seededgraft. Note a microscopicfocusof thrombus (arrow) located at a defectin the endothelial surface ( x 301).
Discussion This experiment demonstrated that endothelial seeding of ePTFE grafts, when combined with a 14-day course of antiplatelet agents, resulted in significantly improved patency when compared to untreated ePTFE grafts. Since the two groups differed in the extent of graft endothelialisation (as measured by thrombus-free surface area, 56% vs 15%), one may conclude that the development of more extensive endothelial surface coverage provided this effect. The thesis that thrombus-free surface area is equivalent to endothelialised surface is supported by the data of Sauvage et a1.11 and by the demonstration (by electron microscopy) of endothelial cells in thrombus-free areas, as shown in Figure 2. In the unseeded grafts, the endothefium was present at the anastomoses and was considered to represent pannus ingrowth, whereas in the seeded grafts it was present in mid-graft locations as well. The effectiveness of seeding may depend on multiple factors, such as size of inoculum, cell viability, strength of initial cellular attachment, and loss of cells after restoration of flow; and variations in some or all of these factors may have accounted for the relatively sparse endothelialisation in this study, as well as for some of the occlusions. In addition, differences in platelet reactivity may have been a factor. 12 None of these parameters was measured in this experiment, and the size of the experimental groups supports our assumption that they were distributed randomly and did not affect results. Although the 10% patency rate in the control animals may seem low, it was not lower than expected in the EurJ VascSurg Vol 2, December 1988
carotid position in mongrel dogs according to a previous study in our laboratories, ~3 in which 6 c m lengths occluded in nearly every case while 50% of 4 cm segments remained patent. These results were similar to those of Schmidt and associates in studies of ePTFE and Dacron, with and without antiplatelet agents. 14 The higher rates of patency for 1 0 c m lengths of 4 mm I.D. ePTFE reported by Kempczinski are with an experimental, porous type of ePTFE; is and he has not reported a direct comparison between it and the material in c o m m o n clinical use. Significant improvements in patency due to endothelial seeding have also been reported for Dacron grafts. Stanley and associates used 1 0 c m lengths of 4 m m I.D. externally supported, weft knitted Dacron in the iliofemoral position in foxhounds, lO Both control and experimental groups received antiplatelet therapy for 2 weeks, beginning preoperatively. Seeding seemed to protect against thrombosis initiated by platelets, as all but one occlusion occurred more than 2 weeks after withdrawal of antiplatelet therapy. For the statistical analysis, the authors chose not to consider three seeded and three control grafts studied 2 weeks after implantation, apparently because all six were patent; but one might question the rationale for that decision. Subsequently, eight of l l seeded grafts were patent, compared to three of l 1 controls; and the significance of this difference was reported as 0.03 by Chi square testing. Fisher's Exact Test might have been used, however, because there were less than five grafts at each data point. In that case, the level of significance would have been 0.043; and if one were to in-
Endothelial Cell Seeded Grafts
clude the grafts studied at 2 weeks, however, the P value would not be significant, 0.06. In a subsequent report from the same group, 12 to 16 cm lengths of 4 m m I.D. ePTFE grafts were implanted in the aorto-iliac position of adult foxhounds, eight with seeded grafts and eight non-seeded controls. 16 All animals received aspirin and dypyridamole beginning preoperatively and for 2 weeks postoperatively. At four weeks, two of eight control and four or eight seeded grafts were patent, an insignificant difference. The only other study comparing two groups which were identical except for seeding is that of Allen and coworkers. 17 They used mongrel dogs with four grafts in each animal, two in the carotid and two in the femoral position. One graft at each position was seeded; all dogs received aspirin for 2 weeks, starting the day before surgery. All grafts were weft knitted, non-supported, external velour Dacron. Patency was evaluated at weekly intervals by palpation for femoral grafts and by transcutaneous Doppler for those in the carotid position. In the experimental group, 27 of 28 were judged to be patent using these methods, compared to 8 of 28 controls. Considering the possibility Of error using these methods of assessing patency, confirmation by gross examination would have been desirable. Some specimens were evaluated histologically, but it is not stated whether or not all grafts were examined for patency when they were removed or, indeed, whether all grafts were removed! The other studies showing significant differences in patency came from Schmidt and co-workers and also involved Dacron. In one paper, they found no significant differences between seeded and unseeded grafts (both Dacron and ePTFE) in mongrel dogs receiving antiplatelet agents. 14 In another, seeded Dacron grafts in several groups of research hounds receiving a variety of antiplatelet agents had higher rates of patency when compared collectively to a single group of research hounds with seeded grafts but no antiplatelet agents. TM A subsequent study confirmed this finding. 19 Finally, they found a significant difference in response to decreased flow in another study using research hounds; seeded Dacron grafts were more likely than unseeded controls to resist thrombosis and return to the same rates of flow which were present prior to manipulation. 2o Since the overall goal of endothelial seeding is to improve patency of small vessel prostheses, it was disconcerting to find that all previous studies showing improved patency used Dacron, which is not generally considered a good small vessel graft or an experimental, porous ePTFE. Another interpretation, of course, is that such a use of Dacron is a rigorous test of patency. For this thesis to be acceptable, however, one must show improved patency on grafts thought to be more favourable, if imperfect, for such use such as ePTFE. This study is the first to support
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that thesis and, consequently, can be considered supportive of using commercially available ePTFE in other studies, as well as in clinical trials of seeding. The troublesome problem that remains, however, is the role of the coagulation system. In studies showing better fates of patency, anti-platelet agents were started preoperatively. Furthermore, four studies used hounds bred for research, though no comments were made about parameters of coagulation in those animals compared to those of mongrel dogs. There is evidence, however, that thrombosis of small vessel grafts in mongrel dogs m a y be predicted on the basis of high or low responses to A D P - induced platelet aggregation from a study using noncrimped, non-supported Dacron grafts in the carotid position in mongrel dogs. 12 In contrast, Shephard used no antiplatelet agents in baboons and yet found 6 of 8 seeded and 6 of 7 unseeded Dacron grafts (5 cm lengths, 4 m m I.D.) patent,21 further emphasising the importance of the experimental model in such studies. Although doubts remain about the role of the coagulation system and though differences in models m a y affect results, it is clear that endothelial seeding has a favourable effect on patency of small vessel prostheses in certain well-defined experimental situations. The task that remains is to understand those situations better and to make subsequent experiments relevant to man.
References 1 GRYGLEWSKIR, KORBUTR, OCETKIEWICZA. Reversal of platelet aggregation by prostacyelin. Pharmacol Res Commun 1978 ; 10:185-190. 2 LOSKUTOFFDJ, MUSSONIL. Interactions between fibrin and plasminogen activators produced by cultured endothelial cells. Blood 1983 ; 62 : 62-68. 3 McCOLLUMCN, KESTERRC, RAJAH SM, LEAROYDP, PETTERM. Arterial graft maturation: the duration of thrombotic activity in Dacron aortobifemoral grafts measured by platelet and fibrinogen kinetics. BrJSurg 1981 ;68:61-64. 4 HARKER LA, SLICHTER SJ, SAUVACELR. Platelet consumption by arterial prosthesis: the effect of endothelialization and pharmacologic inhibition of platelet function. Ann Surg 1977; 186 : 594-601. 5 HERRINGMB, DILLEYRS, ]ERSILDRA, BOXERL, GARDNERA, GLOVER]. Seeding arterial prostheses with vascular endothelium: the nature of the lining. Ann Surg 1979; 190: 84-90. 6 GRAHAMLM, VINTNERDW, FORDJW, KAHN RH, BURKELWE, STANLEY JC. Endothelial cell seeding of prosthetic vascular grafts: early experimental studies with cultured autologous canine endothelium. Arch Surg 1 9 8 0 ; 1 1 5 : 9 2 9 - 9 3 3 . 7 GRAHAMLM, BURKELWE, Ftmo JW, VINTEa DW, KAHN RH, STANLEY JC. Immediate seeding of enzymatically derived endothelium in Dacron vascular grafts: early experimental studies with autologous canine cells. Arch Sury 1980; 115 : 1289-1294. 8 GRAHAMLM, BURKELWE, FORDJW, VINTERDW, KAHNFd-I, STANLEY ]C. Expanded polytetrafluoroethylene vascular prostheses seeded with enzymatically derived and cultured canine endothelial ceils. Surgery 1982 ;91 : 550-559. 9 BURKELWE, FORD]W, VINTERDW, KAHNRH, GRAHAMLM, STANLEY JC. Endothelial seeding. In Stanley JC, ed. Biologic and Synthetic Vascular Prostheses. New York: Grune & Stratton, 1982 ; 631-651.
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10 STANLEYJC. BURKEI,WE, FORD JW, VINTER DW, KAHN RH, WHITEHOUSEWM, GRAHAMLM. Enhanced patency of small-diameter, externally supported Dacron iliofemoral grafts seeded with endothelial cells. Surgery 1 9 8 2 ; 9 2 : 9 9 4 - 1 0 0 5 . 11 SAUVAGELR, WALKERMW, BERGERK, ROBELSB, LISCHKOMM, YATES SG, LOGANGA. Current arterial prostheses: experimental evaluation by implantation in the carotid and circumflex coronary arteries of the dog. Arch Surg 1 9 7 9 ; 1 1 4 : 6 8 7 - 6 9 1 . 12 KAPLANS, MARCOEKF, SAUVAGELR, ZAMMITM, Wu H-D, MATHISEN SR, WALKERMW. The effect of predetermined thrombotic potential of the recipient on small-caliber graft performance. J Vase Surg 1986;3:311-321. 13 HERRINGM, DILLEYPt, PETERSONG, WIGGANSJ, GARDNERA, GLOVER J. Graft material, length, a n d diameter determine the patency of small artery prostheses in dogs. ] Surg Res 1982; 3 2 : 1 3 8 - 1 4 2 . l 4 SCHMIDTSP, HUNTERT], HIRKO M, GELDENTA, EVANCHOMM, SHARP WV, DONOVANDL. Small-diameter vascular prostheses: Two designs of PTFE and endothelial cell-seeded and nonseeded Dacron. J Vase Surg 1 9 8 5 ; 3 : 2 9 2 - 2 9 7 . 15 KEMPCZINSKI RF, ROSEN/elAN JE, PEARCE WH, ROEDERSHE1MERLR, BERLATZKYY, RAMALANJAONAG]. Endothelial cell seeding of a new PTFE vascular prosthetis. Vasc Surg 1985 ; 2 : 242~-429. 16 GRAHAMLM, STANLEY]C, BURKELWE. Improved patency of endo-
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thelial-cell-seeded, long, knitted Dacron and ePTFE vascular prostheses. AS AIO Journal 1985 ;8: 65-73. ALLEN BT, LONGJA, CLARKRE, SICARDGA, HOPKINS KT, WELCHM]. Influence of endothelial cell seeding on platelet deposition and patency in small-diameter Dacron arterial grafts. J Vase Surg 1984; 1:224-233. SCHMIDT SP, HUNTER T], FALKOW LJ, EVANCHO MM, SHARP w e . Effects of antiplatelet agents in combination with endothelial cell seeding on small-diameter Dacron vascular graft performance in the canine carotid artery model. ] Vasc Surg 1985 ; 2 : 8 9 8 - 9 0 6 . SHARP WV, SCHMlDTSP, DONOVANDL. Prostaglandin biochemistry of seeded endothelial cells on Dacron prostheses. J Vasc Surg 1986; 3:256-263. 8CHMIDTSP, HUNTERTJ, SHARE WV, MALINDZARG8, EVANCHOMM. Endothelial cell-seeded four-millimetre Dacron vascular grafts: Effects of blood flow manipulation t h r o u g h the grafts. J Vase Surg 1984; 1 : 4 3 4 - 4 4 1 . SHEPARDAD, ELDRAP-JORGENSEN], KEOUGHEM, FOXALLTF, RAMBERG K, CONNOLLY R, MARKEY WC, GAVRIS V, AUGER KR, LIBBY P, O'DONNELLTF, CALLOWAD. Endothelial cell seeding of small-calibre synthetic grafts in the baboon. Surg 1986 ; 99 : 3 1 8 - 3 2 6 .
Received 25 November 1987
The Influence of Endothelial Seeding and Platelet Inhibition on the Patency of ePTFE Grafts Used to Replace Small Arteries-an Experimental Study d. B. C a m p b e l l , 1 J. L. G I o v e r 2 a n d B. H e r r i n g 3
1Assistant Professor of Surgery, Louisiana State University Medical Center, Shreveport, Louisiana, U.S.A., 2Chief Surgical Services, William Beaumont Hospital, Royal Oak, Michigan 4 8 0 7 2 , U.S.A., 3Associate Clinical Professor of Surgery, Indiana University Medical Center, Indianapolis, Indiana, U.S.A. Previous studies on the influence of endothelial seeding on graft patency have shown that significant improvement has only been achieved with Dacron and an experimental, porous PTFE graft. Methods of assessing patency or showing statistical significance could be questioned in some of these studies. To determine if the combination of endothelial cell seeding and antiplatelet agents would improve patency in small-diameter, commercially available expanded polytetrafluorethylene (ePTFE) grafts, we placed ePTFE grafts into the left carotid position in two groups of mongrel dogs. All grafts were 4 mm internal diameter and 60 mm long, and were interposed in an end-to-end fashion. Both groups received aspirin (80 mg daily) and dipyridamole (25 mg daily) for 14 days, beginning immediately prior to surgery. In Group I (n = 12), the grafts were seeded with enzymatically harvested autogenous endothelium just prior to implanatation; in Group II (n = 10) the grafts were not seeded. AH grafts were removed at 30 days. Seven of 12 (58%) seeded grafts, but only one control graft (10%) remained patent (P=O.03). Six of the seven seeded grafts exhibited surface endothelium, but the single patent control graft did not. The inner capsule of the seeded grafts consisted of a monolayer of endothelium and a thin acellular subendothelial matrix with an average thickness of 8 p. We conclude that a 14-day course of anti-platelet agents combined with endothelial seeding of ePTFE resulted in significantly improved patency compared to controls, with most patent, seeded grafts developing an endothelial lining in 30 days. Key Words: Endothelial seeding; PTFE; platelet inhibitions.
Introduction
Endothelium repairs itselL inhibits platelet aggregation and adherence by the elaboration of prostacyclin,1 and initiates clot lysis by the elaboration of plasminogen activator.2 Assuming that these properties confer resistance to thrombosis, considerable effort has been directed toward the development of endothelialised vascular prostheses by "seeding" grafts with endothelial cells at the time of implantation. Synthetic conduits implanted without endothelial seeding develop a surface consisting of a fibrin-platelet Reprint requests should be sent to: John L. Glover, Department of Surgery, William BeaumontHospital, 3601 West 13 MileRoad, RoyalOak, Michigan 48072, U.S.A. 0950-821X/88/060365 +06 $03,00/0 © 1988 Grune &Stratton Ltd
meshwork; and platelets deposit onto these surfaces for up to nine months, as measured by decreased platelet survival time. s The development of an endothelial lining by these grafts has been shown to decrease platelet consumption in proportion to the amount of surface endothelialised. 4 Consequently, endothelium-lined conduits might be expected to have a higher patency rate than unlined conduits of the same dimensions. Studies of production of such linings show that inoculation of Dacron and ePTFE grafts with autologous endothelium harvested at the time of implantation results in the development of an endothelial lining, usually by four weeks, s s In small-diameter Dacron grafts, however, seeding alone does not improve patency 9 because occlusion occurs before the seeded cells evolve into a functioning lining. The addition of a two-week course of
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antiplatelet agents prevents thrombosis during this interval and results in improved patency in a canine model. TM There was no study, however, which showed that endothelial seeding impro~/es the patency of commercially available PTFE grafts; and this experiment was done for that reason.
M a t e r i a l s and M e t h o d s Twenty-two mongrel dogs, 1 8 - 2 2 kg were anaesthetised with sodium pentothal, intubated, and maintained on mechanical ventilation. Using an aseptic technique, both external jugular veins were removed, everted on stainless steel rods, and manually spun for 1-2 s in a tube of phosphate buffered saline (PBS) to remove loose debris. The veins were immersed in a solution of 0.2% collagenase (Type II, Worthington) for 20 rain at 37°C. The collagenase-cell suspension was decanted into a 5Oral centrifuge tube containing 5 ml of calf serum. The veins were spun again in PBS and washed with a gentle stream of PBS. These solutions were added to the centrifuge tube which was spun at 2000 g for 15 min to form an endothelial cell pellet which was then suspended in 2 ml of serum-free M-199. E-PTFE grafts (W. L. Gore & Assoc., Flagstaff, Arizona) with an average internodal distance of 30~t, an internal diameter of 4 m m and a length of 60 m m were pre-clotted using 5 ml of autologous venous blood. After the blood was forced through the interstices and allowed to clot, the grafts were manually stripped to remove excess coagulum from the lumen. In the seeded group, the grafts were filled with the endothelial cell solution and both ends occluded. After incubation at room temperature for 5 rain, the grafts were rotated 180 ° and incubated for another 5 min. The animals were anticoagulated with intravenous sodium heparin (1 O0 units/kg), and the left carotid arteries were exposed. Four cm of carotid artery was excised and the grafts interposed, end-to-end, with interrupted 6 - 0 Prolene, after which the wounds were closed. Dipyridamole (Boehringer Ingelheim, Elmsford, New York) 25 mg and aspirin (Lilly, Indianapolis, Indiana) 8 0 m g , were given preoperatively and continued daily for 14 days. On the thirtieth postoperative day, the dogs were anaesthetised; the grafts were mobilised; and a catheter was inserted into the carotid artery I cm proximal to the graft. Through the catheter, the graft was perfused with a solution of 100% glutaraldehyde at a pressure of 1 2 0 m m H g until the perfusate leaving the graft was clear. If no flow through the graft occurred, it was designated as occluded. The patency data was analysed by the two-tailed Fisher's Exact Test. Eur] Vasc Surg Vol 2, December 1988
Transverse sections from the mid-graft and a longitudinal section across each anastomosis were randomly selected and examined by light microscopy. The inner capsule was defined as the tissue that formed between the polymer and the blood flow surface. An independent observer, without knowledge of the kind of graft (seeded or not), analysed the inner capsule for thickness and predominant cell type. For those grafts that demonstrated lining cells that looked like endothelium with light microscopy, representative samples were examined by scanning electron microscopy to confirm the identify of the cells by standard morphologic criteria. To obtain thrombus-free surface area, 35 m m transparencies were taken of the inner graft surfaces and projected onto quadrille paper. The percentage of clot-free graft surface was computed by dividing the total number of squares subtended by the clot-free surface by the total n u m b e r of squares subtended by the graft.
Results Seven of the 12 (58%) seeded grafts but only one control graft (10%) was patent at 30 days (P = 0.03). Histological examination of the randomly selected sections of midgraft from each specimen showed that six of the seven patent seeded grafts had a surface endothelium and that the singie patent control graft did not. The inner capsule of the seeded grafts consisted of a monolayer of endothelium and a thin acellular subendothelial matrix with an average thickness of 8 ~ (Fig. 1). Scanning electron microscopy showed multiple areas of confluent endothelium (Fig. 2); there were occasional scattered surface clots overlying gaps in the endothelial surface (Fig. 3). Examination of longitudinal sections across the proximal and distal anastomoses showed that pannus ingrowth extended onto the grafts for 2 - 5 m m in both seeded and unseeded groups. No subintimal hyperplasia was observed in either group. The one patent unseeded graft failed to show any evidence of midgraft endothelial cells. The flow surface appeared to be compacted fibrin. Occluded grafts in both seeded and unseeded groups demonstrated organising thrombus, and no endothelial cells were seen. Thrombus-free surface area was 56.0 + 10.9 in the seeded group and 15.2 + 7.1 ( m e a n + SEM) in the unseeded group; this difference was significant ( P = O.01). In one seeded graft which was occluded by red thrombus for a portion of its length, the clot-free segment had an endothelial lining, but the portion with thrombus did not.
E n d o t h e l i a l Cell Seeded G r a f t s
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Fig. 1. Typical appearance of a patent, seeded graft. Note confluent monolayer of endothelium overlying a thin subendothelial layer (arrow) and the graft wall (W). ( x 301 ).
Fig. 2. Luminal surface of seeded graft. Note confluent endothelial surface. Several red blood cells are present due to fixation artifact (E = endothelial cell, r = red blood cell). (SEM, × 1995). EurJ Vasc Surg Vol 2, December 1988
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Fig. 3. Seededgraft. Note a microscopicfocusof thrombus (arrow) located at a defectin the endothelial surface ( x 301).
Discussion This experiment demonstrated that endothelial seeding of ePTFE grafts, when combined with a 14-day course of antiplatelet agents, resulted in significantly improved patency when compared to untreated ePTFE grafts. Since the two groups differed in the extent of graft endothelialisation (as measured by thrombus-free surface area, 56% vs 15%), one may conclude that the development of more extensive endothelial surface coverage provided this effect. The thesis that thrombus-free surface area is equivalent to endothelialised surface is supported by the data of Sauvage et a1.11 and by the demonstration (by electron microscopy) of endothelial cells in thrombus-free areas, as shown in Figure 2. In the unseeded grafts, the endothefium was present at the anastomoses and was considered to represent pannus ingrowth, whereas in the seeded grafts it was present in mid-graft locations as well. The effectiveness of seeding may depend on multiple factors, such as size of inoculum, cell viability, strength of initial cellular attachment, and loss of cells after restoration of flow; and variations in some or all of these factors may have accounted for the relatively sparse endothelialisation in this study, as well as for some of the occlusions. In addition, differences in platelet reactivity may have been a factor. 12 None of these parameters was measured in this experiment, and the size of the experimental groups supports our assumption that they were distributed randomly and did not affect results. Although the 10% patency rate in the control animals may seem low, it was not lower than expected in the EurJ VascSurg Vol 2, December 1988
carotid position in mongrel dogs according to a previous study in our laboratories, ~3 in which 6 c m lengths occluded in nearly every case while 50% of 4 cm segments remained patent. These results were similar to those of Schmidt and associates in studies of ePTFE and Dacron, with and without antiplatelet agents. 14 The higher rates of patency for 1 0 c m lengths of 4 mm I.D. ePTFE reported by Kempczinski are with an experimental, porous type of ePTFE; is and he has not reported a direct comparison between it and the material in c o m m o n clinical use. Significant improvements in patency due to endothelial seeding have also been reported for Dacron grafts. Stanley and associates used 1 0 c m lengths of 4 m m I.D. externally supported, weft knitted Dacron in the iliofemoral position in foxhounds, lO Both control and experimental groups received antiplatelet therapy for 2 weeks, beginning preoperatively. Seeding seemed to protect against thrombosis initiated by platelets, as all but one occlusion occurred more than 2 weeks after withdrawal of antiplatelet therapy. For the statistical analysis, the authors chose not to consider three seeded and three control grafts studied 2 weeks after implantation, apparently because all six were patent; but one might question the rationale for that decision. Subsequently, eight of l l seeded grafts were patent, compared to three of l 1 controls; and the significance of this difference was reported as 0.03 by Chi square testing. Fisher's Exact Test might have been used, however, because there were less than five grafts at each data point. In that case, the level of significance would have been 0.043; and if one were to in-
Endothelial Cell Seeded Grafts
clude the grafts studied at 2 weeks, however, the P value would not be significant, 0.06. In a subsequent report from the same group, 12 to 16 cm lengths of 4 m m I.D. ePTFE grafts were implanted in the aorto-iliac position of adult foxhounds, eight with seeded grafts and eight non-seeded controls. 16 All animals received aspirin and dypyridamole beginning preoperatively and for 2 weeks postoperatively. At four weeks, two of eight control and four or eight seeded grafts were patent, an insignificant difference. The only other study comparing two groups which were identical except for seeding is that of Allen and coworkers. 17 They used mongrel dogs with four grafts in each animal, two in the carotid and two in the femoral position. One graft at each position was seeded; all dogs received aspirin for 2 weeks, starting the day before surgery. All grafts were weft knitted, non-supported, external velour Dacron. Patency was evaluated at weekly intervals by palpation for femoral grafts and by transcutaneous Doppler for those in the carotid position. In the experimental group, 27 of 28 were judged to be patent using these methods, compared to 8 of 28 controls. Considering the possibility Of error using these methods of assessing patency, confirmation by gross examination would have been desirable. Some specimens were evaluated histologically, but it is not stated whether or not all grafts were examined for patency when they were removed or, indeed, whether all grafts were removed! The other studies showing significant differences in patency came from Schmidt and co-workers and also involved Dacron. In one paper, they found no significant differences between seeded and unseeded grafts (both Dacron and ePTFE) in mongrel dogs receiving antiplatelet agents. 14 In another, seeded Dacron grafts in several groups of research hounds receiving a variety of antiplatelet agents had higher rates of patency when compared collectively to a single group of research hounds with seeded grafts but no antiplatelet agents. TM A subsequent study confirmed this finding. 19 Finally, they found a significant difference in response to decreased flow in another study using research hounds; seeded Dacron grafts were more likely than unseeded controls to resist thrombosis and return to the same rates of flow which were present prior to manipulation. 2o Since the overall goal of endothelial seeding is to improve patency of small vessel prostheses, it was disconcerting to find that all previous studies showing improved patency used Dacron, which is not generally considered a good small vessel graft or an experimental, porous ePTFE. Another interpretation, of course, is that such a use of Dacron is a rigorous test of patency. For this thesis to be acceptable, however, one must show improved patency on grafts thought to be more favourable, if imperfect, for such use such as ePTFE. This study is the first to support
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that thesis and, consequently, can be considered supportive of using commercially available ePTFE in other studies, as well as in clinical trials of seeding. The troublesome problem that remains, however, is the role of the coagulation system. In studies showing better fates of patency, anti-platelet agents were started preoperatively. Furthermore, four studies used hounds bred for research, though no comments were made about parameters of coagulation in those animals compared to those of mongrel dogs. There is evidence, however, that thrombosis of small vessel grafts in mongrel dogs m a y be predicted on the basis of high or low responses to A D P - induced platelet aggregation from a study using noncrimped, non-supported Dacron grafts in the carotid position in mongrel dogs. 12 In contrast, Shephard used no antiplatelet agents in baboons and yet found 6 of 8 seeded and 6 of 7 unseeded Dacron grafts (5 cm lengths, 4 m m I.D.) patent,21 further emphasising the importance of the experimental model in such studies. Although doubts remain about the role of the coagulation system and though differences in models m a y affect results, it is clear that endothelial seeding has a favourable effect on patency of small vessel prostheses in certain well-defined experimental situations. The task that remains is to understand those situations better and to make subsequent experiments relevant to man.
References 1 GRYGLEWSKIR, KORBUTR, OCETKIEWICZA. Reversal of platelet aggregation by prostacyelin. Pharmacol Res Commun 1978 ; 10:185-190. 2 LOSKUTOFFDJ, MUSSONIL. Interactions between fibrin and plasminogen activators produced by cultured endothelial cells. Blood 1983 ; 62 : 62-68. 3 McCOLLUMCN, KESTERRC, RAJAH SM, LEAROYDP, PETTERM. Arterial graft maturation: the duration of thrombotic activity in Dacron aortobifemoral grafts measured by platelet and fibrinogen kinetics. BrJSurg 1981 ;68:61-64. 4 HARKER LA, SLICHTER SJ, SAUVACELR. Platelet consumption by arterial prosthesis: the effect of endothelialization and pharmacologic inhibition of platelet function. Ann Surg 1977; 186 : 594-601. 5 HERRINGMB, DILLEYRS, ]ERSILDRA, BOXERL, GARDNERA, GLOVER]. Seeding arterial prostheses with vascular endothelium: the nature of the lining. Ann Surg 1979; 190: 84-90. 6 GRAHAMLM, VINTNERDW, FORDJW, KAHN RH, BURKELWE, STANLEY JC. Endothelial cell seeding of prosthetic vascular grafts: early experimental studies with cultured autologous canine endothelium. Arch Surg 1 9 8 0 ; 1 1 5 : 9 2 9 - 9 3 3 . 7 GRAHAMLM, BURKELWE, Ftmo JW, VINTEa DW, KAHN RH, STANLEY JC. Immediate seeding of enzymatically derived endothelium in Dacron vascular grafts: early experimental studies with autologous canine cells. Arch Sury 1980; 115 : 1289-1294. 8 GRAHAMLM, BURKELWE, FORDJW, VINTERDW, KAHNFd-I, STANLEY ]C. Expanded polytetrafluoroethylene vascular prostheses seeded with enzymatically derived and cultured canine endothelial ceils. Surgery 1982 ;91 : 550-559. 9 BURKELWE, FORD]W, VINTERDW, KAHNRH, GRAHAMLM, STANLEY JC. Endothelial seeding. In Stanley JC, ed. Biologic and Synthetic Vascular Prostheses. New York: Grune & Stratton, 1982 ; 631-651.
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J.B. Campbell e t a l .
10 STANLEYJC. BURKEI,WE, FORD JW, VINTER DW, KAHN RH, WHITEHOUSEWM, GRAHAMLM. Enhanced patency of small-diameter, externally supported Dacron iliofemoral grafts seeded with endothelial cells. Surgery 1 9 8 2 ; 9 2 : 9 9 4 - 1 0 0 5 . 11 SAUVAGELR, WALKERMW, BERGERK, ROBELSB, LISCHKOMM, YATES SG, LOGANGA. Current arterial prostheses: experimental evaluation by implantation in the carotid and circumflex coronary arteries of the dog. Arch Surg 1 9 7 9 ; 1 1 4 : 6 8 7 - 6 9 1 . 12 KAPLANS, MARCOEKF, SAUVAGELR, ZAMMITM, Wu H-D, MATHISEN SR, WALKERMW. The effect of predetermined thrombotic potential of the recipient on small-caliber graft performance. J Vase Surg 1986;3:311-321. 13 HERRINGM, DILLEYPt, PETERSONG, WIGGANSJ, GARDNERA, GLOVER J. Graft material, length, a n d diameter determine the patency of small artery prostheses in dogs. ] Surg Res 1982; 3 2 : 1 3 8 - 1 4 2 . l 4 SCHMIDTSP, HUNTERT], HIRKO M, GELDENTA, EVANCHOMM, SHARP WV, DONOVANDL. Small-diameter vascular prostheses: Two designs of PTFE and endothelial cell-seeded and nonseeded Dacron. J Vase Surg 1 9 8 5 ; 3 : 2 9 2 - 2 9 7 . 15 KEMPCZINSKI RF, ROSEN/elAN JE, PEARCE WH, ROEDERSHE1MERLR, BERLATZKYY, RAMALANJAONAG]. Endothelial cell seeding of a new PTFE vascular prosthetis. Vasc Surg 1985 ; 2 : 242~-429. 16 GRAHAMLM, STANLEY]C, BURKELWE. Improved patency of endo-
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thelial-cell-seeded, long, knitted Dacron and ePTFE vascular prostheses. AS AIO Journal 1985 ;8: 65-73. ALLEN BT, LONGJA, CLARKRE, SICARDGA, HOPKINS KT, WELCHM]. Influence of endothelial cell seeding on platelet deposition and patency in small-diameter Dacron arterial grafts. J Vase Surg 1984; 1:224-233. SCHMIDT SP, HUNTER T], FALKOW LJ, EVANCHO MM, SHARP w e . Effects of antiplatelet agents in combination with endothelial cell seeding on small-diameter Dacron vascular graft performance in the canine carotid artery model. ] Vasc Surg 1985 ; 2 : 8 9 8 - 9 0 6 . SHARP WV, SCHMlDTSP, DONOVANDL. Prostaglandin biochemistry of seeded endothelial cells on Dacron prostheses. J Vasc Surg 1986; 3:256-263. 8CHMIDTSP, HUNTERTJ, SHARE WV, MALINDZARG8, EVANCHOMM. Endothelial cell-seeded four-millimetre Dacron vascular grafts: Effects of blood flow manipulation t h r o u g h the grafts. J Vase Surg 1984; 1 : 4 3 4 - 4 4 1 . SHEPARDAD, ELDRAP-JORGENSEN], KEOUGHEM, FOXALLTF, RAMBERG K, CONNOLLY R, MARKEY WC, GAVRIS V, AUGER KR, LIBBY P, O'DONNELLTF, CALLOWAD. Endothelial cell seeding of small-calibre synthetic grafts in the baboon. Surg 1986 ; 99 : 3 1 8 - 3 2 6 .
Received 25 November 1987