Prospective clinical study with in vitro endothelial cell lining of expanded polytetrafluoroethylene grafts in crural repeat reconstruction Heinrich Magometschnigg, MD, Margit Kadletz, MD, Martin Vodrazka, MD, Wolfgang Dock, MD, Michael Grimm, MD, Martin Grabenw6ger, MD, Erich Minar, MD, Michael Staudacher, MD, Gabriele Fenzl, MD, and Ernst Wolner, MD, Vienna, Austria A nonrandomized prospective clinical study was undertaken to evaluate the technique and efficacy of in vitro endothelial cell lining o f synthetic grafts, Twenty-six patients (10 men and 16 women with a mean age of 68.4 years; range, 49 to 80 years) with end stage chronic peripheral vascular disease requiring reoperation were entered into the study. In 13 patients venous endothelial cells were harvested 4 to 7 weeks before operation, grown to confluency in culture flasks, and seeded onto the inner surface o f expanded polytetrafluoroethylene grafts. Thirteen patients received untreated expanded polytetrafluoroethylene grafts and served as a control. A scoring system with use of intraarterial angiography was used to assess disease severity. N o statistically significant differences in angiographic score were seen between the two groups, indicating comparable severity of disease. Early secondary graft patency (0 to 30 days) was 92% for the in vitro endothelial cell lining group and 53% for control patients. The amputation rate after 18 months for the in vitro endothelial cell lining group was 15%, with a 31% rate in the control group. The fimctional performance of the in vitro endothelial cell lining bypasses was superior to that of untreated bypass grafts during the observed follow-up period. These early results suggest that in vitro endothelial cell lining is a method that can reduce the early occlusion rate n o w seen after repeat reconstruction of crural vessels. (J VASC SURG 1992;15: 527-35.)
The use of autologous vein grafts indisputably results in the best outcome after surgery to bypass the smaller peripheral vessels. However, because of multiple previous operations, patients with recurrent occlusion in the crural vessel bed often have no remaining suitable veins and must have bypasses with biologic or artificial prostheses. Failure of these artificial prostheses lies ultimately in the blood-contacting surface, which promotes thrombotic occlusion through the activation of platelets. 1 Intraoperative "single stage seeding" of prosthetic grafts has been tested? In their report, From the SecondDepartmentof Surgery,Universityof Vienna (Drs. Magometschnigg, Kadletz, Vodrazka, Dock, Grimm, Grabenw6ger, Staudacher, Fenzl, Wolner), and the First Department of Internal Medicine, Division of Angiology, Universityof Vienna (Dr. Minar). Reprint requests: HeinrichMagometschnigg,MD, II Chirurgische Universit~/tsklinik,Spitalgasse23, I090 Vienna, Austria. 2411/32197
Fasol et al.3 state "The similar results for seeded and nonseeded vascular grafts indicate the failure of seeded grafts to develop a nontraumatizing surface comparable to that ofsaphenous veins." These results could be explained by the two following factors: (1) The number of primarily harvested endothelial cells (ECs) is not sufficient for the formation of a confluent cell layer before implantation. (2) The time between seeding and implantation is too short for effective adhesion of the cells to the graft surface leading to cell loss induced by blood flow.4 The purpose of this study was to test the clinical effectiveness of a new prelining procedure for expanded polytetrafluoroethylene (ePTFE) grafts. The procedure is made up of three individual steps: (1) Cell harvesting from an autologous arm vein performed 4 to 7 weeks before operation, with subsequent cell culture in the laboratory. (2) Seeding of this increased number of autologous ECs onto the prosthesis that has been precoated with fibrin glue. 527
528 Magometschnigg et al.
(3) Cultivation of the cells on the graft surface for 1 additional week to induce expression of the extraccllular matrix that promotes cell adherence.
METHODS Between June 1989 and December 1990, 26 patients with limb-threatening ischemia, both with and without ulceration and gangrene, were entered into the study. All patients were fully informed about the purpose of and methods used in the study. Informed consent was obtained in accordance with the guidelines of the Helsinki Declaration. ~ The study was reviewed and approved by the Ethical Committee of the University of Vienna. Every patient had had failure of one or more previous crural bypass procedures, had no usable autologous vein, and was considered angiographically suitable for revascularization. Patients were divided into two groups, a control group with 13 untreated ePTFE graft implantations and an in vitro endothelial cell lining (IVECL) group with 13 treated ePTFE grafts. Eight men and five women were in both groups. Average age of the control group was 69 years and of the IVECL group 67 years. Risk factors such as tobacco use (IVECL, eight; control patients, five), diabetes (IVECL, nine; control, nine), hypertension (IVECL, three; control, four), and cardiac status were assessed. The assessment of preoperative clinical stage demonstrated advanced peripheral vascular disease. Six control and eight IVECL patients had clinical category 4 (SVS/ISCVS). Seven control and five IVECL patients had clinical category 5 (SVS/ISCVS). All patients in clinical category 5 suffered from leg ulcers. Gangrene of the toes and forefoot was seen in five control and three IVECL patients. The control group had a higher rate of necrosis and a greater number of infected forefoot necroses. Angiography The patients underwent preoperative intraarterial angiography with special attention to the delineation of the calf and foot arteries. Selective orthograde puncture and long delay times were used as were vasodilators. An angiographic score for the description of runoff and resistance according to Ad Hoc Committee on Reporting Standards of the Society for Vascular Surgery/North American Chapter and the International Society for Cardiovascular Surgery (SVS/ISCVS), was calculated, and the groups were compared. 6 This scoring system is based on the patency of the vessels and their relative contribution to the outflow tract. A score of 1 corresponds to excellent runoff and 10 to a blind segment. With use
Journal of VASCULAR SURGERY
of Student's unpaired t test, no significant differences were found between the groups with respect to the morphologic picture or the resistance. The analysis of the preoperative angiogram for each individual patient is shown in Table I.
Vein harvesting Arms veins were harvested as described for saphenous veins according to the "no touch technique. "7 During the procedure the vein was kept moist with saline solution with papaverine at room temperature. A distal cannula was inserted and secured, and the vessel was gently flushed with complete medium. After excision, the cannulated and collagenase filled vessel was transferred to the cell laboratory. Cell culture A standard enzymatic harvest technique with use of 0.1% collagenase (CLS II; Cooper Biomedical, Malvern, Pa.) was applied for 15 minutes at 37 ° C for the establishment of primary cultures from the human venous endothelial cells (VECs). Primary cultures were grown to confluence in 25 cm 2 tissue culture flasks precoated with 30 ~g/rnl human fibronectin and fed two to three times a week. Complete medium was composed of 80% M-199 medium (Gibco Laboratories, Lawrence, Mass.), buffered with 2.2 gm/L sodium bicarbonate, 20% heat inactivated pooled human serum, gentamycin 0.07 mg/ml (Gibco), 70 ~tg/ml of EC growth supplement (ECGS, Collaborative Research Inc., Lexington, Mass.), and 180 ~g/ml preservative-free heparin (Sero-Med, Vienna, Austria), 2 mmol/L L-glutamine (Gibco No. 043 50 30D). When confluence in 25 cm 2 was achieved, the cells were then resuspended by treatment with a solution of 0.05% trypsin (1 : 250) buffered in saline and 0.02% ethylenediamine tetraacetic acid (EDTA) (Inotech, Switzerland) and replaced in two 150 cm 2 tissue culture flasks coated again with human fibronectin. After growth to confluence, second passage VECs were detached from 150 cm 2 culture flasks with trypsin-EDTA, and a complete medium was added to inactivate any residual trypsin before the cell suspension was centrifuged at 800 rpm for 10 minutes in 4 ° C. The supernatant was discarded, and the pellet was resuspended in 20 ml complete medium for the seeding procedure. Graft material Six millimeter reinforced thin-walled PTFE grafts (W. L. Gore & Associates, Elkton, Md.). 80 cm in
V o l u m e 15 Number 3
Endothelial cell lining of ePTFE grafts in humans
March 1992
529
Table I. Influence of preoperative morphology and runoff assessment of crural vessels on postoperative results Preoperative
Group Control
Patient
Runoff score
Resistance score (A G)
Site of distalanastom.
Graft thrombosis
Graft
Early
Late
Patent
Amputation
SA
1
8.5
1
ATA
Yes
-
-
Yes
FM
1
9.0
3
ATA
Yes
-
-
-
UF UH MA
1 1
8.0 8.0
1 1
Tractus Tractus
Yes -
Yes
-
-
2
7,0
1
ATA
-
-
Yes
-
BA
1
11.0
3
ATA
-
-
Yes
-
SA
GJ
1 2
9.5 8.0
2 2
ATA PTA
-
Yes
Yes -
Yes
KA
1
12.5
7
PTA
Yes
-
-
Yes
OR
1 3 1
11.0 6.0 10.5
3 5 3
ATA ATA ATA
Yes -
-
Yes Yes
Yes -
PK GH
1
11.0
3
ATA
Yes
-
Yes
-
1
8.5
1
ATA
-
-
Yes
-
BA FM
1 2
8.0 7.0
3 1
PTA Tractus
-
Yes
Yes -
Yes
ML MF
1 2
10.0 4.5
3 1
ATA Tractus
-
Yes -
Yes
Yes -
NA
1
9.5
2
Tractus
-
-
Yes
-
StR RM
3
5.0
2
Tract-us
-
-
Yes
-
1
9.5
2
ATA
Yes
-
Yes
-
FG
1 2
9.0 9.0
2 2
ATA Tractus
-
Yes
Yes -
-
KE MA IVECL
Patent crural vessels
Postoperative
UH NA
2
10.0
6
PTA
-
-
Yes
-
PW SM
1 2
12,0 10.0
5 5
ATA ATA
-
-
Yes Yes
-
p value
0.371
0.437
-
ATA,
A n t e r i o r t i b i a l a r t e r y ; PTA, p o s t e r i o r t i b i a l a r t e r y ; Tractus, t r a c t u s t i b i o f i b u l a r i s . E a r l y g r a f t t h r o m b o s i s less than 3 0 d a y s ; l a t e g r a f t thrombosis greater than 3 0 d a y s .
length were used for each operation. The internodal distance was 30 txm. For the IVECL group grafts were prepared according to the following method. Precoating procedure for lining o f the IVECL grafts. The fibrinogen compound (Immuno, Austria) contained 3.5 to 5.5 mg/ml fibrinogen, 0.1 to 0.45 mg human fibronectin, 1.4 ~g/ml plasminogen, 10,000 units/ml aprotinin (Trasylol, Bayer, Germany). The thrombin compound contained 20 units/ml thrombin, 475 ng/ml Ca gluconicum, 5000 units/ml aprotinin. Precoating with fibrin glue was performed by forcing this substrate through the interstices of the PTFE graft with 4F Fogarty balloon catheter. 8 After the fibrinogen compound was repeatedly forced through the interstices, the entire graft was subsequently placed into the thrombin solution for 10 minutes at room temperature. Subsequently the graft was rinsed twice with calcium/magnesium-free phosphate-buffered saline (Flow Laboratories Inc., McLean, Va.) before the entire procedure was repeated a second time. Seeding procedure. After surface preparation,
the graft was filled with the VEC suspension. Both ends were occluded with holders from a rotatory device. The prosthesis was incubated in a horizontal position in the rotatory device. An even distribution of VECs was obtained by rotating the graft 6 rph for 3 hours at a constant temperature of 37 ° C in a 5% carbon dioxide atmosphere. At the end of the seeding procedure the graft was gently flushed with 20 ml of M-199. From the VEC count in suspensions before and after rotation the seeding efficiency was calculated. The graft was then placed into a filter-protected glass container and cultivated in 200 ml complete medium for another 7 to 10 days. A 1 cm length was cut from the graft for examination. At the same time, the samples of the graft for bacterial (sterility testincubation period for 10 days), mycotic, and mycoplasmal examination were obtained. Confirmation of endothelial cell identity. Venous endothelial cells were identified by scanning electron microscopy (SEM) examination immediately after the seeding procedure and 2 to 4 hours before the graft implantation. The presence of factor
530
Journal of VAaCULAR SURGERY
Magometschnigg et al.
Table II. Scintigraphic results-visual interpretation and platelet accumulation index 3 Months after operation
1 Week after operation Group
Patient
Proximal*
Control
SA FM UF UH MA BA SA GJ KA OR KE MA PK GH BA FM ML MF NA StR
Amputation (78-89) + (20-43) + (0-17) + (56-78) + (0-68) + (5-46) + Amputation Amputation Amputation (34-39) + (43-57) + (74-89) ÷ (0-9)(33-110) + Amputation (0)(0-10)(0) (2-15> +
RM FG UH NA PW SM
IVECL
Mid*
Distal*
Proximal
Mid
Distal
(120-156) + (34-67) + (42-67) + (47-83) + (71-127) + (54-75) +
(112-198) + (156-209) + (78-123) + (85-143) + (71-186) + (78-89) +
Graft thrombosis Graft thrombosis (31-48) + (51-80) +
(56-78) + (63-79) +
(98-115) + (78-105) +
(6-41) +
(33-45) +
(26-74) +
(46-67) (55-78) (32-57) (0-8)(27-83)
(95-138) ÷ (82-132) + (87-138) + (0-13)(28-92) +
(37-97) + (58-89) + (45-73) + (18-89) ÷ (0-12)-
(77-108) ÷ (121-195) +
(0-6) (o-18)(0-7) (o-19)-
(0-12)(0-6) (0-17)(0-14)-
(o) (0) -
(o) (o)-
(0)
(o-2o)-
+ + + +
(18-37) (46-75) (20-46) (19-53) (0-13)
(0)(0)(0) (11-44) +
(13-60)(0-15)(6-20) (22-38) +
Amputation (0)(0) (0)-
(0-34) = (0-25) -~ (0)-
(7-22) -+ (0)(5-17)-
(18-30) -~ (0-15) -+ (0-27)-
(0)(O)(0)
(0) (0-2)-
(0-7) (0)-
(0-8) (0)-
(0) -
+ + + +
(71-99) + (184-294) +
(8-4o)-
*On the scintigraphic image, the bypass was divided in three parts (proximal, mid, distal). +Positive result by visual interpretation. -Negative result by visual interpretation. ()Results o f the platelet accumulation index range (lowest and highest values in regions o f interest).
VIII-related antigen on the cell surface revealed by immunofluorescent staining was used for EC identification. Transmission electron microscopy (TEM) was not performed routinely. The TEM examination, if it was done, demonstrated the Weibel-Paladies body in ECs. Surgery One operating team performed the operation. Anatomic and extraanatomic tunneling was performed with the help of magnifying glasses with use of careful operative and anastomotic technique. The same surgical techniques were used in control and IVECL groups. During the implantation of IVECL grafts the inner and outer surfaces of the prosthesis were kept moist with complete medium. Anastomoses were performed with 7-0 Prolene (Ethicon Inc., Somerville, N.J.) and 6-0 Prolene sutures for distal and proximal anastomoses, respectively. The site of the proximal anastomosis was always the common femoral artery. The site of the distal anastomosis in the crural region is shown in Table I. Routine administration of 2.2 gm angmentin
(combination of amoxidllin and clavulanate potassium) intravenously (Beecham, Belgium), heparin (Sandoz, Austria) 10,000 I U intravenously, and 250 mg Aspisol (aspirin) intravenously (Bayer) was done before the distal anastomosis had been performed. Antibiotic, anticoagulative, and antiaggregatory therapy with intravenous Augmentin 2 x 2.2 gm, subcutaneous heparin 3 x 5 000 IU, and intravenous aspirin 2 x 250 mg per day was continued for 5 days after surgery. Peroral drug treatment with 100 mg aspirin daily was carried out in both groups after discharge from the hospital. Follow-up After the femorocrural bypass procedure, patients were followed in the vascular clinic and in the noninvasive vascular laboratory every 3 months for the first year and every 6 months thereafter. Graft patency was monitored directly by angiography (intravenous digital subtraction angiography) 1 week after operation and then every 6 months, as well as indirectly by absorption of indium 111 oxine-labeled thrombocytes. Autologous platelets were labeled with indium
Volume 15 Number 3 March 1992
Endothelial cell lining of ePTFE grafts in humans
531
Table III. Life-table analysis of primary patency Group
In~rvM in days
At Hsk
Graft failure
Wi~drawn
Cumu~tive pa~n~
SEM
Con~ol
0 2 3 11 17 19 26 56 83 101 252 339 339 354 0 8 53 55 66
13 12 11 10 9 8 7 6 5 4 3 2 1 0 13 12 11 10 9 8 7 6 5 4 3 2 1 0
0 1 1 1 1 1 1 1 0 1 0 0 0 0 0 1 1 0 0 1 0 0 0 0 0 1 0 0
0 0 0 0 0 0 0 0 1 0 1 1 1 1 0 0 0 1 1 0 1 1 1 1 1 0 1 1
1.0000 0.9231 0.8462 0.7692 0.6923 0.6154 0.5385 0.4615
0.0000 0.0739 0.1001 0.1169 0.1280 0.1349 0.1385 0.1383
0.3692
0.1380
1.0000 0.9231 ,0.8462
0,0000 0.0739 0.1001
0.7521
0.1256
0.5014
0.2212
IVECL
79 114 222 321 325 326 358 405 599
111 oxine according to the method described by Thakur et al.9 Regions of interest (4 x 4 pixels each) encompassing the region of the bypass and the contralateral vessel were drawn on the indium 111 image, and the same was done for the technetium 99m human serum albumin image to allow correction for the blood pool radioactivity. Analysis of scintigrams was performed by visual interpretation and by measurement of platelet accumulation (calculation of platelet accumulation index) in the proximal, mid, and distal parts of the implanted grafts, l°'11 Values were expressed as a range of the lowest and highest values in regions of interest. Postoperative measurement of the ankle-brachial index was not performed because of fear of causing permanent occlusion of the reinforced graft from occlusion during examination.
Statistics The values are expressed as mean + SEM. Differences in mean values between IVECL and control groups for number of patent crural vessels, runoff, and resistance score were assessed by use of Student's unpaired t test. Differences between IVECL and control groups were tested by the life-table method by use of log-rank test for survivorship data. Qualitative comparison with a chi-square test of equality over strata were made. Data were computed with
software SAS-LIFETEST Procedure on an IBM PC (IBM Corp., Armonk, N.Y.) computer. 12 The recommendations of the Ad Hoc Committee on Reporting Standards were followed. 6'13,14
RESULTS Cell culture The inner area of explanted distal arm veins was 7.9 _+ 4.5 cm 2. The number of primary harvested cells was 28 _+ 3 × 103 ceHs/cm2 of vein surface. After a period of 7 to 9 days cell count increased to 9 to 10 × 10 ~. During the second cultivation period (2 to 3 weeks) the total number rose to 5.59 -+ 1.12 × 106 ECs. Seeding density was 43 _+ 11 × 103 ECs/cm 2 of graft surface. Seeding efficiency (number of VECs adherent to the graft surface) was 74% _+ 8%. After seeding, SEM demonstrated a nonconfluent monolayer with good intercellular overlapping. Morphologic control before graft implantation with SEM manifested cobblestone relief, as ECs cells were spread and confluent. Thirty-four patients were initially selected for cell culturing, but only 16 showed well-grown cell cultures with successful lining of PTFE.
Surgery We observed six early and two late graft thromboses in the control group. In the IVECL group one
Journal of VASCULAR
532 Magometschnigg et al.
SURGERY
Table IV. Life-table analysis of secondary patency Group Con~ol
IVECL
In~rvalindays
At Hsk
Graftfailure
Withdrawn
Cumu~tivepaten~
SEM
0 2 3 17 19 19 56 66 83 101 252 339 339 354 0 53 55 66 79 114 222 293 321 325 326 358 405 599
13 12 11 10 9 8 7 6 5 4 3 2 1 0 13 12 11 10 9 8 7 6 5 4 3 2 1 0
0 1 1 1 0 1 1 0 0 1 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 1 0 0
0 0 0 0 1 0 0 1 1 0 1 1 1 1 0 0 1 1 0 1 1 1 1 1 1 0 1 1
1.0000 0.9231 0.8462 0.7692
0.0000 0.0739 0.1001 0.1169
0.6154 0.5385
0.1349 0.1383
0.4308
0.1467
1.0000 0.9231
0.0000 0.0739
0.8308
0.1100
0.5538
0.2377
early and three late graft thromboses occurred. Each case of early graft thrombosis was treated with balloon trombectomy followed by careful revision of the site of the distal anastomosis. Graft failure as the result of technical error was found with an inappropriate site of distal anastomosis in one case in the control group and in one case in the IVECL group. The late graft thromboses were treated with pharmacologic means or amputation. Four patients (30%) in the control group underwent major amputations (above-knee and below-knee) as the ultimate therapy. Amputation was necessary in two patients (15%) in the IVECL group. No deaths occurred within the first 30 days after operation. Three late deaths occurred, all of them with an occluded graft. The influence of angiographic score (number of patent crural vessels, runoff, and resistance) on the prognosis of the reconstruction is given in Table I. No patient in either group had a graft infection. Wound infections developed in six patients in the control, and in three patients in the IVECL group. Complications such as a hematoma, seroma, and lymphfistula were more frequent in the control group. In the follow-up period scintigraphic studies showed higher affinity for accumulation and deposi-
tion of platelets on the inner surface of the ePTFE graft in the control group (Table II). Life-table analysis (primary and secondary patency) of 26 grafts after implantation is shown in Tables III and IV and Figs. 1 and 2. Early secondary graft patency (0 to 30 days) was 92% for the IVECL group and 53% for the control patients. Statistical evaluation for equality of the groups showed the followingp values for the chi-square test over strata for primary patency: log rank, 0.0482; Wilcoxon, 0.0470; and - 2 log (LR), 0.0276; and for secondary patency: log rank, 0.0317; Wilcoxon, 0.0257; - 2 log (LR) 0.0200. DISCUSSION Our study demonstrates that the described multistage technique for EC lining of prosthetic graft material results in a functional graft endothelium. The use of prelined prosthetic grafts for reoperation of crural vessels, followed for i to 18 months, resulted in a markedly decreased rate of early graft occlusion compared to standard ePTFE grafts) s-2° Few noninvasive methods exist to assess the implantation of ECs on a graft and to determine their antithrombotic function. The most commonly used and currently most reliable method is the marking of
Volume 15 Number 3 March 1992
Endothelial cell lining of ePTFE grafls in humans 533
patency
100% ~ IVECL
75% "
--I
oo%1 c.oto i
p < 0.05
25%
I
89
I
179
268
358 days
Fig. 1. Life-table curves (primary patency) for control and WECL groups. patency
100%'
½
IVECL
75%[
control
50%-
25% p < 0.05 • i
89
i
[
179
268
I
358 days
Fig. 2. Life-table curves (secondary patency) for control and IVECL groups. autologous platelets with indium 111 oxine. 1°,11 In our study the platelet accumulation index of the endothelialized prostheses was less than that of the controls at all time points. Direct proof of EC implantation on the graft was successfully obtained by biopsy in one case. Four months after the first operation (aortofemoral IVECL-bypass) it was possible to obtain tissue (5 cm above the distal anastomosis) during the course of a subsequent femoropopliteal vein bypass operation. An EC monolayer could be demonstrated by normal histologic examination, SEM, and TEM (unpubfished data). In this preliminary study, randomization of patients was not attempted. Stage 4 and 5 SVS/ISCVS of chronic peripheral vascular disease is fortunately not frequent. Criteria for the selection of patients (failure of one or more previous crural bypass procedures, no usable autologous vein, angiographically suitable vessels for revascularization, patient consent, only elective surgery) further re-
duced the number of acceptable candidates. Harvesting, cultivation, and lining of ECs is complicated by a variety of factors, and efficiency in our trial was about 50%. Initially 34 patients were selected for cell culturing, but only 16 of them had good cell culture results with excellent graft endothelization. Before graft implantation, three patients with a graft in preparation experienced acute deterioration of the ischemic leg. Therefore an untreated graft was used, and they were discharged from the study. The remaining 18 cell cultures had various problems: infection, insufficient EC growth, and lack of EC purity (fibroblasts, smooth muscle cells). Only eight patients of the "failing culture group" were included in the study as controls, 10 of them dropped out because they were operated on by different surgeons. The final control group was made up of eight patients who failed cell culture and five patients who refused the new method. Despite lack of randomization, patient groups
534 Magometschnigg et al.
were similar when compared both for clinical characteristics and disease severity using the SVS/ISCVS scoring system. The SVS/ISCVS scoring system takes into account the vessel chosen for anastomosis, the severity of disease, the patency of the arch if applicable, and the base resistance. Peterkin et al.21 showed that the system correlates well with the measured peripheral resistance. Almost all patients entering the study were in clinical category 4 SVS/ISCVS. The clinical status of patients between cell harvest and the time of definitive operation is an important factor in determining feasibility of this prelining technique. Patients must wait a number of weeks during which harvested cells are cultured and the graft lined. Intensive theologic therapy was necessary in our patients to avoid acute clinical deterioration. In three cases gangrene developed during the waiting period. Numerous previous studies have dealt with the technique of single stage, intraoperative EC seeding. 2224 The advantage of this technique is that no waiting period is necessary. The crucial weaknesses of this approach, however, have been the low number of initially harvested ECs and insufficient cell attachment to the graft. 4 The relationship between the surface of the donor vein and that of the graft must be at least 0.5 : 1 to ensure a nearly confluent cell layerY Cell culture is able to increase this relationship to at least 1 : 15. A lower arm vein was chosen as the donor vein because the arm veins are large enough (7.9 -+ 4.5 cm 2) and easily accessible. Residual veins in the legs were not used because of both fear of poor wound healing and of potential ischemic injury to the ECs. Based on recent studies we chose fibrin glue to precoat the graft surface since in vitro tests on shear stress resistance of the confluent cell layer revealed this precoating technique to be superior to others. 8 It promotes cell attachment, spreading, and proliferation. The fibrin glue has the additional advantage of clinical applicability. In four IVECL patients angiographic follow-up demonstrated 50% bypass stenoses in the middle part of the graft. Such angiographically seen stenoses in untreated ePTFE grafts is uncommon. Polytetrafluoroethylene grafts with an internodal distance of 30 ~tm acquire intimal hyperplasia in the region of anastomoses. 26 Investigation by TEM and SEM of the stenosed grafts revealed smooth muscle cell proliferation as the cause of this narrowing, and it is of concern that this phenomenon may influence long-term patency. In conclusion, a decreased number of early graft occlusions were seen in the IVECL
Journal of VASCULAR SURGERY
group when compared to controls. This decrease could not be attributed to differences between the groups in operating technique, indications for surgery, morphology or resistance of the peripheral vessels, or perioperative medications. Aside from graft occlusion caused by technical reasons occurring in two patients (one IVECL patient and one control patient), graft trombosis is due to platelet aggregation. 22 Platelet activation by prosthetic graft material is a crucial factor leading to the poor results obtained with synthetic grafts. Antiaggregatory measures, although helpful, are not able to completely neutralize platelet activation. 27 By coating prosthetic grafts with an endothelial layer by use of a multistage technique, platelet aggregation can be diminished, leading to improved early surgical results. CONCLUSION Our study demonstrates that the technique of EC lining in reoperation of crural vessels has a markedly decreased rate of early graft occlusion as compared to standard ePTFE grafts followed for 1 to 18 months after implantation. The tVECL technique in patients without suitable remaining autologous veins is promising. Longer follow-up time is necessary to clarify the questions of EC behavior and the role of smooth muscle cells within the IVECL grafts. REFERENCES 1. Ziats NP, Miller KM, Anderson JM. In vitro and in vivo interactions of cells with biomaterials. Biomaterials 1988;9: 5-13. 2. Herring M, Gardner A, Glover ~A. A single-stage technique for seeding vascular grafts with autogenous endothelium. Surgery 1978;84:498-504. 3. Fasol R, Zilla P, Deutsch M, Grimm M, Fischlein T, Laufer G. Human endothelial cell seeding: evaluation of its effectiveness by platelet parameters after one year. J VASC SURG 1989;9:432-6. 4. Rosenman JE, Kempczinski RF, Pearce WH, Silberstein EB. Kinetics of endothelial cell seeding. J VASC SURG 1985;2: 778-84. 5. Helsiniki Declaration. Ann Intern Med 1966;65:367-8. 6. Rutherford RB, Flanigan DP, Gupta SK, et al. Suggested standards for reports dealing with lower extremity ischernia. VASC SURG 1986;4:80-94. 7. Zilla P, Fasol P, Dudeck U, et al. In situ cannulation, microgrid follow-up and low-density plating provide first passage endothelial cell masscultures for in vitro lining. J VAsc SURG 1990;12:180-9. 8. Zilla P, Fasol R, Preiss P, et al. Use of fibrin glue as a substrate for in vitro endothelialization of PTFE vascular grafts. Surgery 1989;105:515-22. 9. Thakur ML, Walsh L, Malech HL, Gottschalk A. Indium- 111 labeled human platelets: improved method, efficiency and evaluation, l Nucl Med 1981;22:381.
Volume 15 Number 3 March 1992
10. Minar E, Dudczsak 1~ Ehringer H. Postoperative platelet diagnosis: a prerequisite for chnical seeding. In: Zilla P, Fasol R, Deutsch M, eds. Endotheliahzation of vascular grafts, Basel: Karger, 1987:64-70. 11. Isaka Y, Kimura K, Yoneda S, et al. Platelet accumulation in carotid atherosclerotic lesions: semiquantitative analysis with indium-ill platelets and tectmetium-99m human serum albumin. J Nucl Med 1984;25:556-63. 12. SAS Institute Inc, SAS/STAT, User's guide, version 6, fourth ed, vol 2, Cary, NC 1989:846. 13. Peto R, Pike MC, Armitage P, et al. Design and analysis of randomized trials required prolonged observations of each patient. I. Introduction and design. Br J Cancer 1976;34: 585-612. 14. Peto R, Pike MC, Armitage P, et al. Design and analysis of randomized trials required prolonged observations of each patient. Analysis and examples. Br J Cancer 1977;35:1-39. 15. Budd JS, Brennan J, Beard JD, Warren H, Burton PR, Bell PRF. Infrainquinal bypass surgery: factors determining late graft patency. Br J Surg 1990;77:1382-7. 16. Veterans Administration Cooperative Study Group 141. Comparative evaluation of prosthetic, reversed, and in situ vein bypass grafts in distal pophteal and tibial-peroneal revascularization. Arch Surg 1988;123:434-8. 17. Veith FJ, Gupta SK, Ascer E, et al. Six-year prospective multicenter randomized comparison ofautologous saphenous vein and expanded polytetrafluoroethylene grafts in infrainquinal arterial reconstruction. J VASC StJr~G 1986;3:10414. 18. Whittemore AD, Kent KC, Donaldson MC, Couch NP, Mannick JA. What is the proper role of polytetrafluoroethylene grafts in infrainquinal reconstruction? J VASC SURG 1989;10:299-305. 19. Bergan J)', Veith FJ, Bernhard VM, et al. Randomization of
Endothelial cell lining of ePTFE grafts in humans 535
20.
21.
22.
23.
24. 25. 26. 27.
autogenous vein and polytetrafluoroethylene grafts in femoral-distal reconstruction. Surgery 1982;92:921-9. Charlesworth PM, Brewster DC, Darhng RC, Robinson JG, Hallet JW. The fate of polytetrafluoroethylene grafts in lower limb bypass surgery: six-year follow-up. Br • Surg 1985;72: 896-9. Peterkin GA, Manabe S, LaMorte WW, Menzoian JO. Evaluation of a proposed standard reporting system for preoperative angiograms in infrainquinal bypass procedures: angiographic correlates of measured runoff resistance. J VASC SURG 1988;7:379-85. Schneider PA, Hanson SR, Price TM, Harker LA. Performed confluent endothelial cell monolayers prevent early platelet deposition on vascular prostheses in baboons. J VAsc S~RG 1988;8:229-35. 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 Vasc SURG 1984;1:224-33. Ortenwall P, Wadenvik H, Kutti ~, Risberg B. Endothehal cell seeding reduces thrombogenicity of Dacron grafts in humans. J VAsc SURG 1990;11:403-10. Herring M, DiUey R,Cullison T, Gardner A, Glover 5. Seeding endothefium on canine arterial prostheses: the size of inoculum. J Surg Res 1980;28:35-8. Zacharias SRK, Kirkman TR, Clowes AS. Mechanisms of heating in synthetic grafts. T VASC SURG 1987;6:429-36. ZiUa P, Groscurth P, Varga G, Fasol R. PGI2 and PGE1 induce morphological alterations in human platelets similar to those of the initial phase of activation. Exp Hematol 1987;15:741-7.
Submitted Apr. 18, 1991; accepted July 1, 1991.
The use of autologous vein grafts indisputably results in the best outcome after surgery to bypass the smaller peripheral vessels. However, because of multiple previous operations, patients with recurrent occlusion in the crural vessel bed often have no remaining suitable veins and must have bypasses with biologic or artificial prostheses. Failure of these artificial prostheses lies ultimately in the blood-contacting surface, which promotes thrombotic occlusion through the activation of platelets. 1 Intraoperative "single stage seeding" of prosthetic grafts has been tested? In their report, From the SecondDepartmentof Surgery,Universityof Vienna (Drs. Magometschnigg, Kadletz, Vodrazka, Dock, Grimm, Grabenw6ger, Staudacher, Fenzl, Wolner), and the First Department of Internal Medicine, Division of Angiology, Universityof Vienna (Dr. Minar). Reprint requests: HeinrichMagometschnigg,MD, II Chirurgische Universit~/tsklinik,Spitalgasse23, I090 Vienna, Austria. 2411/32197
Fasol et al.3 state "The similar results for seeded and nonseeded vascular grafts indicate the failure of seeded grafts to develop a nontraumatizing surface comparable to that ofsaphenous veins." These results could be explained by the two following factors: (1) The number of primarily harvested endothelial cells (ECs) is not sufficient for the formation of a confluent cell layer before implantation. (2) The time between seeding and implantation is too short for effective adhesion of the cells to the graft surface leading to cell loss induced by blood flow.4 The purpose of this study was to test the clinical effectiveness of a new prelining procedure for expanded polytetrafluoroethylene (ePTFE) grafts. The procedure is made up of three individual steps: (1) Cell harvesting from an autologous arm vein performed 4 to 7 weeks before operation, with subsequent cell culture in the laboratory. (2) Seeding of this increased number of autologous ECs onto the prosthesis that has been precoated with fibrin glue. 527
528 Magometschnigg et al.
(3) Cultivation of the cells on the graft surface for 1 additional week to induce expression of the extraccllular matrix that promotes cell adherence.
METHODS Between June 1989 and December 1990, 26 patients with limb-threatening ischemia, both with and without ulceration and gangrene, were entered into the study. All patients were fully informed about the purpose of and methods used in the study. Informed consent was obtained in accordance with the guidelines of the Helsinki Declaration. ~ The study was reviewed and approved by the Ethical Committee of the University of Vienna. Every patient had had failure of one or more previous crural bypass procedures, had no usable autologous vein, and was considered angiographically suitable for revascularization. Patients were divided into two groups, a control group with 13 untreated ePTFE graft implantations and an in vitro endothelial cell lining (IVECL) group with 13 treated ePTFE grafts. Eight men and five women were in both groups. Average age of the control group was 69 years and of the IVECL group 67 years. Risk factors such as tobacco use (IVECL, eight; control patients, five), diabetes (IVECL, nine; control, nine), hypertension (IVECL, three; control, four), and cardiac status were assessed. The assessment of preoperative clinical stage demonstrated advanced peripheral vascular disease. Six control and eight IVECL patients had clinical category 4 (SVS/ISCVS). Seven control and five IVECL patients had clinical category 5 (SVS/ISCVS). All patients in clinical category 5 suffered from leg ulcers. Gangrene of the toes and forefoot was seen in five control and three IVECL patients. The control group had a higher rate of necrosis and a greater number of infected forefoot necroses. Angiography The patients underwent preoperative intraarterial angiography with special attention to the delineation of the calf and foot arteries. Selective orthograde puncture and long delay times were used as were vasodilators. An angiographic score for the description of runoff and resistance according to Ad Hoc Committee on Reporting Standards of the Society for Vascular Surgery/North American Chapter and the International Society for Cardiovascular Surgery (SVS/ISCVS), was calculated, and the groups were compared. 6 This scoring system is based on the patency of the vessels and their relative contribution to the outflow tract. A score of 1 corresponds to excellent runoff and 10 to a blind segment. With use
Journal of VASCULAR SURGERY
of Student's unpaired t test, no significant differences were found between the groups with respect to the morphologic picture or the resistance. The analysis of the preoperative angiogram for each individual patient is shown in Table I.
Vein harvesting Arms veins were harvested as described for saphenous veins according to the "no touch technique. "7 During the procedure the vein was kept moist with saline solution with papaverine at room temperature. A distal cannula was inserted and secured, and the vessel was gently flushed with complete medium. After excision, the cannulated and collagenase filled vessel was transferred to the cell laboratory. Cell culture A standard enzymatic harvest technique with use of 0.1% collagenase (CLS II; Cooper Biomedical, Malvern, Pa.) was applied for 15 minutes at 37 ° C for the establishment of primary cultures from the human venous endothelial cells (VECs). Primary cultures were grown to confluence in 25 cm 2 tissue culture flasks precoated with 30 ~g/rnl human fibronectin and fed two to three times a week. Complete medium was composed of 80% M-199 medium (Gibco Laboratories, Lawrence, Mass.), buffered with 2.2 gm/L sodium bicarbonate, 20% heat inactivated pooled human serum, gentamycin 0.07 mg/ml (Gibco), 70 ~tg/ml of EC growth supplement (ECGS, Collaborative Research Inc., Lexington, Mass.), and 180 ~g/ml preservative-free heparin (Sero-Med, Vienna, Austria), 2 mmol/L L-glutamine (Gibco No. 043 50 30D). When confluence in 25 cm 2 was achieved, the cells were then resuspended by treatment with a solution of 0.05% trypsin (1 : 250) buffered in saline and 0.02% ethylenediamine tetraacetic acid (EDTA) (Inotech, Switzerland) and replaced in two 150 cm 2 tissue culture flasks coated again with human fibronectin. After growth to confluence, second passage VECs were detached from 150 cm 2 culture flasks with trypsin-EDTA, and a complete medium was added to inactivate any residual trypsin before the cell suspension was centrifuged at 800 rpm for 10 minutes in 4 ° C. The supernatant was discarded, and the pellet was resuspended in 20 ml complete medium for the seeding procedure. Graft material Six millimeter reinforced thin-walled PTFE grafts (W. L. Gore & Associates, Elkton, Md.). 80 cm in
V o l u m e 15 Number 3
Endothelial cell lining of ePTFE grafts in humans
March 1992
529
Table I. Influence of preoperative morphology and runoff assessment of crural vessels on postoperative results Preoperative
Group Control
Patient
Runoff score
Resistance score (A G)
Site of distalanastom.
Graft thrombosis
Graft
Early
Late
Patent
Amputation
SA
1
8.5
1
ATA
Yes
-
-
Yes
FM
1
9.0
3
ATA
Yes
-
-
-
UF UH MA
1 1
8.0 8.0
1 1
Tractus Tractus
Yes -
Yes
-
-
2
7,0
1
ATA
-
-
Yes
-
BA
1
11.0
3
ATA
-
-
Yes
-
SA
GJ
1 2
9.5 8.0
2 2
ATA PTA
-
Yes
Yes -
Yes
KA
1
12.5
7
PTA
Yes
-
-
Yes
OR
1 3 1
11.0 6.0 10.5
3 5 3
ATA ATA ATA
Yes -
-
Yes Yes
Yes -
PK GH
1
11.0
3
ATA
Yes
-
Yes
-
1
8.5
1
ATA
-
-
Yes
-
BA FM
1 2
8.0 7.0
3 1
PTA Tractus
-
Yes
Yes -
Yes
ML MF
1 2
10.0 4.5
3 1
ATA Tractus
-
Yes -
Yes
Yes -
NA
1
9.5
2
Tractus
-
-
Yes
-
StR RM
3
5.0
2
Tract-us
-
-
Yes
-
1
9.5
2
ATA
Yes
-
Yes
-
FG
1 2
9.0 9.0
2 2
ATA Tractus
-
Yes
Yes -
-
KE MA IVECL
Patent crural vessels
Postoperative
UH NA
2
10.0
6
PTA
-
-
Yes
-
PW SM
1 2
12,0 10.0
5 5
ATA ATA
-
-
Yes Yes
-
p value
0.371
0.437
-
ATA,
A n t e r i o r t i b i a l a r t e r y ; PTA, p o s t e r i o r t i b i a l a r t e r y ; Tractus, t r a c t u s t i b i o f i b u l a r i s . E a r l y g r a f t t h r o m b o s i s less than 3 0 d a y s ; l a t e g r a f t thrombosis greater than 3 0 d a y s .
length were used for each operation. The internodal distance was 30 txm. For the IVECL group grafts were prepared according to the following method. Precoating procedure for lining o f the IVECL grafts. The fibrinogen compound (Immuno, Austria) contained 3.5 to 5.5 mg/ml fibrinogen, 0.1 to 0.45 mg human fibronectin, 1.4 ~g/ml plasminogen, 10,000 units/ml aprotinin (Trasylol, Bayer, Germany). The thrombin compound contained 20 units/ml thrombin, 475 ng/ml Ca gluconicum, 5000 units/ml aprotinin. Precoating with fibrin glue was performed by forcing this substrate through the interstices of the PTFE graft with 4F Fogarty balloon catheter. 8 After the fibrinogen compound was repeatedly forced through the interstices, the entire graft was subsequently placed into the thrombin solution for 10 minutes at room temperature. Subsequently the graft was rinsed twice with calcium/magnesium-free phosphate-buffered saline (Flow Laboratories Inc., McLean, Va.) before the entire procedure was repeated a second time. Seeding procedure. After surface preparation,
the graft was filled with the VEC suspension. Both ends were occluded with holders from a rotatory device. The prosthesis was incubated in a horizontal position in the rotatory device. An even distribution of VECs was obtained by rotating the graft 6 rph for 3 hours at a constant temperature of 37 ° C in a 5% carbon dioxide atmosphere. At the end of the seeding procedure the graft was gently flushed with 20 ml of M-199. From the VEC count in suspensions before and after rotation the seeding efficiency was calculated. The graft was then placed into a filter-protected glass container and cultivated in 200 ml complete medium for another 7 to 10 days. A 1 cm length was cut from the graft for examination. At the same time, the samples of the graft for bacterial (sterility testincubation period for 10 days), mycotic, and mycoplasmal examination were obtained. Confirmation of endothelial cell identity. Venous endothelial cells were identified by scanning electron microscopy (SEM) examination immediately after the seeding procedure and 2 to 4 hours before the graft implantation. The presence of factor
530
Journal of VAaCULAR SURGERY
Magometschnigg et al.
Table II. Scintigraphic results-visual interpretation and platelet accumulation index 3 Months after operation
1 Week after operation Group
Patient
Proximal*
Control
SA FM UF UH MA BA SA GJ KA OR KE MA PK GH BA FM ML MF NA StR
Amputation (78-89) + (20-43) + (0-17) + (56-78) + (0-68) + (5-46) + Amputation Amputation Amputation (34-39) + (43-57) + (74-89) ÷ (0-9)(33-110) + Amputation (0)(0-10)(0) (2-15> +
RM FG UH NA PW SM
IVECL
Mid*
Distal*
Proximal
Mid
Distal
(120-156) + (34-67) + (42-67) + (47-83) + (71-127) + (54-75) +
(112-198) + (156-209) + (78-123) + (85-143) + (71-186) + (78-89) +
Graft thrombosis Graft thrombosis (31-48) + (51-80) +
(56-78) + (63-79) +
(98-115) + (78-105) +
(6-41) +
(33-45) +
(26-74) +
(46-67) (55-78) (32-57) (0-8)(27-83)
(95-138) ÷ (82-132) + (87-138) + (0-13)(28-92) +
(37-97) + (58-89) + (45-73) + (18-89) ÷ (0-12)-
(77-108) ÷ (121-195) +
(0-6) (o-18)(0-7) (o-19)-
(0-12)(0-6) (0-17)(0-14)-
(o) (0) -
(o) (o)-
(0)
(o-2o)-
+ + + +
(18-37) (46-75) (20-46) (19-53) (0-13)
(0)(0)(0) (11-44) +
(13-60)(0-15)(6-20) (22-38) +
Amputation (0)(0) (0)-
(0-34) = (0-25) -~ (0)-
(7-22) -+ (0)(5-17)-
(18-30) -~ (0-15) -+ (0-27)-
(0)(O)(0)
(0) (0-2)-
(0-7) (0)-
(0-8) (0)-
(0) -
+ + + +
(71-99) + (184-294) +
(8-4o)-
*On the scintigraphic image, the bypass was divided in three parts (proximal, mid, distal). +Positive result by visual interpretation. -Negative result by visual interpretation. ()Results o f the platelet accumulation index range (lowest and highest values in regions o f interest).
VIII-related antigen on the cell surface revealed by immunofluorescent staining was used for EC identification. Transmission electron microscopy (TEM) was not performed routinely. The TEM examination, if it was done, demonstrated the Weibel-Paladies body in ECs. Surgery One operating team performed the operation. Anatomic and extraanatomic tunneling was performed with the help of magnifying glasses with use of careful operative and anastomotic technique. The same surgical techniques were used in control and IVECL groups. During the implantation of IVECL grafts the inner and outer surfaces of the prosthesis were kept moist with complete medium. Anastomoses were performed with 7-0 Prolene (Ethicon Inc., Somerville, N.J.) and 6-0 Prolene sutures for distal and proximal anastomoses, respectively. The site of the proximal anastomosis was always the common femoral artery. The site of the distal anastomosis in the crural region is shown in Table I. Routine administration of 2.2 gm angmentin
(combination of amoxidllin and clavulanate potassium) intravenously (Beecham, Belgium), heparin (Sandoz, Austria) 10,000 I U intravenously, and 250 mg Aspisol (aspirin) intravenously (Bayer) was done before the distal anastomosis had been performed. Antibiotic, anticoagulative, and antiaggregatory therapy with intravenous Augmentin 2 x 2.2 gm, subcutaneous heparin 3 x 5 000 IU, and intravenous aspirin 2 x 250 mg per day was continued for 5 days after surgery. Peroral drug treatment with 100 mg aspirin daily was carried out in both groups after discharge from the hospital. Follow-up After the femorocrural bypass procedure, patients were followed in the vascular clinic and in the noninvasive vascular laboratory every 3 months for the first year and every 6 months thereafter. Graft patency was monitored directly by angiography (intravenous digital subtraction angiography) 1 week after operation and then every 6 months, as well as indirectly by absorption of indium 111 oxine-labeled thrombocytes. Autologous platelets were labeled with indium
Volume 15 Number 3 March 1992
Endothelial cell lining of ePTFE grafts in humans
531
Table III. Life-table analysis of primary patency Group
In~rvM in days
At Hsk
Graft failure
Wi~drawn
Cumu~tive pa~n~
SEM
Con~ol
0 2 3 11 17 19 26 56 83 101 252 339 339 354 0 8 53 55 66
13 12 11 10 9 8 7 6 5 4 3 2 1 0 13 12 11 10 9 8 7 6 5 4 3 2 1 0
0 1 1 1 1 1 1 1 0 1 0 0 0 0 0 1 1 0 0 1 0 0 0 0 0 1 0 0
0 0 0 0 0 0 0 0 1 0 1 1 1 1 0 0 0 1 1 0 1 1 1 1 1 0 1 1
1.0000 0.9231 0.8462 0.7692 0.6923 0.6154 0.5385 0.4615
0.0000 0.0739 0.1001 0.1169 0.1280 0.1349 0.1385 0.1383
0.3692
0.1380
1.0000 0.9231 ,0.8462
0,0000 0.0739 0.1001
0.7521
0.1256
0.5014
0.2212
IVECL
79 114 222 321 325 326 358 405 599
111 oxine according to the method described by Thakur et al.9 Regions of interest (4 x 4 pixels each) encompassing the region of the bypass and the contralateral vessel were drawn on the indium 111 image, and the same was done for the technetium 99m human serum albumin image to allow correction for the blood pool radioactivity. Analysis of scintigrams was performed by visual interpretation and by measurement of platelet accumulation (calculation of platelet accumulation index) in the proximal, mid, and distal parts of the implanted grafts, l°'11 Values were expressed as a range of the lowest and highest values in regions of interest. Postoperative measurement of the ankle-brachial index was not performed because of fear of causing permanent occlusion of the reinforced graft from occlusion during examination.
Statistics The values are expressed as mean + SEM. Differences in mean values between IVECL and control groups for number of patent crural vessels, runoff, and resistance score were assessed by use of Student's unpaired t test. Differences between IVECL and control groups were tested by the life-table method by use of log-rank test for survivorship data. Qualitative comparison with a chi-square test of equality over strata were made. Data were computed with
software SAS-LIFETEST Procedure on an IBM PC (IBM Corp., Armonk, N.Y.) computer. 12 The recommendations of the Ad Hoc Committee on Reporting Standards were followed. 6'13,14
RESULTS Cell culture The inner area of explanted distal arm veins was 7.9 _+ 4.5 cm 2. The number of primary harvested cells was 28 _+ 3 × 103 ceHs/cm2 of vein surface. After a period of 7 to 9 days cell count increased to 9 to 10 × 10 ~. During the second cultivation period (2 to 3 weeks) the total number rose to 5.59 -+ 1.12 × 106 ECs. Seeding density was 43 _+ 11 × 103 ECs/cm 2 of graft surface. Seeding efficiency (number of VECs adherent to the graft surface) was 74% _+ 8%. After seeding, SEM demonstrated a nonconfluent monolayer with good intercellular overlapping. Morphologic control before graft implantation with SEM manifested cobblestone relief, as ECs cells were spread and confluent. Thirty-four patients were initially selected for cell culturing, but only 16 showed well-grown cell cultures with successful lining of PTFE.
Surgery We observed six early and two late graft thromboses in the control group. In the IVECL group one
Journal of VASCULAR
532 Magometschnigg et al.
SURGERY
Table IV. Life-table analysis of secondary patency Group Con~ol
IVECL
In~rvalindays
At Hsk
Graftfailure
Withdrawn
Cumu~tivepaten~
SEM
0 2 3 17 19 19 56 66 83 101 252 339 339 354 0 53 55 66 79 114 222 293 321 325 326 358 405 599
13 12 11 10 9 8 7 6 5 4 3 2 1 0 13 12 11 10 9 8 7 6 5 4 3 2 1 0
0 1 1 1 0 1 1 0 0 1 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 1 0 0
0 0 0 0 1 0 0 1 1 0 1 1 1 1 0 0 1 1 0 1 1 1 1 1 1 0 1 1
1.0000 0.9231 0.8462 0.7692
0.0000 0.0739 0.1001 0.1169
0.6154 0.5385
0.1349 0.1383
0.4308
0.1467
1.0000 0.9231
0.0000 0.0739
0.8308
0.1100
0.5538
0.2377
early and three late graft thromboses occurred. Each case of early graft thrombosis was treated with balloon trombectomy followed by careful revision of the site of the distal anastomosis. Graft failure as the result of technical error was found with an inappropriate site of distal anastomosis in one case in the control group and in one case in the IVECL group. The late graft thromboses were treated with pharmacologic means or amputation. Four patients (30%) in the control group underwent major amputations (above-knee and below-knee) as the ultimate therapy. Amputation was necessary in two patients (15%) in the IVECL group. No deaths occurred within the first 30 days after operation. Three late deaths occurred, all of them with an occluded graft. The influence of angiographic score (number of patent crural vessels, runoff, and resistance) on the prognosis of the reconstruction is given in Table I. No patient in either group had a graft infection. Wound infections developed in six patients in the control, and in three patients in the IVECL group. Complications such as a hematoma, seroma, and lymphfistula were more frequent in the control group. In the follow-up period scintigraphic studies showed higher affinity for accumulation and deposi-
tion of platelets on the inner surface of the ePTFE graft in the control group (Table II). Life-table analysis (primary and secondary patency) of 26 grafts after implantation is shown in Tables III and IV and Figs. 1 and 2. Early secondary graft patency (0 to 30 days) was 92% for the IVECL group and 53% for the control patients. Statistical evaluation for equality of the groups showed the followingp values for the chi-square test over strata for primary patency: log rank, 0.0482; Wilcoxon, 0.0470; and - 2 log (LR), 0.0276; and for secondary patency: log rank, 0.0317; Wilcoxon, 0.0257; - 2 log (LR) 0.0200. DISCUSSION Our study demonstrates that the described multistage technique for EC lining of prosthetic graft material results in a functional graft endothelium. The use of prelined prosthetic grafts for reoperation of crural vessels, followed for i to 18 months, resulted in a markedly decreased rate of early graft occlusion compared to standard ePTFE grafts) s-2° Few noninvasive methods exist to assess the implantation of ECs on a graft and to determine their antithrombotic function. The most commonly used and currently most reliable method is the marking of
Volume 15 Number 3 March 1992
Endothelial cell lining of ePTFE grafls in humans 533
patency
100% ~ IVECL
75% "
--I
oo%1 c.oto i
p < 0.05
25%
I
89
I
179
268
358 days
Fig. 1. Life-table curves (primary patency) for control and WECL groups. patency
100%'
½
IVECL
75%[
control
50%-
25% p < 0.05 • i
89
i
[
179
268
I
358 days
Fig. 2. Life-table curves (secondary patency) for control and IVECL groups. autologous platelets with indium 111 oxine. 1°,11 In our study the platelet accumulation index of the endothelialized prostheses was less than that of the controls at all time points. Direct proof of EC implantation on the graft was successfully obtained by biopsy in one case. Four months after the first operation (aortofemoral IVECL-bypass) it was possible to obtain tissue (5 cm above the distal anastomosis) during the course of a subsequent femoropopliteal vein bypass operation. An EC monolayer could be demonstrated by normal histologic examination, SEM, and TEM (unpubfished data). In this preliminary study, randomization of patients was not attempted. Stage 4 and 5 SVS/ISCVS of chronic peripheral vascular disease is fortunately not frequent. Criteria for the selection of patients (failure of one or more previous crural bypass procedures, no usable autologous vein, angiographically suitable vessels for revascularization, patient consent, only elective surgery) further re-
duced the number of acceptable candidates. Harvesting, cultivation, and lining of ECs is complicated by a variety of factors, and efficiency in our trial was about 50%. Initially 34 patients were selected for cell culturing, but only 16 of them had good cell culture results with excellent graft endothelization. Before graft implantation, three patients with a graft in preparation experienced acute deterioration of the ischemic leg. Therefore an untreated graft was used, and they were discharged from the study. The remaining 18 cell cultures had various problems: infection, insufficient EC growth, and lack of EC purity (fibroblasts, smooth muscle cells). Only eight patients of the "failing culture group" were included in the study as controls, 10 of them dropped out because they were operated on by different surgeons. The final control group was made up of eight patients who failed cell culture and five patients who refused the new method. Despite lack of randomization, patient groups
534 Magometschnigg et al.
were similar when compared both for clinical characteristics and disease severity using the SVS/ISCVS scoring system. The SVS/ISCVS scoring system takes into account the vessel chosen for anastomosis, the severity of disease, the patency of the arch if applicable, and the base resistance. Peterkin et al.21 showed that the system correlates well with the measured peripheral resistance. Almost all patients entering the study were in clinical category 4 SVS/ISCVS. The clinical status of patients between cell harvest and the time of definitive operation is an important factor in determining feasibility of this prelining technique. Patients must wait a number of weeks during which harvested cells are cultured and the graft lined. Intensive theologic therapy was necessary in our patients to avoid acute clinical deterioration. In three cases gangrene developed during the waiting period. Numerous previous studies have dealt with the technique of single stage, intraoperative EC seeding. 2224 The advantage of this technique is that no waiting period is necessary. The crucial weaknesses of this approach, however, have been the low number of initially harvested ECs and insufficient cell attachment to the graft. 4 The relationship between the surface of the donor vein and that of the graft must be at least 0.5 : 1 to ensure a nearly confluent cell layerY Cell culture is able to increase this relationship to at least 1 : 15. A lower arm vein was chosen as the donor vein because the arm veins are large enough (7.9 -+ 4.5 cm 2) and easily accessible. Residual veins in the legs were not used because of both fear of poor wound healing and of potential ischemic injury to the ECs. Based on recent studies we chose fibrin glue to precoat the graft surface since in vitro tests on shear stress resistance of the confluent cell layer revealed this precoating technique to be superior to others. 8 It promotes cell attachment, spreading, and proliferation. The fibrin glue has the additional advantage of clinical applicability. In four IVECL patients angiographic follow-up demonstrated 50% bypass stenoses in the middle part of the graft. Such angiographically seen stenoses in untreated ePTFE grafts is uncommon. Polytetrafluoroethylene grafts with an internodal distance of 30 ~tm acquire intimal hyperplasia in the region of anastomoses. 26 Investigation by TEM and SEM of the stenosed grafts revealed smooth muscle cell proliferation as the cause of this narrowing, and it is of concern that this phenomenon may influence long-term patency. In conclusion, a decreased number of early graft occlusions were seen in the IVECL
Journal of VASCULAR SURGERY
group when compared to controls. This decrease could not be attributed to differences between the groups in operating technique, indications for surgery, morphology or resistance of the peripheral vessels, or perioperative medications. Aside from graft occlusion caused by technical reasons occurring in two patients (one IVECL patient and one control patient), graft trombosis is due to platelet aggregation. 22 Platelet activation by prosthetic graft material is a crucial factor leading to the poor results obtained with synthetic grafts. Antiaggregatory measures, although helpful, are not able to completely neutralize platelet activation. 27 By coating prosthetic grafts with an endothelial layer by use of a multistage technique, platelet aggregation can be diminished, leading to improved early surgical results. CONCLUSION Our study demonstrates that the technique of EC lining in reoperation of crural vessels has a markedly decreased rate of early graft occlusion as compared to standard ePTFE grafts followed for 1 to 18 months after implantation. The tVECL technique in patients without suitable remaining autologous veins is promising. Longer follow-up time is necessary to clarify the questions of EC behavior and the role of smooth muscle cells within the IVECL grafts. REFERENCES 1. Ziats NP, Miller KM, Anderson JM. In vitro and in vivo interactions of cells with biomaterials. Biomaterials 1988;9: 5-13. 2. Herring M, Gardner A, Glover ~A. A single-stage technique for seeding vascular grafts with autogenous endothelium. Surgery 1978;84:498-504. 3. Fasol R, Zilla P, Deutsch M, Grimm M, Fischlein T, Laufer G. Human endothelial cell seeding: evaluation of its effectiveness by platelet parameters after one year. J VASC SURG 1989;9:432-6. 4. Rosenman JE, Kempczinski RF, Pearce WH, Silberstein EB. Kinetics of endothelial cell seeding. J VASC SURG 1985;2: 778-84. 5. Helsiniki Declaration. Ann Intern Med 1966;65:367-8. 6. Rutherford RB, Flanigan DP, Gupta SK, et al. Suggested standards for reports dealing with lower extremity ischernia. VASC SURG 1986;4:80-94. 7. Zilla P, Fasol P, Dudeck U, et al. In situ cannulation, microgrid follow-up and low-density plating provide first passage endothelial cell masscultures for in vitro lining. J VAsc SURG 1990;12:180-9. 8. Zilla P, Fasol R, Preiss P, et al. Use of fibrin glue as a substrate for in vitro endothelialization of PTFE vascular grafts. Surgery 1989;105:515-22. 9. Thakur ML, Walsh L, Malech HL, Gottschalk A. Indium- 111 labeled human platelets: improved method, efficiency and evaluation, l Nucl Med 1981;22:381.
Volume 15 Number 3 March 1992
10. Minar E, Dudczsak 1~ Ehringer H. Postoperative platelet diagnosis: a prerequisite for chnical seeding. In: Zilla P, Fasol R, Deutsch M, eds. Endotheliahzation of vascular grafts, Basel: Karger, 1987:64-70. 11. Isaka Y, Kimura K, Yoneda S, et al. Platelet accumulation in carotid atherosclerotic lesions: semiquantitative analysis with indium-ill platelets and tectmetium-99m human serum albumin. J Nucl Med 1984;25:556-63. 12. SAS Institute Inc, SAS/STAT, User's guide, version 6, fourth ed, vol 2, Cary, NC 1989:846. 13. Peto R, Pike MC, Armitage P, et al. Design and analysis of randomized trials required prolonged observations of each patient. I. Introduction and design. Br J Cancer 1976;34: 585-612. 14. Peto R, Pike MC, Armitage P, et al. Design and analysis of randomized trials required prolonged observations of each patient. Analysis and examples. Br J Cancer 1977;35:1-39. 15. Budd JS, Brennan J, Beard JD, Warren H, Burton PR, Bell PRF. Infrainquinal bypass surgery: factors determining late graft patency. Br J Surg 1990;77:1382-7. 16. Veterans Administration Cooperative Study Group 141. Comparative evaluation of prosthetic, reversed, and in situ vein bypass grafts in distal pophteal and tibial-peroneal revascularization. Arch Surg 1988;123:434-8. 17. Veith FJ, Gupta SK, Ascer E, et al. Six-year prospective multicenter randomized comparison ofautologous saphenous vein and expanded polytetrafluoroethylene grafts in infrainquinal arterial reconstruction. J VASC StJr~G 1986;3:10414. 18. Whittemore AD, Kent KC, Donaldson MC, Couch NP, Mannick JA. What is the proper role of polytetrafluoroethylene grafts in infrainquinal reconstruction? J VASC SURG 1989;10:299-305. 19. Bergan J)', Veith FJ, Bernhard VM, et al. Randomization of
Endothelial cell lining of ePTFE grafts in humans 535
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autogenous vein and polytetrafluoroethylene grafts in femoral-distal reconstruction. Surgery 1982;92:921-9. Charlesworth PM, Brewster DC, Darhng RC, Robinson JG, Hallet JW. The fate of polytetrafluoroethylene grafts in lower limb bypass surgery: six-year follow-up. Br • Surg 1985;72: 896-9. Peterkin GA, Manabe S, LaMorte WW, Menzoian JO. Evaluation of a proposed standard reporting system for preoperative angiograms in infrainquinal bypass procedures: angiographic correlates of measured runoff resistance. J VASC SURG 1988;7:379-85. Schneider PA, Hanson SR, Price TM, Harker LA. Performed confluent endothelial cell monolayers prevent early platelet deposition on vascular prostheses in baboons. J VAsc S~RG 1988;8:229-35. 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 Vasc SURG 1984;1:224-33. Ortenwall P, Wadenvik H, Kutti ~, Risberg B. Endothehal cell seeding reduces thrombogenicity of Dacron grafts in humans. J VAsc SURG 1990;11:403-10. Herring M, DiUey R,Cullison T, Gardner A, Glover 5. Seeding endothefium on canine arterial prostheses: the size of inoculum. J Surg Res 1980;28:35-8. Zacharias SRK, Kirkman TR, Clowes AS. Mechanisms of heating in synthetic grafts. T VASC SURG 1987;6:429-36. ZiUa P, Groscurth P, Varga G, Fasol R. PGI2 and PGE1 induce morphological alterations in human platelets similar to those of the initial phase of activation. Exp Hematol 1987;15:741-7.
Submitted Apr. 18, 1991; accepted July 1, 1991.
