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Ann Thorac Surg 2001;71:S327-S331
© 2001 The Society of Thoracic Surgeons

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Bioprosthetic valves and conduits: new developments

Clinical autologous in vitro endothelialization of 153 infrainguinal ePTFE grafts

^a First Department of Surgery and Ludwig Boltzman Institute for Applied Cardiovascular Biology, Lainz Hospital, Vienna, Austria
^b Center of Cardiac Surgery, University of Erlangen, Nuremburg, Germany
^c Department of Cardiothoracic Surgery, University of Cape Town, Cape Town, South Africa

Address reprint requests to Dr Zilla, Department of Cardiothoracic Surgery, Cape Heart Centre, University of Cape Town Medical School, Anzio Rd, 7925 Observatory, Cape Town, South Africa
e-mail: zilla{at}capeheart.uct.ac.za

Presented at the VIII International Symposium on Cardiac Bioprostheses, Cancun, Mexico, Nov 3–5, 2000.

	Abstract

Top Abstract Introduction Patients and methods Results Comment References

 
Background. Over the past 17 years, our group has developed and clinically applied an in vitro endothelialization procedure whereby infrainguinal expanded polytetrafluoroethylene (ePTFE) prostheses are confluently lined with cultured autologous endothelial cells before implantation. After a successful randomized pilot study from 1989 to 1993, the procedure was adopted for routine operations.

Methods. Since June 1993, 153 endothelialized ePTFE grafts were implanted in the infrainguinal position in 136 patients (102 above knee (AK) and 51 below knee (BK), 89 men and 47 women, mean age 64.7 ± 9.4 years). Seventeen patients received an endothelialized prosthesis bilaterally. Autologous endothelial cells were harvested from 4- to 5-cm segments of a subcutaneous vein (in 86% the cephalic vein), grown to first-passage mass cultures and confluently lined onto 6- (n = 113) or 7-mm (n = 40) inner diameter (ID) ePTFE grafts, precoated with fibrin glue. The observation period for 6-mm grafts was 7 years, and for 7-mm grafts was 4 years. Patency assessment for Kaplan–Meier survivorship analyses was based on duplex sonography and angiography.

Results. Kaplan–Meier survivorship function revealed a primary patency rate of 62.8% after 7 years (SE = 0.05) for all infrainguinal reconstructions (60% AK/70.8% BK). The primary patency for stage II and III patients was 64.4% after 7 years. The more recent group of 7-mm ID grafts showed a primary patency of 83.7% after 4 years.

Conclusions. Our data provide strong evidence that autologous endothelial cell lining distinctly improves the patency of small diameter vascular grafts.

	Introduction

Top Abstract Introduction Patients and methods Results Comment References

 
Traditionally, the gap between a basic scientific discipline such as cell biology and clinical medicine has been nowhere more unbridgeable than in surgery. Yet, vascular surgery pioneered the field of tissue engineering in the late 1970s. With their concept of "seeding" autologous endothelial cells into a meshwork of a synthetic graft, vascular surgeons such as Malcolm Herring and colleagues [1] and Linda Graham actually implemented the basic cornerstones of today’s tissue engineering by promoting the integration of applied vascular biology into the traditionally hostile realm of surgery.

Unfortunately, regulatory issues were the reason why most of the pioneers shied away from tissue culture techniques. Consequently, too few endothelial cells were inoculated into the prosthesis, resulting in the early desertion of an otherwise highly promising idea [2]. For those who carried the concept further by using culture techniques to increase the endothelial cell numbers before graft lining, the 1980s became a difficult era in which the consequences of the perceived failure of "single-staged" endothelial seeding were acutely felt. Nevertheless, by the end of the decade, sufficient experimental data had been accumulated by a few groups to justify the commencement of three independent clinical pilot studies [3–5]. The convincing results of these early studies coincided with the change of general attitude toward the integration of cell culture into clinical medicine.

Our present report assesses the 7-year results of clinical in vitro endothelialization of 153 infrainguinal expanded polytetrafluoroethylene (ePTFE) grafts, implanted as femoropopliteal bypass grafts.

	Patients and methods

Top Abstract Introduction Patients and methods Results Comment References

 
Encouraged by the 3-year results of our randomized pilot study [5] and the short-term patencies published by Magometschnigg and colleagues [3], we offered in vitro endothelialization to all patients undergoing bypass operations for peripheral arterial occlusion after June 1993. As in the preceding randomized pilot study, the selection criterion was the lack of a suitable saphenous vein or a forthcoming aortocoronary bypass procedure. In the course of 7 years, 136 patients received 153 successfully endothelialized ePTFE grafts (89 men and 47 women, mean age 64.7 ± 9.4 years). In 17 patients an endothelialized prosthesis was bilaterally implanted. One hundred twenty graft implantations were performed for clinical grade I ischemia (84 above knee (AK), 36 below knee (BK)), 15 for grade II (8 AK, 7 BK), and 18 for grade III (10 AK, 8 BK).

Endothelial cell harvest
In patients assigned to in vitro endothelialization, 4- to 5-cm segments of vein were removed under local anesthesia (1% Lignocaine, Astra-Chemie; Linz, Austria) using starch-free gloves to avoid the cytotoxic effect of glove powder. The cephalic vein was used as a cell source in most cases (86.3%), whereas the brachial and the external jugular veins were used in 5.2% and 2.0%, respectively. In 6.5% of cases the saphenous vein was used if the preoperative sonographic assessment confirmed it unsuitable as a conduit. After no-touch dissection veins were cannulated in situ [6], flushed with medium 199 (Gibco, Paisley, UK; 10 mL, 37°C), filled with 0.1% collagenase under slight pressure between two stop-cocks (Worthington Biochemical Corporation, Freehold, NJ; 37°C) and transferred to the laboratory where the cells were harvested after 15 minutes of collagenase exposure at 37°C.

Endothelial cell cultures and graft lining
T 12 filter-protected culture flasks (Falcon, Franklin Lakes, NJ) were used for primary culture. After a single passage to two 162-cm² culture flasks (Costar, Cambridge, MA) at preconfluence [6], a microgrid technique enabled the daily in situ quantification of available endothelial cells [6]. The culture medium consisted of medium 199 supplemented with 10 ng/mL recombinant basic fibroblast growth factor (Boehringer, Ingelheim, Germany; in phase 2), 50 mg/mL gentamycin (Gibco), and 20% of autologous serum. Cells were cultured until the required cell number of approximately 16 x 10⁶ endothelial cells per graft was reached [7]. Based on a significant correlation between serum levels of "lipoprotein a" and growth failure during the preceding randomized pilot study [5], a virologically tested "rescue" serum pool with low lipid levels, including lipoprotein a, replaced the autologous serum at the earliest signs of growth failure.

Before endothelial cell lining, 6-mm or 7-mm expanded PTFE grafts 70 cm long were precoated with fibrinolytically inhibited fibrin glue (Immuno AG, Vienna, Austria) [5, 7]. In contrast to the initial pilot study, neither culture flasks nor grafts could be preincubated with fibronectin, due to its unavailability for clinical use. Grafts were then trimmed to a length of 60 cm and filled with the endothelial cell suspension in culture medium (7.0 ± 1.5 x 10⁵ endothelial cells/cm²). Surface endothelialization was achieved through a microprocessor controlled seeding device (Biegler Electronics, Vienna, Austria) rotating at 6 rph and 37°C for 3 hours, providing a 5% CO₂ atmosphere [7]. After seeding, grafts were further postcultivated for another 8.7 ± 2.3 days to allow the maturation of the cytoskeleton. Immediately after the grafts were removed from the rotation device and before implantation, control specimens were taken for scanning electron and epifluorescence microscopy. Samples for the latter were stained with a vital dye combination (Live/Dead kit, Molecular Probes, Eugene, OR) immediately after rotation.

Graft implantation
Patients were anesthetized with a continuous epidural block using bupivacaine hydrochloride (Carbostesin 0.5%, Astra-Chemie, Linz, Austria). Graft implantation was performed as in the previous study. The culture medium was kept inside the graft without clamping the prosthesis by tilting the operating table. This step was the only one during the operation that differed from a routine implantation of an ePTFE graft. Thin-walled ePTFE (W. L. Gore and Associates, Flagstaff, AZ) was used as graft material for implantation. One hundred thirteen patients received a graft with a 6-mm inner diameter and 40 patients grafts with a 7-mm inner diameter.

All patients received the same antiaggregatory treatment (oral dipyridamole, 75 mg/day and oral acetylsalicylic acid 330 mg/day given as combination drug (Thrombosantin, Boehringer, Ingelheim, Germany) beginning 2 days before implantation and continuing throughout the observation period.

Clinical follow-up
Postoperative control procedures complied with the standards for evaluating results of interventional therapy in peripheral vascular diseases [8]. Follow-up studies were performed after 9 days, 3 months, 6 months, and 1 year. Long-term control investigations were repeated annually. Patency was determined by the ankle brachial index (ABI) and duplex sonography at each time interval. Angiography was performed after 1 year and in patients with suspected graft occlusion. Graft occlusion was suspected if deterioration of the clinical status of the limb was confirmed by a significant drop in the ABI and a lack of flow signal on Duplex sonography. Consent for control angiographies could not be obtained from 20 patients. In those patients the sole means of graft assessment was high-resolution duplex sonography, which is recognized as an accurate and reliable method for the assessment of femoropopliteal arterial disease. Only primary graft patency was considered.

Statistical analysis
Statistical analyses of primary patencies was performed by using the Kaplan–Meier survivorship function (SPSS, Chicago, IL). Patencies were compared by the log-rank test and Breslow test. For group comparisons of culture data Student’s unpaired t test was used. Comparisons were considered to be significant at less than 0.05.

	Results

Top Abstract Introduction Patients and methods Results Comment References

 
Endothelial cell cultures and graft lining
Thirty-six percent of primary cultures showed early signs of growth failure and had the autologous serum exchanged against pooled serum. The "lipoprotein a" level in patients affected by this measure was 35.9 ± 28.3 mg/dL. Over all, in 5% of patients endothelial cells still failed to grow despite the replacement of the autologous serum in the culture medium by pooled serum. The "lipoprotein a" value of these patients was 56.25 ± 16.7 mg/dL compared with 22.2 ± 26.6 mg/dL in patients whose endothelial cells grew in medium with autologous serum (p < 0.05).

The passage of primary cultures from the T 12 filter-protected culture flasks to two 162-cm² flasks was carried out on day 10.3 ± 7.3. An adequate number of endothelial cells for graft lining was available on day 20.8 ± 0.7.8. The delay between vein excision and graft implantation was 29.2 ± 7.5 days. No growth differences were found between endothelial cell cultures from different vein sources. A fully covering endothelium was found in 80% of samples obtained from freshly lined grafts. Ten percent were preconfluently lined and 10% even had some endothelium-free spots. At the time of implantation 90% of control samples showed complete confluence, whereas preconfluent sections were seen in 5% of grafts and small uncovered areas in further 5%.

Clinical follow-up
The observation time ranged from 1 month to 7 years (mean 5.1 years). The Kaplan–Meier survivorship function showed a primary patency rate of 62.8% after 7 years (SE = 0.05) for all infrainguinal reconstructions (Fig 1). Endothelialized grafts implanted in above-the-knee position had a 6-year patency of 60% (SE = 0.12). The patency rate for below-the-knee grafts was 70.8% (SE = 0.06) (Fig 2). This difference was not statistically significant (log-rank test p = 0.26; Breslow test p = 0.32). Primary patency was 83.7% (SE = 0.1) for the 40 grafts with an inner diameter of 7 mm after 4 years, whereas it was 60% (SE = 0.06) for 6-mm grafts (Fig 3). The difference between these two groups was not statistically significant (log-rank test p = 0.29; Breslow test p = 0.31). Thirty-three patients underwent operations for stage II or III peripheral vascular disease. The primary patency rate for this subgroup was 78.0% after 1 year and 64.4% (SE = 0.11) at the end of the observation period (Fig 4).

View larger version (12K): [in this window] [in a new window]   Fig 1. Primary 7-year patency of 153 in vitro endothelialized femoropopliteal ePTFE grafts. Numbers on the graph represent patients at risk.

View larger version (19K): [in this window] [in a new window]   Fig 2. Comparison of 6-year patencies of above-knee versus below-knee grafts. (A) Grafts above the knee joint (n = 102; SE = 0.06). (B) Grafts below the knee joint (n = 51; SE = 0.21). Numbers on graphs represent patients at risk.

View larger version (20K): [in this window] [in a new window]   Fig 3. Influence of graft diameter on the patency of in vitro endothelialized ePTFE grafts. Comparison of 113 6-mm ID grafts (6 years; SE = 0.06) with 40 7-mm ID grafts (4 years; SE = 0.1).

View larger version (19K): [in this window] [in a new window]   Fig 4. Primary patency rate for the subgroup of patients in clinical stage II and III (n = 33; SE = 0.11). Numbers on graph represent patients at risk.

	Comment

Top Abstract Introduction Patients and methods Results Comment References

 
For the past three decades, the high failure rate of prosthetic vascular grafts was almost exclusively explained with the development of anastomotic intimal hyperplasia. Attempts to reduce surface thrombogenicity of the synthetic material were therefore mostly refuted. Eventually, our in vitro endothelialization program has accumulated sufficient numbers of patients over a long enough period to prove that the lack of an endothelium on prosthetic arterial grafts has a much graver consequence on graft performance than generally believed. With an overall experience of 180 in vitro endothelialized ePTFE grafts and a follow-up period of 11 years, we can state with confidence that endothelialized ePTFE grafts perform equally to vein grafts.

After our initial randomized study demonstrated far superior results for the endothelialized group [5], we offered the treatment to all patients who either had no vein graft available or an aortocoronary bypass procedure imminent. This "routine" endothelialization program commenced in 1993 and its 7-year follow-up is summarized in the present study. By now, phase 2 of our program contains 153 endothelialized femoropopliteal bypass grafts. Although the delayed availability of an endothelialized graft restricts the procedure to nonacute interventions, the strict inclusion criteria make it a well-defined nonrandomized cohort study that is based on severe patients with generalized arteriosclerotic disease.

Despite the early reports of distinctly improved graft patencies, most of the vascular surgical community took a long time to recognize this novel approach. Largely, this delayed acceptance was certainly due to the confusion and uncertainties existing within the discipline. Throughout the past 30 years, perceptions rather than generally accepted guidelines existed regarding the long-term performance of bypass procedures. Despite numerous publications promoting standardization of vascular surgical procedures [8], published studies remained heterogenous with regard to most aspects. Especially differences in inclusion criteria, clinical staging, and graft diameters made it difficult to assess generally valid patency guidelines.

For years, Veith’s multicenter study [9] served as the golden standard, reporting a 5-year patency for vein grafts of 68% and for ePTFE of 38%. Reports by other groups varied dramatically, and thus, perceptions often prevailed [10–12]. Therefore, the publication of the Trans Atlantic Inter-Society Consensus (TASC) [13] was widely hailed as a first step toward the creation of a more scientific base line regarding graft patencies. The meta-analyses data for femoropopliteal bypass grafts used in the TASC showed 5-year patencies for vein grafts—both above and below the knee—to be 66% regardless of the position. In critical ischemia, patencies for ePTFE prostheses were reported to be 47% for above-knee grafts and 33% for below-knee grafts. Because most of the major studies had a predominance of stage II and III patients, our results were sometimes disputed because of a bias toward lower stages. However, our current high number of patients eventually makes it meaningful to look into the subgroup of stage II and III patients in our study. Their primary patency rate of 64.4% after 6 years is comparable to Veith’s 5-year primary patency rate of vein grafts in a patient population of equal clinical staging [9]. For a subgroup similar to ours in which no vein grafts were available, Veith and colleagues [9] even reported a 5-year patency of as low as 29% for ePTFE grafts.

A further aspect of improvement was the relatively recent introduction of 7-mm bypass grafts [14]. We began using the wider grafts 4 years ago if anatomic and rheologic considerations such as vessel size and run-off were appropriate. Indeed, the primary patency rate of the 7-mm grafts was considerably higher at 83.7% as compared with 61.0% of the 6-mm grafts. However, statistical tests failed to show any significant difference. This finding is certainly due to the low number of the 7-mm grafts (n = 40) as compared with the 6-mm grafts (n = 113) and also the shorter implantation time of the 7-mm grafts.

Apart from principal doubts concerning the importance of a surface endothelium versus that of anastomotic intimal hyperplasia, methodologic challenges were a major cause for the lack of enthusiasm by other centers. Concerns were particularly directed at the delay associated with the procedure, the failure rate of cell cultures, the higher risk of infection, and the persistence of the lined endothelium. Optimization of the culture procedure has lead to a continuous shortening of the time needed for the completion of the procedure. At present, the total time needed from vein excision to implantation is 29.2 ± 7.5 days, which is 7 days shorter than that required during our randomized phase 1 study. Failure rates of autologous endothelial cell cultures could be reduced from 27% [5] to less than 5% through timely discovery of lipid-induced growth inhibition and replacement of autologous serum supplement to the culture medium. The least justified point of concern is infection: of 180 endothelialized grafts, none was infected before implantation. With regard to the persistence of the lined endothelium on the synthetic blood surface of the prostheses, both clinical explants [15, 16] and nonhuman primate experiments [17] provided sufficient evidence for the long-term stability of the newly created intima.

We conclude that autologous, clinical in vitro endothelialization has reached a stage at which technical challenges of the early years could be successfully reduced to a level at which they no longer pose an insurmountable threshold for other clinical centers. Because the procedure dramatically improves the patency of synthetic grafts to match that of vein grafts, it is hoped that the recently commenced multicenter study will eventually overcome the last resistances of traditional surgeons to the integration of cell biology into clinical surgery.

	References

Top Abstract Introduction Patients and methods Results Comment References

 

1. Herring M., Dilley R., Jersild R.J., Boxer L., Gardner A., Glover J. Seeding arterial prostheses with vascular endothelium. The nature of the lining. Ann Surg 1979;190:84-90.[Medline]
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3. Magometschnigg H., Kadletz M., Vodrazka M., et al. Prospective clinical study with in vitro endothelial cell lining of expanded polytetrafluoroethylene grafts in crural repeat reconstruction. J Vasc Surg 1992;15:527-535.[Medline]
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6. Zilla P., Fasol R., Dudeck U., et al. In situ cannulation, microgrid follow-up and low-density plating provide first passage endothelial cell mass cultures for in vitro lining. J Vasc Surg 1990;12:180-189.[Medline]
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12. Jackson M., Belott T., Dickason T., et al. The consequences of a failed femoropopliteal bypass grafting: comparison of saphenous vein and PTFE grafts. J Vasc Surg 2000;32:498-505.[Medline]
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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Meinhart, J. G. Articles by Zilla, P. Search for Related Content PubMed PubMed Citation Articles by Meinhart, J. G. Articles by Zilla, P. Related Collections Coronary disease Peripheral vascular