Endothelial cell seeding

Endothelial cell seeding is the transplantation of vascular endothelial cells to denuded vascular surfaces. Seeding theoretically reduces the probability of graft or vessel thrombosis and of neointimal fibrous hyperplasia. Thus far, clinical seeding trials disclosed modest improvements in patency and the development of hyperplastic anastomotic lesions in failed grafts. Seeding inefficiency theoretically contributes to anastomotic hyperplasia. The inefficiency is linked to two steps in the seeding process, namely harvesting and cell retention. Of these, cell retention on the seeded surface is the more critical. Priorities for future research should be set first on the retention of seeded endothelium in vitro and second on improved and standardized methods of cell harvesting.     

Endothelial cell seeding

Endothelial cell seeding is a technique that has developed over the past 15 years in response to the need for a high performance synthetic vascular graft. This review details our present knowledge of seeding and examines the various problems that have hampered its introduction into clinical practice.     

Endothelial cell seeding of polytetrafluoroethylene vascular prostheses coated with a fibroblastic matrix

One of the most serious problems with endothelial cell (EC) seeding of prosthetic materials is the poor adhesion and stability of the cells. Although several substrates that improve the initial adhesion have been assayed, the EC are lost within a limited period of time. In this study we attempted to modify the hydrophobic conditions of expanded polytetrafluoroethylene (ePTFE) by treating it with ethanol prior to seeding. In addition, we created a fibroblastic matrix that was also fixed by ethanol to the prosthetic material. In vitro studies were carried out at intervals of 24 hours and 15 days after seeding. EC from umbilical cord vein and fibroblasts from skin were seeded onto disks of PTFE with a porosity of 30 µm. The results obtained show that treatment of ePTFE with ethanol prior to EC seeding modifies its permeability, preventing cellular adhesion. The seeding of fibroblasts onto ePTFE allows a coating to form at 24 hours. The EC seeded onto this matrix adhere to it, forming a monolayer that persisted throughout the entire study period. The fibroblastic matrix allows the long-term survival of the EC on ePTFE.Supported by a grant from the CICYT SAF 92-0875.     

Endothelial cell seeding of polytetrafluoroethylene vascular grafts in humans: a preliminary report

The importance of initial human trials with autologous endothelial seeding lies not only in the implementation of a promising idea but also in the fact that canine data are only partially applicable to humans. The surface area of jugular veins in humans is much smaller than in dogs and considerably longer grafts are needed. Moreover, the reproductive capacity of adult human endothelial cells under in vivo conditions, which probably determines the success of seeding more than the seeding density, is also uncertain. Therefore the efficiency of autologous endothelial seeding in humans was investigated in 18 patients undergoing distal femoropopliteal bypass surgery. The average surface area of the jugular veins was 4.9 +/- 1.7 cm2 with an average cell yield of 32.6 +/- 18.0 x 10(4). The mean number of seeded cells per square centimeter of graft surface was 3.1 x 10(3). In a follow-up extending for 14 weeks, plasma levels of platelet factor 4 and beta-thromboglobulin as well as the platelet function in the whole blood aggregometer showed significantly better results in the seeded group. Plasma thromboxane B2, uptake and survival of indium 111-labeled platelets, and Doppler ultrasound investigations also favored the seeded group, but the results were statistically insignificant. No difference at all was found for the platelet dense granule compounds, releasable adenosine triphosphate and platelet serotonin. Thus our findings did not indicate the development of a closed endothelialized surface after 14 weeks, which is a period three times as long as the one required for confluent endothelial cell coverage in dogs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibronectin coating of an experimental PTFE vascular prosthesis

Because of the known inefficiency of endothelial cell seeding of vascular prostheses, some investigators have pretreated their grafts with fibronectin in an effort to increase endothelial cell adhesion. However, the most efficient method of coating vascular prostheses with fibronectin has not yet been determined and more importantly, the durability of this coating following restoration of pulsatile perfusion has not yet been established. This experiment measured the adsorption of fibronectin which was perfused at three different concentrations through an experimental, highly porous PTFE vascular graft. Most efficient coating of the prosthetic appeared to occur with a 250 mg/ml concentration of fibronectin perfused through the graft for 60 min. Grafts so prepared were subsequently studied in a pulsatile perfusion system which mimicked the canine circulation. Fifteen additional fibronectin-coated grafts were also interposed in the canine carotid artery. Flow was restored, grafts were removed at varying intervals, and fibronectin disappearance was measured. In both the in vitro and in vivo models, three distinct phases of fibronectin dissociation were seen: an initial rapid loss which resulted in an average 30.5% retention of the initial concentration of fibronectin following 30 min of perfusion; a second phase, which occurred from 30 to 120 min, demonstrated an average 2.5% loss of fibronectin per hour; and a final phase, from 2 to 24 hr, during which no significant additional loss of fibronectin was seen. This study describes a new method by which PTFE vascular prostheses can be effectively coated by perfusing them with physiologic concentrations of fibronectin and demonstrates that the resulting bond between fibronectin and prosthetic is stable following restoration of pulsatile flow.     

Endothelial cell seeding: A review

The concept of endothelial cell seeding, designed to provide vascular grafts with a nonthrombogenic lining, has progressed from crude animal experiments during the past two decades to detailed in vitro functional studies using human cells. Although favorable results have been obtained in animal studies this has yet to be translated to humans, where current application of these techniques has been limited to a very few clinical trials. The history, current status and future directions are reviewed herein.     

Biodegradation of a PTFE prosthesis

Processes of biodegradation of PTFE--as they were not seen before for this type of graft--are demonstrated by clinical examples. As it is possible to subsume these results to regularly described processes of incorporation of alloplastic material, it is to be expected that there will be more cases than those observed by us up to now.     

Endothelial cell seeding on prosthetic surfaces

Once thought to be a monolayer of passive cells lining the vasculature, endothelial cells are now known to be important regulators of normal vascular physiology. Unfortunately, these critically important cells are destroyed or removed by interventional and surgical procedures performed to recanalize or bypass vascular obstructions. The loss of these cells contributes to thrombosis and restenosis, the major complications observed after angioplasty, stent deployment, and prosthetic graft implantation. One approach to preventing these complications is the placement of endothelial cells on stents or prosthetic grafts prior to their placement in vivo in the hope that these cells will, after growth and maturation, release the factors necessary to inhibit thrombosis and intimal thickening. The purpose of this review paper is to provide an overview of the physiologic functions of normal and dysfunctional endothelial cells, and to discuss experiments in which endothelial cells have been placed on metallic stents and prosthetic grafts.     

Endothelial cell seeding of prosthetic vascular grafts: early experimental studies with cultured autologous canine endothelium

Thirteen adult dogs underwent thoracoabdominal bypass operations with 6-mm, double-velour Dacron grafts 25 to 30 cm long. Experimental grafts were seeded with cultured autologous endothelial cells (n = 7). Unseeded grafts served as controls (n = 6). Endothelial cells were harvested from external jugular vein segments using 0.1% trypsin and 0.5% collagenase solutions. Grafts were studied at weeks 2 and 4. Endothelial cell coverage of experimental graft surfaces after two weeks was 60% to 70%, and approximately 80% after four weeks. Immunofluorescence using factor VIII-related antigen confirmed the graft's inner surface to be endothelium. Endothelial cell coverage in control grafts occurred as pannus ingrowth, and never exceeded more than 10% of the conduit surface. Generation of an early endothelial surface in prosthetic grafts is possible in a canine model using cultured autologous cells.     

Small artery reconstruction with a new vascular prosthesis

In the Surgical Department of the University of Erlangen, 307 reconstructions in all areas of the arterial tree were performed with PTFE (GORE-TEX^®) grafts, of which 188 were performed in the femoral-popliteal and femoral-crural areas. Femoral-popliteal bypass grafts above the knee joint remained patent in 93% of the patients during an average follow-up time of 6.7 months. Below the knee joint, the patency rate was 85% during an average follow-up time of 5.6 months. In the proximal aorta, aorta-renal, and aorta-femoral areas, long-term results with the PTFE graft have been excellent.     

Endothelial cell harvest for seeding vascular prostheses: the influence of technique on cell function, viability, and number

Direct seeding of endothelial cells onto synthetic vascular prostheses has become the subject of increasing surgical research during the last 5 to 7 years. The currently employed cell harvest techniques are inefficient, resulting in cell counts far below the number of cells calculated to be present on the original donor vein. We have compared two methods of enzymatic endothelial cell harvest: cannulation with flushing and eversion over a stainless steel rod. Harvested cells were plated onto tissue culture plastic and counted after 24 hours of incubation. The methods ensured that only those cells viable and functional enough to adhere to the plastic were being considered. Cells were identified as endothelial by immunohistochemical techniques applying antisera to factor VIII-related antigen. Segments of normal vein and of veins treated by each technique were viewed with a scanning electron microscope. Cannulation was the superior method, providing greater numbers of viable, functional cells. The eversion technique was unreliable and probably injurious to endothelial cells.     

Omniflow: a new vascular prosthesis for hemodialysis access

Between January and November 1988 20 a.-v. shunts for haemodialysis access were performed at the University Hospital of Zurich with the Omniflow, a prosthesis composed of sheep collagen and polyester mesh. The main reason to implant this vascular prosthesis was the failure of Cimino-shunts. In 9 cases a straight and in 11 cases a performed "u" shape graft was used. During the follow-up period of 3-11 months the following 6 complications were observed: 5 graft thrombosis (in 2 cases due to hypotonia, in other 2 cases due to wrong graft selection and in 1 case due to narrowed venous anastomosis), and one graft dysfunction due to a stenosis in the patients vein. In comparison to the vascular prostheses used earlier, the Omniflow graft is characterized by a high patency rate and an easy puncturing.     

Endothelial cell seeding of small-caliber synthetic grafts in the baboon

Endothelial cell (EC) seeding has been proposed as a method to improve the performance of small-caliber synthetic vascular prostheses. Seeding experiments to date have all been carried out in the dog. This study investigates EC seeding of small-caliber Dacron carotid interposition grafts compared with contralateral control grafts in the baboon. Surface thrombogenicity was assessed at 24 hours, 2 weeks, and 4 weeks after implantation with indium 111-labeled autologous platelets. Morphologic and immunohistochemical techniques were used to assess the identity and homogeneity of the EC inoculum before seeding and to identify cell types on the harvested grafts. There was no significant difference in patency rates between seeded and control grafts at 5 weeks. Platelet accumulation on seeded grafts was significantly less (p less than 0.05) than on paired controls at 2 and 4 weeks after implantation. The luminal lining of seeded grafts had more cellular ingrowth, less adherent thrombus, and more surface cells with the morphologic and histochemical characteristics of EC than did the lining of controls. EC seeding reduces the platelet reactivity and accelerates EC coverage of small-caliber grafts in the baboon.     

Endothelial cell seeding reduces thrombogenicity of Dacron grafts in humans

Vascular prostheses in humans do not endothelialize spontaneously. In the present study we explored the feasibility of seeding autologous endothelial cells into prostheses implanted in patients undergoing reconstruction of the infrarenal aorta. In 22 patients one limb of an aortic Dacron bifurcation prosthesis was seeded with autologous endothelial cells harvested from the distal portion of the saphenous vein. The other limb was sham-seeded with culture medium only. The effect of seeding was studied by use of indium 111 radiolabeled platelets and external gamma camera scanning at 1, 4, and 12 months after surgery. No complications ascribable to the seeding procedure were seen. During the first year after surgery a gradual decrease in platelet accumulation occurred over the whole vascular prosthesis. At all time points studied the seeded graft limbs exhibited significantly less deposition of radiolabeled platelets than did control limbs. The observed difference in platelet accumulation on autologous endothelial seeding-treated graft segments merits further investigation of this technique in humans.     

Endothelial seeding of venous prostheses

To evaluate the potential benefit of endothelial seeding of venous prostheses, 20 dogs were subjected to iliocaval reconstruction with expanded polytetrafluoroethylene grafts protected by an arteriovenous fistula. Grafts seeded with enzymatically derived endothelial cells were compared with control grafts that were sham seeded with culture medium and blood. Five seeded and seven sham-seeded grafts remained patient and were perfusion fixed in situ 4 to 6 weeks after operation. Specimens were examined by light, scanning, and transmission electron microscopy. No statistical difference in early patency was noted. The mean thrombus-free surface area was 80% in the seeded and 71% in the sham-seeded group. Light microscopy of these areas revealed a monocellular layer lining the lumen in all grafts. Scanning electron microscopy demonstrated a thin cellular lining covering 50% to 100% of the specimens' surface area in four of the five seeded and five of seven sham-seeded grafts. Transmission electron microscopy revealed these cells to exhibit characteristics typical of endothelial cells. The subcellular layer was equally thin in both groups. Early patency rates were not benefited by endothelial seeding of grafts placed in the venous system. Seeding of grafts with enzymatically derived endothelial cells provides a good endothelial cover with a thin subendothelial layer but not to a greater extent than does sham seeding of the venous prostheses.