Durability of confluent endothelial cell monolayers on small-caliber vascular prostheses in vitro

Since implantation of small-caliber vascular grafts with preformed confluent endothelial cell monolayers (ECMs) may prevent acute platelet deposition and thrombus formation, we have evaluated the conditions necessary to produce durable ECMs. Cultured human umbilical-vein ECs in buffer were attached to vascular grafts--either expanded polytetrafluoroethylene (ePTFE) or knitted Dacron, both with inside diameters of 4 mm--precoated with collaten type I and perfused in vitro with serum-containing culture medium to achieve cell spreading into confluent monolayers. The number of cells attached was quantified by DNA measurements and indium-111 labeling. Morphology was evaluated by scanning electron microscopy. The maximum number of cells that attached acutely was 3.6 X 10(5) cells/cm2 graft, and the minimum number needed for confluence was 1.4 X 10(5) cells/cm2 graft. Confluence was morphologically complete after 2 hours of in vitro perfusion at 15 ml/min. When ECMs were exposed to varying flow rates, cell retention after 1 hour was 96.1% +/- 5.6% at 50 ml/min, 94.6% +/- 6.1% at 100 ml/min, 77.6% +/- 10.8% at 200 ml/min (p less than 0.001), and 40.1% +/- 10.4% at 400 ml/min (p less than 0.0001). Confluence was maintained for all grafts exposed to flows of 100 ml/min or less. Fewer cells were retained when acutely attached, unspread cells (71.5% +/- 11.5%) were compared with established ECMs (89.9% +/- 6.7%) at a flow rate of 100 ml/min (p less than 0.0001). ECMs on knitted Dacron were more durable than on ePTFE (82.7% +/- 5.3% versus 75.5% +/- 4.8% remained attached at 200 ml/min, P less than 0.05). 111Indium-labeled and unlabeled cells were equivalent with respect to saturation level attachment, spreading time to confluence, and durability under flow at 100 ml/min. We conclude that confluent and durable endothelial cell monolayers can be established on small-caliber vascular grafts within 2 hours.     

Effect of defibrinogenation on the early patency rate of experimental small calibre arterial grafts

The effect of defibrinogenation with Arvin was studied in a new animal model of early thrombosis of a 3 mm diameter polytetrafluoroethylene (PTFE) graft with a poor run-off. Fifteen control animals were compared with fourteen animals treated with subcutaneous Arvin 20 units kg-1 body weight day-1, starting 2 days before surgery and continuing for 2 days postoperatively. The peroperative fibrinogen level in the controls was 2.8 +/- 0.9 gl-1 compared with 0.4 +/- 0.3 gl-1 in the treated group. There was no significant difference in the peroperative or postoperative platelet count or haematocrit value between the two groups. Plasma viscosity and whole blood viscosity (at a low shear rate of 0.7s-1) were significantly less during and after surgery in the defibrinogenated group. The degree of defibrinogenation in these animals produced no problems with haemostasis during surgery or in the postoperative period. The cumulative patency rates of the controls at 24 h, 48 h, and 4 days were 43 per cent, 28 per cent and 28 per cent compared with 86 per cent (P less than 0.05), 73 per cent (P less than 0.05) and 73 per cent (P less than 0.05) respectively in the defibrinogenated group. In this model of a narrow PTFE graft with a poor run-off, defibrinogenation was a safe and effective method of improving early patency of small calibre arterial grafts.     

Preformed confluent endothelial cell monolayers prevent early platelet deposition on vascular prostheses in baboons

We assessed the capacity of preformed confluent endothelial cell (EC) monolayers on small-caliber prosthetic grafts to prevent early platelet deposition in a baboon model. Cultured human umbilical vein ECs were attached to expanded polytetrafluoroethylene (Gore-Tex, 4 mm inner diameter, 3 cm length) precoated with type I collagen and perfused in vitro for 2 hours at 15 ml/min with serum-containing culture medium to achieve cell spreading into confluent monolayers. Cell numbers were quantified by deoxyribonucleic acid assay or isotopic counting of indium 111-labeled ECs. Saturation density for cell attachment was 3.55 +/- 0.29 x 10(5) cells per square centimeter of graft. After 1 hour of in vitro perfusion at 100 ml/min, 92.8% +/- 1.8% of cells remained attached and the flow surface was morphologically confluent. When grafts were inserted as extension segments into arteriovenous silicone rubber (Silastic) shunts in baboons, thereby exposing the endothelialized grafts to native flowing blood (100 ml/min) for 1 hour, the EC monolayers remained confluent with 81.05% +/- 5.88% of the cells attached. Indium 111-labeled platelet deposition onto grafts was quantified by dynamic scintillation camera imaging. Platelet deposition on 10 endothelialized grafts was markedly reduced (0.16 +/- 0.04 x 10(9) platelets per graft) compared with 10 untreated control grafts (1.84 +/- 0.59 x 10(9) platelets, p less than 0.02), eight grafts with early attached unspread ECs (2.38 +/- 0.66 x 10(9) platelets, p less than 0.005), and 11 grafts treated with collagen alone (5.93 +/- 0.72 x 10(9) platelets, p less than 0.002).(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced patency of venous dacron grafts by endothelial cell sodding

The effect of microvessel endothelial cell (EC) sodding on the patency of Dacron grafts interposed in canine inferior vena cava was studied. EC were harvested enzymatically from canine omentum and isolated by density gradient centrifugation. Preclotted, knitted Dacron grafts were sodded with >10⁶ EC/cm² surface. The results demonstrate significant improvement in patency of sodded grafts placed in the inferior vena cava as compared with control grafts (p<0.001 in grafts with a distal arteriovenous fistula andp<0.05 in grafts without a distal fistula). The neointima of the sodded grafts were thinner and scanning electron microscopy confirmed the presence of a confluent layer of EC. In addition, the production of prostacyclin but not thromboxane A₂ was significantly enhanced in the sodded grafts as compared with controls. We conclude that microvessel EC sodding of Dacron grafts significantly improves the patency rate and inhibits neointimal thickening of the prosthesis. The mechanism is unknown but may involve a more rapid endothelialization of the graft surface with the potential of producing more prostacyclin and less thromboxane A₂.Supported in part by the Chinese Academic Science Foundation to Zhong-Gao Wang, MD.     

An improved method for endothelial cell seeding on polytetrafluoroethylene small caliber vascular grafts.

The creation of nonthrombogenic synthetic surfaces is a major challenge in biomedical research. The feature that clearly distinguishes natural blood vessels from their artificial counterparts is the presence of endothelial cell lining that besides being nonthrombogenic is capable of repair and renewal. This study describes a method of coating vascular grafts with a uniform, naturally produced subendothelial extracellular matrix before implantation. This substrate provides a most suitable bilayer for endothelial cell adhesion, growth, and differentiation, as compared with grafts coated with fibronectin or basement membrane extracts. It contains both adhesive glycoproteins (fibronectin, laminin, collagen) and proteoglycans (heparan sulfate) as well as endothelial cell growth factors (basic fibroblast growth factor) that support adhesion and normal growth of suboptimal concentrations of endothelial cells. We suggest that the presence in extracellular matrix of both adhesive macromolecules and potent endothelial cell-growth promoting factors will make the extracellular matrix a promising substrate for vascular grafts.     

Forskolin impregnation of small calibre PTFE grafts lowers early platelet graft sequestration and improves patency in a sheep model

Synthetic vascular grafts have a thrombogenic surface which plays a role in graft failure. Systemic pharmacologic interventions have been used to lower platelet sequestration onto the graft surface but are associated with side effects. In this communication we describe the results of a new therapeutic principle of applying forskolin, a powerful cyclic adenosine monophosphate stimulator (cAMP) to the inner surface of PTFE vascular grafts. The grafts were evaluated with Indium-III-oxine labelled platelets and by graft patency on 3 consecutive days after implantation at 1 month and 3 months. Forskolin significantly lowered early platelet sequestration onto the treated graft surface when compared with controls. Graft potency at 1 and 3 months was also significantly higher in the forskolin treated grafts.     

The effect of continued cigarette smoking on the patency of synthetic vascular grafts in Leriche syndrome.

The effects of continued smoking were studied in 187 consecutive patients who underwent aorto-iliac or aorto-femoral grafting because of Leriche disease and who left the hospital with well-functioning grafts. The patients were divided into the following groups: (1) never smoked, (2) stopped smoking after the operation, (3) continued to smoke less than a pack a day and (4) continued to smoke more than a pack a day. The patency of the grafts was evaluated at regular intervals during a follow-up period ranging from 6 months to 10 years. A significant difference in the patency in the favor of the nonsmokers was found, with the "more than one pack a day" group having more than triple the occlusion rate of the nonsmokers, both absolutely and in month-patency time. We recommend that the surgeon make a most sincere effect to induce patients undergoing vascular operations for occlusive vascular diseases to give up smoking. Failure to promise to stop the smoking habit should be regarded as a relatively strong contraindication for surgery in patients not directly threatened with loss of an extremity.     

Reduction of non-endothelial cell contamination of microvascular endothelial cell seeded grafts decreases thrombogenicity and intimal hyperplasia.

INTRODUCTION: fat derived microvascular endothelial cells (MVEC) seeded on prosthetic vascular grafts, improve patency in animals. Results in humans were disappointing, due to thrombogenicity and progressive intimal hyperplasia. Also in animals intimal hyperplasia was found. We postulate that contaminating cells present in the transplant are involved in the intimal hyperplasia. We developed a method to further purify human MVEC from 40-90%. Here we tested the effects of enrichment upon thrombogenicity and seeding-related intimal hyperplasia. METHODS: liposuction fat was enzymatically digested and centrifuged. To enrich MVEC, contaminating macrophages and fibroblasts were removed with dynabeads coated with macrophage- and fibroblast-specific antibodies. Thrombogenicity was assessed by measuring tissue factor and thrombomodulin activity, presence of endothelial nitric oxide synthase and via perfusion of the cells with whole blood. To investigate seeding-related intimal hyperplasia, PTFE grafts were seeded with the cells and cultured for 3 weeks. RESULTS: tissue factor activity of purified cells was reduced compared to nonpurified cells. Purified cells showed thrombomodulin activity and eNOS expression. Fragment 1+2 and Fibrinopeptide A generation after perfusion of purified cells were significantly lower than after perfusion of nonpurified cells, and only nonpurified cells were covered with platelets and fibrin. Prostheses seeded with nonpurified cells showed an EC monolayer above a multilayer of myofibroblasts, prostheses seeded with purified cells only showed a single EC monolayer. Mixing experiments with human umbilical cord EC (HUVEC) and fibroblasts showed that when more than 25% HUVEC were present a confluent EC layer was formed. When the amount of fibroblasts was 25% or less, no development of a subendothelial multilayer of myofibroblasts was found within 3 weeks. CONCLUSION: reduction of non-endothelial cell contamination of microvascular endothelial cell seeded grafts decreases thrombogenicity and might prevent seeding-related intimal hyperplasia.     

Direct platelet effect of low molecular weight dextran in small calibre PTFE grafts.

The thrombogenicity of synthetic vascular grafts is a major factor in occlusion of grafts when they are used to bypass small calibre arteries. In this paper, the effect of low molecular weight dextran (LMWD, Dextran-40) on graft surface-platelet interaction was studied using Indium-III-oxine labelled platelets. It was found that LMWD significantly reduced platelet deposition onto graft surfaces (P less than 0.001). Dextran had a direct antiplatelet effect independent of plasma volume expansion as dextran-soaked grafts significantly reduced platelet deposition when compared to systemic dextran administration (P less than 0.001). We therefore conclude that LMWD has a direct antiplatelet effect which is beneficial in reducing platelet deposition on synthetic PTFE grafts which may improve the early patency of such grafts.     

Improved patency of collagen-impregnated grafts after in vitro autogenous endothelial cell seeding

Currently available prosthetic vascular grafts remain sufficiently thrombogenic to preclude their use as small-caliber arterial substitutes. However, thrombogenicity may be significantly reduced by the presence of an endothelial monolayer on the luminal surface. The present study was undertaken to test the efficacy of lining a small-caliber prosthesis with autogenous endothelial cells in vitro so that the graft may subsequently be implanted with an established confluent endothelial lining. For this purpose, cells were obtained from canine external jugular vein, harvested enzymatically, and passaged in culture. Dacron grafts (4 X 150 mm) were then seeded in vitro and maintained for 48 to 72 hours before implantation in the femoral position of the same animal. Seeded grafts were implanted contralateral to unseeded control grafts and explanted after 1 month. Seeded grafts demonstrated an 86% patency rate at explanation in contrast to the significantly lower 14% patency rate of the unseeded control grafts. This study justifies further investigation directed toward the feasibility of endothelializing intravascular prostheses in vitro before implantation.     

Influence of endothelial cell seeding on platelet deposition and patency in small-diameter Dacron arterial grafts

Serial platelet deposition, surface topography, and patency were evaluated in control (N = 28) and endothelial cell-seeded (N = 28) small-diameter (4 mm inner diameter) USCI Dacron grafts implanted in the carotid and femoral arteries of dogs. All dogs received aspirin (325 mg) daily for 2 weeks starting 24 hours prior to graft implantation. Endothelial cell seeding was performed by mixing suspensions of autologous endothelial cells that had been enzymatically harvested from segments of external jugular vein with blood that was used to preclot the prostheses. The platelet deposition on each graft was quantitated by means of indium 111-labeled platelets and technetium 99m-labeled red cells in a dual-isotope platelet-imaging technique. Platelet deposition on seeded grafts 24 hours after implantation was significantly higher than on the controls (p less than 0.05). Two weeks after implantation platelet deposition on seeded prostheses had decreased to a level significantly lower than that on the controls and continued to decline on serial studies up to 7 months. In contrast to seeded grafts, platelet accumulation on control grafts dramatically increased after the withdrawal of aspirin therapy and was associated with a sharp rise in control graft thromboses. Gross and scanning electron microscopic evaluation of endothelial cell-seeded grafts after 1 month indicated complete neointimal coverage, whereas none of the control grafts explanted at 1 month or later exhibited a continuous neointimal lining. Cumulative 7-month patency for seeded prostheses was significantly higher than for the controls (96% and 29%, respectively; p less than 0.001). We conclude that endothelial cell seeding in combination with short-term aspirin therapy is a simple, reliable diameter Dacron prostheses. Abrupt withdrawal of aspirin therapy may be contraindicated in nonseeded control grafts because it results in increased platelet deposition and thrombosis.     

The influence of endothelial seeding and platelet inhibition on the patency of ePTFE grafts used to replace small arteries--an experimental study

Previous studies on the influence of endothelial seeding on graft patency have shown that significant improvement has only been achieved with Dacron and an experimental, porous PTFE graft. Methods of assessing patency or showing statistical significance could be questioned in some of these studies. To determine if the combination of endothelial cell seeding and antiplatelet agents would improve patency in small-diameter, commercially available expanded polytetrafluorethylene (ePTFE) grafts, we placed ePTFE grafts into the left carotid position in two groups of mongrel dogs. All grafts were 4 mm internal diameter and 60 mm long, and were interposed in an end-to-end fashion. Both groups received aspirin (80 mg daily) and dipyridamole (25 mg daily) for 14 days, beginning immediately prior to surgery. In Group I (n = 12), the grafts were seeded with enzymatically harvested autogenous endothelium just prior to implantation; in Group II (n = 10) the grafts were not seeded. All grafts were removed at 30 days. Seven of 12 (58%) seeded grafts, but only one control graft (10%) remained patent (P = 0.03). Six of the seven seeded grafts exhibited surface endothelium, but the single patent control graft did not. The inner capsule of the seeded grafts consisted of a monolayer of endothelium and a thin acellular subendothelial matrix with an average thickness of 8 mu. We conclude that a 14-day course of anti-platelet agents combined with endothelial seeding of ePTFE resulted in significantly improved patency compared to controls, with most patent, seeded grafts developing an endothelial lining in 30 days.     

Microvascular endothelial cell seeding of small-diameter Dacron vascular grafts

One obstacle to the clinical implementation of endothelial cell seeding of vascular prostheses is the difficulty in derivation of large numbers of autologous endothelial cells from blood vessels of patients requiring vascular grafting. Capillary endothelial cells obtained from fat have been suggested as an abundant alternative to large-vessel endothelium for graft seeding. The object of this study was to evaluate the performance of 4-mm internal diameter (ID) Dacron Microvel grafts seeded with omentally derived microvascular endothelial cells. Six-cm lengths of the test grafts were implanted bilaterally into canine carotid arteries. One of each pair of grafts was seeded with endothelial cells (means = 8.4 x 10(6)) derived from collagenase digestion of autologous omental fat samples. The contralateral graft of each pair was nonseeded. At 5 weeks postoperatively, the grafts were harvested and evaluated. The mean patencies of both the seeded and nonseeded grafts were 89 percent. The mean thrombus-free surface area for seeded grafts was 95 +/- 11 percent. This value was significantly different statistically from the mean thrombus-free surface area of nonseeded grafts, which was 43 +/- 19 percent (P less than .05). Histologically, midgraft regions of seeded grafts were cellular, stained positive for collagen, and were characterized by inner capsules ranging in thickness between 35-94 microns. Luminal cells were identified as endothelial by peroxidase antiperoxidase staining techniques. Midgraft regions of nonseeded grafts demonstrated thrombus accumulation, limited cellularity, and inner capsules between 59-194 microns thick. Scanning electron microscopy of seeded grafts revealed smooth luminal surfaces with tight junctions between adjacent cells; surface cells were not present on midgraft regions of nonseeded grafts. In conclusion, endothelial cells derived from omental fat successfully surfaced on Dacron grafts and imparted characteristics to the graft that would predict long-term graft success.     

Effect of IL-6 on tumor cell invasion of vascular endothelial monolayers

The effect of interleukin-6 (IL-6) on the invasive capacity of B16-F1 mouse melanoma cells into vascular endothelial monolayers was examined, and an in vitro assay system for the quantitative determination of tumor cell invasiveness, using confocal microscopy with a fluorescence image analyzer, was developed. First, the invasive capacity of B16-F1 mouse melanoma cells against bovine vascular endothelial monolayers was estimated; then, the gap junctional intercellular communication (GJIC) of endothelial cells was examined. Treatment of endothelial cells with IL-6 resulted in a remarkable increase in the invasion of tumor cells into the endothelial monolayer, which was found to be significant from 25 ng/ml, and peaked at levels of more than 50 ng/ml. This stimulatory effect of IL-6, which was observed from 3 h after the initiation of treatment and lasted for up to 24 h, was abolished by the addition of the anti-IL-6 antibody. Although phasecontrast microscopy did not reveal any morphological changes in the endothelial cells following treatment with 25–200 ng/ml IL-6 for 24h, the GJIC was observed to be significantly decreased. These findings indicate that the invasive capacity of tumor cells into endothelial cells is affected by IL-6.     

The effect of fibronectin coating on endothelial cell kinetics in polytetrafluoroethylene grafts

Despite the proven experimental success of endothelial cell seeding of prosthetic vascular grafts, the process has not been widely accepted for clinical use because of its complexity and the need for a relatively large length of autologous vein to provide the requisite number of cells. Using autologous endothelial cells radiolabeled with indium 111-oxine, we studied the effect of fibronectin coating of polytetrafluoroethylene grafts in a canine carotid interposition model on initial endothelial cell adherence and subsequent cell retention for up to 72 hours following restoration of blood flow. Since the number of harvested endothelial cells varied widely depending on the diameter and length of available vein, cellular adherence was best expressed as a percentage of the original cell harvest. More than twice as many (46.7% vs. 19.8%) endothelial cells initially adhered to fibronectin-coated grafts compared with uncoated control grafts. Fibronectin did not appear to influence the loss of cells within the first 30 minutes following restoration of blood flow. However, over the next 24 hours, it clearly reduced the mean cell loss from 3.7%/hr to 2.2%/hr and resulted in a sixfold increase (21.3% vs. 3.4%) in the number of retained cells at the end of a 24-hour period. When the endothelial cell preclot was performed with a solution of culture medium 199, as opposed to heparinized whole blood, there was no significant difference in endothelial cell retention.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of wall mechanical properties on patency of arterial grafts

Normal arteries have properties which match the low output impedance of the heart to the high peripheral impedance. These properties can be assessed in terms of compliance (% diameter change per unit pressure change) as well as by other haemodynamic parameters. Experiments were designed using vein, Dacron and expanded polytetrafluoroethylene (PTFE) in a low flow canine femoral artery bypass model. No graft group achieved perfect patency. At twelve weeks 80% of vein grafts, 30% of Dacron grafts, and 15% of PTFE grafts remained patent. The compliance of vein grafts was maintained despite marked thickening of the wall. Patency was correlated at a highly significant level with compliance. The studies demonstrate that the matching of the mechanical properties of grafts to host arteries is important in the design of successful synthetic arterial grafts.     

The effect of vein diameter on patency of in situ grafts

In an attempt to evaluate the effect of vein diameter on early patency and long-term durability of in situ lower limb bypasses, we evaluated 195 femoral-distal, popliteal, and/or tibial bypasses constructed in 189 patients (153 men, 36 women), consisting of tibial bypasses in 116 (60%), and popliteal in 79 (40%). The operative angiograms were reviewed and the vein diameter was measured to the nearest 0.5 mm. Postoperative follow-up consisted of visits every three months where graft patency was assessed by physical examination and measurement of graft flow velocity and ankle-brachial indices. Conduits less than 3 mm had a higher rate of occlusion in the 0-30 day interval, but following that period performed satisfactorily. No conduit less than 2 mm was successfully utilized, because of inability to incise valves without injury in these tiny conduits. Following the perioperative period, conduit diameter does not affect the long-term durability of in situ bypass grafts.     

The effect of flow on vascular endothelial cells grown in tissue culture on polytetrafluoroethylene grafts

Vascular grafts lined with endothelial cells (EC) grown to confluence in culture before implantation may provide a thromboresistant flow surface. Growth of EC on and their adherence to currently available prosthetic materials under conditions of flow are two impediments remaining in the development of such a graft. To address these problems, 22 polytetrafluoroethylene grafts (PTFE) (5 cm by 4 mm inside diameter) were pretreated with collagen and fibronectin, seeded with 2 to 3 X 10(6) bovine aortic EC per graft, and placed in tissue culture (seeded grafts). Twenty-two grafts pretreated with collagen and fibronectin alone served as controls. After 2 weeks morphologic studies revealed that 20/22 seeded grafts were lined with a confluent endothelial layer. Indium 111-oxine was then used to label the EC-seeded grafts. After exposure to either low (25 ml/min) or high (200 ml/min) flow rates for 60 minutes in an in vitro circuit, examination of the luminal surface of the graft by light microscopy and scanning electron microscopy revealed minimal loss of EC. These findings were corroborated by radionuclide scans that showed an insignificant loss of the EC-associated indium label during exposure to flow (7% low flow, 11% high flow). Pretreatment of PTFE grafts with collagen and fibronectin thus promotes both attachment and adherence of EC even under flow conditions.