Enhanced strength of endothelial attachment on polyester elastomer and polytetrafluoroethylene graft surfaces with fibronectin substrate

Successful development of a vascular prosthesis lined with endothelium may depend on the ability of the attached cells to resist shear forces after implantation. The purpose of this article is to describe a model for measurement of endothelial detachment caused by shear stress and to identify biomaterials that resist loss of attached cells as a result of shear stress. With human umbilical venous endothelium labeled with indium 111-oxine, cellular attachment to uncoated and fibronectin-coated polyester elastomer and expanded polytetrafluoroethylene (e-PTFE) graft surfaces was quantified after an 18-hour incubation. PTFE grafts prepared by immediate seeding were also studied. The relative strength of endothelial attachment was determined by the percentage of the original inoculum remaining after the seeded graft surfaces were subjected to a physiologic shear stress of 15 dynes/cm2 during in vitro perfusion. In polyester elastomer grafts, fibronectin did not significantly increase initial attachment but did increase the percentage of inoculum remaining after perfusion (92.1% vs. 39.74%, p = 0.001). A similar relationship existed between fibronectin-coated e-PTFE and immediately seeded e-PTFE preparations with 61.6% and 25.8%, respectively, of the inoculum remaining after perfusion (p = 0.001). Furthermore, the percentage of inoculum retained on fibronectin-coated polyester elastomer was significantly greater than on fibronectin-coated e-PTFE (p = 0.001). In comparing uncoated grafts, polyester elastomer had 39.7% of the inoculum retained after perfusion whereas only 1.8% was remaining on the e-PTFE grafts (p = 0.0001). We conclude that polyester elastomer permits better endothelial cell attachment than e-PTFE and that fibronectin coating enhances the strength of attachment to both graft materials.     

Endothelial cell seeding kinetics under chronic flow in prosthetic grafts

Improved patency of endothelial cell seeded grafts relies on good initial adherence and cell retention when the circulation is restored. In this study human adult endothelial cells (HAECs) were used to evaluate the suitability of commercially available prostheses for seeding. Acutely seeded indum-111 oxine labeled HAECs were used to measure cell adherence to plain and fibronectin (FN)-coated expanded polytetrafluoroethylene (ePTFE), gelatin-impregnated Dacron (Gelseal), and collagen-impregnated Dacron (Hemashield) grafts. Cell loss from FN-coated prostheses, when exposed to a simulated human arterial blood flow of 200 ml/min in an artificial pulsatile circulation, was quantified from the loss of gamma activity from the graft over 24 hours, pressure in the circulation being reduced to 15 mm Hg to reduce fluid loss. Initial HAEC adherence (mean [SD]) to plain grafts was 3(1)%, 47(9)%, and 53(9)% for ePTFE, Gelseal, and Hemashield, respectively. This improved significantly with FN coating (78[6]%, 60[8]%, and 76[4]%). Cell retention after 24 hours of flow to FN-coated grafts was 16(10)%, 25(5)%, and 65(4)% and was confirmed qualitatively by scanning electron microscopy and environmental scanning electron microscopy. FN significantly improved initial cell adherence with Dacron grafts showing the better adherence. Cell retention after 24 hours of flow was better with FN-coated Dacron than with ePTFE but was best with Hemashield grafts.     

Optimal seeding conditions for human endothelial cells

Anin vitro model of endothelial cell seeding has been developed to individually evaluate the steps required for seeding arterial prostheses. Human saphenous vein endothelial cells are radiolabeled with tritiated thymidine and seeded onto 4 mm polytetrafluorethylene grafts. Grafts are then placed into a perfusion circuit for determination of cellular retention. Using this model, the following variables were studied: (1) graft coating (fibronectin versus serum versus plasma); (2) time of incubation of cells with graft (0, 20, 90 minutes); (3) density of the initial seeding solution (4×10³-6×10⁵ cells/cm²). The data suggest that incubation of a graft with plasma provides an adhesive surface that is as effective as fibronectin for enhancing cell retention. With this particular model, seeding densities between 1 and 2 × 10⁵ cells/cm² produce a confluent monolayer with optimal utilization of cells. A shorter 20 minute incubation period resulted in the retention of only half of the seeded cells, while postperfusion attachment increased significantly with a 90 minute incubation period. Data derived from this system can be used to construct a protocol that may be useful for clinicalin vivo seeding trials.     

Endothelial seeding of Dacron and polytetrafluoroethylene grafts: the cellular events of healing

To detect cellular differences in the healing of polytetrafluoroethylene (e-PTFE) and Dacron grafts up to 7 months after implantation, we studied 108 aortic graft interpositions in dogs. Each prosthesis was alternately prepared by endothelial seeding or by an unseeded control method. The grafts were perfusion fixed and studied with light, scanning, and transmission electron microscopy at intervals from before to 221 days after implantation. Seeding resulted in the development of an extensive endothelial flow surface in two out of three of the e-PTFE and none out of four of the Dacron grafts by 10 days after implantation (p = 0.053). After 30 days a microfibrillar subendothelial matrix ranging from 5 to 11 mu formed in all but three grafts with endothelial coverage. The inner capsule of mature Dacron grafts was significantly thicker (169 +/- 143 mu) than in e-PTFE grafts (22 +/- 32 mu; p = 0.002). Seeded and unseeded Dacron grafts had predominantly fibroblasts in the outer capsule of the graft by 10 days. Surface endothelium, vasa vasorum, fibroblasts, and myointimal cells appeared in the inner capsule between 10 and 30 days after implantation. In Dacron grafts, fibroblasts and myointimal cells predominated in the inner capsule at 30 days, with smooth muscle cells not being definitely identifiable until after 150 days. Neither fibroblasts nor myointimal cells were common (present but sparse in one of four e-PTFE grafts) at 30 days, and transmural vasa vasorum were never seen. The seeded endothelial cells migrated rapidly from the sites of initial adhesion near the e-PTFE onto the flow surface. Only one of four of the unseeded e-PTFE grafts had surface endothelium after 30 days, and only moderate coverage developed during 180 days. We conclude that endothelial healing is more rapid in seeded e-PTFE grafts than in seeded Dacron grafts and occurs by a different mechanism.     

Patency in canine inferior vena cava grafting: effects of graft material, size, and endothelial seeding

We studied 117 inferior vena cava (IVC) replacements in dogs to determine the effects of graft material, graft size, endothelial seeding, and cultured endothelial linings on graft patency. As a control, the IVC was removed and reimplanted in 11 dogs. Dacron (n = 7) and expanded polytetrafluoroethylene (e-PTFE) grafts (n = 12) were seeded immediately with the use of enzymatically derived autogenous jugular vein endothelium. Cultured linings were prepared for e-PTFE grafts (n = 9) by inoculating the graft with jugular endothelium and nurturing the lining in tissue culture for 14 to 30 days before implantation. Unseeded grafts (n = 27) were prepared according to the manufacturer's recommendations. These six methods of preparation were tested in grafts measuring 6 mm I.D. and 60 mm in length. Other sizes were tested with a Latin square study design. After 30 to 60 days the grafts were perfusion fixed and studied with light and transmission electron microscopy. Patency was determined by contrast cavography after 7 and 30 days. Patency in the IVC reimplantation was 100% compared with 28.0% of the e-PTFE (p = 0.001) and none of the Dacron grafts that measured 6 mm I.D. and 60 mm long. e-PTFE and Dacron graft patency also differed significantly (p = 0.035). Seeded and culture-lined e-PTFE grafts in that same size were patent in 31.6% compared with 16.7% of unseeded e-PTFE. With grafts measuring 80 mm long, three of the five e-PTFE grafts were patent between 3 and 7 days. All progressed to occlusion by 30 days and compared poorly with all other graft sizes tested (2.6% progression to occlusion [p = 3 X 10(-8)]). Recanalization was not seen in 10 occluded grafts that were followed for 60 days. The histologic features of seeded grafts differed remarkably from grafts previously studied in the arterial circulation and from culture-lined and unseeded venous prostheses in that 60% had prominent large, random, endothelium-lined channels within the inner capsule. Larger graft diameters (p = 0.009) and the omission of an endothelial surface treatment (p = 0.004) were associated with anastomotic subendothelial fibrous hyperplasia. We conclude that graft material is the major determinant of patency in IVC replacements, that an extensive endothelial surface promotes patency, but that simply seeding e-PTFE or Dacron grafts with 10(5) endothelial cells does not provide sufficient endothelium to alter early patency.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of different vascular prostheses and matrices in relation to endothelial seeding.

Thin-walled expanded polytetrafluoroethylene (ePTFE), woven Dacron and gelatin-impregnated Dacron (Gelseal) vascular grafts were compared, the grafts being coated with three different matrices: collagen IV, fibronectin and preclot matrix. In addition, untreated ePTFE and Gelseal were examined. The graft segments, coated with these matrices, were incubated with radiolabelled adult human endothelial cells for 30, 60 and 90 min. Endothelial cell adherence was calculated from the ratio of radioactive counts in the grafts to counts in grafts plus supernatants. Endothelial cell attachment to untreated grafts was poor, but a suitable matrix significantly improved adherence. All three matrices tested gave good results, although preclot was best; 30-60 min incubation was sufficient for optimum cell attachment. Cell adherence to both Dacron and ePTFE was significantly better than to Gelseal. The type of prosthetic polymer and the substrate protein coating used to promote endothelial cell adherence are two important factors which may determine the ultimate success of endothelial seeding in the operating room.     

Clinical experience with a collagen-impregnated knitted Dacron vascular graft

This report describes our first clinical experience with collagen-impregnated Dacron grafts in the aortic position. Fifty-four consecutive patients (43 men and 11 women), average age 67.5 years (44-84), received 11 tube grafts and 23 bifurcated grafts for abdominal aortic aneurysm replacement, 17 bifurcated grafts for aortic occlusive disease, two grafts for aortic pseudoaneurysm, and one graft for thoracoabdominal aneurysm repair. No preclotting maneuvers were used and there was no blood leak from the fabric upon restoration of blood flow, in spite of the use of systemic heparin. The hospital transfusion rate averaged 1.2 units in patients with occlusive disease and 2.7 units in patients undergoing aneurysm resection, which were similar to the transfusion rate in patients receiving standard knitted grafts prior to this study. There were two postoperative deaths from a myocardial infarction and a cerebral hemorrhage and no perioperative thromboembolic events. During a follow-up averaging 41.5 months (26-59), there were two late graft limb thromboses secondary to outflow disease progression. One patient died of aspiration two months postoperatively. Four patients died secondary to cardiac disease in at 13 and 21 months with functioning grafts. The rest of the patients are alive with functioning grafts. We conclude that the collagen-impregnated Dacron graft obviates the need for preclotting maneuvers because of a zero functional porosity.     

Basic fibroblast growth factor coating and endothelial cell seeding of a decellularized heparin-coated vascular graft

The objective of this study was to determine the effect of basic fibroblast growth factor (bFGF) coating on endothelial cell seeding and proliferation on a decellularized heparin coated vascular graft and to determine the retention of seeded cells on the graft under flow conditions. Disks of heparin coated decellularized grafts were incubated for 24 h as controls or with bFGF. Human microvascular endothelial cells (HMECs) or canine peripheral blood endothelial progenitor cells (CEPC) were seeded onto the disks and incubated for 96 h or 48 h, respectively. HMECs were also seeded onto the luminal surfaces of two heparin-coated decellularized grafts for 3 h. One graft was placed in a perfusion culture system and cultured for an additional 6 h with flow and pressure. After culturing, there were 4.7 +/- 1.4 cells/mm(2) HMECs on control grafts and 11.4 +/- 1.4 cells/mm(2) in bFGF treated grafts (P < 0.05). Likewise, with CEPCs, there were 14.8 +/- 4.8 cells/mm(2) in control grafts and 33.3 +/- 7.3 cells/mm(2) in bFGF treated grafts. After only 3 h of cell attachment, 60% of HMECs were retained in the intact graft exposed flow relative to the static control graft, which is an acceptable level. These data demonstrate that bFGF coating on the heparin bound decellularized grafts significantly increases both HMEC and dog EPC proliferation and that seeded cells are stable under perfusion conditions.     

Use of fibrin glue as a substrate for in vitro endothelialization of PTFE vascular grafts

The shear stress resistance of cultured human endothelium was investigated on 6 mm polytetrafluoroethylene vascular grafts. Endothelial cell attachment was promoted by precoating the grafts with fibrin glue, which contained human fibronectin and inhibitors of fibrinolysis (aprotinin and tranexam acid). To evaluate the possible effect of fibrinolysis on cell detachment, seven grafts were lined with adult human saphenous vein endothelial cells (AHSVEC) and 11 with fibrinolytically almost inactive human umbilical vein endothelial cells (HUVEC). Endothelial cell seeding was performed in a microprocessor-controlled rotation device, allowing a low inoculum of 12 X 10(4) endothelial cells/cm2. Grafts were then cultivated for 9 days to enable the maturation of the cytoskeleton, before they were exposed to pulsatile shear stress for 48 hours. A mock circulation simulated the flow patterns and the wall shear forces of the femoral artery. After a 3-hour seeding process, 45% of AHSVEC and 43% of HUVEC were attached to the fibrin matrix, forming a confluent monolayer. After 24 hours of perfusion, a cell loss of 23% in AHSVEC- and of 42% in HUVEC-lined grafts was encountered. In spite of a further cell loss during the following 24 hours of perfusion, the majority of the graft surface was still covered by endothelial cells. Therefore we conclude that fibrin glue is a suitable substrate for the formation of a shear stress-resistant endothelial cell monolayer on polytetrafluoroethylene vascular grafts.     

In vitro adherence and kinetics studies of adult human endothelial cell seeded polytetrafluoroethylene and gelatin impregnated Dacron grafts.

Lining the luminal surface of small diameter vascular prostheses with living endothelial cells reduces thrombogenicity, decreases infection and improves patency. In vitro adherence and kinetics studies of adult human endothelial cell seeded Polytetrafluoroethylene (ePTFE) and Gelatin impregnated Dacron (Gelseal) were performed. Endothelial cell adherence on ePTFE and Gelseal coated with collagen IV, fibronectin and preclot matrices was compared. Untreated ePTFE and Gelseal were also used. Ten graft segments in each group coated with these matrices were incubated with radio-labelled adult human endothelial cells for 30, 60 and 90 min. Labelled endothelial cells seeded in supra-confluent densities on fibronectin coated ePTFE and Gelseal grafts were used for kinetic studies. Resultant endothelial cell monolayers were then exposed to varying shear stress at flow rates of 200 and 300 ml/min in an artificial flow circuit. Endothelial cell attachment to untreated grafts was poor and a suitable matrix significantly improved adherence with fibronectin and preclot but less so with collagen. A 30 min incubation was sufficient for optimum cell attachment. Cell adherence to ePTFE was significantly better than Gelseal. Scanning electron micrographs (SEM) of ePTFE showed preferential attachment to the nodes whilst on Gelseal, cells conformed to Dacron fibres at different levels and directions. Rapidly formed endothelial cell monolayers on ePTFE and Gelseal grafts resisted shear stress of flow with significant cell retention at 2 h. There was patchy coverage of both grafts with evidence of bridging of gaps between individual fibres in Gelseal.     

Development of a "leak-proof," knitted Dacron vascular prosthesis

We have developed a technique to render knitted Dacron prostheses totally impervious to blood leak by impregnating the cloth with collagen. These grafts were prepared by the manufacturer, sterilized, and have an indefinite shelf life. The objective of this study was to determine whether collagen impregnation has a deleterious effect on surface thrombogenicity and graft healing. The infrarenal aortas of 30 mongrel dogs were replaced with 6 cm X 6 mm Dacron grafts (15 collagen-impregnated and 15 control). The collagen-impregnated prostheses were visibly indistinguishable from the control. After blood flow was restored, no interstitial bleeding occurred in the collagen grafts, in contrast to the control grafts that initially leaked profusely. When they recovered, the dogs were divided into three groups: group I (five collagen, five control), group II (five collagen, four control), and group III (five collagen, three control). The grafts were harvested at 3, 6, and 9 months, respectively. Retroperitoneal healing response, capsular thickness and adherence, completeness of neointimal healing, surface fibrinolytic activator activity, and sections taken for light and scanning electron microscopy were studied and compared in a blinded fashion. In a separate set of experiments, experimental and control grafts were compared for in vivo surface thrombogenicity. The healing data were identical in the experimental and control prostheses in all parameters. Of particular interest was that initial capsule adherence was better and surface thrombogenicity less in the collagen-impregnated prosthesis. We conclude that collagen impregnation of a knitted Dacron prosthesis has no deleterious effect for the term of observation of this experiment.     

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.     

Studies in thoracic aortic graft infections: the development of a porcine model and a comparison of collagen-impregnated dacron grafts and cryopreserved allografts.

OBJECTIVE: A porcine model of thoracic aortic graft infection was created, and various anatomic sites and the timing of inoculation of the graft to induce infection were investigated. Ultimately, the ability of cryopreserved allograft to resist infection was compared with that of collagen-impregnated Dacron graft. METHODS: Yorkshire pigs (n = 16) underwent placement of an expanded polytetrafluoroethylene patch graft in the ascending aorta and the left atrial appendage (phase I). Eight animals were immediately given a 50-mL bolus (1 x 10(8) cfu/mL) of Staphylococcus aureus whereas the other 8 received the infusion 24 hours later. Animals were put to death 8 weeks later and the grafts were sterilely explanted and analyzed via microbiologic culture and standard histologic procedures for evidence of infection. The results displayed that the aortic graft and a delay of induced bacteremia of 24 hours were more reliable methods of producing infection. During phase II, 13 pigs were randomized to receive either a collagen-impregnated Dacron graft (n = 6) or a cryopreserved allograft (n = 7) in the ascending aortic position only and infusion of S aureus 24 hours after the operation. The experiment then proceeded to completion. RESULTS: Phase I results displayed that use of an aortic graft and induced bacteremia 24 hours after the operation was a more reliable and reproducible method of producing infection. In phase II, graft infection was present in 38.5% (5/13) of animals, with only 16.7% (1/6) in the collagen-impregnated Dacron graft group and 57.2% (4/7) in the cryopreserved allograft group becoming infected. There was no significant difference between the collagen-impregnated Dacron graft and cryopreserved allograft groups in the incidences of thoracic aortic graft infections (P =.27, Fisher exact test). CONCLUSIONS: This novel porcine model of thoracic aortic graft infection is a reproducible method for the investigation of thoracic aortic graft infections. The phase I study investigated the timing of the induced bacteremia and the most susceptible position of a graft. Phase II demonstrated that collagen-impregnated Dacron grafts are equivalent, if not superior, to cryopreserved allografts in resisting central vascular graft infections in the ascending aorta.     

Adult human endothelial cell seeding using expanded polytetrafluoroethylene vascular grafts: a comparison of four substrates

Prosthetic small-caliber vascular grafts give poorer patency rates than autogenous vein grafts, possibly because the former never spontaneously form endothelium. Animal studies have shown that endothelialization of prosthetic grafts can be encouraged by seeding endothelial cells into the graft at the time of surgery, resulting in improved patency. Information regarding the attachment characteristics of adult human endothelial cells to prosthetic grafts is, however, sparce. Laboratory experiments were performed by use of cell culture techniques to compare the attachment characteristics of adult human endothelial cells to expanded polytetrafluoroethylene graft material, both untreated and treated, with one of four protein substrates--preclotted blood, fibronectin, laminin, and type 4 collagen. Attachment characteristics were compared quantitatively by use of attachment assays and qualitatively by scanning electron microscopy. Attachment to untreated expanded polytetrafluoroethylene was poor but could be greatly improved by preclotting or precoating with any of the proteins, particularly fibronectin. In preclotted grafts seeded endothelial cells formed a virtually confluent monolayer after 1 hour. Cell attachment to the other grafts coated with protein was patchy and inconsistent. It is concluded that a rapid confluent endothelial lining within a prosthetic vascular graft is possible, and of the substrates examined, preclotted blood best encourages cell attachment to expanded polytetrafluoroethylene.     

In vitro endothelialization of small-caliber vascular grafts

To establish the conditions for achieving immediate and complete endothelial cell coverage of the luminal surfaces of small-caliber (internal diameter:4 mm) vascular grafts in vitro, the attachment and spread of endothelial cells cultured from human umbilical veins to expanded polytetrafluoroethylene (PTFE) and knitted Dacron grafts was studied. Cell number was quantified by fluorescent measurements of deoxyribonucleic acid. The completeness of cell coverage and cell junction formation were assessed by means of both scanning and transmission electron microscopy. Cell attachment was compared after expanded PTFE or knitted Dacron grafts were precoated with gelatin, laminin, fibronectin, fibrin, or collagen, singly or in combination. Saturation cell attachment of 3.5 +/- 0.7 X 10(5) cm-2 was completed within 15 minutes when (1) type I collagen was used to form the substrate matrix, (2) human umbilical vein endothelial cells were suspended in phosphate-buffered saline solution supplemented with calcium and magnesium, and (3) the suspended cell number was greater than or equal to 5 X 10(5). In contrast, attachment to untreated or laminin-treated surfaces was 1.3 +/- 0.33 X 10(5) cells cm-2 and attachment to fibrin- or fibronectin-treated surfaces was 2.8 +/- 0.47 and 2.4 +/- 1.1 cells X 10(5) cm-2, respectively. However, to produce a confluent flow surface, the attached cells required several hours in culture medium to spread completely. Maintenance of confluent cell coverage on the graft surface for 3 days in vitro was achieved by means of continuous perfusion with oxygenated tissue culture medium RPMI-1640-HEPES supplemented with 20% fetal bovine serum. We conclude that optimum immediate confluent endothelial coverage of small-caliber vascular grafts requires a high concentration of cells, attachment to collagen-precoated grafts, and several hours of incubation in complete culture medium.     

Evaluation of the thrombogenic potential of three types of arterial graft studied in an artificial circulation.

The thrombogenic potential of woven Dacron, expanded polytetrafluoroethylene (Gore-Tex) and gluteraldehyde-treated human femoral artery (HFA) grafts was evaluated using an artificial circulation. The differences in the platelet parameters (platelet count, adhesion and aggregation) and scanning electron microscopy (SEM) appearances before and after perfusion were used as indices of thrombogenicity. Platelet counts fell significantly with woven Dacron, moderately with HFA and no fall was observed with Gore-Tex. Platelet aggregation was unaffected by any graft, but platelet retention was significantly marked in woven dacron. SEM studies revealed minimal adherence of platelets to Gore-Tex and HFA endothelium, but marked adherences to woven Dacron, accompanied by morphological changes of the adhered platelets. In an artificial circulation, of the three types of arterial graft, woven Dacron has the greatest influence on platelet function, indicating significant thrombogenicity, whereas HFA is intermediate in its effect and Gore-Tex appears to have the lowest thrombogenic potential.     

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.     

Fibronectin Coating of Expanded Polytetrafluoroethylene (ePTFE) Grafts and Its Role in Endothelial Seeding

Although fibronectin's role as a matrix to improve endothelial seeding has been demonstrated by other workers, the optimum concentration for use has never been described. Attachment of fibronectin to expanded polytetrafluoroethylene (ePTFE) was measured, using 125I-radiolabeled protein, at different concentrations and for different time periods. The absolute amount of fibronectin bound to the graft increased with the concentrations used in coating (p less than 0.001) and also with time (p less than 0.01); e.g., at 50 micrograms/ml, 90 min of incubation produced a molecular attachment of 4.0 x 10(11)/cm2 of graft. However, its percentage attachment decreased with a rise in concentration (p less than 0.001). After an initial loss of 22% in 30 min, the fibronectin-graft bond was found to be stable when exposed to a shear stress produced by flow at 200 ml/min. No significant difference in the cell adherence could be found in grafts coated with fibronectin concentrations of 50, 150, and 250 micrograms/ml, although it was significantly less at 10 and 25 micrograms/ml (p less than 0.05).     

Are seeded endothelial cells the origin of neointima on prosthetic vascular grafts?

Although endothelial cell seeding has been demonstrated to be effective in producing uniform endothelial coverage on prosthetic grafts, no one has demonstrated that the seeded cells are the genetic precursors of the endothelial cells that ultimately line the graft. This study was performed to determine whether seeded endothelial cells were the source of the neoendothelial surface on Dacron vascular grafts implanted in the pig. Eight pairs of littermate male and female pigs were selected at birth and allowed to grow until their weight reached 20 to 25 kg. Endothelial cells were enzymatically harvested from the jugular vein of the female siblings and seeded onto 8 mm diameter grafts, 25 cm in length. The grafts were then implanted as a thoracoabdominal bypass into the male siblings. Seven other randomly selected pigs of similar size (four males and three females) had thoracoabdominal bypass with similar grafts, which were preclotted with autologous blood only, but not seeded. After 4 weeks, grafts in both groups were covered with a neointima that was more than 90% thrombus-free. The graft surface cells were grown in culture and were documented to be endothelium. Chromosome analysis demonstrated that the surface cells on the seeded grafts were from the male host and did not originate from the donor female cells. We conclude that seeded endothelial cells are not the source for the neointima on Dacron grafts in the porcine model. In addition, we have again documented that spontaneous endothelial coverage of grafts does occur without endothelial seeding.     

New evidence and new hope concerning endothelial seeding of vascular grafts.

Endothelial cell (EC) seeding of prosthetic bypass grafts has been promoted as a method of improving graft patency. However, an efficient and reliable method of seeding vascular prostheses with ECs is lacking due to inefficient harvesting of ECs and poor attachment and proliferation of cells on the prosthetic surfaces. To investigate the effect of a commonly used prosthetic surface on EC attachment and proliferation, we measured the attachment and proliferation of ECs on polytetrafluoroethylene (PTFE) grafts uncoated or coated with gelatin, laminin, fibronectin, collagen type I and/or III, or RGD (arginine-glycine-aspartate)-containing peptide. EC attachment and proliferation were both significantly decreased on the untreated PTFE graft surface. Conversely, coating of PTFE with fibronectin, RGD, laminin, or gelatin significantly (p less than 0.05) improved the attachment of ECs, with the most striking increases occurring with laminin and gelatin. Similarly, all matrix components in this study improved EC proliferation compared with untreated PTFE, with RGD and gelatin producing the most significant improvement. PTFE adversely effects EC attachment and proliferation. These properties can be improved by treating PTFE graft surfaces with extracellular matrix components in relatively low concentrations. Future investigations are needed to determine whether there are combinations and concentrations of matrix components that will optimize these cellular functions on vascular prostheses.