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.     

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.     

胎儿附属物来源细胞的培养冻存

目的研究从胎儿附属物中分离培养及冻存细胞的较佳方法，以期为组织工程、细胞治疗和基因治疗提供种子细胞。方法取足月产胎儿脐带和胎盘，用酶消化法获得细胞，用相应培养液进行培养传代。将胎盘、脐带组织块冻存，检测加入不同浓度抗冻剂二甲亚砜的组织复苏后的活细胞率，透射电镜下观察其超微结构，并与新鲜组织进行比较；冻存培养所得人脐带血管周细胞和胎膜贴壁细胞的原代细胞，用免疫化学法检测冻存前后各自免疫表型的表达。结果新鲜脐带组织活细胞率为67．0％，抗冻剂体积分数为5％、10％、15％、20％时，冻存复苏脐带组织的活细胞率分别为23．4％、55．5％、48．8％、31．8％。抗冻剂体积分数为10％时，冻存复苏组织与新鲜组织活细胞率最接近（P〉0．05），体积分数为5％和20％时与新鲜组织该指标比较，差异均有统计学意义（P〈0．01）；透射电镜观察结果与之吻合。胎盘组织与脐带组织的情况类同。细胞冻存后免疫表型无明显变化。结论本方法可以从胎儿附属物中分离培养出大量种子细胞，冻存复苏后细胞免疫表型未发生改变，抗冻剂体积分数为10％时效果最好。     

Induction of bone marrow CD34+ cells and endothelialization of polytetrafluoroethylene prostheses

BACKGROUND: The aim of the study was to investigate the endothelialization of prostheses with bone marrow CD34(+) cells. METHODS: CD34(+) cells were isolated from the canine bone marrow by an immune magnetic cell sorting system and induced into endothelial cells (EC) with vascular endothelial growth factor. Cells were seeded to polytetrafluoroethylene prostheses, which were implanted into the abdominal aorta artery and inferior vena cava. RESULTS: The isolated cells derived from bone marrow were CD34(+) cells by flow cytometry, which could differentiate into EC when induced by vascular endothelial growth factor. The neointima on control prostheses was significantly thicker than that on prostheses seeded with EC at the anastomosis and the centre where the prosthesis interposed to aorta. All control prostheses (4/4) interposed to veins were occlusive and two seeded prostheses (2/8) interposed to veins were already occluded. The overall occlusion rate of prostheses interposed to veins in seeded prostheses and control prostheses were 25 and 100%, respectively. However, the neointima on seeded prostheses was significantly thicker in the vein than that in the aorta. CONCLUSION: Bone marrow CD34(+) cells can be induced into EC by vascular endothelial growth factor in vitro. Polytetrafluoroethylene prostheses seeded with CD34(+) cells have the ideal endothelialization and patency.     

Dual cell seeding and the use of zymogen tissue plasminogen activator to improve cell retention on polytetrafluoroethylene grafts

OBJECTIVE: The purpose of this study was to enhance the retention of seeded endothelial cells (EC) on prosthetic vascular grafts. Dual-layer EC and smooth muscle cell (SMC) seeding and gene transfer of a zymogen tissue plasminogen activator gene (tPA) into seeded EC were studied. METHODS: Polytetrafluoroethylene (PTFE) grafts were precoated with fibronectin, seeded with SMC followed by EC a day later, and then, 24 hours later, exposed to an in vitro flow system for 1 hour. Cell retention rates were determined for grafts seeded with EC only, a dual layer of EC on top of SMC, EC transduced with wild-type tPA, and EC transduced with zymogen tPA. RESULTS: Seeding efficiency of PTFE pretreated with fibronectin was 260 +/- 8 cell/mm(2). After exposure to flow, only 39% +/- 14% of the EC were retained when EC were seeded alone, whereas 73% +/- 22% of EC remained on grafts when EC were seeded on top of SMC (P <.001, n = 10). The enzyme activity of a mutant zymogen tPA in absence of fibrin was 14 +/- 1 IU/mL, which is 3.6-fold lower than that in the presence of fibrin (50 +/- 19 IU/mL), whereas fibrin has no effect on the wild-type tPA activity. EC expressing a high level of wild-type tPA had a lower retention rate (37%) when compared with normal EC (45%). EC expressing the mutant zymogen tPA had an improved retention rate (54%, P =.001, n = 10) in absence of fibrin, whereas its retention rate was reduced to 43% when the cells were exposed to fibrin. CONCLUSION: SMC seeded between EC and PTFE improves EC retention in vitro. Transduction of zymogen tPA increases thrombolytic ability of seeded cells with less adverse impact on cell retention than wild-type tPA.     

Smooth muscle cells improve endothelial cell retention on polytetrafluoroethylene grafts in vivo

OBJECTIVE: We investigated the influence of smooth muscle cells (SMC) on endothelial cell (EC) retention on polytetrafluoroethylene (PTFE) grafts and the effect of SMC seeding on intimal hyperplasia in vivo in a rabbit model. METHODS: Fibronectin-coated PTFE grafts (4 mm diameter) were seeded with either EC alone, SMC alone, or SMC followed 24 hours later by EC. The grafts were connected to an extracorporal aortic shunt for 1 hour or were individually implanted for 1, 30, and 100 days into the infrarenal aorta as an end-to-side bypass graft. The number of retained cells was compared at 1 hour and at 1 day after implantation. Neointimal thickness was measured 30 and 100 days after implantation. RESULTS: After 1-hour exposure to blood flow, EC retention rate was greater (P <.005) if seeded on top of SMC (98% +/- 2%; n = 8) versus being seeded alone (65 +/- 11%; n = 8). SMC retention rate was 95 +/- 5% (n = 8) when seeded alone. Similar cell retention was obtained 1 day after implantation. After 30-day implantation the neointima was thicker in grafts seeded with EC and SMC (282 +/- 136 microm; n = 3) than with EC only (52 +/- 45 microm; n = 3; P <.001). However, the neointimal thickness for dual-cell-seeded grafts (126 +/- 60 microm; n = 3) was not significantly different (P =.09) from EC-seeded grafts (79 +/- 48 microm; n = 3) after 100-day implantation. CONCLUSION: EC retention on PTFE grafts in vivo is improved if seeded over a layer of SMC. Further studies are needed to determine whether overlying EC modulate proliferation of underlying SMC.     

Thrombogenesis of different cell types seeded on vascular grafts and studied under blood-flow conditions

Background: Small-diameter vascular grafts tend to have an early and high occlusion rate. Cell seeding on the luminal surfaces of small-diameter vascular prostheses may provide an antithrombotic lining and improve both the short-term and the long-term patency rates. We studied the net results of procoagulant and anticoagulant properties of seeded grafts under blood-flow conditions, and we compared the different available types of donor cells. Methods: Monolayers of liposuction-derived cultured human microvascular endothelial cells (MVECs), human adult endothelial cells (HAECs), human umbilical vein endothelial cells (HUVECs), and human mesothelial cells (MCs) that had been seeded on expanded polytetrafluoroethylene (ePTFE) grafts were perfused with marginally anticoagulated blood (20 U/mL low molecular weight heparin; shear rate, 400/s, 10 minutes) or with non-anticoagulated blood (shear rate, 100/s, 5 minutes). The thrombin and fibrin generation in time was studied with the measurement of the plasma levels of prothrombin fragment 1 and 2 (F 1+2) and of fibrinopeptide A (FPA). The plain ePTFE graft was taken as a control. Results: When the seeded MCs were perfused with recirculating anticoagulated blood, a linear generation of F 1+2 in time was seen, with high levels of F 1+2 and FPA after 10 minutes (4.38 nmol/L and 362 ng/mL, respectively). Allopurinol was added, and the MCs generated less F 1+2 than the HAECs (0.7 nmol/L vs 1.86 nmol/L; P < .05). No fibrin formation was seen. The MVECs generated low amounts of F 1+2 (0.7 nmol/L; 10 minutes), and the HUVECs and the plain ePTFE graft generated the lowest amounts of F 1+2 (0.26 and 0.25 nmol/L, respectively). When the MCs were perfused with non-anticoagulated blood, high amounts of thrombin and fibrin were generated immediately and constantly and could not be decreased with allopurinol. The perfusion of the plain ePTFE graft showed a dramatic increase in F 1+2 and FPA levels towards the end of the experiments. The seeded HAECs, HUVECs, and MVECs inhibited this increase. These results were confirmed by means of scanning electron microscopy. Conclusion: Vascular prostheses that are seeded with cultured MCs are highly procoagulant. Standard ePTFE graft prostheses also initiate coagulation, which supports the idea of cell seeding. The endothelial cells, of which the MVECs are the most readily available, seem to preserve their anticoagulant properties after being seeded on vascular grafts. (J Vasc Surg 1998;28:1094-1103.)     

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.     

Adhesion and Stability of Fibronectin on PTFE Before and After Seeding with Normal and Synchronized Endothelial Cells: In Vitro Study

Abstract: The possible influence of the cell cycle on the efficacy of endothelial cell (EC) seeding onto the surface of polytetrafluoroethylene (PTFE) prostheses was studied. Likewise the ideal fibronectin concentration and optimal incubation time to guarantee the binding of this protein to the prosthetic surface have been calculated. Synchronized ECs, previously labeled with ³H-thymi-dine, were used, and the loss of radioactivity was determined at several times throughout the study. The results showed a progressive loss of cells on the prosthetic surface similar to that occurring with the seeding of un-synchronized ECs. The optimal concentration of fibronectin was 20 μg/ml, and the optimal incubation time was 1 h.     

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.     

Quantitative Analysis of Human Platelet Adhesions Under a Small‐Scale Flow Device

To realize real‐time evaluation of human platelet adhesions onto material surfaces with small volumes of human platelet suspensions, we developed an apparatus consisting of a modified cone and plate‐type viscometer, combined with an upright epi‐fluorescence microscope. The apparatus allowed real‐time evaluation of platelet–material interactions and the initial event of thrombus formation, using small platelet suspension volumes (7.5 µL) under shear flow conditions. To study the dynamic behavior of platelet–material interaction, we chose five representative opaque and transparent materials: acrylate resin (AC), polytetrafluoroethylene (PTFE), polyvynylchrolide (PVC), glass, and a monolayer of human normal umbilical cord vein endothelial cells (EC) on glass under shear flow conditions. The values of adhesiveness of human platelets to the test materials in descending order were as follows: AC > PTFE > PVC > glass > human EC. Under this new small‐scale flow system, we could obtain highly reproducible data, which were comparable with results from a previously developed large‐scale flow system. Therefore, the newly developed cone and plate‐type rheometer is a useful instrument for testing and screening materials, and allows precise quantitative evaluation of human platelet adhesion.     

Polyurethane scaffolds seeded with autologous cells can regenerate long esophageal gaps: An esophageal atresia treatment model

BackgroundPediatric patients suffering from long gap esophageal defects or injuries are in desperate need of innovative treatment options. Our study demonstrates that two different cell sources can adhere to and proliferate on a retrievable synthetic scaffold. In feasibility testing of translational applicability, these cell seeded scaffolds were implanted into piglets and demonstrated esophageal regeneration.MethodsEither porcine esophageal epithelial cells or porcine amniotic fluid was obtained and cultured in 3 dimensions on a polyurethane scaffold (Biostage). The amniotic fluid was obtained prior to birth of the piglet and was a source of mesenchymal stem cells (AF-MSC). Scaffolds that had been seeded were implanted into their respective Yucatan mini-swine. The cell seeded scaffolds in the bioreactor were evaluated for cell viability, proliferation, genotypic expression, and metabolism. Feasibility studies with implantation evaluated tissue regeneration and functional recovery of the esophagus.ResultsBoth cell types seeded onto scaffolds in the bioreactor demonstrated viability, adherence and metabolism over time. The seeded scaffolds demonstrated increased expression of VEGF after 6 days in culture. Once implanted, endoscopy 3 weeks after surgery revealed an extruded scaffold with newly regenerated tissue. Both cell seeded scaffolds demonstrated epithelial and muscle regeneration and the piglets were able to eat and grow over time.ConclusionsAutologous esophageal epithelial cells or maternal AF-MSC can be cultured on a 3D scaffold in a bioreactor. These cells maintain viability, proliferation, and adherence over time. Implantation into piglets demonstrated esophageal regeneration with extrusion of the scaffold. This sets the stage for translational application in a neonatal model of esophageal atresia.     

Specific determination of endothelial cell viability in the whole cell fraction from cryopreserved canine femoral veins using flow cytometry

Abstract: An efficient method for specifically determining the viability of endothelial cells (EC) from cells dissociated from the human saphenous vein was investigated. Three different methods, trypan blue staining assay, [3H]-proline incorporation assay, and flow cytometry (FCM), combined with the fluorescein isothiocyanate conjugated with Griffonia simplicifolia agglutins (GS1-FITC)/propidium iodide (PI) double staining, were used. Both trypan blue staining and [3H] proline incorporation assays demonstrated less sensitivity to determine viability of EC differentially from the other cells. FITC-GS1 showed prominent binding to the vascular EC and could be counted by FCM including PI on dead cells. Following the cryopreservation process, the GS1-FITC/PI FCM analytical method was adopted to test simultaneously the viability of whole cells and EC from the same tissue, human saphenous veins, and mongrel dogs' femoral veins after harvesting, antibiotic solution treatment, and thawing. The viability of the whole cells from veins decreased with a significant difference (p < 0.05) from that of EC after thawing.     

Effect of Cryopreservation and Cell Passage Number on Cell Preparations Destined for Autologous Chondrocyte Transplantation

Autologous chondrocyte transplantation (ACT) is an effective and safe therapy for repairing articular cartilage defects and requires cell preservation and subculture before transplantation. We compared the effects of cryopreservation and passaging on cell viability, proliferation, and maintenance of the function of chondrocytes and synovium-derived mesenchymal stem cells (MSCs) used as sources for ACT. These cells were isolated from the knee joints of rabbits and were cultured, passaged serially, and divided into 2 groups that were either cryopreserved or not. The morphology, viability, gene expression, and differentiation potential of the 2 groups were compared. Maintenance of the potential to undergo chondrogenic differentiation was determined with the use of a 3-dimensional culture method. Passaging and cryopreservation significantly affected the ability of chondrocytes to maintain their morphology, express chondrogenic genes, and differentiate. In contrast, synovium-derived cells were not affected by passaging and cryopreservation. Our results may serve as the foundation for the application of passaged and cryopreserved chondrocyte or other source cells of MSCs in ACT.     

Effects of autologous mesothelial cell seeding on prostacyclin production within Dacron arterial prostheses

Canine abdominal aortas have been replaced with Dacron arterial prostheses to assess the effects of mesothelial cell seeding on graft prostacyclin and thromboxane A2 release. At both 2 weeks and 6 weeks after surgery, three seeded and two unseeded control grafts were examined for prostacyclin release. In addition, thromboxane release was assessed in one seeded and one unseeded graft. Sections of aorta and graft were removed and incubated in PBS containing either 10 microM calcium ionophore A23187 or 20 microM arachidonic acid. The incubation mixture was sub-sampled at 5 min intervals over a 20 min period to assess the progressive release of prostacyclin and thromboxane A2 using a radioimmunoassay for 6-keto-prostaglandin F1 alpha and thromboxane B2 respectively. In seeded grafts, 6-keto-prostaglandin F1 alpha release averaged 15 per cent compared with aorta at 2 weeks and 45 per cent compared with aorta at 6 weeks. By contrast, release from unseeded grafts was undetectable at 2 weeks; however, by 6 weeks there was some release amounting to 15 per cent compared with aorta. There was a statistically significant increase in the release of 6-keto-prostaglandin F1 alpha from mesothelial cell seeded grafts at 6 weeks compared with unseeded grafts (P less than 0.01). Thromboxane release from the graft sections was variable and unrelated to whether the grafts had been seeded or not. These preliminary results, showing that grafts seeded with autologous peritoneal mesothelial cells release more prostacyclin than unseeded grafts, further highlight the role of the mesothelial cell as an alternative to the endothelial cell for improving the patency of arterial Dacron prostheses in the early postoperative days.     

内皮细胞衬里人工血管静脉旁路移植10例

为进一步证实内皮细胞衬里人工血管的临床效果,报告临床应用１０例的长期随访。方法,本组１０例布加综合征病人,行腔房转流术６例,肠房转流术２例,肠颈转流术和肠腔转 流术各１例。应用人工血管内径１４ｍｍ,长８－３０ｃｍ,其中针织涤纶２根,编织涤纶７根,带外支持环ＰＴＦＥ１根。     

Complement activation by synthetic vascular prostheses

Morphologic evaluation of synthetic grafts (both seeded and unseeded) harvested within 2 weeks of implantation has revealed heavy infiltration with polymorphonuclear leukocytes (PMNs). Since complement-activated PMNs are known to damage endothelial cells, we hypothesized that complement activation might prove a barrier to optimal endothelial cell seeding of prosthetic grafts. To determine whether synthetic vascular prostheses activate complement and whether the type of graft material influences the degree of activation, we assayed the plasma of 10 healthy donors for complement activity following incubation with short segments of knitted Dacron and polytetrafluoroethylene (PTFE) graft material. C5a generation was measured by radioimmunoassay and granulocyte aggregometry. Standard hemolytic assays were used to determine depletion of functional classical pathway (CH50 and C4) alternative pathway (APH50) activity. Results indicated substantial complement activation by Dacron and virtually none by PTFE. Activation by Dacron appeared to occur via both complement pathways. Such complement "reactivity" may have important implications for the performance of prosthetic materials as small-caliber vascular grafts, whether seeded with endothelial cells or not.     

Endothelial cell seeding of a new PTFE vascular prosthesis

Previous attempts to line polytetrafluoroethylene (PTFE) prostheses with enzymatically derived endothelial cells have not been as successful as similar work with Dacron grafts because of the failure of such prostheses to develop a satisfactory subendothelium. This article reports our experience with a new, highly porous, unreinforced PTFE prosthesis that appears to circumvent this problem. Segments (4 mm I.D., 10 cm in length) of this new graft were implanted in 41 mongrel dogs as carotid interposition grafts. One graft in each dog was seeded with the dog's own endothelial cells, whereas the contralateral graft was treated in an identical fashion except for the inclusion of endothelium. After a mean period of 7 weeks of implantation, the grafts were harvested, their patencies were noted, the thrombus-free area of their luminal surface was calculated with computerized quantitative planimetry, and graft segments were submitted for scanning and transmission electron microscopy. In seven dogs the luminal surface was scraped from each graft and measured quantitatively. Although seeded grafts failed to show a statistically significant increase in patency during the short course of this experiment, a trend in that direction was quite striking. Furthermore, seeded grafts had a significant increase in thrombus-free area on their luminal surface as well as a significant reduction in the volume of luminal thrombus. Histologically, seeded grafts developed a substantial 75 to 100 microns cellular subendothelium beneath a confluent endothelial lining. No endothelial lining was noted in control grafts. We believe that the superior handling characteristics of this new prosthesis and its ability to develop a substantial subendothelium with a confluent endothelial lining suggest potential future applications and warrant further investigation.     

Precoating substrate and surface configuration determine adherence and spreading of seeded endothelial cells on polytetrafluoroethylene grafts

Primary adherence and attachment area of seeded human endothelial cells (EC) were determined on differently coated polytetrafluoroethylene (PTFE) grafts. Cell counts and morphometric analyses were done immediately after 60 minutes of electronically controlled seeding of 3 x 10(4) EC/cm2, as well as after 3 hours of subsequent incubation. Cell adherence and cell spreading were distinctly superior on two surface-covering substrates: fibronectin-treated type I/III collagen and fibrinolytically inhibited fibrin glue. Uncovered, purely fibronectin- or laminin-coated PTFE or type IV collagen treated with the specifically binding glycoprotein laminin showed a far lower EC attachment rate and less pronounced cell spreading. It appears that not only a high surface content of fibronectin but also a smooth PTFE-covering matrix are prerequisites for optimal primary adherence and cell spreading. Because fibrin glue might be fibrinolytically degraded despite its plasmin-inhibiting epsilon-amino-caproic acid compound, type I/III collagen plus fibronectin could provide an optimal precoating substrate for EC lining of PTFE grafts.