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Seeding of enzymatically derived and subcultivated canine endothelial cells on fibrous polyurethane vascular prostheses F. Hess, R. Jerusalem, 0. Reijnders, C. Jerusalem, S. Steeghs, B. Braun* and P. Grande* Laboratory for Cell Biology and Histology, Netherlands,

and ‘B. Braun Melsungen

University Nijmegen, PO Box 9101, 6500 HB Nijmegen, AG, Carl Braunstrasse 1, 3580 Melsungen, Germany

The

Fibrous polyurethane (FPU) prostheses with or without fibronectin coating and gelatin impregnation and FPU prostheses with or without fibronectin coating were seeded with 4.8 x lo5 subcultivated dog endothelial cells per cm2 prosthesis. Expanded polytetrafluoroethylene (ePTFE) prostheses with and without fibronectin coating served as controls. The numbers of cells retained on uncoated polyurethane prostheses were minimal but increased with fibronectin coating and/or gelatin impregnation. Adhering cells were predominantly round in shape and few cells were seen stretched over the prosthetic fibres. Optimum numbers of cells were found in prostheses impregnated with gelatin and coated with fibronectin, where almost all the cells were stretched forming a confluent monolayer. In ePTFE prostheses only minimal numbers of cells were retained but in the fibronectin-coated prostheses a high cell count was noted. Gelatinimpregnated and fibronectin-coated FPU prostheses, as well as ePTFE prostheses coated with fibronectin, were additionally perfused in vitro after seeding under nearly physiological conditions for 1 h. Cells in the FPU prostheses were still present after perfusion, whereas all the cells in the ePTFE prostheses were lost from the inner surface. It is concluded that FPU prostheses impregnated with gelatin and coated with fibronectin are a suitable substrate for subcultivated endothelial cells to be seeded on. The cells remained at the surface even after 1 h in vitro perfusion with tissue culture medium under nearly physiological conditions. Further research including in vivo implantations is indicated. Keywords:

Vascular prostheses,

endothelium,

ceil-seeding

Received 22 January 1992; revised 11 February 1992; accepted

Lowering the thrombogenicity of synthetic vascular prostheses and increasing the post-operative patency by lining the inner prosthetic surface with endothelial cells before implantation has been a topic of intense research for several years’-“. Up to now, the ideal conditions under which freshly harvested or subcultivated endothelial cells will adhere and spread on to the diverse prosthetic surface structures’, 4q’ and/or coatings39 5-1oas well as the stretching conditions are only poorly defined and are still the subject of much biomedical, biochemical, polymeric and morphological research. Also, the number of endothelial cells which has to be seeded in order to cover a suitable percentage of the prosthetic surface is still unclear’. So far, only a few publications deal with vascular prostheses which were endothelial cell seeded before implantation. There are few reported results on Correspondence 0

1992

to Dr F. Hess.

Butterworth-Heinemann

0142-9612/92/100657-07

Ltd

24 February

1992

improvement in patency due to an endothelial lining”-‘7. Experimentally, most in vitro as well as in viva seedings are carried out using Dacron@ or ePTFE prostheses. It is well documented that spontaneous and complete endothelialization of both types of prostheses after clinical as well as after experimental implantation takes a long time, if it ever occurs18-24. Studies using short fibrous polyurethane vascular prostheses implanted in the rat and dog have shown that endpthelial cells invade this type of prosthesis from the vascular stumps over the anastomotic lines at a continuous speed of 0.3 and 0.1 mm/d, respectively, until complete endothelialization is achievedz3, 24. This means that for clinically implanted prostheses endothelialization will take a long time. Until a complete endothelial lining is present in a vascular prosthesis, the risk of thrombo-occlusive complications remains. It appears that polyurethane may be a suitable Biomaterials

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substrate for cell seeding25-27. The present study investigated the behaviour of subcultivated canine endothelial cells seeded on to fibrous polyurethane prostheses with and without surface coatings, in an attempt to obtain a confluent lining which remains stable and present even during in vitro perfusion.

MATERIALS AND METHODS Endothelial cell harvesting Endothelial cells were harvested from jugular veins of heaIthy Beagle dogs, approximately one year old, as described by Graham et al. 25. Under general anaesthesia (15 mg/kg i.v. sodium thiopenthai) and sterile conditions, both jugular veins were exposed through a mid-line incision in the neck. Both veins were carefully mobilized over a length of approximately 10 cm, They were doubly ligated at the distal end, clamped proximally and, after transection between the double ligature distally, removed. The veins were turned inside out over a stainless steel rod with the same diameter as the vein and secured with a single ligature. To isolate the endothelial cells the following procedure was carried out: (1) washing in Ca- and Mg-free Tyrode for a few minutes at 37’C to remove adhering erythrocytes; (2) incubation in 0.1% trypsin + 0.125% EDTA in Ca- and Mg-free Tyrode for 10min at 37°C; (3) incubation in serum-free RPM1 (Gibco, NY, USA) containing 650 units/ml collagenase for 10 min at 37°C; (4) afterboth incubations, solutions were neutralized with RPM1 + Basal Medium Eagle (BME) (1:l) containing 10% fetal calf serum (FCS); (5) centrifugation of both incubation solutions: (6) resuspension of the cell pellets with the RPM1 + BME + FCS solution; (7) centrifugation; (8) after resuspension the cells were put in tissue culture flasks and then incubated at 37’C in a 5% CO2 atmosphere. Animal care was carried out according to Dutch regulations on animal research.

Subcultivation Flasks free of contamination with fibroblasts and/or smooth muscle cells were used for subcultivation up to 7 or 10 times. When necessary, cells were removed from a culture flask with 0.05% trypsin + 0.02% EDTA in Caand Mg-free Tyrode, washed with RPM1 + BME (1:l) medium containing 10% FCS and divided over new culture flasks, which were put in an incubator at 37°C in a 5% CO, atmosphere.

Seeding procedure Before each seeding procedure, segments of prostheses of the proper length were fixed on a stainless steel conus which was mounted on to a syringe. Fibronectin coating was carried out by filling the piece of prosthesis to be seeded with a solution containing 100 pg fibronectin for 60 min. After removal of the fibronectin solution, prostheses were ready for seeding. Before each seeding procedure, the capacity of the prosthetic segment was calculated and an endothelial cell suspension three times the calculated volume was prepared containing 1.5 X lo6 cells per ml. At first, one-third of the suspension was put into the prosthesis and cells were allowed to settle on the Biomaterials 1992. Vol. 13 NO. 10

Figure 1 In a sterile Petri dish a segment of a vascular prosthesis, fixed on to a stainless steel conus and connected with a syringe, is being incubated during the seeding procedure. Note the marks on the conus used to rotate the prosthesis 120” three times during the seeding procedure.

lower third of the prosthesis for 30 min. During this incubation time the prosthesis was kept in a covered sterile Petri dish at 37°C (Figure I) in an incubator with a 5% CO, atmosphere: the remaining cell suspension to be used for the second and third seeding stage was kept in the same incubator. After 30 min, the prosthesis was emptied, filled with a second one-third aliquot of the cell suspension, turned 120’ on the longitudinal axis (using marks on the conus), and incubated again for 30 min. This procedure was repeated for a third time with the last aliquot of suspension and an additional 30 min. Then, the emptied prosthesis was floated for 1 h in RPM1 + BME (1:l) containing 10% FCS at 37°C to allow the cells to adhere further and/or spread on the inner prosthetic surface. This staged seeding and rotation procedure was carried out to obtain as even as possible a distribution of the cells over the prosthetic surface. The seeding density was determined as follows. From the 3 mm diameter of the prosthesis, per cm length, an inner surface area of 0.94 cm2 and a volume of 0.1 ml was calculated. At each seeding stage, 1,5 X lo5 cells were seeded on 0.31 cm’, resulting in a seeding density of 4.8 X lo5 cells/cm’ inner surface per cm length.

Prostheses The fibrous pol~rethane vascular prostheses used in this study in groups 1-6, were made using the so-called had a fine fibrillar spraying technique 28. Prostheses structure of the wall, consisting of fibres with mean diameter 1 pm, packed together and laying in different directions (Figure &I). They had an inner diameter of 3 mm and were permeable to air and watery liquids. To liquids with decreased viscosity, prostheses were less or even non-permeable. For part of the experiment (groups 3-5), fibrous polyurethane prostheses were used. These were impregnated with gelatin by immersion in a solution containing 5% gelatin and 10% glycol. The prostheses were maintained at 50°C for 20 min under vacuum conditions. After removal from the gelatin solution, prostheses were drained of excessive fluid and air-dried at room temperature for 24 h. To fix the gelatin, prostheses were immersed in 1% glutaraldehyde in distilled water for

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16 h. After washing until no further aldehyde was released, the prostheses were dried at 50°C. This resulted in coating the pol~~thane fibres, sealing off all pores existing throughout the fibrous texture of the wall, and making the prostheses impermeable to air. At the same time most of the microarchitecture of the inner surface structure remained intact (Figure 3a). As control material for the endothelial cell seeding procedures, commercially available expanded PTFE prostheses with inner diameter 3 mm were used in groups 6-8. All groups are listed in Table 1.

in vitro

perfusion

Prostheses of groups 5 and 8 were placed for 1 h in a closed perfusion circuit after the seeding and incubation period. Perfusion was carried out with flow rate 300 ml/ min at 120 mgHg and 37’C. The perfusion medium RPM1 + BME (1:l) containing 10% FCS was used. SEM

and light microscope procedures

After seeding and/or in vitro perfusion, prostheses were fixed in 1.5% glutaraldehyde and divided into two segments for scanning electron microscope (SEM) and light microscope examinations, respectively. For SEM the specimens were post-fixed in osmium tetroxide, dehydrated in increasing ethanol solutions, air-dried and sectioned longitudinally into two parts. The segments were mounted on a specimen holder with silver paste and spattered with gold before examination in a Philips SEM 500. For light microscopy, prostheses were pre-embedded in 3% agar-agar before embedding in paraffin, to fix the cells on the prosthetic inner surface because the polyurethane dissolves during the histological processing. Cross-sections of the prostheses were made and stained with haematoxylin-eosin.

RESULTS

Figure 2 a, Structure of the polyurethane prostheses used in groups 1 and 2. b, Prosthesis of group 1 (non-coated prostheses) after seeding and incubation. Almost no cells have been retained. c, Prosthesis of group 2 (fibronectin coating} after seeding and incubation. A considerable number of predominantly round cells are present. (Scanning electron micrographs, original magnifications x320.)

There were distinct differences between groups l-5 with respect to the number of cells remaining on the prosthetic surface after the seeding and incubation period. without coating almost no cells were retained. However, the combination of gelatin impregnation and fibronectin coating proved to be most effective. As a rule, the cells present on the prosthetic surface in groups l-5 were fairly evenly distributed. After in vitro perfusion (group 5) cells appeared to adhere to the prosthetic surface. In the ePTFE prostheses occasionally a seeded cell could be detected but coating with fibronectin (group 7) yielded rest&s comparable to groups 2 and 3. However, with perfusion (group 8) all cells were lost 60 min after the start of the perfusion. The number of cells in the effluent of each single seeding stage, as indication of the cell-take, could not be determined exactly due to the presence of multiple cell clumps in addition to single cells. Using the cells in the effluent as indicator for the number of cells retained in the prosthesis, the numbers corresponded fairly well with the SEM observations in all groups after seeding. The morphological findings of each group [l-8) are Biomaterials

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Figure 3 a, Structure of a polyurethane prosthesis impregnated with gelatin (SS used in groups 3,4 and 5). Compare to figUr8 2a, and note that the inner architecture is widely preserved. b, Gelatin impregnated prosthesis after seeding and incubation (group 3). A fair number of cells are retained, both stretched and round. c, Prosthesis of group 4, impregnated with gelatin and coated with fibronectin, after seeding and incubation. A high number of stretching ceils are present. Numerous cell-to-cell contacts. d, Prostheses of group 5, 1 h after perfusion with tissue culture medium. An almost confluent monolayer of flat cells is still present. (Light micrographs, original magnifications X320.)

Table 1 Summary of all groups included in this study, presenting the type of prosthesis as well as the type of coating/ impregnation, number of seeding experiments carried out in each group, as well as a score of the results with regard to the cell retention after the seeding (and perfusion) procedure Group

Type of prosthesis

Number of experiments

Cell retention

1

fPU fPU coated with fibronectin fPU coated with gelatin fPU coated with gelatin + fibronectin fPU coated with gelatin + fibronectin followed by 1 h perfusion ePTFE ePTFE coated with fibronectin ePTFE coated with fibronectin followed by 1 h perfusion

4 4 4 4 4 4 4 4

O/f + ++ +++ ++/+++ 0/+ ++ 0

z 4 : 7 8 fPU. fibrous Biomaterials

polyurethane: ePTFE. expanded polytetrafluoroethylene.

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in Table 1 a score

is listed

Group 1: Only a minimal number of cells were seen to adhere to the fibres of the inner prosthetic surface (Figure Zb). Most adhering cells were still round and only a few pseudopods were seen, No cells were seen to spread over the prosthetic fibres. more cells adhered to the prosGroup 2: Significantly thetic material than in group 1 and they were evenly distributed over the total surface area (Figure 2~). However, their number was much lower than the number of cells seeded and little or no spreading of the round cells was noted. Generally, the number of adherent cells was insufficient to cover a significant area of the inner prosthetic surface. Group 3: A moderate number of cells were present on the prostheses [Figure 3b) and they were nearly all pseudopods expressing a beginning of spreading. An insufficient number of cells was present to cover the prostheses completely even in the case of total spreading. Group 4: A high number of cells were found to adhere to the prostheses (Figure 3c) and a significant number of them were seen in different stages of spreading. Cellular contacts were abundantly noted as well as spreading over more than one prosthetic fibre. A sufficiently high number of cells were present and covered almost the complete prosthetic inner surface. group Group 5: The results of this in vitro perfusion resembled greatly those of group 4: a high number of cells were present, and they covered almost all of the prosthetic surface [Figure 34. Numerous cell-to-cell contacts were present and cells were polygonal to spindle shaped. Group 6: Only an occasional cell was found at the inner prosthetic surface. Cells present were round (Figure da). Group 7: A considerable number of cells was present. Most of the seeded cells were stretched but some had maintained a round shape. Cells tended to lay more or less singly or in small clusters. Complete coverage of the prosthesis was not achieved (Figure 4b). Group 8: No cells were present on the inner surface at the end of the perfusion time (Figure 4~). Light microscopy was extremely difficult to interpret, especially in groups 1 and 6 where minimal cells were retained after seeding. With higher numbers of cells present (groups 2, 3, 4, 5, 7, 8), information from the sections could be drawn which paralleled the SEM findings. DISCUSSION One of the main problems in endothelial cell seeding is the inefficiency of the procedure. There are three aspects to this: [l) the number of endothelial cells that can be harvested from donor blood vessels is only a fraction of the number present in those vessels2g-31; (2) the number of

Figure 4 a, ePTFE prosthesis of group 6 (non-coated ePTFE). Sporadic cells can be found. b, After coating with fibronectin (group 7) both stretched and round cells are attached to the wall in high numbers after seeding and incubation. c, Prosthesis coated with fibronectin and perfused for 1 h (group 8). No adhering cells remain. (Scanning electron micrographs, original magnifications X320.)

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cells retained after seeding depends greatly on the physical characteristics of the prosthetic material, the surface structure of the fabric and the presence and type of coating used3$ ‘-s, lo, 32; and (3) the high number of cells necessary to form a continuous lining after seeding, especially in longer prostheses, The last aspect (3) can be reduced and/or solved by subcultivation of the endothelial cells. This is possible under experimental conditions but is clinically possible only in non-emergency cases. If only low numbers of cells adhere after a seeding, no confluent monolayer is obtained. This endangers a successful seeding procedure and subsequent patency of the prosthesis. Therefore, coating of the prosthetic surface is essential to increase retention rates. Seeding of longer prostheses can be performed only with extremely high cell counts, which cannot be obtained readily and requires subcultivation of the primary harvested cells. The seeding density in our study (4.8 X 10’ cells/cm’ prosthesis) is higher than usually reported in the literature (1X lo4 - 2 X 105)‘, 6V8. However, it is not always clear whether a confluent monolayer was achieved with this cell count. On the other hand, calculated from the size of an endothelial cell, approx. 5 X lo5 cells/cm2 vessel wall are found in a normal lining. The seeding density used in our experiments approached this number of cells very closely. One should also take into account that a certain number of cells in an inoculum are dead or less vital. Less vital cells need more time than others in the competition of settling at the surface of the prosthesis after seeding, To achieve a confluent monolayer in a seeded prosthesis, a sufficient number of cells has to be provided. Previous experiments in our laboratory have shown that 4.8 X lo5 subcultivated cells were adequate to form a monolayer of 1 cm’ in approx. 1 h. Based on this experience, we used this number of cells per cm length of prosthesis with an inner diameter of 3 mm, equal to approx. 1 cm2 inner surface and the 3 X 30 + 60 min seeding and incubation times sequence. For successful seeding, the physical characteristics of the prostheses have to be taken into account and, if necessary, it should be provided with a surface coating. Coating with cell attachment proteins, such as fibronectin, improved results considerably” ‘-‘* lo. The uncoated polyurethane prostheses used in this study retained more cells after seeding than uncoated ePTFE prostheses, indicating a more hydrophilic nature of the pol~~thane. l3ecause of the impe~eable nature of the ePTFE prostheses, it was impossible to coat this type of prosthesis without altering the microarchitecture of the inner surface. Since we observed that seeding 4.8 X 10’ cells per cm2 polyurethane prosthesis impregnated with gelatin and coated with fibronectin [group 4), as well as ePTFE prostheses coated with fibronectin (group 7), resulted in an almost confluent monolayer, in vitro perfusion may be regarded as a vital step towards practical use of this seeding procedure. Although the perfusion time was relatively short (1h), no significant cell loss could be detected in the polyurethane prostheses, while perfusion conditions approached physiological values and forces. This indicates that the seeded cells were very firmly anchored on the prosthetic surface and able to withstand considerable shear forces. The perfusion Biomaterials

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medium did not contain red blood cells. These observations present a strong contrast to the results of the perfused ePTFE prostheses, in which no cells remained adherent to the inner surface after perfusion. This indicates that, although high numbers of cells will adhere after fibronectin coating, the anchoring of the cells is insufficient. Cell loss of seeded cells in ePTFE and Dacron@ prostheses coated with fibronectin has also been observed by others32-34. The perfusion medium used in this study was cell-free and may have enhanced the retention of seeded cells because of the lesser shear forces exerted on the prosthetic inner surface compared to those present in a cell-containing medium such as blood3’. The firm anchoring of the seeded cells in group 5, paralleled observations of the permanent presence and stability of a cellular lining developed in this kind of prosthesis post-implantation in rats and dogsz3* 24,36, In conclusion, we have found that fibrous polyurethane prostheses impregnated with gelatin and coated with fibronectin are a suitable substrate for cell seeding. The three-staged 30 min seeding and 120” rotation procedure provided ample time for the cells to adhere to the prosthetic surface and spread evenly in the following incubation period of 1 h. The seeding density of 4.8 X 105/cmz prosthesis established a confluent monolayer which remained stable under perfusion conditions. In viva experiments using this type of prosthesis, coating and seeding procedure are strongly indicated.

REFERENCES Herring, M., Dilley, R., Cullison, T., Gardner, A. and Glover, J., Seeding endothelium on canine arterial prostheses, 1. Snrg. Res. 1960, 28, 35-38 Schmidt, S., Hunter, T., Hirko, M., Belden, T., Evancho, M. and Sharp, W., Small diameter vascular prostheses: two designs of PTFE and endothelial cell-seeded and non-seeded Dacron@, J. Vase. Surg. 1985, 2, 293497 Seeger, J. and Klingman, N., Improved endothelial cell seeding with cultured cells and fibronectin-coated grafts, J, Surg. Res. 1985, 38, 641-647 Boyd, K., Schmidt, S., Pippert, T., Hite, S. and Sharp, W., The effects of pore size and endothelial cell seeding upon the performance of small-diameter ePTFE vascular grafts under controlled flow conditions, J. Biomed. Mater. Res. 1968, 22, 163-177 Budd, J., Allen, K., Bell, P. and James, R., The effect of va~ing fibronectin concent~tion on the attachment of endothelial cells to polytetrafluomethylene vascular grafts, J. Vast. Surg. 1990,12,126-130 Kaehler, J., Zilla, P,, Fasol, R., Deutsch, M. and Kadletz, M., Precoating substrate and surface configuration determine adherence and spreading of seeded endothelial cells on polytetrafluoroethylene grafts, J. Vast. Surg. 1989,9,535-541 Vohra, R., Thomson, G., Carr, H., Sharma, H. and Walker, M., Comparison of different vascular prostheses and matrices in relation to endothelial seeding, Br. J. Surg. 1991,78,417-420 Seeger, J. and Klingman, N., Improved in vivo endothelialization of prosthetic grafts by surface modification with fibronectin, J. Vase. Surg. 1988,8,476-482 Dalsing, M., Kevorkian, M., Raper, B., Nixon, C., Laika, S., Cikrit, D,, Unthank, J. and Herring, M., An experi-

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mental collagen-impregnated Dacron@ graft: potential for endothelial seeding, Ann. Vast. Surg. 1989,3,127-133 Patterson, R., Keller, J., Silberstein, E. and Kempczinski, R., A comparison between fibronectin and matrigel pretreated ePTFE vascular grafts, Ann. Vast, Surg. 1989, 3(2),180-166 Fasol, R., Zilla, P., Deutsch, M., Grimm, M. and Fischlein, T., Human endothelial cell seeding: evaluation of its effectiveness by platelet parameters after one year, J. Vast. Surg. 1989, 9, 432-436 Herring, M., Compton, R., LeGrand, D., Gardner, A., Madison, D. and Glover, J., Endothelial seeding of pol~etrafluo~ethylene popliteal bypasses, J. Vast. Surg. 1987,6,114-118 Park, P., Jarrell, B., Williams, S., Carter, T., Rose, D., Martinez-Hernandez, A. and Carabusi, R., Thrombus free human endothelial surface in the midregion of a Dacron@ vascular graft in the splanchnic venous circuit: observations after nine months implantation, J. Vast. Surg. 1990, l&488-477 brtenwall, P., Wadenvik, H. and Risberg, B., Reduced platelet deposition on seeded versus unseeded segments of expanded polytetrafluoroethylene grafts: clinical observations after a 6-months follow-up, J. Vast. Surg. 1989,10,374-380 Rupnick, M., Hubbard, A., Pratt, K., Jarrell, B. and Williams, S,, Endothelialization of vascular prosthetic surfaces after seeding or sodding with human microvascular endothelial cells, J. Vast. Surg. 1969, 9, 788795 Stanley, J., Burkel, W., Ford, J*, Vinter, Ft., Kahn, R., Whitehouse, W. and Graham, L., Enhanced patency of small diameter, externally supported dacron iliofemoral grafts seeded with endothelial cells, Surgery 1982,92, 994-1001 Herring, M. and LeGrand, D., The histology of seeded PTFE grafts in humans, Ann. Vast. Surg. 1989, 3(2), 96-103 Sauvage, L., Berger, K., Wood, S., Yates, S., Smith, J, and Mansfield, P., Interspecies healing of porous arterial prostheses, Arch. Surg. 1974,109,698-705 Berger, K., Sauvage, L., Rao, A. and Woods, S., Healing of arterial prostheses in man, Ann. Surg. 1972, 176, 18-27 Ratto, G., Lunghi, C., Spinelli, E., Agati, R., Tomellini, M. and Motta, G., Scanning electron microscopy evaluation of porous and nonporous arterial substitutes, Surg. GynecoI. Obstet. 1962,155,358-362 Hertzer, N., Regeneration of endothelium in knitted and velour dacron vascular grafts in dogs, J. Cardiovasc. Surg. 1981,22,223-230 Hess, F., Braun, B., Jerusalem, C., van Det, R., Steeghs, S. and Skotnicki, S., Endothelialization of polyurethane vascular prostheses implanted in the dog carotid and femoral artery, J. Cardiovasc. Surg. 1988,4, 458-463 Hess, F., Steeghs, S., Braun, B., van Det, R., Grande, P., Jerusalem, C. and Skotnicki, S., Improved patency rate of small caliber polyurethane vascular prostheses implanted in the dog carotid and femoral artery by use of acetyl-

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salicylic acid and dipyridamol, J. Thorac. Cardiovasc. Surg. 1988,36,221-226 Hess, F., Jerusalem, C., Steeghs, S., Reijnders, O., Braun, B. and Grande, P., Development and long-term fate of a cellular lining in fibrous polyurethane vascular prosthesis implanted in the dog carotid and femoral artery, J. Cardiovasc. Surg., 1992, 33,358-365 Graham, L., Burkel, W., Ford, J., Vinter, D., Kahn, R. and Stanely, J., Immediate seeding of enzymatically derived endothelium on dacron vascular grafts: early experimental studies with autogenous canine cells, Arch. Surg. 1980,115,1289-1294 Gerlach, J., Schauwecker, H., Hennig, E. and Biicherl, E., Endothelial celt seeding on different polyurethanes, Artif., Organs 1989, 13,144-147 Gerlach, J., Schauwecker, H. and Planck, H., Polyurethanes and their cytocompatibility for cell seeding, in Medical Textiles for Implantation (Eds H. Planck, M. Dauner and M. Renardy), Springer Verlag, Berlin, 1990, pp 187-191 Braun, B., Grande, P., Lehnhardt, F., Jerusalem, C. and Hess, F., Herstellung und tie~xperimentelle Untersuchung einer kleinlumigen mikroporijsen Polyurethan Gefassprothese, Vasa Suppl. 1988, 22 Pearce, W., Rutherford, R. and Whitehill, T., Successful endothelial seeding with omentally derived microvascular endothelial cells, J. Vast. Surg. 1987, 5, 203-206 Jarrell, B., Williams, S., Carabasi, R. and Hubbard, F., Immediate vascular graft monolayers using microvessel endothelial cells, in ~ndotheliai Seeding in Vascular Surgery (Eds M. Herring and J. Glover), Grune & Stratton, Orlando, USA, 1987,pp 37-55 Rosenman, J., Kempczinski, R., Pearce, W. and Silberstein, E., Kinetics of endothelial cell seeding, J Vast. Surg. 1985,3,776-784 Vohra, R., Thomson, G., Sharma, H., Carr, H. and Walker, M., Effects of shear stress on endothelial cell monolayer5 on expanded polytetrafluoroethylene fePTFE) grafts using precfot and fibronectin matrices, Eur. J. Vase. Surg. 1990, 4, 33-41 Greisler, H., Endean, E., Klosak, J., Ellinger, J., Henderson, S., Pham, S., Durham, S., Showalter, D., Levine, J. and Borovetz, H., Hemodynamic effects on endothelial cell monolayer detachment from vascular prostheses, Arch. Surg. 1989, 124, 429-433 Greisler, H., Johnson, S., Joyce, K., Henderson, S., Patel, N., Alkhamis, T., Beissinger, R. and Unkim, D., The effects of shear stress on endothelial cell retention and function on expanded polytetrafluoroethylene, Arch. Surg. 1990, 126,1623-1625 Gourevitch, D., Jones, L., Cracker, J. and Goldman, M., Endothelial cell adhesion to vascular prosthetic surfaces, ~iomaterials 1988, 9, 97-100 Hess, F., Jerusalem, C. and Braun, B., The endothelialization process of a fibrous polyurethane microvascular prosthesis of the implantation in the abdominal aorta of the rat. A scanning electron microscopic study, J. Cardiovasc. Surg. 1963, 24, 516-524

Biomaterials

1992. Vol. 13 No. 10

Seeding of enzymatically derived and subcultivated canine endothelial cells on fibrous polyurethane vascular prostheses.

Fibrous polyurethane (FPU) prostheses with or without fibronectin coating and gelatin impregnation and FPU prostheses with or without fibronectin coat...
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