Comparison of immediate seeding of endothelial cells with culture lining of small diameter ePTFE carotid interposition grafts

This study is the first to compare chronic healing characteristics of immediately seeded grafts with those of grafts lined by autogenous venous endothelial cells in tissue culture prior to implantation. Ten mongrel dogs had a segment of external jugular vein excised for enzymatic harvest of endothelial cells. After approximately 21 days growth in tissue culture, 4 X 10(6) cells/ml were inoculated into a 6-cm length of 4 mm i.d. ePTFE for formation of a confluent lining in culture media. The remaining external jugular vein had its endothelial cells enzymatically harvested for immediate seeding of an identical length of preclotted ePTFE. Both grafts were implanted end-to-end in the carotid position and excised after 30 days. In 6 of the 10 dogs, grafts were patent bilaterally; all others were occluded. Planimetric measurements on patent grafts with immediate seeding showed a thrombus-free surface area of 56 +/- 39% compared to 86 +/- 15% for culture-lined grafts (P = 0.046). Endothelial coverage was 70 +/- 24% for immediately seeded grafts and 29 +/- 21% for culture-lined grafts (P = 0.016). We conclude that immediate seeding and culture lining of autogenous endothelial cells in small diameter ePTFE grafts produce equivalent short-term patency. While culture-lined grafts have an initially less thrombogenic luminal surface, subsequent development of a confluent endothelial lining is slower than that with an immediate seeding preparation, and thus would appear to offer no significant clinical benefit, especially in light of the complexity culture lining adds to the procedure.     

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)     

Viability of engineered vessels as arterial substitutes.

The search for vessel substitutes to replace small-/medium-caliber vessels is an ongoing concern for vascular surgeons. Engineered vessels were designed for use as arterial equivalents and assessed in an in vivo model in dog. Three study groups were established: clinical expanded polytetrafluoroethylene (ePTFE; control, n = 24), ePTFE seeding with endothelial cells (EC graft, n = 12), and ePTFE with a fibroblast matrix seeded with EC (FM+EC graft, n = 12). Grafts were subjected to a custom-designed femoral ex vivo circuit and implanted in the carotid artery for 60 days. The viability of the prosthetic grafts was evaluated. The ex vivo circuit revealed that the presence of a fibroblast matrix induced over double the cell retention compared to EC grafts. A significant reduction in platelet adhesion in EC grafts was observed. After their in vivo implantation, the engineered vessels were more efficient at avoiding occlusion than the prosthetic grafts. The FM+EC grafts induced more endothelialization than those seeded with ECs alone. Intimal hyperplasia response was reduced in the EC substitutes. Significant differences in apoptotic cells emerged between the EC and control ePTFE grafts. In conclusion, engineered vessels showed improved initial patency over ePTFE grafts. The EC graft was best at combating restenosis, a good indicator of the long-term efficiency of the graft.     

Synthetic vascular grafts seeded with genetically modified endothelium in the dog: Evaluation of the effect of seeding technique and retroviral vector on cell persistence in vivo

Unique characteristics of endothelium make it an attractive target cell for gene transfer. Genetically modified endothelial cells (ECs) seeded on synthetic vascular grafts offer the potential to control neointimal hyperplasia, decrease graft thrombogenicity and improve small diameter graft patency. This study addresses the issue of synthetic vascular graft colonization with endothelial cells transduced with noninducible retroviral marker genes in the dog. Autologous endothelial cells were enzymatically harvested and transduced with either the bacterial Neo^R gene or human growth hormone gene using retroviral vectors. All transduced cells were positive by polymerase chain reaction (PCR) amplification for the transduced gene sequence prior to graft seeding. Transduced ECs were seeded on Dacron grafts (n = 3) preclotted with autologous blood. These grafts exhibited complete endothelialization at times from 250 to 360 days. Recovered DNA, however, was negative for the transduced gene sequence when analyzed by PCR and Southern blotting. Expanded polytetrafluoroethylene (ePTFE) was evaluated (n = 8) using several different cell seeding protocols. Grafts were seeded at 3 densities (ranging from 6 × 10³ to 1.5 × 10⁵ cells/cm²) and 2 different adherence times. Seeding substrate was also evaluated. Grafts were either preclotted with whole blood or incubated with 20 or 120 μg/ml fibronectin for 60 min. Graft biopsies were evaluated from 2 to 52 wk. Limited endothelialization was present in 4 dogs as early as 2 wk, but never progressed to full luminal coverage. The remaining dogs failed to ever exhibit any luminal EC adherence. Two dogs with limited EC coverage had positive DNA by PCR for the Neo^R gene sequence at 2 and 3 wk. In contrast to transduced EC's, nontransduced EC colonization of ePTFE was complete at 2 wk when seeded under conditions that transduced cells had failed to persist. Neither seeding density, adherence time, seeding substrate or retroviral vector used influenced the uniformly poor graft coverage seen with transduced cells. Results of this study indicate that despite successful gene transfer using 4 different retroviral vectors, transduced endothelial cells seeded under varying conditions appear altered in their ability to stably adhere and colonize synthetic vascular grafts in vivo.     

The effect of preformed confluent endothelial cell monolayers on the patency and thrombogenicity of small calibre vascular grafts

Endothelial cell seeding has been proposed as a method to improve the patency rates in small calibre prosthetic vascular grafts. The seeding methods used at present leave much of the graft luminal surface devoid of endothelial cells and thus still significantly thrombogenic. We have developed a method to preform confluent endothelial cell monolayers, on the grafts prior to implantation, and this study investigates the effect of these monolayers on the early thrombogenicity and patency of polytetrafluoroethylene (PTFE) grafts. Small diameter PTFE grafts were seeded with canine endothelial cells obtained from the external jugular vein. Each of five dogs then received a graft seeded with its own cells and a contralateral, non-seeded control graft. At 1 and 10 weeks after graft implantation graft thrombogenicity was assessed by the use of Indium labelled platelets. The thrombogenicity index (TI) of each graft was determined from counts of gamma activity recorded over a period of 7 days. Grafts were subsequently removed at 12 weeks. At 1 week the mean TI for the seeded grafts was 0.123 (SD 0.019) and that for the controls 0.183 (SD 0.017) (p = 0.005). At 10 weeks only the seeded grafts could be assessed because all of the control grafts had occluded. At this point in time the seeded grafts had a mean TI of 0.159 (SD 0.011) (p = 0.047 vs. seeded at 1 week). By the time of removal at 12 weeks, all control grafts were occluded but only one of the seeded grafts had occluded (p = 0.025). In conclusion, the use of preformed, confluent endothelial cell monolayers for seeding prosthetic grafts significantly reduces the early graft thrombogenicity and improves graft patency. It does not, however, completely halt the increase in thrombogenicity which occurs during the early post-implantation period.     

Endothelial seeding of polytetrafluoroethylene femoral popliteal bypasses: The failure of low-density seeding to improve patency

Purpose: We compared 66 seeded polytetrafluoroethylene and 53 autologous vein grafts to determine whether endothelial seeding could improve the patency of polytetrafluoroethylene femoral popliteal bypass grafts and to determine whether endothelial seeding could be performed consistently in multiple institutions. Methods: Nine surgeons at four hospitals randomized patients to receive either a seeded polytetrafluoroethylene or a vein graft, but if no satisfactory vein (n = 26) existed, an "obligatory" seeded polytetrafluoroethylene graft was used. Results: Scanning electron microscopy confirmed satisfactory initial attachment of endothelium on the discarded ends of the grafts. Patency was compared with the use of log rank analysis and revealed better patency in vein grafts at 30 months (vein = 91.6% ± 4.1%; seeded polytetrafluoroethylene = 37.8% ± 9.4%; p = 0.006). Failed grafts revealed anastomotic hyperplasia. Conclusions: (1) Vein graft patency was better than seeded polytetrafluoroethylene grafts; (2) seeding did not improve patency in below-the-knee bypasses as suggested by pilot studies; (3) the failure of seeded grafts was associated with anastomotic hyperplasia but not with the failure of initial endothelial attachment; and (4) each institution reported similar results. (J VASC SURG 1994;20:650-5.)     

Improved in vivo endothelialization of prosthetic grafts by surface modification with fibronectin

Endothelial cell growth in vitro is enhanced by coating with fibronectin the surface on which cells grow. Similar coating of prosthetic arterial grafts may promote in vivo graft endothelialization if graft patency is not adversely affected. In each of 15 dogs, two fibronectin-coated polytetrafluoroethylene grafts and two grafts that were not coated were implanted. One graft in each pair was seeded with autologous endothelial cells, so that four different grafts were studied in each animal: a coated, seeded graft; a coated graft that was not seeded; a seeded graft that was not coated; a graft that was neither coated nor seeded. At 2, 4, and 8 weeks, grafts from five animals were examined for patency, surface endothelialization, and indium 111 platelet reactivity. After seeding, surface coverage by endothelium of coated grafts was more complete and more rapid than in uncoated grafts (64% ± 23% vs 31% ± 13% at 4 weeks, p < 0.05). Without seeding, coated grafts also appeared to have increased endothelial cell ingrowth compared with plain grafts (48.8% ± 15.1% vs 37.6% ± 1.5% at 8 weeks). Early (2-week) platelet reactivity of coated grafts was increased (p = 0.06), but patency was not adversely affected. Thus fibronectin coating of prosthetic grafts promotes surface endothelialization in vivo without altering graft patency. (J VASC SURG 1988;8:476-82.)     

Enhanced patency of small-diameter, externally supported Dacron iliofemoral grafts seeded with endothelial cells

Fourteen adult foxhounds underwent bilateral iliofemoral bypasses with externally supported, knitted Dacron grafts measured 4 mm in internal diameter and 10 cm in length. These conduits were preclotted with 10 ml of blood mixed with 0.5 ml of culture medium. Autologous endothelial cells, enzymatically derived from external jugular veins, were added to blood and medium used to preclot one graft in each dog. The other, unseeded graft served as a control. Grafts were anastomosed, end to end, to the iliac and femoral arteries. All dogs received dipyridamole, 50 mg twice a day for 4 days preoperatively, and aspirin, 5 grains four times a day for 1 day preoperatively. Both drugs were continued 14 days after operation. Grafts were removed from three dogs at 2 and 4 weeks and from four dogs at 8 and 16 weeks. All grafts were patent at 2 weeks during drug administration. Cumulative patency rates beyond 2 weeks were 73% in 11 seeded grafts and 27% in 11 control grafts, a statistically significant difference (P = 0.03). Seeded grafts were completely surfaced with a monolayer of endothelium between 2 and 4 weeks. Small-graft patency appeared related to evolution of endothelial surfaces, the development of which was clearly facilitated by seeding with autologous endothelium.     

Delayed exposure to pulsatile shear stress improves retention of human saphenous vein endothelial cells on seeded ePTFE grafts

Since significant loss of endothelial cells (ECs) from the surface of a seeded prosthetic graft occurs after implantation, improved cell retention following exposure to flow should increase the likelihood of long-term success with this technology. An in vitro pulsatile flow circuit was developed to study the effects of two variables on cell retention: cell density at the time of seeding and postseeding incubation time. Fibronectin-coated ePTFE grafts (4 mm x 5 cm) were seeded with human saphenous vein ECs at two densities, confluent (1 x 10(5) cells/cm2) or subconfluent (2 x 10(4)), and incubated in vitro for varying time intervals (90 min, 1, 3, or 7 days). Test grafts were exposed to 90 min of pulsatile flow in an in vitro flow circuit, then fixed, and stained, and in situ cell counts (cells/cm2) were determined for nine representative fields per graft. Paired control grafts were treated identically but were not exposed to flow. Cell retention was calculated using the formula: % retention = cells/cm2 perfused graft divided by cells/cm2 control graft. Grafts exposed to flow 90 min after seeding demonstrated significantly lower cell retention when compared to later time points. When cells were seeded at confluent density, maximal retention (92 +/- 3%) occurred 24 hr after seeding. Prolonged culture of cells seeded on ePTFE grafts at confluent density resulted in increased cell loss. In contrast, on grafts seeded at subconfluent density, retention improved as cells grew to confluence (16 +/- 4.5% initially to 82 +/- 7% at 7 days).(ABSTRACT TRUNCATED AT 250 WORDS)     

内皮化小口径人工血管的研究

目的 探讨成人大隐静脉内皮细胞(HSVECs)种植到人工血管内表面的可行性.方法 酶消化法获取成人大隐静脉内皮细胞,在体外扩增培养13～15 d,将扩增培养的内皮细胞种植于纤维蛋白胶预衬的聚四氟乙烯(ePTFE)人工血管内表面,继续体外培养9～12 d.在不同的时间点,分别剪取部分内皮化的人工血管,行荧光显微镜和扫描电镜检查.结果 内皮细胞种植于人工血管后,扫描电镜下可见细胞在血管表面黏附、生长、增殖.平均孵育12 d后,人工血管腔面见一层均匀的基质,其表面有内皮细胞单层,内皮细胞排列紧密,呈梭形.结论 成人大隐静脉内皮细胞可以种植到人工血管,在体外增殖形成内皮细胞单层,达到内皮化的效果.     

In vivo patency of endothelial cell-lined expanded polytetrafluoroethylene prostheses in an ovine model

The performance of small-diameter vascular prostheses may be improved by implantation of grafts lined with endothelial cells. Expanded polytetrafluoroethylene (ePTFE) prostheses (4 mm x 40 mm) were coated with fibronectin (20 micrograms/ml), seeded with endothelial cells, and cultured for 48 h to produce a confluent, autologous endothelial cell lining. They were implanted as carotid interposition grafts in sheep. Seeded ePTFE grafts were compared with nonseeded ePTFE grafts and autologous carotid artery grafts. No anticoagulant or antiplatelet therapy was administered, making this a stringent test model for the thromboresistance of a small-diameter prosthesis. After 13 weeks the patencies of seeded, nonseeded, and autologous artery grafts were 16% (1/6), 0% (0/6), and 100% (6/6), respectively. The one seeded graft that was patent was fully lined with endothelial cells and showed no stenosis. The remaining five seeded grafts were occluded by fibrous tissue and displayed substantial spindle cell hyperplasia. There was no apparent difference between the autologous artery grafts and normal arterial tissue, and the anastomoses showed no stenosis. The ovine model provides a conservative test of prosthesis survival and may be useful for study of graft failure.     

Endothelial seeding of polytetrafluoroethylene popliteal bypasses. A preliminary report

At St. Vincent Hospital, Indianapolis, 17 patients have undergone femoropopliteal bypass operations with polytetrafluoroethylene (PTFE) grafts that were seeded with enzymatically harvested, autogenous endothelium. Three patients received seeded grafts because satisfactory veins were not available. Twenty-eight patients alternately received seeded or unseeded, externally supported e-PTFE grafts. Graft patency was evaluated by clinical criteria and changes in the Doppler ankle-brachial systolic pressure ratios at 2 and 30 days postoperatively and at 3-month intervals thereafter. Occlusions were defined arteriographically if the clinical situation or the Doppler findings deteriorated. Smoking histories were taken, and carboxyhemoglobin (COHgb) levels were sampled 1 month postoperatively. Cumulative patency after 3 months was 93.3% +/- 6.5% for seeded and 84.0% +/- 10.4% for unseeded grafts. After 1 year it was 81.6% +/- 12.3% for seeded grafts and 30.8% +/- 18.7% for unseeded grafts (p = 0.02). Thus far all but one of the occlusions have occurred in patients with a history of smoking or with a COHgb level greater than 1.5%, whereas all of the seeded grafts in nonsmokers with COHgb levels less than or equal to 1.5% are patent. We conclude that endothelial seeding of PTFE femoropopliteal grafts is feasible. During this preliminary study period, a small number of patients had favorable patency rates with seeded as compared with unseeded grafts, especially among smokers. A multiple-institution study will be needed to establish the role of endothelial cell seeding in the treatment of vascular occlusions of the femoral and popliteal arteries.     

Host response to autologous endothelial seeding

Dacron and expanded polytetrafluoroethylene grafts, seeded with autologous venous endothelial cells at the time of implantation, subsequently develop endothelial linings. However, it has not been shown whether the endothelial cells in these linings are derived from the seeded cells, or whether the seeding process itself stimulates host endothelial cells to proliferate and cover the grafts. As a first step in testing this hypothesis, bilateral end-to-side aortoiliac expanded polytetrafluoroethylene grafts with an internal diameter of 6 mm, an internodal distance of 22 microns, and an average length of 9.2 cm were placed in 10 adult mongrel dogs weighing 20 to 25 kg; the aorta was ligated just distal to the origin of the grafts. The graft on one side, chosen at random, was seeded with autologous endothelium that was harvested by enzyme single-stage technique from external jugular veins; the other side was not seeded. After 4 weeks the animals were anesthetized and heparinized, and the grafts were fixed by perfusion in vivo with 2.5% glutaraldehyde solution before they were removed. Both grafts occluded in two animals, and both grafts were patent in five animals. In two animals the seeded grafts were open and the unseeded grafts were occluded. In one the seeded graft was occluded and the unseeded graft was patent. There was no significant difference in clot-free surface area between seeded (29% +/- 18%) and unseeded (31% +/- 11%) grafts. Scanning electron microscopy showed the presence of an endothelial monolayer that averaged 39% +/- 20% and 36% +/- 26% coverage, respectively, in the clot-free midgraft portions of all seeded and unseeded patent grafts.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Xenograft seeding of Dacron grafts in dogs

The benefit of autologous endothelial cell seeding in dogs has been widely accepted. This experiment seeks to determine if a similar effect accompanies the use of xenograft (porcine) endothelial cells in dogs. Thirty-two mongrel dogs underwent thoracoabdominal aortic bypass with 25- to 30-cm segments of double velour Dacron 8-mm grafts. Endothelial cell seeding was performed with whole blood suspensions of either autologous endothelial cells (Group I) or heterologous endothelial cells from pigs (Group II). Cell-free culture medium was added to whole blood for preclotting in unseeded control animals (Group III). Ten animals in each group were sacrificed at 30 days and all grafts were patent. Thrombus-free surface areas were Group I = 61.7 (+/- 6.4%, SEM); Group II = 53.2 (+/- 6.8%, SEM); and Group III 42.2 (+/- 8.0%, SEM). There was a significant difference between autologous-seeded and unseeded grafts (P less than 0.04). Endothelialization was confirmed by scanning and transmission electron microscopy in all groups but was better in both seeded groups: Group I-9/10 grafts, Group II-8/10 grafts, and Group III-4/10 grafts. Factor VIII immunofluorescent staining confirmed the presence of endothelium on selected grafts in each group. These results raise questions concerning the proposed mechanism of endothelial seeding since xenograft cells seem to facilitate healing in the canine model. If heterograft cells can be effective, it may not be necessary to harvest cells from the recipient in order to achieve the benefits of seeding.     

Aspirin improves the patency rate of seeded vena cava grafts

The purpose of this study was to evaluate the effectiveness of aspirin (ASA) and porcine endothelial cell seeding in improving the patency rate of vena cava grafts. Thirty-nine dogs underwent infrarenal vena cava replacement by 10 cm lengths of 8 mm I.D. ringed polytetrafluoroethylene grafts. Thirty-one grafts were seeded with 1-1.5 x 10(6) porcine aortic endothelial cells while eight were not (GIII). Of the seeded group, 16 animals received no ASA (GI), while 15 others (GII) were given ASA (325 mg) daily starting two days preoperatively and continuing until sacrifice. Venograms were performed on the fourth postoperative day. Grafts were harvested 32 days after insertion and evaluated for patency rate and endothelialized surfaces. The 32-day patency rate was significantly higher for GII than for GI and III animals (67% vs. 13 and 25% respectively). Endothelialized surface was higher in GII than Gi and III (67% vs. 16% and 18% respectively). We conclude that endothelial cell seeding alone does not prevent graft closure and that a combination of ASA and cell seeding significantly increases the patency rate of vena cava grafts.     

Smooth muscle cell seeding in biodegradable grafts in rats: a new method to enhance the process of arterial wall regeneration

In this study a smooth muscle cell seeding technique was developed and evaluated in biodegradable vascular grafts in rats. Cultured smooth muscle cells (3.5 to 5 X 10(6) in 0.1 ml whole blood) were seeded by means of a one-step preclotting technique in microporous, compliant, biodegradable vascular grafts (internal diameter 1.3 mm, length 1 cm; n = 16). Four seeded grafts as well as four nonseeded preclotted control grafts were histologically evaluated immediately after seeding and on preclotting; the other seeded grafts (n = 12) as well as nonseeded preclotted control grafts (n = 12) were implanted into the abdominal aorta of rats and were evaluated after 2 hours (n = 4), 2 days (n = 4), and 1 week (n = 4) with regard to the process of arterial wall regeneration. All grafts, except one seeded graft harvested at 1 week, were patent. All nonseeded control grafts showed no (at 2 hours and at 2 days) or limited (at 1 week) smooth muscle cell ingrowth. Of the 11 patent seeded grafts three also showed poor or limited neomedia development, probably because of improper preclotting. However, eight of the 11 patent seeded grafts showed fast and uniform noemedia development (clearly discernible at 2 days). Moreover, in two patent 1-week seeded grafts there were neomedia comparable in thickness and architecture (circularly arranged smooth muscle cells) to that of normal rat media, and a neointima had already lined 30% to 40% of the lumen. These results clearly demonstrate that smooth muscle cell seeding in biodegradable grafts is feasible and indicate that this seeding can enhance the process of arterial wall regeneration in these grafts.     

Acutely sodded expanded polytetrafluoroethylene grafts produce only prostacyclin: a qualitative difference from saphenous veins

Select subsets of patients require prosthetic graft material for revascularization. Although arterial prosthetic grafts of large caliber perform acceptably, grafts of <6 mm exhibit a high attrition rate. Microvessel endothelial sodding, a method resulting in the lining of prosthetic grafts with autologous endothelium, improves graft patency; however, aggressive antiplatelet therapy is still required, because terminating an antiplatelet regimen accelerates graft attrition. The present investigation was designed to address the acute production of vasoactive substances in microvessel endothelial cell sodded expanded polytetrafluoroethylene (ePTFE) grafts in an attempt to delineate a possible mechanism behind the continued requirement for antiplatelet therapy. Equal lengths of acutely sodded ePTFE grafts (canine falciform ligament source) and saphenous veins (SV) (canine source) were evaluated by superfusion bioassay. Basal secretion from ePTFE grafts relaxed the biodetector ring 1 +/- 3%, whereas SV relaxed the ring 10 +/- 3% (p < 0.05, ePTFE vs. SV). Relaxation with acetylcholine stimulation was 49 +/- 7% in grafts and 50 +/- 10% in veins (p = NS). Calcium ionophore stimulation produced relaxation of 37 +/- 9% from ePTFE grafts and 100 +/- 23% from SV (p < 0.05). Indomethacin added to perfusate reduced relaxations from sodded ePTFE grafts to 20.2 +/- 9.2% with acetylcholine stimulation and 12.5 +/- 4.3% with calcium ionophore (p < 0.05 vs. control); addition of N(G)-monomethyl-L-arginine (L-NMMA) had no effect on the release of vasoactive substances from ePTFE grafts. In contrast, relaxations of effluent from SV stimulated by acetylcholine and calcium ionophore were significantly attenuated with indomethacin and L-NMMA (p < 0.05 vs. control). Scanning electron microscopy demonstrated confluent endothelium in SV and a nonconfluent endothelial cell layer in grafts. Acutely sodded ePTFE grafts produce vasoactive substances that quantitatively and qualitatively differ from those produced by canine SV. The ePTFE grafts produce mainly prostanoids, whereas SV produce both nitric oxide and prostanoids. The endothelial cell isolation procedure and absence of immediate graft luminal confluence may contribute to the observed differences.     

Rapid cellular luminal coverage of Dacron inferior vena cava prostheses in dogs by immediate seeding of autogenous endothelial cells derived from omental tissue: results of a preliminary trial

Endothelial cell seeding methods might reduce the high failure rates of venous vascular prostheses, but low flow rates in venous vascular prostheses impose a need to protect early patency and to attain early endothelial cell coverage without waiting several weeks for relatively small endothelial cell innocula from autologous veins to form confluent linings. To obtain large number of autologous endothelial cells for high-density seeding, canine omental microvascular endothelial cells were harvested by collagenase digestion and density gradient centrifugation, with yields of 1.34 +/- 0.24 (SD) X 10(6) cells/gm of omentum (N = 8 harvests). Primary culture of a subfraction from each harvest showed the cell population to be dominated by factor VIII-related antigen-positive endothelial cells with only a few nonstaining cells (estimated to be 10% or less of total cell number) visible. Freshly harvested omental cells were seeded onto double velour knitted Dacron prostheses at densities of 5 X 10(5) cells/cm2 of graft luminal surface in an autologous plasma suspension by use of prior preclotting with cell-free autologous plasma, followed by endothelial cell seeding in autologous plasma, with plasma recalcification during endothelial cell instillation. Six seeded and two control (sham-seeded) vascular prostheses 5 cm long with 10 mm inner diameter were used as inferior vena cava interposition grafts. A distal arteriovenous fistula and aspirin (300 mg) and dipyridamole (50 mg orally every day) starting 3 days before surgery were used to protect early patency of all grafts. Seeded venous vascular prostheses were explanted for study at intervals of 1,5, and 10 days after surgery (N = 2 prostheses at each time); the two control venous vascular prostheses were explanted at 10 days. All venous vascular prostheses were patent at time of removal. In seeded venous vascular prostheses, light, scanning, and transmission electron microscopy showed emergence of numerous flattened endothelial cell-like cells on the luminal surface 24 hours after surgery, followed by formation of a confluent cellular lining without adherent platelets by 5 to 10 days after surgery. Control venous vascular prostheses, in contrast, remained covered by an irregularly thickened fibrin and red cell thrombus, which sometimes encroached on the lumen. Our results suggest that (1) omental tissue can furnish endothelial cells for high-density immediate seeding of venous vascular prostheses, and (2) that the method we used to combine features of both so-called high density "seeding" and "sodding" techniques offers both more rapid prosthesis coverage than the former and shorter intraoperative times for cell attachment to prostheses than the latter.(ABSTRACT TRUNCATED AT 400 WORDS)     

Seeding of ePTFE carotid interposition grafts in sheep and dogs: species-dependent results

Autologous endothelial seeding of thin-walled ePTFE vascular prostheses (I.D.4 mm), interposed in the carotid artery, was performed in 10 dogs and 14 sheeps. Aspirin (250 mg/day) and dipyridamole (75 mg/twice daily) were given throughout the study as antiplatelet therapy. Animals were killed 2 and 5 weeks after surgery. Patency rates for seeded grafts in dogs were 75% (6:8) and 83% (10:12) at 2 and 5 weeks, respectively. In control grafts the patency rates were identical. Patency rates for seeded grafts in sheep were 0% (0:5) and 11% (1:9) at 2 and 5 weeks, respectively. Control grafts in sheep had a patency rate of 40% (2:5) and 0% (0:9) at corresponding times. Scanning electron microscopy showed an almost complete endothelialization of seeded grafts in dogs after 5 weeks. Platelet deposition was studied in the dogs by means of chromium-51-labeled autologous platelets. Significantly fewer platelets accumulated on seeded grafts, and the ratio of 6-keto-PCF1 alpha to thromboxane B2 was significantly higher, compared with unseeded grafts, which indicated the presence of a functionally active endothelial lining in seeded grafts. Differences in the hemostatic system could account for the high clotting incidence in sheep, compared with that in dogs. Such species differences make extrapolations to the clinical situation from autologous endothelial seeding in experimental animals hazardous.