Thrombus-free Area Research Articles

Mesothelial seeding of knitted Dacron.

Bilateral superficial femoral artery replacement using knitted Dacron was performed in 38 dogs. One side was seeded with omental mesothelium and the other acted as an unseeded control. 111In-labelled platelet accumulation on grafts was measured at 5 days and 2 months and the thrombogenicity index of seeded and unseeded grafts calculated. Patency was monitored for 2 months, at which time grafts were removed and luminal thrombus, ultrastructural cell cover and prostacyclin release were measured. Cell seeding did not influence the mean(s.e.m.) thrombogenicity index of 0.95(0.25) and 0.88(0.24) at 5 days in control and seeded grafts respectively; nor was there any difference between the groups at 2 months. Occlusion occurred in six control and four seeded grafts. Seeding did not significantly improve the percentage thrombus-free area or luminal cell cover. Neither did it enhance mean(s.e.m.) luminal 6-keto-prostaglandin F1 alpha release of 2.58(0.80) pg cm-2 in controls and 2.63(0.78) pg cm-2 in seeded grafts. Further studies demonstrated that only a mean(s.e.m.) of 4.4(1.9) per cent of the seeded inoculum was present on grafts 48 h after implantation, providing too few cells to achieve confluent cover. Mesothelial cell seeding might be useful in promoting a healed graft surface but critical levels of seeding density must be achieved before the technique can be properly evaluated.

Age dependency of neointima formation on vascular prostheses in dogs.

Neointima formed quickly on vascular prostheses implanted in young dogs but not in aged dogs. Previously, we found that impeding neointima formation on vascular prostheses occurred more frequently in aged animals. From these observations, we hypothesized that neointima formation was age-dependent in dogs. To test the hypothesis, 26 fabric Dacron vascular prostheses were analyzed. Half of them were retrieved from aged dogs (more than 13 years old) while the other half were from young ones (less than 1 year old). The grafts were harvested at 8 weeks and 3 months after implantation. The graft surfaces were photographed and analyzed by computer for the ratio of the areas with and without thrombus. Light and scanning electron microscopic observation revealed that most of the thrombus-free areas were lined with endothelial cells. Then the endothelialized areas were calculated. Using data obtained from macroscopic, light microscopic, and scanning electron microscopic observations, the arithmetic means were calculated as the degree of neointima formation. In young animals, the degrees at 8 weeks and at 3 months were 89.1 +/- 8.5% (mean +/- SD) and 95.7 +/- 3.3%, respectively. In old animals, they were 27.9 +/- 5.9% and 31.5 +/- 6. 8%, respectively. From these results, we concluded that neointima formation was age-dependent in dogs.

Experimental replacement of the thoracic inferior vena cava with a high-porosity expanded polytetrafluoroethylene graft wrapped in an omental pedicle flap.

We examined the patency and healing of a high-porosity expanded polytetrafluoroethylene (ePTFE) graft implanted as an interposition graft in the thoracic inferior vena cava (IVC) and wrapped in an omental pedicle flap. High-porosity ePTFE grafts of 60 microns fibril length, with an internal diameter of 10 mm and a length of 4 cm, were implanted in 12 mongrel dogs. In 6 dogs, the grafts were wrapped in omental pedicle flap, and in the remaining 6 the grafts were unwrapped. The animals were killed 4 weeks after the replacement and the grafts were removed for examination. Patency of the graft in both groups was 100%; however, the thrombus-free area in the omentum-wrapped group was significantly larger (P < 0.05) than that in the unwrapped group. Light microscopy revealed the marked infiltration of cells and capillaries within the graft interstices in the omentum-wrapped group. These findings suggest that encapsulation of the high-porosity ePTFE graft is promoted by an omental pedicle flap.

Expanded polytetrafluoroethylene grafts for portal vein replacement: use of omentum wrap to promote graft healing.

This study was designed to reexamine the healing process of expanded polytetrafluoroethylene (EPTFE) grafts with standard porosity (30 microm) and high porosity (60 microm) in portal vein replacement, and to evaluate the effect of an omentum wrap, which has certain functions that promote healing, on graft healing. These grafts, either wrapped by the omentum or not, were placed as portal vein replacements in 24 mongrel dogs. After 1 month, the grafts were retrieved and examined for patency, thrombus-free areas, thickness of the pseudointima, and the total number of cells growing into the graft wall. There were no statistical differences in the patency rates. The high-porosity grafts had a significantly larger thrombus-free area, a thicker pseudointima, and a larger growth of cells than the standard-porosity grafts. The omentum wrap significantly increased the thrombus-free area and stimulated a larger growth of cells in both grafts. The high-porosity grafts plus omentum demonstrated a thrombus-free area of 82.2% vs 27.3% in the standard-porosity grafts. In addition, the migration of fibroblasts and macrophages was most evident in the high-porosity grafts wrapped by the omentum. In conclusion, graft healing enhancement was observed in the high-porosity EPTFE grafts wrapped by the omentum. It is thus suggested that transmural cellular migration plays an important role in the process of graft healing.

Evaluation of the healing of precoated vascular dacron prostheses

Knitted and woven Dacron grafts commercially coated with bovine collagen, gelatin and human albumin were implanted end-to-side between the infrarenal aorta and the bifurcation in 35 growing pigs. Grafts were explanted after 4, 8 and 12 weeks and compared to 6 uncoated knitted prostheses preclotted with blood that served as a control. Uncoated grafts rapidly developed a firmly attached neointima lined with endothelium. Compared with coated grafts the thrombus-free area of uncoated grafts was significantly larger (P less than 0.05). The slow resorption of albumin resulted in a delayed and incomplete neointimal healing and failing graft incorporation. Although the bovine collagen was only minimally cross-linked by formaldehyde, healing of the neointima was compromised in a thin woven graft that demonstrated peeling of the inner capsule even after 12 weeks. The identical collagen as well as bovine gelatin were quickly degraded in knitted grafts and both types showed transprosthetic infiltration at 4 weeks. All knitted grafts coated with either collagen or gelatin, however, were occluded after 8 and 12 weeks. Light microscopy revealed hyperplasia of smooth-muscle cells within the thickened distal anastomotic region. These results demonstrate that a timely return of porosity is mandatory for the development and maintenance of an intact neointima. Both the structure of the fabric as well as the method of preparing the coating are crucial variables to determine the rate of biodegradation.

Comparison of two techniques to isolate microvascular endothelial cells from the omentum

The purpose of the study was to compare two different techniques for isolation of omental microvascular endothelial cells (ECs). Segments of unreinforced polytetrafluoroethylene (PTFE) grafts, 9 cm long and 6 mm in diameter, were implanted in 22 dogs as an aortic interposition. Fourteen grafts were seeded with a mean of 7 × 10 5 viable ECs, derived from the microvessels of the omentum: eight grafts (group A) were seeded with ECs obtained by collagenase digestion and by filtration through a pore mesh; six grafts (group B) were seeded with ECs obtained by collagenase digestion and by Percoll gradient separation. In eight grafts (group C), the ECs were not added to the preclot mixture and served as a control. Animals were sacrificed 5 weeks after surgery. The percentage of thrombus-free area was 65 ± 22% for group A grafts and 74 ± 15% for group B grafts (NS). The subendothelial layer was 280 ± 60 μm thick in group A and 220 ± 30 μm thick in group B ( P < 0.05). Seeded grafts showed a higher production of 6-keto-PGF 1α after addition of sodium arachidonate than control grafts. Percoll gradient separation allows isolation of a more purified suspension of ECs. Refinements in omental EC procurement are still required to minimize contamination with other types of cells.

The use of split-thickness skin as an autogenous arterial conduit.

Eight mongrel dogs were anesthetized using general anesthesia, and graft consisting of split-thickness skin obtained from their abdomen was devised and inserted in each of their infrarenal aortas. Six of the grafts were reinforced with meshed polytetrafluoroethylene (PTFE) as an outer layer to prevent potential aneurysmal dilatation of the skin graft. None of the dogs received aspirin at any time. The dogs were sacrificed at one, four, and six weeks. The grafts were examined with light microscopy and three of them were analyzed with scanning electron microscope. All grafts remained patent and none formed aneurysmal dilatations. None of the dogs developed infections or aortoenteric fistulas. Of the three grafts that showed thrombus-free areas, all were oriented with the epidermis facing the lumen. The three grafts where the epidermis formed the external surface did not degenerate in any area. Endothelium-like cells continuous with the host vessel were present on all grafts at 4 and 6 weeks by light or scanning electron microscopy. Preliminary data from this study indicated that split-thickness skin may be a suitable arterial conduit. Even if it does not prove to have long-term patency rates, it may be a suitable temporary conduit until infections resolve and synthetic prostheses can be reinserted.

Seeding with endothelial cells derived from the microvessels of the omentum and from the jugular vein: A comparative study

Segments of an experimental polytetrafluoroethylene graft (9 cm long, 6 mm I.D.) of high porosity were implanted in 25 dogs as aortic interposition grafts. Nine grafts were seeded with a mean of 7 × 105 viable endothelial cells (ECs) derived from the jugular vein (group A) and eight were seeded with a mean of 7 × 105 viable ECs derived from the microvessels of the omentum (group B). Eight grafts were not seeded and they served as controls (group C). Animals were put to death 5 weeks after graft implantation. The thrombus-free area was measured at 81% ± 10% in group A, 65% ± 22% in group B, and 25% ± 13% in group C (p < 0.05, group A vs group B; p < 0.05, group B vs group C). The thickness of the subendothelial layer was 151 ± 60 μm in group A, 280 ± 60 μm in group B, and 100 ± 75 μm in group C (p < 0.001, group B vs groups A and C). The production of 6-keto-PGF1α in the presence of sodium arachidonate was higher in seeded grafts (p < 0.05). Omentally derived microvessel ECs can be seeded in vascular grafts; refinements in the technique of EC procurement are required to minimize contamination and to obtain ECs with more effective biologic activity.

Bacterial infectibility of chronically implanted endothelial cell-seeded expanded polytetrafluoroethylene vascular grafts.

Vascular prosthetic grafts become more resistant to infection as the interval between implantation and bacteremic challenge increases. Endothelial cell (EC) seeding of such grafts has been shown to improve measurably their ability to resist a bacteremic challenge several weeks after implantation, presumably by reducing the amount of thrombus-free area (TFA) on their luminal surface. However, no investigators have reported the impact of EC seeding on the ability of chronically implanted vascular prostheses to resist a late bacteremic challenge. Bilateral common carotid interposition grafts were placed in 15 adult mongrel dogs with a 4 mm internal diameter, experimental, expanded polytetrafluoroethylene (ePTFE) prosthesis. One animal died shortly after operation and the grafts in two dogs thrombosed, thereby leaving 12 animals with at least one patent graft for subsequent study. At a mean interval of 45 weeks after implantation, five dogs (seven patent grafts) were challenged with an intravenous infusion of 3 X 10(8) radiolabeled Staphylococcus aureus; bacterial adherence and the TFA of the graft's luminal surface were determined for each of the patent grafts. There was no statistically significant difference in bacterial adherence or TFA between EC-seeded and control grafts. At a mean interval of 53 weeks after implantation, the remaining seven dogs (14 patent grafts) received a similar bacterial infusion and the animals were allowed to recover. Five days later, the grafts were harvested and cultured. Once again, there was no significant difference in the infectibility of EC-seeded vs. control grafts.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial infectibility of chronically implanted endothelial cell—seeded expanded polytetrafluoroethylene vascular grafts

Vascular prosthetic grafts become more resistant to infection as the interval between implantation and bacteremic challenge increases. Endothelial cell (EC) seeding of such grafts has been shown to improve measurably their ability to resist a bacteremic challenge several weeks after implantation, presumably by reducing the amount of thrombus-free area (TFA) on their luminal surface. However, no investigators have reported the impact of EC seeding on the ability of chronically implanted vascular prostheses to resist a late bacteremic challenge. Bilateral common carotid interposition grafts were placed in 15 adult mongrel dogs with a 4 mm internal diameter, experimental, expanded polytetrafluoroethylene (ePTFE) prosthesis. One animal died shortly after operation and the grafts in two dogs thrombosed, thereby leaving 12 animals with at least one patent graft for subsequent study. At a mean interval of 45 weeks after implantation, five dogs (seven patent grafts) were challenged with an intravenous infusion of 3 × 108 radiolabeled Staphylococcus aureus; bacterial adherence and the TFA of the graft's luminal surface were determined for each of the patent grafts. There was no statistically significant difference in bacterial adherence or TFA between EC-seeded and control grafts. At a mean interval of 53 weeks after implantation, the remaining seven dogs (14 patent grafts) received a similar bacterial infusion and the animals were allowed to recover. Five days later, the grafts were harvested and cultured. Once again, there was no significant difference in the infectibility of EC-seeded vs. control grafts. Although several investigators have shown that EC seeding appears to increase the ability of prosthetic vascular grafts to resist a bacteremic challenge within the first 4 to 6 weeks after implantation, we documented that if such grafts remain patent beyond 10 to 12 months, a luminal flow surface develops that is equally effective in resisting late graft infection regardless of whether they had been initially seeded with ECs.

Impact of endothelial cell seeding on long-term patency and subendothelial proliferation in a small-caliber highly porous polytetrafluoroethylene graft

Previous reports have demonstrated that endothelial cell seeding of polytetrafluoroethylene (PTFE) grafts enhances short-term patency. This experiment was undertaken to study its impact on the long-term patency of a highly porous, experimental PTFE graft and to determine whether increasing the internodal distance of the graft material resulted in increased proliferation of the subendothelium. Ten centimeter long, 4 mm internal diameter segments of an unreinforced, experimental PTFE graft were implanted into 36 mongrel dogs as carotid interpositions. In each animal, one graft was seeded with autologous endothelial cells, enzymatically derived from the external jugular veins, whereas the contralateral graft was treated in identical fashion except that endothelial cells were not added to the preclot mixture. Nineteen animals were killed at 12 weeks; six at 22 weeks; eight at 26 weeks; and three at 52 weeks. The mean follow-up period was 20.1 weeks. The overall patency rate was 58.3% (21 of 36 grafts) for seeded grafts vs. 27.8% (10 of 36 grafts) for control grafts (p < 0.01). The thrombus-free area was planimetrically measured at 83.4% ± 4.5% in seeded grafts vs. 55.1% ± 9.7% in control grafts (p < 0.05). Scanning electron microscopy confirmed the presence of a confluent cellular monolayer in seeded grafts, whereas control grafts exhibited a variable coagulum of fibrin, platelets, and endothelial cells. The thickness of the subendothelial layer varied from 56 to 95 μm with no progressive increase in thickness between 12 and 52 weeks. The presence of endothelium was confirmed by immunoperoxidase staining for canine factor VIII—related antigen in both seeded and control grafts. Endothelial seeding of the porous, experimental PTFE graft in this model significantly increased thrombus-free, luminal surface area and long-term patency compared with nonseeded control grafts. The increased porosity of our graft was not associated with progressive subendothelial proliferation, as had been feared, and may instead have been partially responsible for the finding of endothelium in the midportion of those long-term, control grafts that remained patent.

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 μm 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.