To study healing and endothelialization of vascular grafts, microporous polytetrafluoroethylene (PTFE) prostheses 2 mm in inner diameter and 5 mm long were implanted into the infrarenal aorta or caval vein of the rat. Patency was assessed in six rats from each group at days 3, 7, 14, 28, and 56 after implantation. Four grafts were occluded, two in the aorta (56 days) and two in the caval vein (3 and 14 days). The prostheses were examined via scanning electron and light microscopy for evaluation of endothelialization. At 3 days, the inner surface of the aortic grafts was covered by a plasma proteinaceous layer and that of the caval vein grafts by a thin mural thrombus. Endothelial cells then migrated from aortakaval vein edges over the graft. At 14 days, the caval vein grafts were completely reendothelialized, and, at 28 days, the mural thrombus in these grafts was replaced by neointima. In contrast, endothelialization of the aortic grafts had advanced only 1 mm at about 56 days, never forming a complete endothelial layer. We conclude that endothelialization of microporous PTFE prostheses is more rapid and complete in the caval vein than in the aorta of the rat. o 1992 wiley-Liss, Inc. MICROSURGERY 13:277-286

1992

ENDOTHELIALIZATION OF MICROPOROUS POLYTETRAFLUOROETHYLENE GRAFTS IN THE INFRARENAL AORTA AND CAVAL VEIN OF THE RAT ZBlGNlEW ZDANOWSKI, Y.D., ELSE RIBBE, Y.D., Ph.D., and STlG BENGMARK, M.D., Ph.D.

Microvascular grafting is clinically well established for procedures such as replantation of amputed digits or hands, free intestine and skin flap transfers, and revascularization of coronary and cerebral vessels.’32Both autogenous vein and arterial grafts have been used with varying patency rates. Since harvesting of such grafts can be time consuming, use of prefabricated grafts would be advantageous. An ideal synthetic graft should be impervious to blood, nonthrombogenic, and durable; have viscoelastic properties similar to those of the host artery; and be easy to handle. In addition, it should resist infection and bi~degradation.~,~ So far, no available graft material meets all these requirements, and graft failure, often due to thrombosis or infection, is still an important complication in vascular surgery. It was reported that endothelial cell (Ec) seeding on the inner surface of synthetic grafts decreased their thrombogenicity and susceptibility to hematogenous i n f e ~ t i o n . ~Thus, ’ ~ the de-

From the Department of Surgery, Lund University, Lund, Sweden. Acknowledgments: We thank Jarl Ekstrand, Department of Medical Microbiology: Erik Hallberg, Department of Zoology; and Ragnar MArtensson, Departrnent of Dermatology, Lund, for providing facilities for the SEM and LM studies. PTFE grafts were kindly provided by W.L. Gore and Assoc., Flagstaff, Arizona. This study was financially supported by a grant from the Medical Faculty, Lund University. Address reprint requests to Dr. Zbigniew Zdanowski. Department of Surgery, Lund University, 5-221 85 Lund. Sweden. Received for publication April 3, 1991; revision accepted March 19, 1992. 0 1992 Wiley-Liss, Inc.

gree and rate of in vivo endothelialization of synthetic grafts give significant information for the development of suitable vascular grafts. Endothelialization of arterially placed polytetrafluoroethlylene (PTFE)prostheses, seems to be slow and incomplete in both humans and animals.7-8 Although healing of synthetic venous grafts is less well documented, complete endothelialization of microporous PTFE grafts was found in the femoral vein of the rat at 40 days and in the canine inferior vena cava at 70 days following implantat i ~ n . ’ ” Others ~ have shown that neointimal formation was limited to the area of anastomosis between PTFE grafts and rat caval vein at 100 days.” In contrast, rapid endothelization of microporous PTFE grafts (2 mm inner diameter, 5 mm length) in the caval vein of the rat was observed in our previous study.’* Though microvascular prostheses have been implanted both into the aorta and into the caval vein with good patency rate^,^'^'-'^ direct comparisons of the endothelialization of prostheses in the venous and arterial systems are lacking. The present aim was to evaluate more systematically the healing of microporous FTFE grafts in the infrarenal aorta and the caval vein of the rat. MATERIALS AND METHODS

Sixty male Sprague-Dawley rats, weighing between 220 and 280 g, were used. FTFE grafts (Gore-Tex; W.L. Gore & Associates) with an inner diameter of 2 mm, 5 mm

Endothelialization of Microporous PTFE Grafts length, and porosity (internodular distance) of 30 pm were implanted into the infrarenal aorta (30 rats) or the infrarenal caval vein (30 rats) of rats under ether anesthesia. Endto-end anastomoses were performed using a continuous suture technique with 9-0 Ethilon (Ethicon) sutures. The mean clamping time of the aorta or the caval vein was 21 min. Implantations were carried out using an operating microscope and sterile instruments. Patency of the prostheses was verified by the visible and palpable presence of blood flow distally in the aorta and proximally in the caval vein. No anticoagulants were administered before or after implantation. Rats were kept under standard laboratory conditions with food (pellets) and water ad libitum. On days 3, 7, 14, 28, and 56 after graft implantation, six rats in each group were reoperated on while under ether anesthesia. The femoral vein was dissected and ligated, and a cannula was inserted proximally. The rat was sacrificed by an overdose of ether, and perfusion was immediately performed, as previously described by Ribbe et al.,14 with 200 ml of a solution containing 0.08 M sodium phosphate buffer, 2.5% glutaraldehyde, and 0.02 g/liter dextran T 70. The femoral artery was cut to let out excess fluid. The mean perfusion time was 20 min. The graft was dissected and excised, and patency was verified visually. The graft was kept in the perfusion solution for 12 hr, washed five times with Milloning's phosphate buffer, and cut in half longitudinally. One-half of the graft was prefixed in 3% glutaraldehyde and 0.1 M cacodylate buffer for 3 hr, postfixed in 1% osmium tetroxide, dehydrated with ethanol in increasing concentration, dried at the critical point, mounted on a specimen holder with silver paste, and sputter coated with goldpaladium. All specimens were examined in an SEM. The second one-half of the graft was fixed in 4% formaldehyde, embedded in paraffin, microtome sectioned to a thickness of 4 pm, and stained with hematoxylin-eosin. These specimens were examined with a light microscope. The Wilcoxon nonparametric, one-tailed, signed-rank test was used for statistical analysis of graft endothelialization.

Figure 1. SEM photographs of luminal surface of microporous PTFE graft after implantation into the aorta in the rat. A: At 3 days, anastomosis without signs of endothelialization. Note suture (S) and red blood cells (r), whereas structure of luminal surface is not visible due 8:At 14 days, anastomosis covered by ento mural thrombus dothelium (E). Note completely endothelialized suture (S). Several red blood cells are present. C: At 28 days, ingrowth of endothelium (E) from the anastomotic side onto the graft. Structure of the graft (G) exposed in the nonendothelialized areas. A-C: x 7,000.D: At 56 days, the middle of the graft's inner surface is without signs of endothelialization. Red blood cells are present between the fibrils. D: x 2,000.

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RESULTS

All animals survived the experiment. Four grafts, two in the aorta (56 days) and two in the caval vein (3 and 14 days), were occluded due to thrombosis. Pseudoaneurysm in one aortic anastomosis and stenosis in one venous anastomosis were found. All grafts were well retroperitonealized and incorporated into the surrounding tissue. No abscesses or other gross signs of infection were noted at excision of the grafts. Scanning Electron Microscope Aortic grafts. Three days after implantation, the sutures

were exposed, the anastomoses were covered by thin mural thrombi (Fig. lA), and the inner surface of the graft was covered by a proteinaceous layer. At 7 days, Ec passed the anastomotic line, which was, however, still visible. Sutures were exposed. At 14 days, Ec, migrating from the aortic edges, covered the anastomoses (Fig. 1B). At 28 days, the anastomoses were bridged, sutures were covered by endothelium, and Ec had advanced -0.85 mm from the anastomotic line. In the middle portion of the graft, the inner surface structure was still visible between the endothelialized areas (Fig. IC). At 56 days, the endothelialized zone, though reaching -1 mm into the prosthesis, was still incomplete in some prostheses, and the inner surface was exposed in nonendothelialized areas (Fig. 1D). Caval vein grafts. Three days after implantation, the tran-

sition zone was smooth, and sutures were covered by endothelium (Fig. 2A). The inner surface of the prosthesis was generally covered by a thin, continuous mural thrombus containing platelets. The inner surface structure of the graft was not visible. Ec, migrating from the vein edges, growing onto the mural thrombus, passed the anastomotic line (Fig. 2B). At 7 days, the anastomoses were totally overbridged by Ec, reaching the middle of the grafts. The mural thrombus was still observed in nonendothelialized areas (Fig. 2C). At 14 days, endothelialization was complete (Fig. 2D), and, at 28 days, mural thrombi were no longer observed. At 56 days, the inner surface of the graft was covered by a complete neointimal lining. Light Microscopy Aortic grafts. At 3 and 7 days, anastomoses were covered

by a thin mural thrombus, whereas on the remaining inner surface no lining or only a proteinaceous layer was present (Fig. 3a,b). At 14 days, the anastomoses were covered by Ec ingrowing onto mural thrombus from the aortic edges (Fig. 3c), but the middle part of the grafts appeared similar as at 7 days. On the outside, an adventitial capsule was observed. At 28 and 56 days, anastomoses were covered by a multilayered neointima, which gradually became thinner until it remained monolayered at the growth zone (Fig. 3d).

Endothelialization of Microporous PTFE Grafts

Table 1. Distance of Endothelialitationof Microporous PTFE Grafts (of 5 mm Length and 2 mm Inner Diameter) in the Aorta and the Caval Vein in the Rat 3, 7, 14, 28, and 56 Days Following Implantation (complete = 2,500 wrn).e Time (days)

Aorta grafts (Fm)

3

0

7 14 28 56

98 2 49 521 2 129 850 207 1,053 356

*

(6) (6)

(6) (6) (4)

Caval vein grafts (Fm)

385 2 90b 1,516 f 30gb Completeb Completeb Completeb

(5) (6) (5) (6) (6)

aThe distance from anastomosis was measured. In individual grafts, the endothelialization at proximal and distal anastomoses did not differ. bSignificant/ygreater.

Nonendothelialized areas were still naked or covered only by a proteinaceous layer. Caval vein grafts. At 3 days, the inner prosthetic surface

was totally covered with a thin mural thrombus (Fig. 4a). At 7 days, anastomoses were covered by endothelium (Fig. 4b). The mural thrombus persisted in the middle of the graft and in the growth zone but not at the anastomoses. At 14 days, grafts were completely endothelialized, though underlying mural thrombus was still present in the middle portion (Fig. 4c). At 28 and 56 days, grafts were covered by multilayered neointima placed directly onto the inner surface of the prostheses (Figure 4d). As is shown in Table 1, the distances of reendothelialization of venous compared to aortic grafts were significantly higher at all time points (P < 0.OOOl).There were no differences in endothelialization progress between proximal and distal anastomoses of individual grafts. DISCUSSION

Failure of synthetic vascular grafts with small inner diameters (ID) (

Endothelialization of microporous polytetrafluoroethylene grafts in the infrarenal aorta and caval vein of the rat.

To study healing and endothelialization of vascular grafts, microporous polytetrafluoroethylene (PTFE) prostheses 2 mm in inner diameter and 5 mm long...
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