Heparin coating of vascular prostheses reduces thromboemboli

Heparin coating of vascular prostheses reduces thromboemboli Edmond F. Ritter, MD, Yong Bae Kim, MD, Helmut P. Reischl, MD, Donald Serafii, MD, Adam M. Rudner, BS, and Bruce Klitzman, PhD, Durham, N. C., Seoul, Korea, and Erlangen, Germany

Backqound. Synthetic conduits madefrom currently available materials are suboptimal for use in small-diameter vascular reconstructionbecause of their high surface thrombogenicity, which leads to failure. Methods. In this study control, heflarin-irrigated, or heparin-bonded expanded PolytetraJuoroethylene (ePTFE) grafts (4 mm long by 1 mm inner diameter) were implanted to reconstruct the iliac artery in male rats. The cremaster muscle was isolated as an islandjap based on branches of the iliac artery downstream from the graft. Emboli were quantitated by using intravital fluorescent microscopy of the cremastermuscle? microcirculation. Results.The mean number of emboli observed per animal during a 20-minuteperiod was 91 for the control group, 84 for the heparin-irrigated group, and 22 for the tm’dodecylmethylammonium chloride (TDMAC)-heparin group. The mean area of each embolus was 1057 pm2 for control, 940 pm2 for heparin-irrigated, and 808 pm2 for TDiVAC-heparin-coated grafts ($I c 0.05 for TDMAC-he$arin versus control or heparin-irrigated). Conclusions.A TDMAC-heparin coating of ePTm microvascularprostheses sigkficantly reduces downstream microemboli. (Surgery 1997;122:888-92.) From the Department of Surpy, Division of Plastic and Reconstructive Surgery, the Plastic Surgery Research Laboratories and Department of Cell Biology, Duke University Medical Center; Durham, N.C., the Department of Surgery, College of Medicine, Soonchunhyang University, Seoul, Korea, and the Ins&t fiir Physiologic, Universitiit Erlangen/Niirnberg, Erlangen, Germany

VASCULAR CONDUITS HAVE substantial applicability in clinical medicine, but the need exists for a prosthesis with compatibility and performance comparable to autogenous vascular grafts. The acute thrombogenecity of currently available biomaterials has limited their use for small-diameter reconstructions. Even when a vascular prosthesis remains patent, downstream microemboli may adversely affect tissue perfusion and reduce flow through the graft. In some situations the downstream events may be more important than the actual accumulation of thrombi on the surface of the pr0sthesis.l A parallel may be drawn between this and a similar situation in which patients with a popliteal aneurysm sustain chronic embolization from the aneurysm, producing a reduced vascular outflow bed. In turn, these patients sustain a high rate of thrombosis of their

PROSTHETIC

Accepted for publication Feb. 18, 1997. Reprint requests: Bruce K&man, PhD, Plastic Surgery Research Laboratories, Duke University Medical Center, Durham, NC 27710-3906. Copyright 0 1997 by Mosby-Year Book, Inc. 0039-6060/97/$5.00+0 11/56/82340 888

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vascular reconstruction.2 We undertook this study to develop a model to quantify production of microemboli by vascular prostheses and to determine whether heparin bonding would reduce the formation of these downstream microemboli. METHODS Animal preparation. Animals were cared for as specified in the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” (publication number 86-23, revised 1985), and the animal protocol was approved by the Duke University Institutional Animal Care and Use Committee. Male CD virus antibody-free rats (Dominion Laboratories, Dublin, Va.) weighing 100 to 150 gm were anesthetized with intraperitoneal sodium pentobarbital (Nembutal; Abbott Labs, Abbott Park, Ill.), 60 mg/kg, with supplemental doses as required. The entire lower abdomen and scrotal area were shaved. Each rat was placed on a heating pad, and rectal temperature wasmaintained at 37” ‘1 1” c. Anatomy and dissection. The cremaster muscle was isolated on its vascular supply, as described by

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Fig. 1. Schematized drawing of the rat with an ePTFE microvascular prosthesis implanted and the cremaster muscle exposed for intravital microscopy. First-order and second-order arterioles are indicated by A, and A,, respectively.

Fig. 2. Image shows bifurcation of a cremaster muscle first-order arteriole into two second-order arterioles. A, No emboli in the field. B, A large embolus visible at the bifurcation (original magnification x200).

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* p < 0.05

* p c 0.01

Mean ““’ Thrombuus Size

W-3 5oo 1

i

1

1

Control

HeparinIrrigated

TDMACHeparin

Control

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Fii. 3. Total number of microemboli per animal observed (during 20-minute period) in arterioles after implantation of control, heparin-irrigated, and TDMAGheparin-coated ePTFE grafts (bars, f SE).

TDMACHeparin

’

Fig. 4. Average sizes of microemboli observed in the cremaster microcirculation after implantation of 1 mm X 4 mm control, heparin-irrigated, and TDMAC-heparin-coated ePTFE grafts (bars, + SE).

200,000

* p < 0.01

150,000 1

Cumulative Thrombus loo~ooo1 Area (pm*) 50,000

HeparinIrrigated

T

1 T

* T 0

, Control

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Fig. 5. Total cross-sectional area of microemboli observed (during implantation of control, heparin-irrigated, and TDMAC-heparin-coated

et a1.3and Grant4 (Fig. 1). An incision was made from the anterior superior iliac spine to the upper scrotum. The hypogastric artery and all small branches of the right iliac artery were ligated with 8-O nylon except for the pudic-epigastric artery and the superior pudendal artery, which supply the cremaster muscle. The right iliac artery was transected, and an expanded polytetrafluoroethylene (ePTFE) interpositional graft (4 mm long, 1 mm inner diameter) was implanted in the iliac artery (Fig. 1) under optical magnification provided by a Week operating microscope using eight 9-O nylon sutures for each anastomosis. Platelet labeling. To facilitate visualization of microemboli, platelets were labeled with acridine red (dimethyl [6-( methylamino)-SH-xanthen-3-yliden] ammonium chloride; Pfaltz and Bauer,

Anderson

20-minute period) in arterioles ePTFE grafts (bars, k SE).

after

Waterbury, Conn.) by the method of Tangelder et a1.5 The rat received a bolus injection (1 to 3 mg/kg) followed by a continuous infusion (0.5 mg/kg per hour) of a solution prepared by dissolving 50 mg of acridine red in 0.5 ml ethanol and diluting to a total volume of 10 ml with saline solution. Preparation of vascular grafts. All ePTFE grafts (W.L. Gore & Associates, Flagstaff, Ariz.) were sterilized with ethylene oxide before implantation. Control. The six control grafts were obtained by transversely dividing stock grafts with a singleedged razor and were irrigated with sterile saline solution. Hepatin-irrigated grafts. Heparin grafts were irrigated with heparinized saline solution (100 units/ml) before implantation.

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Volume122, Number 5 TDMAC-hefiarin-coated grafts. Grafts were immersed in a solution of 25 mg/ml tridodecylmethylammonium chloride (TDMAC) in 100% ethanol for 30 minutes and air dried overnight. Grafts were washed in distilled water and then immersed in a 10,000 IU/ml heparin solution for 30 minutes and again air dried. Finally, grafts were immersed in saline solution for 10 minutes before implantation. Intravital microscopy. Observations were made with a Zeiss ACM fluorescence microscope (Carl Zeiss, Inc., Rockleigh, NJ.) with a 75 W xenon arc lamp. A ZeissFT 520-560 excitation filter and an LP 590 barrier filter were used. Images were recorded with a Cohu 4410 (Cohu, San Diego, Calif.) siliconintensified target camera and Toshiba DX-7 videocassette recorder and were viewed on a Panasonic WV-5410 video monitor. Time accurate to 0.01 second was encoded on the recorded image with a FOR-A VTG33 video timer. The diameter of each vessel was measured with a video caliper (Microcirculation Research Institute, Texas A & M University, College Station, Texas) Image analysis. Videotapes were replayed, and the number of microemboli passing a reference point on the vessel was counted during each lminute period. Fields containing microemboli were digitized (PCVisiont frame grabber; Imaging Technology, Inc.), and the size of each microembolus was determined by computerized planimetry with the image analysis program JAVA (‘Handel Scientific). Statistical analysis. An analysis of variance was performed to detect differences among groups, followed by pairwise comparisons performed by using a Wilcoxon rank-sum test. A p value of 0.05 or lesswas required for statistical significance. RESULTS

Blood flow resumed immediately in all animals on release of the microvascular clamps. A “no reflow” phenomenon was not observed, Fluorescent emboli were initially observed in all casesmoving with the blood stream. The microemboli appeared to be most clearly visible at the bifurcation of first- to second-order arterioles (Fig. 2). The number and sizes of the emboli were measured proximal to bifurcations. Not all individual platelets were routinely detectable, but small aggregates of only a few platelets were clearly evident. The mean number of microemboli seen per animal during the 20-minute observation period after clamp removal (n = 6 animals per group) was 91 in the control group, 84 in the heparin-irrigated coating group, and 22 in the TDMAC-heparin

group (p < 0.01 versus control or heparin-irrigated, Wilcoxon rank-sum test; Fig. 3). The mean area of each embolus image was 1057 pm2 for control, 940 pm2 for heparin-irrigated, and 808 urn* for TDMAC-heparin-coated grafts (p < 0.05 versus control or heparin-irrigated; Fig. 4). By multiplying the total number of microemboli by the average area per microembolus, the total embolus area observed per animal was calculated as 137,660 ,um2 in the control group, 79,040 pm2 in the heparinirrigated group, and 17,498 pm2 in the TDMACheparin group (p < 0.05 versus control or heparinirrigated; Fig. 5). DISCUSSION

The high acute and subacute occlusion rates of small-diameter prosthetic grafts limit their clinical use. The paramount reason for acute occlusion of vascular prostheses is graft-induced thrombosis.@ This phenomenon is related to graft-mediated activation of clotting mechanisms.8Jg Many techniques have been used to improve the performances of vascular prostheses. Systemic administration of agents including aspirin, aspirin plus dipyridamole, heparin, ibuprofen, and prostacyclin has demonstrated limited clinical efficacy.10-16 Physical and biologic surface modifications of graft material have effected incremental, but likewise limited, improvements in performance.17w-34This study suggests that the technique of bonding TDMAC-heparin to the intraluminal surface of the graft significantly reduces microemboli from ePTFE grafts. Previous studies from our laboratory have suggested that even when grafts remain patent, there may be significant differences in tissue oxygen tension, reflecting differences in microemboli and resulting in reduced perfused capillary density. In an epigastric flap model perfused with a 1 mm by 4 mm ePTFE graft, heparin-bonded grafts had significantly higher oxygen tension than flaps perfused by untreated ePTFE (p < 0.05) .2j The vascular isolation technique allows continuous monitoring of cremaster muscle microcirculatory blood flow downstream from a vascular reconstruction and facilitates quantification of microemboli. Other studies have quantified the perfused capillary density downstream from a microsurgical anastomosisand report a decrease in the 2 hours after operation.26 Another study from our laboratory quantified a decrease in blood flow through first-, second-, and third-order arterioles downstream from ePTFE vascular prostheses (1 mm inner diameter) and improvement of flow by reduction of surface thrombogenicity.27

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Although all grafts in this study remained patent, this was not unexpected because of the short lengths of the grafts and the brevity of the observation period.. Other studies of longer graft lengths and durations have demonstrated uniformly low patency rates of untreated ePTFE.20>23>24 In summary, downstream embolic events may be important to the quality of microvessel perfusion downstream from vascular prostheses. This model permits quantitative testing of the effect of surface modifications of vascular prostheses on microemboli formation. Further, the apparent thromboresistant properties of TDMAC-heparin coating may have a role in improving the performances of currently available small-diameter vascular prostheses. The assistance of Drs. Mark Dewhirst and Edgardo Ong, Division of Radiation Oncology, Duke University Medical Center, in the use of the JAVA analysis program is gratefully acknowledged. The technical support of Richard Sepka, Lucille Smith, and Nell Schrader was extremely beneficial. The ePTFE microvascular graft material was a generous gift of W.L. Gore & Associates, Flagstaff, Ariz.

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JA. Prevention of platelet aggregation and adherence to prosthetic vascular grafts by aspirin and dipyridamole. Surgery 1978;84:37-44. Hancock JB, Forshaw PL, Kaye MP. Gore-tex (polytetrafluoroethylene) in canine coronary artery bypass. J Thorac Cardiovasc Surg 1980;80:94101. Barry KJ, Scott RM, Keough EM. Heparin and small caliber polytetrafluoroethylene grafts in the carotid arteries of rats. J Microsurg 1981;3:72-6. Kaye MP, Gloviczki P, Dewanjee MK, Claus PL, Lovaas ME. Ibuprofen in experimental vascular surgery. Am J Med 1984;77:95-101. Claus PL, Gloviczki P, Hollier LH, Kaye MP. Patency of polytetrafluoroethylene microarterial prostheses improved by ibuprofen. Am J Surg 1982;144:180-5. Callow AD, Connolly R, O’Donnell TF Jr, Gembarowicz R, Keough E, Ramberg-Laskaris K, et al. Platelet-arterial synthetic graft interaction and its modification. Arch Surg 1982;117:1447-55. Allen BT, Sparks RE, Welch MJ, Mason NS, Mathias CJ, Clark RE. Reduction of platelet deposition on vascular grafts using an antiplatelet graft coating technique. J Surg Res 1984;36:80-8. Herring MB, Dilley R, Jersild RA, Boxer L, Gardner A, Glover J. Seeding arterial prostheses with vascular endothelium. Ann Surg 1979;190:8490. Schmidt SP, Hunter TJ, Falkow LJ, Evancho MM, Sharp WV. Effects of antiplatelet agents in combination with endothelial cell seeding on small-diameter Dacron vascular graft performance in the canine carotid artery model. J Vast Surg 1985;2:898-906. Greco RS, Kim HC, Donetz AP, Harvey RA. Patency of a small vessel prosthesis bonded to tissue plasminogen activator and iloprost. Ann Vast Surg 1995;9:1405. Seeger JM, Klingman N. Improved in vivo endothelialization of prosthetic grafts by surface modification with fibronectin. J Vast Surg 1988;8:476-82. Madras PN, Ward CA, Johnson WR. Enhanced thromboresistance of surfaces by denucleation. Trans Am Sot Artif Intern Organs 1980;26:153-8. Demas CP, Vann R, Ritter E, Sepka RS, Klitzman B, Barwick WJ. Decreased thrombogenicity of vascular prostheses following gas denucleation by hydrostatic pressure. Plast Reconstr Surg 1988;82:1042-5. Ritter EF, Vann RD, Wyble C, Barwick WJ, Klitzman B. Hydrostatic pressure reduces thrombogenicity of polytetrafluoroethylene vascular grafts. Am J Physiol 1989;257:H107681. Skarada DJ, duLaney TV, Steele TM, Ritter EF, Klitzman B. Continual in vivo assesment of 1 mm diameter vascular prostheses [abstract]. Transactions of Fifth World Biomaterials Congress 1996;1:278. Acland RD, Anderson G, Siemionow M, McCabe S. Direct in vivo observations of embolic events in the microcirculation distal to a small-vessel anastomosis. Plast Reconstr Surg 1989;84;280-8. Li Z, Serafin D, Klitzman B. Arteriolar hemodynamics downstream from implanted microvascular prostheses [abstract]. Transactions of the Plastic Surgery Research Council 1992;37:167-9.