Formation of a Functional Endothelium on Vascular Grafts STUART K. WILLIAMS, TIMOTHY SCHNEIDER, BARBARA KAPELAN, AND BRUCE E. JARRELL Department of Surgery, Jefferson Medical College, Philadelphia, Pennsylvania I9107


Cell lining, Artificial polymeric vascular grafts, Small caliber blood vessels

The lack of a functional endothelial cell lining on artificial polymeric vascular ABSTRACT grafts severely reduces their effectiveness in replacing small caliber (< 6 mm) blood vessels. Techniques have now been developed t o transplant autologous endothelial cells from one site in the body onto the surface of grafts prior to implantation. Pre-clinical animal trials provide evidence that grafts sodded with autologous, fat-derived, microvessel endothelial cells exhibit a stable, antithrombogenic lining of endothelium. The new endothelial cell lining exhibits morphologies identical with endothelium on native blood vessels. The effectiveness of endothelial cell sodding techniques in pre-clinical animal trials provides support for expanded clinical trials. INTRODUCTION The major clinical treatment of occlusive arterial disease has historically involved the use of replacement grafts to bypass the obstructed portion of the blood vessel. The effectiveness of these bypasses is assessed by the maintenance of normal blood perfusion to the previously ischemic tissue. Loss of patency in these grafts most often results in the return of symptoms or conditions which prompted the original bypass surgery. Therefore, a major goal continues t o be the development of bypass grafts with improved long-term patency rates. Evaluation of the patency rates of currently used bypass grafts has provided insight into parameters important to long-term graft function. Vascular grafts can be categorized into three general classes: polymeric grafts (e.g., ePTFE and Dacron), non-autologous natural vessels (e.g., tanned bovine aorta and umbilical vein), and autologous vessels (e.g., saphaneous vein and internal mammary artery). Several generalized conclusions can be made from the long history of the use of these grafts as arterial replacements. First, grafts placed in large-diameter, high flow rate positions (e.g., aortic replacements) exhibit very acceptable patency rates regardless of the type of graft used. Second, a significant drop in patency is observed in grafts placed in small-diameter (less than 6 mm) positions, especially under conditions of low run-off. Finally, natural autologous vessel replacements exhibit superior patency rates as compared to all other classes of grafts (Loop et al., 1986; Veith et al., 1986; Veterans Administration Study, 1988). This superior patency is believed to result from the “living” nature of autologous grafts since they comprise natural cellular elements. Most significant is the presence of a natural endothelial cell lining on the lumenal surface of these autologous vessels. A major hypothesis in our laboratory is that the creation of a natural endothelial cell lining on the lumenal surface of a polymeric vascular graft would replace the highly thrombogenic polymeric surface with an antithrombogenic cellular lining. Furthermore, this antithrombogenic endothelialized vascular graft would


exhibit improved short-term patency rates as compared to non-endothelialized polymeric grafts placed in identical anatomic positions. The endothelialized grafts would be expected to exhibit long-term patency rates similar to autologous vessel replacements. In order to test this hypothesis, our laboratory has been conducting in vitro and in vivo experiments to evaluate the efficacy of transplanting endothelial cells from one site in a donor to the surface of a vascular graft. In vitro studies have focused on maximizing the interaction of endothelial cells with both artificial and natural polymeric surfaces. In vivo studies have focused on optimizing an operating room compatible methodology that establishes a living endothelial cell lining on vascular grafts implanted into animals as well as humans. GRAFT HEALING IN ANIMALS AND MAN Spontaneous Endothelialization A remarkable difference exists between the healing of grafts placed as arterial substitutes in animals as compared to identical graft materials placed into humans. Sauvage et al. (1974) first described the lack of spontaneous formation of an endothelial cell layer on grafts explanted from humans after years of implantation. Identical grafts placed in animals have been shown by numerous authors to form a lumenal endothelialized surface after implantation for as little as 2 weeks (Graham et al., 1982;Herring et al., 1984;Kempczinski et al., 1985; Schmidt et al., 1985; Stanley et al., 1985). One major requirement for spontaneous endothelialization in animals is that the graft be porous in construction and thus exhibit the ability to allow cellular migration through the graft interstices (Clowess et al., 1986). Figure 1 illustrates an island of canine endothelial cells forming spontaneously on the surface of a control graft implanted as a carotid artery interpositional graft for 5 weeks. While nonendothelialized

Received July 15, 1989; accepted in revised form September 26, 1990. Address reprint requests to Stuart K. Williams, Ph.D., Department of Surgery, University of Arizona Health Sciences Center, Tucson, AZ 85724.



Fig. 1. Scanning electron micrograph of an island of endothelial cells on the surface of a vascular graft. These antithrombogenic cells have resulted from the process known as spontaneous endothelialization. The arrowhead illustrates an area onto which endothelium have not migrated.This area remains thrombogenic as evidenced by the deposition of platelets. Bar = 5.7 km.

areas in close association with EC remain thrombo- lular elements with the lumenal surface, the ablumegenic, the island of endothelial cells exhibits an- nal tissue surface, and at the anastomotic site. As tithrombogenic characteristics as evidenced by the lack discussed, animals and man exhibit a differential celof platelet, fibrin, and white blood cell deposition. In lular response on the lumenal and ablumenal surface. addition to differences in the rate of spontaneous en- Conversely, cellular healing at the anastomotic site exdothelialization, major differences exist in the coagu- hibits fewer species-dependent variations. The most lation and fibrinolytic system between different ani- notable reaction is the growth of tissue from the native mals and between animals and man (National Heart, blood vessel onto the surface of the vascular prosthesis; Lung, and Blood Institute Working Group, 1985). forming what is termed a pannus. Figure 2 is a light Nevertheless, animal studies have provided invaluable photomicrograph which illustrates the anastomotic information on the biostability, infectivity, inflamma- area of a graft implanted in a canine carotid artery for tory reactivity, and cellular reactivity of all the pros- 5 weeks. After this time period the cells which may thetic materials clinically in use as well as numerous have originated presumably from both the intima and materials currently in development. More importantly, the media have begun to migrate and take residence o n animal models continue to provide an irreplaceable the polymeric graft surface. A scanning electron micromeans to evaluate the two major causes of graft failure, graph of this region of an implanted graft (Fig. 3) ilearly thrombogenicity and intimal hyperplasia. lustrates the presence of cells on the lumenal surface which exhibit a morphology and antithrombogenicity similar to the endothelium which lines a native artery. Pannus Formation The rate of migration of these endothelial cells onto Following implantation of vascular grafts the natu- polymeric graft surfaces is initially believed to be quite ral healing process involves the interaction of host cel- rapid, in the range of 0.2 mm per day (Berger et al.,



Fig. 2. Light micrograph of the anastomotic region of a control graft (ePTFE) implanted for 5 weeks. The ablumenal surface of the graft (arrows) exhibits incorporation with the ingrowth of tissue into the graft interstices. The lumenal surface of the graft (arrowheads)illustrates the process known as pannus formation with the migration and proliferation of cells onto the surface. Bar = 200 pm.

1972; Sauvage et al., 1974). However, for reasons that are currently unknown, this migratory activity of the endothelium ceases after the cells have migrated approximately 1 cm (Berger et al., 1972; Sauvage et al., 1974). This cessation of endothelial cell migratory activity appears to be a phenomenon observed in all animal species including man. The formation of this pannus results in an antithrombogenic surface spanning about 1 cm a t both the proximal and distal ends of implanted grafts. This formation of an endothelialized pannus does not appear to affect the inherent thrombogenicity of the nonendothelialized portion of the vascular graft especially in grafts which span distances greater than 50 cm (e.g., femoral tibia1 bypass grafts). While cellular migratory activity is a function shared by numerous cells, the endothelium has a unique ability to exhibit migratory activity in the presence of high arterial shear stresses. Endothelial cells must therefore exhibit two cellular functions simultaneously, specifically motility and shear-resistant adherence.

Graft Seeding Due to the inherent thrombogenicity of currently available vascular grafts and our evolving knowledge of the importance of the vascular endothelium in main-

taining an antithrombogenic layer on the lumenal surface of blood vessels, a major hypothesis of many laboratories has been that establishing an endothelial cell lining on a graft will improve long-term patency. Dr. Malcolm Herring first put this technique to practice when he seeded vascular grafts with a patient's own vein-derived endothelium and implanted the graft in an arterial position (Herring et al., 1978). Since this pioneering work, Herring and a large group of other investigators have been conducting seeding trials in both animals and humans (Allen et al., 1984; Douville et al., 1987; Graham et al., 1979, 1980, 1982; Herring et al., 1984, 1986; Hirko et al., 1987; Kempczinski et al., 1985; Park et al., 1989; Reisberg et al., 1986; Rosenman et al., 1985; Schmidt et al., 1984,1985; Sharefkin et al., 1982; Stanley et al., 1985; Tannenbaum et al., 1987; Zilla et al., 1987). These trials have established that endothelial cell seeding does result in the accelerated formation of an endothelial cell lining on grafts as compared to control grafts prepared for implantation without the addition of cells. Several investigators questioned whether the newly formed monolayer of endothelium was truly derived from the cellular inoculum or whether seeding just accelerated the process of spontaneous endothelialization (Hollier et al., 1986). A



Fig. 3. The lumenal surface of the pannus is covered by endothelial cells which exhibit morphologies similar to endothelium on native vessels. Scanning electron micrograph. Bar = 23.8 pm.

Fig. 4. Microvascularized fat represents a n easily obtainable source of large quantities of endothelial cells. These low (a)and high (b) power scanning electron micrographs illustrate the cellular nature of human fat removed by the process known as liposuction. a: Bar = 66.7 km. b: Bar = 38.5 pm.

Fig. 5. Scanning electron micrograph illustrating the spreading of a human adult endothelial cell on a polymeric surface. A single cell has the ability to rapidly cover a large surface area. Bar = 2.9 pm.

Fig. 6. Scanning electron micrograph of the midportion of a vascular graft which was sodded with autologous fat microvessel endothelial cells at the time of implantation. This graft was explanted after 5 weeks in a carotid artery. Bar = 15.4 Fm.

Fig. 7. A scanning electron micrograph of the midportion of a vascular graft which was implanted as a carotid interposition graft in a canine model, using standard procedures without endothelial cell treatment. This graft exhibits a normal thrombogenic reaction as evidenced by the deposition of cellular and proteinaceous material. Bar = 15.4 pm.



Fig. 8. Transmission electron micrograph through the midportion of a control vascular graft after 5 weeks of implantation. This region illustrates the deposition and tight packing of platelets (P) on the surface which by gross morphologicalexamination will give the appearanceof a thrombus-freearea. Bar = 0.6 pm.

partial answer to this question has been provided recently through molecular biologic techniques. Several investigators have now shown that autologous endothelium transfected to carry a genetic marker and subsequently seeded onto vascular flow surfaces could be morphologically identified on the lumenal surface of the vessel weeks after implantation (Nabel et al., 1989; Wilson et al., 1989). MICROVESSEL ENDOTHELIAL CELL SODDING Research efforts in our laboratory have focused on two specific questions. First we have evaluated different sources on endothelial cells to establish endothelial cell monolayers on vascular grafts, and second we have evaluated procedures to improve the adherence of human endothelial cells to commercially available as well as experimental vascular graft surfaces. After studying techniques for the isolation and long-term cultivation of human adult endothelial cells from large blood vessels we decided t o explore alternate sources of endothelium to be used in vascular graft seeding techniques. We focused on techniques for the isolation of endothelial cells from microvascularized fat with the

belief that this source of endothelium may have advantages over macrovascular endothelium (Jarrell et al., 1986,1987; Radomski et al., 1987; Rupnick et al., 1989; Williams, 1987; Williams et al., 1989). Some of these reasons include the following:

1. Fat-derived microvessel endothelium represents an easily obtainable autologous source of endothelial cells. 2. Almost all patients who would be candidates for vascular procedures have sufficient fat for endothelial cell procurement. 3. The techniques for isolation of microvessel endothelium are well established and have been highly reproducible in our laboratory. 4. The surgical technique required for the aspiration of a small sample of subcutaneous fat is relatively noninvasive. 5. Endothelial cell monolayers which form during spontaneous endothelialization are presumably derived from microvascularized tissue which surrounds the implanted graft. Therefore, microvessels exhibit the ability to phenotypically differentiate and assume the role of large-vessel endothelium.



Fig. 9. This series of scanning electron micrographs illustrates the numerous morphologies that endothelial cells exhibit on the lumenal surface of microvessel endothelial cell sodded grafts. a: Bar = 7.7 pm. b Bar = 7.7 pm. c: Bar = 15.4 pm. d Bar = 15.4 pm.

During our development of the microvessel endothelial cell procurement and deposition technology we decided to use a new terminology to discriminate between endothelial cell seeding techniques using large-vesselderived endothelium, and cell deposition techniques using microvessel-derived endothelium. Since sufficient endothelial cells are derived from fat sources to seed surfaces a t confluent cell densities (>lo5 cells/

cm2) we have adopted the term sodding to describe high-density deposition (Rupnick et al., 1989).In addition, where seeding classically involved the deposition of cells onto grafts within a solution which would subsequently form a fibrin clot (e.g., whole blood or plasma), sodding involves direct application of cells onto a natural or artificial polymer. Sodding may also have the advantage that a functional monolayer of



Fig. 10. Scanning electron micrograph of the anastomosis arrows between a native artery (N)and a vascular graft (G).Bar = 227.3 Fm.

cells is created on the surface of a graft prior to its implantation into an animal or human. Thus, the antithrombogenic qualities of an endothelial lining may be established using microvessel sodding techniques at the time of graft implantation.

MICROVESSEL ISOLATION TECHNIQUES The methods to isolate fat-derived microvessel endothelial cell are essentially modifications of the original technique described by Wagner et al. (1972, 1975). Briefly, the technique utilizes a bacterial collagenase to digest adipose tissue resulting in a slurry of cells. Vascular endothelial cells are removed from buoyant adipocytes via centrifugation. If cells are to be cultured, the vascular pellet is subjected t o further centrifugation in a density gradient material to remove cells of different inherent densities (Madri and Williams, 1983; Williams et al., 1981, 1987). For endothelial cell sodding, the source of fat is extremely important since certain fat sources are contaminated with a higher concentration of nonendothelial cells (Williams et al., 1989). Figure 4a,b illustrates the structure of human adipose tissue obtained via liposuction. This source of fat is predominantly adipocytes and endothe-

lium with minimal contamination by mesothelium, pericytes, and smooth muscle cells. Once isolated, cells are then used immediately for graft sodding.

IN VITRO STUDIES OF ENDOTHELIUM-POLYMER INTERACTION With the development of techniques to isolate and culture both animal and human adult endothelial cells, numerous studies have evaluated the interaction of endothelium with both natural and artificial polymeric surfaces (Van Wachem, 1987; Watkins et al., 1984; Williams et al., 1985). Human adult endothelial cells appear to exhibit the most stringent substrate and growth factor requirements for their adherence and long-term growth (Jarrell et al., 1984; Maciag et al., 1981; Thornton et al., 1983). An inclusive study of this field is outside the scope of this review. Our laboratory has evaluated human adult endothelial cell interaction with polymeric surfaces using biochemical as well as morphological techniques with particular focus on evaluating the physical forces that regulate ECpolymer interaction. As shown in Figure 5 , individual human endothelial cells exhibit the ability to adhere and spread on artificial polymers. We have observed



Fig. 11. Higher-magnification scanning electron micrograph of the anastornotic region of a control graft illustrates the migration edge (arrows) of endothelial cells as they populate the surface of this polymeric graft. Bar = 22.7 pm.

that both the adherence and spreading phenomena is highly dependent upon the state of the endothelium, the type of polymer, and the surface coatings on the polymer (Baker et al., 1985; Hoch et al., 1989;Jarrell et al., 1988, 1989; Pratt et al., 1988; Radomski et al., 1989). Extensive research is ongoing to develop surfaces which are highly attractive to the adherence and spreading of human adult endothelial cells. CANINE SODDING TRIALS We have evaluated the effectiveness of autologous microvessel endothelial cell sodding techniques in establishing endothelial cell monolayers on vascular grafts implanted in a canine model. Our model utilizes 4 mm i.d. grafts which are 6 cm in length and therefore represent a system which is highly susceptible to loss of patency due to early thrombogenicity. Microvessel endothelial cells are derived from falciform ligament fat and sodded onto the graft surface at an initial sodding density of 2 x lo5 cellskm'. Sodded and control (sham sodded) grafts are implanted as paired interpositional grafts in the carotid position and explanted a t appropriate time periods. We subsequently evaluate grafts

morphologically at both the light and electron microscopic level. MATERIALS AND METHODS Light Microscopy Methods Samples removed from canine carotid arteries for evaluation by light microscopy were fixed in 4% paraformaldehyde for 2 hours followed by dehydration and paraffin embedding in an automated tissue processor. Paraffin sections were obtained and stained with either hematoxylin and eosin or with trichrome. Stained sections were evaluated and photomicrographs were obtained on a Nikon optiphot microscope. Electron Microscopy Methods Samples for scanning and transmission electron microscopy were fixed with 3% glutaraldehyde buffered with 0.05 M PIPES at pH 7.4. Samples for scanning electron microscopy were fixed for 2 hours, dehydrated in a graded series of acetone, and critical point dried in a Polaron CPD with carbon dioxide. Dried samples were mounted on SEM specimen stubs and sputter coated using a gold target. Samples were subsequently



Fig. 12. Light photomicrographs of the lumenal surface (L)of a control grafi (9) at the anastomotic site. Cellular material has migrated and proliferated on this surface resulting in organized cellular layers. This region is thicker closer to the native blood vessel (A), and becomes a single cell layer at the leading edge of migration (B). A Bar = 40 pm. B: Bar = 40 wm.

examined in either a JEOL JSM 35C or an AMRAY 1200 scanning electron microscope. Samples for transmission electron microscopy were glutaraldehyde fixed for 1hour and post-fixed with 1% osmium tetroxide in 0.05 M PIPES buffer at pH 7.4 for 1 hour. Following dehydration in a graded series of acetone, samples were embedded in Spurr’s lowviscosity media. Silver sections were obtained with a Diatome diamond knife on a Sorvall MT2-B microtome and picked up on uncoated copper grids. Sections were stained with 1%uranyl acetate and counter stained with lead. Sections were examined and photomicrographs were obtained in a JEOL 100 CXII transmission electron microscope. Morphology of Sodded Vascular Grafts Following 5 weeks of implantation as carotid interposition grafts in a canine model, a dramatic difference is seen in the morphology of the midportion of a microvessel sodded graft (Fig. 6) as compared to a control graft implanted without cell treatment (Fig. 7). The control graft exhibits the typical reaction of blood with a thrombogenic surface, as illustrated by adherent white cells, red cells, platelets, and fibrin strands. This surface is often described as a pseudointima, but bears little resemblance to the natural cell lining of a native blood vessel. Transmission electron microscopy reveals that apparent smooth “cellular areas” are often composed of tightly packed deposits of platelets (Fig. 8). This platelet deposition may continue, leading to eventual occlusion of the lumen or a segment of platelets may dislodge in the form of a microembolus. What is

apparent is the highly reactive nature of a nonendothelialized thrombogenic surface which probably never becomes quiescent but rather is highly reactive even after years of implantation. The reactivity of vascular grafts is not as important in large-diameter positions, such as aortic replacements. The high flow rate in large-diameter grafts will tend to maintain patency by effectively removing large aggregates of material due to the action of shear stress at the vessel wall. In smalldiameter positions the flow rate is much lower resulting in the more rapid deposition of clots. This more rapid deposition is probably the result of less efficient, removal rather than increased deposition The more ob-, vious reason for the loss of graft patency in small-di-. ameter positions is that these grafts have less crosssectional area, thus less adherent material is required t o occlude them. Midgraft regions of microvessel endothelial cell sodded grafts exhibit a well-developed continuous layer of highly attenuated cells with morphological features identical with the endothelial cells which line native blood vessels (Fig. 6). Moreover, these cells are an-. tithrombogenic as evidenced by the lack of cellular and. proteinaceous elements deposited on their surface. We have observed a wide variety of morphologies of cells which line the midportion of a vascular graft following: sodding (Fig. 9a-d). Figure 9a illustrates an endothelial lining which exhibits a high degree of orientation. Often this elongated morphology is attributed to flow dynamics; however, we often see this form of orientation perpendicular to the lumenal flow direction s u g gesting either that a different force is affecting cells or



Fig. 13. The vascular grafts in these studies were connected to native vessel using proline sutures (p). These scanning electron micrographs illustrate a segment of this suture material which has not been incorporated with tissue but remains open to the blood. Endothelial cells have migrated onto this suture resulting in an antithrombogenic surface. A: Bar = 76.9 km. B Bar = 38.5 Fm.

that the localized flow patterns are different at the surface of the graft. An additional factor regulating cellular surface morphology is the nature of the underlying cellular or polymeric structures, Certain dacron grafts are crimped, resulting in a highly uneven surface topology. The endothelial cell layer formed conforms to this uneven structure while subendothelial cellular proliferation is enhanced, leading to the creation of a flat surface topology (Herring et al., 1984; Schmidt et al., 1985).

Morphologic Observations of the Anastomotic Region Following implantation of a vascular graft a dynamic healing process is initiated involving the interaction of tissue with the ablumenal surface of the graft and the interaction of the host blood vessel with the cut ends of the polymeric graft. A native blood vessel is a multicellular tissue comprising the endothelial intimal lining, a smooth-muscle-rich media, and a surrounding adventitia. All three of these layers interact with the polymeric material during healing. The anastomotic healing process is illustrated in Figure 10 which shows a nonendothelialized graft implanted in an animal for 5 weeks. The ablumenal surface of the graft has been incorporated into the surrounding tissue although a constant foreign body reaction continues for months after graft implantation. The medial layer of the native vessel has no cellular counterpart in this implant but is replaced by the graft itself. The lumenal surface of the native vessel maintains a continuum with the artificial graft surface; however, the cellularity of this neointima

rapidly decreases on the graft surfaces. As previously discussed, this pannus rarely extends farther than 1 cm in all species. At medium magnification the migrating sheet of endothelial cells can be identified due to their characteristic tight packing (Fig. 11). The underlying cellular elements are presumably of muscle cell or fibroblast origin and extend a few millimeters farther than the endothelial cell layer in this explant. Of interest, these underlying cells also exhibit a degree of nonthrombogenicity due either to the close proximity of endothelium or their own inherent antithrombogenic functions under these conditions. High-magnification light microscopy illustrates that at least two cellular types predominate in the anastomotic region (Fig. 12). The lumenal surface of the graft is covered by a thin layer of attenuated cells which, although not unequivocally identified by light microscopy, are most likely endothelial cells that have migrated onto the lumenal surfaces of the graft. The underlying cellular layers are characterized as a phenotypically diverse population, most likely myofibroblasts. These cells exhibit a rapid reduction in thickness and are in continuity with the medial layers of the native blood vessel. This layer is disorganized as compared to normal media. Intensive research has focused on this anastomotic region since anastomotic hyperplasia represents a major cause of long-term graft failure. Of interest is the fact that the area of most active hyperplasia is composed of rapidly proliferatory cells, presumably of smooth muscle cell origin, underlying a layer of intact endothelium. Our original belief that an intact endo-



and Stanley, J.C. (1982) Expanded polytetrafluoroethylene vascular prostheses seeded with enzymatically derived and cultured canine endothelial cells. Surgery, 91:550-559. Graham, L.M., Vinter, D.W., Ford, J.W., Kahn, R.H., Burkel, W.E., and Stanley, J.C. (1979) Cultured autogenous endothelial cell seeding of prosthetic vascular grafts. Surg. Forum, 30204-306. Graham, L.M., Vinter, D.W., Ford, J.W., Kahn, R.H., Burkel, W.E., and Stanley, J.C., (1980) Endothelial cell seeding of prosthetic vascular grafts: Early experimental studies with cultured autologous canine endothelium. Arch. Surg., 115:929-933. Herring, M., Baughman, S., Glover, J., Kesler, K., Jesseph, J., Campbell, J., Dilley, R., Evan, A., and Gardner, A. (1984) Endothelial seeding of Dacron and polytetrafluoroethylene grafts: The cellular events of healing. Surgery, 96745-755. Herring, M., Cardner, A,, and Glover, J. (1978) A single staged tech., nique for seeding vascular grafts with autogenous endothelium. Surgery, 84498-504. Herring, M., Compton, R.S., Gardner, A.L., and LeGrand, D.R. (1986) Clinical experiences with endothelial seeding in Indianapolis. In: Endothelialization of Vascular Grafts. P. Zilla, R. Fasol, and M. Deutsch, eds. Karger Publishing Co., Switzerland pp. 218-224. Hirko, M.K., Schmidt, S.P., Hunter, T.J., Evancho, M.M., Sharp, W.V., and, Donovan, D.L. (1987) Endothelial cell seeding improveis SUMMARY AND CONCLUSIONS 4 mm ePTFE vascular graft performance in antiplatelet medicated Native blood vessels represent a complex organ sysdogs. Artery, 14(3):137-153. tem comprising multicellular elements. These cells col- Hoch, J., Jarrell, B.E., Schneider, T., and Williams, S.K. (1989) Endothelial cell interactions with native surfaces. Ann. Surg., 3(2): lectively maintain a structural and functional conduit 153-159. for the uninterrupted flow of blood. 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Pathol., 123220-230. Douville, E.C., Kempczinski, R.F., Birinyi, L.K., and Ramalanjaona, Park, P.K., Jarrell, B.E., Williams, S.K., Carter,T.L., Rose, D.g., MarG.R. (1987) Impact of endothelial cell seeding on long-term patency tinez-Hernandez, A., and Carabasi, R.A. (1989) Achievement of a and subendothelial proliferation in a small-caliber highly porous thrombus-free, human endothelial surface in the mid-region of a polytetrafluoroethylene graft. J. Vasc. Surg., 5544-550. Dacron vascular graft. J . Vasc. Surg., in press. Graham, L.M., Burkel, W.E., Ford, J.W., Vinter, D.W., Kahn, R.H., Pratt, K.J., Jarrell, B.E., Williams, S.K., Carabasi, R.A., Rupnick,

thelial cell monolayer inhibits the growth of underlying smooth muscle must therefore be reviewed and amended. Scanning electron microscopic evaluation of the anastomotic site provides an example of the multifactorial processes that take place as this site heals. At low magnification (Fig. 10) the lumenal surface exhibits numerous irregularities. The general size mismatch between the native vessel and prosthetic graft can be appreciated. A mismatch of vessel caliber is nearly always observed in arterial replacements, especially when end-to-side or side-to-side anastomoses are performed. All observed in this micrograph is suture material used to form the anastomosis. A higher-magnification micrograph of these sutures illustrates their total endothelialization presumably due to migration of cells onto their surface (Fig. 13).

FUNCTIONAL ENDOTHELIUM ON VASCULAR GRAFTS M.A., and Hubbard, F.A. (1988) Kinetics of endothelial cell-surface attachment forces. J. Vasc. Surg., 7(4):591-599. Radomski, J.S., Jarrell, B.E., Pratt, K.J., and Williams, S.K. (1989) Effects of in vitro aging on human endothelial cell adherence to Dacron vascular graft material. J . Surg.Res., in press. Radomski, J.S., Jarrell, B.E., Williams, S.K., Koolpe, E.A., Greener, D.A., and Carabasi, R.A. (1987) Initial adherence of human capillary endothelial cells to Dacron. J. Surg. Res., 42:133-140. Reisberg, B., Ortenwall, P., Wadenvik, H., and Kutti, J . (1986) Endothelial cell seeding: Experience and first clinical results in Gotchborg. In: Endothelialization of Vascular Grafts. P. Zilla, R. Fasol, and M. Deutsch, eds. Karger Publishing, Switzerland Co., pp. 225232. Rosenman, J.E., Kempczinski, R.F., Pearce, W.H., and Silberstein, E.B. (1985) Kinetics of endothelial cell seeding. J. Vasc. Surg., 2(6): 778-784. Rupnick, M.A., Hubbard, A., Pratt, K., Jarrell, B.E., and Williams, S.K. (1989) Endothelialization of vascular prosthetic surface following seeding or sodding techniques using human microvessel endothelial cells. J . Vasc. Surg., 9788-795. Sauvage, L.R., Berger, K.E., Wood, S.J., Yates, S.G., Smith, J.C., and Mansfield, P.B. (1974) Interspecies healing of porous arterial prostheses. Arch. Surg., 109:698-705. Schmidt, S.P., Hunter, T.J., Hirko, M., Belden, T.A., Evancho, M>M., Sharp. W.V., and Donovan, D.L. (1985) Small diameter vascular prostheses: two designs of FTFE and endothelial cell-seeded and nonseeded Dacron. J . Vasc. Surg., 2(2):292-297. Schmidt, S.P., Hunter, T.J., Sharp, W.V., Malindzak, G.S., and Evancho, M.M. (1984) Endothelial cell-seeded four-millimeter Dacron vascular grafts. J. Vasc. Surg., 1(3):434-441. Sharefkin, J.B., Latker, C., Smith, M., Cruess, D., Clagett, C.P., and Rich, N.M. (1982) Early normalization of platelet survival by endothelial seeding of Dacron arterial prostheses in dogs. Surgery, 92:385-393. Stanley, J.C., Burkel, W.E., and Lindblad, B. (1985) Endothelial cell seeding of synthetic vascular prostheses. Acta Chir. Scand. [Suppl.], 529:17-27. Tannenbaum, G., Ahlborn, T., Benvenisty, A., Reemstma, K., and Nowygrod, R. (1987) High density seeding of cultured endothelial cells leads to rapid coverage of polytetrafluoroethylene grafts. Curr. Surg. 44:318-321. Thornton, S., Mueller, S., and Levine, E. (1983) Human endothelial cells: cloning and long-term serial cultivation employing heparin. Science, 222:623-625. Van Wachem, P.B. (1987) The influence of protein adsorption on interactions of cultured human endothelial cells with polymers. J . Biomed. Mater. Res., 21:701-718. Veith, F.J., et al. (1986) Six-year prospective multicenter randomized

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comparison of autologous saphenous vein and expanded polytetrafluoroethylene grafts in infrainguinal arterial construction. J. Vasc. Surg., 3(1):104-114. Veterans Administration Cooperative Study Group 141 (1988) Comparative evaluation of prosthetic, reversed, and in situ bypass grafts in distal popliteal and tibial-peroneal revascularization. Arch. Surg., 123:434-438. Wagner, R.C., Kriener, P., Barrnett, R.J., and Betensky, M.W. (1972) Biochemical characterization and cytochemical localization of catecholamine-sensitive adenylate cyclase in isolated capillary endothelium. Proc. Natl. Acad. Sci. U.S.A., 69:3175. Wagner, R., and Matthews, M. (1975) The isolation and culture of capillary endothelium from epidymal fat. Microvasc. Res., 10:286297. Watkins, M.T., Sharefkin, J.B., Zajtchuk, R., Maciag, T.M., DAmore, P.A., Ryan, US.,Wart, H.V., and Rich, N.M. (1984) Adult human saphenous vein endothelial cells: Assessment of their reproductive capacity for use in endothelial seeding of vascular prostheses. J. Surg. Res., 36588-596. Williams, S.K. (1987) Isolation and culture of microvessel and large vessel endothelial cells: their use in transport and clinical studies. In: Microvascular Perfusion and Transport in Health and Disease. P. McDonaugh, ed. Karger Publishing Co., Switzerland, pp. 204245. Williams, S.K., Devenney, J.J., and Bitensky, M.W. (1981) Micropinocytic ingestion of glycosylated albumin by isolated microvessels: Possible role in the pathogenesis of diabetic microangiopathy. Proc. Natl. Acad. Sci. U.S.A., 78(4):2393-2397. Williams, S.K., Jarrell, B.E., Friend, L., Carabasi, R.A., Radomski, J.S., Koolpe, E., Mueller, S.N., Thornton, S.C., Marinucci, T., and Levine, E. (1985) Adult endothelial cell compatibility with prosthetic graft material. J. Surg. Res., 38:618-629. Williams, S.K., Jarrell, B.E., and Rose, D.G. (1987) The isolation of human fat-derived microvessel endothelial cells for use in vascular graft endothelialization In: Endothelialization of Vascular Grafts. P. Zilla, R. Fasol, and M. Deutsch, eds. Karger Publishing Co., Switzerland, pp. 211-217. Williams, S.K., Jarrell, B.E., Rose, D.G., Pontell, J., Kapelan, B.A., Park, P.K., and Carter, T.L. (1989) Human microvessel endothelial cell isolation and vascular graft sodding in the operating room. Ann. Vasc. Surg., 3(2):146-152. Wilson, J.M., Birinyi, L.K., Salomon, R.N., Libby, P., Callow, A.D., and Mulligan, R.C. (1989) Implantation of vascular grafts lined with genetically modified endothelial cells. Science, 244:13441346. Zilla, P., Fasol, R., Deutsch, M., Fischlein, T., Minar, E., Hammerle, A,, Krupicka, O., and Kadletz, M. (1987) Endothelial cell seeding of polytetrafluoroethylene vascular grafts in humans: A preliminary report. J . Vasc. Surg., 6535-541.

Formation of a functional endothelium on vascular grafts.

The lack of a functional endothelial cell lining on artificial polymeric vascular grafts severely reduces their effectiveness in replacing small calib...
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