Polypropylene small-diameter vascular grafts Howard P. Greisler,+ Charles W. Tattersall, Scott C. Henderson, and Emma A. Cabusao Loyola University Medical Center, Maywood, Illinois, and Hines VA Hospital, Hines, Illinois Jacqueline D. Garfield and Dae Un Kim Saint Barnabas Medical Center, Livingston, New Jersey Polypropylene’s physical properties (e.g., high tensile strength) and relatively inert behavior suggest that fabrication into an arterial substitute may result in an efficacious prosthesis. Grafts were woven from polypropylene yarn into conduits 4 mm I.D. X 50 mm in length. Control grafts were Dacron and ePTFE. Baseline platelet aggregometry on all dogs was performed M ADP. Aspirin and dipyridawith mole were given for three days preoperatively and maintained for 2 weeks after surgery. Fifty-four grafts were placed into the aortoiliac position, two different graft materials per dog. The grafts were explanted at intervals of 2 weeks through 16 months; photographed for thrombusfree surface area determinations; and preserved for light, scanning, and transmission electron microscopy. Late (416 month) patency was 81% (13/16) for polypropylene, 69% (9/13) for Dacron, and
20% (1/5) for ePTFE. These data include one year patencies of 11/12 (92%) for polypropylene and 7/10 (70%) for Dacron. Late patency for polypropylene grafts w a s b e t t e r t h a n for PTFE (p < 0.05). Platelet aggregation status did not predict g r a f t patency. Light microscopy of 2week polypropylene explants showed inner capsules composed of myofibroblasts and macrophages, with patchy areas of endothelial cells lining the lumen. By 1 month, a confluent endothelialized surface was seen in all polypropylene explants. Progressive thickening of inner capsules with myofibroblasts and collagen continued through 4 months, reachi n g a mean thickness of 142 t 50 p m (compared to 150 t 30 p m for Dacron). These findings suggest potential clinical efficacy for polypropylene as an arterial substitute. 0 1992 John Wiley & Sons, Inc.
IN TRODUCTION
The use of polypropylene as a suture material in vascular anastomoses and as a mesh in hernia repairs is well established. Desirable physical properties of this polymer include its high tensile strength,’,’ low fatigability, and its resistance to degradation by inflammatory proce~ses.~ It is adaptable to both monofilamentous and polyfilamentous fibers. Polypropylene suture is relatively inert, eliciting little tissue reaction in vascular anastomoses. These properties of polypropylene as a biomaterial suggest that its fabrication into a “To whom correspondence should be addressed at Loyola University Medical Center, Department of Surgery, 2160 South First Avenue, Maywood, Illinois 60153. Journal of Biomedical Materials Research, Vol. 26, 1383-1394 (1992) 0 1992 John Wiley & Sons, Inc. CCC 0021-9304/92/101383-12$4.00
GREISLER ET AL.
1384
vascular graft may result in an effective arterial prosthesis. Dacron, by contrast, elicits a chronic low-grade inflammatory reaction, which may theoretically decrease the potential for endothelialization due to neutrophil and macrophage release of proteolytic enzymes and products of oxygen metabolism. Previous work from this laboratory has demonstrated the efficacy of rabbit aortic interposition grafts woven from compound yarns containing polypropylene in combination with the bioresorbable copolymer polyglactin 910 (PG910).4,5The current study evaluates vascular grafts composed entirely of polypropylene implanted into the aor toiliac position in canines, and compares these to Dacron and expanded polytetraf luoroethylene (ePTFE), the two currently available graft materials.
METHODS
Preoperative platelet aggregation studies Mongrel dogs weighing 14-25 kg were phlebotomized of 30 mL of blood from a peripheral vein on three different days for platelet aggregation studies. Baseline aggregometry in response to adenosine diphosphate (ADP) was then performed on a dual channel Bio-Data Platelet Aggregation Profiler (Bio-Data Corporation, Hatboro, PA) using our previously described method! Briefly, blood was collected into tubes containing 0.109M sodium citrate. After centrifugation at l20g for 10 min, the platelet-rich plasma (PRP) was transferred to a plastic tube, capped, and allowed to stand for 30 min. Platelet-poor plasma (PPP) was obtained by centrifugation of the remaining blood sample at 150008 for 20 min. Next, one channel of the aggregometer was calibrated using a blank consisting of 360 y L of PPP plus 40 y L of saline. The other channel received 360 y L of PRP and was run for 4 min, following which 40 p L of saline (control) or W 5 M ADP was added and run for another 4 min. A positive aggregation study was defined as either greater than 60% platelet aggregation to ADP or an aggregation to ADP 40% above that to saline control. All dogs, irrespective of baseline platelet aggregation status, were then medicated with aspirin, 325 mg daily and dipyridamole 25 mg three times daily for 3 days prior to operation. Animal care complied with the ”Principles of Laboratory Animal Care” and the “Guide for The Care and Use of Laboratory Animals” (NIH publication No. 85-23, Revised 1985). Platelet aggregation studies were repeated on the day of surgery.
Operative technique Vascular grafts, 4 mm I.D. X 50 mm in length, were woven from polypropylene or Dacron yarns (woven and provided by Ethicon, Inc., Somerville, NJ), or were commercially available ePTFE (W.L. Gore & Assoc., Flagstaff, AZ). Both textile grafts were similarly woven from yarns of two denier per filament (denier/number of filaments: polypropylene = 50/24, Dacron =
POLYPROPYLENE SMALL-DIAMETER GRAFTS
1385
55/27) with the water porosities measuring 500-600 mL/cm2/min at 200 mm Hg pressure. The polypropylene weave density measured 87 X 64 warp ends X picks/inch and the yarn textile strength measured 23 i 1 kg. No materials were velour-treated. The contralateral iliac artery of some dogs was replaced by different experimental grafts not included in the current study. The dogs were fasted overnight and then anesthetized with intravenous thiopental 30 mg/kg, intubated, and maintained with nitrous oxide and halothane. Cefazolin, 1 g, was administered intravenously preoperatively. Using a midline abdominal incision, and after intravenous heparinization (100 U/kg), the grafts were interposed between the distal infrarenal aortas (end-to-side) and the distal common iliac arteries (end-to-end) bilaterally. The transected proximal iliac arteries were ligated. Each graft type was placed alternately on left and right sides. Blood flow through the operated iliac arteries was quantitated prior to and following graft implantation using a Could-Statham Model SP2204 electromagnetic f lowmeter (Gould, Inc. Medical Products Div., Oxnard, CA). All dogs were maintained on aspirin and dipyridamole for 2 weeks after surgery. The grafts were explanted from anesthetized animals after intervals of 2 weeks through 16 months (Table I). Explanted specimens were opened longitudinally, flushed with 10 mL of saline, pinned flat, and photographed. Thrombus-free surface area was determined by computerized planimetry for all patent specimens (Jameco Computer, Jameco Electronics, Belmont, CA; Pencept PenPad Graphics Tablet, Pencept Inc., Waltham, MA). Three blinded observers performed the planimetry and the results were averaged. The grafts were perserved in 10% buffered formalin.
Light microscopy Formalin-fixed specimens were then cut transversely to divide each graft into three segments: proximal, middle, and distal. Portions of each segment were then embedded in paraffin, and 5-pm-thick longitudinal sections were prepared of the proximal and distal segments of each graft. Cross sections were similarly prepared of the middle segment of each graft. All sections were stained with hematoxylin and eosin. Inner capsule thickness (ICT) measurements were performed under light microscopy every 2.5 mm along the length of-the specimens, but not with 2.5 mm of the anastomoses. Measurements were averaged over the length of each proximal, middle, and distal
TABLE I Explantation Times 2 Weeks 1 Month 2 Months 4 Months 6 Months 12 Months 16 Months
Polypropylene Dacron ePTFE
2 2 2
2 1 1
4 3
3
2 1 1
2 2 2
12 10 -
-
2
GREISLER ET AL.
1386
graft segment. The extent of the macrophage and foreign body reactions were compared.
Scanning electron microscopy Portions of the formalin fixed grafts which were not embedded in paraffin were fixed again with 2.5% purified glutaraldehyde in 0.1M cacodylatehydrochloride-buffer, then postfixed in 1% osmium tetroxide with 0.1M cacodylate. After staining with tannic acid-osmium-thiocarbohydrazideosmium, the specimens were dehydrated in graded alcohols, critical-point dried, and carbon and palladium evaporated. A JSM-35 JEOL (USA Electron Optics Division, Peabody, MA) scanning electron microscope was used to image the preparations.
Transmission electron microscopy Fixed specimens as described above were stained en bloc with tannic acid, dehydrated in graded alcohols, and embedded in epoxy resin (Epon EM bed 812). Semithin sections of approximately 2 pin thickness were stained with toluidine blue and reviewed by light microscopy to select suitable areas for ultrathin sections. These ultrathin (about 70 nm) sections were stained on grids with uranyl acetate and with lead citrate, then examined at 80 KV with a transmission electron microscope (model EM-109, Carl Zeiss, Inc., Thornwood, NJ).
Statistical methods Grafts were randomly implanted into dogs regardless of aggregometry status and any given graft type was implanted alternately into right and left sides. Differences in patency rates and in thrombus free surface areas were analyzed by two way analysis of variance and by paired and unpaired Student t tests.
RESULTS
No significant differences in handling characteristics between polypropylene and Dacron grafts were discerned by the surgeons. Electromagnetic f lowmetry demonstrated no significant blood flow differences among the groups. Prior to graft implantation the iliac artery blood flows ranged from 78.1 -+ 63 to 87.6 2 67 mL/min among the graft types. Following implantation blood flows measured 202.3 -+ 102 to 223.2 k 146 mL/ min. The increased blood flow following implantation reflects the hyperemia that occurs following removal of arterial crossclamps.
POLYPROPYLENE SMALL-DIAMETER GRAFTS
1387
Overall patency rates and thrombus free surface area for each graft type are shown in Tables I1 and 111. Early (2 weeks-2 months) patency was 100% (8/8) among the polypropylene grafts, 67% (4/6) among the Dacron grafts, and 83% (5/6) among the ePTFE grafts. Late (4-16 months) patency was 81% (13/16) among polypropylene, 69% (9/13) among the Dacron, and 20% (1/5) among the ePTFE grafts. The single late patent ePTFE graft was explanted at 4 months. Late patency for polypropylene grafts was significantly greater than for ePTFE ( p < 0.05). Of the polypropylene grafts explanted after 1 year, 11 of 12 (92%) were patent compared to 7/10 (70%) Dacron grafts patent at 1 year. Percentage thrombus free surface area did not differ significantly among the three graft types. There were no aneurysms, perigraft hematomas, or infections. Histologically, at 2 weeks polypropylene grafts demonstrated inner capsules composed primarily of fibrin coagula with occasional myofibroblasts and macrophages. Patchy areas of endothelial cells were seen at 2 weeks which achieved confluence by 1 month. Pannus ingrowth of endothelial cells and smooth muscle cells was observed at 2 weeks. At 1 month, the inner capsules evidenced maturation and contained numerous myofibroblasts and abundant collagen beneath a confluent endothelialized blood-contacting surface. No significant differences were seen comparing peri-anastomotic vs. central zones after this time point. Macrophage and foreign body reaction were seen predominantly in the outer capsules subjacent to the prosthetic material. The extent of the foreign body reaction after 1 month diminished slightly, then remained stable. Dacron explants appeared remarkably similar to those of polypropylene at each time point. Inner capsules were moderately cellular, much more so than TABLE I1 Patency Rates and Thrombus-Free Surface Areas Explantation Time 2 Weeks-2 Months
Polypropylene Dacron ePTFE
4 Months-16 Months
Patent
% TFS"
Patent
% TFS"
818 (100%) 416 (67%) 516 (83%)
96 2 4 88 ? 12 56 2 35
13/16 (81%) 9/13 (69%) 115 (20%)
99 ? 2 98 ? 3 100 5 0
"Percent thrombus-free surface area.
TABLE 111 Patency Rates -Results Explantation Time 2 Weeks 1 Month 2 Months 4 Months 6 Months 12 Months 16 Months
Polypropylene Dacron ePTFE
212 212 212
212
011
111
414 213 213
212 111 111
012 1I 2 012
11/12 7/10 -
-
012
1388
GREISLER ET AL.
are seen typically in Dacron grafts explanted from human applications. An endothelial cell layer was present by 1 month. Expanded PTFE grafts likewise had well-developed inner capsules composed of myofibroblasts and collagen by 2 months. Examples of the histologic appearance of each graft type explanted after 4 months are shown in Figure 1. Measurements of inner capsule thickness found progressive thickening through 4 months and then stabilized. No significant differences were found along the lengths of the explanted grafts of any group. Mean ICT at 2 months measured 93 t 11 p m for polypropylene, 80 5 50 for Dacron, and 90 +- 40 for ePTFE. Mean ICT of 1-year explants of polypropylene was 142 50 pm of Dacron was 150 t 30. No ePTFE grafts were patent at 1year so this comparison was not possible. The ePTFE grafts, unlike the polypropylene or Dacron grafts, resulted in inner capsules which varied widely in thickness along the length of each graft. ICT for the patent 4-month ePTFE explant ranged from 50 to 240 pm, with a mean of 190 It_ 70 pm. Scanning electron microscopy of two week polypropylene explants demonstrated patchy areas of cuboidal-shaped cells lining the luminal surfaces. By 1 month, this layer was confluent. Beyond 2 months, explanted specimens showed an endothelial cell layer which had progressed to spindle shapes, with the long axis of cells oriented to the direction of the flow of blood (Fig. 2). Transmission electron microscopy of the cellular surface of 1 month and later polypropylene explants revealed tight junctions, pinocytotic vesicles,
*
Figure l(a). Midportion of a polypropylene graft explanted after 4 months, showing a well developed neointima (inner capsule), composed of myofibroblasts and lined by an endothelial cell flow surface (H&E, original magnification X130).
POLYPROPYLENE SMALL-DIAMETER GRAFTS
Figure l(b). Midportion of a Dacron graft explanted after 4 months, showing a well developed neointima composed of myofibroblasts and lined by an endothelial ceIl flow surface (H&E, original magnification X130).
Figure l(c). Midportion of an ePTFE graft explanted after 4 months, showing a well developed neointima. Macrophages and myofibroblasts are seen within the interstices of the prosthetic material (H&E, original magnification X130).
1389
1390
GREISLER ET AL.
Figure 2. Scanning electron micrograph of the luminal surface of a polypropylene 2-month explant showing confluent endothelium (original magnification X750).
and Weibel-Palade bodies, confirming that these are endothelial cells [Fig. 3(a)-(c)]. Cells beneath the endothelial cell layer in grafts removed after 1 month appeared fibroblastlike, with few myofilaments. After 2 months these had the characteristic appearance of myofibroblasts with abundant myofilaments as well as rough endoplasmic reticulum [Fig. 3(c)]. One-year specimens contained myofibroblasts which phenotypically resembled smooth muscle cells, with extensive myofilaments and dense bodies throughout the cytoplasm. Extracellular collagen matrix was seen in the inner capsules of 2-week explants and increased in 2- and 4-month specimens, but then stabilized and appeared no different at 1 year. Baseline platelet aggregation studies determined that of the 54 dogs, 28 were nonaggregators, nine were aggregators, and 17 were indeterminant (Table IV). Indeterminant animals were those which had different aggregation reactions from one preoperative testing date to the next. All dogs classified as aggregators by baseline analysis changed to nonaggregator status after the 3 days of aspirin and dipyridamole administration. In this study, baseline platelet aggregation status did not predict graft patency (Table IV). However, the intrinsic tendency toward thrombosis of individual dogs did have an effect. Among the 40 patent grafts, the contralateral graft was patent in 39 (97.5%).Among the 14 occluded grafts, only 5 (35.7%) (p < 0.02) had patent contralateral grafts. Of interest is that among the 5 dogs with occluded ePTFE grafts, 3 had patent grafts contralaterally. No significant difference was found between thrombus free surface areas of patent grafts as a function of platelet aggregation status.
POLYPROPYLENE SMALL-DIAMETER GRAFTS
Figure 3(a). Photomicrograph of a 1-year polypropylene explant. A smooth confluent layer of endothelium is present on the luminal surface. The neointima is composed of myofibroblasts and extracellular matrix (H&E, original magnification X540).
Figure 3(b). Transmission electron micrograph of the midportion of a polypropylene 1-year explant showing a luminal surface of endothelium (E) with tight junctions (arrow), and a neointima composed of myofibroblasts (MF), and collagen fibers (original magnification X7200).
1391
GREISLER ET AL.
1392
Figure 3(c). Transmission electron micrograph from the inner capsule of a polypropylene 1 year explant showing myofibroblasts with abundant myofilaments, with dense bodies (arrowhead), along with rough endoplasmic reticulum (arrow). The extracellular matrix is predominantly collagen fibers (original magnification X9600). TABLE IV Platelet Aggregometry vs. Patency Nonaggregator
Aggregator
Indeterminant ~
Polypropylene Dacron ePTFE All grafts
9/11 6/7 5/10 20/28
(82%) (86%) (50%) (71%)
6/7 (86%) 1/2 (50%)
o/o
719 (78%)
~
~~~~
6/6 (100%) 6/10 (60%) 1/1 (100%) 13/17 (76%)
DISCUSSION
Synthetic arterial prostheses have been fabricated from a wide range of materials. Dacron and ePTFE grafts are commercially available in a number of forms, e.g., knitted or woven Dacron, reinforced or nonreinforced ePTFE. Grafts composed of either material are highly satisfactory in large-diameter clinical applications such as to the aortoiliac position. In these high-flow systems, patency is primarily determined not by the surface characteristics of the graft, but rather by the adequacy of the runoff. In small-diameter applications such as for femoral-crural bypass, the nature of the blood-contacting surface and the biomechanical properties of the graft become important variables in determining patency. Both Dacron and ePTFE have been disappointing in these small diameter bypasses.
POLYPROPYLENE SMALL-DIAMETER GRAFTS
1393
Polypropylene, since its introduction in the 1970s, has been the favored suture material for vascular anastomoses. It is a polymer of high tensile strength' and is not deteriorated by exposure to body tissues3 Polypropylene induces a relatively low inflammatory reaction and also relatively low levels of platelet adhesion and aggregation.' For these reasons, polypropylene was considered a logical choice as a yarn from which to fabricate an arterial substitute. Results showed a highly satisfactory overall patency of 88% in this canine model, including 92% patency rate among l-year explants. Histologically, the polypropylene grafts demonstrated relatively cellular inner capsules which showed progressive thickening through 4 months and then stabilized. Foreign body reaction was approximately that seen with Dacron and ePTFE, and was confined predominantly to the region of the outer capsule. A conf luent endothelialized luminal surface was seen by 1 month. Transmission electron microscopy revealed myofibroblasts in the inner and outer capsules which appeared to be immature in 2-week and l-month explants, but differentiated into a contractile phenotype by 2 months. At 1 year, the myofibroblasts had ultrastructural characteristics resembling contractile mature smooth muscle cells. An emphasis of this laboratory has been the search for an arterial substitute which results in more favorable prosthesisltissue and prosthesis/blood interfaces.*Our laboratory has tested a variety of grafts composed of totally resorbable materials in rabbit and dog models. After resorption of the grafts, there remain endothelialized conduits with walls composed predominantly of myofibroblasts and collagen. Although the incidence of aneurysmal dilatation in many of these studies approached zero,'-'' this remains a theoretical concern as regards clinical implantation. Attempts to obviate this problem have included the construction of compound grafts containing both a resorbable and a nonresorbable component. Dacron woven with polyglactin 910 was tested in a rabbit model." It was found, however, that the presence of Dacron inhibited the macrophage mediated arterial regeneration stimulated by the resorbable component. Polypropylene was then used as the nonresorbable component of grafts woven from compound yarns containing 70% PG910 and 30% polypropylene, and inhibition of cellular regeneration in the same rabbit model was not d e t e ~ t e d . ~ In a related study,' we evaluated the effects of physical properties of polypropylene on the extent of tissue ingrowth. Prostheses were woven from compound yarns containing 69% PG910 and 31% polypropylene. Grafts contained either two deniers per filament (2 dpf) polypropylene (Group l), or 15 deniers per filament (15 dpf) polypropylene (Group 2). The polypropylene of Group 1 elongated 93% at break, in contrast to the polypropylene of Group 2 which elongated 26% at break. The PG910 component initially had the highest elastic modulus, and with resorption the elasticity became defined by the polypropylene component. These grafts, implanted into the rabbit aorta model, showed a significantly thicker and more cellular inner capsule ( p 5 0.01, Student's unpaired t test) and significantly more occlusion ( p 5 0.02, x2 test) among those (Group 1) with the more elastic polypropylene.
GREISLER ET AL.
1394
The physical properties and biocompatibility characteristics of polypropylene resulted in woven vascular grafts which were efficacious in the canine aortoiliac model. Further evaluation of polypropylene as an arterial substitute is warranted, and may result in a small-diameter vascular graft which is clinically advantageous. References 1. 2.
3. 4. 5. 6.
7.
8. 9.
10. 11.
12.
P. B. Dobrin, ”Surgical manipulation and the tensile strength of polypropylene sutures,” Arch. Surg., 124, 665-668 (1989). P. B. Dobrin, ”Polypropylene suture stresses following closure of longitudinal arteriotomy,” 1. Vasc. Surg., 7, 423-428 (1988). T. Nilsson, ”Mechanical properties of Prolene@and Ethilon@sutures after 3 weeks i n vivo,” Scand. J Reconstr. Surg., 16, 11-15 (1982). H. P. Greisler, D.U. Kim, J.W. Dennis, J. J. Klosak, K. A. Widerborg, E. D. Endean, and R. M. Raymond, “Compound polyglactin 9lO/polypropylene small vessel prosthesis,” J. Vasc. Surg., 5, 572-583 (1987). H. I? Greisler, ”Effects of polypropylene’s mechanical properties on histologic and functional reactions to polyglactin 910/polypropylene vascular prostheses,” Surg. Forum, 38, 323-326 (1987). H. I? Greisler, J. F. McGurrin, J. J. Klosak, C.W. Tattersall, J. Ellinger, S.C. Henderson, and E. A. Cabusao, “The validity of canine platelet aggregometry i n predicting vascular graft patency,” J. Cardiovasc. Suri., 31. 712-718 (1990). H: Dahlke, N. Dociu, and K. Thurau, “Thrombogenicity of different suture materials as revealed by scanning electron microscopy,” 1. Biomed. Muter. Xes., 14, 251-268 (1980). H. P. Greisler, ”Macrophage/biomaterial interactions with bioresorbable vascular prostheses,” ASAIO Trans., 34,1051-1059 (1988). H. P. Greisler, ’Arterial regeneration over absorbable prostheses,” Arch. S U Y ~ .117,1425-1431 , (1982). H. P. Greisler, J. Ellinger, T. H. Schwarcz, J. Golan, R. M. Raymond, and D.U. Kim, “Arterial regeneration over polydioxanone prostheses in the rabbit,” Arch. Surg., 122, 715-721 (1987). H.P. Greisler, E.D. Endean, J. J. Klosak, J. Ellinger, J.W. Dennis, K. Buttle, and D.U. Kim, ”Polyglactin YlO/polydioxanone biocomponent totally resorbable vascular prostheses,” I. Vasc. Surg., 7, 697-705 (1988). H. P. Greisler, T. H. Schwarcz, J. Ellinger, and D.U. Kim, ”Dacron inhibition of arterial regenerative activities,” J. Vasc. Surg., 3, 747-756 (1986).
Received August 1, 1991 Accepted March 17, 1992
20% (1/5) for ePTFE. These data include one year patencies of 11/12 (92%) for polypropylene and 7/10 (70%) for Dacron. Late patency for polypropylene grafts w a s b e t t e r t h a n for PTFE (p < 0.05). Platelet aggregation status did not predict g r a f t patency. Light microscopy of 2week polypropylene explants showed inner capsules composed of myofibroblasts and macrophages, with patchy areas of endothelial cells lining the lumen. By 1 month, a confluent endothelialized surface was seen in all polypropylene explants. Progressive thickening of inner capsules with myofibroblasts and collagen continued through 4 months, reachi n g a mean thickness of 142 t 50 p m (compared to 150 t 30 p m for Dacron). These findings suggest potential clinical efficacy for polypropylene as an arterial substitute. 0 1992 John Wiley & Sons, Inc.
IN TRODUCTION
The use of polypropylene as a suture material in vascular anastomoses and as a mesh in hernia repairs is well established. Desirable physical properties of this polymer include its high tensile strength,’,’ low fatigability, and its resistance to degradation by inflammatory proce~ses.~ It is adaptable to both monofilamentous and polyfilamentous fibers. Polypropylene suture is relatively inert, eliciting little tissue reaction in vascular anastomoses. These properties of polypropylene as a biomaterial suggest that its fabrication into a “To whom correspondence should be addressed at Loyola University Medical Center, Department of Surgery, 2160 South First Avenue, Maywood, Illinois 60153. Journal of Biomedical Materials Research, Vol. 26, 1383-1394 (1992) 0 1992 John Wiley & Sons, Inc. CCC 0021-9304/92/101383-12$4.00
GREISLER ET AL.
1384
vascular graft may result in an effective arterial prosthesis. Dacron, by contrast, elicits a chronic low-grade inflammatory reaction, which may theoretically decrease the potential for endothelialization due to neutrophil and macrophage release of proteolytic enzymes and products of oxygen metabolism. Previous work from this laboratory has demonstrated the efficacy of rabbit aortic interposition grafts woven from compound yarns containing polypropylene in combination with the bioresorbable copolymer polyglactin 910 (PG910).4,5The current study evaluates vascular grafts composed entirely of polypropylene implanted into the aor toiliac position in canines, and compares these to Dacron and expanded polytetraf luoroethylene (ePTFE), the two currently available graft materials.
METHODS
Preoperative platelet aggregation studies Mongrel dogs weighing 14-25 kg were phlebotomized of 30 mL of blood from a peripheral vein on three different days for platelet aggregation studies. Baseline aggregometry in response to adenosine diphosphate (ADP) was then performed on a dual channel Bio-Data Platelet Aggregation Profiler (Bio-Data Corporation, Hatboro, PA) using our previously described method! Briefly, blood was collected into tubes containing 0.109M sodium citrate. After centrifugation at l20g for 10 min, the platelet-rich plasma (PRP) was transferred to a plastic tube, capped, and allowed to stand for 30 min. Platelet-poor plasma (PPP) was obtained by centrifugation of the remaining blood sample at 150008 for 20 min. Next, one channel of the aggregometer was calibrated using a blank consisting of 360 y L of PPP plus 40 y L of saline. The other channel received 360 y L of PRP and was run for 4 min, following which 40 p L of saline (control) or W 5 M ADP was added and run for another 4 min. A positive aggregation study was defined as either greater than 60% platelet aggregation to ADP or an aggregation to ADP 40% above that to saline control. All dogs, irrespective of baseline platelet aggregation status, were then medicated with aspirin, 325 mg daily and dipyridamole 25 mg three times daily for 3 days prior to operation. Animal care complied with the ”Principles of Laboratory Animal Care” and the “Guide for The Care and Use of Laboratory Animals” (NIH publication No. 85-23, Revised 1985). Platelet aggregation studies were repeated on the day of surgery.
Operative technique Vascular grafts, 4 mm I.D. X 50 mm in length, were woven from polypropylene or Dacron yarns (woven and provided by Ethicon, Inc., Somerville, NJ), or were commercially available ePTFE (W.L. Gore & Assoc., Flagstaff, AZ). Both textile grafts were similarly woven from yarns of two denier per filament (denier/number of filaments: polypropylene = 50/24, Dacron =
POLYPROPYLENE SMALL-DIAMETER GRAFTS
1385
55/27) with the water porosities measuring 500-600 mL/cm2/min at 200 mm Hg pressure. The polypropylene weave density measured 87 X 64 warp ends X picks/inch and the yarn textile strength measured 23 i 1 kg. No materials were velour-treated. The contralateral iliac artery of some dogs was replaced by different experimental grafts not included in the current study. The dogs were fasted overnight and then anesthetized with intravenous thiopental 30 mg/kg, intubated, and maintained with nitrous oxide and halothane. Cefazolin, 1 g, was administered intravenously preoperatively. Using a midline abdominal incision, and after intravenous heparinization (100 U/kg), the grafts were interposed between the distal infrarenal aortas (end-to-side) and the distal common iliac arteries (end-to-end) bilaterally. The transected proximal iliac arteries were ligated. Each graft type was placed alternately on left and right sides. Blood flow through the operated iliac arteries was quantitated prior to and following graft implantation using a Could-Statham Model SP2204 electromagnetic f lowmeter (Gould, Inc. Medical Products Div., Oxnard, CA). All dogs were maintained on aspirin and dipyridamole for 2 weeks after surgery. The grafts were explanted from anesthetized animals after intervals of 2 weeks through 16 months (Table I). Explanted specimens were opened longitudinally, flushed with 10 mL of saline, pinned flat, and photographed. Thrombus-free surface area was determined by computerized planimetry for all patent specimens (Jameco Computer, Jameco Electronics, Belmont, CA; Pencept PenPad Graphics Tablet, Pencept Inc., Waltham, MA). Three blinded observers performed the planimetry and the results were averaged. The grafts were perserved in 10% buffered formalin.
Light microscopy Formalin-fixed specimens were then cut transversely to divide each graft into three segments: proximal, middle, and distal. Portions of each segment were then embedded in paraffin, and 5-pm-thick longitudinal sections were prepared of the proximal and distal segments of each graft. Cross sections were similarly prepared of the middle segment of each graft. All sections were stained with hematoxylin and eosin. Inner capsule thickness (ICT) measurements were performed under light microscopy every 2.5 mm along the length of-the specimens, but not with 2.5 mm of the anastomoses. Measurements were averaged over the length of each proximal, middle, and distal
TABLE I Explantation Times 2 Weeks 1 Month 2 Months 4 Months 6 Months 12 Months 16 Months
Polypropylene Dacron ePTFE
2 2 2
2 1 1
4 3
3
2 1 1
2 2 2
12 10 -
-
2
GREISLER ET AL.
1386
graft segment. The extent of the macrophage and foreign body reactions were compared.
Scanning electron microscopy Portions of the formalin fixed grafts which were not embedded in paraffin were fixed again with 2.5% purified glutaraldehyde in 0.1M cacodylatehydrochloride-buffer, then postfixed in 1% osmium tetroxide with 0.1M cacodylate. After staining with tannic acid-osmium-thiocarbohydrazideosmium, the specimens were dehydrated in graded alcohols, critical-point dried, and carbon and palladium evaporated. A JSM-35 JEOL (USA Electron Optics Division, Peabody, MA) scanning electron microscope was used to image the preparations.
Transmission electron microscopy Fixed specimens as described above were stained en bloc with tannic acid, dehydrated in graded alcohols, and embedded in epoxy resin (Epon EM bed 812). Semithin sections of approximately 2 pin thickness were stained with toluidine blue and reviewed by light microscopy to select suitable areas for ultrathin sections. These ultrathin (about 70 nm) sections were stained on grids with uranyl acetate and with lead citrate, then examined at 80 KV with a transmission electron microscope (model EM-109, Carl Zeiss, Inc., Thornwood, NJ).
Statistical methods Grafts were randomly implanted into dogs regardless of aggregometry status and any given graft type was implanted alternately into right and left sides. Differences in patency rates and in thrombus free surface areas were analyzed by two way analysis of variance and by paired and unpaired Student t tests.
RESULTS
No significant differences in handling characteristics between polypropylene and Dacron grafts were discerned by the surgeons. Electromagnetic f lowmetry demonstrated no significant blood flow differences among the groups. Prior to graft implantation the iliac artery blood flows ranged from 78.1 -+ 63 to 87.6 2 67 mL/min among the graft types. Following implantation blood flows measured 202.3 -+ 102 to 223.2 k 146 mL/ min. The increased blood flow following implantation reflects the hyperemia that occurs following removal of arterial crossclamps.
POLYPROPYLENE SMALL-DIAMETER GRAFTS
1387
Overall patency rates and thrombus free surface area for each graft type are shown in Tables I1 and 111. Early (2 weeks-2 months) patency was 100% (8/8) among the polypropylene grafts, 67% (4/6) among the Dacron grafts, and 83% (5/6) among the ePTFE grafts. Late (4-16 months) patency was 81% (13/16) among polypropylene, 69% (9/13) among the Dacron, and 20% (1/5) among the ePTFE grafts. The single late patent ePTFE graft was explanted at 4 months. Late patency for polypropylene grafts was significantly greater than for ePTFE ( p < 0.05). Of the polypropylene grafts explanted after 1 year, 11 of 12 (92%) were patent compared to 7/10 (70%) Dacron grafts patent at 1 year. Percentage thrombus free surface area did not differ significantly among the three graft types. There were no aneurysms, perigraft hematomas, or infections. Histologically, at 2 weeks polypropylene grafts demonstrated inner capsules composed primarily of fibrin coagula with occasional myofibroblasts and macrophages. Patchy areas of endothelial cells were seen at 2 weeks which achieved confluence by 1 month. Pannus ingrowth of endothelial cells and smooth muscle cells was observed at 2 weeks. At 1 month, the inner capsules evidenced maturation and contained numerous myofibroblasts and abundant collagen beneath a confluent endothelialized blood-contacting surface. No significant differences were seen comparing peri-anastomotic vs. central zones after this time point. Macrophage and foreign body reaction were seen predominantly in the outer capsules subjacent to the prosthetic material. The extent of the foreign body reaction after 1 month diminished slightly, then remained stable. Dacron explants appeared remarkably similar to those of polypropylene at each time point. Inner capsules were moderately cellular, much more so than TABLE I1 Patency Rates and Thrombus-Free Surface Areas Explantation Time 2 Weeks-2 Months
Polypropylene Dacron ePTFE
4 Months-16 Months
Patent
% TFS"
Patent
% TFS"
818 (100%) 416 (67%) 516 (83%)
96 2 4 88 ? 12 56 2 35
13/16 (81%) 9/13 (69%) 115 (20%)
99 ? 2 98 ? 3 100 5 0
"Percent thrombus-free surface area.
TABLE 111 Patency Rates -Results Explantation Time 2 Weeks 1 Month 2 Months 4 Months 6 Months 12 Months 16 Months
Polypropylene Dacron ePTFE
212 212 212
212
011
111
414 213 213
212 111 111
012 1I 2 012
11/12 7/10 -
-
012
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are seen typically in Dacron grafts explanted from human applications. An endothelial cell layer was present by 1 month. Expanded PTFE grafts likewise had well-developed inner capsules composed of myofibroblasts and collagen by 2 months. Examples of the histologic appearance of each graft type explanted after 4 months are shown in Figure 1. Measurements of inner capsule thickness found progressive thickening through 4 months and then stabilized. No significant differences were found along the lengths of the explanted grafts of any group. Mean ICT at 2 months measured 93 t 11 p m for polypropylene, 80 5 50 for Dacron, and 90 +- 40 for ePTFE. Mean ICT of 1-year explants of polypropylene was 142 50 pm of Dacron was 150 t 30. No ePTFE grafts were patent at 1year so this comparison was not possible. The ePTFE grafts, unlike the polypropylene or Dacron grafts, resulted in inner capsules which varied widely in thickness along the length of each graft. ICT for the patent 4-month ePTFE explant ranged from 50 to 240 pm, with a mean of 190 It_ 70 pm. Scanning electron microscopy of two week polypropylene explants demonstrated patchy areas of cuboidal-shaped cells lining the luminal surfaces. By 1 month, this layer was confluent. Beyond 2 months, explanted specimens showed an endothelial cell layer which had progressed to spindle shapes, with the long axis of cells oriented to the direction of the flow of blood (Fig. 2). Transmission electron microscopy of the cellular surface of 1 month and later polypropylene explants revealed tight junctions, pinocytotic vesicles,
*
Figure l(a). Midportion of a polypropylene graft explanted after 4 months, showing a well developed neointima (inner capsule), composed of myofibroblasts and lined by an endothelial cell flow surface (H&E, original magnification X130).
POLYPROPYLENE SMALL-DIAMETER GRAFTS
Figure l(b). Midportion of a Dacron graft explanted after 4 months, showing a well developed neointima composed of myofibroblasts and lined by an endothelial ceIl flow surface (H&E, original magnification X130).
Figure l(c). Midportion of an ePTFE graft explanted after 4 months, showing a well developed neointima. Macrophages and myofibroblasts are seen within the interstices of the prosthetic material (H&E, original magnification X130).
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Figure 2. Scanning electron micrograph of the luminal surface of a polypropylene 2-month explant showing confluent endothelium (original magnification X750).
and Weibel-Palade bodies, confirming that these are endothelial cells [Fig. 3(a)-(c)]. Cells beneath the endothelial cell layer in grafts removed after 1 month appeared fibroblastlike, with few myofilaments. After 2 months these had the characteristic appearance of myofibroblasts with abundant myofilaments as well as rough endoplasmic reticulum [Fig. 3(c)]. One-year specimens contained myofibroblasts which phenotypically resembled smooth muscle cells, with extensive myofilaments and dense bodies throughout the cytoplasm. Extracellular collagen matrix was seen in the inner capsules of 2-week explants and increased in 2- and 4-month specimens, but then stabilized and appeared no different at 1 year. Baseline platelet aggregation studies determined that of the 54 dogs, 28 were nonaggregators, nine were aggregators, and 17 were indeterminant (Table IV). Indeterminant animals were those which had different aggregation reactions from one preoperative testing date to the next. All dogs classified as aggregators by baseline analysis changed to nonaggregator status after the 3 days of aspirin and dipyridamole administration. In this study, baseline platelet aggregation status did not predict graft patency (Table IV). However, the intrinsic tendency toward thrombosis of individual dogs did have an effect. Among the 40 patent grafts, the contralateral graft was patent in 39 (97.5%).Among the 14 occluded grafts, only 5 (35.7%) (p < 0.02) had patent contralateral grafts. Of interest is that among the 5 dogs with occluded ePTFE grafts, 3 had patent grafts contralaterally. No significant difference was found between thrombus free surface areas of patent grafts as a function of platelet aggregation status.
POLYPROPYLENE SMALL-DIAMETER GRAFTS
Figure 3(a). Photomicrograph of a 1-year polypropylene explant. A smooth confluent layer of endothelium is present on the luminal surface. The neointima is composed of myofibroblasts and extracellular matrix (H&E, original magnification X540).
Figure 3(b). Transmission electron micrograph of the midportion of a polypropylene 1-year explant showing a luminal surface of endothelium (E) with tight junctions (arrow), and a neointima composed of myofibroblasts (MF), and collagen fibers (original magnification X7200).
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Figure 3(c). Transmission electron micrograph from the inner capsule of a polypropylene 1 year explant showing myofibroblasts with abundant myofilaments, with dense bodies (arrowhead), along with rough endoplasmic reticulum (arrow). The extracellular matrix is predominantly collagen fibers (original magnification X9600). TABLE IV Platelet Aggregometry vs. Patency Nonaggregator
Aggregator
Indeterminant ~
Polypropylene Dacron ePTFE All grafts
9/11 6/7 5/10 20/28
(82%) (86%) (50%) (71%)
6/7 (86%) 1/2 (50%)
o/o
719 (78%)
~
~~~~
6/6 (100%) 6/10 (60%) 1/1 (100%) 13/17 (76%)
DISCUSSION
Synthetic arterial prostheses have been fabricated from a wide range of materials. Dacron and ePTFE grafts are commercially available in a number of forms, e.g., knitted or woven Dacron, reinforced or nonreinforced ePTFE. Grafts composed of either material are highly satisfactory in large-diameter clinical applications such as to the aortoiliac position. In these high-flow systems, patency is primarily determined not by the surface characteristics of the graft, but rather by the adequacy of the runoff. In small-diameter applications such as for femoral-crural bypass, the nature of the blood-contacting surface and the biomechanical properties of the graft become important variables in determining patency. Both Dacron and ePTFE have been disappointing in these small diameter bypasses.
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Polypropylene, since its introduction in the 1970s, has been the favored suture material for vascular anastomoses. It is a polymer of high tensile strength' and is not deteriorated by exposure to body tissues3 Polypropylene induces a relatively low inflammatory reaction and also relatively low levels of platelet adhesion and aggregation.' For these reasons, polypropylene was considered a logical choice as a yarn from which to fabricate an arterial substitute. Results showed a highly satisfactory overall patency of 88% in this canine model, including 92% patency rate among l-year explants. Histologically, the polypropylene grafts demonstrated relatively cellular inner capsules which showed progressive thickening through 4 months and then stabilized. Foreign body reaction was approximately that seen with Dacron and ePTFE, and was confined predominantly to the region of the outer capsule. A conf luent endothelialized luminal surface was seen by 1 month. Transmission electron microscopy revealed myofibroblasts in the inner and outer capsules which appeared to be immature in 2-week and l-month explants, but differentiated into a contractile phenotype by 2 months. At 1 year, the myofibroblasts had ultrastructural characteristics resembling contractile mature smooth muscle cells. An emphasis of this laboratory has been the search for an arterial substitute which results in more favorable prosthesisltissue and prosthesis/blood interfaces.*Our laboratory has tested a variety of grafts composed of totally resorbable materials in rabbit and dog models. After resorption of the grafts, there remain endothelialized conduits with walls composed predominantly of myofibroblasts and collagen. Although the incidence of aneurysmal dilatation in many of these studies approached zero,'-'' this remains a theoretical concern as regards clinical implantation. Attempts to obviate this problem have included the construction of compound grafts containing both a resorbable and a nonresorbable component. Dacron woven with polyglactin 910 was tested in a rabbit model." It was found, however, that the presence of Dacron inhibited the macrophage mediated arterial regeneration stimulated by the resorbable component. Polypropylene was then used as the nonresorbable component of grafts woven from compound yarns containing 70% PG910 and 30% polypropylene, and inhibition of cellular regeneration in the same rabbit model was not d e t e ~ t e d . ~ In a related study,' we evaluated the effects of physical properties of polypropylene on the extent of tissue ingrowth. Prostheses were woven from compound yarns containing 69% PG910 and 31% polypropylene. Grafts contained either two deniers per filament (2 dpf) polypropylene (Group l), or 15 deniers per filament (15 dpf) polypropylene (Group 2). The polypropylene of Group 1 elongated 93% at break, in contrast to the polypropylene of Group 2 which elongated 26% at break. The PG910 component initially had the highest elastic modulus, and with resorption the elasticity became defined by the polypropylene component. These grafts, implanted into the rabbit aorta model, showed a significantly thicker and more cellular inner capsule ( p 5 0.01, Student's unpaired t test) and significantly more occlusion ( p 5 0.02, x2 test) among those (Group 1) with the more elastic polypropylene.
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The physical properties and biocompatibility characteristics of polypropylene resulted in woven vascular grafts which were efficacious in the canine aortoiliac model. Further evaluation of polypropylene as an arterial substitute is warranted, and may result in a small-diameter vascular graft which is clinically advantageous. References 1. 2.
3. 4. 5. 6.
7.
8. 9.
10. 11.
12.
P. B. Dobrin, ”Surgical manipulation and the tensile strength of polypropylene sutures,” Arch. Surg., 124, 665-668 (1989). P. B. Dobrin, ”Polypropylene suture stresses following closure of longitudinal arteriotomy,” 1. Vasc. Surg., 7, 423-428 (1988). T. Nilsson, ”Mechanical properties of Prolene@and Ethilon@sutures after 3 weeks i n vivo,” Scand. J Reconstr. Surg., 16, 11-15 (1982). H. P. Greisler, D.U. Kim, J.W. Dennis, J. J. Klosak, K. A. Widerborg, E. D. Endean, and R. M. Raymond, “Compound polyglactin 9lO/polypropylene small vessel prosthesis,” J. Vasc. Surg., 5, 572-583 (1987). H. I? Greisler, ”Effects of polypropylene’s mechanical properties on histologic and functional reactions to polyglactin 910/polypropylene vascular prostheses,” Surg. Forum, 38, 323-326 (1987). H. I? Greisler, J. F. McGurrin, J. J. Klosak, C.W. Tattersall, J. Ellinger, S.C. Henderson, and E. A. Cabusao, “The validity of canine platelet aggregometry i n predicting vascular graft patency,” J. Cardiovasc. Suri., 31. 712-718 (1990). H: Dahlke, N. Dociu, and K. Thurau, “Thrombogenicity of different suture materials as revealed by scanning electron microscopy,” 1. Biomed. Muter. Xes., 14, 251-268 (1980). H. P. Greisler, ”Macrophage/biomaterial interactions with bioresorbable vascular prostheses,” ASAIO Trans., 34,1051-1059 (1988). H. P. Greisler, ’Arterial regeneration over absorbable prostheses,” Arch. S U Y ~ .117,1425-1431 , (1982). H. P. Greisler, J. Ellinger, T. H. Schwarcz, J. Golan, R. M. Raymond, and D.U. Kim, “Arterial regeneration over polydioxanone prostheses in the rabbit,” Arch. Surg., 122, 715-721 (1987). H.P. Greisler, E.D. Endean, J. J. Klosak, J. Ellinger, J.W. Dennis, K. Buttle, and D.U. Kim, ”Polyglactin YlO/polydioxanone biocomponent totally resorbable vascular prostheses,” I. Vasc. Surg., 7, 697-705 (1988). H. P. Greisler, T. H. Schwarcz, J. Ellinger, and D.U. Kim, ”Dacron inhibition of arterial regenerative activities,” J. Vasc. Surg., 3, 747-756 (1986).
Received August 1, 1991 Accepted March 17, 1992
