- Email: [email protected]
Impact of endothelial cell seeding on long-term patency and subendothelial proliferation in a small-caliber highly porous polytetrafluoroethylene graft
Impact of endothelial cell seeding on long-term patency and subendothelial proliferation in a small-caliber highly porous polytetrafluoroethylene graft E. Charles DouviUe, M.D., Richard F. Kempczinski, M.D., Louis K. Birinyi, M.D., and Georges R. Ramalanjaona, M.D., Cincinnati, Ohio Previous reports have demonstrated that endothelial cell seeding of polytetrafluoroethylene (PTFE) grafts enhances short-term patency. This experiment was undertaken to study its impact on the long-term patency of a highly porous, experimental PTFE graft and to determine whether increasing the internodal distance of the graft material resulted in increased proliferation of the subendothelium. Ten centimeter long, 4 mm internal diameter segments of an unreinforced, experimental PTFE graft were implanted into 36 mongrel dogs as carotid interpositions. In each animal, one graft was seeded with autologous endothelial cells, enzymatically derived from the external jugular veins, whereas the contralateral graft was treated in identical fashion except that endothelial cells were not added to the preclot mixture. Nineteen animals were killed at 12 weeks; six at 22 weeks; eight at 26 weeks; and three at 52 weeks. The mean follow-up period was 20.1 weeks. The overall patency rate was 58.3% (21 of 36 grafts) for seeded grafts vs. 27.8% (10 of 36 grafts) for control grafts (p < 0.01). The thrombus-free area was planimetrically measured at 83.4%-+ 4.5% in seeded grafts vs. 55.1%-+ 9.7% in control grafts (p < 0.05). Scanning electron microscopy confirmed the presence of a confluent cellular monolayer in seeded grafts, whereas control grafts exhibited a variable coaguhim of fibrin, platelets, and endothelial cells. The thickness of the subendothefial layer varied from 56 to 95 ~m with no progressive increase in thickness between 12 and 52 weeks. The presence of endothelium was confirmed by immunoperoxidase staining for canine factor V I I I related antigen in both seeded and control grafts. Endothelial seeding of the porous, experimental PTFE graft in this model significantly increased thrombus-free, luminal surface area and long-term patency compared with nonseeded control grafts. The increased porosity of our graft was not associated with progressive subendothelial proliferation, as had been feared, and may instead have been partially responsible for the finding of endothelium in the midportion of those long-term, control grafts that remained patent. (J VAsc SuRG 1987;5:544-50.)
Autogenous saphenous vein remains the preferred conduit for bypass of occluded arteries below the knee. Unfornmately, the vein may be unavailable in a significant percentage of patients who need it, which explains the recent interest in methods to reduce the inherent thrombogenicity of synthetic vas-
From the Division of Vascular Surgery,Departmentof Surgery, Universityof CincinnatiMedicalCenter. Presented at the Tenth AnnualMeetingof the MidwesternVascular SurgicalSociety,Indianapolis,Ind., Sept. 19-20, 1986. Supported in part by a graft from the W. L. Gore Co., Flagstaff, Ariz. Reprint requests: Richard F. Kempczinski,M.D., Universityof CincinnatiMedicalCenter, Divisionof Vascular Surgery,231 Bethesda Ave., Cincinnati,OH 45267-0558. 544
cular prostheses.1 In addition to endothelial ceil seeding, 2 some of the other techniques have included coating the luminal surface of the prosthesis with aspirin-impregnated polyglycolic acid, 3 albumin, 4 or carbon, s Endothelial cell seeding of Dacron grafts was first described by Herring et aLe Subsequent reports have demonstrated improved short-term patency of smallcaliber (4 mm) Dacron grafts seeded with enzymatically derived endothelial cellsY However, polytetrafluoroethylene (PTFE) grafts offer several important advantages over Dacron grafts. These include ease of handling, increased resistance to bacteremic infectibility,s and the ability to be inserted without the need for preclotting. However, endothelial cell
Volume 5 Number 4 April 1987
Endothelial seeded PTFE grafts
Table I. Patency of seeded vs. control grafts Seeded No. o f wk after implantation 12 22 26 52 Overall patency
12/19 4/6 2/8 3/3 21/36 (58.3%)
Control
6/19 1/6 1/8 2/3 10/36 (27.8%)
NOTE: p < 0.01.
seeding of PTFE has been less satisfactory than similar work with Dacron grafts because the new intimal lining that formed appeared to be loosely adherent and was easily denuded. 9 A previous report from this laboratory confirmed successful seeding of a new, highly porous, 4 mm PTFE graft with a trend toward improved patency at 6 weeks? ° This investigation was undertaken to examine the effect of endothelial cell seeding on the long-term patency of this graft and to determine whether increasing its porosity resulted in proliferation of the subendothelium during prolonged implantation. MATERIAL A N D M E T H O D S Ten centimeter lengths of an experimental, highly porous (internodal distance 45 Ixm), unreinforced PTFE vascular prosthesis (W. L. Gore & Assoc., Inc., Flagstaff, Ariz.) with an internal diameter of 4 mm was used for all grafts.~° They were implanted in large (25 to 30 kg) conditioned mongrel dogs by means of standard vascular techniques with the dogs given pentobarbital anesthetic. The animals were cared for by our Department of Laboratory Animal Medicine in compliance with the "Principles of Laboratory Animal Care" and the "Guide for the Care and Use of Laboratory Animals" (NIH Publication No. 80-23, revised 1978). Each animal received a 48-hour perioperative course of prophylactic antibiotics. Systemic anticoagulation was achieved before arterial clamping with parenteral beef-lung heparin (2 mg/kg). Platelet function was suppressed for 6 weeks postoperatively with 325 mg of aspirin and 50 mg of dipyridamole daily. Through a midline cervical incision, both external jugular veins were exposed and a 10 cm segment was removed from each vein and everted over sterile glass rods. The veins were secured in place and immediately immersed in cold Hank's calcium and magnesium-free, balanced salt solution (CMF-HBSS). After brief rinsing in this solution, they were incubated for 10 minutes at 37 ° C in 0.1% trypsin and 0.125% EDTA in CMF-HBSS at pH 8.1 followed by incu-
545
Table II. Thrombus-free area in seeded vs. control grafts Seeded (N = 19) Control (N = 10) Significance
83.4% _+ 4.5% 55.1% ± 9.7% p < 0.05
NOTE: Data are expressed as mean _+ standard error.
Table IlL Thickness of subendothelium Wk after implantation 12 22 26 52 Mean
Seeded Ozm) 73.6 56.2 62.5 87.4 71.9
± ± ± ± ±
22.2 4.3 12.5 31.1 11.9
(N (N (N (N (N
Control Ozm) = = = = =
7) 4) 2) 3) 16)
68.8 _+ 11.4 (N 50.0 (N 100.0 (N 95.0 ± 35.1 (N 75.0 ± 11.8 (N
= = = = =
4) l) 1) 2) 8)
bation in 0.5% collagenase in HBSS with calcium and magnesium at pH 7.4 for 10 minutes. They were then immersed in cold medium 199 and mechanically rotated for 5 minutes. The cells suspended in both enzyme solutions as well as in the culture medium were combined and centrifuged at 600 rpm in a refrigerated centrifuge. The resulting endothelial cell pellet (average size 7.5 × 10 s cells) was washed in cold medium 199 and recentrifuged. The final cell pellet was resuspended in 1 ml of cold cukure medium and added to 10 ml of the dog's own heparinized blood. This mixture was then used to seed the experimental graft by forcibly injecting it through the interstices of the graft 10 times. The control graft was treated in an identical manner except that 1 ml of cell-free culture medium, instead of an endothelial cell suspension, was added to 10 ml of autologous blood. Both carotid arteries were exposed through the same incision and a 10 cm segment of each artery was isolated and excised. On one side, randomly chosen, a seeded graft was interposed in the carotid artery in end-to-end fashion with continuous 6-0 polypropylene suture. A control graft was inserted into the opposite side and blood flow was reestablished. Meticulous hemostasis was achieved and the incision was closed. The animals were killed at varying intervals after graft implantation. Nineteen animals were killed at 12 weeks; six at 22 weeks; eight at 26 weeks; and three at 52 weeks. The mean follow-up period was 20.1 weeks. In all cases, the animals were reanesthetized and were given parenteral beef-lung heparin (2 mg/kg). Their previous surgical wounds were reexplored and the grafts were exposed. Their pa-
Journal of VASCULAR SURGERY
546 Douville et al.
Fig. 1. Scanning electron micrograph of cut edge of a seeded PTFE graft (GF) at 52 weeks after implantation demonstrates an intact endothelial layer (EC). Note orifice of a luminal capillary (arrow) and a uniform 80 to 90 p~m thick subendothelial matrix (SE). (Original magnification × 160.) tency was determined by palpation of a pulse in the graft and in the distal artery and was confirmed by direct inspection after graft removal. The entire graft and a segment of normal artery were removed and flushed gently with normal saline solution to eliminate gross, intralluminal blood. The grafts were opened longitudinally, pinned to a polystyrene board, and photographed. The percentage of the luminal surface covered by red thrombus was quantitated by means of computerized planimetry (Videoplanimeter, Carl Zeiss, Inc., Thornwood, N.Y.). The grafts were divided into 1 cm segments and sections from the midportion were immersed into 10% neutral buffered formalin, 2.5% glutaraldehyde (in 0.1 mol/L phosphate buffer [pH 7.14]) and Zamboni's solution, embedded in paraffin, and critical-point freeze dried for scanning electron microscopy. The grafts from the three animals at 52 weeks were perfusion-fixed at 100 mm H g with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) and representative sections were submitted for light and electron microscopy. The presence of endothelium
was confirmed by means of direct immunofluorescence and peroxidase-antiperoxidase staining for factor VIII-related antigen according to a method described elsewhere. ~ The observations recorded for each graft included its patency, the percentage of thrombus-free luminal surface area, the thickness of the subendothelial layer, and the presence and extent of endothelium. Statistical analysis included the chisquare test for patency and the Mann-Whitney test for unpaired data for the percentage of thrombusfree, luminal surface area in seeded vs. control grafts. The small sample size of several of our groups precluded the valid use of the Mantel-Haenszel test. RESULTS Patency. The overall patency rate for seeded grafts was 58.3% (21 of 36 grafts) vs. 27.8% (10 of 36 grafts) for control grafts (Table I). This difference was statistically significant (p < 0.01). The mean follow-up period for all grafts was 20.1 weeks. Thrombus-free area. The thrombus-frec area was calculated for each graft patent at the time the
Volume 5 Number 4 April 1987
Endothelial seeded PTFE graf~s 547
Fig. 2. Light photomicrograph of a seeded graft recovered 52 weeks after implantation demonstrates capillaries, fibrous ingrowth, and macrophages throughout graft wall. (Milligan's trichrome stain; original magnification x 25.) animals were killed and averaged 83.4% + 4.5% (standard error) in seeded grafts vs. 55.1% + 9.7% in control grafts (Table II). This difference was statistically significant (p < 0.05). Subendothelial thickness. Representative cross sections of patent seeded and control grafts were examined by means of scanning electron microscopy to evaluate the thickness of the subendothelial layer (Table III). It averaged 73.6 +_ 22.2 p~m at 12 weeks and did not increase significantly over time. Graft morphology. Scanning electron microscopy revealed a confluent layer of endothelial-like cells on the luminal surface of seeded grafts at 12 weeks (Fig. 1). Several of the patent, long-term control grafts also demonstrated a confluent cellular monolayer on their luminal surface. Immunohistochemical staining for canine factor VIII-related antigen confirmed that these were indeed endothelial cells. Three animals survived to 52 weeks and were available for study. All three seeded grafts and two of three control grafts in these animals were patent
at the time o f death. The visual similarity between seeded and control grafts at 1 year was striking. Endothelial coverage and thickness of the subendothelial matrix were similar in both groups. In addition, transmural ingrowth of fibroblasts and interstitial capillaries was common and macrophages frequently filled the outer half of the graft wall (Fig. 2). Smooth muscle-like cells were identified in the subendothelium of both control and seeded grafts (Fig. 3). Areas of degeneration with focal, heterotopic ossification of the inner capsule were noted in one control and two seeded grafts. Scanning electron micrographs of the luminal surface at i year showed that the seeded and control grafts that had remained patent were virtually indistinguishable. DISCUSSION
Efforts to develop a prosthetic conduit for the replacement of occluded small to medium-sized arteries in low flow situations that will offer patency rates comparable to those seen with autogenous sa-
548
Journal of VASCULAR SURGERY
Douville et al.
Fig. 3. High-power, light photomicrograph of a seeded graft at 1 year demonstrates endothelial cell monolayer subtended by a cellular layer of smooth muscle-like cells beneath which there is a thicker, but less cellular,layer of fibroblasts and collagen. (Hematoxylin-eosinstain; original magnification x 200.) phenous vein continue. The antithrombogcnic character of the flow surface of native vessels is a direct consequence of the ability of the endothelial cells that form this layer to resist thrombus formation. Presumably, the presence of an endothelial lining on the luminal surface of a synthetic graft would confer the same advantages it provides in native vessels, that is, the synthesis of glycoproteins at the blood-tissue interface, which in turn would prevent activation of blood coagulation, the binding of thrombin, the inhibition of platelet activation, the synthesis of prostacyclin, and the activation of plasminogen, m'~2 Recently, Sharefldn et al.13 demonstrated that endothelial cell seeding of Dacron prostheses reduced platelet-prosthetic interactions and normalized platelet survival times more rapidly than in nonsceded control grafts. Endothelial cell seeding of Dacron prostheses has been shown to improve patency of 4 mm grafts. Stanley et al.6 reported a cumulative patcncy rate in 4 mm, 10 cm long, endothelial cell-seeded Dacron grafts of 73% vs. 27% in control grafts followed for 16 weeks. Allen et al.7 reported a cumulative patency rate of 96% in seeded grafts compared with 29% in control grafts with 4 mm, 6 cm long Dacron grafts in a canine model followed to 7 months. However, work with PTFE has been less successful. Herring, Gardner, and Glover ~4reported that endothelial cellseeded PTFE grafts demonstrated poor adherence of the inner capsule to the graft material. With externally wrapped PTFE, others have confirmed a prom-
inent failure of these less porous grafts to develop a suitable subendothelial matrix and postulated that this deficiency could adversely affect the durability of the inner lining. 9 Previous work in our laboratory has demonstrated successful seeding of this highly porous (45 txm) unwrapped PTFE graft and the development of a 75 to 100 txm thick, subendothelial matrix composed of collagen and smooth musclc cells. 1° The present study reaffirms that seeding of prosthetic grafts does significantly enhance their long-term patency. Although the concept of "maximum critical length" would predict that a 4 mm prosthesis more than 6 cm in length would invariably occlude, is Schmidt et al.16 demonstrated that the initiation of antiplatelet agents immediately after graft implantation until graft healing occurred played an important role in preventing graft thrombosis, both in control and seeded PTFE grafts. The cumulative patency rate of 28% in 10 cm long control grafts that we have observed further supports this conclusion. A surprising observation in this experiment has been the finding of extensive endothelial coverage on control grafts that maintained long-term patency. Kusaba et al. ~r had previously suggested that merely increasing the porosity of PTFE grafts by creating multiple perforations through the wall resulted in the early formation of ncointima and complete reendothelialization by 12 weeks. In keeping with this hypothesis, our demonstration of endothcliaMikc cells that stained positive for factor VIII-related an-
Volume 5 Number 4 April 1987
tigen on control grafts may in part be due to the greater porosity of our graft. However, the precise origin of these cells remains unclear since pannus ingrowth is usually limited to 1 cm from either anastomosis, 9':° and transmural ingrowth would be unlikely after only 93 days. :8 Recent work with unseeded, 4 mm PTFE grafts (internodal distance 30 I~m) implanted in baboons has further advanced our understanding of the cellular details of graft healing. ~9 The endothelial cells that covered these grafts appeared to arise from each of the arterial anastomoses. Unlike our observations, transmural capillary ingrowth was limited to the perianastomotic area and was not found in the midportion of the graft. These authors also confirmed our observation that the thickness of the subendothelium remained surprisingly stable despite continued smooth muscle cell proliferation and remodeling, z° The presence of heterotopic bone and degeneration in several of our long-term grafts is a new finding in our model and lends support to the hypothesis that there may be chronic, unidentified endothelial cell injury in these grafts, resulting in smooth muscle cell proliferation. 2° Such injury could result in dystrophic calcification and heterotopic bone formation. A recent case report of similar degenerative changes in an expanded PTFE femoropopliteal graft suggests that this problem may also occur in humans, z: Clearly, factors other than the mere presence or absence of endothelium influenced the patency of the prosthetic grafts in our canine model. Among these were individual platelet aggregation responses to arachidonic acid and thromboxane-prostacyclin ratios. 2z Since endothelial cell seeding of small-caliber, low-flow PTFE grafts in experimental models clearly improves long-term patency and since endothelial cell seeding has recently been shown to increase the spread of endothelium in vascular prostheses implanted in humans, continued investigation in this area is certainly warranted. 23 However, graft composition and porosity as well as the biochemical characteristics of the flow surface may all be equally important in determining a graft's ability to develop and maintain a viable, intact endothelial layer. The technical assistance of Judi Falk, Mary Hehemann, Carole Vireday, Mary DuBay, and Tony Hartman is gratefully acknowledged. W. Jean Dodds (Albany, N.Y.) supplied the antibody to canine factor VIII-related antigen. REFERENCES
1. Veith FJ, Moss CM, Sprayregen S, Montefusco C. Preoperative saphenous venography in arterial reconstructive surgery of the lower extremity. Surgery 1979;85:253-6.
Endothelial seeded PTFE grafts
549
2. Herring MB, Gardner AL, Glover J. A single-stage technique for seeding vascular grafts with autogcnous endothelium. Surgery 1978;84:498-504. 3. Allen BT, Sparks RE, Welch MJ, Mason NS, Mathias CJ, Clark RE. Reduction of platelet deposition on vascular grafts using an antiplatelct graft coating technique. J Surg Res 1984;36:80-8. 4. Sharp WV, Teague PC, Richenbacher WE. Thrombogenic potential of Dacron grafts after prior exposure to whole blood, plasma or albumin. Trans Am Soc Artif Intern Organs 1979;25:275-8. 5. Goldfarb D, Houk JA, Moore L, Gain DL. Graphite expanded polytetrafluoroethylene: an improved small artery prosthesis. Trans Am Soc Artif Intern Organs 1977;23: 268-74. 6. Stanley JC, Burkel WE, Ford JW, et al. Enhanced patency of small diameter, externally supported Dacron iliofcmoral grafts seeded with endothelial cells. Surgery 1982;92:994-1005. 7. Allen BT, Long JA, Clark RE, Sicard GA, Hopkins KT, Welch MJ. Influence of endothelial cell seeding on platclct deposition and patency in small-diameter Dacron arterial grafts. J VASCSURG 1984;1:224-33. 8. Rosenman JE, Pcarce WH, Kempczinski RF. Bacterial adherence to vascular grafts after in vitro bactercmia. J Surg Res I985;38:648-55. 9. Graham LM, Burkel WE, Ford JW, Vinter DW, Kahn RH, Stanley JC. Expanded polytetrafluorocthylene vascular prostheses seeded with enzymatically derived and cultured canine endothelial cells. Surgery, 1982;91:550-9. 10. Kempczinski RF, Rosenman JE, Pearce WH, Roedershcimer LR, Beflatzk-yY, Ramalanjaona GR. Endothelial cell seeding of a new PTFE vascular prosthesis. J VASC SUP,G 1985;2: 424-9. 11. Nakanc PK, True CD. The current status of the enzymelabeled antibody method at the tissue level. In: Nakamura RM, ed. Immunoassays in the clinical laboratory. Proceedings of the First Annual Conference on Immunoassays in the Clinical Laboratory. New York: Alan R Liss, Inc, 1979:139-47. 12. Lang WE. Characteristics of plasminogen activation secreted by endothelial cells in vivo. Blood 1979;54(Suppl 1):287a. 13. Sharefldn JB, Latker C, Smith M, Cruess D, Clagctt GP, Rich NM. Early normalization of platelet survival by endothelial seeding of Dacron arterial prostheses in dogs. Surgery 1982;92:385-93. 14. Herring MA, Gardner A, Glover J. Seeding endothelium onto canine arterial prostheses: the effects of graft design. Arch Surg 1979;114:679-82. 15. Herring MB, Dilley R, Peterson G, Wiggens J, Gardner A, Glovcr J. Graft material, length and diameter determine patency of small arterial prostheses in dogs. J Surg Res 1982;32:138-42. 16. Schmidt SP, Hunter TJ, Hirko M, et al. Small-diameter vascular prostheses: two designs of PTFE and endothelial cellseeded and nonsecded Dacron. J VASCSURG 1985;2:292-7. 17. Kusaha A, Fischer CR, Matulewski TJ, Matsumoto T. Experimental study of the influence of porosity on development of neointima in Gore-Tex grafts: a method to increase longterm patency rate. Am Surg 1981;47:347-54. 18. Herring M, Baughman S, Glover J, et al. Endothelial seeding of Dacron and polytetrafluoroethylene grafts: the cellular events of healing. Surgery 1984;96:745-54. 19. Clowcs AW, Gown AM, Hanson SR, Rcidy MA. Mechanisms of arterial graft failure. I. Role of cellular proliferation
550
Journal of VASCULAR SURGERY
Douville et al.
in early healing of PTFE prostheses. Am J Pathol 1985; 118:43-54. 20. Clowes AW, Kirkman TR, Clowes MM. Mechanisms of arterial graft failure. II. Chronic endothelial and smooth muscle cell proliferation in healing polytetrafluoroethylene prostheses. J Vasc SURG 1986;3:877-84. 21. Eggleton SPH, Palmer J, Stamp G, Bain JR, Settlage RA, Newcombe JF. Heterotopic ossification of an expanded
polytetrafluoroethylene vascular graft. Br J Surg 1986;73: 159-60. 22. Zammit M, Kaplan S, Sauvage LR, Marcoe KF, Wu H-D. Aspirin therapy in small-caliber arterial prostheses: long-term experimental observations. J VAsc SURG 1984;1:839-51. 22. Herring M, Baughman S, Glover J. Endothelium develops on seeded human arterial prostheses: a brief clinical note. J VAsc SURG 1985;2:727-30.
B O U N D VOLUMES AVAILABLE TO SUBSCRIBERS Bound volumes of the J o u ~ d , ~ oF VASCULARSURGERY for 1987 are available to subscribers only. They may be purchased from the publisher at a cost of $44.00 ($58.00 international) for Vol. 5 (January to June) and Vol. 6 (July to December). Price includes shipping charges. Each bound volume contains a subject and author index, and all advertising is removed. Copies are shipped within 60 days after publication of the last issue in the volume. The binding is durable buckram with the journal name, volume number, and year stamped in gold on the spine. Payment must accompany all orders. Contact Circulation Fulfillment, The C. V. Mosby Company, 11830 Westline Industrial Drive, St. Louis, MO 63146, USA. In the United States call toll free: (800)325-4177, ext. 351. In Missouri call collect: (314)872-8370, ext. 351. Subscriptions must be in force to qualify. Bound volumes are not available in place o f a regular JOURNALsubscription.
Autogenous saphenous vein remains the preferred conduit for bypass of occluded arteries below the knee. Unfornmately, the vein may be unavailable in a significant percentage of patients who need it, which explains the recent interest in methods to reduce the inherent thrombogenicity of synthetic vas-
From the Division of Vascular Surgery,Departmentof Surgery, Universityof CincinnatiMedicalCenter. Presented at the Tenth AnnualMeetingof the MidwesternVascular SurgicalSociety,Indianapolis,Ind., Sept. 19-20, 1986. Supported in part by a graft from the W. L. Gore Co., Flagstaff, Ariz. Reprint requests: Richard F. Kempczinski,M.D., Universityof CincinnatiMedicalCenter, Divisionof Vascular Surgery,231 Bethesda Ave., Cincinnati,OH 45267-0558. 544
cular prostheses.1 In addition to endothelial ceil seeding, 2 some of the other techniques have included coating the luminal surface of the prosthesis with aspirin-impregnated polyglycolic acid, 3 albumin, 4 or carbon, s Endothelial cell seeding of Dacron grafts was first described by Herring et aLe Subsequent reports have demonstrated improved short-term patency of smallcaliber (4 mm) Dacron grafts seeded with enzymatically derived endothelial cellsY However, polytetrafluoroethylene (PTFE) grafts offer several important advantages over Dacron grafts. These include ease of handling, increased resistance to bacteremic infectibility,s and the ability to be inserted without the need for preclotting. However, endothelial cell
Volume 5 Number 4 April 1987
Endothelial seeded PTFE grafts
Table I. Patency of seeded vs. control grafts Seeded No. o f wk after implantation 12 22 26 52 Overall patency
12/19 4/6 2/8 3/3 21/36 (58.3%)
Control
6/19 1/6 1/8 2/3 10/36 (27.8%)
NOTE: p < 0.01.
seeding of PTFE has been less satisfactory than similar work with Dacron grafts because the new intimal lining that formed appeared to be loosely adherent and was easily denuded. 9 A previous report from this laboratory confirmed successful seeding of a new, highly porous, 4 mm PTFE graft with a trend toward improved patency at 6 weeks? ° This investigation was undertaken to examine the effect of endothelial cell seeding on the long-term patency of this graft and to determine whether increasing its porosity resulted in proliferation of the subendothelium during prolonged implantation. MATERIAL A N D M E T H O D S Ten centimeter lengths of an experimental, highly porous (internodal distance 45 Ixm), unreinforced PTFE vascular prosthesis (W. L. Gore & Assoc., Inc., Flagstaff, Ariz.) with an internal diameter of 4 mm was used for all grafts.~° They were implanted in large (25 to 30 kg) conditioned mongrel dogs by means of standard vascular techniques with the dogs given pentobarbital anesthetic. The animals were cared for by our Department of Laboratory Animal Medicine in compliance with the "Principles of Laboratory Animal Care" and the "Guide for the Care and Use of Laboratory Animals" (NIH Publication No. 80-23, revised 1978). Each animal received a 48-hour perioperative course of prophylactic antibiotics. Systemic anticoagulation was achieved before arterial clamping with parenteral beef-lung heparin (2 mg/kg). Platelet function was suppressed for 6 weeks postoperatively with 325 mg of aspirin and 50 mg of dipyridamole daily. Through a midline cervical incision, both external jugular veins were exposed and a 10 cm segment was removed from each vein and everted over sterile glass rods. The veins were secured in place and immediately immersed in cold Hank's calcium and magnesium-free, balanced salt solution (CMF-HBSS). After brief rinsing in this solution, they were incubated for 10 minutes at 37 ° C in 0.1% trypsin and 0.125% EDTA in CMF-HBSS at pH 8.1 followed by incu-
545
Table II. Thrombus-free area in seeded vs. control grafts Seeded (N = 19) Control (N = 10) Significance
83.4% _+ 4.5% 55.1% ± 9.7% p < 0.05
NOTE: Data are expressed as mean _+ standard error.
Table IlL Thickness of subendothelium Wk after implantation 12 22 26 52 Mean
Seeded Ozm) 73.6 56.2 62.5 87.4 71.9
± ± ± ± ±
22.2 4.3 12.5 31.1 11.9
(N (N (N (N (N
Control Ozm) = = = = =
7) 4) 2) 3) 16)
68.8 _+ 11.4 (N 50.0 (N 100.0 (N 95.0 ± 35.1 (N 75.0 ± 11.8 (N
= = = = =
4) l) 1) 2) 8)
bation in 0.5% collagenase in HBSS with calcium and magnesium at pH 7.4 for 10 minutes. They were then immersed in cold medium 199 and mechanically rotated for 5 minutes. The cells suspended in both enzyme solutions as well as in the culture medium were combined and centrifuged at 600 rpm in a refrigerated centrifuge. The resulting endothelial cell pellet (average size 7.5 × 10 s cells) was washed in cold medium 199 and recentrifuged. The final cell pellet was resuspended in 1 ml of cold cukure medium and added to 10 ml of the dog's own heparinized blood. This mixture was then used to seed the experimental graft by forcibly injecting it through the interstices of the graft 10 times. The control graft was treated in an identical manner except that 1 ml of cell-free culture medium, instead of an endothelial cell suspension, was added to 10 ml of autologous blood. Both carotid arteries were exposed through the same incision and a 10 cm segment of each artery was isolated and excised. On one side, randomly chosen, a seeded graft was interposed in the carotid artery in end-to-end fashion with continuous 6-0 polypropylene suture. A control graft was inserted into the opposite side and blood flow was reestablished. Meticulous hemostasis was achieved and the incision was closed. The animals were killed at varying intervals after graft implantation. Nineteen animals were killed at 12 weeks; six at 22 weeks; eight at 26 weeks; and three at 52 weeks. The mean follow-up period was 20.1 weeks. In all cases, the animals were reanesthetized and were given parenteral beef-lung heparin (2 mg/kg). Their previous surgical wounds were reexplored and the grafts were exposed. Their pa-
Journal of VASCULAR SURGERY
546 Douville et al.
Fig. 1. Scanning electron micrograph of cut edge of a seeded PTFE graft (GF) at 52 weeks after implantation demonstrates an intact endothelial layer (EC). Note orifice of a luminal capillary (arrow) and a uniform 80 to 90 p~m thick subendothelial matrix (SE). (Original magnification × 160.) tency was determined by palpation of a pulse in the graft and in the distal artery and was confirmed by direct inspection after graft removal. The entire graft and a segment of normal artery were removed and flushed gently with normal saline solution to eliminate gross, intralluminal blood. The grafts were opened longitudinally, pinned to a polystyrene board, and photographed. The percentage of the luminal surface covered by red thrombus was quantitated by means of computerized planimetry (Videoplanimeter, Carl Zeiss, Inc., Thornwood, N.Y.). The grafts were divided into 1 cm segments and sections from the midportion were immersed into 10% neutral buffered formalin, 2.5% glutaraldehyde (in 0.1 mol/L phosphate buffer [pH 7.14]) and Zamboni's solution, embedded in paraffin, and critical-point freeze dried for scanning electron microscopy. The grafts from the three animals at 52 weeks were perfusion-fixed at 100 mm H g with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) and representative sections were submitted for light and electron microscopy. The presence of endothelium
was confirmed by means of direct immunofluorescence and peroxidase-antiperoxidase staining for factor VIII-related antigen according to a method described elsewhere. ~ The observations recorded for each graft included its patency, the percentage of thrombus-free luminal surface area, the thickness of the subendothelial layer, and the presence and extent of endothelium. Statistical analysis included the chisquare test for patency and the Mann-Whitney test for unpaired data for the percentage of thrombusfree, luminal surface area in seeded vs. control grafts. The small sample size of several of our groups precluded the valid use of the Mantel-Haenszel test. RESULTS Patency. The overall patency rate for seeded grafts was 58.3% (21 of 36 grafts) vs. 27.8% (10 of 36 grafts) for control grafts (Table I). This difference was statistically significant (p < 0.01). The mean follow-up period for all grafts was 20.1 weeks. Thrombus-free area. The thrombus-frec area was calculated for each graft patent at the time the
Volume 5 Number 4 April 1987
Endothelial seeded PTFE graf~s 547
Fig. 2. Light photomicrograph of a seeded graft recovered 52 weeks after implantation demonstrates capillaries, fibrous ingrowth, and macrophages throughout graft wall. (Milligan's trichrome stain; original magnification x 25.) animals were killed and averaged 83.4% + 4.5% (standard error) in seeded grafts vs. 55.1% + 9.7% in control grafts (Table II). This difference was statistically significant (p < 0.05). Subendothelial thickness. Representative cross sections of patent seeded and control grafts were examined by means of scanning electron microscopy to evaluate the thickness of the subendothelial layer (Table III). It averaged 73.6 +_ 22.2 p~m at 12 weeks and did not increase significantly over time. Graft morphology. Scanning electron microscopy revealed a confluent layer of endothelial-like cells on the luminal surface of seeded grafts at 12 weeks (Fig. 1). Several of the patent, long-term control grafts also demonstrated a confluent cellular monolayer on their luminal surface. Immunohistochemical staining for canine factor VIII-related antigen confirmed that these were indeed endothelial cells. Three animals survived to 52 weeks and were available for study. All three seeded grafts and two of three control grafts in these animals were patent
at the time o f death. The visual similarity between seeded and control grafts at 1 year was striking. Endothelial coverage and thickness of the subendothelial matrix were similar in both groups. In addition, transmural ingrowth of fibroblasts and interstitial capillaries was common and macrophages frequently filled the outer half of the graft wall (Fig. 2). Smooth muscle-like cells were identified in the subendothelium of both control and seeded grafts (Fig. 3). Areas of degeneration with focal, heterotopic ossification of the inner capsule were noted in one control and two seeded grafts. Scanning electron micrographs of the luminal surface at i year showed that the seeded and control grafts that had remained patent were virtually indistinguishable. DISCUSSION
Efforts to develop a prosthetic conduit for the replacement of occluded small to medium-sized arteries in low flow situations that will offer patency rates comparable to those seen with autogenous sa-
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Fig. 3. High-power, light photomicrograph of a seeded graft at 1 year demonstrates endothelial cell monolayer subtended by a cellular layer of smooth muscle-like cells beneath which there is a thicker, but less cellular,layer of fibroblasts and collagen. (Hematoxylin-eosinstain; original magnification x 200.) phenous vein continue. The antithrombogcnic character of the flow surface of native vessels is a direct consequence of the ability of the endothelial cells that form this layer to resist thrombus formation. Presumably, the presence of an endothelial lining on the luminal surface of a synthetic graft would confer the same advantages it provides in native vessels, that is, the synthesis of glycoproteins at the blood-tissue interface, which in turn would prevent activation of blood coagulation, the binding of thrombin, the inhibition of platelet activation, the synthesis of prostacyclin, and the activation of plasminogen, m'~2 Recently, Sharefldn et al.13 demonstrated that endothelial cell seeding of Dacron prostheses reduced platelet-prosthetic interactions and normalized platelet survival times more rapidly than in nonsceded control grafts. Endothelial cell seeding of Dacron prostheses has been shown to improve patency of 4 mm grafts. Stanley et al.6 reported a cumulative patcncy rate in 4 mm, 10 cm long, endothelial cell-seeded Dacron grafts of 73% vs. 27% in control grafts followed for 16 weeks. Allen et al.7 reported a cumulative patency rate of 96% in seeded grafts compared with 29% in control grafts with 4 mm, 6 cm long Dacron grafts in a canine model followed to 7 months. However, work with PTFE has been less successful. Herring, Gardner, and Glover ~4reported that endothelial cellseeded PTFE grafts demonstrated poor adherence of the inner capsule to the graft material. With externally wrapped PTFE, others have confirmed a prom-
inent failure of these less porous grafts to develop a suitable subendothelial matrix and postulated that this deficiency could adversely affect the durability of the inner lining. 9 Previous work in our laboratory has demonstrated successful seeding of this highly porous (45 txm) unwrapped PTFE graft and the development of a 75 to 100 txm thick, subendothelial matrix composed of collagen and smooth musclc cells. 1° The present study reaffirms that seeding of prosthetic grafts does significantly enhance their long-term patency. Although the concept of "maximum critical length" would predict that a 4 mm prosthesis more than 6 cm in length would invariably occlude, is Schmidt et al.16 demonstrated that the initiation of antiplatelet agents immediately after graft implantation until graft healing occurred played an important role in preventing graft thrombosis, both in control and seeded PTFE grafts. The cumulative patency rate of 28% in 10 cm long control grafts that we have observed further supports this conclusion. A surprising observation in this experiment has been the finding of extensive endothelial coverage on control grafts that maintained long-term patency. Kusaba et al. ~r had previously suggested that merely increasing the porosity of PTFE grafts by creating multiple perforations through the wall resulted in the early formation of ncointima and complete reendothelialization by 12 weeks. In keeping with this hypothesis, our demonstration of endothcliaMikc cells that stained positive for factor VIII-related an-
Volume 5 Number 4 April 1987
tigen on control grafts may in part be due to the greater porosity of our graft. However, the precise origin of these cells remains unclear since pannus ingrowth is usually limited to 1 cm from either anastomosis, 9':° and transmural ingrowth would be unlikely after only 93 days. :8 Recent work with unseeded, 4 mm PTFE grafts (internodal distance 30 I~m) implanted in baboons has further advanced our understanding of the cellular details of graft healing. ~9 The endothelial cells that covered these grafts appeared to arise from each of the arterial anastomoses. Unlike our observations, transmural capillary ingrowth was limited to the perianastomotic area and was not found in the midportion of the graft. These authors also confirmed our observation that the thickness of the subendothelium remained surprisingly stable despite continued smooth muscle cell proliferation and remodeling, z° The presence of heterotopic bone and degeneration in several of our long-term grafts is a new finding in our model and lends support to the hypothesis that there may be chronic, unidentified endothelial cell injury in these grafts, resulting in smooth muscle cell proliferation. 2° Such injury could result in dystrophic calcification and heterotopic bone formation. A recent case report of similar degenerative changes in an expanded PTFE femoropopliteal graft suggests that this problem may also occur in humans, z: Clearly, factors other than the mere presence or absence of endothelium influenced the patency of the prosthetic grafts in our canine model. Among these were individual platelet aggregation responses to arachidonic acid and thromboxane-prostacyclin ratios. 2z Since endothelial cell seeding of small-caliber, low-flow PTFE grafts in experimental models clearly improves long-term patency and since endothelial cell seeding has recently been shown to increase the spread of endothelium in vascular prostheses implanted in humans, continued investigation in this area is certainly warranted. 23 However, graft composition and porosity as well as the biochemical characteristics of the flow surface may all be equally important in determining a graft's ability to develop and maintain a viable, intact endothelial layer. The technical assistance of Judi Falk, Mary Hehemann, Carole Vireday, Mary DuBay, and Tony Hartman is gratefully acknowledged. W. Jean Dodds (Albany, N.Y.) supplied the antibody to canine factor VIII-related antigen. REFERENCES
1. Veith FJ, Moss CM, Sprayregen S, Montefusco C. Preoperative saphenous venography in arterial reconstructive surgery of the lower extremity. Surgery 1979;85:253-6.
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2. Herring MB, Gardner AL, Glover J. A single-stage technique for seeding vascular grafts with autogcnous endothelium. Surgery 1978;84:498-504. 3. Allen BT, Sparks RE, Welch MJ, Mason NS, Mathias CJ, Clark RE. Reduction of platelet deposition on vascular grafts using an antiplatelct graft coating technique. J Surg Res 1984;36:80-8. 4. Sharp WV, Teague PC, Richenbacher WE. Thrombogenic potential of Dacron grafts after prior exposure to whole blood, plasma or albumin. Trans Am Soc Artif Intern Organs 1979;25:275-8. 5. Goldfarb D, Houk JA, Moore L, Gain DL. Graphite expanded polytetrafluoroethylene: an improved small artery prosthesis. Trans Am Soc Artif Intern Organs 1977;23: 268-74. 6. Stanley JC, Burkel WE, Ford JW, et al. Enhanced patency of small diameter, externally supported Dacron iliofcmoral grafts seeded with endothelial cells. Surgery 1982;92:994-1005. 7. Allen BT, Long JA, Clark RE, Sicard GA, Hopkins KT, Welch MJ. Influence of endothelial cell seeding on platclct deposition and patency in small-diameter Dacron arterial grafts. J VASCSURG 1984;1:224-33. 8. Rosenman JE, Pcarce WH, Kempczinski RF. Bacterial adherence to vascular grafts after in vitro bactercmia. J Surg Res I985;38:648-55. 9. Graham LM, Burkel WE, Ford JW, Vinter DW, Kahn RH, Stanley JC. Expanded polytetrafluorocthylene vascular prostheses seeded with enzymatically derived and cultured canine endothelial cells. Surgery, 1982;91:550-9. 10. Kempczinski RF, Rosenman JE, Pearce WH, Roedershcimer LR, Beflatzk-yY, Ramalanjaona GR. Endothelial cell seeding of a new PTFE vascular prosthesis. J VASC SUP,G 1985;2: 424-9. 11. Nakanc PK, True CD. The current status of the enzymelabeled antibody method at the tissue level. In: Nakamura RM, ed. Immunoassays in the clinical laboratory. Proceedings of the First Annual Conference on Immunoassays in the Clinical Laboratory. New York: Alan R Liss, Inc, 1979:139-47. 12. Lang WE. Characteristics of plasminogen activation secreted by endothelial cells in vivo. Blood 1979;54(Suppl 1):287a. 13. Sharefldn JB, Latker C, Smith M, Cruess D, Clagctt GP, Rich NM. Early normalization of platelet survival by endothelial seeding of Dacron arterial prostheses in dogs. Surgery 1982;92:385-93. 14. Herring MA, Gardner A, Glover J. Seeding endothelium onto canine arterial prostheses: the effects of graft design. Arch Surg 1979;114:679-82. 15. Herring MB, Dilley R, Peterson G, Wiggens J, Gardner A, Glovcr J. Graft material, length and diameter determine patency of small arterial prostheses in dogs. J Surg Res 1982;32:138-42. 16. Schmidt SP, Hunter TJ, Hirko M, et al. Small-diameter vascular prostheses: two designs of PTFE and endothelial cellseeded and nonsecded Dacron. J VASCSURG 1985;2:292-7. 17. Kusaha A, Fischer CR, Matulewski TJ, Matsumoto T. Experimental study of the influence of porosity on development of neointima in Gore-Tex grafts: a method to increase longterm patency rate. Am Surg 1981;47:347-54. 18. Herring M, Baughman S, Glover J, et al. Endothelial seeding of Dacron and polytetrafluoroethylene grafts: the cellular events of healing. Surgery 1984;96:745-54. 19. Clowcs AW, Gown AM, Hanson SR, Rcidy MA. Mechanisms of arterial graft failure. I. Role of cellular proliferation
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in early healing of PTFE prostheses. Am J Pathol 1985; 118:43-54. 20. Clowes AW, Kirkman TR, Clowes MM. Mechanisms of arterial graft failure. II. Chronic endothelial and smooth muscle cell proliferation in healing polytetrafluoroethylene prostheses. J Vasc SURG 1986;3:877-84. 21. Eggleton SPH, Palmer J, Stamp G, Bain JR, Settlage RA, Newcombe JF. Heterotopic ossification of an expanded
polytetrafluoroethylene vascular graft. Br J Surg 1986;73: 159-60. 22. Zammit M, Kaplan S, Sauvage LR, Marcoe KF, Wu H-D. Aspirin therapy in small-caliber arterial prostheses: long-term experimental observations. J VAsc SURG 1984;1:839-51. 22. Herring M, Baughman S, Glover J. Endothelium develops on seeded human arterial prostheses: a brief clinical note. J VAsc SURG 1985;2:727-30.
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