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Autologous vein supported with a biodegradable prosthesis for arterial grafting
Autologous Vein Supported With a Biodegradable Prosthesis for Arterial Grafting Hans-Peter Zweep, MD, Shinichi Satoh, MD, PhD, Berend van der Lei, MD, PhD, Wouter L. J. Hinrichs, PhD, Freerk Dijk, Jan Feijen, PhD, a n d Charles R. H. Wildevuur, MD, PhD Cardiopulmonary Surgery Research Division and Laboratory for Histology and Cell Biology, University Hospital Groningen, Groningen, and Department of Biomaterials, University Twente, Enschede, the Netherlands
To evaluate the potential of a supporting, compliant, biodegradable prosthesis to function as a temporary protective scaffold for autologous vein grafts in the arterial circulation, we implanted vein grafts into the carotid arteries of rabbits, either with (composite grafts) or without (control grafts) such a supporting prosthesis, and evaluated them up to 6 weeks. The control vein grafts showed edema and severe medial disruption with infiltration of polymorphonuclear cells on day 1. Over the study, irregular fibrocyte formation resulted in the
formation of a fibrotic vein wall. In contrast, the composite vein grafts showed preservation of smooth muscle cell layers and elastic laminae with a minor inflammatory response. Regular proliferation of fibroblasts, which in some areas were circularly oriented, was observed. We conclude that a supporting, compliant, biodegradable prosthesis can function as a protective scaffold for vein grafts in the arterial circulation, thus reducing damage to the vein graft wall and allowing gradual arterialization.
hen used as an arterial substitute, autologous vein grafts undergo degenerative changes during longterm implantation [l, 21. This is reflected by their low long-term patency rate compared with the patency rate of arterial autografts [3]. The degenerative changes in the vein wall might have their origin in the early postoperative period. After implantation, the vein wall shows a certain degree of deendothelialization and medial damage, including disruption, fibroblast transformation of smooth muscle cells, and an inflammatory response in the vein wall with edema [4-61. All these changes are alleged to lead to progressive fibrosis, intimal hyperplasia, and atherosclerosis of the vein wall [4,51. Factors such as exposure to storage media [7, 81, surgical trauma [9, 101, ischemia resulting from the interruption of vasa vasorum [5, 61, and sudden exposure to the higher arterial pressure, resulting in overstretching of the vein wall [9, 11, 121, are involved in early damage and changes of the vein wall. With special storage media and a surgical "no-touch'' technique, this "iatrogenic" damage can be reduced [9, 13, 141. However, damage to the vein graft caused by unavoidable ischemia and overstretching of the vein wall remains. This study was undertaken to evaluate the potential of a supporting, compliant, biodegradable prosthesis to function as a temporary protective scaffold for autologous vein grafts when implanted into the arterial system. Such a protective scaffold might prevent overstretching and allow gradual adaptation to arterial pressures, ie, gradual
arterialization of the vein graft. In previous studies, we [15-191 have already demonstrated the good healing characteristics of such biodegradable prostheses.
Accepted for publication May 21, 1992. Address reprint requests to Dr Wildevuur, Cardiopulmonary Surgery Research Division, University Hospital Groningen, Oostersingel 59, 9713 EZ Groningen, the Netherlands.
0 1993 by
The Society of Thoracic Surgeons
(Ann Thorac Surg 1993;55:427-33)
Material and Methods Male Chinchilla rabbits (n = 34) weighing 3.5 to 4 kg were used for microsurgical implantation of both a control autologous vein graft and a composite vein graft composed of autologous vein supported with a compliant, biodegradable prosthesis [ 151. The side of implantation (ie, right or left carotid artery) was randomized. All animals used in this study received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985).
Biodegradable Prosthesis The compliant, biodegradable prostheses were prepared from 90/10 (wt%) polyurethane/poly-L-lactic acid. The prostheses had an internal diameter of 2.5 mm, a length of 1.5 cm, and a pore size gradient in their 0.3-mm-thick graft wall ranging from 30 pm in the inner region of the graft lattice to 100 pm in the outer region. All prostheses were sterilized with ethylene oxide and evacuated for 24 hours under high vacuum (10 to 5 mm Hg). Degradation of these prostheses is primarily a matter of fragmentation [201.
Surgical Technique The rabbit was anesthetized by an intravenous injection of sodium pentobarbital (Nembutal; Abbott Laboratories, 0003-4975/93/$6.00
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ZWEEPETAL SUPPORTED VEIN GRAFT
North Chicago, IL; 30 mg/kg) into the marginal ear vein. The animal also was intubated, and inhalation anesthesia was accomplished with 1% halothane (Fluothane; Wyerth-Ayerst). Under sterile conditions, a ventral midline incision was made in the neck, and the carotid arteries were carefully exposed. A I-cm segment with a diameter of 2 mm (kO.1 mm) from a branch of the external jugular vein at the mandibular area was used as the control vein graft. This vein graft matched in size with the carotid artery after clamp release. A 1-cm segment with a diameter of 3.2 mm (20.1 mm) from the internal jugular vein was used as the autologous vein graft to create the composite vein graft. All vein grafts were stored for 5 minutes in heparinized saline solution. Then all vein grafts were implanted in the carotid arteries with end-to-end anastomoses by means of standard microsurgical techniques ~91.
To implant the composite vein graft, the exposed carotid artery was first clamped proximally and distally and cut in the middle. Then the distal clamp was released, and the prosthesis (internal diameter, 2.5 mm) was slipped over the distal part of the carotid artery. After implantation of the vein graft, the prosthesis was shifted back over the vein graft, including both anastomoses, thus reducing the diameter of the vein graft from 3.2 mm ( k O . 1 mm) to 2.5 mm and forming a composite vein graft. Interrupted sutures of 9-0 Ethilon on a BV-4 needle (Ethicon, Somerville, NJ) were used to make the vessel anastomoses. The mean carotid artery cross-clamp time was 25 minutes. After hemostasis had been secured by gentle pressure, a solution of lidocaine hydrochloride was applied at the proximal and distal anastomoses to prevent spasm of the carotid artery. Patency was determined by direct inspection for arterial pulsation. After the operation on both carotid arteries, the wound was irrigated with saline solution and closed with 2-0 Dexon. The rabbits were housed individually under controlled environmental conditions with free access to water and normal L.K.K. pellet rabbit food (Hope Farms BV, Woerden, the Netherlands). They did not receive any anticoagulants.
Graft Evaluation and Harvesting Procedure The grafts were evaluated at 1day (n = 6 rabbits), 1 week (n = 6), 2 weeks (n = 6), 3 weeks (n = 8), and 6 weeks (n = 8) after operation. The rabbits were then anesthetized with pentobarbital (Nembutal; 30 mg/kg body weight, administered intravenously). The composite vein grafts and control vein grafts were carefully dissected free, and heparin sodium (1,000 IU, intravenously) was given to prevent clotting caused by the harvesting procedure. All grafts were gently perfused with 6.8% sucrose solution in 0.1 moYL cacodylate buffer and then fixed by pressurecontrolled perfusion (100 mm Hg) with 2% glutaraldehyde in 0.1 m o m cacodylate buffer. Each of the fixed specimens was divided into two equal segments. Each segment was then prepared for and evaluated by routine light or scanning electron microscopy. Both forms of microscopy were used for morpho-
Ann Thorac Surg 1!?93;55:42733
logic evaluation of the endothelial lining. Light microscopy was used for morphologic evaluation of the graft walls, the subendothelial smooth muscle cell layers as well as the adventitia.
Preparation for Scanning Electron Microscopy The grafts, fixed by pressure-controlled perfusion, were dissected and left in the same fixative for 24 hours at 4°C. Next, the specimens were rinsed for 30 minutes in 6.8% sucrose solution in 0.1 mom cacodylate buffer, pH 7.4, to remove excess glutaraldehyde and postfixed for 3 hours in 1% osmium tetroxide in 0.1 m o m cacodylate buffer, pH 7.4, at 4°C. After dehydration in an ethanol series up to 100%, the specimens were dried to the critical point with liquid carbon dioxide. Then they were cut into two or three longitudinal segments and sputter coated with gold (15 nm). These segments were examined in an ISI-Ds-130 scanning electron microscope operated at 7 to 10 kV.
Preparation for Light Microscopy The specimens were dehydrated in alcohol. After immersion in Technovit 8100 (Kulzer GmbH, Wehrheim, Germany) overnight, they were embedded in 40 mL of Technovit 8100 (solution A) and in 1mL of the hardener Technovit 8100 (solution B). After polymerization at room temperature, semithin (2 pm) sections were routinely cut and stained with hematoxylin and eosin and elastin stain (Verhoeff's stain).
Results Unimplanted Vein Grafts Unimplanted external (control) and internal jugular vein grafts (as in the composite vein grafts) consisted of an endothelial layer, an incomplete internal elastic lamina, a media with one to two layers of smooth muscle cells, and an adventitia composed of collagen. Both media and adventitia contained thin elastic laminae.
Control Vein Grafts On day 1, the control vein grafts were deendothelialized over approximately 80% of the graft surface. The thin smooth muscle cell layers were completely disrupted, and many polymorphonuclear cells (PMNs) had infiltrated into the edematous wall (Fig la). Fragments of thin elastic laminae were observed in the vein wall (Fig lc). At 1 week, reendothelialization had occurred over approximately 50% of the graft surface. Disruption of the vein wall and edema with many PMNs were still observed (Fig 2a). At 2 weeks, all control vein grafts were completely reendothelialized. At this time, as well as at 3 weeks, irregularly oriented fibrocytes had partly replaced the areas of disruption with PMNs (Fig 3a). Elastic laminae were no longer seen (Fig 3c). At 6 weeks, all areas of inflammation in the control vein grafts were completely replaced by mature fibrous tissue with an irregular structure (Fig 4a).
Composite Vein Grafts The composite vein grafts were also deendothelialized over approximately 80% of the graft surface on day 1.
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Fig I. Light micrographs of (a, c) a control vein graft and (b, d ) a composite vein graft 1 day after implantation. (a) In the control vein graft, there is complete disruption of smooth muscle cell layers with infiltration of many polymorphonuclear cells into the edematous vein wall. (b) In the composite vein graft, most smooth muscle cell layers are preserved. The vein wall is edematous. (c) In the control vein graft, only fragments of elastic laminae are present. (d) Elastic laminae are preserved in the composite vein graft. (a and b, hematoxylin and eosin; c and d, elastin stain lverhoeff's stain]; all, X250.) (E = elastic laminae;'I = prosthesis; V = vein wall.)
Although partial disruption of the smooth muscle cell layers was observed, most of them were still preserved and were arranged longitudinally. The vein wall had an edematous appearance in the deep layers but showed significantly less PMN infiltration than the control vein
grafts (Fig lb). The thin elastic laminae normally present in the vein wall were preserved (Fig Id). At 1 week, the composite vein grafts were reendothelialized over approximately 75% of their surface. The vein wall by then showed regular layers of longitudinally
430
ZWEEPETAL SUPPORTED VEIN CRAFT
Ann Thorac Surg 1993;5542733
Fig 2 . Light micrographs of (a) a control vein graft and (b) a composite vein graft 1 week after implantation. (a) Note the disruption of the smooth muscle cell layers in the control vein graft. Polymorphonuc[ear cells (arrows) are abundant in the edematous vein wall. (b) In contrast, the composite vein graft shows regular layers of immature fibroblasts with only a few polymorphonuclear cells. (Hematoxylin and eosin; x2.50.) = prosthesis; V = vein wall.)
oriented, immature fibroblasts with few PMNs (Fig 2b). Edema had disappeared, and thin elastic laminae were present. The prosthesis was surrounded and infitrated by many macrophages and multinuclear giant cells. At 2 weeks, endothelial coverage was complete. The vein wall had regular layers of longitudinally oriented, immature fibroblasts with only a very few PMNs. Thin elastic laminae were still present in the vein wall. Many macrophages and multinuclear giant cells were observed in and around the prosthetic material. At 3 weeks, the vein wall had a similar aspect, but no PMNs were present (Figs 3b, 3d). By then, the prosthesis had started to fragment. At 6 weeks, the vein wall had gradually arterialized; it had increased in thickness and consisted of eight to 12 layers of immature fibroblasts. Moreover, in some areas, these layers had a prevalence for circular orientation (Fig 4b). Thin elastic laminae were observed but were less pronounced. Fragmentation of the prosthesis had continued slowly, and many macrophages and multinuclear giant cells were present.
Comment The present study demonstrates that a compliant, biodegradable prosthesis can indeed function as a protective scaffold for autologous vein grafts when implanted into the arterial circulation: the prosthesis prevents overstretching of vein grafts, preventing subsequent damage [21]. We observed the preservation of smooth muscle cell layers and elastic laminae and only a minor inflammatory
(P
reaction with few PMNs in the vein wall after 1 day of implantation. Moreover, the slowly degrading prosthesis allowed regular and gradual arterialization of the vein wall. In contrast, the unsupported control vein graft wall was completely disrupted after 1 day of implantation and showed severe inflammatory reaction of PMNs and edema. The walls healed with the formation of mature fibrous tissue without elastic laminae, resulting in fibrosis of the vein wall at 6 weeks. The early changes observed in our control vein grafts resemble the changes described in coronary artery vein grafts in humans. After implantation, a large degree of deendothelialization occurs, and the media shows disruption of smooth muscle cell layers with edema and leukocyte infiltration [MI.All these changes in the vein wall might originate from damage to the vein graft during preparation [7,81 or implantation [9, 101 and from ischemia [5, 61 and overstretching by the higher arterial pressures [9, 11, 121. Regeneration occurs with reendothelialization and progressive medial fibrosis [MI,as was observed in our control vein grafts. Later changes include intimal hyperplasia and atherosclerosis [4, 51. Although many growth factors and enzymes might be involved in this process, a key role in the pathogenesis of vein graft degeneration is attributed to platelet-derived growth factor. It can be released not only by platelets but also by endothelial cells, smooth muscle cells, and monocytes [22-241. To date, little attention has been paid to the influence on vein graft degeneration of the inflammatory reaction of
Ann Thorac Surg 1993;55427-33
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431
Fig 3. Light micrographs of (a, c) a control vein graft and (b, d ) a composite vein graft 3 weeks after implantation. (a) Note the irregular arrangement offibrocytes in the control vein graft. (b) The composite vein graft shows regular proliferation of immature fibroblasts. (c) In the control vein graft, elastic laminae have completely disappeared. (d) In the composite vein graft, elastic laminae are present. (a and b, hematoxylin and eosin; c and d , elastin stain [Verhoeff's stain]; all, ~ 2 5 0 . (P ) = prosthesis; V = vein wall.)
PMNs in the vein wall caused by the disruption of the smooth muscle cell layers after implantation. In our rabbit model, the severe inflammatory reaction with PMNs in the disrupted layers of the control vein grafts was especially striking and lasted for the first 3 weeks in contrast to
the composite vein grafts. These activated PMNs release several enzymes, including elastase and collagenase [25]. These enzymes lyse, among others, elastic laminae and collagen, further destroying the integrity of the vein graft matrix. These inflammatory processes lead to fibrosis, as
432
ZWEEPETAL SUPPORTED VEIN GRAFT
Ann Thorac Surg
1993:55427-33
Fig 4. Light micrographs of (a) a control vein graft and fb) a composite oern graft 6 weeks after implantation. (a) Note the irregular orientation of fibrocytes in the control vein graft. (b) The vein wall of the composite vein graft has increased in thickness and shows several layers of circularly oriented, immature fibroblasts (*) underneath the regular, longitudinally oriented layers. fHematoxylin and eosin; X2.50.) fP = prosthesis; V = vein wall.)
observed in our unsupported control vein grafts, and might therefore play an additional role in the development of degenerative changes in vein grafts. In contrast, the composite vein grafts showed no complete disruption of the smooth muscle cell layers and only a minor inflammatory reaction on 1 day. Already after 1 week, the regular, longitudinally oriented layers of immature fibroblasts had regenerated, most likely from transformed preserved smooth muscle cell layers in the vein graft wall [4,6]. This transformation into fibroblasts might be induced by ischemia resulting from the unavoidable interruption of vasa vasorum [6,26]. The organized structure of the supported vein graft walls might be due to the preservation of its matrix function, as demonstrated by the preservation of elastic laminae. During the following weeks, the vein wall gradually increased in thickness by regular proliferation of fibroblasts, which in some areas had a prevalence for circular orientation. Elastic laminae were less pronounced at 6 weeks, probably because of elastase produced by macrophages 1271 that were present in the chronic reaction against the prosthetic material. The fibroblasts remained immature and did not become mature, ie, fibrocytes, as in the unsupported vein grafts. Long-term studies are required to see if these immature fibroblasts will differentiate into myofibroblasts and myoblasts and will produce elastin, thus resulting in the formation of an arteriallike structure. In conclusion, this study demonstrated that a compliant, biodegradable prosthesis can function as a protective scaffold for autologous vein grafts. The reduced damage is
especially expressed by a reduced infiltration of PMNs. Our rabbit model appeared to be very sensitive for evaluating damage to vein grafts, resulting in already great short-term consequences for the control vein grafts. Longterm studies are warranted to determine the ultimate role of the supporting prosthesis in terms of the function and biological behavior of autologous vein grafts implanted into the arterial system of rabbits. This study has been supported by The Netherlands Technological Foundation STW No. 88, 1482. We thank J. Elstrodt for microsurgical assistance, H. van Goor for the light microscopic handling procedures, and H. R. A. Meiborg for the photography.
References 1. Sottiurai VS, Batson RC. Autogenous vein grafts: experimental studies. In: Stanley JC, Burkel WE, Lindenauer SM,
Bartlett RH, Turcotte JG, eds. Biologic and synthetic vascular prostheses. New York: Grune & Stratton, 1982311. 2. Stanley JC, Graham LM, Whitehous WM, Lindenauer SM, Zelenock GB, Cronenwett JL. Autogenous saphenous vein as an arterial graft: clinical status. In: Stanley JC, Burkel WE, Lindenauer SM, Bartlett RH, Turcotte JG, eds. Biologic and synthetic vascular prostheses. New York: Grune & Stratton,
1982333. 3. Lytle BW, Loop FD, Cosgrove DM, Ratliff NB, Easley K, Taylor PC. Long-term (5 to 12 years) serial studies of internal
mammary artery and saphenous vein coronary bypass grafts.
J Thorac Cardiovasc Surg 1985;89:24&58. 4. Dilley RJ, McGeachie JK, Prendergast FJ. A review of the
Ann Thorac Surg 1993;55427-33
histologic changes in vein-to-artery grafts, with particular reference to intimal hyperplasia. Arch Surg 1988;123:691-6. 5. Fonkalsrud EW, Sanchez M, Zerubavel R. Morphological evaluation of canine autogenous vein grafts in the arterial circulation. Surgery 1978;84:25%64. 6. Brody WR, Angell WW, Kosek JC. Histologic fate of the venous coronary artery bypass in dogs. Am J Pathol1972;66: 111-9. 7. Abbot WM, Mundth ED, Austen WG. Autogenous vein grafts. Effects of simple storage on structural properties. Surg Forum 1973;2426&2. 8. Baumann FG, Catinella FP, Cunningham JN, Spencer FC. Vein contraction and smooth muscle cell extension as causes of endothelial damage during graft preparation. Ann Surg 1981;194199-21 1. 9. Adcock OT, Adcock GLD, Wheeler JR, Gregory RT, Snyder SO, Gayle RG. Optimal techniques for harvesting and preparation of reversed autogenous vein grafts for use as arterial substitutes: a review. Surgery 1984;96:886-94. 10. King P, Royle JP. Autogenous vein grafting in atheromatous rabbits. Cardiovasc Res 1971;6:627-33. 11. Ramos JR, Berger K, Mansfield PB, Sauvage LR. Histologic fate and endothelial changes of distended and nondistended vein grafts. Ann Surg 1976;183:205-28. 12. Angelini GD, Passani SL, Breckenridge IM, Newby AC. Nature and pressure dependence of damage induced by distension of human saphenous vein coronary artery bypass grafts. Cardiovasc Res 1987;21:902-7. 13. Gottlot R. The preservation of venous endothelium by dissection without touching and by an atraumatic technique of vascular anastomosis. Minerva Chir 1977;32:69%700. 14. LoGerfo FW, Quist WC, Cantelmo NL, Haudenschild CC. Integrity of vein grafts as a function of initial intimal and medial preservation. Circulation 1983;68(Suppl2):117-24. 15. Van der Lei B, Bartels HL, Nieuwenhuis P, Wildevuur CRH. Microporous, compliant, biodegradable vascular grafts for the regeneration of the arterial wall in rat abdominal aorta. Surgery 1985;98:955-63. 16. Van der Lei B, Wildevuur CRH, Dijk F, Bhauw EH, Molenaar I, Nieuwenhuis P. Sequential studies of arterial wall regeneration in microporous, compliant, biodegradable small-
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17. 18.
19.
20.
21.
22.
23. 24.
25. 26. 27.
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caliber vascular grafts in rats. J Thorac Cardiovasc Surg 1983;9369.!%707. Van der Lei B, Wildevuur CRH. From synthetic, microporous, compliant, biodegradable small-caliber vascular graft to a new artery. Thorac Cardiovasc Surg 1989;3733747. Van der Lei B, Wildevuur CRH. The use of biodegradable grafts for arterial reconstructions. A review of the Groningen experience. In: Kogel HC, ed. The prosthetic substitution of blood vessels: actual state and future development. Miinchen: Quintessenz Verlags-GmbH, 1991:67. Bartels HL, Van der Lei B. Small-caliber vascular grafting in the rat abdominal aorta with biodegradable vascular prostheses. Lab Anim 1988;22:1224. Hinrichs WLJ, Kuit J, Feil H, Wildevuur CRH, Feijen J. The in vivo fragmentation of microporous polyurethane- and copolyesterether elastomer-based vascular prostheses. Biomaterials (in press). Barra JA, Volant A, Leroy JP, et al. Constrictive perivenous mesh prosthesis for preservation of vein integrity. Experimental results and application for coronary bypass grafting. J Thorac Cardiovasc Surg 1986;92:330-6. Wilcox JN, Smith KM, Williams LT, Schwartz SM, Gordon D. Platelet-derived growth factor mRNA detection in human atherosclerotic plaques by in situ hybridization. J Clin Invest 1988;82:1134-43. Ross R. The pathogenesis of atherosclerosisan update. N Engl J Med 1986;314:488-500. Walker LN, Bowen-Pope DF, Ross R, Reidy MA. Production of platelet-derived growth factor-like molecules by cultured arterial smooth muscle cells accompanies proliferation after arterial injury. Cell Biol 1986;83:7311-5. Smolen JE, Boxer LA. Function of neutrophils. In: Williams WJ, Beutler E, Erslev AJ, Lichtman MA, eds. Hematology. 4th ed. New York: McGraw-Hill, 1990:780. McCabe M, Cunningham GJ, Wyatt AP, Rothnie NG, Taylor GW. A histological and histochemical examination of autogenous vein grafts. Br J Surg 1967;56:147-55. Groopman JE, Golde DW. Biochemistry and function of monocytes and macrophages. In: Williams WJ, Beutler E, Erslev AJ, Lichtman MA, eds. Hematology. 4th ed. New York McGraw-Hill, 1990:869.
To evaluate the potential of a supporting, compliant, biodegradable prosthesis to function as a temporary protective scaffold for autologous vein grafts in the arterial circulation, we implanted vein grafts into the carotid arteries of rabbits, either with (composite grafts) or without (control grafts) such a supporting prosthesis, and evaluated them up to 6 weeks. The control vein grafts showed edema and severe medial disruption with infiltration of polymorphonuclear cells on day 1. Over the study, irregular fibrocyte formation resulted in the
formation of a fibrotic vein wall. In contrast, the composite vein grafts showed preservation of smooth muscle cell layers and elastic laminae with a minor inflammatory response. Regular proliferation of fibroblasts, which in some areas were circularly oriented, was observed. We conclude that a supporting, compliant, biodegradable prosthesis can function as a protective scaffold for vein grafts in the arterial circulation, thus reducing damage to the vein graft wall and allowing gradual arterialization.
hen used as an arterial substitute, autologous vein grafts undergo degenerative changes during longterm implantation [l, 21. This is reflected by their low long-term patency rate compared with the patency rate of arterial autografts [3]. The degenerative changes in the vein wall might have their origin in the early postoperative period. After implantation, the vein wall shows a certain degree of deendothelialization and medial damage, including disruption, fibroblast transformation of smooth muscle cells, and an inflammatory response in the vein wall with edema [4-61. All these changes are alleged to lead to progressive fibrosis, intimal hyperplasia, and atherosclerosis of the vein wall [4,51. Factors such as exposure to storage media [7, 81, surgical trauma [9, 101, ischemia resulting from the interruption of vasa vasorum [5, 61, and sudden exposure to the higher arterial pressure, resulting in overstretching of the vein wall [9, 11, 121, are involved in early damage and changes of the vein wall. With special storage media and a surgical "no-touch'' technique, this "iatrogenic" damage can be reduced [9, 13, 141. However, damage to the vein graft caused by unavoidable ischemia and overstretching of the vein wall remains. This study was undertaken to evaluate the potential of a supporting, compliant, biodegradable prosthesis to function as a temporary protective scaffold for autologous vein grafts when implanted into the arterial system. Such a protective scaffold might prevent overstretching and allow gradual adaptation to arterial pressures, ie, gradual
arterialization of the vein graft. In previous studies, we [15-191 have already demonstrated the good healing characteristics of such biodegradable prostheses.
Accepted for publication May 21, 1992. Address reprint requests to Dr Wildevuur, Cardiopulmonary Surgery Research Division, University Hospital Groningen, Oostersingel 59, 9713 EZ Groningen, the Netherlands.
0 1993 by
The Society of Thoracic Surgeons
(Ann Thorac Surg 1993;55:427-33)
Material and Methods Male Chinchilla rabbits (n = 34) weighing 3.5 to 4 kg were used for microsurgical implantation of both a control autologous vein graft and a composite vein graft composed of autologous vein supported with a compliant, biodegradable prosthesis [ 151. The side of implantation (ie, right or left carotid artery) was randomized. All animals used in this study received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985).
Biodegradable Prosthesis The compliant, biodegradable prostheses were prepared from 90/10 (wt%) polyurethane/poly-L-lactic acid. The prostheses had an internal diameter of 2.5 mm, a length of 1.5 cm, and a pore size gradient in their 0.3-mm-thick graft wall ranging from 30 pm in the inner region of the graft lattice to 100 pm in the outer region. All prostheses were sterilized with ethylene oxide and evacuated for 24 hours under high vacuum (10 to 5 mm Hg). Degradation of these prostheses is primarily a matter of fragmentation [201.
Surgical Technique The rabbit was anesthetized by an intravenous injection of sodium pentobarbital (Nembutal; Abbott Laboratories, 0003-4975/93/$6.00
428
ZWEEPETAL SUPPORTED VEIN GRAFT
North Chicago, IL; 30 mg/kg) into the marginal ear vein. The animal also was intubated, and inhalation anesthesia was accomplished with 1% halothane (Fluothane; Wyerth-Ayerst). Under sterile conditions, a ventral midline incision was made in the neck, and the carotid arteries were carefully exposed. A I-cm segment with a diameter of 2 mm (kO.1 mm) from a branch of the external jugular vein at the mandibular area was used as the control vein graft. This vein graft matched in size with the carotid artery after clamp release. A 1-cm segment with a diameter of 3.2 mm (20.1 mm) from the internal jugular vein was used as the autologous vein graft to create the composite vein graft. All vein grafts were stored for 5 minutes in heparinized saline solution. Then all vein grafts were implanted in the carotid arteries with end-to-end anastomoses by means of standard microsurgical techniques ~91.
To implant the composite vein graft, the exposed carotid artery was first clamped proximally and distally and cut in the middle. Then the distal clamp was released, and the prosthesis (internal diameter, 2.5 mm) was slipped over the distal part of the carotid artery. After implantation of the vein graft, the prosthesis was shifted back over the vein graft, including both anastomoses, thus reducing the diameter of the vein graft from 3.2 mm ( k O . 1 mm) to 2.5 mm and forming a composite vein graft. Interrupted sutures of 9-0 Ethilon on a BV-4 needle (Ethicon, Somerville, NJ) were used to make the vessel anastomoses. The mean carotid artery cross-clamp time was 25 minutes. After hemostasis had been secured by gentle pressure, a solution of lidocaine hydrochloride was applied at the proximal and distal anastomoses to prevent spasm of the carotid artery. Patency was determined by direct inspection for arterial pulsation. After the operation on both carotid arteries, the wound was irrigated with saline solution and closed with 2-0 Dexon. The rabbits were housed individually under controlled environmental conditions with free access to water and normal L.K.K. pellet rabbit food (Hope Farms BV, Woerden, the Netherlands). They did not receive any anticoagulants.
Graft Evaluation and Harvesting Procedure The grafts were evaluated at 1day (n = 6 rabbits), 1 week (n = 6), 2 weeks (n = 6), 3 weeks (n = 8), and 6 weeks (n = 8) after operation. The rabbits were then anesthetized with pentobarbital (Nembutal; 30 mg/kg body weight, administered intravenously). The composite vein grafts and control vein grafts were carefully dissected free, and heparin sodium (1,000 IU, intravenously) was given to prevent clotting caused by the harvesting procedure. All grafts were gently perfused with 6.8% sucrose solution in 0.1 moYL cacodylate buffer and then fixed by pressurecontrolled perfusion (100 mm Hg) with 2% glutaraldehyde in 0.1 m o m cacodylate buffer. Each of the fixed specimens was divided into two equal segments. Each segment was then prepared for and evaluated by routine light or scanning electron microscopy. Both forms of microscopy were used for morpho-
Ann Thorac Surg 1!?93;55:42733
logic evaluation of the endothelial lining. Light microscopy was used for morphologic evaluation of the graft walls, the subendothelial smooth muscle cell layers as well as the adventitia.
Preparation for Scanning Electron Microscopy The grafts, fixed by pressure-controlled perfusion, were dissected and left in the same fixative for 24 hours at 4°C. Next, the specimens were rinsed for 30 minutes in 6.8% sucrose solution in 0.1 mom cacodylate buffer, pH 7.4, to remove excess glutaraldehyde and postfixed for 3 hours in 1% osmium tetroxide in 0.1 m o m cacodylate buffer, pH 7.4, at 4°C. After dehydration in an ethanol series up to 100%, the specimens were dried to the critical point with liquid carbon dioxide. Then they were cut into two or three longitudinal segments and sputter coated with gold (15 nm). These segments were examined in an ISI-Ds-130 scanning electron microscope operated at 7 to 10 kV.
Preparation for Light Microscopy The specimens were dehydrated in alcohol. After immersion in Technovit 8100 (Kulzer GmbH, Wehrheim, Germany) overnight, they were embedded in 40 mL of Technovit 8100 (solution A) and in 1mL of the hardener Technovit 8100 (solution B). After polymerization at room temperature, semithin (2 pm) sections were routinely cut and stained with hematoxylin and eosin and elastin stain (Verhoeff's stain).
Results Unimplanted Vein Grafts Unimplanted external (control) and internal jugular vein grafts (as in the composite vein grafts) consisted of an endothelial layer, an incomplete internal elastic lamina, a media with one to two layers of smooth muscle cells, and an adventitia composed of collagen. Both media and adventitia contained thin elastic laminae.
Control Vein Grafts On day 1, the control vein grafts were deendothelialized over approximately 80% of the graft surface. The thin smooth muscle cell layers were completely disrupted, and many polymorphonuclear cells (PMNs) had infiltrated into the edematous wall (Fig la). Fragments of thin elastic laminae were observed in the vein wall (Fig lc). At 1 week, reendothelialization had occurred over approximately 50% of the graft surface. Disruption of the vein wall and edema with many PMNs were still observed (Fig 2a). At 2 weeks, all control vein grafts were completely reendothelialized. At this time, as well as at 3 weeks, irregularly oriented fibrocytes had partly replaced the areas of disruption with PMNs (Fig 3a). Elastic laminae were no longer seen (Fig 3c). At 6 weeks, all areas of inflammation in the control vein grafts were completely replaced by mature fibrous tissue with an irregular structure (Fig 4a).
Composite Vein Grafts The composite vein grafts were also deendothelialized over approximately 80% of the graft surface on day 1.
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Fig I. Light micrographs of (a, c) a control vein graft and (b, d ) a composite vein graft 1 day after implantation. (a) In the control vein graft, there is complete disruption of smooth muscle cell layers with infiltration of many polymorphonuclear cells into the edematous vein wall. (b) In the composite vein graft, most smooth muscle cell layers are preserved. The vein wall is edematous. (c) In the control vein graft, only fragments of elastic laminae are present. (d) Elastic laminae are preserved in the composite vein graft. (a and b, hematoxylin and eosin; c and d, elastin stain lverhoeff's stain]; all, X250.) (E = elastic laminae;'I = prosthesis; V = vein wall.)
Although partial disruption of the smooth muscle cell layers was observed, most of them were still preserved and were arranged longitudinally. The vein wall had an edematous appearance in the deep layers but showed significantly less PMN infiltration than the control vein
grafts (Fig lb). The thin elastic laminae normally present in the vein wall were preserved (Fig Id). At 1 week, the composite vein grafts were reendothelialized over approximately 75% of their surface. The vein wall by then showed regular layers of longitudinally
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Fig 2 . Light micrographs of (a) a control vein graft and (b) a composite vein graft 1 week after implantation. (a) Note the disruption of the smooth muscle cell layers in the control vein graft. Polymorphonuc[ear cells (arrows) are abundant in the edematous vein wall. (b) In contrast, the composite vein graft shows regular layers of immature fibroblasts with only a few polymorphonuclear cells. (Hematoxylin and eosin; x2.50.) = prosthesis; V = vein wall.)
oriented, immature fibroblasts with few PMNs (Fig 2b). Edema had disappeared, and thin elastic laminae were present. The prosthesis was surrounded and infitrated by many macrophages and multinuclear giant cells. At 2 weeks, endothelial coverage was complete. The vein wall had regular layers of longitudinally oriented, immature fibroblasts with only a very few PMNs. Thin elastic laminae were still present in the vein wall. Many macrophages and multinuclear giant cells were observed in and around the prosthetic material. At 3 weeks, the vein wall had a similar aspect, but no PMNs were present (Figs 3b, 3d). By then, the prosthesis had started to fragment. At 6 weeks, the vein wall had gradually arterialized; it had increased in thickness and consisted of eight to 12 layers of immature fibroblasts. Moreover, in some areas, these layers had a prevalence for circular orientation (Fig 4b). Thin elastic laminae were observed but were less pronounced. Fragmentation of the prosthesis had continued slowly, and many macrophages and multinuclear giant cells were present.
Comment The present study demonstrates that a compliant, biodegradable prosthesis can indeed function as a protective scaffold for autologous vein grafts when implanted into the arterial circulation: the prosthesis prevents overstretching of vein grafts, preventing subsequent damage [21]. We observed the preservation of smooth muscle cell layers and elastic laminae and only a minor inflammatory
(P
reaction with few PMNs in the vein wall after 1 day of implantation. Moreover, the slowly degrading prosthesis allowed regular and gradual arterialization of the vein wall. In contrast, the unsupported control vein graft wall was completely disrupted after 1 day of implantation and showed severe inflammatory reaction of PMNs and edema. The walls healed with the formation of mature fibrous tissue without elastic laminae, resulting in fibrosis of the vein wall at 6 weeks. The early changes observed in our control vein grafts resemble the changes described in coronary artery vein grafts in humans. After implantation, a large degree of deendothelialization occurs, and the media shows disruption of smooth muscle cell layers with edema and leukocyte infiltration [MI.All these changes in the vein wall might originate from damage to the vein graft during preparation [7,81 or implantation [9, 101 and from ischemia [5, 61 and overstretching by the higher arterial pressures [9, 11, 121. Regeneration occurs with reendothelialization and progressive medial fibrosis [MI,as was observed in our control vein grafts. Later changes include intimal hyperplasia and atherosclerosis [4, 51. Although many growth factors and enzymes might be involved in this process, a key role in the pathogenesis of vein graft degeneration is attributed to platelet-derived growth factor. It can be released not only by platelets but also by endothelial cells, smooth muscle cells, and monocytes [22-241. To date, little attention has been paid to the influence on vein graft degeneration of the inflammatory reaction of
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Fig 3. Light micrographs of (a, c) a control vein graft and (b, d ) a composite vein graft 3 weeks after implantation. (a) Note the irregular arrangement offibrocytes in the control vein graft. (b) The composite vein graft shows regular proliferation of immature fibroblasts. (c) In the control vein graft, elastic laminae have completely disappeared. (d) In the composite vein graft, elastic laminae are present. (a and b, hematoxylin and eosin; c and d , elastin stain [Verhoeff's stain]; all, ~ 2 5 0 . (P ) = prosthesis; V = vein wall.)
PMNs in the vein wall caused by the disruption of the smooth muscle cell layers after implantation. In our rabbit model, the severe inflammatory reaction with PMNs in the disrupted layers of the control vein grafts was especially striking and lasted for the first 3 weeks in contrast to
the composite vein grafts. These activated PMNs release several enzymes, including elastase and collagenase [25]. These enzymes lyse, among others, elastic laminae and collagen, further destroying the integrity of the vein graft matrix. These inflammatory processes lead to fibrosis, as
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Fig 4. Light micrographs of (a) a control vein graft and fb) a composite oern graft 6 weeks after implantation. (a) Note the irregular orientation of fibrocytes in the control vein graft. (b) The vein wall of the composite vein graft has increased in thickness and shows several layers of circularly oriented, immature fibroblasts (*) underneath the regular, longitudinally oriented layers. fHematoxylin and eosin; X2.50.) fP = prosthesis; V = vein wall.)
observed in our unsupported control vein grafts, and might therefore play an additional role in the development of degenerative changes in vein grafts. In contrast, the composite vein grafts showed no complete disruption of the smooth muscle cell layers and only a minor inflammatory reaction on 1 day. Already after 1 week, the regular, longitudinally oriented layers of immature fibroblasts had regenerated, most likely from transformed preserved smooth muscle cell layers in the vein graft wall [4,6]. This transformation into fibroblasts might be induced by ischemia resulting from the unavoidable interruption of vasa vasorum [6,26]. The organized structure of the supported vein graft walls might be due to the preservation of its matrix function, as demonstrated by the preservation of elastic laminae. During the following weeks, the vein wall gradually increased in thickness by regular proliferation of fibroblasts, which in some areas had a prevalence for circular orientation. Elastic laminae were less pronounced at 6 weeks, probably because of elastase produced by macrophages 1271 that were present in the chronic reaction against the prosthetic material. The fibroblasts remained immature and did not become mature, ie, fibrocytes, as in the unsupported vein grafts. Long-term studies are required to see if these immature fibroblasts will differentiate into myofibroblasts and myoblasts and will produce elastin, thus resulting in the formation of an arteriallike structure. In conclusion, this study demonstrated that a compliant, biodegradable prosthesis can function as a protective scaffold for autologous vein grafts. The reduced damage is
especially expressed by a reduced infiltration of PMNs. Our rabbit model appeared to be very sensitive for evaluating damage to vein grafts, resulting in already great short-term consequences for the control vein grafts. Longterm studies are warranted to determine the ultimate role of the supporting prosthesis in terms of the function and biological behavior of autologous vein grafts implanted into the arterial system of rabbits. This study has been supported by The Netherlands Technological Foundation STW No. 88, 1482. We thank J. Elstrodt for microsurgical assistance, H. van Goor for the light microscopic handling procedures, and H. R. A. Meiborg for the photography.
References 1. Sottiurai VS, Batson RC. Autogenous vein grafts: experimental studies. In: Stanley JC, Burkel WE, Lindenauer SM,
Bartlett RH, Turcotte JG, eds. Biologic and synthetic vascular prostheses. New York: Grune & Stratton, 1982311. 2. Stanley JC, Graham LM, Whitehous WM, Lindenauer SM, Zelenock GB, Cronenwett JL. Autogenous saphenous vein as an arterial graft: clinical status. In: Stanley JC, Burkel WE, Lindenauer SM, Bartlett RH, Turcotte JG, eds. Biologic and synthetic vascular prostheses. New York: Grune & Stratton,
1982333. 3. Lytle BW, Loop FD, Cosgrove DM, Ratliff NB, Easley K, Taylor PC. Long-term (5 to 12 years) serial studies of internal
mammary artery and saphenous vein coronary bypass grafts.
J Thorac Cardiovasc Surg 1985;89:24&58. 4. Dilley RJ, McGeachie JK, Prendergast FJ. A review of the
Ann Thorac Surg 1993;55427-33
histologic changes in vein-to-artery grafts, with particular reference to intimal hyperplasia. Arch Surg 1988;123:691-6. 5. Fonkalsrud EW, Sanchez M, Zerubavel R. Morphological evaluation of canine autogenous vein grafts in the arterial circulation. Surgery 1978;84:25%64. 6. Brody WR, Angell WW, Kosek JC. Histologic fate of the venous coronary artery bypass in dogs. Am J Pathol1972;66: 111-9. 7. Abbot WM, Mundth ED, Austen WG. Autogenous vein grafts. Effects of simple storage on structural properties. Surg Forum 1973;2426&2. 8. Baumann FG, Catinella FP, Cunningham JN, Spencer FC. Vein contraction and smooth muscle cell extension as causes of endothelial damage during graft preparation. Ann Surg 1981;194199-21 1. 9. Adcock OT, Adcock GLD, Wheeler JR, Gregory RT, Snyder SO, Gayle RG. Optimal techniques for harvesting and preparation of reversed autogenous vein grafts for use as arterial substitutes: a review. Surgery 1984;96:886-94. 10. King P, Royle JP. Autogenous vein grafting in atheromatous rabbits. Cardiovasc Res 1971;6:627-33. 11. Ramos JR, Berger K, Mansfield PB, Sauvage LR. Histologic fate and endothelial changes of distended and nondistended vein grafts. Ann Surg 1976;183:205-28. 12. Angelini GD, Passani SL, Breckenridge IM, Newby AC. Nature and pressure dependence of damage induced by distension of human saphenous vein coronary artery bypass grafts. Cardiovasc Res 1987;21:902-7. 13. Gottlot R. The preservation of venous endothelium by dissection without touching and by an atraumatic technique of vascular anastomosis. Minerva Chir 1977;32:69%700. 14. LoGerfo FW, Quist WC, Cantelmo NL, Haudenschild CC. Integrity of vein grafts as a function of initial intimal and medial preservation. Circulation 1983;68(Suppl2):117-24. 15. Van der Lei B, Bartels HL, Nieuwenhuis P, Wildevuur CRH. Microporous, compliant, biodegradable vascular grafts for the regeneration of the arterial wall in rat abdominal aorta. Surgery 1985;98:955-63. 16. Van der Lei B, Wildevuur CRH, Dijk F, Bhauw EH, Molenaar I, Nieuwenhuis P. Sequential studies of arterial wall regeneration in microporous, compliant, biodegradable small-
ZWEEPETAL SUPPORTED VEIN GRAFT
17. 18.
19.
20.
21.
22.
23. 24.
25. 26. 27.
433
caliber vascular grafts in rats. J Thorac Cardiovasc Surg 1983;9369.!%707. Van der Lei B, Wildevuur CRH. From synthetic, microporous, compliant, biodegradable small-caliber vascular graft to a new artery. Thorac Cardiovasc Surg 1989;3733747. Van der Lei B, Wildevuur CRH. The use of biodegradable grafts for arterial reconstructions. A review of the Groningen experience. In: Kogel HC, ed. The prosthetic substitution of blood vessels: actual state and future development. Miinchen: Quintessenz Verlags-GmbH, 1991:67. Bartels HL, Van der Lei B. Small-caliber vascular grafting in the rat abdominal aorta with biodegradable vascular prostheses. Lab Anim 1988;22:1224. Hinrichs WLJ, Kuit J, Feil H, Wildevuur CRH, Feijen J. The in vivo fragmentation of microporous polyurethane- and copolyesterether elastomer-based vascular prostheses. Biomaterials (in press). Barra JA, Volant A, Leroy JP, et al. Constrictive perivenous mesh prosthesis for preservation of vein integrity. Experimental results and application for coronary bypass grafting. J Thorac Cardiovasc Surg 1986;92:330-6. Wilcox JN, Smith KM, Williams LT, Schwartz SM, Gordon D. Platelet-derived growth factor mRNA detection in human atherosclerotic plaques by in situ hybridization. J Clin Invest 1988;82:1134-43. Ross R. The pathogenesis of atherosclerosisan update. N Engl J Med 1986;314:488-500. Walker LN, Bowen-Pope DF, Ross R, Reidy MA. Production of platelet-derived growth factor-like molecules by cultured arterial smooth muscle cells accompanies proliferation after arterial injury. Cell Biol 1986;83:7311-5. Smolen JE, Boxer LA. Function of neutrophils. In: Williams WJ, Beutler E, Erslev AJ, Lichtman MA, eds. Hematology. 4th ed. New York: McGraw-Hill, 1990:780. McCabe M, Cunningham GJ, Wyatt AP, Rothnie NG, Taylor GW. A histological and histochemical examination of autogenous vein grafts. Br J Surg 1967;56:147-55. Groopman JE, Golde DW. Biochemistry and function of monocytes and macrophages. In: Williams WJ, Beutler E, Erslev AJ, Lichtman MA, eds. Hematology. 4th ed. New York McGraw-Hill, 1990:869.