Structural and functional changes in vein bypass graft

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1 Reduction of Intimal and Medial Thickening in Sheathed Vein Grafts Heinz Robert Zurbrügg, MD, Markus Wied, Gianni D. Angelini, FRCS, and Roland Hetzer, MD, PhD Bristol Heart Institute, University of Bristol, Bristol, England Background. Arterial pressures are described as an important factor in the development of graft degeneration and in reduced patency rate in vein bypass grafts. Sheathing of the graft with a pressure resistant mesh tubing might slow down this development. Methods. Saphenous vein grafts were implanted into the carotid arteries of five pigs in order to evaluate the influence on myointimal hyperplasia of a compliant Phynox mesh tubing (a wrought Cobalt-Chromium- Nickel-Molybdenum-Iron Alloy), which surrounded autologous vein grafts that were exposed to arterial pressure. Each pig was operated on using a sheathed vein graft (biocompound-graft, a hybrid vascular prosthesis) on one side and an untreated saphenous vein on the other. Results. After 4 weeks intimal hyperplastic changes were found in all histological sections. The wall thickness (medial and intimal layer) varied from 351 m to 432 m in the biocompound-graft and from 391 m to 1196 m in the native vein grafts (p < 0.05, n 5). Severe myocytial and fibroblast proliferation was only found in the control grafts. Cellularity of the medial layer differed at sites of maximal cellular density and ranged from 11 to 12 cells in the biocompound-graft and from 17 to 18 cells per counting field in the native vein grafts (p < 0.05, n 5). Conclusions. External support of vein grafts reduces intimal and medial layer proliferation. The findings of this study are in accordance with the results reported by other research groups. (Ann Thorac Surg 1999;68:79 83) 1999 by The Society of Thoracic Surgeons Structural and functional changes in vein bypass graft walls might be seen as a symptom or a pathogenic factor for graft degeneration and reduced patency rate. Brody and associates described arterial pressure as an important factor in the development of these changes [1]. Angelini and associates revealed reduced adenosine triphosphate-diphosphate concentration ratios in graft veins that was caused by distention before implantation [2]. Various attempts have been made to cope with arterial pressures as a wall stress factor for venous graft walls [3 5]. The biocompound-graft (Alpha Research) (sheathed graft), a hybrid prosthesis intraoperatively assembled out of the patients vein and a highly flexible Phynox mesh (Alpha Research, Luterbach, Switzerland) mesh, also seeks to overcome this problem. During the process of assembly a balloon catheter is inserted into the graft vein. Then a fine mesh tubing is pushed onto the vein and they are glued together with fibrin sealant [6, 7]. In this study intimal and medial layer changes, together with aspects of biocompatibility where close contact of mesh material to the adventitial tissue in a pulsating Accepted for publication Jan 22, Adress reprint requests to Dr Zurbrügg, Deutches Herzzentrum Berlin, Augustenburger Platz Berlin, Germany; zurbruegg@dhzb.de. environment occurs, were examined. Therefore the use of human fibrin sealant in pigs has been omitted. Material and Methods Five white race pigs, weighing 20 to 25 kg, were operated on (Bristol Heart Institute, University of Bristol, Bristol, England). All animals were handled under humane conditions in accordance with the Home Office Animals (Scientific Procedures) Act 1998 (HMSO 1990). They were subjected to the common carotid artery surgery medication and anesthesia. A detailed description of the animal model has been previously described [8]. An unmanipulated saphenous vein graft was interposed into one carotid artery, and for the other carotid artery the graft vein was sheathed with 7.8-mm mesh tubing. A compliant, braided Phynox mesh tubing, with fiber diameters of 32 m, was slipped over the vein (sheathed graft). Vein grafts of at least 25 mm in length were used. The principle behind the mesh tubing is that of a Chinese finger, which decreases in diameter when longitudinal tension is applied. The mesh tubing was smoothed out over the vein in order to make it fit evenly over the vein wall. The steps of construction are described elsewhere [9]. The original bonding procedure with human fibrin sealant was not performed so as to avoid impairment of the biocompatibility examinations by The Society of Thoracic Surgeons /99/$20.00 Published by Elsevier Science Inc PII S (99)00452-X

2 80 ZURBRUGG ET AL Ann Thorac Surg REDUCTION OF WALL CHANGES IN SHEATHED GRAFTS 1999;68:79 83 Pressure Fixation Four weeks after implantation histological sections of the native veins were made using elastic van Gieson s stain. Toluidine blue stain was used for the sheathed veins because of the special treatment applied (see below). An equal number of control sections (unsheathed grafts) were made and stained with toluidine blue in order to achieve comparability for the cell counts and for the wall thickness comparisons. All grafts were divided into proximal, medial, and distal pieces and a randomly selected section was made for each piece, which was at least 5 mm away from the anastomosis and the next section. Six sections per pig (for each 3 biocompound-graft and each 3 saphenous vein control sections) were evaluated. To avoid the destruction of the sheathed samples during the cutting process, caused by the fibers of the mesh tubing, the tissue was embedded into Epon Araldit and cut with a diamondcoated microtome saw into 70 m slices. Transverse sections were selected at the midportion of each piece. Toluidine blue was used for staining as described by Hehrlein and associates [10, 11]. Because toluidine blue only stains the core, the distinction of cell types is impaired. Therefore, the total number of cells was counted in both intimal and medial layers in five microscopic fields (32 m 32 m 0.65 m depth 1 ) per section at 400 magnification. The narrowest parts of the grafts are decisive for the fate of the graft. Therefore the maximal and minimal values of wall thickness (medial and intimal together) and cellularity found in each sheathed graft were compared with their corresponding unsheathed control grafts. s were used for presentation of the data and the Wilcoxon s matched pairs test for statistical comparisons was used. Means and standard deviations are shown in the tables for completeness reasons only. Fig 1. Histological section (toluidine blue stain; 10 before % reduction) of a sheathed vein graft (pig) 4 weeks after implantation into the arterial circuit. The Phynox fibers are visible in the adventitial layer. 748 m (median 528 m) for vein grafts (p 0.05, n 5). Maximal medial layer thickness varied not significantly from 167 m to210 m, (median 180 m) for sheathed grafts and from 180 m to448 m (median 403 m) for vein grafts. The measurements are given in Table 1. Minimal wall thickness per section site went from 134 m to 170 m with a median of 160 m in the sheathed vessels and from 160 m to 261 m with a median 173 m in unsheathed veins. The minimal intimal thickness varied from 68 m to83 m with a median of 76 m in the sheathed grafts and from 76 mto177 m with a median of 86 m for the unsheathed veins. This Results Graft vessel diameters varied from approximately 6 mm for the sheathed grafts to approximately 8 mm for the control grafts. Whereas areas of intimal hyperplastic changes were found in all histological sections (Figs 1, 2) the cell counts of the medial layer as an indicator of inflammatory response differed significantly (Figs 3, 4). The maximal wall thickness at each section site for the sheathed grafts (intimal and medial layer) varied from 351 m to432 m (median 379 m), whereas the unsheathed grafts ranged from 391 m to 1196 m with a median value of 939 m (p 0.05, n 5 ). Maximal intimal layer thickness varied from 191 m to222 m (median 199 m) for sheathed grafts, and from 224 m to 1 A Leica DMRBE Microscope (Leica Mikrosystem GmbH, Bensheim, Germany) was used and only focused within the cell cores were counted. The depth can be calculated as follows: 0.5 D a 2, where D depth, wavelength (550nm) and a aperture of the objective (for 400 magnification : 0.65). Fig 2. Histological section (elastic van Gieson s stain; 10 before % reduction) of a vein graft (pig) 4 weeks after implantation into the arterial circuit.

3 Ann Thorac Surg ZURBRUGG ET AL 1999;68:79 83 REDUCTION OF WALL CHANGES IN SHEATHED GRAFTS 81 Fig 3. Wall segment of a sheathed graft (toluidine blue stain; 100 before % reduction) with areas of intimal and medial layer thickening 4 weeks after implantation into a pig carotid artery. The section shows an irregular distribution of cells with a concentration around the Phynox fibers. Fig 4. Histological section (elastic van Gieson s stain; 100 before % reduction) of a vein segment with low intimal and significant medial layer thickening 4 weeks after implantation into a pig carotid artery. A loss of myocytes with a concentration of inflammatory cells in the medial layer is visible. difference was found to be significant (p 0.05, n 5). In the medial wall layer minimal wall thickness per section site varied from 66 m to97 m (median 82 m) in the sheathed grafts and 69 m to108 m (median 82 m) in the unsheathed veins. The measurement results are given in Table 2. The maximal intimal layer cellularity for each section site ranged from 6 cells to 8 cells (median 7 cells) in the sheathed grafts and from 7 to 8 cells (median 8 cells) in the unsheathed veins. The maximal cellularity for the medial layer varied from 11 to 12 cells (median 12 cells) in the sheathed grafts and 17 to 18 cells (median 18 cells) for the native vein grafts (p 0.05, n 5, Table 3). Minimal cellularity values per section site in the inti- Table 1. Results of the Measurements of Wall Thickness Which Give the Maximal Values for - in the Intimal and Medial Wall Layer of Biocompound-Grafts (bcg) and Unsheathed Veins (nv) 4 Weeks After Implantation in Pigs That Underwent Surgery on Both Carotid Arteries. The bcg Significantly Reduced Overall Wall Thickness (Medial and Intimal Layer) as Well as Intimal Layer Thickness. Wilcoxon s Matched Pairs Test was Applied for p-values. Maximal Values at Maximum Deviation Thickness of medial intimal layer bcg m m 379 m p 0.043, n 5 nv m m 939 m Thickness of intimal layer bcg m m 199 m p 0.043, n 5 nv m m 528 m Thickness of medial layer bcg m m 180 m p (not significant) nv m m 403 m

4 82 ZURBRUGG ET AL Ann Thorac Surg REDUCTION OF WALL CHANGES IN SHEATHED GRAFTS 1999;68:79 83 Table 2. Results of the Measurements of Wall Thickness Which Give the Minimal Values for each Section Site - in the Intimal and Medial Wall Layer of Biocompound-Grafts (bcg) and Unsheathed Veins (nv) 4 Weeks After Implantation in Pigs That Underwent Surgery on Both Carotid Arteries. The bcg Significantly Reduced Overall Wall Thickness (Medial and Intimal Layer). Wilcoxon s Matched Pairs Test Was Applied for p-values. Minimal Values At Minimum ( m) Deviation ( m) ( m) Thickness of medial intimal layer bcg p (not significant) nv Thickness of intimal layer bcg p 0.043, n 5 nv Thickness of medial layer bcg p 0.89 (not significant) nv mal wall layer ranged from 4 to 5 cells (median 4 cells) in sheathed graft samples and from 4 to 5 cells (median 5 cells) in the unsheathed veins. The minimal values in the medial layer went from 5 to 6 cells (median 5 cells) and 12 to 15 cells (median 14 cells). These differences were found to be significant (p 0.05 and n 5). These results are given in Table 4. One to 2 giant cells were found in half of the sheathed sections. Low rates of foreign body giant cells, together with moderate inflammatory cell infiltration adjacent to the fibers of the mesh, indicate that a mild foreign body response took place. Inflammatory cells were limited to an area of about 30 m around the fibers, whereas generalized proliferative activity was to be seen in the unsheathed graft sections (Figs 3, 4). Comment A significant reduction of intimal and overall wall thickness from the maximal values at each section site was found. As the maximal wall thickness is crucial to the outcome of the bypass graft, the introduction of a sheath for a graft may reduce the risk of occlusion. Whereas there was an obvious and significant difference in the intimal and overall wall thickness, the values for the medial tissue did not differ significantly. Equalizing processes might be the reason for this effect. The mesh tubing prevented the veins from becoming extensively distended by the arterial pressure and thus prevented the medial layer from becoming thinner. As wall distention occurs in the unsheathed grafts, the wall injury may induce a proliferation of the thin medial layer and therefore equalize the differences when compared with sheathed grafts. This coincides with the increased medial layer cellularity, which was not observed in the sheathed veins. Significantly less inflammation occurred in the sheathed vein grafts, which supports the concept that less wall injury and reparative activity took place. Decreased intimal proliferation, observed in the sheathed vein grafts, might consequently be the result of the optimization of tangential stress on the endothelial cell layer due to an almost regular inner diameter of the sheathed vein. The adventitial foreign body reaction around the mesh fibers did not induce proliferation in the medial layer. The inflammatory cells are in close distance to the fibers, which suggests a high degree of histocompatibilty with the mesh graft employed. Overall external support of vein grafts reduces intimal and medial layer proliferation. The findings of this study Table 3. Results of the Cellularity Count Which Give the Maximal Values for - in the Intimal and Medial Wall Layer of Biocompound-Grafts (bcg) and Unsheathed Veins (nv) 4 Weeks After Implantation in Pigs That Underwent Surgery on Both Carotid Arteries. The bcg Significantly Reduced Medial Wall Layer Cellularity. Wilcoxon s Matched Pairs Test was Used for p-values. Maximal Values at Maximum Deviation Cellularity (intimal layer) bcg p (not significant) nv Cellularity (medial layer) bcg p 0.043, n 5 nv

5 Ann Thorac Surg ZURBRUGG ET AL 1999;68:79 83 REDUCTION OF WALL CHANGES IN SHEATHED GRAFTS 83 Table 4. Results of the Cellularity Count Which Give the Minimal Values for - in the Intimal and Medial Wall Layer of Biocompound-Grafts (bcg) and Unsheathed Veins (nv) 4 Weeks After Implantation in Pigs That Underwent Surgery on Both Carotid Arteries. The bcg Significantly Reduced Medial Wall Layer Cellularity. Wilcoxon s Matched Pairs Test was Used for p-values. Minimal Values at Minimum Deviation Cellularity (intimal layer) bcg p (not significant) nv Cellularity (medial layer) bcg p 0.043, n 5 nv are in accordance with the results of other groups [4, 5, 12 14]. References 1. Brody WR, Kosek JC, Angell WW, Stanford PA. Changes in vein grafts following aorto-coronary bypass induced by pressure and ischemia. J Thorac Cardiovasc Surg 1972;64: Angelini GD, Bryan AJ, Williams HMJ, Morgan R, Newby AC. Distension promotes platelet and leukocyte adhesion and reduces short-term patency in pig arteriovenous bypass grafts. J Thorac Cardiovasc Surg 1990;99: Zweep HP, Satoh S, van der Lei B, Hinrichs WLJ, Dijk F, Feijen J, Wildevuur CRH. Autologous vein supported with a biodegradable prostheses for arterial grafting. J Thorac Surg 1993;55: Barra A, Bolant A, Leroy JP, et al. Constrictive perivenous mesh prosthesis for preservation of vein integrity. J Thorac Cardiovasc Surg 1986;92: Moritz A, Raderer F, Magometschnigg H, et al. The use of mesh-tube-constricted dilated or varicose veins as an arterial bypass conduit. Thorac Cardiovasc Surg 1992;40: Zurbrügg HR, Hetzer R. The use of biocompound-grafts together with varicose veins. J Cardiovasc Surg 1996; 37(6 Suppl 1): Zurbrügg HR, Wied M, Wenzel K, Hetzer R. Die Biocompound-Gefässprothese in der aorto-koronaren Bypasschirurgie. Z. Herz-, Thorax-, Gefässchir 1997;11: Angelini GD, Izzat MB, Bryan AJ, Newby AC. External stenting reduces early medial and neointimal thickening in a pig model of arteriovenous bypass grafting. J Thorac Cardiovasc Surg 1996;112: Zurbrügg HR, Wied M, Hetzer R. Complete revascularisation with the biocompound-graft procedure in patients with varicose veins. Arch Chir Thorac Cardiovasc 1997;19: Hehrlein C, Gollan C, Dönges K, et al. Low-dose radioactive endovascular stents prevent smooth muscle cell proliferation and neointimal hyperplasia in rabbits. Circulation 1995; 92: Hehrlein C, Stintz M, Kinscherf R, et al. Pure -particleemitting stents inhibit neointima formation in rabbits. Circulation 1966;93: Karayannacos PE, Hostetler JR, Bond MG, et al. Mediating factors in subendothelial fibromuscular hyperplasia. Ann Surg 1978;187: Batellier J, Tedgui A. Atherogenesis reduction of an arterialized venous graft using an external sleeve. Chirurgie 1992; 118: Batellier J, Wassef M, Merval R, Duriez M, Tedgui A. Protection from atherosclerosis in vein grafts by a rigid external support. Arterioscler Thromb 1993;13: