Surgical sealant in the prevention of early vein graft injury in an ex vivo model

Cardiovascular Pathology 12 (2003) 202 – 206

Surgical sealant in the prevention of early vein graft injury in an ex vivo model W. Stooker a,*, H.W.M. Niessen b,c,1, E.K. Jansen a, J. Fritz b,c, W.R. Wildevuur a, V.W.M. Van Hinsbergh d,e, Ch.R.H. Wildevuur f, L. Eijsman a a

Department of Cardiac Surgery, VU Medical Centre, Amsterdam, The Netherlands b Institute of Pathology, VU Medical Centre, Amsterdam, The Netherlands c Institute of Cardiovascular Research, VU Medical Centre, Amsterdam, The Netherlands d Institute of Cardiovascular Research, VU Medical Centre, Amsterdam, The Netherlands e Gaubius Laboratory TNO-PG, Leiden, The Netherlands f Department of Experimental Thoracic Surgery, University Hospital Groningen, Groningen, The Netherlands Received 28 June 2002; received in revised form 24 February 2003; accepted 5 March 2003

Abstract Background: The amelioration of the adaptation process (arterialisation) of the vein graft wall to the arterial circulation in coronary artery bypass surgery by using extravascular support is clearly established in animal models and in in vitro and ex vivo set-ups. This support consists of some form of external graft-supporting modality like a prosthetic graft of stent. The clinical application of perivenous support, however, is hampered due to the fact that no easy applicable external support is available. Considering that application in the form of a spray is the most convenient modality, we evaluated whether polyethylene glycol is capable of providing adequate perivenous support. Polyethylene glycol is a synthetic, biodegradable product, used in cardiac surgery as a sealant, and is commercially available in the form of a spray. Methods: Segments of human saphenous vein graft obtained during coronary artery bypass graft (CABG) procedures were placed in an ex vivo model, a side loop of the extracorporeal perfusion circuit, and perfused with autologous blood, making the circumstances identical to the implanted saphenous vein grafts concerning pressure, temperature, level of complement and leukocyte activation and blood pressure. Alternately around every other study vein graft segment polyethylene glycol was applied. Unsupported grafts served as control. After 1 min of solidification, perfusion was started with a pressure of about 60 mmHg (nonpulsatile flow). Perfusion was maintained for 60 min, after which the grafts were collected for light microscopy and electron microscopy. Results: Light microscopy and electron microscopy showed remarkable attenuation of endothelial cell loss and less injury of smooth muscle cells of the circular and longitudinal layer of the media in the supported group compared to the nonsupported vein graft segments. Conclusion: Polyethylene glycol is able to provide adequate external vein graft support, preventing overdistension, in an ex vivo model. This provides a basis for clinical application. Further investigation is warranted to evaluate long-term effects. D 2003 Elsevier Inc. All rights reserved. Keywords: Vein graft; Perivenous support; Endothelium; Smooth muscle; Polyethylene glycol

1. Introduction The patency rates of human vein grafts following coronary artery bypass grafting (CABG) are generally less favourable than those of selected arterial grafts [1 –3]. This difference in patency is in part attributable to the sudden * Corresponding author. Onze Lieve Vrouwe Gasthuis, 1e Oosterparkstraat 279, 1091 HA Amsterdam, The Netherlands. Tel.: +31-2059-93669; fax: +31-2059-93675. E-mail address: [email protected] (W. Stooker). 1 Dr. Niessen is a recipient of the Dr. E. Dekker Program of the Netherlands Heart Foundation (D99025). 1054-8807/03/$ – see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1054-8807(03)00058-9

exposure of the vein graft to the arterial circulation, which results in overdistention, due to the higher and pulsatile pressure. Furthermore, changes in flow and shear stress play an important role in the vessel wall injury [4]. The benefit of perivenous support, preventing overdistension, on future patency of human saphenous vein grafts is clearly established in experimental models [4 –10], but clinical application is hampered because of the lack of easy applicable forms of support. Based on animal studies [7,11,12], perivenous support should be elastic, biodegradable, nonrestrictive and permeable to provide adequate arterialisation. In our opinion, an effective modality to provide this

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support may be the perivenous application of a spray, after completion of the distal and proximal anastomoses but before exposure to the high arterial pressure. To elude whether this concept of perivenous sprayable support might work, we evaluated whether polyethylene glycol, used in cardiac surgery as a sealant, and commercially available in the form of a spray (CoSeal, Cohesion Technologies, Pablo Alto, USA) provides adequate perivenous support and subsequent attenuation of early vein graft wall injury. CoSeal surgical sealant is a synthetic, nontoxic hydrogel that is formed at the time of administration of the two components, one containing thoroughly mixed polyethylene glycol and sodium phosphate buffer and the other containing polyethylene glycol and a sodium phosphate/sodium carbonate buffer provided by the manufacturer in an application system, and solidifies within 60 s.

2. Materials and methods CABG patients were included in the study after informed consent. The study was approved by the local ethical committee. Anaesthesia and cardiopulmonary bypass (CPB) were performed according to the routine protocol. The ex vivo model is a modification of the set-up that we described earlier [10] and consists of a small roller pump (Sto¨ckert) and a vein irrigation set (Bentley Laboratories Europe, Uden, The Netherlands), connected to the extracorporeal circuit with an inflow cannula attached to the arterial filter of the arterial line, and an outflow cannula connected to the cardiotomy reservoir (Fig. 1). The pressure in the ex vivo system is equalized with the blood pressure of the patients by varying the distal occlusion and by adjusting the flow. During bypass, a blood pressure of 60 mmHg mean was aimed at. As the ex vivo system is a side loop of the extracorporeal circuit and the experiments are performed during the CABG procedure, the study vein grafts are perfused with autologous blood. After the start of the extracorporeal bypass, the study saphenous vein graft with a length of about 3 cm was mounted in the ex vivo perfusion system. At no point during the harvesting procedure, distension was allowed before perfusion in the perfusion system. Before pressurizing the vein graft segment, but after start of the perfusion with a pressure just enough to counteract the collapse of the vein (usually about 8 mmHg), alternately in consecutive patients, polyethylene glycol hydrogel (CoSeal), was administered according to the directions of the manufacturer, or no perivenous support was applied. After the application of polyethylene glycol and after 1 min of solidification, the intraluminal pressure was allowed to approximate the arterial pressure of the patient. The study vein graft was placed in the pericardium during perfusion. In this way, the vein graft segments were exposed to exactly the same conditions like the vein grafts implanted in the patients. After 60 min, the study vein graft perfusion was terminated, and the vein graft was collected for light and electron microscopy. All

Fig. 1. Schematic representation of ex vivo perfusion model.

specimens for morphologic analysis were taken some distance from either cannulation site to ensure that the segments were free of trauma caused by ligation or injury from possible flow disturbances at the cannula tip. 2.1. Immunohistochemistry The vein graft segments were fixed in formaldehyde and embedded in low melting point paraffin wax. Transverse sections of formaldehyde embedded tissue samples were cut at 5 Am and were stained with hematoxylin –eosin and with Elastica Von Giesson. For immunohistochemistry, endogenous peroxidase activity was blocked by 0.3% (v/v) H2O2 in methanol. After preincubation with normal rabbit serum (Dakopatts, Glostrup, Denmark) during 15 min (1:50), the slides were incubated for 60 min with the antibody CD34 (1:25) (Becton and Dickinson). 2.2. Electron microscopy The vein wall segments were fixed in 2% (v/v) gluteraldehyde for 30 min and 1.5% (w/v) osmium tetroxide for 10 min, dehydrated with acetone and embedded in Epon 812. Ultrathin sections were collected on 300-mesh Formavar-coated Nickel-grids. The sections were contrasted with uranyl acetate and lead citrate and were examined in a Jeol 1200 EX electron microscope. 2.3. Histological analysis The light and electron microscopical sections were assessed by two independent investigators (HWMN and WS).

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A numeric grading system according to Griffith et al. [13] was used to score the uniformity, continuity and integrity of the histological structures: 0 (intact, no disruption), 1 ( < 10% disruption), 2 (10 – 25% disruption), 3 (25 – 50%) and 4 ( > 50% disruption). In this way, the endothelial layer, the internal lamina, medial smooth muscle and connective tissue were assessed. 2.4. Statistics Statistical analysis of the histological scores was carried out using the Kruskal – Wallis test for nonparametric data. All data are given as mean F S.D. and were analysed using the SPPS 9.0.1 software. A probability value of P less than or equal to .05 was considered statistically significant upon two-tailed testing.

3. Results Upon pressure perfusion of the vein grafts, a clear distension of the nonsupported graft segments was noticed, whereas the polyethylene glycol supported segments showed no distension during the perfusion (not shown). Vascular damage was further analysed and quantified by histological scoring (Table 1). All nonsupported vein graft segments showed extensive areas of deendothelialisation (Fig. 2A; Table 1) and fragmentation of the basal lamina (Table 1). The endothelial layer and basal lamina were preserved in all supported vein graft segments (Fig. 2B; Table 1). In the longitudinal smooth muscle cell layer of the media, slight injury of smooth muscle cells and some disorganisation of collagen fibres and edema were found both in the nonsupported (Fig. 3A) and supported group (Fig. 3B). Although the injury in the supported group seemed less pronounced, the difference in injury score is not significant (Table 1). Table 1 With support (N = 9) Loss of endothelium Disruption of basal lamina Disorganisation of collagen fibres and edema Damage of SMC in the longitudinal layer Damage of SMC in the circular layer

b

No support (N = 6)

Pa

0.44 F 0.53

4.0 F 0.00

.001

0.11 F 0.33

3.83 F 0.41

< .001

1.11 F 0.33

3.83 F 0.41

< .001

1.67 F 0.50

2.0 F 0.00

.127

1.78 F 0.44

4.0 F 0.00

.001

SMC = smooth muscle cell. a Statistical significance was determined using the Kruskal – Wallis (rank-sum) test for nonparametric data. b The graded score represents the percent disruption expressed as mean F S.D. and was scored according to the scale described by Griffith et al.: 0 (no disruption), 1 ( < 10% disruption), 2 (10 – 25% disruption), 3 (25 – 50% disruption) and 4 (>50% disruption).

Fig. 2. Endothelial coverage in vein specimens after 1 h of ex vivo perfusion. (A) CD34 staining of a microscopical section of nonsupported vein graft after 60 min of perfusion. Extensive areas of deendothelialisation are observed. Magnification 400  . (B) CD34 staining of a microscopical section of a polyethylene glycol hydrogel supported vein graft showing an intact endothelial layer. Magnification 250  .

An impressive difference in media damage was found in the circular muscle layer (Fig. 3; Table 1). Severe vacuolisation, indicative for important smooth muscle cell damage, and extensive edema and disorganisation of collagen fibres were found in the nonsupported vein graft segments (Fig. 3C), whereas only tiny vacuoles were found in the vein graft segments of the supported group with preservation of collagen fibres and absence of edema (Fig. 3D).

4. Discussion This study demonstrates the beneficial effect of perivenous polyethylene glycol support on human saphenous vein graft segments in an ex vivo model. The attenuation of the injury in the supported vein graft segments compared to the injury encountered in nonsupported vein grafts after exposure to arterial pressure is a consistent phenomenon. The injury pattern in the nonsupported vein graft segments corresponds to the postmortem findings in saphenous vein coronary artery bypass grafts mentioned by Kockx et al.

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Fig. 3. (A, B) Electron microscopy photomicrograph showing a representative cross-section of a longitudinal muscle layer of a vein graft after 1 h of ex vivo perfusion without support (A) and with polyethylene glycol support (B). Magnification 3600  . Although more smooth muscle cell damage is found in this nonsupported vein graft compared to the supported vein graft, the overall difference in injury score is not significant (Table 1). (C, D) Electron microscopy photomicrograph of the circular muscle layer of a vein graft showing severe injury in the nonsupported group (C) and important attenuation of smooth muscle cell injury of smooth muscle cells and preservation of collagen in the supported group (D). Magnification 3600  .

[14]. In their study, a complete deendothelialisation was found within 24 h and extensive smooth muscle cell injury especially in the circular smooth muscle layer of the media. The protective effect of polyethylene glycol is comparable to the effect of perivenous PTFE graft support that we have previously found in the perfused vein model [10]. A significant attenuation of smooth muscle cell injury was shown in the circular layer, which indicates a protective effect of perivenous polyethylene glycol support concerning smooth muscle cell injury. The injury scores of the longitudinal muscle layer, however, show no significant difference in the supported group compared to the unsupported vein grafts. This result differs from the results in our earlier experiments with perivenous PTFE graft support [10] and fibrin glue support [16], in which no longitudinal smooth muscle cell injury was noticed in the supported group, and might be attributable to differences in supportive properties. Polyethylene glycol has certain characteristics that suggest its suitability to be used as perivenous support. It is a

nontoxic, completely synthetic, elastic, nonrestrictive biodegradable product, which is easy to apply around the vein graft segments and solidifies within 1 min. It is translucent, which makes optical control of the vein graft during CABG procedures possible after application. Its biodegradation after about 30 days warrants more adequate support during the phase of smooth muscle cell rearrangement in the arterialisation process as demonstrated in animal studies [11,12] compared to fibrin glue, which is expected to biodegrade within a few days. In the animal studies the optimal support period was between 3 and 6 weeks. Another advantage of polyethylene glycol is the fact that no human or animal blood products form the basis of the product. Compared to fibrin glue, manufactured from human plasma, the synthetic basis of polyethylene glycol eliminates the possible risk of viral transmission. Polyethylene glycol is used in pharmacology as a carrier of different kinds of medication [15]. In this respect, the possibility to, for example, add smooth muscle cell

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proliferation inhibitors to the perivenous supporting polyethylene glycol might be an interesting concept to further modulate the smooth muscle cell rearrangement and, in this way, direct the remodelling of the vein graft wall towards an artery-like vessel wall. We limited our observation period to 1 h based on our earlier experiments [10] in which a complete deendothelialisation and severe injury of smooth muscle cells was found in unsupported vein graft segments within 1 h of perfusion. Although longer-term experiments, concerning the feasibility and safety of external polyethylene glycol application for this indication, are necessary, our study shows the possibility to provide adequate external vein graft support with a biodegradable spray.

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