Proefschrift Fahner

Experim

ental Stu

dies o

n G

lycerol Preserved

Vascu

lar Allo

grafts

Peter Fahn

er

Experimental Studies on Glycerol Preserved Vascular Allografts

Peter Fahner

UITNODIGING

voor het bijwonen van

de openbare verdediging

van het proefschrift

Experimental Studieson Glycerol Preserved

Vascular Allografts

op vrijdag 6 juni 2014

om 13.00 uur

in de Aula van de

Universiteit

van Amsterdam

Oude Lutherse Kerk

Singel 411, hoek Spui

1012 WN Amsterdam

Aansluitend receptie in de

naastgelegen

Tetterode Bibliotheek

Paranimfen:

Hanneke van Krimpen

André Janse

[email protected]

Peter [email protected]

Experimental studies

on glycerol preserved vascular allografts

Peter Jurriaan Fahner

Fahner.indd 1 5-5-2014 14:35:07

This study was performed at Department of Experimental Surgery in collaboration with

Department of Vascular Surgery of the Academic Medical Center (AMC), University of

Amsterdam, The Netherlands

Cover: Front: Stairwell Hotel Ohla, Barcelona, Spain

Back: AMC/ Inst. voor Wetenschappelijk Onderzoek, Amsterdam,

The Netherlands

Printed by: Gildeprint, Enschede, The Netherlands

ISBN: 9789461086815

Printing of this thesis was financially supported by

University of Amsterdam, The Netherlands

Glaxo Smith Kline, Zeist, The Netherlands

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Experimental studies

on glycerol preserved vascular allografts

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus

prof. dr. D.C. van den Boom

ten overstaan van een door het college voor promoties

ingestelde commissie

in het openbaar te verdedigen in de Aula van de Universiteit

op vrijdag 6 juni 2014 om 13.00 uur

door

Peter Jurriaan Fahner

geboren te Angguruk, Indonesië

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Promotores: Prof. dr. D.A. Legemate

Prof. dr. T.M. van Gulik

Copromotor: Dr. M.M. Idu

Overige leden: Prof. dr. E.T. van Bavel

Dr. M. Heger

Prof. dr. B.A.J.M. de Mol

Dr. J.I. Rotmans

Prof. dr. B.H. Walpoth

Prof. dr. C.J.A.M. Zeebregts

Faculteit der Geneeskunde

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In memory of my father Dr. Chr. Fahner

In memory of my father in law F. Janse

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Fahner.indd 6 5-5-2014 14:35:07

CONTENTS

Contents

Chapter 1 General introduction and outline of the thesis 9

Chapter 2 Systematic review of preservation methods and clinical outcome of

infrainguinal vascular allografts 23

J Vasc Surg 2006;44:518-24

Chapter 3 Morphological and functional alterations in glycerol preserved rat aortic

allografts 39

Int J Art Org 2004;27:979-89

Chapter 4 Glycerol preserved arterial allografts evaluated in the infrarenal rat aorta 57

Eur Surg Res 2009;42:78-86

Chapter 5 Comparison of preserved vascular allografts using glycerol and University

of Wisconsin solution in a goat carotid artery transplantation model 77

Eur Surg Res 2012;48:64-72

Chapter 6 Summary and conclusions

Samenvatting en conclusies 95

Chapter 7 General discussion and future perspectives 101

Dankwoord 107

Curriculum Vitae 111

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Fahner.indd 8 5-5-2014 14:35:07

1General introduction and outline of the thesis

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10 | Chapter 1

Introduction

The experiments described in this thesis focus on the applicability of glycerol preservation of

vascular allografts as an alternative to the existing vascular preservation methods and synthetic

conduits in peripheral arterial revascularization. As the synthetic prosthesis performs well in

central arterial reconstructions, the long term results in small caliber reconstructions of less than

6 mm are disappointing. Despite advances in secondary prevention, medical treatment, and

interventional technology (1;2), the incidence of peripheral arterial disease requiring vascular

reconstructions is increasing (3;4). Therefore we need to further pursue the development of a

durable patent vascular graft for peripheral vascular reconstructions.

Due to the limited results of the small diameter synthetic grafts, the focus for a vascular graft

has widened to biological allografts. Two obstacles in the development of a suitable biologic

vascular graft are the thrombogenicity of the graft lumen and the immunogenicity of the

transplanted structure. Furthermore, the vascular graft should have compliance characteristics

comparable to the native vessel. It is thought that preserved biological grafts could serve as a

scaffold for autologous cells including endothelial cells, vascular smooth muscle cells (SMC) and

fibroblasts to repopulate the graft, thereby improving its biocompatibility.

As an alternative to allografts, the use of xenografts such as the bovine venous graft has been

explored, however with disappointing long-term results in peripheral arterial reconstruction (5;6).

Allografts therefore are considered more suitable for reconstructive purposes.

Biological scaffolds have the advantage of possessing an extra cellular matrix which expresses

signaling components essential for cell adhesion, migration, proliferation and differentiation of

ingrowing recipient cells (7). Preservation of the collagen and elastin scaffold ensure retention of

tensile strength, and the glycosaminoglycans play a crucial role in endothelial cell adhesion and

proliferation, inhibition of SMC proliferation and migration in addition to their antithrombotic

properties (8;9). It has been shown that the production of endothelial mediators involved in

coagulation homeostasis was significantly greater in human saphenous vein endothelial cells

grown on biological matrices compared to those grown on polytetrafluoroethylene (PTFE) (10).

Another advantage of biological vascular grafts compared to synthetic conduits is the greater

resistance to infections. In a small-intestinal, submucosal biological scaffold used as an iliac patch

both infection and aneurysm formation were significantly lower as compared to PTFE patches

(11). The process of remodeling and earlier development of vasa vasora in biological scaffolds

enable a more rapid and effective recruitment of circulating immune system components,

thereby preventing graft infection (12). The early formation of a confluent endothelium layer

prevents formation of thrombi and disturbs bacterial adherence and, therefore, establishment

of infection (13).

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General introduction and outline of the thesis | 11

1The aims of the studies in this thesis were 1) to find evidence based on clinical studies in

literature for use of the best alternative vascular allograft in peripheral reconstructions 2) to

establish a standard method for glycerol preservation of arterial allografts and 3) to investigate

graft patency, morphological alterations and functionality of glycerol preserved allografts in two

animal models.

Historical perspective of vascular transplantation

The first records of arterial and venous transplantation were probably the reconstructions

performed during World War I when battle victims provided a readily accessible source of

vascular allografts (14). While initially autologous veins and arteries were employed in vascular

reconstructions, in the late 1940s a shift to allogeneic sources began with the work of Hufnagel

and Gross et al (15-17). Human arterial allografts, harvested at autopsies and sterilized by cobalt

irradiation, were used in reconstructions for thoracic aorta coarctations. In 1951, abdominal

aortic surgery was performed by Oudot when an aortic allograft was used for an occluded aortic

bifurcation. In that same period, Dubost et al replaced an abdominal aortic aneurysm by an

allograft (18;19). In the mid- 1950s, arterial allografts were frequently used after vascular graft

banks had been established (20). Initial enthusiasm for these grafts diminished, however, as

degeneration and graft failure were increasingly observed (21). After the addition of a synthetic

mesh around vascular allografts, these vascular grafts became the preferred material for vascular

reconstructions in the second half of the twentieth century (22-24). It was recognized that fresh

allografts suffered of a strong immune response, and, therefore, research focused on different

preservation methods to diminish rapid rejection and dissolution. Different preservation methods

were examined in the past decades using formalin, alcohol, ethylene oxide, betapropriolactone,

irradiation and freeze drying (17;25-31).

The above mentioned studies mainly dealt with vascular grafts for central reconstructions.

After the introduction of prosthetic vascular grafts made of PTFE and Dacron, showing good

results in aortic repair, the need for vascular allografts for central reconstructions diminished.

Nowadays, a few indications exist for the use of allografts in central reconstructions such as in

prolonged antibiotic resistant prosthetic graft infections.

For reconstructions of small and intermediate caliber arteries the use of autologous vein as

primary choice is generally accepted. Nevertheless, even autologous vein has its shortcomings

in that it is not always available, it may be diseased, and it requires additional time and surgical

dissection for preparation. Furthermore, the vein obviously can be inadequate in terms of length

or luminal diameter for use in a lower-limb revascularization procedure. Therefore, alternative

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12 | Chapter 1

biological resources have been explored as a potentially useful alternative for substitution of

small and intermediate caliber arteries.

Preservation methods of vascular allografts

Several methods have been explored for preservation of vascular allografts. The most commonly

used methods comprising treatment with gutaraldehyde, cold storage, cryopreservation and

lyophilization, are described below. Glycerol preservation is subsequently introduced as a

potential alternative for arterial allograft preservation.

Glutaraldehyde stabilized umbilical cord vein

Glutaraldehyde has been used to preserve human umbilical vein grafts, harvested after delivery.

These umbilical vein grafts (Biograft, Bio Vascular, Inc. St Paul, MN) consisted of veins tanned

with glutaraldehyde as described by Dardik. The conduit is supported by a polyester (Dacron)

mesh placed around the vein, sterilized and stored in 50% alcohol.

The use of fresh umbilical vein as a vascular graft was first attempted in the 1950s and 1960s

(32-34). In the following years, complications as early thrombosis, necrosis, microabcesses and

cellular infiltration hampered initial success of the vein graft. Based on studies by Rosenberg

et al and Carpentier et al (35;36), glutaraldehyde tanning of the umbilical vein was adopted

and applied in both animal and clinical studies (37-40). The aldehyde tanning process resulted

in cross-linkage of protein moieties in the vessel wall, especially the collagenous components,

which in turn increased tensile strength of the vessel wall and at the same time, sterilized the

tissue (41).

Cold storage

Cold storage, i.e. hypothermic preservation, is the standard method for preservation of solid

organ grafts. Vessels harvested during multi-organ donation are likewise stored and used when

reconstructive procedures are necessary to revascularize the graft in the recipient. Procured

arterial and venous segments are rinsed in saline and placed in a preservation medium such as

University of Wisconsin solution (UW). The usual storage temperature is 40C.

Experiences in liver and kidney transplant surgery show that arteries supplying organ

transplants rarely become thrombosed or aneurysmatic (42). Endothelial coverage of the

lumen of the transplanted vessel probably improves long-term patency as seen in autologous

and alloplastic bypass grafts (43-45). Although this concept seems promising when applied in

peripheral revascularization using arterial allografts even under immunosuppressive regimen,

graft rejection may still lead to graft occlusion. Prager et al reached a primary patency rate of

Fahner.indd 12 5-5-2014 14:35:07


154% after one year in a study of 13 patients in which immunosuppression was used in fresh

arterial homografts in peripheral arterial reconstructions (46). The experimental evaluation of

allogeneic vein transplantation began with the experiments of Alexis Carrel who transplanted

jugular vein allografts in the thoracic aorta in dogs (47). After follow-up of 2 years and 2 months,

the vein maintained the same size, but the vessel wall was replaced with connective tissue.

Since that time, numerous experimental and clinical studies have been performed to evaluate

transplantation of venous allografts (48-56). The immunologic basis for graft occlusion in fresh

cold-stored veins was supported by the study of Schwartz et al who demonstrated the antigenicity

of canine allograft veins (57). Degenerative changes occur first in the adventitia and the adjacent

media, but viable muscle cells were still identified 119 days after allografting. Graft failure usually

results from vessel occlusion, which is presumably due to the combined effect of fibrotic stenosis

of the vessel and thrombosis. Thrombosis is a prominent feature in graft rejection.

Cryopreservation

Cryopreservation is a technique used to preserve vascular allografts by cooling the tissue to

temperatures far below 00C. Using cryoprotectants, the tissue is gradually cooled to temperatures

of -70 0C with a rate of -2 0C/min. Finally, the tissue is rapidly cooled to -196 0C.

Early reports on the clinical use of cryopreserved veins indicated results inferior to autologous

vein grafts with patency rates of 25% to 33% after a follow-up to 20 months and 66% after

12 months in the aortacoronary position (58-63).

These poor patency rates are probably due to several causes. Carpenter and Tomaszewski have

demonstrated a sustained immunogenic activity in cryopreserved venous allografts and suggested

that the rejection response may contribute to venous graft failure (64;65). Loss of functional

endothelial lining in cryopreserved venous allografts has also been indicated as a cause of graft

failure. It has been estimated that cryopreservation yields preservation rates of approximately

50% - 80% of intact endothelial cells (66). These cells have decreased thrombomodulin activity

and decreased nitric oxide production resulting in impaired anticoagulative function (67).

Therefore, anticoagulation therapy would probably improve allograft patency. Posner et al

combined an anticoagulation protocol with moderate immunosuppressive therapy and obtained

improved patencies but on the other hand, had substantial complications with pseudo-aneurysm

formation and hemorrhage (68).

Arterial allografts were used for revascularization in peripheral reconstructions in the early

1950s (69;70). These consisted of large and medium-sized cadaveric arteries, but initial results

were discouraging. Subsequent advances in the design of synthetic prostheses led to the disuse

of these allografts. Recent improvements in organ harvesting methods, along with progress in

cryobiology, has however, renewed interest in the use of arterial allografts (71). Rigorous control

of the curve of cooling and the use of a penetrating cryoprotectant such as dimethylsulfoxide for

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14 | Chapter 1

partial preservation of cellular viability greatly improved the success of cryopreservation. Clinical

studies of cryopreserved cadaveric arteries in infrageniculate reconstructions showed patencies

of 42% - 68% during 1 - 2 years of follow-up (72-74).

Although some authors found almost the same features of rejection for fresh and

cryopreserved allografts, others suggest that cryopreservation may decrease antigenicity of

arterial allografts (75;76). The prevention of rejection in cryopreserved allografts, however,

remains a challenge. Even though the use of immunosuppressive drugs could maintain structural

integrity of cryopreserved allografts, long-term immunosuppression does not seem a desirable

option in patients with critical leg ischemia (77;78).

Lyophylization

Since it is generally accepted that extracellular ice formation presents a hazard for biopreservation

by freezing of multicellular tissues, a major focus was set on developing low-temperature

preservation techniques that avoid ice crystallization.

Lyophilization has shown to be an effective technique to provide preservation of a number of

cells including monocytes, ova, early embryos and pancreatic islets. Cryoprotectants are added,

and the tissue is fast-cooled stepwise to temperatures around -100 ºC and then more slowly to

the final temperature between -135 ºC and -160 ºC via vapor nitrogen storage. Re-warming of

the tissue before transplantation is performed in a similar manner; slowly to -100 ºC and then

rapidly to 0 ºC.

Greater saphenous veins obtained from multi organ donations and preserved through

lyophylization were thought to be an attractive source of venous allografts for arterial

reconstructions (79;80). The freeze-drying process diminished immunogenicity and maintained

structural integrity (81-84). Despite the lack of antigenicity and satisfactory early graft patency

observed in animal studies, these results could not be reproduced in infrainguinal arterial

reconstructions using lyophilized saphenous allografts in humans (85).

Marrangoni et al introduced freeze-drying for preservation of arterial allografts in a clinical

study (86). Although the incidence of aneurysm development was relatively low, intimal

hyperplasia was observed. There are still controversies that make one hesitate to launch clinical

trials with lyophilized arterial allografts.

Glycerol preservation

Glycerol preservation of human amnion

Preservation of human amnion in 85% glycerol at 40C for over a year was performed as an

effective biological dressing in treatment of skin burns in rats. Microscopic examination of

Fahner.indd 14 5-5-2014 14:35:08


1preserved amnion revealed the maintenance of structural integrity comparable to that of fresh

amnion. No immunological rejection was observed with the use of glycerol-preserved amnion

on partial-thickness wounds in clinical cases (87). These observations encouraged further studies

on glycerol preservation of connective tissue structures.

Glycerol preservation of skin allografts

Glycerol preservation of skin was first implemented by Basile in 1982 (88) in a study of glycerol

preserved porcine skin. In 1984, the Dutch Skin Bank, later re-named Euro Skin Bank in 1992,

performed an extensive amount of research on glycerol preservation of skin allografts (89;90).

In the last twenty years, this skin bank delivered large quantities of glycerolized allografts to

burn centers throughout Europe. Clinical experiences at numerous burn centers proved the

effectiveness of glycerol preserved allografts as a temporary coverage in burn injuries and as

biological closure of various skin defects (91-93). The mechanism of glycerol preservation is

based on dehydratation of tissues by physically replacing most of the intracellular water while

maintaining the cell’s ionic concentration and preserving tissue by protecting cell integrity (94).

Advantages of glycerol preservation

I) Immunological

Glycerol preservation, by removing all cellular elements, results in a non-viable tissue matrix

which is considered less antigenic and less liable to rejection than cryopreserved or fresh skin

allografts (95).

II) Compliance

For optimal prolonged graft function, it appears to be important to have comparable compliance

of graft material and native vessel in preventing intimal hyperplasia, graft occlusion and aneurysm

formation (96-101). Compliance mismatch as a cause of intimal hyperplasia and graft failure may

still play an important role in failure of peripheral vascular grafting. This may cause excessive

stretching of SMC’s resulting in their proliferation (102). Abbot et al (103) demonstrated the effect

of compliance mismatch on graft patency in both arterial segments treated with glutaraldehyde

and untreated segments. Patency rates of the treated, less compliant segments were significantly

lower. The relevance of compliance match is also demonstrated by the substantial differences in

10 year patency rates between vein grafts (30-50%) and internal mammary artery grafts (80-

90%) used for coronary bypasses (104;105).

III) Antimicrobial properties

Previous experiments of storage of skin allografts in glycerol shows antiviral properties of glycerol

preservation dependent on concentration, time and temperature (106;107).

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16 | Chapter 1

IV) Storage duration

In comparison with other methods of tissue preservation, glycerolization has proven to be a

relatively simple and inexpensive procedure. Furthermore, it was able to store skin tissue for

periods up to 5 years while maintaining the morphological and structural integrity of the skin

(108).

Outline of the thesis

In the last decades, different methods have been used for vascular allograft preservation. The

most important preservation methods of venous and arterial allografts have been applied in

clinical trials. These include cryopreservation, cold storage, lyophilization and glutaraldehyde

preservation of vascular grafts. Chapter 2 offers a systematic review of the literature from 1966

to 2004 of clinical studies in which vascular allografts were used for infrainguinal reconstruction.

The goal of this review was to find evidence for the preservation of vascular allografts showing

the best long term patencies without aneurysmal changes. It was concluded that no systematic

review of randomized controlled trials of different allograft preservation methods had been

performed. Therefore, the evidence for the best alternative to autologous vein could only be

based on single randomized trials.

A great deal of research has been performed on the preservation of skin allografts for

the treatment of patients with extensive burn wounds when autologous tissue is lacking for

complete wound coverage. These studies convincingly demonstrated the superiority of glycerol

preservation compared to other methods for skin allograft preservation including glutaraldehyde

tanning, lyophilization, cold storage and cryopreservation. These results warranted research

on potential application of glycerol preservation in vascular allografts. Since the biomechanical

alterations of vascular tissue after glycerol preservation and the optimal glycerol preservation

protocol for preservation of vascular allografts were not established, experimental studies were

developed to investigate breaking strength, bursting pressure and functional alterations after

glycerol preservation in rat aorta segments. These experiments are described in chapter 3.

After having proved that glycerol preserved allografts could resist physiological blood

pressure, these allografts were tested in an infrarenal, rat aorta transplantation model as

described in chapter 4. The feasibility of implantation of a glycerol preserved allograft was

thereby examined in an animal model. Glycerol preserved aortic allografts of donor rats were

implanted in 18 recipients. Another group of 18 rats received an autotransplantation of the infra

renal aorta as controls. Graft surveillance with duplex sonography was performed on a regular

basis, and angiographic images were produced before harvesting the grafts after a follow-up

period of 3 months.

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1After the application of glycerol preserved allografts was shown to be feasible in a small-

animal transplantation model, these results needed to be evaluated in a large animal model to

enable extrapolation to the clinical setting. Chapter 5 discusses experiments performed in the

goat carotid artery transplantation model. The experiments consisted of three animal groups.

Firstly, carotid allografts preserved in University of Wisconsin (UW) solution were transplanted

bilaterally. In the second group, glycerol preserved allografts were used as bilateral interposition

grafts in the common carotid artery. The third group was considered to represent the golden

standard of autologous vein grafting. The jugular vein was used for unilateral autotransplantation.

A summary of the studies described in this thesis is provided in chapter 6. This thesis is

concluded with a general discussion and future perspectives in chapter 7.

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18 | Chapter 1

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1(66) Bambang LS, Mazzucotelli JP, Moczar M, Beaujean F, Loisance D. Effects of cryopreservation on

the proliferation and anticoagulant activity of human saphenous vein endothelial cells. J Thorac Cardiovasc Surg 1995 Oct:998-1004.

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(69) De Bakey ME. Les greffes d’aorte. In: Fontaine R, Dubost C, editors. Les Greffes Vasculaires. Paris: Presse Universitaire de France; 1954, p. 264-5.

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(71) Gournier JP, Adham M, Favre JP, Raba M, Bancel B, Lepetit JC, et al. Cryopreserved arterial homografts: preliminary study. Ann Vasc Surg 1993 Nov;7(6):503-11.

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(77) Callow AD. Arterial homografts. Eur J Vasc Endovasc Surg 1996 Oct;12(3):272-81.

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(79) Foster JH, Lance EM, Scott HW, Jr. Experience with ethylene oxide treated freeze-dry arterial homografts in 110 consecutive patients. Ann Surg 1958 Aug;148(2):230-8.

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(86) Marrangoni AG, Cecchini LP. Homotransplantation of arterial segments preserved by the freeze-drying method. Ann Surg 1951 Dec;134(6):977-83.

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22 | Chapter 1

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Fahner.indd 22 5-5-2014 14:35:08

2Systematic review of preservation methods and

clinical outcome of infrainguinal vascular allografts

P.J. Fahner, M.M. Idu, T.M. van Gulik, D.A. Legemate

J Vasc Surg 2006;44:518-24

Department of SurgeryAcademic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

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24 | Chapter 2

Abstract

Objective. We systematically reviewed clinical studies on the use of venous and arterial allografts

for infrainguinal revascularization in the absence of suitable autologous vein. We attempted to

find evidence for the best infrainguinal vascular allograft by a systematic review of the available

literature.

Methods. An electronic search of the Medline, Embase and Cochrane databases was employed

to determine key-articles from studies on the different types of vascular allograft used in

infrainguinal reconstruction from 1966 to 2004. Articles were independently reviewed using

previously defined inclusion and exclusion criteria. Study results were gathered with cumulative

primary patency as most important endpoint. Secondary end points were major complications,

graft disintegration, and major limb loss. Quantitative analysis was performed on the prospective

randomized trials and linear regression analysis was performed on cumulative primary patency.

Fontaine’s classification system was applied.

Results. No systematic review of randomized controlled trials (RCT) was found. Five RCTs,

three prospective cohort or case series and fifteen retrospective case series with 3837 vascular

allografts were found. Methods of allograft preservation were cryopreservation (CP) (5 studies),

cold storage (CS) (3 studies) and glutaraldehyde preservation (GA) (15 studies). One-year

cumulative primary patency rates were 13-79% for CP, 63-80% for CS and 40-91% for GA. The

weighted mean one-year cumulative primary graft patency rate was 41% for CP, 71% for CS and

70% for GA allografts. Four randomized trials on femoropopliteal bypasses demonstrated higher

patency rates of glutaraldehyde-preserved human umbilical veins than in PTFE-grafts. Statistical

heterogeneity between studies (I2 = 91.4%) was too high to perform a formal meta-analysis.

The rate of major limb loss was 20-58% for CP, 10-69% for CS and 0-65% for GA and the

percentage of graft disintegration was 2-6% for CP, 4-15% for CS and 0-11% for GA.

Conclusions. A firm conclusion could not be made because there were no studies available in

which direct comparison was performed between different preservation methods of vascular

allografts. In addition, heterogeneity of the individual studies hampered direct comparison of

different types of vascular allografts. However, the overall graft performance of glutaraldehyde-

preserved human umbilical vein allografts may be superior to that of other vascular allografts.

Fahner.indd 24 5-5-2014 14:35:08

Systematic review of preservation methods and clinical outcome of infrainguinal vascular allografts | 25

2

Introduction

Autologous vein is the conduit of choice in patients who need a bypass for critical lower leg

ischemia. However, suitable autologous veins may be of inferior quality or absent as a result

of prior use. Alternatives include prosthetic grafts (Polytetrafluoroethylene [PTFE] and Dacron

[DuPont, Wilmington, Del]) or vascular allografts. Prosthetic vascular grafts have disappointing

patency rates of approximately 30% at 5 years in distal reconstructions and cannot prevent limb

loss in many patients with critical lower limb ischemia (1).

Transplantation of a vascular allograft is an attractive alternative in patients with no suitable

autologous vein. The technique of vascular transplantation was introduced by Carrel and Guthrie

in their classical studies of 1908 (2). In the mid-19th century, studies reported that large-caliber

vascular allografts could result in graft disruption, calcification and occlusion. As a consequence

large-caliber allografts were abandoned in favor of prosthetic grafts, which performed

satisfactorily in non-infected operative fields. Currently, the large majority of allografts are used

in infrainguinal vascular reconstructions.

Transplantation of blood vessels requires a suitable graft preservation method. Improvements

in tissue preservation techniques allow a considerable number of blood vessels of different

lengths and diameters to be stored and used whenever necessary (3). Over the past two decades

this has lead to a renewed interest in vascular allograft transplantation of small caliber grafts

(< 6 mm) for peripheral vascular reconstructions.

The aim of this systematic review was to evaluate the results of clinical studies in which

vascular allografts were used in the management of patients needing an infra inguinal bypass

operation and to find evidence for the best vascular preservation technique.

Methods

Inclusion and exclusion criteria

Clinical studies of patients with vascular allografts for infra-inguinal arterial obstructive disease

(acute and chronic) were included. In accordance with the recommendations of the Ad Hoc

Committee for Reporting Standards of the Society for Vascular Surgery of North America, only

series of at least 40 procedures were included (4;5). Requirements were that the donor vessels be

either arterial or venous segments and that the method of preservation of the allograft should be

mentioned. The arterial donor vessels had to be taken from the iliac or femoropopliteal arteries,

and the venous donor vessels had to be taken from the saphenous vein or human umbilical vein

(HUV).

Fahner.indd 25 5-5-2014 14:35:08

26 | Chapter 2

For inclusion, information on patency, location of the anastomosis, and length of follow-up

period had to be retrievable. Patency rates had to be based on objective findings and had to

have been demonstrated by an accepted vascular imaging technique such as duplex scanning,

arteriography or magnetic resonance imaging. Composite conduits were excluded. However,

we did include the Biograft® (Meadox Medicals, Oakland, NY) – an HUV allograft externally

reinforced by Dacron mesh – because there is no contact between the luminal blood flow and

the prosthetic material. Studies on xenografts were excluded, as were studies that included

reconstructions for aneurysmal disease, access for hemodialysis, or vascular trauma.

Cumulative primary graft patency was the primary endpoint. Secondary endpoints were

major complications, and major limb loss. Major limb loss was defined as a below-knee or more

proximal amputation.

Search strategy

Two authors (P.J.F and M.M.I) independently undertook an electronic search of Medline (January

1966 to January 2004), Embase (January 1988 to December 2003), the Cochrane Clinical

Trials Register, and the Cochrane Database of Systematic Reviews. No language restrictions

were enforced, and a manual cross-reference search of key articles was performed to identify

additional relevant articles. The following keywords and Medical Subject Heading terms were

used: peripheral arterial occlusive disease, peripheral ischemic disease, critical limb ischemia,

infra-inguinal repair, arterial reconstruction, vascular reconstruction, transplantation, arterial

graft, venous graft, arterial homograft, vein homograft, arterial allograft, venous allograft,

autologous vein, patency, limb salvage, reoperation, redo, amputation, fresh, cold storage, 40C,

cryopreservation, lyophilization and glutaraldehyde. The search commands were combined with

the maximally sensitive qualifying string for randomized controlled trials as defined by the UK

Cochrane Centre (6). The keywords and Medical Subject Heading terms were combined with

different suffixes to define the best fit for the search question.

Data collection

Extracting of patient data and graft characteristics and deciding wether the studies met the

inclusion criteria were performed independently by two authors (P.J.F and M.M.I). Patency data,

as primary outcome, were collected at 1, 6, 12 months and 2-3 years after surgery. The checklists

of three working groups on reporting standards served as guidelines to ensure that all relevant

data were gathered from the articles. These groups were the Meta-analysis Of Observational

Studies in Epidemiology Group (7), the Quality of Reporting of Meta-analyses Group (8) and the

Consolidated Standards of Reporting Trials (9).

Fahner.indd 26 5-5-2014 14:35:08


2

Statistical analysis

Quantitative data from the four prospective randomized controlled trials were entered into

Cochrane RevMan 4.2.7 software (The Cochrane Collaboration, Oxford, England) and analyzed

with MetaView. If statistical heterogeneity (I2) less than 60%, meta-analysis was planned (10).

The weighted mean was calculated for one-year and two- three year cumulative primary graft

patency for the non-randomized cohort studies. Weighted means were calculated by multiplying

the outcome concerned by the number of allografts included in each single study. These results

were added up and divided by the total number of allografts in the relevant allograft preservation

group.

Linear regression analysis was performed on the cumulative primary patency rates of all studies

after 24 months, and the percentage included patients with Fontaine stage II limb ischemia. The

Pearson correlation coefficient was calculated by use of SPSS 6.2 software (SPSS Inc, Chicago, Ill).

Results

Studies included

A total of 1260 publications were found. They were all screened by title to see whether they

matched the inclusion criteria. If the relevance of an article was unclear, the abstract was read

and information on patients, type of bypass surgery and graft type was collected. This search

yielded 81 articles that were then reviewed in detail on inclusion and exclusion criteria. A final set

of 23 key articles was obtained (11-33).

No systematic review of RCTs on vascular allograft preservation methods was retrieved.

Four RCTs, were retrieved that compared vascular allografts with alternative conduit material

for peripheral vascular bypass surgery. These trials compared HUV allografts with PTFE and

autologous vein grafts (29;30;32;33), and they provided the highest level of evidence available

for the comparison of the HUV allograft and prosthetic bypass (level Ib according to the Level

of Evidence and Grades of Recommendation as provided by the National Health Service Centre

for Evidence-Based Medicine). One RCT concerning venous allografts looked at the effect of

immunosuppressive therapy on graft patency (13). Three studies were prospective cohort or case

series, and the remaining studies were retrospective case series containing level IV evidence.

Studies excluded

If patient series were published more than once due to prolonged follow-up, only the article

with the longest follow-up was reviewed. Four review articles dealing with synthetic grafts,

xenografts and the financial aspects of vascular disease were excluded.

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28 | Chapter 2

If identical studies from the same institution were reported by different authors, they were

included only once. Two articles on lyophilization were excluded because of small study groups.

Studies on peripheral arterial reconstructions using vascular allografts in combination with

non-allograft material were also excluded. One Japanese and one Czech article were excluded

because of translation complications.

Vascular allograft preservation methods.

Three vascular allograft preservation methods were used in the selected studies: cryopreservation,

cold storage and glutaraldehyde preservation. Cryopreservation was used for both arterial (15)

and venous (11-14) allograft preservation. In all cases cryopreservation was performed in a

dimethyl sulfoxide containing solution by using a programmable control-rate freezer. Storage

temperatures ranged from –120ºC to –196ºC. Most grafts were obtained from Cryolife,

Inc (Kennesaw, Ga) and had an outer diameter of more than 3.5 mm. The storage, thawing,

and handling protocols provided by Cryolife have been established during the last two decades.

Cold storage was used in three studies, and all of these were on venous allografts that were

harvested during varicose vein surgery (16-18). Diameters ranged from 6 to 9 mm, and lengths

from 30 to 65 cm. The grafts were stored at 4ºC in a saline solution containing antibiotics

(chloromycetin and amphotericin). The veins were examined under aseptic conditions for wall

quality and diameter measurement and were ready for use after 10 to 21 days.

All articles about glutaraldehyde vascular allograft preservation were studies in which the

HUV was used as a conduit. These grafts (Biograft, Meadox Medicals, Oakland, NJ, or Bio-

Vascular Inc., St. Paul, Minn.) were manually prepared, tanned with glutaraldehyde, and

covered with a polyester Dacron mesh. Storage was performed in 50% aqueous ethanol. Just

before implantation, grafts underwent a rinse procedure. In most studies the glutaraldehyde

preservation protocol itself was not described in detail.

Baseline and outcome characteristics

Table 1 describes the baseline characteristics of the 23 key articles. The publication year of

the studies ranged from 1968 to 2002. Fifteen of these studies (68%) were published after

1990. A total of 3263 patients with 3837 vascular allografts were included in this systematic

review. Critical limb ischemia (CLI; Fontaine III-IV) was the indication for vascular reconstruction

in 78% (2927/3762) of the vascular allografts. The distal anastomosis was located crurally in

38% (1340/3527) and in the popliteal region in 57% (2034/3592) of the procedures. Crural

locations included the tibial, peroneal and pedal arteries. A popliteal anastomosis was defined as

being either above the knee or below the knee.

Fahner.indd 28 5-5-2014 14:35:08


2

Table 1. General characteristics of included studies of arterial and venous infrainguinal allografts

Study Year Inclusion period

No. grafts

Critical ischemia

Crural anastomosis

Anti- coagulation

Study design

Cryopreservation venous allografts

Brockbank (11) 1992 1985-1990 75 NR 39 % NR retrosp case serie

Martin (12) 1994 1987-1993 115 80 % 88 % AP or AC retrosp case serie

Carpenter (13) 1997 2 year 40 100 % 93 % AP prosp RCT

Farber(14) 2003 1992-2002 240 89 % 65 % AP or AC retrosp case serie

Cryopreservation arterial allografts

Branchereau (15) 2000 1991-1997 165* 100% 79 % AP and/or AC

retrosp case serie

Cold storage venous allografts

Reedt Dortland (16)

1991 1984-1989 156 67 % 33 % AC retrosp case serie

Rebane (17) 1997 1978-1993 107 100 % 63 % AC retrosp case serie

De Leersnijder (18)

1992 1982-1989 100 41 % NR AC (26%) retrosp case serie

Glutar aldehyde venous allografts (HUV)

Dardik (19) 1988 1975-1985 907 94 % 36 % NR retrosp case serie

Dardik (20) 1995 1985-1993 167 89 % 51 % NR retrosp case serie

Dardik (21) 2002 1990-2000 283 89 % 61 % AC prosp case serie

Robison (22) 1983 1976-1981 99 91 % 79 % BK NR retrosp case serie

Hirsch (23) 1984 5 year 133 63 % 0 % NR retrosp case serie

Boontje (24) 1986 1978-1984 257 26 % 38 % NR retrosp case serie

Jarrett (25) 1989 1977-1986 211 73 % 0 % NR retrosp case serie

Batt (26) 1990 1978-1987 105 95 % 50 % AC retrosp case serie

Sato (27) 1995 1977-1993 111 31 % NR NR retrosp case serie

Comparison HUV with other conduits

Weisel (28) 1981 1976-1979 66 68 % 18 % NR prosp cohort study

Eickhoff (29) 1987 1980-1981 50 81 % 0 % NR prosp RCT

McCollum (30) 1991- 1984-1981 87 73 % 0 % AP prosp RCT

Johnson (31) 1991 NR 55 85 % 25 % NR prospect case serie

Johnson (32) 2000 1983-1988 261 68 % 0 % AP prosp RCT

Aalders (33) 1992 1983-1984 47 23 % 0 % AC prosp RCT

NR = Not reported, retrosp = retrospective, CS = case serie, AP = antiplatelet therapy (aspirin and/ or clopridogrel), AC = anitcoagulant therapy (warfarin), CHS = prospective cohort study, BK = below knee prosp RCT = prospective randomized clinical trial, * = study included 45 grafts preserved by cold storage.

Fahner.indd 29 5-5-2014 14:35:08

30 | Chapter 2

Most studies were retrospective case series. Of the prospective series five were RCTs, two were

case series and one was a cohort study. The only RCT performed on cryo-preserved allografts

was that of Carpenter et al (13) which, after vascular allograft implantation, randomized patients

to either immunosuppressive therapy with azathioprine (17 patients) or no immunosuppressive

therapy (23 patients). The overall cumulative primary graft patency rate at 12 months was 13%,

and there was no significant difference between the groups. Four RCTs were retrieved on studies

in which HUV allografts were compared with other conduits in femoropopliteal bypasses. Eickhoff

et al (29) published a multicenter trial comparing HUV and PTFE-grafts. They demonstrated a

1 year cumulative primary patency rate of 74% for the HUV grafts compared with 55% for the

PTFE grafts (81% CLI). Aalders et al (33) reported a study of above-knee femoropopliteal bypass

grafts only (23% CLI) in which the 6 year cumulative primary patency rates of PTFE and HUV

grafts were 38% and 71% respectively. Johnson et al (32) compared above-knee femoropopliteal

bypasses using autologous saphenous vein, PTFE and HUV. The cumulative primary patency rates

after 5 years were 73%, 39% and 53% respectively. McCollum et al (30) compared HUV grafts

with PTFE grafts (CLI 73%). The 1 year cumulative primary patency rates in this study were 68%

for HUV and 61% for PTFE.

Table 2 describes the clinical outcomes of the allografts in the 23 key articles. The data on

graft patency were extracted directly from the text or graphs. Only five articles had follow-

up periods of longer than five years. The 1 year cumulative primary patency of cryopreserved

venous allografts ranged from 13% to 79%. There was only one key article on cryopreserved

arterial allografts, with a reported 1 year cumulative primary patency of 49%. Key articles on

cold storage as a method of vascular preservation concerned venous allografts only. The 1 year

0 10 20 30 40 50 60 70 80 900

25

50

75

100

% patients with Fontaine stage II

Gra

ft p

aten

cy

Figure Plot of linear regression analysis of number of patients with Fontaine stage II and overall 2 year cumulative primary graft patency rate of all included studies. Pearson correlation coefficient = 0.58, (P = 0.01).

Fahner.indd 30 5-5-2014 14:35:09


2

Table 2. Clinical characteristics of allografts and patient outcomes

Study Cum prim (sec) patency Mortality 30d

Major comp

Majorlimb loss

Graft desint

1m 6m 1y 2-3y

Cryopreservation venous allografts

Brockbank (11) 94 87 79 79 NR NR NR NR

Martin (12) - 59(59) 37(40) 19(28) 0 % 0 % 27 % 2 %

Carpenter (13) - - 13 - 3 % 15 % 58 % 3 %

Farber (14) 83(83) 50(52) 30(30) 18(20) 6 % 11 % 33 % 4 %

Cryopreservation arterial allograftsBranchereau (15) 83(90) - 49(60) 35(42) 3 % 1 % 20 % 6 %

Weighted mean one-year cum prim patency

41

Cold storage venous allograftsReedt Dortland (16)

- 89 80 62 NR 4 % 10 % 15 %

Rebane (17) (83) - (65) (38) 4 % 1 % 69 % 4 %

De Leersnijder (18) - 79 63 46 4 % NR 15 % 15 %


71

Glutar aldehyde venous allograftsDardik 1988 (19) 83 67 59 43 NR 4 % 35 % 2 %

Dardik 1995 (20) (86) (84) (80) (65) NR 4 % 38 % 3 %

Dardik 2002 (21) - - 84(87) 67(74) NR 3 % 35 % 0.4 %

Robison (22) 86 69 64 43 2 % NR 29 % NR

Hirsch (23) 91 81 73 69 7 % 2 % NR 2 %

Boontje (24) 96 - 86 70 1 % NR NR 1 %

Jarrett (25) 90(85) 83(78) 78(70) 63(59) 1 % 6 % 10 % NR

Batt (26) 61 - 42 33 6 % 7 % 65 % 8 %

Sato (27) 94 87 86 79 NR 1 % NR 10 %

Comparison HUV with other conduitsWeisel (28) 70 49 40 36 5 % NR NR NR

Eickhoff (29) - 78 75 53 NR 0 % 4 % 0 %

McCollum (30) - 78 68 57 NR 1 % 14 % NR

Johnson 1991 (31) - - 63 49 NR NR 5 % 7 %

Johnson 2000 (32) - 77 74 64 1 % 3 % 10 % 0.4 %

Aalders (33) - 96 (96) 91 (92) 86 (89) 0 % 0 % 0 % 11 %


70

Cum prim (sec) patency = cumulative primary (secondary) patency, 30d = 30 days, comp = major complications, disint = disintegration, NR = Not reported

Fahner.indd 31 5-5-2014 14:35:09

32 | Chapter 2

cumulative primary patency of cold-stored venous allografts ranged from 63% to 80% and for

glutaraldehyde preserved venous allografts, it ranged from 40% to 91%. The weighted mean

1 year and 2-3 year cumulative primary graft patency rate was 41% and 31% for cryopreserved

allografts, 71% and 51% for cold-stored allografts, and 70% and 56% for glutaraldehyde

allografts, respectively.

Postoperative follow-up information on 30-day postoperative mortality, major complications,

major limb loss, and graft disintegration was collected. Postoperative mortality was not reported

in 9 of the key-articles. The overall postoperative mortality in the remaining 14 articles was

2.9% (51/1744). Major complications such as infection requiring graft removal, hemorrhage that

necessitated reoperation, and non-graft-related complications such as myocardial infarction,

pulmonary embolism, stroke, deep venous thrombosis, and gastro-intestinal hemorrhage. The

reported incidences of major complications were between 0% and 15%. Major limb loss occurred

in 0-69% of the patients after graft implantation. Graft disintegration (aneurysm formation or

graft rupture) was not reported in 5 of the 23 key articles but occurred in 0-15% of grafts.

In the analysis of the four RCTs concerning HUV allografts versus PTFE and saphenous vein, the

weighted mean difference could not be calculated in two RCTs because the standard deviation

was not reported. Statistical heterogeneity (I2) was 91.4%. This was far above the accepted

maximum level of 60% for performing a meta-analysis; therefore, it was not appropriate to

perform a meta-analysis.

A significant positive correlation existed between the percentage of patients with Fontaine

stage II disease and the 2 year cumulative primary graft patency (Fig). The Pearson correlation

coefficient was 0.58, significant at the 0.01 level.

Discussion

In this review we included 23 studies in which 3 different methods of vascular allograft

preservation were used. Because 15 of the 23 studies were retrospective case series, the level of

evidence of most studies in this review was low, and, as a consequence, results are susceptible

to bias. We tried to minimize bias by using objective criteria for important outcome parameters

and did not impose any restrictions on the language of publication in the initial search. Because

of the heterogeneity in various clinical factors, such as indication for operation, level of distal

anastomosis, anticoagulation use, and high statistical heterogeneity, it was not appropriate to

perform an overall meta-analysis of the studies.

The reported incidences of major complications were relatively low (0-15%). Two studies on

cryopreserved venous allografts (13;14) showed the highest major complication rates to be 11%

and 15% (Table 2). This is reflected by the high mean age and large number of patients with

Fahner.indd 32 5-5-2014 14:35:09


2

substantial renal dysfunction in the series of Farber et al (14) and the high percentage of CLI

(100%) and previous peripheral bypass procedures shown in the series of Carpenter et al (13).

In most series (12/21), the distal anastomosis was situated in the infrapopliteal or cruropedal

artery in more than 50% of cases. Reported major limb loss varied widely from 0-69% and was

highest in cold storage venous allografts described by Rebane et al (17).

Albers et al (1) performed a meta-analysis on 43 studies of PTFE bypass grafts to infrapopliteal

arteries and reported a pooled estimate of foot preservation of 66% after 3 years follow-up. Of

the studies in our review, 67% (12/18) reported a higher percentage of foot preservation. In

another meta-analysis, Albers et al (34) reviewed 33 studies of patients with an infrapopliteal

allograft bypass, and the pooled 1 year foot preservation rate was between 70% and 80%.

If a patient with limb ischemia needs a bypass to the popliteal artery, the most suitable type of

graft to use is still open to debate. In The Cochrane Review, Mamode and Scott (35) were unable

to demonstrate any clear evidence in support of the use of a particular type of graft at this level.

Yet a recent randomized trial clearly favored autologous saphenous vein over PTFE grafts (36).

If a patient needs a crural bypass the Transatlantic Inter-Society Consensus Working Group (37)

recommend the use of good-quality autologous vein to construct the bypass. They based their

findings on evidence from a meta-analysis by Hunink et al (38), with 5-year primary patency rates

for infrageniculate reconstructions of 66% for autologous vein and 33% for PTFE. Whether the

addition of a distal cuff in PTFE grafts or a distal arteriovenous fistula will ultimately result in an

improved patency of infrainguinal PTFE grafts is still to be defined (39-41).

Fifteen of the 23 series included looked at venous allografts preserved in glutaraldehyde

(HUV), and the highest patency rates were reported in this group (range 33-89%). With the

exception of Brockbank et al (11), studies in which cryopreserved venous allografts were used

reported the lowest patency rates. Because of the high percentage of patients lost to follow-up

(92% after 2 years), the results of this study should be interpreted cautiously. No studies that

included different types of allografts or graft preservation methods were available, so direct

comparison between various vascular allografts or preservation techniques was not possible.

Because of heterogeneity among studies, as reflected in differing levels of preoperative

critical ischemia, percentages of crural anastomosis, and differences in number of patients

lost to follow-up, it can be argued that weighted mean cumulative primary patency rate is

inappropriate for comparing the results of the different allografts. However, comparison of

patency rates is essential when deciding which allograft to use. Because the patency rate is

an important characteristic of graft function, the weighted mean primary patency used in this

review is currently the best approach when comparing the results of these allografts with the

data available in the literature.

Graft disintegration is a particular problem in cold-stored venous allografts. The degree of

antigenicity of the vascular allograft plays a role in the process of biodegradation, as shown in

Fahner.indd 33 5-5-2014 14:35:09

34 | Chapter 2

reconstructions with bovine heterografts. The preservation technique is important in diminishing

the rejection response to the allograft. It has been shown that the glutaraldehyde molecule

masks the histocompatibility antigen sites on vascular tissue (21), and it has been suggested that

cryopreservation decreases the acceptor immunological response against venous allografts (42).

This probably explains the lower biodegradation rate of glutaraldehyde and cryopreserved

vascular allografts in comparison with cold-stored grafts.

Tissue matching and immunosuppressive therapy are important issues in vascular

transplantation. Most studies in our review report the results of allotransplantations performed

without ABO blood type matching. The role of tissue matching on the function of vascular

allografts is still to be defined. Only one study randomized for immunosuppressive therapy (13),

and it was demonstrated that immunosuppression had no effect on the patency of cryopreserved

allografts. It has been demonstrated that cryopreserved and cold-stored allografts retain cell-

surface structures such as the major histocompatibility complex that are involved in the process

of allograft rejection.

The ideal vascular allograft should have a high graft patency rate, a low graft disintegration

rate, need no imunnosuppressive therapy, be available off the shelf in different diameters and

lengths, and be able to be stored for long periods. This systematic review addressed the various

aspects of vascular allografts. Because of recent advances in preservation of vascular allografts

for use in clinical vascular surgery, their results needs to be compared with those described in

this systematic review (43, 44).

In conclusion, this systematic review assessed the results of studies on infrainguinal vascular

allografts. Different allograft preservation methods were compared, and it was demonstrated that

heterogeneity among studies was high and excluded a formal meta-analysis. A firm conclusion

could not be made because there were no studies available in which direct comparison was

performed between different preservation methods of vascular allografs. However, this review

revealed that patency rates for infrainguinal reconstructions performed by using allografts were

satisfactory. If the results of major complications, limb loss, and graft disintegration were also

taken into account, glutaraldehyde preservation of HUV allografts seemed superior to the other

methods of vascular allograft preservation.

Acknowledgement

We are grateful to Dr D. Th. Ubbink for assistance in statistical and meta-analysis.

Fahner.indd 34 5-5-2014 14:35:09


2

Reference List

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(2) Guthrie CC. Structural changes and survival of cells in transplanted bloodvessels. J Am Med Assoc 1908;50:1035-6.

(3) da Gama AD. The fate of arterial transplantation or the death of the microcosm. Leriche memorial lecture. Cardiovasc Surg 1999;7(7):671-80.

(4) Myers KA. Reporting standards and statistics for evaluating intervention. Cardiovasc Surg 1995 Oct;3(5):455-61.

(5) Rutherford RB, Becker GJ. Standards for evaluating and reporting the results of surgical and percutaneous therapy for peripheral arterial disease. J Vasc Interv Radiol 1991 May;2(2):169-74.

(6) Greenhalgh T. How to read a paper. The Medline database. BMJ 1997 Jul 19;315(7101):180-3.

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(9) Moher D, Schulz KF, Altman D. The CONSORT statement: revised recommendations for improving the quality of reports of parallel-group randomized trials. JAMA 2001 Apr 18;285(15):1987-91.

(10) Higgins JP, Thompson SG, Deeks JJ, Altman DG. Measuring inconsistency in meta-analyses. BMJ 2003 Sep 6;327(7414):557-60.

(11) Brockbank KG, McNally RT, Walsh KA. Cryopreserved vein transplantation. J Card Surg 1992 Jun;7(2):170-6.

(12) Martin RS, III, Edwards WH, Mulherin JL, Jr., Edwards WH, Jr., Jenkins JM, Hoff SJ. Cryopreserved saphenous vein allografts for below-knee lower extremity revascularization. Ann Surg 1994 Jun;219(6):664-70.

(13) Carpenter JP, Tomaszewski JE. Immunosuppression for human saphenous vein allograft bypass surgery: a prospective randomized trial. J Vasc Surg 1997 Jul;26(1):32-42.

(14) Farber A, Major K, Wagner WH, Cohen JL, Cossman DV, Lauterbach SR, et al. Cryopreserved saphenous vein allografts in infrainguinal revascularization: analysis of 240 grafts. J Vasc Surg 2003 Jul;38(1):15-21.

(15) Branchereau A, Albertini JN, Magnan PE, Barral X, Favre JP, Guidicelli H, et al. Arterial allografts as a conduit for surgical reconstruction. Crit Ischemia 2000;10(3):71-5.

(16) Reedt Dortland RW, van Leeuwen MS, Steijling JJ, Theodorides T, van Vroonhoven TJ. Long-term results with vein homograft in femoro-distal arterial reconstructions. Eur J Vasc Surg 1991 Oct;5(5):557-64.

(17) Rebane E, Tikko H, Tunder E, Lepner U, Helberg A, Pulges A, et al. Venous allografts for infrainguinal vascular bypass. Cardiovasc Surg 1997 Feb;5(1):21-5.

(18) De Leersnijder D, Willocx P, Van Marck E, Vanmaele R. Venous homografts in infra-inguinal procedures: an eight years experience. J Cardiovasc Surg (Torino) 1992 Nov;33(6):633-40.

(19) Dardik H, Miller N, Dardik A, Ibrahim I, Sussman B, Berry SM, et al. A decade of experience with the glutaraldehyde-tanned human umbilical cord vein graft for revascularization of the lower limb. J Vasc Surg 1988 Feb;7(2):336-46.

(20) Dardik H. The second decade of experience with the umbilical vein graft for lower-limb revascularization. Cardiovasc Surg 1995 Jun;3(3):265-9.

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(21) Dardik H, Wengerter K, Qin F, Pangilinan A, Silvestri F, Wolodiger F, et al. Comparative decades of experience with glutaraldehyde-tanned human umbilical cord vein graft for lower limb revascularization: an analysis of 1275 cases. J Vasc Surg 2002 Jan;35(1):64-71.

(22) Robison JG, Brewster DC, Abbott WM, Darling RC. Femoropopliteal and tibioperoneal artery reconstruction using human umbilical vein. Arch Surg 1983 Sep;118(9):1039-42.

(23) Hirsch SA, Jarrett F. The use of stabilized human umbilical vein for femoropopliteal bypass. Experience with 133 operations with 5-year follow-up. Ann Surg 1984 Aug;200(2):147-52.

(24) Boontje AH. Angiographic assessment of biografts for femoropopliteal bypass. J Cardiovasc Surg (Torino) 1986 Mar;27(2):136-40.

(25) Jarrett F, Hirsch SA. Reoperation for complications of stabilized human umbilical vein grafts. Am J Surg 1989 Nov;158(5):438-42.

(26) Batt M, Gagliardi JM, Avril G, Guzman R, Guidoin R, Hassen-Khodja R, et al. Human umbilical vein grafts as infrainguinal bypasses: long-term clinical follow-up and pathological investigation of explanted grafts. Clin Invest Med 1990 Aug;13(4):155-64.

(27) Sato O, Okamoto H, Takagi A, Miyata T, Takayama Y. Biodegradation of glutaraldehyde-tanned human umbilical vein grafts. Surg Today 1995;25(10):901-5.

(28) Weisel RD, Johnston KW, Baird RJ, Drezner AD, Oates TK, Lipton IH. Comparison of conduits for leg revascularization. Surgery 1981 Jan;89(1):8-15.

(29) Eickhoff JH, Broome A, Ericsson BF, Buchardt Hansen HJ, Kordt KF, Mouritzen C, et al. Four years’ results of a prospective, randomized clinical trial comparing polytetrafluoroethylene and modified human umbilical vein for below-knee femoropopliteal bypass. J Vasc Surg 1987 Nov;6(5):506-11.

(30) McCollum C, Kenchington G, Alexander C, Franks PJ, Greenhalgh RM. PTFE or HUV for femoro-popliteal bypass: a multi-centre trial. Eur J Vasc Surg 1991 Aug;5(4):435-43.

(31) Johnson WC, Squires JW. Axillo-femoral (PTFE) and infrainguinal revascularization (PTFE and umbilical vein). Vascular Registry of the New England Society for Vascular Surgery. J Cardiovasc Surg (Torino) 1991 May;32(3):344-9.

(32) Johnson WC, Lee KK. A comparative evaluation of polytetrafluoroethylene, umbilical vein, and saphenous vein bypass grafts for femoral-popliteal above-knee revascularization: a prospective randomized Department of Veterans Affairs cooperative study. J Vasc Surg 2000 Aug;32(2):268-77.

(33) Aalders GJ, van Vroonhoven TJ. Polytetrafluoroethylene versus human umbilical vein in above-knee femoropopliteal bypass: six-year results of a randomized clinical trial. J Vasc Surg 1992 Dec;16(6):816-23.

(34) Albers M, Romiti M, Pereira CA, Antonini M, Wulkan M. Meta-analysis of allograft bypass grafting to infrapopliteal arteries. Eur J Vasc Endovasc Surg 2004 Nov;28(5):462-72.

(35) Mamode N, Scott RN. Graft type for femoro-popliteal bypass surgery. Cochrane Database Syst Rev 2000;(2):CD001487.

(36) Klinkert P, Schepers A, Burger DH, van Bockel JH, Breslau PJ. Vein versus polytetrafluoroethylene in above-knee femoropopliteal bypass grafting: five-year results of a randomized controlled trial. J Vasc Surg 2003 Jan;37(1):149-55.

(37) Management of peripheral arterial disease (PAD). TransAtlantic Inter-Society Consensus (TASC). Eur J Vasc Endovasc Surg 2000 Jun;19 Suppl A:Si-250.

(38) Hunink MG, Wong JB, Donaldson MC, Meyerovitz MF, Harrington DP. Patency results of percutaneous and surgical revascularization for femoropopliteal arterial disease. Med Decis Making 1994 Jan;14(1):71-81.

(39) Oderich GS, Panneton JM, Yagubyan M, Bower TC, Hofer J, Noel AA, et al. Comparison of precuffed and vein-cuffed expanded polytetrafluoroethylene grafts for infragenicular arterial reconstructions: a case-matched study. Ann Vasc Surg 2005; 19:49-55.

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2

(40) Kreienberg PB Darling RC 3rd, Chang BB, Paty PS, Lloyd WE, Shah DM. Adjunctive techniques to improve patency of distal prosthetic bypass grafts: polytetrafluoroethylene with remote arteriovenous fistulae versus vein cuffs. J Vasc Surg 2000;31:696-701.

(41) Panneton JM, Hollier LH, Hofer JM. Multicenter randomized prospective trial comparing a pre-cuffed polytetrafluoroethylene graft to a vein cuffed polytetrafluoroethylene graft for infragenicular arterial bypass. Ann Vasc Surg 2004;18:199-206.

(42) Weber TR, Dent TL, Salles CA, Ramsburgh SR, Fonseca FP, Lindenauer SM. Cryopreservation of venous homografts. Surg Forum 1975;26:291-3

(43) Fahner PJ, Idu MM, Legemate DA, Vanbavel E, Borstlap J, Pfaffendorf M, et al. Morphological and functional alterations in glycerol preserved rat aortic allografts. Int J Artif Organs 2004;27:979-89.

(44) Martin ND, Schaner PJ, Tulenko TN, Shapiro IM, Dimatteo CA, Williams TK, et al. In vivo behavior of decellularized vein allograft. J Surg Res 2005;129:17-23.

Fahner.indd 37 5-5-2014 14:35:09

Fahner.indd 38 5-5-2014 14:35:09

3Morphological and functional alterations in

glycerol preserved rat aortic allografts

P.J. Fahner, MD 1, M.M. Idu*, MD, PhD 1, D.A. Legemate, MD, PhD 1, E. VanBavel, PhD2,J. Borstlap 1, M. Pfaffendorf, PhD

3, J. van Marle, PhD 4, T.M. van Gulik MD, PhD1

Department of Surgery and Surgical laboratory1, Department of Medical Physics 2, Department of Pharmacotherapy 3, Center for Microscopic Research 4 Academic

Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Int J Art Org 2004;27:979-89

Fahner.indd 39 5-5-2014 14:35:09

40 | Chapter 3

Abstract

Glycerol preservation is an effective method for long-term preservation of skin allografts and has

a potential use in preserving arterial allografts. We evaluated the effect of glycerol concentration

and incubation period on vessel-wall integrity of rat aortic allografts. No significant differences

were measured in breaking strength (2.3 ± 0.3 N) and bursting pressure (223 ± 32 kPa) between

standard glycerolized and control segments

(1.7 ± 0.3 N, 226 ± 17 kPa). Isometric tension measurements showed complete lack of

functional contraction and relaxation capacity in allograft segments prepared according to all

preservation protocols. Morphologically, thickness of the vessel-wall media diminished after

preservation using low (30/50/75 %) or high (70/85/98 %) concentrations of glycerol, as

compared to control segments (i.e. 81 ± 2.4 μm, 95 ± 5.6 μm and 125 ± 3.5 μm, respectively).

Confocal microscopy and Fourier analysis demonstrated that vascular collagen and elastin bundle

orientation had remained unaltered. Electron microscopy showed defragmentation of luminal

endothelial cells.

In conclusion, glycerol preservation of rat aorta resulted in an acellular tissue matrix, which

maintained biomechanical integrity and extracellular matrix characteristics. The next step in the

investigation will be to test the concept of glycerol preservation of arterial allografts in a vascular

transplantation model.

Fahner.indd 40 5-5-2014 14:35:09

Morphological and functional alterations in glycerol preserved rat aortic allografts | 41

3

Introduction

Peripheral bypass surgery is usually the final therapeutic option for patients with extensive lower

limb arterial occlusive disease to prevent impending major limb loss. The autologous vein is

the material of choice for distal vascular reconstruction owing to its good long-term patency

(1). However, in about one-third of patients the autologous vein cannot be used because of

earlier harvesting or poor quality. Alternative materials such as synthetic grafts (Dacron,

Polytetrafluoroethyleen) and biological grafts (human umbilical vein) are commonly used as

peripheral arterial conduits. However, these alternative grafts are hampered by poor long-term

patency. Due to advanced preservation methods, vascular allotransplantation became an option

for patients who need a peripheral arterial reconstruction. In the past, different preservation

methods were developed for storage of vascular transplants and other biomaterials (2-5). Many of

these preservation methods were applied to and studied in skin allografts. Glycerol preservation,

relatively simple and cheap, proved to be a superior preservation method for skin grafts and is

currently the preservation technique of first choice for skin allograft preservation and storage

within the Euro Skin Bank in the Netherlands (6-9).

The main features of glycerol preservation of skin allografts are that the basic architecture

of the skin remains unaffected, glycerol is an effective antibacterial and antiviral agent, and it

diminishes glycerol diminishes antigenicity of the tissues used for transplantation (10-13). These

characteristics held promise for the preservation of vascular allografts and therefore we decided

to evaluate glycerol preservation of rat aortic vascular grafts. The aim of this study was two-fold:

(i) to determine the biomechanical properties and functional characteristics of rat aortic grafts

after preservation with different glycerol concentrations, and (ii) to assess possible morphological

alterations of rat aortic grafts after glycerol preservation.

Materials and methods

Glycerol-preservation protocol

Glycerol preservation was performed by successively immersing the grafts in solutions with

different glycerol concentrations using different incubation times. This preservation protocol was

based on the currently used standard protocol for preserving skin grafts by the Euro Skin Bank

(13). This glycerol-preservation protocol, which had been developed empirically, was extensively

tested and has now been used for many years. Briefly, the protocol consists of three aseptic

stages, i.e. the skin grafts are firstly immersed in a 50% concentrated glycerol solution for

4 hours at room temperature, then in a 70% glycerol solution for 3 hours at 33oC, and finally in

a 85% concentrated glycerol solution for another 3 hours at 33oC. After preservation the grafts

are stored at 4oC.

Fahner.indd 41 5-5-2014 14:35:09

42 | Chapter 3

Several experimental groups were studied. In the first group, the effects of storage on

biomechanical and morphological characteristics of the vessel wall were investigated (group

1A-C in Table Ι). In the second group, the effects of different glycerol concentrations were

examined (group 2A-C in Table Ι), and in the last group the influence of incubation time was

studied (group 3A-F in Table Ι). The incubation temperatures of all three stages of glycerolization

in all experimental groups were identical to the protocol used by the Euro Skin Bank. After

preservation, all glycerol-preserved grafts were cold-stored at 4 0C for 12 hours while remaining

immersed in their final glycerol solution until testing. Rat aorta segments tested immediately

after harvesting served as controls, and breaking strength was measured after incubation in

saline as well (group C1-2 in Table Ι).

Different glycerol concentrations were obtained by dilution of glycerol 98% (Genfarma Corp.

Maarssen, The Netherlands) with demiwater. To obtain a 0.9% NaCl concentration we added

0.45g of sodium chloride to 50 ml of glycerol solution.

Animals

Male Wistar rats (Charles Rivers, Maastricht, The Netherlands) weighing 240-290 g had a period

of acclimatization of at least five days before they were sacrificed by stunning and decapitation

for retrieval of the thoracic and abdominal aorta. The protocol was approved by the Animal

Ethics Committee of the University of Amsterdam, The Netherlands.

Biomechanical experiments

Breaking- strength experiments

Ring segments of 2 mm of descending thoracic aorta, devoid of intercostal arteries, were

harvested from eighteen rats. Each study group contained six aortic segments. The segments

were mounted on two metal hooks.

If a blood vessel is tested for a load-elongation curve, the internal structure of the tissue will

change with each cycle of loading and unloading. By repeated cycling, eventually a steady state is

reached, rendering the specimen into a preconditioned state (14). Therefore, in our experiments,

the tension-testing machine (Newport, Every Cedex, France) was loaded and unloaded ten times

up to 1 N. The segments were subsequently stretched at a constant speed of 0.1 mm/sec until

breaking. The load and deformation were continuously recorded on an X-Y recorder (Newport,

Every Cedex, France), and load-deformation curves were generated by computer software with

which the maximum strength at the breaking point, i.e. the maximum load, could be recorded.

Fahner.indd 42 5-5-2014 14:35:09


3

Tab

le I.

Gly

cero

l inc

ubat

ion

prot

ocol

s an

d re

sult

s of

exp

erim

enta

l gro

ups.

Exp

erim

enta

lg

rou

pG

lyce

rol

con

cen

trat

ion

(%

)

Incu

bat

ion

tim

es (

h)

Bre

akin

gst

ren

gth

(N

)B

urs

tin

gp

ress

ure

(k

Pa)

Med

iath

ickn

ess

(μm

)

End

oth

elia

llin

ing

Ad

ven

titi

alco

mp

actn

ess

Cel

ln

ucl

eus

Ori

enta

tio

n in

dex

(%)

med

iaad

ven

titi

a

Biom

echa

nica

l exp

erim

ents

Mor

phol

ogic

al a

sses

smen

t

1 A

(ESB

)50

/70/

854

/3/3

2.29

(0.3

0)22

3.3

(31.

52)

117.

70

(11.

48)

inte

rmin

term

pykn

otic

54.

10 (3

.83)

78.2

0 (1

.43)

1 B

1 w

k st

orag

e)50

/70/

854

/3/3

2.6

4 (1

.12)

1 C

(3

m s

tora

ge)

50/7

0/85

4/3

/32.

28 (0

.42)

2 A

30/5

0/65

4/3

/32.

96 (0

.71)

232.

5 (4

5.81

)8

0.60

(2

.35)

lost

inte

rm/lo

wpy

knot

ic62

.96

(3.9

6)72

.41

(1.6

6)

2 B

40/

60/7

54

/3/3

1.81

(0.1

7)20

3.2

(11.

12)

2 C

50/7

0/85

4/3

/31.

79 (0

.17

204.

1 (2

8.01

)9

4.99

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Fahner.indd 43 5-5-2014 14:35:09

44 | Chapter 3

Bursting-pressure experiments

Pilot study

A pilot experiment was performed to investigate if a significant difference existed in bursting

pressures between thoracic and abdominal aortic segments. Aortas from seven rats were

harvested proximally just below the thoracic arch and distally just above the bifurcation. Each

aorta was divided into five segments of one cm, three of them thoracic and two abdominal.

For the different thoracic and abdominal positions, the bursting pressures (n=6/ position) were

measured as described for the main study (Figure 1).

Main study

Segments of one cm of descending thoracic and abdominal aorta were harvested from sixteen

rats and randomly allocated to different experimental groups. In the first group (group 1A in

Table Ι), the Euro Skin Bank incubation protocol was tested and in the second group (group 2A-C

in Table Ι), the effect of incubation times was investigated. Fresh segments, which were tested

directly after harvesting, served as controls (group C1 in Table Ι). Each study group consisted of

three aortic segments.

The aortic segments were stretched to their original length in situ and fixed by two needle

tips. A 3 F balloon catheter (Le Maitre Vasc Inc., Bad Soden, Germany) was placed intraluminally

and connected to a pressure controller (Braun Medical, Melsungen, Germany) and a pressure

transducer (Baxter, Deerfield, Illinois, USA). The balloon catheter was inflated until the vessel wall

ruptured. The pressure transducer was linked to a computer for continuous registration of the

changes in pressure. A time/pressure curve was automatically generated. Pressure measurements

with the transducer were linear and reproducible for pressures up to 280 kPa.

thor 1 thor 2 thor 3 abd 1 abd 20

100

200

300

Anatomic position

Burs

ting

pres

sure

(kP

a)

Figure 1. Bursting-pressure results of a pilot experiment of unpreserved, sequential thoracic (thor) and abdominal (abd) aortic segments of seven rats. Bars represent means ± SEM.

Fahner.indd 44 5-5-2014 14:35:09


3

Viability testing, contraction and relaxation properties

Viability testing was performed on rat aortic segments preserved by two different glycerolization

protocols indicated as low and high protocol. The glycerol concentrations used for the low

protocol were 30%, 50% and 65%, and for the high protocol 70%, 85%, 98%. Incubation

periods, temperatures and storage times were the same as for group 1A in Table Ι. Fresh

segments, tested immediately after harvesting served as controls. To discriminate among effects

on viability induced by glycerol or incubation periods, temperatures and storage times, segments

preserved in saline for the same incubation periods, temperatures and storage times as the

glycerolization protocols, were also tested (Figure 2).

Aortic ring segments of three mm were vertically mounted between stainless-steel hooks

in a five-ml organ bath. Each study group consisted of six aortic rings. The upper hook was

attached to an isometric force transducer (Kyowa™, Tokyo, Japan) connected to a Power Lab/8s

data acquisition system (AD Instruments, Australia). The organ bath contained Tyrode’s solution

consisting of 124 mM NaCl, 4.0 mM KCl, 0.9 mM CaCl2, 1.1 mM MgCl2, 0.42 mM NaH2PO4,

24.9 mM NaHCO3 and 5.5 mM glucose. The solution was maintained at 37 °C and pH = 7.4 by

bubbling the solution with carbogen O2/CO2 (95:5).

The contraction ability of the aortic segments was tested by applying 100 μM L-phenylephrine,

an α1-adrenoceptor agonist. The relaxation capacity was measured in two functional pathways: a)

the endothelium-dependent pathway using methacholine (100 µM) to stimulate the endothelial

1.0e

-9

3.0e

-9

1.0e

-8

3.0e

-8

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- 7

3.0e

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6

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0.0

2.5

5.0

7.5

10.0freshNaCllow glycerolhigh glycerol

Na+- Nitroprusside concentration

Con

trac

tion

forc

e (m

N)

Figure 2. Viability tests, results of contraction and relaxation experiments. Control rat aortic ring segments tested immediately after harvesting (fresh) and segments stored in 0.9 % saline (NaCl) showed complete maintenance of relaxation capacity. Note the absence of endothelium-independent relaxation capacity of segments after both low and high glycerol concentration preservation protocols (see Materials and Methods). Bars represent means ± SEM.

Fahner.indd 45 5-5-2014 14:35:10

46 | Chapter 3

cell to release endothelium-derived relaxing factor (EDRF), and b) the endothelium-independent

pathway using Na+ -nitroprusside (100 µM), an NO-donor (15).

The initial resting tension was set at 10 mN and was adjusted throughout the experiment.

After 1 hour of equilibration, the ring segments were exposed to a high potassium concentration

(60 mM) for 10min to depolarise the cells, followed by contraction. When maximum contraction

had been reached, the segment was rinsed with Tyrode’s solution. The potassium contraction

was repeated twice with a 15-min interval. Afterwards, a dose-response curve was constructed

for L-phenylephrine (1.10-8 – 1.10-5 M) and methacholine (1.10-8 – 1.10-5 M) with a potassium

contraction, Tyrode’s rinsing, and a L-phenylephrine (1.10-6) contraction in between measurements.

To determine the endothelium-independent relaxation of the smooth-muscle cells, the NO-

donor Na+-nitroprusside (1.10-9 – 1.10-6 M) was added to the organ bath, after the potassium

contraction, Tyrode’s rinsing and the L-phenylephrine (1.10-6) contraction, in order to be able to

construct the dose-response curve.

Morphological assessment

Morphological assessment was performed in samples prepared according to the same

preservation protocols as for bursting-pressure experiments, corresponding to group 1A, 2A and

2C in Table Ι. Fresh segments served as controls (group C1 in Table Ι).

low intermed high control0

25

50

75

100media

adventitia

Glycerol concentration

Orie

ntat

ion

inde

x (%

)

Figure 3. Orientation index of the media extracellular matrix (media) and adventitial extracellular matrix (adventitia) of rat aortic segments preserved with low, intermediate or high glycerol concentration protocols. Fresh segments served as controls (control). Bars represent means ± SEM.

Fahner.indd 46 5-5-2014 14:35:10


3

Histology

Five-mm aortic ring segments were used, three specimens in each study group. After

preservation, the segments were fixed in 10% formaline solution (Mallinckrodt Baker, Deventer,

The Netherlands), dehydrated, and embedded in paraffin. Transverse sections (5 μm) were

stained with hematoxylin-eosin (HE), Elastin von Gieson, van Gieson, and picro sirius red, and

were mounted on slides for microscopic examination.

A Leica LB 30 S light microscope (Leica, Wetzlar, Germany) was used to view the slides. The

Qwin Image pro analysis software (version 2.6, Leica Imaging Systems Ltd., Cambridge, UK) was

used to measure elastin bundle width and distance between elastin bundles of the media, as

well as media thickness.

A scoring system was used to describe alterations in endothelial lining, adventitial compactness,

and features of cell nuclei as follows. Endothelial lining was scored as complete (all endothelial

cells attached to the media), intermediate (endothelial cells disconnected from the media for

less than 75%), or lost (endothelial cells disconnected from the media for more than 75%).

Compactness of the extracellular matrix of the tunica adventitia was graded as high (compact

collagen bundles), intermediate (low fragmentation of collagen bundles and loose matrix), or low

(high fragmentation of collagen bundles and loose matrix). The aspect of cell nuclei in intima,

media and adventitia was described as sparse chromatin (nuclei with low chromatin density) or

pyknotic (small nuclei with high chromatin density).

Extracellular matrix orientation

The same images as used for the assessment of bundle width and distance were used for matrix

orientation of measurements of the media and adventitia. Image analysis was done with the Fast

Fourier Transform (FFT) module of the same Qwin Image pro analysis software as used for the

histological measurements. To compare the elastin-bundle orientation and the collagen-bundle

orientation in the vessel wall after glycerol preservation with the same orientation in unpreserved

tissue, an orientation index was determined as described below.

Determination of orientation index

Images (512 * 512 pixels) were acquired with a Leica SP2 confocal system making use of the

fluorescent properties of eosin in the HE-stained histological slides: excitation 488 nm / detection

550-590 nm, using a 40* immersion objective and a pinhole setting corresponding with 1 Airy

disc. An orientation index was defined by dividing the short axis of the first-order maximum by

the long axis of the first-order maximum expressed as %. Consequently, a totally parallel structure

gives an orientation index close to zero, whereas as totally random orientation is represented by

an orientation index of 100%. This method was earlier described in detail for skin tissue (16;17).

Fahner.indd 47 5-5-2014 14:35:10

48 | Chapter 3

Each cross section of the aorta was scanned with the microscope to obtain four images (0°, 90°,

180°, 270°) of the media as well as of the adventitia.

Scanning electron microscopy

To examine the effect of glycerol preservation on the endothelial cell lining, scanning electron

microscopy was performed. Five-mm aortic segments were used, three specimens in each study

group.

The segments were rinsed in saline and fixed for 24 hours in McDowall fixative (4%

paraformaldehyde and 1% glutaraldehyde in 0.1M phosphate buffer). After fixation, the

segments were dehydrated in graded ethanols, placed in 1,1,1,3,3,3 hexamethyl disilazan (Merck-

Schuchardt), and dried at room temperature for 12 hours. Subsequently, they were mounted on

stubs with conductive carbon cement, sputter-coated with 20 nm gold-palladium, and scanned

with an electron microscope (Philips SEM 525 equipped with an Orion frame grabber) operated

at 10 kV and a spot size of 50 nm.


Continuous data are presented as means, and non-parametric statistical tests were used for

analysis: the Mann-Whitney test was used to determine differences between two unrelated

groups and the Kruskas-Wallis test was used to determine differences between more than

two unrelated groups. A difference with a p-value of 0.05 or less was considered a statistically

significant difference. The statistical analysis was performed with Statistical Package for the

Social Sciences version 11.01. for Windows ® (SPSS ®, Chicago, Illinois, USA).

Results

Biomechanical experiments

There were no significant differences (p = 0.27) in breaking-strength values measured in the

different glycerol-preserved experimental groups. There were also no significant differences

between the experimental and control groups (group C1-2 in Table Ι). The vessel wall ruptured

at a mean diameter of 17.4 (2.8) mm and 15.2 (2.2) mm in the glycerol-preserved groups and in

the control groups, respectively.

In the pilot study no significant differences (p = 0.31) were found among the mean bursting

pressures of the aortic segments of different locations (Figure 1). In the main study were no

significant differences (p = 0.84) within the glycerol-preserved experimental groups, the mean

bursting pressure tested being comparable to that of unpreserved grafts in the control group

(group C1 in Table Ι).

Fahner.indd 48 5-5-2014 14:35:10


3

Viability testing: contraction and relaxation

The control segments showed a contraction capacity of 41% (9.23 N (±1.85) against 3.79 N

(±1.24)) after 12 hours of storage. Although the endothelium-dependent relaxation capacity

was absent, the endothelium-independent relaxation capacity was still complete (Figure 2).

The functional contraction capacity of the aortic segments preserved in glycerol was found

to be completely lost. The same was true for the endothelium-dependent and endothelium-

independent relaxation capacities (Figure 2).

Morphological assessment

Histology

The mean elastin-bundle width in the media in the control segments was 4.16 μm. There were

no significant differences (p = 0.56) between the mean elastin-bundle widths in the media in the

experimental groups, and there were no differences between the experimental groups and the

control group (see Materials and Methods).

The mean distances between elastin bundles in the media in the control segments was 9.25

μm. There were no significant differences (p = 0.63) between the mean elastin-bundle distances

in the media in the experimental groups, nor when this was compared to the control group.

The media thickness was significantly less in the segments preserved at low glycerol

concentrations (p = 0.05) and in those preserved at high glycerol concentrations (p = 0.05)

than in the control group (group C1 in Table Ι). For the segments incubated at intermediate

concentrations, no significant alterations were found in media thickness.

Endothelial lining, compactness of adventitia and nuclear aspects were assessed using the

scoring system described above. The endothelial lining of the aortic segments was invariably

lost after glycerol preservation. The adventitial compactness was diminished after glycerol

preservation and the cell nucleus of all glycerol-preserved cells were pyknotic (Table Ι). These

features demonstrate that after glycerol preservation, an extracellular tissue matrix without living

cells is all that is left.

Orientation Index

There were no significant differences (p = 0.21) in the orientation indices of the media within the

experimental groups, nor when these were compared to the index of the control group (group

C1 in Table Ι). There were no significant differences (p = 0.22) in the orientation indices of the

adventitia within the experimental groups, also not when these were compared to that of the

control group (Figure 3).

Fahner.indd 49 5-5-2014 14:35:10

50 | Chapter 3


In the control segments, a confluent layer of endothelial cells was present (Figure 4a). The

segments preserved in glycerol showed a flattened layer of endothelial cell remnants with

defragmentation of cellular membranes (Figure 4b). Detachment of the intimal layer from the

internal elastic lamina was evident and fenestration of the intima was shown. These features

were more obvious in the segments preserved with the high glycerol concentrations than in the

segments preserved in low concentrations.

The layers of elastin, which tended to be arranged cylindrically around the lumen, were not

changed in any group.

Figure 4a. Electron micrograph of fresh rat aorta showing the luminal surface with a confluent layer of endothelial cells.

Figure 4b. Electron micrograph of rat aorta after preservation with high-concentration glycerol. Endothelial cells are flattened and partially detached from the underlying internal elastic lamina.

Discussion

For patients who need a peripheral vascular reconstrunction and who do not have an autologous

vein available, vascular transplantation is a reasonable alternative. To be able to prepare and store

appropriate biological grafts for a long time, an efficient preservation method is a prerequisite.

In the past, various methods of preserving arterial and venous grafts have been developed, such

as cryopreservation (18), glutaraldehyde tanning (19), lyophilization (20) and simple cold storage

(21). However, these methods did not result in successful vascular grafts for femorodistal bypass

grafting. Investigations of alternative preservation methods are, therefore, still ongoing.

Because the literature on glycerol preservation of arterial allografts was limited (22),

we decided to examine the alterations in vessel-wall function and architecture after glycerol

preservation of rat aorta arterial allografts.

Fahner.indd 50 5-5-2014 14:35:10


3

The incubation protocol for glycerol preservation from the Euro Skin Bank that we followed

is based on empirism. During years of development, the glycerol concentrations and incubation

periods were established. It was noted that high glycerol concentrations (85%) combined with

short incubation periods induced a quick dehydratation of skin grafts, resulting in a parchment-

like structure unsuitable for clinical use. Preservations starting with lower concentrations (50%)

proved to be more successful. In our present study this protocol was applied to vascular grafts

for the first time, and the influence was investigated of alterations in concentrations of glycerol

and incubation periods on vessel-wall characteristics.

Although most of the forces on a vessel are dilatational, vessels are also subject to longitudinal

stress.

We used the bursting-pressure test because the vessel wall is loaded in all directions, and this

test best represents the physiological situation. In addition, the breaking-strength experiments,

provide information about the diameter at which the vessel wall ruptures. Therefore, besides

bursting pressure, it is useful to measure longitudinal stress resistance as well (Table Ι).

Glycerol-preserved aortic allografts stored in glycerol at 4oC maintained tensile strength

up to 3 months after preservation, indicating that structural integrity of the vessel wall was

maintained. The breaking-strength values in our experiments were comparable to the control

values of 1.55 N in the study of Brüel et al (23) who investigated the role of cross-links in collagen

and elastin and their relation to mechanical stability in Wistar rats. As there were no significant

differences in tensile strength between the groups with different glycerol incubation periods

(group 3A-3F Table Ι), we continued to use the standard incubation times of the Euro Skin Bank.

Bursting-pressure measurements, often used for testing an anastomosis (24;25), are

classically performed by inflating the vessel through an intraluminal cannula after ligation of all

side branches (26-28). This method leads to a wide variation in results and has many confounding

factors, such as the presence of side branches and differences in vessel length. Therefore, we

used an intraluminally placed balloon catheter for determination of the bursting pressures. The

mean bursting pressures measured in this study were in the range of 203.2 kPa to 232.5 kPa,

corresponding to about 13 times the normal rat arterial systolic blood pressure. Since we could

not demonstrate a significant difference (p = 0.27) in the mean bursting pressures of the thoracic

and abdominal segments in our pilot study, we did not discriminate between these two locations

in the remaining part of the study. The biomechanical tests used in our study demonstrated

that glycerol preservation preserved the biomechanical characteristics of the rat aorta and we

could conclude that the extracellular matrix of the vessel wall was still able to withstand more

than physiological pressures after preservation according to the different glycerol preservation

protocols. Loss of extracellular matrix integrity may lead to serious complications, which was

illustrated by Noel et al (29) who reported 9% perianastomotic hemorrhage and graft rupture in

patients who had in situ aortic replacements with cryopreserved aortic allografts.

Fahner.indd 51 5-5-2014 14:35:10

52 | Chapter 3

We examined functional changes in the aortic segments in vitro using isometric tension

measurements. The experiments of Moriyama et al (30) showed a negative correlation between

the duration of warm ischemia in rat aortic tissue and cell viability. Segments of the thoracic aorta

were excised from rats at different postmortem intervals and the maximal contractile response of

freshly isolated arteries decreased to 50% after 6 to 8 hours of warm ischemia. Vischjager et al

(31) measured a 68% decrease in contractile responses of rat aortic segments after seven days of

preservation in University of Wisconsin preservation solution at 4 oC. Cryopreservation of vascular

grafts also influenced cell function (32). The relaxation capacity of human mesenteric artery

ring segments diminished to 29% after cryopreservation (33). We expected to find complete

absence of contraction and relaxation of the aortic segments after glycerol preservation because

of the known loss of viable cells in skin grafts after glycerol preservation. Although viable

endothelial cells play an important role in long-term graft performance by providing nutrition

to the fibroblasts in the vessel wall and by preventing thrombosis, enhanced endothelial viability

may, on the other hand, lead to increased antigenicity of the allograft after implantation

(34). The endothelial cell plays a central role in antigen presentation and is probably the most

immunogenic component of the vascular transplant (35). The low antigenic properties of a

glycerol-preserved skin allograft are probably due to the absence of viable cells. Although an

acellular matrix remained after glycerolization of rat aortic arteries, the structural architecture

of the vessel wall was unaffected. Preservation of only an acellular structure probably leads to

better graft function after implantation, because the endothelial and smooth muscle cells are

major causes of arterial graft rejection (36;37). The extracellular matrix, consisting of elastin and

collagen networks is obviously less immunogenic (38). Although the quality of endothelial and

smooth muscle cells is reduced after cryopreservation and cold storage, the remaining viable cells

still induce an immunological reaction. From this point of view, the absence of viable cells after

glycerol preservation is an advantage compared to other preservation methods.

The elastin bundle width and distance between bundles were measured to examine wether

the extracellular matrix was altered due to the glycerolization process. Alterations in the

extracellular matrix could have implications for the ingrowth of fibroblasts, smooth muscle cells

and endothelial cells (39).

The elastin bundle distances measured in our present study were similar to the results of

Berry et al (40) who found approximately 7.5 µm as mean distance between two elastic lamellae

in the aorta of adult rats. The results from our study confirm that the extracellular achitecture of

the vessel wall after glycerolization was preserved.

We used confocal laser scanning microscopy to analyse the orientation of the extracellular

matrix components after glycerol preservation. This technique was previously applied in our

institution to study collagen bundle architecture and orientation in normal and scar skin tissue.

The Fast Fourier Transformation is a mathematical method to calculate the periodicity and

Fahner.indd 52 5-5-2014 14:35:10


3

orientation of structures, e.g. collagen bundles, and can be represented as a power plot (41).

Fourier image analysis was shown to be a superior method to measure collagen orientation

compared to subjective histological evaluation (42). In our present study, we demonstrated that

glycerol preservation did not disturb the collagen bundle orientation in the extracellular matrix

in the rat aorta.

Due to the observed defragmentation of the endothelial cells, they were no longer able to

initiate any contraction or relaxation response, which was in accordance with the results of the

viability testing experiments. Futher experiments need to be performed in vivo to show whether

or not these endothelial cell remnants still induce any immunological reaction and how they may

have modified thrombogenicity of the luminal surface.

Conclusions

In this study, we demonstrated that glycerol preservation of rat aortic allografts resulted

in a nonviable structure that maintained the mechanical integrity and extracellular matrix

characteristics of fresh aorta segments. A significant decrease in media thickness was shown in

aorta segments after preservation in low (30/50/75 %) as well as high (70/85/98 %) concentrations

of glycerol, compared to intermediate (50/70/85 %) and control segments. Alterations in glycerol

concentration and incubation periods did not significantly influence breaking strength and

bursting-pressure. These results encourage us to test the concept of glycerol preservation of

arterial allografts in a vascular transplantation model.

Acknowledgements

The authors thank Ms M-J Mathy for excellent assistance with the isometric tension measurements

and Ms G. E. E. van Noppen for editorial comments.

Fahner.indd 53 5-5-2014 14:35:10

54 | Chapter 3

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(28) Kolb S, Wray RC, Weeks PM. Bursting strength of microarterial repairs. Surg Forum 1979; 30:531-533.

(29) Noel AA, Gloviczki P, Cherry KJ, Jr., Safi H, Goldstone J, Morasch MD et al. Abdominal aortic reconstruction in infected fields: early results of the United States cryopreserved aortic allograft registry. J Vasc Surg 2002; 35(5):847-852.

(30) Moriyama S, Utoh J, Murai Y, Hagiwara S, Kunitomo R, Nishi K et al. Functional, metabolic, and histological changes of vascular tissues after warm ischemia. Ann Thorac Cardiovasc Surg 2001; 7(3):143-149.

(31) Vischjager M, Van Gulik TM, Kromhout JG, Van Marle J, Pfaffendorf M, Klopper PJ et al. Morphology and function of preserved microvascular arterial grafts: an experimental study in rats. Ann Vasc Surg 1997; 11(3):284-291.

(32) Vischjager M, Van Gulik TM, Van Marle J, Pfaffendorf M, Jacobs MJ. Function of cryopreserved arterial allografts under immunosuppressive protection with cyclosporine A. J Vasc Surg 1996; 24(5):876-882.

(33) Muller-Schweinitzer E, Mihatsch MJ, Schilling M, Haefeli WE. Functional recovery of human mesenteric and coronary arteries after cryopreservation at -196 degrees C in a serum-free medium. J Vasc Surg 1997; 25(4):743-750.

(34) Christy JP, Lupinetti FM, Mardan AH, Thompson SA. Endothelial cell viability in the rat aortic wall. Ann Thorac Surg 1991; 51(2):204-207.

(35) Pober JS, Collins T, Gimbrone MA, Jr., Libby P, Reiss CS. Inducible expression of class II major histocompatibility complex antigens and the immunogenicity of vascular endothelium. Transplantation 1986; 41(2):141-146.

(36) Salomon RN, Hughes CC, Schoen FJ, Payne DD, Pober JS, Libby P. Human coronary transplantation-associated arteriosclerosis. Evidence for a chronic immune reaction to activated graft endothelial cells. Am J Pathol 1991; 138(4):791-798.

(37) Hayry P, von Willebrand E, Parthenais E, Nemlander A, Soots A, Lautenschlager I et al. The inflammatory mechanisms of allograft rejection. Immunol Rev 1984; 77:85-142.

(38) Ellingsworth LR, DeLustro F, Brennan JE, Sawamura S, McPherson J. The human immune response to reconstituted bovine collagen. J Immunol 1986; 136(3):877-882.

(39) Walles T, Herden T, Haverich A, Mertsching H. Influence of scaffold thickness and scaffold composition on bioartificial graft survival. Biomaterials 2003; 24(7):1233-1239.

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(40) Berry CL, Sosa-Melgarejo JA, Greenwald SE. The relationship between wall tension, lamellar thickness, and intercellular junctions in the fetal and adult aorta: its relevance to the pathology of dissecting aneurysm. J Pathol 1993; 169(1):15-20.

(41) Russ JC, editor. Processing images in frequency space. The image processing handbook. Boca Raton: CRC Press, 1995: 283-346.

(42) van Zuijlen PP, de Vries HJ, Lamme EN, Coppens JE, van Marle J, Kreis RW et al. Morphometry of dermal collagen orientation by Fourier analysis is superior to multi-observer assessment. J Pathol 2002; 198(3):284-291.

Fahner.indd 56 5-5-2014 14:35:10

4Glycerol preserved arterial allografts

evaluated in the infrarenal rat aorta

P.J. Fahner1, M.M. Idu1, T.M. van Gulik1, B. van Wijk1, A.C. van der Wal2, D.A. Legemate1

1Department of Surgery and 2Department of PathologyAcademic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Eur Surg Res 2009;42:78-86

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58 | Chapter 4

Abstract

Background. Vascular transplantation has become an alternative for prosthetic grafts. Suitable

storage methods for vascular allografts are therefore necessary. For small calibre arterial

allografts, cryopreservation and cold storage showed discouraging results. Since glycerol

preservation proved effective for the storage of skin allografts, this preservation method was

investigated for vascular allografts using a rat aortic transplantation model.

Methods. Glycerol preserved allografts (GA) were transplanted to the infrarenal aorta (n=18) in

Wistar rats. A control group (n=18) underwent immediate auto transplantation (AU) of an equal

length of aorta.

Results. Cumulative graft patency at 90 days follow-up was 93% for AU and 78% for GA (ns).

No aneurysm formation was detected in both groups. Intraluminal endothelial cell coverage,

integrity of the media and smooth muscle cell repopulation were comparable in both groups.

Intimal thickness was less in GA compared to AU and inflammatory reaction in the adventitia

was diminished in GA.

Conclusion. Glycerol preserved allografts were successfully grafted with an acceptable patency

rates if compared with autografts, while intima hyperplasia and adventitial inflammatory reaction

were less.

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Glycerol preserved arterial allografts evaluated in the infrarenal rat aorta | 59

4

Introduction

Transplantation of small diameter allografts is an alternative for patients who need an infrainguinal

arterial bypass graft, especially if autologous veins cannot be used. Vascular allografts require

proper storage techniques to ensure instantaneous availability. Owing to lower or absent

antigenic potential, preserved vascular allografts are by far superior to fresh allografts. Fresh

Brown Norway rat aorta segments induced a higher inflammatory reaction when subcutaneously

implanted in Lewis recipients compared to cryopreserved and glutaraldehyde preserved segments.

Cryopreserved aorta segments were stored for two weeks and glutaraldehyde segments for

three days before implantation (1). Different preservation methods such as cryopreservation,

glutaraldehyde tanning and cold storage have been the focus of extensive research for several

decades (2). Allograft function is hampered by antigenicity and graft rejection which can lead to

graft dilatation, intimal cell proliferation and graft rupture. Studies on cold-stored allografts have

been unsuccessful, due to disintegration and rapid rejection of the graft material. In the study

of van Reedt Dortland et al (3), cold-stored venous homografts, denatured for at least 6 weeks,

were used in femorodistal arterial reconstructions in patients when a suitable autologous vein was

not available. These grafts developed aneurysms in 58% after 5 years. Rebane et al performed

infrainguinal reconstructions for limb salvage in 107 patients. The venous allografts were cold

stored up to 10 days. Early thrombosis, indicating acute rejection, occurred in 16% and 5 year

graft patency rate was only 20% (3;4). Cryopreservation on the other hand has been extensively

used as a preservation method in the clinical setting, but mainly for large calibre arterial vessels

such as aortic allografts. Long-term patency of small calibre cryopreserved vascular grafts is poor

with reported patency rates of less than 50% at one year (5-7). Therefore, better preservation

techniques are necessary to improve the patency rate of small diameter vascular allografts.

Research on preservation techniques of skin allografts (8-10) showed that glycerol preservation

is superior to cryopreservation in regard to graft acceptance and immunogenicity. Glycerol skin

allografts, with a storage life of at least 2 years, were evaluated in 39 patients with extensive

third-degree burns. Cryopreserved allografts failed before epithelialisation was established

and glycerol allografts results in a complete graft take in 73% at one week postoperative. If

glycerol allografts were applied, wound epithelialisation by autologous epithelium was enhanced

compared to fresh-frozen allografts (11). Glycerol preservation of skin allografts attenuates

allograft reaction and has antiviral and antibacterial properties (12-17). The fact that glycerol

preservation results in a tissue matrix without living cells diminishes immunogenicity of the graft.

Previous experiments in our laboratory demonstrated that glycerol preservation of the rat aorta

preserves the biomechanical characteristics and architecture of the vessel wall (18).

The aim of this study was to evaluate in an aortic transplantation model in the rat i) long-term

patency of glycerol preserved arterial allografts in the infrarenal aortic position ii) aneurysmal

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60 | Chapter 4

degeneration of glycerol preserved arterial allografts, iii) histological features of the glycerol

preserved vessel wall after implantation, with emphasis on restoration of endothelial cell lining,

inflammatory response, degenerative changes of the vessel wall and intimal hyperplasia.


Preparation of glycerol preserved allografts

The glycerolization protocol consists of three stages;

1. Incubation in a glycerol 50% solution for 4 hours at room temperature

2. Incubation in a glycerol 70% solution for 3 hours at 33°C

3. Incubation in a glycerol 85% solution for 3 hours at 33°C

After completion of the protocol, the grafts were stored in glycerol 85% for 12 hours at 4°C.

Previous in-vitro experiments in our laboratory showed that this protocol resulted in optimal

glycerol preservation of arterial allografts (18).

Experimental design

The experimental protocol was approved by the Animal Ethics Committee of the Academic

Medical Centre, University of Amsterdam, The Netherlands. Male outbred Wistar rats (Unilever

and Harlan), 300-370 g, were used as donors and recipients of aortic grafts (Charles Rivers,

Maastricht, The Netherlands). The animals were housed one week prior to the experiments,

had free access to water and chow and maintained on a 12-hour light-dark cycle. All operative

procedures were performed under clean but not sterile conditions. In the experimental (allograft)

group a 10 mm long segment of the infrarenal abdominal aorta was excised and a donor glycerol

preserved abdominal aortic allograft of similar length was implanted as an interposition graft. In

the control (autograft) group a segment of 10 mm of the infrarenal abdominal aorta was excised

and immediately reanastomosed to restore aortic continuity. Before implantation the glycerol

preserved aortic allograft was immerged in saline to diminish the tissue content of glycerol in the

graft at 33°C for at least 20 minutes. Both control and experimental groups consisted of eighteen

rats. Scheduled sacrifice was on post-operative day 1, 3, 7, 14, 30 and 90 respectively. On these

days three animals were sacrificed in both experimental and control group.

Aortic allograft implantation and harvesting

All animals were anesthetized by inhalation of a mixture of O2/N2O 1:1 and isoflurane 0.8 - 2.0%.

After endotracheal intubation the rats were ventilated and anaesthesia was maintained with

the same mixture. The rats were placed on a heating pad and positioned under a heating lamp

Fahner.indd 60 5-5-2014 14:35:11


4

to maintain a rectally measured core temperature between 36°C and 37°C. After a midline

laparotomy, the infrarenal abdominal aorta was exposed, clamped and a 10 mm segment was

excised using an operation microscope (Zeiss™, Germany). The grafts and the aortic stumps were

flushed with saline. No heparin or anticoagulant medication was used in the study. The end-

to-end anastomoses were performed with interrupted 9.0 sutures (Ethylon™). After restoring

the blood flow intra-operative patency was determined by visualization of a pulse distally to

the graft. Operation time and aortic clamping time averaged 90 and 45 minutes, respectively.

After sacrifice of the rat the graft was harvested en-bloc, flushed with saline and fixed in 10%

formaldehyde.

Duplex scanning and angiography

To assess graft patency and aneurysm formation, defined as a consistent 50% increase in graft

diameter, colour duplex scanning (19) and angiography were performed. Duplex scanning

(Hewlett Packard Sonos 5000) was performed under general anaesthesia using a miniature

15 MHz probe at post-operative day 1, 3, 7, 14, 30 and 90 respectively. The abdominal aorta was

examined from the renal arteries to the aortic bifurcation. Diameters of the aorta were measured

in B-mode on the following five locations; proximal native vessel, proximal anastomosis, mid-

graft, distal anastomosis and distal native vessel. To investigate the presence of flow-limiting

lesions peak systolic velocity (PSV) was measured. The PSV-max was defined as the maximum

peak systolic velocity in a stenosis, and the PSV-ratio as the PSV in the stenosis divided by the PSV

in the pre- or poststenotic region. A significant stenosis was defined as a PSV-ratio of ≥ 2.5 or a

PSV-max of ≥ 250 cm/sec. Before excision of the aortic allograft a digital subtraction angiography

(DSA) was performed (Philips™) by injection of three ml radio contrast solution (Visipaque™

320 mg I/ml, Nycomed, The Netherlands) through a catheter introduced into the carotid artery.

Preparation and histological staining of specimens

All explanted graft segments were flushed with 10% buffered formaldehyde, dehydrated and

embedded in paraffin for light microscopic examination. Grafts were divided into three equal

segments of 3-4 mm length: one proximal segment including the proximal anastomosis, one

midgraft segment and one distal segment including the distal anastomosis. Of each segment,

5 µm sagittal sections were cut and stained with haematoxylin-eosin (H&E), picosirius red (PSR)

and Elastica van Gieson (EvG) respectively. PSR stains all types of collagen bright red and EvG

stains elastic fibres deep black. Two additional sections were mounted for immunohistochemistry.

We used Anti-vWF (von Willebrand factor) antibody (DAKO, dilation 1: 100, visualizes all

endothelial cells) and anti-alpha-actin-antibody (DAKO, dilution 1: 400, visualizes vascular smooth

muscle cells at all stages of maturation) as primary antibodies. For detection of immunoreactivity

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62 | Chapter 4

we applied a streptavidin biotin complex method with DAB as substrate. Fresh full thickness

arterial wall was used as positive control tissue.

Histopathologic evaluation of grafts

Endothelial cell lining, integrity of elastin meshwork of the media, presence of medial smooth

muscle cells and adventitial inflammation were assessed on a semi quantitative basis. Two

observers (PJF and BvW) independently scored all sections which were blinded for study group

and follow-up period.

Morphometry

For morphometric analyses we used Image Pro-4 software (Image Pro-plus 4.5) (20). The

specimens were evaluated with a 10 times magnification objective of the light microscope

(Olympus, BX60). Intima thickness (distance between inner surface and internal elastica lamina)

and media thickness (distance between inner and outer elastica lamina) were automatically

measured at 6 places in each specimen and mean values were calculated.

Collagen content of the media and adventitia were quantified planimetrically on Picosirius

red stains for which we used grey scale detection with fixed threshold.


All statistical analyses were performed with GraphPad Prism 4.00 programme (GraphPad

Software, San Diego, USA). For comparison of cumulative graft patency data the log rank test

was used. Differences between groups in the morphological and morphometric analyses were

tested with Student-t test for continuous data and with the Mann-Whitney U test for comparison

of the semi-quantitative scores. The Kruskal-Wallis test was used to compare more than two

unpaired groups. A p-value of less than 0.05 was considered statistical significant.

RESULTS

Animal survival and graft patency

All animals survived the postoperative period until scheduled sacrifice, except one in the glycerol

group which was sacrificed at day 6 postoperatively because of severe weight loss. Macroscopic

examination of the harvested aorta revealed occlusion of the allograft. Of all other animals,

4 grafts occluded, one in the control group and three in the glycerol group. All occlusions

occurred within the first 3 postoperative days as was determined by duplex scanning. Cumulative

graft patency at 3 months follow-up for the autografts and the glycerol preserved allografts was

93% and 78% respectively (p = 0.14).

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4

PNal PNau PAal PAau MGal MGau DAal DAau DNal DNau0

25

50

75

100

125

150

175

localization of measurement in native vessel and graft

mea

n PS

V a

t da

y 7(

cm/s

ec)

Figure 1. Mean peak systolic velocities (cm/sec) of glycerol allografts and autografts after 7 days follow-up. Error bars depict SEM. PN = proximal native vessel, PA = proximal anastomosis, MG = midgraft, DA = distal anastomosis, DN = distal native vessel, al = glycerol allograft, au = autograft. (MGal and MGau; p< 0.01).

= autograft = glycerol allograft

PNal PNau PAal PAau MGal MGau DAal DAau DNal DNau0

102030405060708090

100110120130

localization of measurement in native vessel and graft

mea

n PS

V a

t da

y 9

0 (c

m/s

ec)

Figure 2. Mean peak systolic velocities (cm/sec) of glycerol allografts and autografts after 90 days follow-up. Error bars depict SEM. PN = proximal native vessel, PA = proximal anastomosis, MG = midgraft, DA = distal anastomosis, DN = distal native vessel, al = glycerol allograft, au = autograft. (PNal and PNau; p< 0.01).


Fahner.indd 63 5-5-2014 14:35:11

64 | Chapter 4

Duplex sonography and angiographic parameters

The mean PSV at the different sites of the graft in both groups are represented in figure 1 and

2. Two autografts and one allograft showed a significant stenosis. In the midgraft part of the

transplanted allografts the mean PSV after 7 days follow-up was significantly lower compared

to the autografts (p< 0.01). No significant difference was detected after one months and after

3 months follow-up the mean PSV was consistently lower in the allografts at all locations although

only significantly different in the proximal native vessel in the transplanted allografts (p< 0.01).

No aneurysm formation or graft-disintegration was found during follow-up. After 7 and

90 days follow-up no significant difference in graft diameter was measured between both

graft types. After one month the mean diameter of the allografts was significantly higher at

the proximal anastomosis (p< 0.01) and midgraft (p= 0.02) as shown in figure 3. Percentage

graft stenosis on angiograms was not encountered in the midgraft segments, neither in the

allografts or autografts. In 4 autografts a stenosis was measured at the proximal anastomosis

and in 6 autografts at the distal anastomosis. Three glycerol allografts developed a stenosis at

the proximal anastomosis and one at the distal anastomosis. No significant difference existed

between autografts and allografts (p= 0.17).

Morphological and morphometric results

Intima

Endothelial cell (EC) coverage was assessed with anti-Vwf immunostaining. When pooling

the scores of the endothelial cell coverage at all time points for both graft types, intraluminal

endothelial cell coverage was more abundant in the proximal and distal segments compared to

the midgraft segments (p= 0.26). Endothelial cell coverage is presented in figure 4 in relation

with implantation time. There are no consistent differences in endothelial cell coverage between

autografts and glycerol grafts during the implantation period.

Intimal thickness was evaluated in the same segments using H&E stained sections. Increase in

intimal thickness, interpreted as intimal hyperplasia, was first observed in the glycerol allografts

at day 3 and in the autografts at day 14. When pooling the neo-intimal thickness of all graft

segments, intimal thickness was significantly greater in the autografts compared to the glycerol

preserved allografts after 1 and 3 months follow- up (figure 5, p< 0.01).

Media

The integrity of the elastic meshwork of the media, evaluated in Elastica van Gieson stained

sections, did not differ significantly between both types of grafts (p = 0.64). The length of

implantation period did not affect elastin network integrity which was comparable in all graft

segments.

Fahner.indd 64 5-5-2014 14:35:11


4PAal PAau MGal MGau DAal DAau

0

1

2

3

localization in graft

graf

t d

iam

eter

at

day

30

(mm

)

Figure 3. Mean graft diameter measurements (mm) of allografts and autografts after 30 days follow-up. Error bars depict SEM. PA = proximal anastomosis, MG = midgraft, DA = distal anastomosis, al = glycerol allograft, au = autograft. (PAal and PAau; p> 0.01, MGal and MGau; p= 0.02).


gl1 a1 gl3 a3 gl7 a7 gl14 a14 gl30 a30 gl90 a90

0

1

2

3

4

follow up (days)

mea

n to

tal s

core

Figure 4. Mean score of intraluminal endothelial cells (Factor VIII staining) after pooling of results for proximal, midgraft and distal segments. Error bars depict SEM. Score 0 = complete coverage, 1 = some endothelial lining, 2 = no endothelial cells. gl = glycerol allograft, a = autograft.


Fahner.indd 65 5-5-2014 14:35:11

66 | Chapter 4

0

10

20

30

40

50

60

follow up (days)

intim

al t

hick

ness

(μm

)

1 3 7 14 30 90

Figure 5. Pooled results of proximal, midgraft and distal mean intimal thickness of allografts and autografts. Error bars depicts SEM.


Medial width in proximal, mid and distal graft segments was significantly less in the glycerol

allografts compared to the autografts (p<0.01). Figure 6a shows a mid segment of a glycerol

allograft and figure 6b shows a mid segment of an autograft. Differences were most prominent

after day-1 and day-3 postoperatively and diminished during further follow-up. The pooled mean

thickness (SD) was 52.2 (12.9) μm for glycerol allografts and 71.1 (27.3) μm for autografts.

In all segments, apart from scant mononuclear infiltration around the sutures, no inflammatory

reaction was observed in both graft types.

The pooled mean collagen content of the media was lower in autografts compared to

allografts at all time points. In the autografts a decrease of the mean collagen content was

measured in the first three days postoperatively which however recovered, and after 14 days

exceeded the amount at day-1. For the glycerol allografts collagen content remained almost

similar at all time points (figure 7).

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4

Figure 6A. Haematoxylin-eosin staining of a glycerol allograft midgraft segment (10x).

Figure 6B. Haematoxylin-eosin staining of an autograft midgraft segment (10x). Arrows point to the media in both fi gures.

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

follow-up (days)

perc

enta

ge c

olla

gen

in m

edia

(%)

1 3 7 14 30 90

Figure 7. Results of pooled data of percentage collagen in media of glycerol allografts and autografts, referring to proximal, midgraft and distal graft segments.


Fahner.indd 67 5-5-2014 14:35:12

68 | Chapter 4

gl1 a1 gl3 a3 gl7 a7 gl14 a14 gl30 a30 gl90 a900

1

2

3

4

5

6

7

*

follow up (days)

mea

n to

tal s

core

Figure 8. Mean score of α-smooth muscle cell (smc) staining of medial cells after pooling of results for proximal, midgraft and distal segments. Scores of all three segments were added and the mean calculated (score for single segment; 0 = > 50% smc ingrowth, 1 = < 50% smc ingrowth, 2 = some smc, n=3 for each follow-up). Mean maximal score is 3x2, mean minimal score is 3x0. * p= 0.01. Error bars depict SEM.


Presence of smooth muscle cells in the extra cellular tissue matrix was evaluated with anti

α- smooth muscle actin immunostaining. Both graft types showed slightly lower numbers of

smooth muscle cells in the distal segments compared to the proximal and medial segments. As

expected, the highest amount of positively stained cells was found in the autograft segments

at day-1 postoperatively. In the glycerol allografts this amount diminished in the first week

postoperatively and recovered to almost the level of day three after three months follow-up

(figure 8). No consistent differences in smooth muscle cell repopulation between autografts and

glycerol allografts exist during follow-up.

Adventitial parameters

Quantification of collagen in the adventitia of both graft types and in the different graft segments

showed equal results.

Both graft types showed a higher score of inflammatory reaction at the sites of anastomosis.

The pooled score at the proximal and distal segments was lower for the glycerol allografts

Fahner.indd 68 5-5-2014 14:35:12


40 10 20 30 40 50 60 70 80 90 100

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

follow up (days)

tota

l sco

re f

or in

flam

mat

ion

Figure 9. Results of adventitial inflammation scoring in haematoxylin-eosin stained sections. Data shown are the result of pooling of the proximal, medial and distal segments ( = allografts, ----- = autografts). Score 0 = some lymphocytes, 1 = clusters of lymphocytes, 2 = diffuse infiltration.

compared to the autografts. This indicates a less extensive inflammatory reaction in the

glycerol allografts after implantation. The pooled score of the midgraft segments was slightly

lower in the allografts. The calculated total inflammatory score for proximal, medial and distal

segments reached its maximum earlier in glycerol grafts compared to autografts (day 14 and day

30 respectively). During follow-up a decrease in inflammation was measured in both groups,

inflammation being less in the allografts (figure 9).

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70 | Chapter 4

DISCUSSION

The infrarenal rat aortic replacement model is well established in the investigation of alternatives

for the venous or arterial autograft (21-24). It is clear that for the reconstruction of large arteries

synthetic vascular grafts (e.g. ePTFE or Dacron) are the conduits of choice. However, for small

diameter reconstructions such prosthetic grafts have low cumulative patencies, ranging from

24% to 58% after three years (25;26). These results have stimulated research in alternatives to

prosthetic grafts such as preserved vascular allografts and xenografts, and the use of biomaterials

to serve as a vascular tissue matrix (27;28). Extra cellular matrix scaffolds can serve as templates

for cellular attachment and ingrowth of recipient cells (29;30). Main efforts in this field have been

directed to minimizing immunogenicity and inflammation of the graft, while preserving extra

cellular matrix integrity and mechanical properties (31).

More details of the glycerol preservation process in skin allografts were clarified by Huang

et al and Ross et al. In the permeation of glycerol in skin a process of diffusion and binding is

involved. Both histological and ultra structural analysis showed that the integrity of skin structure

was maintained and degradation of the skin avoided due to effective sequestration of water

(32;33). The preservation of connective tissue after glycerolization was confirmed by glycerol

preservation of ovine cardiac valve allografts and mechanical properties maintained for 6 months

after implantation in the ovine descending aorta (34;35).

In the present study, glycerol preserved aortic allografts were examined as an alternative

biomaterial for arterial grafting. Patency rate after 3 months was 78% for glycerol preserved

allografts. These results are in accordance with the high patency rates after 100 days reported

by Wolff et al who transplanted rat aortic allografts and femoral veins preserved in 98% glycerol

(36). In the study of Wolff, however, no angiography or sonography was performed rendering

reliability of patency detection uncertain. Also possible aneurysm formation could have easily

been missed in this report. In our study aneurysm formation could be ruled out and owing to

the histological examinations new insight was provided into the performance of the glycerol

preserved allografts after implantation. The results of the present study and that of the study of

Wolff are different from the total graft occlusions after two weeks reported by Bishop et al (37).

In this study glycerol preserved DA strain rat femoral vein was transplanted into the common iliac

artery of Lewis rats. In the same study hind leg and foreleg veins were preserved in glycerol 98%

and transplanted to the common carotid artery. After six month of follow up a patency rate of

64% was reached. They reported a very strong allograft reaction as a result of the two rat strains

which obviously influenced patency rates.

Two pathways are involved in the immune response against allografts in transplant

vasculopathy. At first, donor endothelial cells and antigen presenting cells (APC) of the allograft

induces proliferation of recipient T- cells. Donor MHC I molecules on the surface of transplanted

Fahner.indd 70 5-5-2014 14:35:12


4

cells induces T- cell activation (38). Secondly, recipient APC’s present donor MHC and minor

antigens from the allograft to recipient T- cells. This requires recognition of the allogens as

peptides bound to recipient MHC II molecules (39;40).

Direct sensitization of the recipient by Langerhans cells, which have a strong expression

of MHC II molecules and are the APC’s of the skin, will be blocked in glycerol preserved skin

allografts since active migration of Langerhans cells from the allograft is no longer possible due

to cell death after glycerolization.

In the current experiments we used outbred Wistar rats. It could be debated if intimal

hyperplasia would have been more distinct when immunologically more different animal strains

had been used for our aortic transplantation experiments. Osako et al (41) demonstrated more

intimal hyperplasia in fresh and cryopreserved allografts compared to fresh and cryopreserved

isografts in their Lewis rat aortic transplantation model. However, Takeishi et al (42), who

performed cryopreserved femoral artery transplantation in Lewis and Brown Norway rats, found

quite similar patency rates for isografts (100% and 87%) and allografts (100% and 78%) after 1

and 3 months follow up.

Although it is difficult to assess the effect of genetically incompatibility between different

rat strains in relation to patency rates, one should be aware of the genetically relation when

comparing results of vascular implantation studies in which different rat strains were used. The

difference in above mentioned patencies is in agreement with the lower genetically uniformity

between DA and Lewis rats compared to BN and Lewis rat strains. This is confirmed by the higher

amount of cellular damage in DA donor livers compared to BN donor livers in an orthotopic rat

liver transplantation model (43).

The role of intraluminal endothelial cells is crucial in vascular graft patency. Several studies

have been performed to examine preservation methods for maintaining endothelial cell viability.

Preservation of luminal endothelial cell lining in University of Wisconsin (UW) solution appears

more successful compared to phosphate buffered saline (PBS) for up to seven days of cold storage

of rat carotid arteries (44). Longer periods of cold storage however led to endothelial cell death

and incomplete luminal coverage, which resulted in intimal hyperplasia and graft occlusion in

transplanted rabbit femoral arteries (45). Although on the one hand, the best thromboresistant

graft surface is luminal lining with host endothelial cells, transplanted donor endothelial cells on

the other hand, evoke an immunogenic response that will induce pathologic changes in graft

wall architecture (46;47). Furthermore, initial absence of allograft endothelium did not seem to

influence graft patency in the experiments of Komorowska et al (48) in which cryopreserved

femoral arteries were transplanted in a rat model. The absence of endothelium is probably

an advantage of glycerol preservation since no viable endothelial cells are transplanted while

the extracellular tissue matrix is used as template for recipient cell attachment and ingrowth

(49;50). Our study reveals endothelial cells in glycerol preserved allografts after transplantation

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72 | Chapter 4

which obviously originate from repopulating cells of the recipient. Theoretically this has the

advantage of the development of an antithrombotic luminal surface without the disadvantage

of immunologic reactions after transplantation of viable donor cells (51). Another important

finding is the lesser intimal hyperplasia as measured after one and three months and the lower

inflammatory response after one month follow-up in glycerol preserved allografts. Since intimal

hyperplasia is the common healing response to arterial wall injury and occurs subsequent to

immunoinflammatory endothelial injury, glycerol preservation has potential advantages (52).

The absence of living endothelial cells in the glycerol allografts unable those to induce the

cascade of cellular damage after transplantation which results in proliferation of smooth muscle

cells and fibroblasts involved in intimal hyperplasia. This will be valuable in the application of

glycerol allografts in humans since endothelialization is prolonged in humans (53;54). Although

the endothelialization of prosthetic grafts is at least 7.5 times more pronounced in any animal

model compared to human (including rat, dog and baboon models), interpretation of graft

endothelialization in rat models to humans is complicated by graft dimension difference (55).

In conclusion, in our aortic transplantation model in the rat, glycerol preserved allografts

were successfully grafted and had acceptable graft patencies if compared with autografts in

conjunction with diminished intimal hyperplasia and in the absence of aneurysmatic matrix

degeneration. These results warrant further investigation of this preservation method in a

clinically relevant, large animal model.

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4

Reference List

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(2) Dardik H, Wengerter K, Qin F, Pangilinan A, Silvestri F, Wolodiger F, et al. Comparative decades of experience with glutaraldehyde-tanned human umbilical cord vein graft for lower limb revascularization: an analysis of 1275 cases. J Vasc Surg 2002 Jan;35(1):64-71.

(3) van Reedt Dortland RW, van Leeuwen MS, Steijling JJ, Theodorides T, van Vroonhoven TJ. Long-term results with vein homograft in femoro-distal arterial reconstructions. Eur J Vasc Surg 1991 Oct;5(5):557-64.

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(8) Brown JB, Fryer MP, Randall P, Lu M. Postmortem homografts as biological dressings for extensive burns and denuded areas; immediate and preserved homografts as life-saving procedures. Ann Surg 1953 Oct;138(4):618-30.

(9) Robson MC, Krizek TJ. Predicting skin graft survival. J Trauma 1973 Mar;13(3):213-7.

(10) Schechter I, Belldegrin A, Ben Basat M, Kaplan I. Prolonged retention of glutaraldehyde-treated skin homografts in humans. Br J Plast Surg 1975 Jul;28(3):198-202.


(12) Basil A. A comparative study of glycerinized and lyophilized porcine skin in dressings for third-degree burns. Plast Reconstr Surg 1982;69:969-74.

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(17) van Baare J, Cameron PU, Vardaxis N, Pagnon J, Reece J, Middelkoop E, et al. The 1998 Lindberg Award. Comparison of glycerol preservation with cryopreservation methods on HIV-1 inactivation. J Burn Care Rehabil 1998 Nov;19(6):494-500.

(18) Fahner PJ, Idu MM, Legemate DA, Vanbavel E, Borstlap J, Pfaffendorf M, et al. Morphological and functional alterations in glycerol preserved rat aortic allografts. Int J Artif Organs 2004 Nov;27(11):979-89.

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(19) Wessely R, Paschalidis M, Wagenpfeil S, Wegener F, Neumann FJ, Theiss W. A comprehensive approach to visual and functional assessment of experimental vascular lesions in vivo. Am J Physiol Heart Circ Physiol 2004 Jun;286(6):H2461-H2467.

(20) Blatt RJ, Clark AN, Courtney J, Tully C, Tucker AL. Automated quantitative analysis of angiogenesis in the rat aorta model using Image-Pro Plus 4.1. Comput Methods Programs Biomed 2004 Jul;75(1): 75-9.

(21) Qin F, Dardik H, Pangilinan A, Robinson J, Chuy J, Wengerter K. Remodeling and suppression of intimal hyperplasia of vascular grafts with a distal arteriovenous fistula in a rat model. J Vasc Surg 2001 Oct;34(4):701-6.

(22) Ribbe EB, Holmin T, Lowenhielm PC. Microvascular polytetrafluoroethylene (Gore-Tex) grafts in the infrarenal rat aorta. Microsurgery 1987;8(2):48-53.

(23) Tizian C, Glass KD, Demuth R. Using expanded polytetrafluoroethylene for bridging arterial microvascular defects: an experimental study with evaluation in the scanning electron microscopy. Thorac Cardiovasc Surg 1980 Aug;28(4):273-6.

(24) Zelder O, Werner HH, Jerusalem CR. The use of microvascular graft as an arterial substitute in the abdominal aorta of the rat. Microsurgery 1983;4(3):157-63.

(25) Eickhoff JH, Broome A, Ericsson BF, Buchardt Hansen HJ, Kordt KF, Mouritzen C, et al. Four years’ results of a prospective, randomized clinical trial comparing polytetrafluoroethylene and modified human umbilical vein for below-knee femoropopliteal bypass. J Vasc Surg 1987 Nov;6(5):506-11.

(26) Johnson WC, Lee KK. A comparative evaluation of polytetrafluoroethylene, umbilical vein, and saphenous vein bypass grafts for femoral-popliteal above-knee revascularization: a prospective randomized Department of Veterans Affairs cooperative study. J Vasc Surg 2000 Aug;32(2):268-77.

(27) Hilbert SL, Ferrans VJ, Jones M. Tissue-derived biomaterials and their use in cardiovascular prosthetic devices. Med Prog Technol 1988;14(3-4):115-63.

(28) Zdrahala RJ. Small caliber vascular grafts. Part I: state of the art. J Biomater Appl 1996 Apr;10(4):309-29.

(29) Stansby G, Berwanger C, Shukla N, Hamilton G. Endothelial cell seeding of vascular grafts: status and prospects. Cardiovasc Surg 1994 Oct;2(5):543-8.

(30) Williams SK, Schneider T, Kapelan B, Jarrell BE. Formation of a functional endothelium on vascular grafts. J Electron Microsc Tech 1991 Dec;19(4):439-51.

(31) Schmidt CE, Baier JM. Acellular vascular tissues: natural biomaterials for tissue repair and tissue engineering. Biomaterials 2000 Nov;21(22):2215-31.

(32) Huang Q, Pegg DE, Kearney JN. Banking of non-viable skin allografts using high concentrations of glycerol or propylene glycol. Cell Tissue Bank 2004;5(1):3-21.

(33) Ross A, Kearney JN. The measurement of water activity in allogeneic skin grafts preserved using high concentration glycerol or propylene glycol. Cell Tissue Bank 2004;5(1):37-44.

(34) Aidulis D, Pegg DE, Hunt CJ, Goffin YA, Vanderkelen A, Van HB, et al. Processing of ovine cardiac valve allografts. Effects of preservation method on structure and mechanical properties. Cell Tissue Bank 2002;3(2):79-89.

(35) Neves J, Abecassis M, Santiago T, Ramos T, Melo J, Gruys E, et al. Processing of ovine cardiac valve allografts. Implantation following antimicrobial treatment and preservation. Cell Tissue Bank 2002;3(2):105-19.

(36) Wolff KD, Dienemann D. Vessel preservation with glycerol: an experimental study in rats. J Oral Maxillofac Surg 1990 Sep;48(9):914-8.

(37) Bishop AJ, Glasby MA, Houlton JE. A morphological assessment of vein allografts preserved in glycerol and used for arterial replacement. J Cardiovasc Surg (Torino) 1987 Sep;28(5):491-7.

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(38) Denton MD, Davis SF, Baum MA, Melter M, Reinders ME, Exeni A, et al. The role of the graft endothelium in transplant rejection: evidence that endothelial activation may serve as a clinical marker for the development of chronic rejection. Pediatr Transplant 2000 Nov;4(4):252-60.

(39) Watschinger B, Gallon L, Carpenter CB, Sayegh MH. Mechanisms of allo-recognition. Recognition by in vivo-primed T cells of specific major histocompatibility complex polymorphisms presented as peptides by responder antigen-presenting cells. Transplantation 1994 Feb 27;57(4):572-6.

(40) Zdoroveac A, Doebis C, Laube H, Brosel S, Schmitt-Knosalla I, Volk HD, et al. Modulation of Graft Arteriosclerosis in a Rat Carotid Transplantation Model. J Surg Res 2007 Sep 29.

(41) Osako M, Otani H, Yamamura T, Nakao Y, Hattori R, Omiya H, et al. Alloimmune response may be involved in neointimal hyperplasia in cryopreserved aortic allografts. Transplant Proc 2001 Jun;33(4):2566-70.

(42) Takeishi M, Hirase Y, Kojima T. Experimental study of cryopreserved allogeneic transfer of vessel: preliminary report 4. Microsurgery 1994;15(1):55-62.

(43) Mollevi DG, Ginesta MM, Domenech P, Serrano T, Figueras J, Jaurrieta E. Hemostatic status in long-term surviving xenografts. Surgery 2006 Jun;139(6):775-81.

(44) Vischjager M, van Gulik TM, Kromhout JG, van MJ, Pfaffendorf M, Klopper PJ, et al. Morphology and function of preserved microvascular arterial grafts: an experimental study in rats. Ann Vasc Surg 1997 May;11(3):284-91.

(45) Crowe DM, Hurley JV, Mitchell GM, Niazi Z, Morrison WA. Long-term studies of cold-stored rabbit femoral artery and vein autografts. Br J Plast Surg 1998 Jun;51(4):291-9.

(46) Marois Y, Wagner E, Paris E, Roy R, Douville Y, Guidoin R. Comparison of healing in fresh and preserved arterial allografts in the dog. Ann Vasc Surg 1999 Mar;13(2):130-40.

(47) Zhao XM, Green M, Frazer IH, Hogan P, O’Brien MF. Donor-specific immune response after aortic valve allografting in the rat. Ann Thorac Surg 1994 May;57(5):1158-63.

(48) Komorowska-Timek E, Zhang F, Shi DY, Lineaweaver WC, Buncke HJ. Effect of cryopreservation on patency and histological changes of arterial isogeneic and allogeneic grafts in the rat model. Ann Plast Surg 2002 Oct;49(4):404-9.



(51) Borschel GH, Huang YC, Calve S, Arruda EM, Lynch JB, Dow DE, et al. Tissue engineering of recellularized small-diameter vascular grafts. Tissue Eng 2005 May;11(5-6):778-86.

(52) Gomes D, Louedec L, Plissonnier D, Dauge MC, Henin D, Osborne-Pellegrin M, et al. Endoluminal smooth muscle cell seeding limits intimal hyperplasia. J Vasc Surg 2001 Oct;34(4):707-15.

(53) Dixit P, Hern-Anderson D, Ranieri J, Schmidt CE. Vascular graft endothelialization: comparative analysis of canine and human endothelial cell migration on natural biomaterials. J Biomed Mater Res 2001 Sep 15;56(4):545-55.

(54) Rezvan A, Allen FD, Lelkes PI. Steady unidirectional laminar flow inhibits monolayer formation by human and rat microvascular endothelial cells. Endothelium 2004 Jan;11(1):11-6.

(55) Davids L, Dower T, Zilla P. The lack of healing in conventional vascular grafts. In: Zilla PP, Greisler HP, editors. Tissue engineering of vascular prosthetic grafts. Austin, Texas: R.G. Landes; 1999, p. 3-44.

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5Comparison of preserved vascular allografts using

glycerol and University of Wisconsin solution in

a goat carotid artery transplantation model

P.J. Fahner1, D.A. Legemate1, A.C. van der Wal2, J. van Marle3, S.L.M. Peters4, C.F. van Eck1, T.M. van Gulik1, M.M. Idu1

1Department of Surgery and 2Department of PathologyAcademic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

2 Department of Pathology, AMC3 Department of Microscopic Research, AMC

4 Department of Pharmacology & Pharmacotherapy, AMC

Eur Surg Res 2012;48:64-72

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78 | Chapter 5

Abstract

Background. Prosthetic grafts have poor patency rates in peripheral arterial reconstructions.

Glycerol (GL)-preserved grafts are an alternative. The aim of this study was to examine patency,

graft morphology and function of GL-preserved allografts in a goat carotid artery animal

model.

Methods. The first group (n=7) underwent bilateral replacement of the carotid artery by a carotid

allograft that was preserved in GL for 1 week. In the second group (n=5), a carotid artery allograft

that was preserved in University of Wisconsin solution (UW) for 48 h was used. In the third group

(n=5), the jugular vein (autologous vein, AU) was used. The follow-up was 3 months.

Results. One UW graft and 1 GL graft occluded in the first 24 h postoperatively. Three-month

primary patency rates for GL, UW and AU grafts were 93%, 100% and 80% respectively (p =

0.39). Graft diameter was increased in UW allografts (p < 0.005), whereas GL allografts remained

unchanged. After explantation, GL allografts demonstrated contraction and relaxation capacity

and lower intimal thickness (p < 0.001).

Conclusion. GL preservation has proven to be a feasible method for arterial allograft transplantation

in a large animal model with decreased intimal hyperplasia and renewed functional capacity.

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solution in a goat carotid artery transplantation model | 79

5

Comparison of preserved vascular allografts using glycerol and University of Wisconsin

Introduction

Prosthetic vascular grafts have proven less successful when used as small caliber grafts (< 6mm)

in peripheral arterial reconstructions compared to great diameters in aortic repair. Therefore

research in small-diameter, biological or tissue-engineered vascular constructs has continued.

To minimize the immunological response to allogeneic vascular grafts, current research has

focused on the use of decellularized tissue matrices which are potentially repopulated by host

derived endothelial and smooth muscle cells after implantation (1-4). Biological tissues provide an

extracellular matrix scaffold for migrating host cells using host specific adhesion receptors. This

results in diminished or absent immunological response to the grafts and enhanced ingrowth of

host endothelial and smooth muscle cells (5).

The use of biological vascular grafts requires appropriate techniques for graft preservation

and storage. Various preservation methods for vascular allografts have been examined in the

past two decades, of which cryopreservation, glutaraldehyde tanning and cold storage have

found clinical application. Early thrombosis, intimal hyperplasia and aneurysmal degeneration,

however, have hampered optimal performance. Glycerol (GL) preservation of skin allografts is

successfully in the management of burn injuries (6-9). Glycerol, a non-toxic intermediate of the

fatty acids metabolism, leaves the structural integrity of the skin unaffected and after application

on wounds, generates an unspecific host inflammatory reaction rather than an immune response

leading to rejection (10;11). An additional advantage of the use of glycerol is its potency to

eliminate micro-organisms (12). In vitro experiments using infected cadaveric split skin grafts

showed inactivation of herpes simplex virus, polio virus, HIV-1 and elimination of bacterial

growth after prolonged storage (13-15).

The efficacy of glycerol for preservation of skin allografts has encouraged examination of

glycerol for preservation of vascular allografts. Previous in vitro experiments in our laboratory

on glycerol preservation of rat aortic allografts confirmed maintenance of mechanical integrity

and extracellular matrix characteristics after three months preservation (16). In an in vivo rat

aortic transplantation model, glycerol preserved allografts showed a three months patency

rate comparable to autografts, with decreased intima hyperplasia and adventitial inflammatory

reaction (17).

The primary aim of the current study was to assess the use of glycerol preserved arterial

allografts in a preclinical, large animal model, with special emphasis on patency rate, vessel

wall function and graft morphology after implantation. Our secondary aim was to compare

glycerolization of vascular allografts with cold storage preservation using the University of

Wisconsin (UW), as widely used in most types of solid organ transplantation (18).

Because UW preservation is applicable for relatively short preservation times in contrast to

glycerol preservation we hypothozised that glycerol preservation was a feasible preservation

Fahner.indd 79 5-5-2014 14:35:13

80 | Chapter 5

method for vascular allografts with patency rates comparable to UW preserved allografts and

venous autografts.


Animals and experimental groups

Dutch female milk goats (65kg - 104kg) were used, obtained from an official breeder. The

animals were housed five days prior to the experiments and maintained on a 12-hour light-dark

cycle. The animals had free access to water, bix and hay. The experimental protocol was approved

by the Animal Ethics Committee of the Academic Medical Center, University of Amsterdam,

The Netherlands. This protocol was in accordance with EU regulation on the care and use of

laboratory animals. In addition, the investigation conforms with the Guide for the Care and Use

of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 84-

23, revised 1996).

The study consisted of three experimental groups. One group (n = 7) underwent bilateral

replacement of the common carotid artery by a glycerol preserved carotid allograft (glycerol

group, GL). In the second group (n = 5) carotid allografts cold preserved in UW solution, were

used for bilateral replacement of the common carotid artery (University of Wisconsin group,

UW). In animals of the third group (n = 5) unilateral autotransplantation of the jugular vein to the

carotid artery was performed (autologous vein group, AU). This group served as golden standard

since autologous venous allografts reached highest patency rates in peripheral arterial bypass

surgery. At the beginning of the study one animal was sacrificed to serve as allograft donor.

In subsequent experiments, the segments of carotid artery which were excised in order to be

replaced by preserved allografts, were preserved and stored until implantation in the following

animal. All grafts were harvested after 3 months follow-up.

Surgical protocol and graft preservation

The animals were premedicated with 20 mg/kg Ketamine and 0.5 mg/kg Midazolam i.m. and

were ventilated using a mixture of O2 / N 2O, 1:1 L and Isoflurane 1-1.5% after endotracheal

intubation and placement of a nasogastric tube. Postoperatively, buprenorfine 5.0 µg/ kg and

finadyne 1.0 mg/kg was administered i.m. for control of pain. Finadyne 1.0 mg/kg injection was

repeated on a weekly basis. Ampicilline (1 g) was given 24 hours preoperatively and on day 1

and 3 postoperatively. All operations were performed under sterile conditions. Using a midline

neck incision, the carotid artery and jugular vein were identified and dissected over a length of

10 cm. Three minutes before clamping of the carotid artery, heparin (100 IU/ kg) was infused

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5


intravenously. A vascular allograft or autograft of 6.0 cm length was implanted as interposition

graft with end-to-end anastomosis using a 6.0 Prolene running suture.

The preservation protocol for the glycerol allografts was in accordance with the protocol

routinely used at the Euro Skin Bank (Beverwijk, The Netherlands) and was optimized for

preservation of arterial allografts (16). Briefly, the grafts were incubated in glycerol solution

(Glycerolum, Genfarma BV, Maarssen, The Netherlands) of increasing concentration as follows:

1. Incubation in a glycerol 50% solution for 4 hr at room temperature

2. Incubation in a glycerol 70% solution for 3 hr at 330C

3. Incubation in a glycerol 85% solution for 3 hr at 330C

After completion of the protocol, the grafts were stored in glycerol 85% for one week at 40C.

Cold storage was performed using University of Wisconsin preservation solution (ViaSpanR,

Barr Laboratories, Inc., NY) in which the grafts were immersed for 48 hr at 40C. Before

implantation all grafts were rinsed in saline for 20 min.

Graft surveillance

Routine color duplex scanning (Hewlett Packerd Sonos 5000) of the grafts was performed at

post-operative day 1, 3, 7 and week 2, 3, 4, 8 and 12. Peak systolic velocity (PSV), end diastolic

velocity (EDV) and vessel diameter were measured. Significant stenosis was arbitrarily defined as a

PSVmax which was 2.5 times increased. Just before explantation, a digital subtraction angiography

(DSA) was performed by puncture of the femoral artery and placement of a 5 Fr angiographic

catheter (Imager IITM, Boston Scientific Corporation, Watertown) in the aortic arch. A PTFE coated

steerable guide wire (2.7 Fr, Back-up Meier, Boston Scientific Corparation, Natick, USA) was

manipulated into the proximal carotid artery. Five ml of radio contrast solution (VisipaqueTM

320mg I/ml, Amersham Cygne BV, Eindhoven, The Netherlands) was manually injected.

Contraction and relaxation experiments

Segments of glycerol allografts (n=10) were tested for functional responses immediately following

explantation. In these experiments, the nonpreserved carotid artery proximal and distal to the

graft (n=12) served as fresh controls. After explantation, contraction and relaxation responses

were studied in 4 mm segments in an organ bath set-up as described previously (16). The initial

resting tension was set at 20 mN and was adjusted throughout the experiment. After one hour

of equilibration, the segments were exposed three times to a 50 mM depolarizing potassium

chloride solution for 5- 10 min intervals. After a rinsing procedure a concentration-response

curve was constructed for L-phenylephrine (1.10-8 – 1.10-4 M) to study receptor-dependent

contractile responses.

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82 | Chapter 5

To examine endothelium-dependent relaxation of the smooth muscle cells, a concentration-

response curve was constructed for methacholine (1.10-7 – 1.10-4 M) after rinsing with Tyrode’s

solution and precontraction with L-phenylephrine (1.10-5 M). Subsequently, the segments were

exposed to the NO-donor sodium nitroprusside (1.10-5 – 1.10-4 M) to determine endothelium-

independent relaxation.

Histological and electronmicroscopical examination

Histology

Five-mm ring samples were used for histological examination. Of each graft two samples

were examined in the proximal, mid and distal part of the graft, respectively. Sections of the

native vessel proximal to the implanted graft served as controls. Immediately after harvesting,

the segments were rinsed in saline, fixed in 10% formaldehyde solution (Mallinckrodt Baker,

Deventer, The Netherlands), dehydrated and embedded in paraffin. Transverse sections (5µm)

were stained with hematoxylin-eosin (HE) and Elastin van Gieson (EvG) and were mounted on

slides for microscopic evaluation. Of each slide three measurements of intima and media were

performed at 3, 6, 9 and 12 hour on a clockwise grid. A Leica LB 30 S light microscope (Leica,

Wetzlar, Germany) was used in combination with the Qwin Image pro analysis software (version

2.6, Leica Imaging Systems Ltd., Cambridge).


Luminal coverage of the graft with endothelial cells was examined using scanning electron

microscopy. Ring segments were rinsed in saline and fixed for 24 hours in McDowell fixative

(4% paraformaldehyde and 1% glutaraldehyde in 0.1M phosphate buffer). The samples were

dehydrated in graded ethanol’s, immersed in 1,1,1,3,3,3 hexamethyl disilazan (Merck- Schuchardt)

and dried at room temperature for 12 hours. Finally, the tissue segments were mounted on stubs

with conductive carbon cement, sputter-coated with 20 nm gold-palladium and scanned with an

electron microscope (Philips SEM 525 equipped with an Orion frame grabber) operated at 10 kV

and a spot size of 50 nm.

Angiographies, functional and morphological examinations were evaluated by blinded

investigators.

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5



One way analysis of variance analysis was performed when means of three or more groups were

compared. If only means of two groups were analyzed the t-test was used. To test if time of

follow-up and graft location had effects on PSVmax values, a two way ANOVA analysis was used.

The log-rank test was used to compare patency rates. All statistical analysis was performed using

the GraphPad Prism 4.0 program (GraphPad Software, San Diego).

RESULTS

Animal follow-up and graft patency

Thirteen out of seventeen animals underwent surgery without any complication. Two animals

developed a graft occlusion within the first 24 hours postoperatively. Both suffered from

postoperative complications. One in the autologous vein autograft group had a postoperative

hemorrhage and thrombosis at the anastomosis requiring thrombectomy and reanastomosis.

The other animal with occlusion of a glycerol allograft developed a postoperative cardiac arrest,

was resuscitated and survived. No graft occlusions were observed after postoperative day

one. Two animals of the UW allograft group were euthanized on postoperative day 14 and 16

respectively, due to severe respiratory distress. The grafts were harvested and autopsy revealed

bilateral pneumonia with abcess formation in both animals. The 90-days primary patency rate

for UW, glycerol and autologous vein grafts was 100%, 93% and 80% respectively (p = 0.39).

Peak systolic velocity and graft diameter

When all measurements at the different locations in the grafts were pooled, the maximal mean

peak systolic velocity (PSVmax) could be depicted for all time-points during follow-up (Fig 1). No

significant stenosis, was detected in any of the experimental groups. As expected, the mean

PSVmax pooled for all time-points at follow-up was lowest in the midgraft segments for all the

three groups (Fig 2). Both graft location and time of follow-up did affect PSV max (p = 0.03 and

p = < 0.001). Flow velocity was significantly lower in the midgraft segments of the autologous

vein grafts whereas flow velocity was higher at the proximal anastomosis of the venous allografts

(p < 0.001). No differences in flow velocity were found between the UW and glycerol allografts.

Results of pooled diameter measurements of each graft during follow-up are shown in

figure 3. The diameter of autologous vein grafts placed as interposition graft in the carotid artery

was increased compared to native, non-operated jugular vein. A significant increase in diameter

was shown during follow-up (p < 0.005) only for the UW preserved allografts. The diameter of

glycerol allografts remained unchanged during 3 months follow-up.

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84 | Chapter 5

1d 3d 1w 2w 3w 1m 2m 3m60

65

70

75

80

85

90

95

100UW

GL

AU

follow up

mea

n PS

V (c

m/s

ec)

Figure 1. Maximal mean peak systolic velocity (PSVmax) in cm/sec for carotid artery allografts preserved in University of Wisconsin solution (UW, n = 10), in glycerol (GL, n = 14) and jugular vein autografts (AU, n = 5). 1d = 1 day, 1w = 1 week, 1m = 1 month.

pv pa mg da dv pv pa mg da dv pv pa mg da dv0

10

20

30

40

50

60

70

80

90

100

110

120

AU vein UW GLYCgraft locations in research groups

mea

n PS

V (c

m/s

ec)

Figure 2. Mean peak systolic velocity (PSVmax) in cm/sec for jugular vein autografts (AU vein, n = 5), carotid artery allografts preserved in University of Wisconsin solution (UW, n = 10) and glycerol preserved allografts (Glyc, n = 14). Error bars depict SEM. pv = proximal native vessel, pa = proximal anastomosis, mg = mid graft, da = distal anastomosis, dv = distal native vessel.

Fahner.indd 84 5-5-2014 14:35:13


5


Angiography

Angiography performed after three months follow-up showed a midgraft stenosis of

approximately 50% in one glycerol allograft. In none of the other glycerol or UW allografts a

stenosis could be detected. The diameter of the venous allografts was 2.3 times the diameter

of the native carotid artery. No aneurysmatic dilatation could be demonstrated in any of the

preserved allografts.

1d 3d 7d 2w 3w 1m 2m 3m 1d 3d 7d 2w 3w 1m 2m 3m 1d 3d 7d 2w 3w 1m 2m 3m0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

AU vein UW GLYC

follow up for different research groups

mea

n di

amet

er (c

m)

Figure 3. Mean diameter (cm) of jugular vein autografts (AU vein, n = 5), carotid artery allografts preserved in University of Wisconsin solution (UW, n = 10) and glycerol preserved allografts (GLYC, n = 14) during follow-up. Error bars depict SEM. 1d = 1 day, 1w = 1 week, 1m = 1 month.

Contraction and relaxation response

All grafts showed a modest contractile response to KCL and after stimulation with KCL and

phenylephrine. A dilatory response was measured after administration of both Na+ - nitroprusside

and methacholine. The response to methacholine was limited in both carotid allografts and

control grafts probably explained by damage to the endothelial cells during explantation.

The contractile and dilatory responses were more substantial in the control carotid grafts

(Fig 4).

Morphological and morphometrical analysis

Mean intimal thickness was less in the glycerol allografts in both proximal and midgraft segments

compared to the UW preserved grafts albeit that this difference was statistically significant only

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86 | Chapter 5

-20

-10

0

10

20

30

carotid graft

carotid control

KCl

SNPMET

50

60

70

80

PHE

∆ c

ontr

actil

e fo

rce

(mN

)

Figure 4. Differences in isometric contractile and relaxation force responses (mN) assessed in ring segments of fresh carotid arteries (carotid control, n = 12) and glycerol preserved allografts (carotid graft, n = 10). Error bars depict SEM. KCl = after stimulation with 50mM potassiumchlorite, SNP = after addition of Na+ - nitroprusside (1.10-5 – 1.10-4M) to determine endothelium-independent relaxation, MET = after addition of methacholine (1.10-7 – 1.10-4M) to examine endothelium-dependent relaxation and PHE = after addition of the α1-adrenoceptor agonist L-phenylephrine (1.10-7.5 – 1.10-4 M)

in the midgraft position (p < 0.001). At the distal anastomosis, the intima was thicker in the

glycerol allografts (Fig 5, p = 0.02). As expected, the autologous vein grafts and control carotid

artery segments showed only thin intimal layers. The results of media thickness are presented in

figure 6. At all locations the glycerol allografts had the smallest thickness of media which was

significantly different compared to the UW allografts (Fig 6, p < 0.01). Pooling of the results at all

locations showed no significant difference in mean intimal thickness between glycerol allografts

and UW allografts (34µm (SD = 5) and 37µm (SD = 4)). For the pooled results of media thickness,

a significant difference (p < 0.001) was found between mean thickness of glycerol and UW

segments (124µm (SD = 56) vs 161µm (SD = 4)).


The scanning electron micrographs of the UW allografts showed a confluent layer of endothelial

cells somewhat higher compared to the control carotid artery endothelial cells (Fig 7A and B).

The endothelial cells were partly shriveled up and became less strongly attached to the sub-

endothelial medial layer (Fig 7B insert). Confluent intraluminal coverage was also determined

in the glycerol preserved allografts. A neo-intima was formed (Fig 7C) and the intraluminal

surface of the autologous vein grafts demonstrated a diminished profile compared to the arterial

allografts as seen in Fig 7D. Some damage to the confluent layer of endothelial cells was seen as

small cracks in the intima of the transplanted venous autografts (Fig 7D insert).

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5


gl uw au gl uw au gl uw au ca 0

50

100

150

200

250

prox distmid

med

ia t

hick

ness

(µm

)

Figure 6. Results of mean media thickness (µm) of glycerol preserved carotid allografts (gl, n = 14), carotid allografts preserved in University of Wisconsin solution (uw, n = 10), jugular venous autografts (au, n = 5) and fresh carotid arteries (ca, n = 5). Error bars depict SEM. prox = proximal part of graft, mid = middle part of graft and dist = distal part of graft.

gl uw au gl uw au gl uw au ca 0

10

20

30

40

50

60

prox distmid

intim

al t

hick

ness

(µm

)

Figure 5. Results of mean intimal thickness (µm) of glycerol preserved carotid allografts (gl, n = 14), carotid allografts preserved in University of Wisconsin solution (uw, n = 10), jugular venous autografts (au, n = 5) and fresh carotid arteries (ca, n = 5). Error bars depict SEM. prox = proximal part of graft, mid = middle part of graft and dist = distal part of graft.

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88 | Chapter 5

Figure 7. Scanning electon micrographs with high-power inserts of luminal coverage with endothelial cells in fresh carotid arteries (a), carotid allografts preserved in UW (b), GL-preserved carotid allografts (c) and AU autografts (d).

A B

C D

DISCUSSION

This study demonstrates the feasibility of glycerol preservation of vascular allografts with

diameters comparable to human peripheral arteries. No signifi cant stenosis was observed in the

glycerol allografts. When compared to UW preserved allografts, glycerol allografts demonstrate

less intimal hyperplasia in midgraft segments and repopulation of the glycerolized tissue matrix

by recipient cells resulting in renewed contraction and relaxation capability of the vessel wall

after transplantation.

The bilateral carotid artery transplantation model used in the current study, is successfully used

for the assessment of pseudo-intima proliferation, for examination of the effect of antiplatelet

drugs, and for assessment of arterial conduits and intravascular thrombectomy devices (19-22).

An important advantage of our model is the similarity of vessel diameter and hemodynamic

properties of the carotid artery in the goat when compared to human peripheral arteries (23).

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5


Limitations of the study include the time of follow up. A prolonged follow up period for up to

6 or 12 months would have facilitate the extrapolation to the clinical setting with patency rates

after 1 or 2 years follow up. Although this had lead to high costs.

Another limitation is that we did not transplantate the upper or lower limb arteries. These are

mainly involved in human peripheral arterial disease. Transplantation of leg arteries in an animal

model is not favourable due to higher risk of infection and traumatic events which hampers

wound healing.

During three months follow up, the mean diameter of the glycerol preserved allografts

stayed close to the initial value of almost 6.0 millimeter. A significant increase in arterial diameter

however, was shown in the UW preserved allografts which consistently increased after one

month of implantation. The increase in diameter potentially leads to aneurysm formation and

ultimately, vessel wall disintegration (24). In a review of explanted synthetic and biological

grafts in clinical peripheral arterial reconstructions, aneurysmal degeneration was the reason

for graft explantation in 72% of allografts used as a blood access for hemodialysis (25). In

general, aneurysmatic dilatation develops in the long term, therefore we are cautious with final

conclusions from our experiments because of the relatively short follow-up. Future research is

necessary to study the long-term risk of aneurysmal degeneration.

Endothelial dysfunction followed by endothelial cell-leucocyte interaction, endothelial

destruction and smooth muscle cell (SMC) loss are processes which play a central role in vascular

allograft rejection and graft dysfunction (26;27). To overcome this cascade, re-endothelialization

and neo-intimal formation, most likely the result of the proliferation of SMCs of recipient origin,

is generated in transplanted vascular allografts (28). Functional endothelial cells and SMCs are

necessary in maintaining a good vasomotor function as alterations in the nitric oxide-endothelin

homeostasis play an important role in the development of allograft transplant vasculopathy (29).

In a rat aorta interposition model transplanted cold stored allografts remain as nonfunctional

conduits after one and two months when they no longer respond to acetylcholine and

phenylephrine (30).

In a previous study glycerol preserved allografts were transplanted in a rat aorta transplantation

model. Anti-von Willebrand factor antibody immunostaining was used to assess endothelial graft

coverage and anti-α-actin antibody to visualize vascular SMC. No differences were found in

endothelial cell coverage and SMC repopulation between GL allografts and autografts after 3

months of follow-up. Given the available techniques in goats, it is not feasible to differentiate

between persisting cells of the donor in the vascular graft after transplantation or ingrowing

cells from the recipient. A mononuclear infiltration was observed only around the sutures of a

GL preservated vascular graft (17). Therefore, we assessed repoplulation of the non-viable tissue

matrix after GL preservation in our study, using the contraction and relaxation experiments.

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90 | Chapter 5

GL allografts did respond to vasodilating and vasconstricting agents demonstrating neo-

endothelial cell and SMC interactions resulting again in some of the original functionality. This

means ingrowth of recipient endothelial and SMCs in the donor extracellular tissue matrix. GL

preservation results in pyknotic nuclear cells and complete loss of contraction and relaxation

capacity (16).

Bishop et al (31) reported ten days glycerol 98% preservation of venous allografts used as

interposition graft in the common carotid artery in a goat model. Six months patency was 64%

compared to 88% for autografts. Handling and suturing qualities were as good as untreated

fresh vein and aneurysm formation in the vessel wall or at the anastomosis was not detected

suggesting adequate strength and durability of the allografts. Although scanning electron

microscopy and histology revealed endothelial disruption after preservation it apparently did not

result in a sufficiently thrombogenic graft surface to compromise long-term graft function (31).

Differences in patency rates between this experiment and the current study can, apart from the

longer observation time (i.e. 6 months), be explained by a better compliance match between

native artery and glycerol preserved artery compared to glycerol preserved vein.

Neo-intimal thickness was comparable at both proximal and distal anastomoses in both UW

and glycerol preservation groups and was very limited in autologous venous grafts.

In contrast, glycerol allografts showed a significantly thinner intima in the midgraft segments

compared to the UW allografts after three months follow-up. Although a short period of UW

preservation preserves vasomotor activity after cold storage which means that endothelial

cells and SMCs will survive the storage period to some extent, these cells will have sustained

damage due to the 48 hours of cold ischemia (26). These injured cells induce cellular proliferation

probably from surviving native graft SMCs, recipient endothelial cells from both anastomoses

and circulating endothelial progenitor cells (32;33). Extensive neo-intimal formation in UW grafts

can occur secondary to the immunological reaction to graft endothelial cells which results in

chronic intimal injury and enhanced neo-intimal proliferation (34). The diminished neo-intimal

formation in the glycerol preserved allografts in the current study is probably explained by the

fact that glycerol preservation results in a non-viable extracellular tissue from which all living

endothelial cells have been eliminated (16).

An intact intraluminal coverage by an endothelial monolayer in the transplanted vascular

grafts is important in prevention of intravascular coagulation (35). In the current study this

monolayer was present in the control carotid arteries and could also be demonstrated in the

autologous vein grafts. Flat endothelial cells with a smooth surface and without pores and

microvilli are criteria for cells in normal condition (36). Endothelial cells were flattened in the

glycerol allografts too, comparable with the endothelial cells in the autologous veins. However,

the endothelial cells preserved in UW seemed to have decreased in size and have lost cellular

attachment. Endothelial cell remnants seen on top of the neo-endothelial cells in the glycerol

Fahner.indd 90 5-5-2014 14:35:15


5


allografts suggest temporary luminal coverage by donor endothelial cells to enable recipient neo-

endothelial cells to proliferate and constitute a new bond with the graft internal elastic lamina.

GL-preserved arterial allografts can probably be used for small caliber vascular bypasses when

AU specimens are not available. UW preserved allografts could only be preserved for a short

period of time (1-2 weeks), whereas GL-preserved allografts could be stored for at least 3 months

with maintenance of structural properties. In view of these prolonged storage opportunities, iliac

and femoral arteries could be procured in multi-organ harvest procedures, preserved in GL and

stored in a vascular tissue bank. The absence of aneurysmal degeneration and the lesser degree

of intimal hyperplasia and inflammatory reaction in GL allografts, as compared to UW allografts,

render GL allografts a feasible alternative to cold-stored vascular grafts.

In conclusion, glycerol preservation of arterial allografts in this large animal model of carotid

artery transplantation, showed a promising patency rate after 3 months. Vessel wall diameter

of the preserved allografts was maintained as well as several functional characteristics owing to

repopulation of the vascular tissue matrix by host cells.

These results encourage us to devise a pilot study using glycerol preserved arterial allografts

in a clinical setting.

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92 | Chapter 5

Reference List

(1) Amiel GE, Komura M, Shapira O, Yoo JJ, Yazdani S, Berry J, et al. Engineering of blood vessels from acellular collagen matrices coated with human endothelial cells. Tissue Eng 2006 Aug;12(8):2355-65.

(2) Kaushal S, Amiel GE, Guleserian KJ, Shapira OM, Perry T, Sutherland FW, et al. Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo. Nat Med 2001 Sep;7(9):1035-40.


(4) Shum-Tim D, Stock U, Hrkach J, Shinoka T, Lien J, Moses MA, et al. Tissue engineering of autologous aorta using a new biodegradable polymer. Ann Thorac Surg 1999 Dec;68(6):2298-304.


(6) Mackie D. Postal survey on the use of glycerol-preserved allografts in clinical practice. Burns 2002 Oct;28 Suppl 1:S40-S44.


(8) Vloemans AF, Middelkoop E, Kreis RW. A historical appraisal of the use of cryopreserved and glycerol-preserved allograft skin in the treatment of partial thickness burns. Burns 2002 Oct;28 Suppl 1:S16-S20.

(9) Vloemans AF, Schreinemachers MC, Middelkoop E, Kreis RW. The use of glycerol-preserved allografts in the Beverwijk Burn Centre: a retrospective study. Burns 2002 Oct;28 Suppl 1:S2-S9.

(10) Richters CD, Hoekstra MJ, du Pont JS, Kreis RW, Kamperdijk EW. Immunology of skin transplantation. Clin Dermatol 2005 Jul;23(4):338-42.

(11) Richters CD, Hoekstra MJ, van BJ, du Pont JS, Kamperdijk EW. Immunogenicity of glycerol-preserved human cadaver skin in vitro. J Burn Care Rehabil 1997 May;18(3):228-33.

(12) Ben-Bassat H. Performance and safety of skin allografts. Clin Dermatol 2005 Jul;23(4):365-75.

(13) Cameron PU, Pagnon JC, van BJ, Reece JC, Vardaxis NJ, Crowe SM. Efficacy and kinetics of glycerol inactivation of HIV-1 in split skin grafts. J Med Virol 2000 Feb;60(2):182-8.

(14) van Baare J, Ligtvoet EE, Middelkoop E. Microbiological evaluation of glycerolized cadaveric donor skin. Transplantation 1998 Apr 15;65(7):966-70.

(15) van Baare J, Buitenwerf J, Hoekstra MJ, du Pont JS. Virucidal effect of glycerol as used in donor skin preservation. Burns 1994;20 Suppl 1:S77-S80.

(16) Fahner PJ, Idu MM, Legemate DA, Vanbavel E, Borstlap J, Pfaffendorf M, et al. Morphological and functional alterations in glycerol preserved rat aortic allografts. Int J Artif Organs 2004 Nov;27(11):979-89.

(17) Fahner PJ, Idu MM, van Gulik TM, van Wijk B, van der Wal AC, et al. Glycerol preserved arterial allografts evaluated in the infrarenal rat aorta. Eur Surg Res 2009 Feb;42(2):78-86.

(18) Corner JA, Berwanger CS, Stansby G. Preservation of vascular tissue under hypothermic conditions. J Surg Res 2003 Jul;113(1):21-5.

(19) Hilbert SL, Boerboom LE, Livesey SA, Ferrans VJ. Explant pathology study of decellularized carotid artery vascular grafts. J Biomed Mater Res A 2004 May 1;69(2):197-204.

(20) Pichakron KO, Bui PT, Pickard BJ, Perlstein J, Mathis D, Muir-Padilla J, et al. The application of split-thickness skin graft as an autogenous arterial conduit in a goat (Capra hircus) model. Vasc Endovascular Surg 2006 May;40(3):213-22.

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5


(21) Rainwater LM, Plate G, Gloviczki P, Bahn RC, Hollier LH, Kaye MP. Morphologic quantitation of pseudointima and effects of antiplatelet drugs on vascular prostheses in goats. Am J Surg 1984 Aug;148(2):195-202.

(22) van Ommen VG, van der Veen FH, Geskes GG, Daemen M, Habets J, Dassen WR, et al. Comparison of arterial wall reaction after passage of the Hydrolyser device versus a thrombectomy balloon in an animal model. J Vasc Interv Radiol 1996 May;7(3):451-4.

(23) Lemson MS, Daemen MJ, Kitslaar PJ, Tordoir JH. A new animal model to study intimal hyperplasia in arteriovenous fistulas. J Surg Res 1999 Jul;85(1):51-8.

(24) Berry CL, Sosa-Melgarejo JA, Greenwald SE. The relationship between wall tension, lamellar thickness, and intercellular junctions in the fetal and adult aorta: its relevance to the pathology of dissecting aneurysm. J Pathol 1993 Jan;169(1):15-20.

(25) Wagner E, Guidoin R, Marois M, Mantovani D, Roy R, Ricci M, et al. Histopathologic findings in synthetic and biologic explanted grafts used in peripheral arterial reconstruction. ASAIO J 1994 Jul;40(3):M279-M283.

(26) Gohra H, McDonald TO, Verrier ED, Aziz S. Endothelial loss and regeneration in a model of transplant arteriosclerosis. Transplantation 1995 Jul 15;60(1):96-102.

(27) Bigaud M, Schraa EO, Andriambeloson E, Lobstein V, Pally C, Kobel T, et al. Complete loss of functional smooth muscle cells precedes vascular remodeling in rat aorta allografts. Transplantation 1999 Dec 15;68(11):1701-7.

(28) Aziz S, McDonald TO, Gohra H. Transplant arterial vasculopathy: evidence for a dual pattern of endothelial injury and the source of smooth muscle cells in lesions of intimal hyperplasia. J Heart Lung Transplant 1995 Nov;14:S123-S136.

(29) Ramzy D, Rao V, Tumiati LC, Xu N, Miriuka S, Delgado D, et al. Role of endothelin-1 and nitric oxide bioavailability in transplant-related vascular injury: comparative effects of rapamycin and cyclosporine. Circulation 2006 Jul 4;114(1 Suppl):I214-I219.

(30) Andriambeloson E, Bigaud M, Schraa EO, Kobel T, Lobstein V, Pally C, et al. Endothelial dysfunction and denudation in rat aortic allografts. Arterioscler Thromb Vasc Biol 2001 Jan;21(1):67-73.

(31) Bishop AJ, Glasby MA, Houlton JE. A morphological assessment of vein allografts preserved in glycerol and used for arterial replacement. J Cardiovasc Surg (Torino) 1987 Sep;28(5):491-7.

(32) Yokote K, Take A, Nakaseko C, Kobayashi K, Fujimoto M, Kawamura H, et al. Bone marrow-derived vascular cells in response to injury. J Atheroscler Thromb 2003;10(4):205-10.

(33) Xiong W, Gao CJ, Lu XF, Cheng JQ, Li YP. Prolonged cold preservation promotes the recipient’s cell participating in neointima formation but delays the later graft arteriosclerosis in rat model. Transplant Proc 2005 Jan;37(1):312-5.

(34) Lagaaij EL, Cramer-Knijnenburg GF, van Kemenade FJ, van Es LA, Bruijn JA, van Krieken JH. Endothelial cell chimerism after renal transplantation and vascular rejection. Lancet 2001 Jan 6;357(9249):33-7.

(35) Bombeli T, Mueller M, Haeberli A. Anticoagulant properties of the vascular endothelium. Thromb Haemost 1997 Mar;77(3):408-23.

(36) van Leeuwen EB, Molema G, van Luyn MJ, de Jong KP, Dijk F, Slooff MJ, et al. Scanning electron microscopic analysis of endothelial cell coverage and quality in large vessels from multi-organ donors: effects of preservation on endothelial cell integrity. Clin Transplant 2000 Jun;14(3):246-51.

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6Summary and conclusions

Samenvatting en conclusies

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96 | Chapter 6

SUMMARY AND CONCLUSIONS

In chapter 1, the topic of this thesis is introduced and an outline of the thesis presented.

Chapter 2 provides a systematic review of preservation methods of vascular allografts.

No systematic review of randomized controlled trials concerning the use of preserved vascular

allografts was found in literature. Five randomized controlled trials, 3 prospective cohort or

case series and 15 retrospective case series were identified. Cryopreservation, cold storage and

glutaraldehyde preservation have been methods used for allograft preservation. A wide range

in one-year cumulative primary patency rates and major limb loss was reported for the different

preservation methods used in these studies.

The heterogeneity of studies hampered a formal meta-analysis. The overall graft performance

of glutaraldehyde-preserved human umbilical vein allografts, however, seemed superior to the

other preservation methods. These results underscore the importance of a uniform design in

clinical trials ensuring that patient characteristics, level of anastomosis and anticoagulation

therapy are comparable. No clinical studies on glycerol preservation of vascular grafts were

found. Therefore, a preclinical study measuring the quality of glycerol preservation of arteries in

the ex vivo and in vivo setting, was undertaken in this thesis

In chapter 3, morphological and functional alterations in glycerol preserved allografts were

assessed. Breaking strength, bursting pressure and functional alterations were determined in

glycerol preserved, rat aortic segments. Breaking-strength, bursting pressure and the diameter

of the ruptured vessel wall were not significantly different between glycerol preserved allografts

and control vessels. Although endothelium-dependent or endothelium-independent contraction

or relaxation was abolished, no alterations in elastin-bundle width or mean bundle distance

were detected. The orientation index of the media was, likewise, preserved. Scanning electron

microscopy revealed flattening of the endothelial cells and defragmentation of cellular membranes

in the glycerol preserved allografts. It was concluded that the structural integrity of the aorta was

well preserved after glycerol preservation and that the glycerol preserved aortic wall was strong

enough to withstand normal physiological blood pressure. These results justified further testing

of glycerol preserved arteries in vascular transplantation experiments.

In chapter 4, the outcome of glycerol preserved allografts was studied in a rat aorta transplantation

model. A rat allograft transplantation model was developed in which wdonor rat aortas were

preserved and subsequently transplanted into recipient rats. After three months of follow-up,

the glycerol preserved allografts had a lower peak systolic velocity (PSV) than autografts. No

aneurysms were detected in the allografts implying that glycerol preservation maintained the

Fahner.indd 96 5-5-2014 14:35:15

Summary and conclusions | 97

6

structural integrity of the vessel wall. Furthermore, no significant differences were found in graft

stenosis between the groups. Thickness of the intimal layer was greater in autografts than in

allografts after 1 and 3 months follow-up. Width of the media was less in glycerol allografts and

in both groups, no inflammatory reaction was found in the media. Cumulative graft patency

rates showed no significant differences between both groups. The observation that all viable

cells had died during glycerol preservation implies that the endothelial cells no longer play a role

in the immunological response of the recipient due to the lack of antigen presentation.

Since the results of rat models cannot be directly applied to human vascular transplantation

because of obvious dimensional differences, the next step entailed experiments in a larger animal

model.

Chapter 5 deals with arterial allografts preserved in glycerol or University of Wisconsin (UW)

solution transplanted in a goat model. Donor goat carotid arteries were preserved with glycerol

or with UW solution. The latter is a well established organ preservation solution based on a

colloid component and impermeants. Three months primary patency rates for glycerol allografts,

UW allografts and jugular vein autografts were 93%, 100% and 80%, respectively. Although

graft diameter of UW allografts increased significantly during three months follow-up, the

glycerol allografts showed no aneurysm formation. Glycerol better preserved the matrix of the

vessel wall as compared to UW.

Isometric contraction and relaxation tests in these experiments demonstrated renewed graft

functional capacity after implantation which implicated ingrowth of recipient endothelial and

smooth muscle cells into the preserved vascular tissue matrix. Scanning electron microscopy

showed a confluent layer of endothelial cells in both glycerol and UW allografts.

These results demonstrate the feasibility of a glycerol preserved allograft in a large animal

model.

In Chapter 6 a summary of the studies is presented along with the main concluions.

Chapter 7 provides an overall discussion of the main conclusions of this thesis and ends with a

view on future perspectives concerning the topic.

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98 | Chapter 6

SAMENVATTING EN CONCLUSIES

In hoofdstuk 1 wordt het onderwerp van dit proefschrift geïntroduceerd en wordt een overzicht

van het proefschrift gepresenteerd.

Hoofdstuk 2 handelt over een systematisch overzicht van preservatie methoden van vasculaire

transplantaten. In de literatuur werd geen systematisch overzicht van gerandomiseerd

gecontroleerde studies gevonden betreffende het gebruik van gepreserveerde vasculaire

transplantaten. Vijf gerandomiseerd gecontroleerde studies, 3 prospectieve cohort of

enkelvoudige studies en 15 retrospectieve enkelvoudige studies werden geïdentificeerd.

Cryopreservatie, koude opslag en glutaaraldehyde preservatie zijn technieken die werden

gebruikt voor transplantaat preservatie. Er bestond een grote spreiding binnen deze studies

waarin verschillende preservatie technieken werden gebruikt voor wat betreft de cumulatieve

primaire patency rate en het verlies van een onderbeen of bovenbeen. De heterogeniteit van

de studies stond het uitvoeren van een formele meta-analyse in de weg. Echter de algehele

prestatie van de glutaaraldehyde gepreserveerde humane navelstreng vene leek superieur ten

opzichte van de andere preservatie technieken. Deze resultaten onderstrepen het belang van

een uniforme studieopzet bij klinische studies waarbij zorg wordt gedragen voor het feit dat

patiënt karakteristieken, het niveau van de anatomose en antistolling therapie met elkaar kunnen

worden vergeleken. Een klinische studie naar het gebruik van een glycerol gepreserveerd vasculair

transplantaat werd niet gevonden. Daarom werd in dit promotie onderzoek een analyse verricht

naar de kwaliteit van glycerol preservatie van arteriën in een ex vivo en in vivo onderzoeksmodel.

In hoofdstuk 3 worden de morfologische en functionele veranderingen in een glycerol

gepreserveerd transplantaat geanalyseerd. Breekkracht, scheurkracht en functionele

veranderingen werden onderzocht in glycerol gepreserveerde aorta segmenten van de

rat. Breekkracht, scheurkracht en de diameter van het vat waarbij de vaatwand scheurde

was niet significant verschillend tussen glycerol gepreserveerde transplantaten en controle

vaatsegmenten. Hoewel endotheel-afhankelijke of endotheel-onafhankelijke contractie en

relaxatie werd uitgeschakeld na glycerolpreservatie werden er geen veranderingen vastgesteld

in breedte van de elastine bundels of gemiddelde onderlinge afstand tussen deze bundels. Ook

de oriëntatie index in de media bleef onaangetast. Scanning elektronen microscopie toonde

een afvlakking van de endotheel cellen en fragmentatie van het celmembraan van het glycerol

gepreserveerde transplantaat. Er werd geconcludeerd dat de structurele integriteit van de aorta

goed bewaard bleef en de vaatwand bestand bleek tegen normale fysiologische bloeddrukken

na glycerol preservatie. Deze resultaten rechtvaardigden vervolg onderzoek naar een glycerol

gepreserveerde arterie in vaat transplantatie experimenten.

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Samenvatting en conclusies | 99

6

In hoofdstuk 4 worden de resultaten van een glycerol gepreserveerd transplantaat in

een rat aorta transplantatie model beschreven. Een ratten model voor vaattransplantaties

werd ontwikkeld waarbij de aorta van een donor rat werd gepreserveerd en vervolgens

getransplanteerd naar een ontvangende rat. Na een evaluatie periode van drie maanden toonden

de glycerol gepreserveerde transplantaten een lagere piek systolische snelheid (PSV) vergeleken

met de autografts. Er werd geen aneurysma formatie waargenomen in de transplantaten wat

betekende dat na glycerol preservatie de structurele integriteit van de vaatwand bewaard bleef.

Verder bleken er geen significante verschillen te bestaan tussen de groepen voor wat betreft

het optreden van transplantaat stenose. De intima was dikker in de autotransplantaten dan

in de glycerol gepreserveerde transplantaten na een vervolg periode van 1 en 3 maanden. De

media was dunner in de glycerol gepreserveerde transplantaten en in beide groepen werd er

geen ontstekingsreactie gezien in de media. De cumulatieve graad waarin de transplantaten

patent waren, bleek niet verschillend voor beide groepen. De wetenschap dat er geen levende

cellen meer over bleven na de glycerol preservatie betekende dat de endotheel cellen geen

rol meer speelden in de immunologische response van de transplantaat ontvanger vanwege

de afwezigheid van antigeen presentatie. Omdat er natuurlijk duidelijke verschillen zijn in

dimensies tussen rat en mens, kunnen de resultaten vanuit het ratten model niet direct worden

geëxtrapoleerd naar vaat transplantaties bij de mens. Daarom werd als volgende stap een serie

experimenten uitgevoerd in een groter proefdier model.

Hoofdstuk 5 handelt over arteriële transplantaten in een geiten model welke zijn gepreserveerd

in glycerol of in een University of Wisconsin (UW) oplossing. De arteria carotis van donor geiten

werd gepreserveerd in glycerol of UW oplossing. De laatste is een weid geaccepteerd orgaan

preservatie medium gebaseerd op een colloïde en dragerstof. De primaire patency rate na 3

maanden bleek voor glycerol transplantaten, UW transplantaten en autotransplantaten van de

vena jugularis, 93%, 100% en 80% respectievelijk. Hoewel de diameter van de UW transplantaten

na 3 maanden controle significant was toegenomen, werd bij de glycerol transplantaten geen

aneurysma vorming waargenomen. In vergelijk met UW bleef bij glycerolpreservatie de matrix

van de vaatwand beter behouden. De isometrische contractie en relaxatie experimenten

binnen deze studie lieten zien dat er opnieuw functionele contractie en relaxatie mogelijk werd

in de vaatwand na implantatie. Dit impliceerde ingroei van endotheel en gladde spiercellen

van de getransplanteerde dieren in de gepreserveerde vasculaire weefsel matrix. Met behulp

van scanning elektronen microscopie werd aangetoond dat er een aaneengesloten laag van

endotheel cellen was ontstaan in zowel de glycerol als UW transplantaten. De resultaten uit deze

studie toonde aan dat een glycerol gepreserveerd transplantaat kon worden toegepast in een

groot dier model.

Fahner.indd 99 5-5-2014 14:35:15

100 | Chapter 6

In hoofdstuk 6 wordt een overzicht gegeven van de studies aangevuld met de belangrijkste

conclusies.

Hoofdstuk 7 geeft een algemene discussie over de belangrijkste conclusies van dit proefschrift

en sluit af met een visie op mogelijke ontwikkelingen in de toekomst aangaande het onderwerp.

Fahner.indd 100 5-5-2014 14:35:15

7General discussion and future perspectives

Fahner.indd 101 5-5-2014 14:35:15

102 | Chapter 7

Update of systematic review of preservation methods of vascular allografts

The systematic review described in chapter 2, was published in 2006 and included studies

from 1966 until 2004. Therefore, on completion of this thesis in 2014, a renewed systematic

review of clinical studies on peripheral reconstructions using preserved vascular allografts was

performed, covering the last ten years. The same search strategies were followed and the same

in- and exclusion criteria were used to determine key articles in which different types of vascular

allografts were used in arterial reconstructions. A total of twelve clinical studies were retrieved

in the last search. Seven studies were excluded, of which five because of the small number of

patients included. One Czech article was excluded because of translation problems and one

multi-center study evaluated reconstructions with cryopreserved allografts in the aortic position.

Hence, a final set of five additional key articles was obtained (1-5).

Three studies used cryopreserved venous allografts, one retrospective series described

reconstructions using cold stored venous allografts and in one clinical study, the human umbilical

vein (HUV) graft was evaluated. A total of 202 patients were included in the studies using

cryopreserved venous allografts; 43 and 197 patients were enrolled in the cold stored allograft

study and HUV allograft series, respectively.

The weighted mean 1-year cumulative primary graft patency rate was 35% for the

cryopreserved venous allografts, 46% for the cold stored allografts while the 5-year cumulative

primary graft patency rate for the HUV allografts was 54%. The results of the cryopreserved

venous allografts and HUV allografts were comparable to the previous review, showing a

weighted mean 1-year cumulative primary graft patency rate for venous allografts of 41% and

5-year cumulative primary graft patency rate for the HUV allografts of 53%. The patency results

for the cold stored allografts were slightly lower in the recent series since patency rates in the

previous review ranged from 63% to 80%.

This update shows that the conclusion of the former systematic review was confirmed by

the current review. Because studies in which a specific preservation method is compared, are

still lacking, a firm conclusion cannot be made. HUV allografts however, seemed superior to the

other preservation methods showing high 5-year patency-rates.

Transplantation of glycerol preserved allografts.

The implantation studies presented in chapters 4 and 5 demonstrated the feasibility of glycerol

preserved allografts for vascular substitution. It is however important to take caution when

extrapolating patency rates from animal models to humans. The process of endothelialization

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General discussion and future perspectives | 103

7

for example, is more rapid and complete in animals and different in prosthetic vascular grafts

(6-11) and in animal studies, anticoagulant therapy is usually not used. In clinical application of

glycerol preserved arterial allografts however, anticoagulants are probably needed at least until

intraluminal reendothelialization has been completed. Secondly, in the rat aorta transplantation

model, young animals are used as allograft donors. These animals do not suffer from alterations

in vessel wall constitution such as in atherosclerosis, diabetes mellitus, nicotine abuse or ageing.

These would be all potential factors influencing the results of graft function and patency rates

after application of human glycerol preserved arterial allografts.

Tissue engineering of vascular grafts

While the aforementioned studies concern transplantation of biological vascular tissue, another

area of research is the tissue engineering of synthetic vascular conduits.

As allografts appear to have the advantages of easier placement, lower complication rate

and better resistance to infections, it can be postulated that allografts perform superiorly to

synthetic conduits in revascularization of lower limbs. Future experiments should clarify the

advantages of allografts to synthetic polymers used in combination with biological matrices.

The synthetic polymers include polyglycolic acid, poly-L-lactic acid, poly-DL-lactic-co-glycolic acid

and polyurethanes, the first three being biodegradable (12-15). The degradation process should

follow a timeline that enables the recipient cells to form a functional conduit resistant to the

biomechanical stress and strain imposed on a native vessel. Synthetic scaffolds have the advantage

of reproduction on an industrial scale and the possibilities of quality control at the point of

manufacture (16). A disadvantage of synthetic scaffolds is the lack of biological, cell attachment

factors present in naturally derived scaffolds while their relation with the development of intimal

hyperplasia is uncertain (17;18).

The first tissue-engineered blood vessel was developed by Weinberg and Bell who used

cultured bovine endothelial cells, vascular smooth muscle cells and fibroblasts on a collagen

scaffold (19). Also biodegradable scaffolds seeded with autologous mononuclear cells have been

used in vascular constructs (20). To enable autologous cells to attach to the biological scaffold a

suitable bioreactor system is required. Niklason et al successfully developed the first bioreactor

for vascular tissue engineering (21). Another limitation of this approach is the long culture time of

approximately 8 weeks between the harvest of autologous cells and the culturing of a mature and

durable conduit (22-26). An alternative to endothelialization of the tissue-engineered vascular

graft probably is the utilization of in situ endothelial cells as was reviewed by Melchiorri et al (27).

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104 | Chapter 7

Future perspectives

Since the applicability of glycerol preservation of arterial allografts has been shown in animal

studies, the next step is the preparation of a glycerol allograft for clinical use.

Although extensive research has been performed in the elimination of bacterial and viral

contamination in glycerol preserved skin allografts, experiments will be needed to examine if

bacterial and/or viral contamination jeopardizes the use of glycerol preserved vascular allografts.

Likewise, contamination studies will be needed looking at human immunodeficiency virus

(HIV), hepatitis B and C, Treponema pallidum, Cytomegalovirus and Toxoplasma. Furthermore,

the duration to safely eliminate these viruses in the process of glycerol preservation must be

established.

Knowing that the attachment of recipient endothelial cells to biological scaffolds is superior

if compared to synthetic constructs, it is interesting to investigate the potential improvements of

complete luminal endothelial cell lining of glycerol preserved arterial allografts via cell seeding.

A potential next step is the development of a vascular tissue bank for glycerol arterial allografts

in collaboration with the established tissue banks using existing infrastructure for cadaveric donor

harvesting protocols. Teams involved in the harvesting of organs and tissues can be trained

to harvest the arteries between the aortic bifurcation and the infragenual popliteal arteries

for glycerol preservation and storage. Ultimately, the efficacy of glycerol preserved allografts

needs to be assessed in properly designed, randomized trials using the venous autograft as gold

standard. Uniform requirements for allograft quality surveillance and anticoagulation therapy

should be established in such protocols.

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General discussion and future perspectives | 105

7

Reference List

(1) Zehr BP, Niblick CJ, Downey H, Ladowski JS. Limb salvage with CryoVein cadaver saphenous vein allografts used for peripheral arterial bypass: role of blood compatibility. Ann Vasc Surg 2011 Feb;25(2):177-81.

(2) Randon C, Jacobs B, De RF, Beele H, Vermassen F. Fifteen years of infrapopliteal arterial reconstructions with cryopreserved venous allografts for limb salvage. J Vasc Surg 2010 Apr;51(4):869-77.

(3) Bannazadeh M, Sarac TP, Bena J, Srivastava S, Ouriel K, Clair D. Reoperative lower extremity revascularization with cadaver vein for limb salvage. Ann Vasc Surg 2009 Jan;23(1):24-31.

(4) Matia I, Janousek L, Marada T, Adamec M. Cold-stored venous allografts in the treatment of critical limb ischaemia. Eur J Vasc Endovasc Surg 2007 Oct;34(4):424-31.

(5) Neufang A, Espinola-Klein C, Dorweiler B, Messow CM, Schmiedt W, Vahl CF. Femoropopliteal prosthetic bypass with glutaraldehyde stabilized human umbilical vein (HUV). J Vasc Surg 2007 Aug;46(2):280-8.

(6) Burkel WE, Graham LM, Stanley JC. Endothelial linings in prosthetic vascular grafts 4. Ann N Y Acad Sci 1987;516:131-44.

(7) Clagett GP, Burkel WE, Sharefkin JB, Ford JW, Hufnagel H, Vinter DW, et al. Platelet reactivity in vivo in dogs with arterial prostheses seeded with endothelial cells. Circulation 1984 Mar;69(3):632-9.

(8) Merzkirch C, Davies N, Zilla P. Engineering of vascular ingrowth matrices: are protein domains an alternative to peptides? Anat Rec 2001 Aug 1;263(4):379-87.

(9) Stanley JC, Burkel WE, Graham LM, Lindblad B. Endothelial cell seeding of synthetic vascular prostheses. Acta Chir Scand Suppl 1985;529:17-27.

(10) Wu MH, Shi Q, Wechezak AR, Clowes AW, Gordon IL, Sauvage LR. Definitive proof of endothelialization of a Dacron arterial prosthesis in a human being. J Vasc Surg 1995 May;21(5):862-7. (11)

(11) Burkel WE, Ford JW, Vinter DW, Kahn RH, Graham LM, Stanley JC. Fate of knitted dacron velour vascular grafts seeded with enzymatically derived autologous canine endothelium 4. Trans Am Soc Artif Intern Organs 1982;28:178-84.

(12) Kim BS, Mooney DJ. Development of biocompatible synthetic extracellular matrices for tissue engineering 4. Trends Biotechnol 1998 May;16(5):224-30.

(13) Niklason LE, Abbott W, Gao J, Klagges B, Hirschi KK, Ulubayram K, et al. Morphologic and mechanical characteristics of engineered bovine arteries. J Vasc Surg 2001 Mar;33(3):628-38.

(14) Tiwari A, Salacinski H, Seifalian AM, Hamilton G. New prostheses for use in bypass grafts with special emphasis on polyurethanes. Cardiovasc Surg 2002 Jun;10(3):191-7.



(17) Kim BS, Mooney DJ. Development of biocompatible synthetic extracellular matrices for tissue engineering. Trends Biotechnol 1998 May;16(5):224-30.

(18) Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993 Apr 29;362(6423):801-9.

(19) Weinberg CB, Bell E. A blood vessel model constructed from collagen and cultured vascular cells. Science 1986 Jan 24;231(4736):397-400.

(20) Hibino N, Villalona G, Pietris N, Duncan DR, Schoffner A, Roh JD, et al. Tissue-engineered vascular grafts form neovessels that arise from regeneration of the adjacent blood vessel. FASEB J 2011 Aug;25(8):2731-9.

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(21) Niklason LE, Gao J, Abbott WM, Hirschi KK, Houser S, Marini R, et al. Functional arteries grown in vitro. Science 1999 Apr 16;284(5413):489-93.

(22) Anders E, Alles JU, Delvos U, Potzsch B, Preissner KT, Muller-Berghaus G. Microvascular endothelial cells from human omental tissue: modified method for long-term cultivation and new aspects of characterization. Microvasc Res 1987 Sep;34(2):239-49.

(23) Pronk A, Leguit P, Hoynck van Papendrecht AA, Hagelen E, van Vroonhoven TJ, Verbrugh HA. A cobblestone cell isolated from the human omentum: the mesothelial cell; isolation, identification, and growth characteristics. In Vitro Cell Dev Biol 1993 Feb;29A(2):127-34.

(24) Sharp WV, Schmidt SP, Meerbaum SO, Pippert TR. Derivation of human microvascular endothelial cells for prosthetic vascular graft seeding. Ann Vasc Surg 1989 Apr;3(2):104-7.

(25) Tiwari A, Salacinski HJ, Hamilton G, Seifalian AM. Tissue engineering of vascular bypass grafts: role of endothelial cell extraction. Eur J Vasc Endovasc Surg 2001 Mar;21(3):193-201.

(26) Williams SK. Human clinical trials of microvascular endothelial cell sodding. In: Zilla P, Greisler HP, editors. Tissue Engineering of Vascular Prosthetic Grafts. Landes: Austin: 1999, p. 143-7.

(27) Melchiorri AJ, Hibino N, Fisher JP. Strategies and techniques to enhance the in situ endothelialization of small-diameter biodegradable polymeric vascular grafts. Tissue Eng Part B Rev 2013 Aug;19(4):292-307.

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Dankwoord | 107

Dankwoord

In de overtuiging dat elke nieuwe dag een geschenk is uit Gods hand zijn wij Hem dankbaar voor

al die gaven die we in de achterliggende jaren mochten ontvangen.

Prof. dr. T.M. van Gulik, beste Thomas. Zeer dank voor de mogelijkheid die jij gaf om te werken

op het Chirurgisch Laboratorium van het AMC. Een enthousiaste en inspirerende omgeving

wist jij daar te creëren om de experimenten op te zetten en uit te voeren. Je ontwapenende

presentatie gaf altijd het gevoel dat ik bij je welkom was. Dank voor de uitmuntende lezing van

de engelse teksten.

Prof. dr. D.A. Legemate, beste Dink. Dankzij de mede door jou opgezette samenwerking

tussen de experimentele chirurgie en vaatchirurgie werd het onderzoek mogelijk. Dank voor je

begeleiding in de opzet van de verschillende onderzoeken en het bewaken van de voortgang. Je

grote kennis over de opzet en analyse van een systematic review bewonder ik.

Dr. M.M. Idu, beste Mirza. Veel hulp heb je gegeven bij de praktische uitvoering van de

experimenten. We hebben heel wat hechtdraadjes verbruikt voor alle anastomosen. Nederland

zijn we doorgereisd van noord naar zuid ter voorbereiding van de volgende studie. Dank voor je

niet afnemende doorzettingsvermogen.

Prof. dr. E.T. van Bavel, Dr. M. Heger, Prof. dr. B.A.J.M. de Mol, Dr. J.I. Rotmans, Prof. dr. C.J.A.M.

Zeebregts, geachte leden van de promotiecommissie. Dank voor uw bereidheid het manuscript

te willen beoordelen en van commentaar te voorzien. Prof. dr. B.H. Walpoth, dear Beat. Thank

you very much for your valuable discussions we have had in Szeged, Hungary and in Athens,

Greece on the Congress of the European Society for Surgical Research. Thanks a lot for your

introduction in your research laboratory at Hôpitaux Universitaires de Genève and for your

willingness to participate in the doctoral committee.

Beste IWO bewoners, dank voor de plezierige samenwerking. Beste kamergenoten, Ivo

Schoots, Geert Koffeman, Bob Heijnen en Suzanne van Veen. Dank jullie zeer voor de

gezellige tijd samen. Collega onderzoekers, Maud Bessems, Oktay Camlidag, Sander Dinant,

Benedict Doorschot, Maarten Jansen, Maarten-Paul van de Kerkhove, Jeroen Lips, Paul Poyck,

Reeta Vetelainen, Annebeth de Vries. We hebben heel veel teamvergaderingen, retraites,

onderzoekervergaderingen en buitenlandse reizen met elkaar kunnen beleven. Dank voor de

Fahner.indd 107 5-5-2014 14:35:16

108 | Dankwoord

stimulerende ontmoetingen. Beste (bio)technici en analisten, Adri Maas, Goos Huijzer, Henk de

Wit, Marloes Klein, Peter Schneider, Robin Hartman, Maringa Emons, Esther Posno en Albert van

Wijk. Zonder jullie ondersteuning waren de experimenten nooit van de grond gekomen. Dank

voor jullie altijd durende behulpzaamheid. Beste Postdoc´s, Irene Straatsburg, Ruurtje Hoekstra

en Leo Abrahamse. Dank voor jullie wetenschappelijke support.

Beste Wouter Florijn, Henriette Griffioen, Ties van de Berg en Kor Brandsma. Dank voor jullie hulp

bij het toezicht op de naleving en correcte uitvoering van de onderzoeksprotocollen.

Beste onderzoeksstudenten, Jaap Borstlap, Bram van Wijk en Carola van Eck. Dank voor jullie

hulp bij de experimenten, data invoer en analyse. Het was een eer jullie begeleider te mogen

zijn. Dear Dora Bedreaga, it was a great pleasure to me that you have participated as a research

student in our group. Best Kitty Cisse en Andrea de Leeuw. Hartelijk dank voor de vele manieren

waarop jullie secretariële ondersteuning hebben verleend.

Medewerkers van het vaatcentrum en de afdeling radiologie van het AMC, beste Johan van

Gurp, Dianne Hanson, Ineke Zwiers en Theo van de Storm. Hoe vaak zijn jullie niet met je echo

apparaat op pad gegaan door alle gangetjes richting het IWO? Jullie lieten het gewoon nooit

afweten. Hartelijk dank voor de inzet. Theo bedankt voor je hulp bij de angio´s.

Medewerkers van de afdeling Medische fysica, Farmacotherapie, Pathologie en Microscopisch

onderzoek van het AMC. Prof. Dr. Pfaffendorf, MJ Mathy, Stephan Peters, Allard van der Wal

en Jan van Marle. Dank jullie voor de expertise die jullie hebben ingebracht en de enthousiaste

bereidheid tot samenwerking.

Mevrouw G.E.E. van Noppen, beste Wendy. Dank voor je mooie cursus en begeleiding van de

eerste stappen bij het schrijven van engelse teksten.

Medewerkers van het Rode Kruis Ziekenhuis en de Euro Skin Bank te Beverwijk. Prof. Dr. R.W. Kreis,

Dr. R.S. Breederveld, Paul van Zuijlen, Jeroen van Baare, Hans Hoekstra, Esther Middelkoop,

Nelleke Richters en Jos Vloemans. Door jullie bereidheid tot inzicht in het preservatieprotocol

van de glycerol donorhuiden werd het onderzoek op vaten mogelijk gemaakt. Dank voor jullie

discussies en getoonde interesse in het verloop van het onderzoek.

Beste Adelheid Schokker, dank voor je hulp bij de invoer van data en je blijvende interesse

voor het onderzoek. Dank ook voor je grote gastvrijheid en de gezellige ontmoetingen in de

Fahner.indd 108 5-5-2014 14:35:16

Dankwoord | 109

achterliggende jaren. In zeer dierbare herinnering aan je echtgenoot Bob Schokker die ik heb

mogen leren kennen als kundig arts, tactisch bestuurder en bovenal als waardevolle vriend.

Dr C.F. Gattuso van de Kring Oost van de Rijksdienst voor de keuring van vee en vlees voor het

verlenen van toestemming om 1 geitenkop te vervoeren vanuit Slachthuis Midden Nederland

te Twello naar het AMC. Hierdoor werd het mogelijk mij voorafgaand aan de experimenten te

verdiepen in de halsanatomie van de geit.

Beste collegae en medewerkers van ziekenhuis De Sionsberg. Bedankt voor de wijze waarop jullie

me in jullie midden hebben opgenomen. Dank aan het hele team van de afdeling radiologie voor

jullie betrokkenheid. Het is me een groot genoegen met jullie te mogen samenwerken.

Geliefde familie en vrienden. Dank voor jullie getoonde interesse in de onderzoeksjaren. Al moest

ik nog wel eens verstek laten gaan bij feesten en partijen, de onderlinge banden bleven bestaan.

Beste Hanneke van Krimpen en André Janse, dank voor jullie langdurige vriendschap. Het is

prachtig dat jullie paranimf willen zijn.

Lieve kinderen, Anne-Fleur, Joachim, Jean-Luc en Florentijn. We mochten jullie ontvangen in de

achterliggende jaren en genieten elke dag van jullie aanwezigheid. Misschien raken jullie later

ook wel gemotiveerd voor het doen van onderzoek. Lieve Anneke, je bent van onschatbare

waarde gebleken. Niet in het minst in de jaren waarin je heel wat keren de honneurs alleen

moest waarnemen. Ik blijf je voor altijd dankbaar.

Fahner.indd 109 5-5-2014 14:35:16

Fahner.indd 110 5-5-2014 14:35:16

Curriculum Vitae | 111

Curriculum Vitae

The author was born on February 18th, 1972 in Angguruk, Indonesia. He graduated from high

school, Van Lodestein College, in 1991 and obtained his medical degree at Erasmus University

Rotterdam in 1998. He was resident in internal medicine (Ziekenhuis Velp, Velp) urology (Canisius

Wilhelmina Ziekenhuis, Nijmegen) and surgery (Eemland Ziekenhuis, Amersfoort, Rode Kruis

Ziekenhuis, Beverwijk and St Lukas Andreas Ziekenhuis, Amsterdam). From 2001 until 2004 he

was PhD fellow at the Surgical Laboratory of the Academic Medical Center, Amsterdam under

supervision of Prof. dr. D.A. Legemate and Prof. dr. T.M. van Gulik. From 2006 until 2012 he

followed his residency in radiology at the University Medical Center Groningen under supervision

of Prof. dr. M. Oudkerk, Prof. dr. E.J. van der Jagt and Dr. L.M. Kingma. Since 2012 he works as

a radiologist in Ziekenhuis de Sionsberg, Dokkum.

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Experim

ental Stu

dies o

n G

lycerol Preserved

Vascu

lar Allo

grafts

Peter Fahn

er

Experimental Studies on Glycerol Preserved Vascular Allografts

Peter Fahner

UITNODIGING

voor het bijwonen van

de openbare verdediging

van het proefschrift

Experimental Studieson Glycerol Preserved

Vascular Allografts

op vrijdag 6 juni 2014

om 13.00 uur

in de Aula van de

Universiteit

van Amsterdam

Oude Lutherse Kerk

Singel 411, hoek Spui

1012 WN Amsterdam

Aansluitend receptie in de

naastgelegen

Tetterode Bibliotheek

Paranimfen:

Hanneke van Krimpen

André Janse

[email protected]

Peter [email protected]

Proefschrift Fahner

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