Addressing thrombogenicity in vascular graft construction

Addressing Thrombogenicity in Vascular Graft Construction

Sandip Sarkar,1 Kevin M. Sales,2 George Hamilton,3 Alexander M. Seifalian1

1 Biomaterials and Tissue Engineering Centre (BTEC), Academic Division of Surgical and Interventional Sciences,University College London, London, United Kingdom

2 Advanced Nanomaterials Laboratory (ANL), BTEC, University College London, London, United Kingdom

3 Department of Vascular Surgery, Royal Free Hospital Hampstead NHS Trust, London, United Kingdom

Received 13 March 2006; revised 24 July 2006; accepted 8 August 2006Published online 31 October 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.30710

Abstract: Thrombosis is a major cause of poor patency in synthetic vascular grafts for small

diameter vessel (<6 mm) bypass. Arteries have a host of structural mechanisms by which they

prevent triggering of platelet activation and the clotting cascade. Many of these are present in

vascular endothelial cells. These mechanisms act together with perpetual feedback at different

levels, providing a constantly fine-tuned non-thrombogenic environment. The arterial wall

anatomy also serves to promote thrombosis as a healing mechanism when it has been severely

injured. Surface modification of synthetic graft surfaces to attenuate the coagulation cascade

has reduced thrombosis levels and improved patency in vitro and in animal models. Success in

this endeavor is critically dependent on the methods used to modify the surface. Platelets adhere

to positively charged surfaces due to their own negative charge. They also preferentially attach

to hydrophobic surfaces. Therefore synthetic graft development is concerned with hydrophilic

materials with negative surface charge. However, fibrinogen has both hydrophilic and

hydrophobic binding sites–amphiphilic materials reduce its adhesion and subsequent platelet

activation. The self-endothelializing synthetic graft is an attractive proposition as a confluent

endothelial layer incorporates many of the anti-thrombogenic properties of arteries. Surface

modification to promote this has shown good results in animal models. The difficulties

experienced in achieving spontaneous endothelialisation in humans have lead to the

investigation of pre-implantation in vitro endothelial cell seeding. These approaches ultimately

aim to result in novel synthetic grafts which are anti-thrombogenic and hence suitable for

coronary and distal infrainguinal bypass. ' 2006Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl

Biomater 82B: 100–108, 2007

Keywords: thrombogenicity; vascular bypass graft; surface modification; vascular endothe-

lium; blood compatibility; platelet adhesion; hydrophilic; electronegative

INTRODUCTION

The main determinants of patency of vascular bypass grafts

are thrombogenic potential and level of intimal hyperplasia

(IH). Of these two, the former is arguably more important as

it is responsible for earlier graft occlusion. Also IH is associ-

ated with the abrupt change in distensibility between native

vessel and stiff prosthesis at the anastomosis,1 but this causal

link appears to be less clearly demonstrated for blood compat-

ible vascular conduits than for thrombogenic graft surfaces.2

The currently used grafts for vascular bypass in small cali-

bre (<6 mm), low flow states are autologous vessels or if these

are not available, polytetrafluoroethylene (PTFE) prostheses.3

The autologous vessels used are long saphenous vein in the

case of infrainguinal bypass; for coronary artery bypass the

preferred alternative to saphenous vein is the internal mam-

mary (or internal thoracic) artery. The results of bypass pro-cedures with autologous vessels are considerably better than

when synthetic alternatives are used (Figure 1) and every

attempt is made to utilize the former. However, in as many as

one third of cases, this is not possible due to previous use,

inadequate calibre or diseased vessel.

Considerable research effort is being made to develop syn-

thetic graft materials with a favorable blood compatibility.

This is hampered by the relatively poor understanding of how

surface properties relate to thrombogenic potential as well as

the difficulties faced in surface modification technology.

On implantation of a vascular graft, plasma proteins im-

mediately adsorb to the wall. They present binding sites for

integrin receptors, which are to be found on platelets and

Correspondence to: A. M. Seifalian (e-mail: [email protected])

' 2006 Wiley Periodicals, Inc.

100

many cells.4 Adhesion to these as well as to the vessel wall

itself may cause activation of the platelets. Surface modifi-

cation to improve blood compatibility includes methods to

reduce both plasma protein adsorption and platelet adhesion

to the prosthesis wall. In addition, the interactions between

plasma proteins and platelets that ultimately lead to platelet

activation have also been targeted.

This review aims to present what is already known about

blood/surface interactions as well as discuss the main contro-

versies in vascular surface thrombogenicity, illustrating with

examples of the approaches used to address these issues.

Search Methods

A medline search was undertaken for original articles using

the keywords: thrombogenicity; blood compatibility; endo-

thelialisation; surface modification; platelet; vascular graft.

Further searches were undertaken as required to aid under-

standing of the articles produced from the original searches.

THE ARTERIAL SURFACE

The arterial wall is composed of a number of histologically

separate layers, which allow a multitude of specialized func-

tions, only one of which is blood compatibility (Figure 2). The

innermost layer is a single layer of vascular endothelial cells

(vEC) attached to a basement membrane. This layer is in close

contact with the blood flowing through the vessel and so is

intimately involved in preventing thrombosis. The vascular en-

dothelium is non-thrombogenic. The basement membrane

attaching it has moderate thrombogenicity; but it is the highly

thrombogenic collagen5 of the subendothelial connective tis-

sue, which rapidly instigates platelet adhesion followed by the

cascade of events leading to thrombus formation (Figure 3).

Additional subendothelial components promoting platelet ad-

herence include fibronectin, laminin, and thrombospondin. Ad-

herence is facilitated by the presence of Von Willebrand

Factor, a platelet adhesive protein synthesized by vEC. Expo-

sure of the subendothelium to blood components also reduces

the expression and activity of antithrombin III. This design is

to ensure speedy coagulation in the event of serious arterial

damage, which in conjunction with vasospasm prevents heavy

bleeding.

There are numerous biochemical pathways involving mul-

tiple cell surface receptors and intracellular signalling path-

Figure 2. The multilaminar composite tissue organization of the arterial wall. [Color figure can be

viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 1. The relative primary patency of PTFE and vein grafts inperipheral bypass.

101THROMBOGENICITY IN VASCULAR GRAFT CONSTRUCTION

Journal of Biomedical Materials Research Part B: Applied BiomaterialsDOI 10.1002/jbmb

ways by which vEC modulate plasma protein and platelet ad-

hesion. Even with no physiological stimulus, vEC inhibit pla-

telet aggregation. To some extent, this is prostacyclin

mediated and can be reversed by application of non-steroidal

anti-inflammatory drugs.6 The remainder of this platelet

aggregation inhibition is mediated by endothelium derived

relaxing factor (EDRF). Anticoagulant factors present in the

vEC membrane include heparin-like glycosaminoglycans

(heparans), thrombomodulin, and adenosine diphosphatase

(ADPase). The heparans moiety is essential for antithrombin

III activity. vEC also express the procoagulant Tissue Factor

(TF) to exert fine control over overall thrombotic tendency.

Any trigger for thrombosis such as vascular injury tilts the

homeostasis towards increased TF expression and a downre-

gulation of membrane anticoagulants, particularly thrombo-

modulin.7 This latter forms complex with circulating

thrombin, reducing its availability for fibrinogenesis. TF acts

by binding Factor VII/VIIa causing the subsequent activation

of Factors IX and X. Circulating low density lipoproteins

(LDL), desmopressin,8 and homocysteine enhance this activ-

ity. The oxidized form of LDL further enhances platelet ad-

hesion.9 To counteract TF and regulate its activation, the

serine protease inhibitor Tissue Factor Pathway Inhibitor

(TFPI) circulates alone or in complexes with plasma lipopro-

teins and acts to inhibit Factor X directly10 by binding to the

TF/Factor VIIa complex. Its activity is reduced by LDL. Ni-

tric oxide (NO) which has an anti-aggregatory effect on pla-

telets is also released by vECs as well as by platelets

themselves.

These are the principle mechanisms amongst many uti-

lized by the vEC to regulating thrombogenicity.

SURFACE MODIFICATION TO ATTENUATE THECOAGULATION CASCADE

Attempts have been made to reduce the inherent thrombo-

genicity of synthetic graft surfaces by incorporating some

of the mechanisms employed by vECs.11 Conversely, the

use of collagen as a graft sealant as the blood graft inter-

face has had to be tempered due to it’s thrombogenicity.12

Membrane glycosaminoglycans may be simulated by the

incorporation of heparin onto the graft surface. Results were

initially mixed and dependent on the method of heparin

bonding13 with covalent bonds with long half-life reducing

the thrombogenicity of both PTFE14 and Dacron.15 A tran-

sient coating however is one reason for failure of heparin to

improve short term patency.16 Current technology for hepa-

rin bonding has resulted in promising results for many

groups (Table I). As well as discouraging coagulation hepa-

TABLE I. Summary of Research into Heparin-Bonded Vascular Grafts in the Last 2 Years

Year Graft Study Type Outcome Reference

2006 Polycaprolactone in vitro ; vascular smooth muscle cell growth 17

2005 Thermoplastic

polyurethane

in vitro ;platelet adhesion; thrombin inactivation 13

2005 PTFE Retrospective

clinical trial

92% 2 year primary patency for

below knee bypass

18

2004 Wire coiled

polymer coated

in vitro vEC layer promotion; ;thrombus

formation

19,20

2004 PTFE animal model ;platelet adhesion; ;IH 21

2004 Carbon coated PTFE animal model ;early platelet adhesion; ;late thrombus;

:patency22

2004 PTFE animal model ;platelet adhesion; ;IH 23

2004 Collagen in vitro ;thrombin generation 24

Figure 3. An overview of the coagulation cascade.

102 SARKAR ET AL.


rin also reduces the proliferation of vascular smooth muscle

cells in the media and intima, thus abating neointimal

hyperplasia.17 A recent clinical trial has shown heparin-

bonded Dacron to be as good as PTFE in the long term for

Femoropopliteal bypass.25

Heparin elution from graft surfaces has also been con-

sidered, partly as a result of the observation that the anti-

proliferative effect of drug-eluting stents (medium term)

seems to outlast the drug elution. However, in the case of

stents, it is now becoming apparent that this benefit may

not persist in the longer term with delayed thrombosis

being increasingly recognised,26 possibly in association

with endothelial cell dysfunction and slow vessel wall heal-

ing.27 Hence, anticoagulant elution may be more suited to

intra-vascular catheters.28

Additionally, platelet activity has been directly targeted

with covalently bonded dipyridamole (Persantin) attached

to the polycarbonate urethane graft (Chronoflex),29 which

acts by preventing cyclic adenosine monophosphate (cAMP)

formation, an essential intraplatelet messenger signalling

platelet aggregation. Although there was reduced platelet

aggregation and a delayed thrombin reaction in vitro, thisstill translated to five goat carotid grafts being occluded

within 10 weeks. However, chronoflex may be unsuited to

implantation due to considerable biodegradation noted

within 10 weeks in sheep.

ADPase (adenosine diphosphatase) was first noted as a

natural antithrombogenic agent in the renal glomerular cap-

illary wall where blood is in contact with the relatively

thrombogenic glomerular basement membrane. In addition,

a form of ADPase is secreted in the saliva of ticks. Platelet

derived ADP (adenosine diphosphate) interacts with vEC at

the CD39 receptor to cause release of EDRF. ADPase

coated polyurethane grafts have better patency in a rabbit

carotid model than polyurethane alone.30 However, recent

indications are that the ADP effect on vEC is more impor-

tant in small mammals than in man.31 It is also becoming

clear that the platelet inhibitory effect of ADPase does not

solely function through vEC EDRF release. Costa et al.

have demonstrated the anti-platelet effects of ADPase in

rabbit arteries where the endothelium has been destroyed

beforehand.32

Factor X inhibition by TFPI has been exploited by attach-

ing it to Dacron grafts. On implantation in animal models,

reduced fibrinogen adherence was noted33 as well as a thin-

ner neointima and lower thrombosis levels34 compared with

controls. However, the numbers included were small and it is

unclear that a benefit remains in the long term as the half-life

of TFPI is less than 2 hours, requiring constant replenishment

whilst at the same time, the expression of TF increases over

12 months following implantation.35

Hirudin is a thrombin inhibitor, which is secreted in the

saliva of leeches and genetically engineered recombinant

varieties have been used therapeutically as an anticoagu-

lant. As with heparin, its presence at vascular anastomoses

reduces IH,36 suggesting thrombin inhibition is directly

linked to prevention of IH. Dacron with covalently bonded

hirudin shows less thrombus formation and thinner neoin-

tima than Dacron alone.37

Nitric oxide(NO) is released by platelets to form a micro-

environment that is unfavorable to platelet adhesion.38

Recently attempts have been made to manufacture NO-eluting

vascular grafts due to its antithrombogenic and anti-IH

effects.39 The preliminary results are encouraging in animal

models but the challenge of long-term NO production from a

synthetic conduit needs to be addressed. NO effects are medi-

ated by the cyclic 3050-guanosine monophosphate (cGMP)

secondary messenger pathway. Phosphodiesterase inhibitors

such as sildenafil cause the accumulation of cGMP, thus

potentiating NO effect40 and the two may have a synergistic

role in future anti-platelet grafts. However, as with TFPI, the

importance of the role of NO as a platelet inhibitor has con-

siderable interspecies variability with it having little relevance

in mouse models.41

Electrical Charge

Amongst the esoteric properties which afford thrombo-

genicity, the issue of surface charge is refreshingly simple.

Platelets have net electronegative surface charge, and it has

been known since the 1950s that the blood vessel wall also

exhibits a similar surface charge, as one would expect for

their mutual repulsion for each other. Sawyer demonstrated

that injury to the vessel wall lead to it gaining positive

charge.42

PTFE has a weak negative charge too, but this is further

enhanced by the fibrinous layer that forms on its luminal

surface after implantation. As well as contributing to anti-

thrombogenicity, this characteristic also has antibacterial

properties, as those bacteria which are associated with graft

infection have electronegative surface properties.43 Carbon

coating to PTFE further increases its electronegativity, and

is in commonplace clinical use.

However negative electropotential alone will not ensure

an antithrombogenic surface. The effect of surface chemis-

try is of paramount importance too. Collagen is very

thrombogenic despite electronegativity. Therefore although

electronegativity is a prerequisite for a non-thrombogenic

surface, additional surface properties are also critical in

determining the platelet reaction.

Surface Wettability

As with negative electrical charge, a hydrophilic surface

may confer thromboresistance but that is not to say that all

hydrophilic surfaces are thromboresistant.44 In fact, similar

materials with different wettability may show no correla-

tion with thromboresistance.45 Van Kampen’s46 assessment

of poly(aminoacid) grafts revealed that platelet adherence

to the wall was competing with leucocyte adherence which

favored fibrinolysis and eventual leucocytic endothelial

transformation. Which component would prevail could not

be predicted with surface characterization.



PTFE’s advantageous qualities include its biocompati-

bility, non-degradation, and strength. It is mildly electroneg-

ative, but unfortunately is hydrophobic and prone to

thrombosis in low flow states. One method of reducing the

hydrophobicity of a material is to covalently bond hydro-

philic groups to its surface. Heparin is hydrophilic, contribut-

ing to its anti-platelet adherence properties and reducing the

hydrophobic nature of collagen24 and PTFE (Table I). Simi-

larly, the use of compliant silicone rubber as a vascular con-

duit has been hampered by its hydrophobicity, affording it

high affinity for platelets and fibrinogen. Julio et al.47 Cova-

lently bonded hydrophilic monomers to these grafts to reduce

short term thrombogenicity.

Polyethylene glycol (PEG) and polyethylene oxide (PEO)

are hydrophilic flexible polymers which resist protein adsorp-

tion when bonded to the vascular surface. This translates to

reduced thrombogenicity when they are used to coat vascular

grafts.48 The length and degree of branching of these poly-

mers may correlate with the degree of platelet repulsion, due

to their flexibility increasing thermodynamic interactions at

the surface.49 The great challenge with respect to these groups

has been firm bonding to grafts with no translation to clinical

application despite in-vitro and in-vivo study for over a dec-

ade.

However before platelet interactions with the prosthesis,

fibrinogen may adsorb to the blood contact surface, and

become activated as well as activating platelets. Fibrinogen

has bonding sites for both hydrophilic and hydrophobic sur-

faces.50 Amphiphilic surfaces can discourage fibrinogen

binding.51 This may be due to the different microdomains

effectively canceling each others effects.52 They are also

implicated in preventing platelet activation,53 possibly by

interfering with platelet binding with fibrinogen. Co-poly-

merization of hydrophilic polymers such as PEO54 and

PEG53 with hydrophobic counterparts result in materials

which demonstrate these antithrombogenic properties. Poly-

siloxane and polycaprolactone-based amphiphilic copoly-

mers have been commercially available for use as surface

modifying additives for the extracorporeal circuits of car-

diopulmonary bypass.55

Fibrinogen adherence may be discouraged by creating

conditions favorable to albumin attachment. The phospho-

lipid membrane has a preference for albumin due to fatty

acid chains, and in particular C16 chains. Sixteen carbon

acyl groups56 or alkyl groups form a characteristic steric

conformation57 which confers thromboresistance. A similar

effect is seen with other specific configurations of carbon

chains (C1058 and C1859).

MECHANICAL CONSIDERATIONS

Turbulent haemodynamics as well as excessive shear stress

promote platelet activation and thrombogenesis60 and graft

selection needs to minimize these. There is considerable

overlap with the mechanisms responsible for IH which is re-

sponsible for graft occlusion in the medium and long term.

The factors involved can be separated into geometric factors

and mechanical characteristics. The former includes accurate

graft calibre matching61 and consideration of the angle of

take off at an end to side anastomosis. Trials of tapering

grafts62 (to reflect the native artery) have not been successful

as they ignore the relative overall increase in vessel surface

area brought about by increasing branching at the same time

as diameter reduction distally. Graft kinking (a particular

problem in long grafts where inadvertent twisting at the time

of implantation is possible,63 as well as those which cross

joints) results in gross turbulence as well as stagnant areas,

providing a highly thrombogenic environment, so this needs

to be minimized by the use of relatively stiff materials and

external reinforcing rings. However, very stiff materials lack

the viscoelasticity of arteries, promoting IH and occlusion.

The anastomosis itself is paramount–suturing itself causes

stress concentrations and poor surgical technique may leave

suture material within the graft wall as a thrombogenic nidus.

These mechanical issues are discussed more fully in our

recent review.64

ENDOTHELIALIZATION

Rather than mimicking the vEC, encouraging a confluent

endothelial lining to develop on the graft surface results in

antithrombogenicity as well as a physiological layer that

can respond to the shear forces exerted by the prevailing

hemodynamic conditions.

Spontaneous endothelialisation of a synthetic prosthesis

occurs by direct migration from the anastomotic edge, trans-

mural migration of endothelial cells and by cell transforma-

tion from endothelial progenitor stem cells (EPC). The first of

these is very limited in extent, with PTFE showing creeping

confluent endothelialisation at a rate of 0.1 mm per week from

the anastomosis with rat aorta.65 The second is responsible for

the majority of confluent endothelium forming quickly in ani-

mal models. The third is only now starting to be appreciated

as a mechanism of endothelial layer ‘housekeeping’ in native

arteries, being responsible for repair of minor damage to the

intima.66 This field of study is in early infancy–the identifica-

tion and origins of EPCs are yet to be defined. Despite some

evidence of the endotheliazation of vascular prosthesis in

some animal models this is limited in the clinical situation.

There are few large animal studies such as sheep and primate

models, compared with small animals such as rat and rabbit,

which endothelialize more readily, and so are a poor reflection

of prosthesis behavior in humans, where the major cell type

migrating into the graft are smooth muscle cells. Unlike EC

smooth muscle cells do not prevent thrombogenesis but rather

promote IH.

Endothelial cell adherence and differentiation is not

likely to occur in high flow conditions. This means that the

blood in the vessel lumen is not an efficient source of

vECs. A more likely source is newly formed capillaries as

a result of perigraft tissue infiltration. In addition, endotheliali-

sation is promoted by basic fibroblast growth factor which is

104 SARKAR ET AL.


secreted at the site of angiogenesis.67 A graft that is porous

throughout its wall will endothelialize faster and with more

confluence than a non-porous tube. PTFEs poor endothelialisa-

tion is due to its small pore size. Hazama et al. found that

standard PTFE with fibril length of 30 mm did not allow for

any transmural angiogenesis in rat aortas, whereas increased

fibril length (and hence pore size) allowed capillaries to infil-

trate the wall. The amount of cellular infiltration which corre-

lated with thrombus free area was greatest for an intermediate

pore size (fibril length 60 mm). Even longer fibrils lead to a

reduction of transmural infiltration. In humans there is little

evidence of any endothelialisation of PTFE.68 Knitted Dacron

is more porous than PTFE and forms a patchy endothelial

layer after implantation in humans.69

Animal models show spontaneous confluent endotheliali-

sation much more readily than humans70 despite human

vECs demonstrating good migration potential.71 Apart from

porosity studies, efforts have been made to enhance this in

animals in the hope that it will translate to a greater endo-

thelial coverage in human implanted grafts. Also, aware-

ness of the difficulty of spontaneous endothelialisation has

promoted the concept of endothelial cell seeding of grafts.

Successful seeding requires a high uptake of vECs and has

required a time consuming three stage procedure. However,

a viable single stage seeding technique may be possible if

a graft with a high capacity to firmly attach endothelial

cells is developed. The various surface modifications under-

taken to promote spontaneous and seeded endothelialization

are summarized in Table II.

CONCLUSION

The currently available synthetic graft materials give poor

results as small calibre cardiovascular bypass conduits due to

unfavorable interactions between the luminal surface and

blood components, causing a high rate of thrombosis. In pro-

ducing the new generation of blood compatible grafts, surface

properties must be carefully considered. Either a material with

integral low thrombogenic characteristics or surface modifica-

TABLE II. Summary of Surface Modification to Enhance Endothelialization (in Chronological Order)

Graft Modified Additive Study Type Outcome Reference

PTFE P15 peptide in vitro :endothelialisation; ;IH 72

PTFE Anti-CD34 antibodies animal model Rapid endothelialisation; :IH 73

PTFE Vascular Endothelial, GF animal model :endothelialisation; :IH 74

Poly(ether) urethane

(Tecothane)

Cholesterol in vitro :endothelialisation and

resistance to shear stress;

:endothelial precursor cell adherence

75

PTFE Poly(amino acid) urethane animal model :endothelialisation 76

Fibrin Endothelial cell GF in vitro :vEC proliferation; ;early platelet

adhesion; ;late thrombus; :patency77

Polyurethaneurea YIGSRa in vitro :endothelialisation;:transmural cell migration;

:hydroxyproline productionb

78

PTFE Tumorigenic human

squamous cell line

animal model Confluent endothelialisation

by 5 weeks

79

Dacron, PTFE, polypropylene,

silicone, polyurethane

Titanium in vitro :vEC adhesion; no increase

in inflammation

80

Dacron Collagen film in vitro Confluent endothelialisation 81

Dacron Collagen animal model Largely endothelialised and

thrombus free at 3 weeks

82

Collagen Heparin in vitro :basic fibroblast growth

factor (bFGF) binding and release

83

Polyurethane bFGF/Heparin animal model :endothelialisation, neovascularisation 84

PTFE Nitrogen, oxygen

(:hydrophilicity)in vitro :endothelialisation,

:plasma protein adsorption

85

PTFE RGD cell adhesion peptide in vitro :immediate vEC adhesion;

no long term advantage compared

with fibronectin coating

86

Dacron Carbon in vitro vEC proliferation 87

Polyurethanes and silicones Extracellular matrix;

fibronectin;gluteraldehyde

preserved matrix (GPM)

in vitro GPM provides optimal

vEC proliferation

88

PTFE Fibronectin animal model :endothelialisation(both spontaneous and seeded)

89

a Endothelial cell adhesive peptide sequence.bMark of collagen synthesis.

Keys: GF, growth factor.



tion and composite coated grafts need to be considered. These

modifications take the form of enhancing physical properties

by addressing surface charge and hydrophilicity or by incor-

porating the biochemical mechanisms employed by arteries.

This latter can be approached by assembling individual anti-

thrombogenic constructs or by encouraging confluent endo-

thelialisation.

However, unlike many animal models, spontaneous endo-

thelialisation of long graft segments does not readily occur

in humans. Therefore further surface modification to form a

microenvironment favorable to endothelialisation has been

considered, including microporosity, extracellular matrix

deposition, and specific factors which promote endothelial

cell adherence and proliferation. Most recently, the peptide

sequences involved in the latter have been isolated and

used as surface modifiers.

It is hoped that enhancing endothelialisation in animals

will result in a viable endothelializing synthetic graft for

humans. A concerted move away from the use of small ani-

mals as models of vascularization and towards higher spe-

cies will better represent potential graft behavior before

clinical trials. Tissue engineering advances the possibility

of in-vitro endothelialisation of grafts before implantation–

this technology will also benefit greatly from surface modi-

fication to facilitate rapid viable endothelialisation.

FUTURE DIRECTIONS

The use of vECs from other sources has become an impor-

tant field of study in the search for a better vascular pros-

thesis. Mechanisms for this have included the harvest of

cells from augmental fat and the use of endothelial progen-

itor cells from circulating blood.90 Studies using mesenchy-

mal stem cells from the blood have proved useful in canine

models and could provide a useful source of cells for clini-

cal applications. The study of tissue engineered vascular

grafts has shown many advantages over synthetic grafts but

cell sources remain uncertain.

It is this group’s belief, however, that the potential of seg-

mented polyurethanes has not been fully realized. In particu-

lar, manipulation of the hard segment to optimize hydrogen

bonding and van der Vaal interactions with the adjacent soft

segment could result in improved mechanical properties and

hemocompatibility. To this end, we have demonstrated a

novel nanocomposite polyurethane with antithrombogenic

properties by incorporating polyhedral oligomeric silses-

quioxane into the hard segment.91

REFERENCES

1. Tiwari A, Cheng KS, Salacinski HJ, Hamilton G, SeifalianAM. Improving the patency of vascular bypass grafts: Therole of suture materials and surgical techniques on reducinganastomotic compliance mismatch. Eur J Vasc Endovasc Surg2003;25:287–295.

2. White RA, Klein SR, Shors EC. Preservation of compliancein a small diameter microporous, silicone rubber vascularprosthesis. J Cardiovasc Surg 1987;28:485–490.

3. Kannan RY, Salacinski HJ, Butler PE, Hamilton G, SeifalianAM. Current status of prosthetic bypass grafts: A review.J Biomed Mater Res B: Appl Biomater 2005;74:570–581.

4. Kidane AG, Salacinski HJ, Tiwari A, Bruckdorfer KR, Seifa-lian AM. Anticoagulant and antiplatelet agents: Their clinicaland device application(s) together with usages to engineer sur-faces. Biomacromolecules 2003;5:798–813.

5. Francoise F-L. Microfibrils from the arterial subendothelium.Int Rev Cytol 1999;188:1–40.

6. Forstermann U, Mugge A, Alheid U, Bode SM, Frolich JC.Endothelium-derived relaxing factor (EDRF): A defencemechanism against platelet aggregation and vasospasm inhu-man coronary arteries. Eur Heart J 1989;10:36–43.

7. Fungaloi P, Waterman P, Nigri G, Statius-van Eps R, SluiterW, van Urk H, LaMuraglia G. Photochemically modulatedendothelial cell thrombogenicity via the thrombomodulin-tis-sue factor pathway. Photochem Photobiol 2003;78:475–480.

8. Galvez A, Gomez-Ortiz G, Diaz-Ricart M, Escolar G, Gonzalez-Sarmiento R, Zurbano MJ, Ordinas A, Castillo R. Desmopressin(DDAVP) enhances platelet adhesion to the extracellular matrixof cultured human endothelial cells through increased expressionof tissue factor. Thromb Haemost 1997;77:975–980.

9. Dardik R, Varon D, Tamarin I, Zivelin A, Salomon O, Shenk-man B, Savion N. Homocysteine and oxidized low densitylipoprotein enhanced platelet adhesion to endothelial cellsunder flow conditions: Distinct mechanisms of thrombogenicmodulation. Thromb Haemost 2000;83:338–344.

10. Banfi C, Camera M, Giandomenico G, Toschi V, Arpaia M,Mussoni L, Tremoli E, Colli S. Vascular thrombogenicityinduced by progressive low density lipoprotein oxidation: Pro-tection by antioxidants. Thromb Haemost 2003;89:544–553.

11. Kannan RY, Salacinski HJ, Vara DS, Odlyha M, SeifalianAM. Principles and applications of surface analytical techni-ques at the vascular interface. J Biomater Appl 2006;21:5–32.

12. Guidon R, Marois Y, Deng X, Chakfe N, Marois M, Roy R,King MW, Douville Y. Can collagen impregnated polyesterarterial prostheses be recommended as small diameter bloodconduits? ASAIO J 1996;42:974–983.

13. Lin W-C, Tseng C-H, Yang M-C. In-vitro hemocompatibilityevaluation of a thermoplastic polyurethane membrane withsurface-immobilised water-soluble chitosan and heparin. Mac-romol Biosci 2005;5:1013–1021.

14. Iwai Y. Development of a thermal cross-linking hepariniza-tion method and its application to small caliber vascular pros-theses. ASAIO J 1996;42:M693–M697.

15. Mohamed MS, Mukherjee M, Kakkar VV. Thrombogenicityof heparin and non-heparin bound arterial prostheses: An invitro evaluation. J R Coll Surg Edinb 1998;43:155–157.

16. Bergqvist D, Jensen N, Persson N. Heparinization of polytet-rafluoroethylene (ePTFE) grafts. The effect on pseudointimalhyperplasia. Int Angiol 1988;7:65–70.

17. Luong-Van F, Grondahl L, Chua KN, Leong KW, Nurcombe V,Cool SM. Controlled release of heparin from poly(epsilon-capro-lactone) electrospun fibers. Biomaterials 2006;27:2042–2050.

18. Walluscheck KP, Bierkandt S, Brandt M, Cremer J. Infrain-guinal ePTFE vascular graft with bioactive surface heparinbonding. First clinical results. J Cardiovasc Surg (Torino)2005;46:425–430.

19. Knetsch MLW, Aldenhoff YBJ, Schraven M, Koole LH. Humanendothelial cell attachment and proliferation on a novel vasculargraft prototype. J BiomedMater Res A 2004; 71:615–624.

20. Aldenhoff YBJ, Knetsch MLW, Hanssen JH, Lindhout T,Wielders SJ, Koole LH. Colis and tubes releasing heparin.Studies on a new vascular graft prototype. Biomaterials 2004;25:3125–3133.

106 SARKAR ET AL.


21. Lin PH, Chen C, Bush RL, Yao Q, Lumsden AB, Hanson SR.Small caliber heparin coated ePTFE grafts reduce plateletdeposition and neointimalhyperplasia in a baboon model. JVasc Surg 2004;39:1322–1328.

22. Laredo J, Xue L, Husak VA, Ellinger J, Singh G, ZamoraPO, Greisler HP. Silyl-heparin bonding improves the patencyand in vivo thromboresistance of carbon coated polytetrafluoro-ethylene vascular grafts. J Vasc Surg 2004;39:1059–1065.

23. Lin PH, Bush RL, Yao Q, Lumsden AB, Chen C. Evaluationof platelet deposition and neointimal hyperplasia of heparincoated small caliber ePTFE grafts in a canine femoral arterybypass model. J Surg Res 2004;118:45–52.

24. Keuren JF, Wielders SJ, Driessen A, Verhoeven M, Hendriks M,Lindhout T. Covalently bound heparin makes collagen throm-boresistant. Arterioscler Thromb Vasc Biol 2004;24: 613–617.

25. Devine C, McCollum C, NorthWest Femoro-Popliteal Trial Par-ticipants. Heparin-bonded Dacron or polytetrafluoroethylene forfemoropopliteal bypass: Five-year results of a prospective ran-domized multicentre clinical trial. J Vasc Surg 2004; 40:924–931.

26. Iakovou I, Schmidt T, Bonizzoni E, Ge L, Sangiorgi GM,Stankovic G, Airoldi F, Chieffo A, Montorfano M, CarlinoM, Michev I, Corvaja N, Briguori C, Gerckens U, Grube E,Colombo A. Incidence, predictors and outcome of thrombosisafter successful implantation of drug-eluting stents. JAMA2005;293:2126–2130.

27. Maekawa K, Kawamoto K, Fuke S, Yoshioka R, Saito H, Sato T,Hioka T. Severe endothelial dysfunction after sirolimus-elutingstent implantation. Circulation 2006;113:e850–e851.

28. Lee KH, Han JK, Byun Y, Moon HT, Yoon CJ, Kim SJ, Choi BI.Heparin-coated angiographic catheters: An in vivo comparisonof three coating methods with different heparin release profiles.Cardiovasc Intervent Radiol 2004;27:507–511.

29. Aldenhoff YBJ, van der Veen FH, ter Woorst J, Habets J, Poole-Warren LA, Koole LH. Performance of a polyurethane vascularprosthesis carrying a dipyradamole (Persantin) coating on itslumenal surface. J BiomedMater Res 2001;54:224–233.

30. van der Lei B, Bartels HL, Robinson PH, Bakker WW.Reduced thrombogenicity of vascular prostheses by coatingwith ADP-ase. Int Angiol 1992;11:268–271.

31. Nylander S, Mattsson C, Lindahl TL. Characterisation of spe-cies differences in the platelet ADP and thrombin response.Thromb Res 2005;117:543–549.

32. Costa AF, Gamermann PW, Picon PX, Mosmann MP, KettlunAM, Valenzuela MA, Sarkis JJ, Battastini AM, Picon PD. In-travenous apyrase administration reduces arterial thrombosisin a rabbit model of endothelial denudation in vivo. BloodCoagul Fibrinolysis 2004;15: 545–551.

33. Rubin BG, Toursarkissian B, Petrinec D, Yang LY, EisenbergPR, Abendschein DR. Preincubation of Dacron grafts withrecombinant tissue factor pathway inhibitor decreases theirthrombogenicity in vivo. J Vasc Surg 1996;24:865–870.

34. Sun LB, Utoh J, Moriyama S, Tagami H, Okamoto K, KitamuraN. Pretreatment of a Dacron graft with tissue factor pathway in-hibitor decreases thrombogenicity and neointimal thickness: Apreliminary animal study. ASAIO J 2001;47:325–328.

35. Kowalewski R, Zimnoch L, Wojtukiewicz MZ, Glowinski S,Glowinski J. Coagulation activators and inhibitors in the neo-intima of polyester vascular grafts. Blood Coagul Fibrinolysis2003;14:433–439.

36. Mureebe L, Turnquist SE, Silver D. Inhibition of intimalhyperplasia by direct thrombin inhibitors in an animal veinbypass model. Ann Vasc Surg 2004;18:147–150.

37. Wyers MC, Phaneuf MD, Rzucidlo EM, Contreras MA,LoGerfo FW, Quist WC. In vivo assessment of a novel dacronsurface with a covalently bound recombinant hirudin. Cardio-vasc Pathol 1999;8:153–159.

38. Williams RH, Nollert MU. Platelet derived NO slows thrombusgrowth on a collagen type III surface. Thromb J 2004;2:11.

39. Fleser PS, Nuthakki VK, Malinzak LE, Callahan RE, Sey-mour ML, Reynolds MM, Merz SI, Meyerhoff ME, BendickPJ, Zelenock GB, Shanley CJ. Nitric oxide-releasing bio-polymers inhibit thromus formation in a sheep model of arte-riovenous bridge grafts. J Vasc Surg 2004;40:803–811.

40. Gudmundsdottir IJ, McRobbie SJ, Robinson SD, Newby DE,Megson IL. Sildenafil potentiates nitric oxide mediated inhibi-tion of human platelet aggregation. Biochem Biophys ResCommun 2005;337:382–385.

41. Ozuyaman B, Godecke A, Kusters S, Kirchhoff E, E ScharfR, Schrader J. Endothelial nitric oxide synthase plays a minorrole in inhibition of arterial thrombus formation. Thromb Hae-most 2005;93:1161–1167.

42. Sawyer PN, Pate JW, Weldon CH. Relations of abnormal andinjury electropotential differences to intravascular thrombosis.Am J Physiol 1953;175:108–116.

43. Jucker BA, Harms H, Zehnder AJ. Adhesion of the positivelycharged bacterium Stenotrophomonas (xanthomonas) malti-philia 70401 to glass and teflon. J Bacteriol 1996;178:5472–5479.

44. Kallmes DF, McGraw JK, Evans AJ, Mathis JM, HergenrotherRW, Jensen ME, Cloft HJ, Lopes MB, Dion JE. Thrombogenic-ity of hydrophilic and nonhydrophilic microcatheters and guid-ing wires. Am J Neuroradiol 1997;18:1243–1251.

45. Lin J-C, Cooper SL. Surface characterization and ex vivoblood compatibility study of plasma-modified small diametertubing: Effect of sulphur dioxide and hexamethyldisiloxaneplasmas. Biomaterials 1995;16:1017–1023.

46. van Kampen CL, Gibbons DF. Effect of implant surfacechemistry upon arterial thrombosis. J Biomed Mater Res1979;13:517–541.

47. Julio CA, de-Queiroz AA, Higa OZ, Marques EF, MaizatoMJ. Blood compatibility of tubular polymeric materials stud-ied by biological surface interactions. Braz J Med Biol Res1994;27:2565–2568.

48. Karrer L, Duwe J, Zisch AH, Khabiri E, Cikirikcioglu M,Napoli A, Goessi A, Schaffner T, Hess OM, Carrel T, Kalan-gos A, Hubbell JA, Walpoth BH. PPS-PEG surface coating toreduce thrombogenicity of small diameter ePTFE vasculargrafts. Int J Artif Organs 2005;28:993–1002.

49. Bergstrom K, Osterberg E, Hoffman AS, Schuman TP, Koz-lowski A, Harris JM. Effects of branching and molecularweight of surface-bound poly(ethylene oxide) on proteinrejection. J Biomater Sci Polym Ed 1994;6:123–132.

50. Roach P, Farrar D, Perry CC. Interpretation of protein adsorp-tion: Surface-induced conformational changes. J Am ChemSoc 2005;127:8168–8173.

51. Freij-Larson C, Nylander T, Jannasch P, Wesslen B. Adsorptionbehaviour of amphiphilic polymers at hydrophilic surfaces:Effects on protein adsorption. Biomaterials 1996;17:2199–2207.

52. Rubens FD, Mesana T. The inflammatory response to cardio-pulmonary bypass: A therapeutic overview. Perfusion 2004;19:S5–S12.

53. Ahmed F, Alexandridis P, Shankaran H, Neelamegham S.The ability of poloxamers to inhibit platelet aggregation de-pends on their physicochemical properties. Thromb Haemost2001;86:1532–1539.

54. Grainger DW, Noriji C, Okano T, Kim SW. In vitro and exvivo platelet interactions with hydrophilic-hydrophobic poly(ethylene oxide)-polystyrene multiblock copolymers. J BiomedMater Res 1989;23:979–1005.

55. Gu YJ, Boonstra PW, Rijnsburger AA, Haan J, van OeverenW. Cardiopulmonary bypass circuit treated with surface-modi-fying additives: A clinical evaluation of blood compatibility.Ann Thorac Surg 1998;65:1342–1347.

56. Tsai CC, Huo HH, Kulkarni P, Eberhart RC. Biocompatiblecoatings with high albumin affinity. ASAIO J 1990;36:M307–M310.



57. Frautschi JR, Munro MS, Lloyd DR, Eberhart RC. Alkyl deriv-atized cellulose acetate membranes with enhanced albumin af-finity. Trans Am Soc Artif Intern Organs 1983;29:242–244.

58. Frautschi JR, Eberhart RC, Hubbell JA. Alkylated cellulosicmembranes with enhanced albumin affinity: Influence of com-peting proteins. J Biomater Sci Polym Ed 1995;7:563–575.

59. Tsai CC, Frautschi JR, Eberhart RC. Enhanced albumin affin-ity of silicone rubber. ASAIO J 1988;34:559–563.

60. Karino T, Goldsmith HL. Role of blood cell-wall interactionsin thrombogenesis and atherogenesis: A microrheological study.Biorheology 1984;21:587–601.

61. Longest PW, Kleinstreuer C. Particle-hemodynamics model-ing of the distal end-to-side femoral bypass: Effects of graftcaliber and graft-end cut. Med Eng Phys 2003;25:843–858.

62. Van Tricht I, De Wachter D, Tordoir J, Verdonck P. Hemody-namics in a compliant hydraulic in vitro model of straightversus tapered PTFE arteriovenous graft. J Surg Res 2004;116:297–304.

63. Dobrin PB, Hodgett D, Canfield T, Mrvicka R. Mechanical deter-minants of graft kinking. Ann Vasc Surg 2001;15:343–349.

64. Sarkar S, Salacinski HJ, Hamilton G, Seifalian AM. The me-chanical properties of infrainguinal vascular bypass grafts:Their role in influencing patency. Eur J Vasc Endovasc Surg2006;31:627–636.

65. Zhang Z, Wang Z, Liu S, Kodama M. Pore size, tissue ingrowthand endothelialization of small diameter microporous polyur-ethane vascular prostheses. Biomaterials 2004;25:177–187.

66. Roberts N, Jahangiri M, Xu Q. Progenitor cells in vasculardisease. J Cell Mol Med 2005;9:583–591.

67. Tomizawa Y, Noishiki Y, Okoshi T, Nishida H, Endo M,Koyanagi H. Endogenous basic fibroblast growth factor forendothelialization due to angiogenesis in fabric vascular pros-theses. ASAIO J 1996;42:M698–M702.

68. Guidon R, Chakfe N, Maurel S, How T, Batt M, Marois M.Expanded polytetrafluoroethylene arterial prostheses in humans:A histopathological study of 298 surgically excised grafts. Bio-materials 1993;14:678–693.

69. Shi Q, Wu MH-D, Sauvage LR. Clinical and experimentaldemonstration of complete healing of porous Dacron patchgrafts used for closure of the arteriotomy after carotid endar-terectomy. Ann Vasc Surg 1999;13:313–317.

70. Rashid ST, Salacinski HJ, Hamilton G, Seifalian AM. Theuse of animal models in developing the discipline of cardio-vascular tissue engineering: A review. Biomaterials 2004;25:1627–1637.

71. Dixit P, Hern-Anderson D, Ranieri J, Schmidt CE. Vasculargraft endothelialization: Comparative analysis of canine andhuman endothelial cell migration on natural biomaterials.J Biomed Mater Res 2001;56:545–555.

72. Li C, Hill A, Imran M. In vitro and in vivo studies of ePTFEvascular grafts treated with P15 peptide. J Biomater Sci PolymEd 2005;16:875–891.

73. Rotmans JI, Heyligers JM, Verhagen HJ, Velema E, Nagte-gaal MM, de Kleijn DP, de Groot FG, Stroes ES, PasterkampG. In vivo cell seeding with anti-CD34 antibodies success-fully accelerates endothelialization but stimulates intimalhyperplasia in porcine arteriovenous expanded polytetrafluoro-ethylene grafts. Circulation 2005;112:12–18.

74. Randone B, Cavallaro G, Polistena A, Cucina A, Coluccia P,Graziano P, Cavallaro A. Dual role of VEGF in pretreated exper-imental ePTFE arterial grafts. J Surg Res 2005;127:70–79.

75. Stachelek SJ, Alferiev I, Choi H, Kronsteiner A, Uttayarat P,Gooch KJ, Composto RJ, Chen I-W, Hebbel RP, Levy RJ. Cho-lesterol-derived polyurethane: Characterization and endothelialcell adhesion. J BiomedMater Res Pt A 2005;72A:200–212.

76. Wang C, Zhang Q, Uchida S, Kodama M. A new vascular prosthe-sis coated with polyamino-acid urethane copolymer (PAU) toenhance endothelialization. J Biomed Mater Res 2002;62:315–322.

77. Santhosh Kumar TR, Krishnan LK. Endothelial cell growthfactor (ECGF) enmeshed with fibrin matrix enhances prolifer-ation of EC in vitro. Biomaterials 2001;22:2769–2776.

78. Jun H-W, West JL. Endothelialization of microporous YIGSR/PEG-modified polyurethaneurea. Tissue Eng 2005;11: 1133–1140.

79. Kidd KR, Patula VB, Williams SK. Accelerated endotheliali-zation of interpositional 1mm vascular grafts. J Surg Res2003;113:234–242.

80. Lehle K, Buttstaedt J, Birnbaum DE. Expression of adhesionmolecules and cytokines in vitro by endothelial cells seededon various polymer surfaces coated with titaniumcarboxoni-tride. J Biomed Mater Res A 2003;65:393–401.

81. Pollara P, Alessandri G, Bonardelli S, Simonini A, CabibboE, Portolani N, Tiberio GA, Giulini SM, Turano A. Completein vitro prosthesis endothelialization induced by artificialextracellular matrix. J Invest Surg 1999;12:81–88.

82. Noishiki Y, Ma XH, Yamane Y, Satoh S, Okoshi T, Takaha-shi K, Iwai Y, Kosuge T, Ichikawa Y, Yamazaki I, Mo M.Succinylated collagen crosslinked by thermal treatment forcoating vascular prostheses. Artif Organs 1998;22:672–680.

83. Wissink MJB, Beernink R, Poot AA, Engbers GHM, Beugel-ing T, van Aken WG, Feijen J. Improved endothelializationof vascular grafts by local release of growth factors from hep-arinized collagen matrices. J Control Rel 2000;64:103–114.

84. Doi K, Matsuda T. Enhanced vascularization in a microporouspolyurethane graft impregnated with basic fibroblast growthfactro and heparin. J Biomed Mater Res 1997;34:361–370.

85. Dekker A, Reitsma K, Beugeling T, Bantjes A, Feijen J, vanAken WG. Adhesion of endothelial cells and adsorption of se-rum proteins on gas plasma-treated polytetrafluoroethylene.Biomaterials 1991;12:130–138.

86. Mazzucotelli JP, Moczar M, Zede L, Bambang LS, LoisanceD. Human vascular endothelial cells on expanded PTFE pre-coated with an engineered protein adhesive factor. Int J ArtifOrgans 1994;17:112–117.

87. Bernex F, Mazzucotelli JP, Roudiere JL, Benhaiem-Sigaux N,Leandri J, LoisanceD. In vitro endothelialization of carbon-coatedDacron vascular grafts. Int J Artif Organs 1992;15:172–180.

88. Zilla P, Fasol R, Grimm M, Fischlein T, Eberl T, Preiss P, Kru-picka O, von Oppell U, Deutsch M. Growth properties of cul-tured human endothelial cells on differently coated artificialheart materials. J Thorac Cardiovasc Surg 1991;101:671–680.

89. Seeger JM, Klingman N. Improved in vivo endothelializationof prosthetic grafts by surface modification with fibronectin.J Vasc Surg 1988;8:476–482.

90. Alobaid N, Salacinski HJ, Sales KM, Ramesh B, Kannan RY,Hamilton G, Seifalian AM. Nanocomposite containing bioac-tive peptides promote endothelialisation by circulating progen-itor cells: An in vitro evaluation. Eur J Vasc Endovasc Surg2006;32:76–83.

91. Kannan RY, Salacinski HJ, De Groot J, Clatworthy I, BozecL, Horton M, Butler PE, Seifalian AM. The antithrombogenicpotential of a polyhedral oligomeric silsesquioxane (POSS)nanocomposite. Biomacromolecules 2006;7:215–223.

108 SARKAR ET AL.


Addressing thrombogenicity in vascular graft construction

Documents

Transcript of Addressing thrombogenicity in vascular graft construction