Enhanced blood compatibility of polymers grafted by sulfonated PEO via a negative cilia concept

Biomaterials 24 (2003) 2213–2223

Enhanced blood compatibility of polymers grafted by sulfonatedPEO via a negative cilia concept

Young Ha Kima,*, Dong Keun Hana, Ki Dong Parkb, Soo Hyun Kima

aBiomaterials Research Center, Korea Institute of Science and Technology, Seoul 130-650, Republic of KoreabDepartment of Molecular Science and Technology, University of Ajou, Suwon 442-749, Republic of Korea

Received 29 October 2002; accepted 19 January 2003

Dedicated to Dr. Un Young Kim, on the occasion of his retirement from KIST

Abstract

In our laboratory sulfonated PEO (PEO-SO3) was designed as a ‘‘negative cilia model’’ to investigate a synergistic effect of PEO

and negatively charged SO3 groups. PEO-SO3 itself exhibited a heparin-like anticoagulant activity of 14% of free heparin.

Polyurethane grafted with PEO-SO3 (PU-PEO-SO3) increased the albumin adsorption to a great extent but suppressed other

proteins, while PU-PEO decreased the adsorption of all the proteins. The platelet adhesion was decreased on PU-PEO but least on

PU-PEO-SO3 to demonstrate an additional effect of SO3 groups. The enhanced blood compatibility of PU-PEO-SO3 in the ex vivo

rabbit and in vivo canine implanting tests was confirmed. Furthermore, PU-PEO-SO3 exhibited an improved biostability and

suppressed calcification in addition to the enhanced antithrombogenicity. The in vivo antithrombogenicity and biostability were

improved in the order of PUoPU-PEOoPU-PEO-SO3. The calcium amounts deposited was decreased in the order of PU>PU-

PEO>PU-PEO-SO3 in spite of the possible attraction between negative SO3 groups and positive calcium ions. The bioprosthetic

tissue (BT) was grafted with H2N-PEO-SO3 via glutaraldehyde (GA) residues after conventional GA fixation. BT-PEO-SO3 also

displayed the decreased calcification by in vivo animal models. The application of PEO-SO3 was extended by designing amphiphilic

copolymers containing PEO-SO3 moiety and hydrophobic long alkyl groups as anchors. The superior effect of PEO-SO3 groups on

thromboresistance compared to PEO was confirmed also in the case of copolymers coated or blended with other polymers and the

systems coupled by UV irradiation, photoreaction or gold/sulfur or silane coupling technology, and therefore it might be very useful

for the medical devices.

r 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Sulfonated PEO grafting; Thromboresistance; Biostability; Calcification; Modified tissue

1. Introduction

Polymeric materials have contributed significantly tothe development and improvement of devices andsystems in medicine. Nevertheless, there are still threemajor complications impeding widespread applicationto blood contacting devices; thrombosis, calcification,and infection. Surface-induced thrombus formation is aserious problem in surgical therapy and application ofartificial organs. This is a result of the fact that therelationship between surface properties and surface-induced thrombosis has not been thoroughly evaluated.In order to develop better blood compatible materials, a

vast number of researches have been directed towardthree aspects: endothelial cell lining and recently tissueengineering, the utilization of biologically active mole-cules such as heparin, and chemical modification [1].Bioengineering approaches such as endothelial cellseeding or tissue engineering may solve the problemassociated with thrombosis ultimately but in the future[2]. Immobilized or slow released systems incorporatingheparin [3] or other biological components have broughtonly several practical devices useful in the clinic. Therehave been a number of contributions to improve theblood compatibility of materials by chemical ap-proaches, and the current hypotheses were explainedin the literature [4,5], but the results are still notconclusive [6]. Many hydrogels or hydrophilized sur-faces exhibit good blood compatibility, but most of

*Corresponding author.

E-mail address: [email protected] (Y.H. Kim).

0142-9612/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0142-9612(03)00023-1

these might be not truly antithrombogenic but only anti-thromboadhesive [6] as they interact with platelets andconsume them. The grafting of poly(ethylene oxide)(PEO) was reported to be more blood compatible due toan additional chain motion (molecular cilia) effect [7].However, very hydrophobic (low surface energy) inertsurfaces such as silicones and fluoropolymers are alsoknown to be hemocompatible. Hydrophilic/hydropho-bic microdomain structures such as polyurethane (PU)were also proven to be more blood compatible. All themodified surfaces above have more or less evidences thatare sometimes controversial, especially with respect tohydrophilicity or hydrophobicity. It was also reportedthat a negatively charged surface was more bloodcompatible than a positive one [8]. Especially manypolymers containing sulfonate groups showed more orless enhanced blood compatibility. Recently, a biomem-brane-like surface containing phospholipid groupsshowed excellent blood compatibility [9,10].

Among the above hypotheses, surface modificationusing PEO has shown to decrease protein adsorptionand platelet or cell adhesion on biomaterials contactedwith blood or tissue. The role of PEO was explained byits unique properties such as the excluded volume on thesurface and the flexible hydrophilic chain motion toexpel proteins/cells [7,11–23]. The theoretical analysiswas summarized in detail in the literature [22]. Methodsfor the surface modification by PEO included a simplephysical adsorption [16], a self-assembled monolayer(SAM) [15,20], or a chemical bond formation, such aschemical coupling [17,18] or graft polymerization [7].Whitesides et al. demonstrated that SAM composed ofoligo-PEO decreased protein adsorption [15,20]. Allthese studies demonstrated the more or less decreasedadsorption of proteins and adhesion of platelets, andtherefore improved blood compatibility.

Meanwhile, it was also postulated that positivelycharged surfaces are generally thrombogenic, whileuniformly negatively charged surfaces tend to beantithrombogenic [8]. It is now accepted that the uniqueanticoagulant activity of heparin, a naturally occurringpolysaccharide, results from its negatively chargedsulfonate and aminosulfate groups. A number ofpolymers containing sulfonate groups, so called hepar-in-like materials (heparinoid), were studied to supportthe concept. Sulfonated polystyrenes [24] or othersexhibited an improved blood compatibility. In addition,PU was sulfonated at the site of diamine chain extenderor urethane hydrogens, and these anionic PUs werereported to show a much improved thromboresistancewith a decreased platelet adhesion and an enhancedfibrinogen adsorption [25–27].

Accordingly, it is highly probable that PEO contain-ing sulfonate end groups, sulfonated PEO (PEO-SO3),may exhibit an anticoagulant activity such as heparin.Furthermore, the surfaces grafted with PEO-SO3 may

enhance their blood compatibility significantly by meansof a synergistic effect of the dynamic mobility of PEOchains and the negatively charged sulfonate groups. Thismodel, called as a ‘‘negative cilia model’’ shown inFig. 1, was intensively investigated in our lab for the lastyears and the results are summarized here. First of all,PEO-SO3 was grafted to PU to study the effect onthromboresistance and protein adsorption. In addition,copolymers containing PEO-SO3 segments have beenstudied.

On the other hand, calcification is another seriousproblem that may occur when biomaterials are appliedin devices over a long period. Calcification, defined asthe deposition of calcium compounds, occurs with awide spectrum of medical devices [28]. The leading causeof failure of artificial heart valves is attributed to thecalcification. The actual mechanism of calcification isnot completely understood. It is related to hostmetabolism and to implant chemistry and structure.Therefore, the effect of polymers or tissues grafted withPEO-SO3 on calcification was also investigated.

2. Results and discussions

2.1. Blood compatibility of sulfonated PEO (PEO-SO3)

and PU grafted with PEO-SO3 (PU-PEO-SO3)

2.1.1. Preparation and surface characteristics of PEO-

SO3 and PU-PEO-SO3

Polyurethanes (PUs) are widely applied to medicaldevices due to the excellent mechanical property andgood biocompatibility. The classical PUs of medicalgrade are composed of polyether-based polyols (usuallyPTMG, poly(tetramethylene oxide)) and MDI (methy-lene bis(p-phenyl isocyanate)) and extended by adiamine or diol. However, it suffers from deteriorationdue to biodegradation and calcification when implantedin the body for a long time [29].

The modification scheme of PU is illustrated in Fig. 2.PU (Pellethane) has urethane and urea –CO–NH–groups which can be reacted with diisocyanates, and

PlateletCell

FlexiblePEO chain

Surface

SO3-

SO3- SO3

-

protein

Fig. 1. Negative cilia model.

Y.H. Kim et al. / Biomaterials 24 (2003) 2213–22232214

one free isocyanate group was utilized for furthercoupling with PEO derivatives (path a). The �OH or�NH2 end groups of corresponding PEO derivativeswere converted into sulfonate groups by the reactionwith propane sultone (PST). Or, the only one end groupof PEO was first reacted with propane sultone and thenthe other end group with diisocyanates to prepare OCN-PEO-SO3 which was coupled to PU (path b). Thevarious molecular weight (MW) of PEO was applied tostudy its effect. The detailed procedure and the surfacecharacteristics have been reported elsewhere [30,31]. Thereaction could be carried out on surfaces in the non-solvent media [30] or by a bulk one in the solution state[31,38].

All the reactions shown in Fig. 2 were confirmed byATR-IR and NMR. The enrichment of oxygen afterPEO grafting or the appearance of sulfur peak afterintroducing SO3 groups was observed at ESCA analysis.The contact angles were changed along the reactions;the receding angle of PU measured by a dynamicWilhelmy plate method was 59� but decreased to 20� forPU-PEO200 and 14� for PU-PEO2000, and presented acomplete wetting behavior after the introduction ofionic SO3 groups [30]. Therefore, it was proved that thehydrophilicity of PU surfaces was increased in a greatextent after coupling PEO, especially PEO-SO3. Inaddition, the surface smoothness was also enhancedafter grafting of PEO, especially PEO-SO3 as observedby SEM [30].

2.1.2. Anticoagulant activity of sulfonated

PEO (PEO-SO3)

As explained briefly above, a number of studies onheparin-like polymers containing sulfonate or sulfategroups have been carried out. However, their resultinganticoagulant activities were reported more or less (1–16% of heparin) depending on the chemical nature. Weprepared PEO-SO3 and PU-PEO-SO3 by the bulksolution reactions as shown in Fig. 2, and their antic-oagulant activities were evaluated by the measurementof activated partial thromboplastin time (APTT),thrombin time (TT), reptilase (a thrombin-like snake

venom enzyme) time (RT) and Factor Xa assay, andcompared with those of free heparin [31].

Heparin binds to specific lysine residues in ATIII(antithrombin III) and cause a conformational changeof ATIII, thereby the complex formed readily reactswith thrombin to inhibit the formation of fibrin networkwhich is an important step for the thrombosis. Table 1lists the anticoagulant activities of various polymers,which was determined by APTT at various concentra-tions. It was very interesting to find that PEO-SO3 itselfdisplayed 14% of anticoagulant activity as compared tofree heparin, whereas the original PEO showed nosignificant bioactivity in the blood clotting system. Inaddition, PU-PEO-SO3 exhibited also a small extent ofactivity, about 2% of heparin, maybe due to therelatively low SO3 concentration and decreased chainmobility of PEO-SO3 grafted to high MW PU. Thebioactivity of PEO-SO3 and PU-PEO-SO3 estimated byTT reached about 80% of APTT, while Factor Xa datadid not show any significant increase in the prolongationof clotting time. This suggests that the bioactivity ofPEO-SO3 and PU-PEO-SO3 might be resulted by aninteraction with mainly thrombin rather than withFactor Xa. PEO-SO3 inhibited thrombin up to 66%and PU-PEO-SO3 up to 13%, as evaluated by extendedTT at various thrombin concentrations. Furthermore,PEO-SO3 and PU-PEO-SO3 exhibited prolonged TT butnormal RT as the concentrations of SO3 groups wasincreased. This result implies that the system containssome heparin-like activity because reptilase is notinhibited by heparin. Moreover, TT was extended withincreasing concentrations of SO3 in the presence ofATIII rather than in the absence of ATIII, which issimilar to the case of heparin and ATIII. As aconclusion, PEO-SO3 exhibited a heparin-like antic-oagulant activity of 14% of free heparin, as expected, aswell as PU-PEO-SO3 in a small extent [31].

2.1.3. In vitro platelet interactions with PU-PEO and

PU-PEO-SO3

Fig. 3 shows the effect of modified PU surfaces onplatelet adhesion. It was confirmed here that PEO-grafted PUs (PU-PEO) displayed less platelet adhesionthan the untreated PU, and the adhesion was decreased

PU PU-NCO + HO-PEO-OH PU-PEOPST

PU-PEO-SO3

or H2 N-PEO-NH 2

(a)

H2N-PEO-NH2 O3S-PEO-NH2 O3S-PEO-NCOHDI

PU-NH2PST

PU-SO3

(b)

OSO2

PST

Fig. 2. Modification scheme of PU to couple PEO or sulfonated PEO:

HDI=hexamethylenediisocyanate, PST=propane sultone.

Table 1

The anticoagulant activity of PEO-SO3 and PU-PEO-SO3 evaluated by

APTT

Polymer SO3 concentration Anticoagulant activity

(mol/g) (unit/mg) (% of heparin)

Heparin 3.05� 10�3 173 100

PEO1000 — o0.2 o0.1

PEO-SO3 3.86� 10�4 24.2 14

PU-PEO-SO3 1.33� 10�4 3.5 2

Y.H. Kim et al. / Biomaterials 24 (2003) 2213–2223 2215

with increasing PEO MW from 200 to 2000. Inparticular, sulfonated PEO-grafted PU-PEO-SO3 ex-hibited a much lower degree of platelet adhesioncompared to PU and even PU-PEO. In addition, theshape change of platelets adhered was least on PU-PEO-SO3 and most on untreated PU [32]. The decrease ofplatelet adhesion on the surfaces grafted with variousPEO derivatives are now well accepted by a number ofinvestigators and explained by the excluded volumeeffect and the dynamic mobility of PEO chains grafted.Also, the in vitro behavior of platelets on sulfonatedpolymers has been studied in the literatures. PUcontaining just SO3 groups [25–27] or poly (hydro-xyethyl methacrylate)-sulfoalkyl(meth)acrylates [33]showed less platelet adhesion. Such depressed interac-tion of the sulfonated polymers with platelets wasexplained by electrical repulsions between SO3 groupsand negatively charged proteins as well as platelets. Inour study on PU-PEO-SO3 surfaces, both the hydro-philic PEO and the negatively charged SO3 groupsseemed to be affected in a synergistic way to exhibit theleast adhesion and shape change of platelets.

The depressed interaction of platelets with PU-PEO-SO3 was investigated again in the study by measuringcytoplasmic free calcium levels in platelets, whichindicate a degree of interaction with platelets, afterbeing contacted with modified surfaces [34]. Thecytoplasmic free calcium level of PU-PEO1000-SO3

remained relatively constant and low, in contrast withthe significant increase observed for PU-PEO1000-OH,PU-PEO1000-NH2, and control PU, as shown in Fig. 4.The effect of the PEO-SO3 grafting and especially theunique platelet-suppressive property of SO3 end groupscompared to OH or NH2 ones [34] was confirmed.

2.1.4. Protein adsorption of modified PUs

Proteins are adsorbed on the materials instantly whencontacted with foreign materials and deformed to reactfurther with blood or tissue components, and thereforeprotein adsorption might regulate all the subsequent

body–material interactions. The protein adsorption topolymer surfaces is proceeded by ionic, hydrophilic or/and hydrophobic interactions and thus influenced byvarious factors, including surface compositions, wett-ability, surface charges, and roughness. Among anumber of proteins in blood, fibrinogen is high surfaceactive and thus plays an important role in thrombusformation. In general, fibrinogen and gamma globulinhave been shown to adsorb more strongly on thesurfaces that are believed to be thrombogenic, whilealbumin tends to adsorb to more antithrombogenicsurfaces. The hydrophilic surfaces or hydrogels andPEO-grafted surfaces have been reported to suppress theprotein adsorption as well as platelet adhesion in anumber of studies, while the relatively hydrophobicsurfaces enhance the interactions. Several modifiedpolymers exhibited the unique selective adsorptionamong various proteins. Sulfonated PU [26,27] showedmore fibrinogen adsorption and less platelet adhesionthan untreated PU, but they enhanced blood compat-ibility. C-18 alkyl coupled PU showed an increase inalbumin adsorption due to a specific affinity [35].

The PU pellets were surface modified along thescheme in Fig. 2, and incubated in each proteinsolutions radiolabeled. The proteins adsorbed undervarious protein concentrations and periods were mea-sured by a scintillation counter [36]. The adsorptionresults after 5min were presented in Fig. 5 where 3 kindsof protein adsorbed on each PU were compared on thesame scale. On the untreated PU (Fig. 5a), albumin (A)was adsorbed most and followed by fibrinogen (F) andgamma globulin (G). On the PU-PEO all the proteinadsorptions decreased substantially to confirm thedepression effect of PEO on protein adsorption. In thecase of PU-SO3, directly sulfonated one without PEOunit, only fibrinogen adsorption was enhanced whileother proteins did not, indicating a specific affinity ofsulfonate groups to fibrinogen as reported in theliteratures. However, it was really interesting to findthat only albumin adsorption was increased in a great

0

1

2

3

4

5

6

7

8

9

10

PU PU-PEO-NH2 PU-PEO-OH PU-PEO-SO3

Cyt

opla

smic

[C

a2+ ]

10 min Incubation20 min Incubation

Fig. 4. Cytoplasmic free calcium levels in platelets contacted with

modified PUs.

Pla

tele

t ad

hesi

on (

% o

f P

RP

)

PU PU-PEO1000PU-PEO200

PU-PEO

PU-PEO-SO3

PU-PEO2000

40

30

20

10

0

Fig. 3. The platelet adhesion on modified PUs; the effect of PEO or

sulfonated PEO grafted. (mean7SD, n ¼ 3).


extent on PU-PEO-SO3, while the amount of fibrinogenand gamma globulin was suppressed as for PU-PEO[36]. It is very difficult to explain the specific affinity ofPU-PEO-SO3 to albumin as well as that of sulfonategroups to fibrinogen. The total amount of proteinsadsorbed on each modified PU was in the order of PU-SO3>PUXPU-PEO-SO3>PU-PEO. The antithrombo-genicity evaluated by in vitro platelet adhesion in thepart 1.3. and ex vivo or in vivo animal model (see thenext parts) was in the order of PU-PEO-SO3bPU-PEO>PU-SO3XPU. These results indicate that notonly the amount of proteins adsorbed but also the kindof proteins and/or the extent of deformation of proteinsadsorbed is important for the improved antithrombo-genicity. That PU-PEO-SO3 enhanced albumin absorp-tion but depressed other proteins may improve theantithrombogenicity of the system in a great extent inaddition to the less adhesion of platelets on it.

2.1.5. Ex vivo and in vivo blood compatibility of PU-

PEO-SO3

Finally, the blood compatibility of PU-PEO-SO3 wasevaluated by several ex vivo and in vivo animal models.

A rabbit arterio–arterial (A–A) shunt test is a simpleand rapid method to evaluate the blood compatibility ofmaterials [37]. The luminal surfaces of PU tubing (innerdiameter was 2mm) was modified, and it was connectedto the carotid arteries under the conditions of low bloodflow. The extension of the occlusion time measuredindicates the improved non-thrombogenicity. The occlu-sion time was just 50min for PU control, 90min for PU-SO3, prolonged to 140min for PU-PEO2000 butenormously extended to 360min for PU-PEO-SO3

demonstrating the excellent non-thrombogenicity ofPU-PEO-SO3 [32]. These occlusion time results revealthe contribution of each SO3 or PEO concept onantithrombogenicity and agree with the in vitro plateletadhesion test.

In addition, the bulk-modified PU-PEO-SO3 wasapplied to a polymeric heart valve implanted by acanine RV–PA (right ventricle-pulmonary artery) shuntmodel (Fig. 6b). A new type of a sink-hole valve(Fig. 6a) was composed of a sink-hole like strut and 2PU leaflet membranes attached along the axis, and PUuntreated and PU-PEO-SO3 was coated on each side tocompare directly [38]. After 24 days implantation, on

0 2 4 6 8 10

0.00

0.07

0.14

0.21

0.28

AlbFibIgG

PU

Plasma concentration (% normal)

Am

ount

ads

orbe

d (u

g/cm

2 )

A

F

G

0 2 4 6 8 10

0.00

0.05

0.10

0.15

0.20

0.25

PU-PEO


Am

ount

ads

orbe

d (u

g/cm

2 )

A

F

G

0 2 4 6 8 10

0.00

0.05

0.10

0.15

0.20

0.25


Am

ount

ads

orbe

d (u

g/cm

2 )

A

F

G

0 2 4 6 8 10

0.00

0.07

0.14

0.21

0.28

PU-PEO-SO3


Am

ount

ads

orbe

d (u

g/cm

2 ) A

F

G

PU-SO3

(a) (b)

(c) (d)

Fig. 5. The protein adsorption on modified PUs.


the untreated PU leaflet it was observed some thrombusand micro-cracks formed (Fig. 7a), while it was rela-tively clean on PU-PEO-SO3 leaflet (Fig. 7b). Inaddition, a thick layer of protein adsorbed was formedon PU (Fig. 7c), while that on PU-PEO-SO3 was thin(Fig. 7d). Therefore, the excellent blood compatibility ofPU-PEO-SO3 was clearly demonstrated by showingfewer thrombus formation and protein absorption onthe surface than the untreated PU did. The micro-cracksobserved on PU should be resulted from the degrada-tion during the implantation. However, there were no

cracks on PU-PEO-SO3. This reveals that PU-PEO-SO3

exhibited also an improved biostability in addition tothe enhanced antithrombogenicity compared to PU.Furthermore, the PU-PEO-SO3 exhibited the lessamount of calcium deposited in the specimens comparedto PU. The amount of Ca deposited was about 250 mg/cm2 for PU untreated, however decreased to 100 mg/cm2

for PU-PEO-SO3. This result was very interesting whenwe think of the fact that the SO3 groups havenegative groups capable of attracting more positivecalcium ions.

SVC

IVC

RA

RV

PA

valve

PU PU-PEO-SO3

(b)(a)

Fig. 6. The canine RV-PA model (b) implanting a new Sinkhole heart valve (a).

Protein layer [Ca]=~250 ug/cm2

[Ca]=~100 ug/cm2

(a) (c)

(b) (d)

Fig. 7. The explanted PUs after 24 days implantation in the canine RV-PA model; SEM morphology of PU (a) and PU-PEO-SO3 (b), and TEM

pictures of PU (c) and PU-PEO-SO3 (d).


2.1.6. Enhanced in vivo biostability and suppressed

calcification of PU-PEO-SO3

The above results of PU-PEO-SO3 on biostability andcalcification encouraged us to conduct another implantstudy using a rat subdermal model. The surface-modified PUs were implanted subcutaneously for upto 6 months [39]. A SEM study demonstrated thatextensive surface cracks were revealed on the PU surfaceafter 2 months and these cracks were considerablycovered all over the surface. However, PU-PEO-SO3

exhibited very few cracks up to 4 months. The degree ofsurface cracking on explanted PUs was decreased in theorder of PU>PU-PEO>PU-PEO-SO3. The calciumcontents deposited, regardless of implantation time,were also increased in the same order. The deposition ofCa was found abundantly, but that of phosphorous washardly in existence in all implanted PUs, suggesting thatthe calcium compound is not a hydroxyapatite [39]. Itwas reported that PU is degraded in the body by ahydrolysis and enzymatic interactions [29]. Severalhypotheses have been suggested for the mechanism forcrack formation and degradation of PUs. It wasreported that PU surface crack is produced by adherentleukocytes, a localized chemical degradation and re-sidual stress [29], and neutrophil played an importantrole. It was also observed that the soft polyether-basedpolyol segment is susceptible to chemical degradation orbiodegradation by enzymes and metabolic products(hydride ions or peroxides) [29,40]. Unfortunately, thephagocytic cells, such as macrophages or foreign bodygiant cells, were not found on our implanted PUspecimens after several months, as these cells act in theinitial step in inflammatory reactions. Nevertheless, theenhanced biostability of PU-PEO and especially PU-PEO-SO3 can be attributed to that the PEO or PEO-SO3

segments introduced on the surfaces might havedecreased the interactions with those phagocytic cellsor enzymes to retard the biodegradation. In addition,the SO3 groups contributed an additional effect on thesecell material interactions.

Table 2 presented the calcium contents deposited inthe specimens during the implantation. The values wereincreased gradually on time, regardless of the kind ofspecimens. However, there was a clear difference todemonstrate the effect of PEO and especially PEO-SO3.The Ca amount was decreased in the same order asbiostability to indicate a close relationship each other.Nevertheless, this study is the first example to demon-strate the difference in calcification on the differentlysurface-modified polymers.

2.2. Calcification resistance of bioprosthetic tissues (BT)

grafted by PEO-SO3

Calcification is associated with actually all the softimplants such as bioprosthetic heart valves, polymeric

blood pumps and heart valves, contact lens, etc. Theleading cause of failure of artificial heart valves isattributed to the calcification. It is known that theimplanted medical devices lead to the most dystrophiccalcification, where the tissues are necrotic or otherwisealtered in normocalcemic subjects. The calcificationmechanism of bioprosthetic heart valves is discussed indetail in the literature [28]. The glutaraldehyde (GA)treatment of tissues for the crosslinking would be themajor cause by deteriorating the tissue structure, whilefresh tissues do not calcify. It was often reported that theGA residue unreacted might induce the calcification. Inaddition, glycoaminoglycan components in tissues areextracted during the GA treatment to provide the sitesfor the initial nucleation of calcium compounds. Bloodcomponents or lipids deposited would have a contribu-tion too. There have been a number of approaches toprevent the calcification, and aminooleic acid after-treatment or heparin coupling, etc. was the representa-tive one.

In our study, PEO-SO3 derivative ended with anamino group was coupled to the tissue after theconventional GA fixation (actually crosslinking) [41–43]. BT like porcine aortic valve leaflets or bovinepericardium was treated with GA solution at 4�C for 1week then incubated in NH2-PEO-SO3 solution for 2days. Coupling of NH2-PEO-SO3 occurs throughresidual GA groups via Schiff base formation to yieldBT-PEO-SO3. BT-PEO-SO3 was compared with the BTcontrol which was prepared by exposing it to similarconditions but in the absence of PEO-SO3. BT-PEO-SO3

had the greater resistance to collagenase digestion thanBT control did. The calcification of the modified tissueswas investigated by in vivo rat subdermal, and twocanine circulatory implantation models such as aorta-illiac (A-I) shunt and RV-PA shunt. As summarized inTable 3, BT-PEO-SO3 exhibited the decreased calciumamounts compared to the control in all in vivo animalexperiments. Such a decreased calcification might beexplained by several effects as followings; the amino endgroups of NH2-PEO-SO3 were coupled to the residualGA groups to remove the possible contribution of GAresidues for the calcification. PEO-SO3 segments wouldhave filled the space in the collagen matrix which wasreported as nucleating sites for calcification. The

Table 2

The calcium contents (mg/cm2) in the modified PU implanted

subdermally in rat, mean7SD (n ¼ 5–7)

Polymer 2 months 4 months 6 months

PU 79724 154743 221743

PU-PEO1000 6677 120727 162735

PU-PEO1000-SO3 57717n 71724n 93724n

nSignificance level using an unpaired Student’s t-test when compar-

ing modified PUs to PU (po0.05).


enhanced blood compatibility of PEO-SO3 mightdecrease the adhesion of blood or cellular components.This noble method can be a useful anticalcificationtreatment for implantable tissue valves and pericardiumtissue patch.

2.3. The mechanism of enhanced blood compatibility of

PEO-SO3 grafted system (negative cilia concept)

The results of our study on PU grafted by PEO orPEO-SO3 can be summarized as followings;

* The surface hydrophilicity was increased by PEOgrafting, especially displayed a complete wettingbehavior by PEO-SO3 grafting.

* The surface smoothness was improved.* PEO-SO3 exhibited a heparin-like anticoagulant

activity (14% of free heparin) as well as PU-PEO-SO3 (2% of free heparin).

* Platelet adhesion was decreased by PEO grafting andfurther by PEO-SO3 grafting. The higher MW ofPEO exhibited the larger effect than low MW did.

* The surface grafted with PEO suppressed the totalprotein absorption. PEO-SO3 grafted PU exhibitedthe specific affinity for albumin, while SO3 groupsthemselves showed the affinity for fibrinogen.

* The biostability was improved by PEO grafting andfurther by PEO-SO3 grafting.

* The calcification was decreased by PEO grafting andfurther by PEO-SO3 grafting.

Therefore, the synergistic effect of the hydrophilic anddynamic PEO chain and the negatively charged SO3 endgroups, ‘‘negative cilia model’’, would be very useful forthe enhanced blood compatibility. In this model, thepossible interactions are as follows: the hydrated flexiblePEO chain motion suppresses protein adsorption andplatelet adhesion, where the perpendicular orientationof PEO chains would be increased by the electricrepulsion between SO3 end groups with each other. In

addition, the negatively charged SO3 end groups of PEOchains expel proteins/platelets (except albumin) bearingalso negative charge further by an electric repulsion.PEO-SO3 chain grafted has a specific affinity to albuminwhich is antithrombogenic. Moreover, the SO3 groupsexhibited a heparin-like anticoagulant activity contri-buting for better blood compatibility. Through suchinteractions, the antithrombogenicity of PU-PEO-SO3

was enhanced to a great extent.It was very often reported that for the improvement of

blood compatibility by PEO grafting it is essential toprovide a uniform and complete coverage of PEO-grafted surface. Furthermore, the repulsive force by thegrafted PEO layers should be large enough to preventadhesion of platelet aggregates formed rather than ofindividual platelet [23]. The SO3 groups in our studymight have provided the additional repulsive forces. Themore quantitative investigation on the amount andfrequency of PEO-SO3 introduced is on going.

The additional effect of SO3 end groups was demon-strated also for improving the biostability and suppres-sing the calcification of PU. Maybe the highlyhydrophilic nature of the PEO-SO3 surface provides arapid and firm hydration and the negatively chargedsurfaces might decrease the adsorption and activation ofphagocytic cells. In addition, the surface has the mostsmoothness which can suppress the adsorption ofphagocytic cells. The calcification mechanism of poly-meric implants should be different from the case ofmodified tissue as it is not associated with GAtreatment. Therefore, for the polymeric implants thebiodegradation or the deposition of proteins or lipids onthe surfaces might play a major role for the calcification.In our study on modified PUs, PU-PEO-SO3 systemdisplayed the least calcification by an additional effect ofSO3 groups in spite of the possible attraction betweennegative SO3 groups and positive calcium ions. Inseveral reports, the complexation of calcium ions withPEO chain segments was hypothesized for the calcifica-tion of PU as in the case of PEO-polybutyleneter-ephthalate containing PEO main chain [44]. However,our study on PEO-grafted system indicates that thecalcification may not proceed by a simple ionic orcomplexing interaction but should be associated withcell material interaction involved by various phagocyticcells.

2.4. Blood compatibility of copolymers or coupled system

containing PEO-SO3

The enhanced blood compatibility of PU-PEO-SO3

shown above might be influenced by a microdomainstructure of PU, one of the concepts for improvingblood compatibility. Therefore, it was extended to othersystems than PU. Copolymers or other coupling

Table 3

The calcium contents (mg/cm2) in the modified tissues implanted in

animals mean7SD (n ¼ 4–5)

Rat subdermal,

3 week

Canine A-I

shunt, 6 week

Canine RV-PA

shunt, 8 week

Porcine valve

(PV) control

15.575.0 16.875.9 20.073.0

PV-PEO-SO3 1.970.1 3.570.3 3.370.2

yyyyyyyyyyyyyyyyyyyyyyyyyy

Bovine

pericardium

(BP) control

116.571.1

BP-PEO-SO3 17.970.1

BP fresh 0.4570.04


methods have been investigated to study the effect ofPEO-SO3 introduced.

2.4.1. (Meth) acrylates copolymers containing PEO or

PEO-SO3 side chain

Amphiphilic copolymers of hydrophobic octadecylacrylate and hydrophilic PEO-acrtylates were designedin our lab. These polymers can be applied to coating oradditives to modify the surfaces of biomaterials. Whenthese polymeric surfaces have a contact with body fluid,the hydrophilic PEO- or PEO-SO3 moiety will orient tothe surfaces to express the activity, while the hydro-phobic octadecyl unit will play as an anchor not to bewashed away. When such polymers of low MW added inthe other polymers, the added one would migrate to thesurfaces to reveal the same effect. Random copolymersof octadecyl acrylate (OA) and PEO(MW 1000–4000)-acrylate (PEOA) or sulfonated PEO-acrylate (PEO-SO3A) were prepared with various PEO or PEO-SO3

composition, and their blood compatibility was inves-tigated [45,46]. Fig. 8 shows the platelet adhesion of PUsurfaces coated with the copolymer P(PEOA/OA) (a) orP(PEO-SO3A/OA) (b), respectively, on the same scale,where the P(PEO-SO3A/OA) coating decreased theadhesion in the larger extent than the substrate andeven P(PEOA-OA) coating did [46].

2.4.2. Sulfonated PEO-PPO-PEO derivatives as a

surface modifying additives

Amphiphilic block copolymers of hydrophobic PPOand hydrophilic PEO (PEO-PPO-PEO) were added toPU to modify the surface property, and the effect wascompared in the presence of SO3 end groups. Thecopolymer composed of short MW PEO (below 880)was not effective at all to decrease the platelet adhesion,while disulfonated one suppressed the platelet adhesionsubstantially. The copolymer containing longer MWPEO was effective to decrease the platelet adhesionregardless of the kind of end groups [47].

2.4.3. Coupling of PEO-SO3 by UV irradiation or ozone

treatment

In addition to the usual chemical modificationdescribed above, coupling of PEO-SO3 by UV irradia-tion or ozone treatment was studied too. A PEO-SO3

derivative ended by an azide group was prepared andcoupled on PU by a UV irradiation [48]. Then, FactorXa bioactivity and platelet adhesion using a epifluoscentvideo microscopy were conducted to compare with PUuntreated or PU/polyethylenimine(PEI)/heparin. FactorXa bioactivity of PU/PEI/heparin and PU-PEO-SO3

was 0.21 and 0.011 IU/cm2, respectively. PU-PEO-SO3

and PU/PEI/heparin decreased the platelet adhesion inthe same extent compared to PU to confirm the validityof PEO-SO3 coupling [48]. In addition, PEO-SO3 wascoupled directly on PU [48], PE [49] or silicone [49]

utilizing radicals evolved after an ozone treatment. Thecoupling yield seemed to be smaller than the aboveazide-UV coupling method [48]. Nevertheless, thesuperior effect of PEO-SO3 groups to decrease theplatelet adhesion compared to PEO itself was confirmedagain [49].

Very recently, PEO-SO3 was coupled on the metallicsurfaces by utilizing the technology including a goldlayer deposition, chemisorption of sulfur compoundscontaining functional groups, and subsequently cou-pling PEO-SO3 [50]. The modified metallic surfacesexhibited the decreased platelet adhesion or proteinadsorption as well as vascular smooth muscle cellinteractions.

Therefore, the superior effect of PEO-SO3 groups onthromboresistance compared to PEO was confirmedalso in the case of copolymers or other systemscontaining the PEO-SO3 moiety. PEO-SO3 can beintroduced not only by chemical reactions but also bygrafting via UV irradiation, photoreaction or gold/sulfur or silane coupling technology, which might bevery useful applications as coatings or additives for themedical devices.

PU control

poly (APEG-OA) = 1:9



80

60

40

20

0

80

60

40

20

0

0 20 40 60 80 100 120

Incubation time (min)

0 20 40 60 80 100 120

Incubation time (min)

PU control

poly (APEG-SO3-OA) = 1:9



Plat

elet

adh

esio

n (%

)Pl

atel

et a

dhes

ion

(%)

(a)

(b)

Fig. 8. Platelet adhesion on PU films coated with poly(APEG-OA)

(a) or poly(APEG-SO3-OA) and (b) copolymers.


Acknowledgements

The authors appreciate the scientists involved in thesestudies for their great contributions. We thank Dr. UnYoung Kim who retired from KIST and Prof. SungWan Kim, University of Utah for their encouragements.This work was supported by Korea MOST NationalLaboratory Projects.

References

[1] Kim YH, Park KD, Han DK. Blood compatible polymers. In:

Salamone JC, editor. Polymeric materials encyclopedia, vol. 1.

Boca Raton: CRC Press, 1996. p. 825–35.

[2] Langer R, Vacanti JP. Tissue engineering. Science 1993;260:

920–6.

[3] Olsson P, Sanchez J, Mollnes TE, Riesenfeld J. On the blood

compatibility of end-point immobilized heparin. J Biomater Sci

Polym Ed 2000;11:1261–73.

[4] Andrade JD, Nagaoka S, Cooper SL, Okano T, Kim SW.

Surfaces and blood compatibility-Current hypotheses. ASAIO J

1987;10:75–84.

[5] Ishihara K. Blood compatible polymers. In: Tsuruta T, Hayashi

T, Kataoka K, Ishihara K, Kimura Y, editors. Biomedical

applications of polymeric materials. Boca Raton: CRC Press,

1993. p. 89–117.

[6] Ratner BD. Blood compatibility—a perspective. J Biomater Sci

Polym Ed 2000;11:1107–19.

[7] Mori Y, Nagaoka S, Takiuchi H, Kikuchi T, Noguchi N,

Tanzawa H, Noishiki Y. A new antithrombogenic material

with long poly(ethylene oxide) chains. ASAIO J 1982;28:

459–63.

[8] Srinivasan S, Sawyer PN. Correlation of the surface charge

characteristics of polymer with their antithrombogenic character-

istics. In: Rembaum A, Shen M, editors. Biomedical polymers.

New York: Marcel Dekker, 1971. p. 51–66.

[9] Ishihara K, Aragaki R, Ueda T, Watanabe A, Nakabayashi N.

Reduced thrombogenicity of polymers having phospholipid polar

groups. J Biomed Mater Res 1990;24:1069–77.

[10] Ishihara K, Fujita H, Yoneyama T, Iwasaki Y. Antithrombogenic

polymer alloy composed of 2-methacryloxyethyl phosphorylcho-

line polymer and segmented PU. J Biomater Sci Polym Ed

2000;11:1183–95.

[11] Merill EW, Salzman EW. Poly(ethylene oxide) as a biomaterial.

ASAIO J 1983;6:60–5.

[12] Jeon SI, Lee JH, Andrade JD, De Gennes PG. Protein surface

interactions in the presence of PEO I. Simplified theory. J Colloid

Interface Sci 1991;142:149–58.

[13] Jeon SI, Andrade JD. Protein surface interactions in the presence

of PEO 2. Effect of protein size. J Colloid Interface Sci

1991;142:159–66.

[14] Gombotz WR, Guanghui W, Horbett TA, Hoffman AS. Protein

adsorption to PEO surfaces. J Biomed Mater Res 1991;25:

1547–62.

[15] Prime KL, Whitesides GM. Adsorption of proteins onto surfaces

containing end-attached oligo(ethylene oxide)—a model system

using self-assembled monolayers. J Am Chem Soc 1993;

115:10714–21.

[16] Claesson P. PEO surface coatings: relation between intermole-

cular forces, layer structure and protein repellency. Colloids Surf

A 1993;77:109–18.

[17] Lee JH, Lee HB, Andrade JD. Blood compatibility of

poly(ethylene oxide) surfaces. Prog Polym Sci 1995;20:1043–79.

[18] McPherson TB, Shim HS, Park K. Grafting of PEO to glass,

Nitinol, and pyrolytic carbon surfaces by gamma-irradiation.

J Biomed Mater Res Appl Biomater 1997;38:289–302.

[19] Szleifer I. Protein adsorption on surfaces with grafted polymers: a

theoretical approach. Biophys J 1997;72:595–612.

[20] Harder P, Grunze M, Dahint R, Whitesides GM, Laibinis PE.

Molecular conformation in oligo(ethylene glycol)-terminated self-

assembled monolayers on gold and silver surfaces determines their

ability to resist protein adsorption. J Phys Chem B 1998;102:

426–36.

[21] Halperin A. Polymer brushes that resist adsorption of model

proteins: design parameters. Langmuir 1999;15:2525–33.

[22] Archambault JG. Protein adsorption to PEO-grafted surfaces.

Ph.D. thesis, McMaster University, Canada, 2002.

[23] Park K, Shim HS, Dewanjee MK, Eigler NL. In vitro and in vivo

studies PEO-grafted blood contacting cardiovascular protheses.

J Biomater Sci Polym Ed 2000;11:1121–34.

[24] Jozefonvicz J, Mauzac M, Aubert N, Jozefowicz M. Antithrom-

bogenic activity of polysaccharide resins. In: Williams DF, editor.

Biocompatibility of tissue analogs, vol. 2. Boca Raton: CRC

Press, 1985. p. 133–52.

[25] Lelah MD, Pierce JA, Lambrecht LK, Cooper SL. Polyether-

urethane ionomers: surface property/ex vivo blood compatibility

relationships. J Colloid Interface Sci 1985;104:422–39.

[26] Grasel TG, Cooper SL. Properties and biological interaction of

polyurethane anionomers: effect of sulfonate incorporation.

J Biomed Mater Res 1989;23:311–38.

[27] Santerre JP, Van der Kamp NH, Brash JL. Effect of sulfonation

of segmented polyurethanes on the transient adsorption of

fibrinogen from plasma: possible correlation with anticoagulant

behavior. J Biomed Mater Res 1992;26:39–57.

[28] Schoen FJ, Levy RJ. Tissue heart valves: current challenges

and future research perspectives. J Biomed Mater Res

1999;47:439–65.

[29] Zhao Q, Agger MP, Fitzpatrick M, Anderson JM, Hiltner A,

Strokes K, Urbanski P. Cellular interaction with biomaterials: in

vivo cracking of pre-stressed pellethane 2363-80A. J Biomed

Mater Res 1990;24:621–37.

[30] Han DK, Jeong SY, Ahn K-D, Kim YH, Min BG. Preparation

and surface properties of PEO-sulfonate grafted polyurethanes

for enhanced blood compatibility. J Biomater Sci Polym Ed

1993;4:579–89.

[31] Han DK, Lee NY, Park KD, Jeong SY, Kim YH, Cho HI,

Min BG. Heparin-like anticoagulant activity of sulfonated

PEO and PEO-SO3 grafted polyurethane. Biomaterials

1995;16:467–71.

[32] Han DK, Jeong SY, Kim YH, Min BG, Cho HI. Negative cilia

concept for thromboresistance: synergistic effect of PEO and

sulfonate groups grafted onto PU. J Biomed Mater Res

1991;25:561–75.

[33] Chen WY, Xu BZ, Feng XD. Synthesis of polysulfohexylmetha-

crylate with anticoagulant activity. J Polym Sci: Polym Chem Edn

1982;20:547–54.

[34] Park KD, Suzuki K, Lee WK, Lee JE, Kim YH, Sakurai Y,

Okano T. Platelet adhesion and activation on PEG modified PU

surfaces: measurement of cytoplasmic calcium. ASAIO J

1996;42:876–7.

[35] Munro M, Quattrone AJ, Ellsworth SR, Kulkarni P, Eberhart

RC. Alkyl substituted polymers with enhanced albumin affinity.

ASAIO J 1981;27:499–503.

[36] Han DK, Ryu GH, Park KD, Kim UY, Min BG, Kim YH.

Plasma protein adsorption to sulfonated PEO-grafted PU surface.

J Biomed Mater Res 1996;30:23–30.

[37] Nojiri C, Okano T, Grainger D, Park KD, Nakahama S, Suzuki

K, Kim SW. Evaluation of nonthrombogenic polymers in a new

rabbit A–A shunt model. ASAIO J 1987;33:596–601.


[38] Han DK, Lee KB, Park KD, Kim CS, Jeong SY, Kim YH, Kim

HM, Min BG. In vivo canine studies of sinkhole valve and

vascular graft coated with biocompatible PU-PEO-SO3. ASAIO J

1993;39:537–41.

[39] Han DK, Park KD, Jeong SY, Kim YH, Kim UY, Min BG. In

vivo biostability and calcification-resistance of surface-modified

PU-PEO-SO3. J Biomed Mater Res 1993;27:1063–73.

[40] Zhao Q, Topham N, Anderson JM, Hiltner A, Lonoen G, Payet

CR. Foreign-body giant cells and PU stability: in vivo correlation

of cell adhesion and surface cracking. J Biomed Mater Res

1991;25:177–83.

[41] Park KD, Yun JY, Han DK, Jeong SY, Kim YH, Choi KS, Kim

HM, Kim HJ, Song SS. Chemical modification of implantable

biological tissue for anticalcification. ASAIO J 1994;40:377–82.

[42] Park KD, Lee WK, Yun JY, Han DK, Kim SH, Kim YH, Kim

HM, Kim KT. Novel anticalcification treatment of biological

tissues by grafting of sulphonated PEO. Biomaterials 1997;18:

47–51.

[43] Lee WK, Park KD, Kim YH, Suh H, Park JC, Lee JE, Sun K,

Baek MJ, Kim HM, Kim SH. Improved calcification resistance

and biocompatibility of tissue patch grafted with sulfonated PEO

or heparin after glutaraldehyde fixation. J Biomed Mater Res

Appl Biomater 2001;58:27–35.

[44] van Blitterswijk CA, van der Brink J, Leenders H, Hessling SC,

Bakker D. Polyactive: a bone bonding polymer effect of PEO/

PBT proportion. Trans Soc Biomater 1991;14:11.

[45] Lee HJ, Park KD, Han DK, Kim YH, Cho I. Synthesis and

characteristics of thromboresistant copolymers having PEG/

PEG-SO3. Polymer (Korea) 1997;21:1045–52.

[46] Lee HJ, Park KD, Park HD, Lee WK, Han DK, Kim SH, Kim

YH. Platelet and bacterial repellance on sulfonated PEG-acrylate

copolymer surfaces. Colloid Surf B: Biointerfaces 2000;18:355–70.

[47] Lee JH, Ju YM, Lee WK, Park KD, Kim YH. Platelet adhesion

onto segmented PU surfaces modified by PEO- and sulfonated

PEO-containing block copolymer additives. J Biomed Mater Res

1998;40:314–23.

[48] Nojiri C, Kuroda S, Saito N, Park KD, Hagiwara K, Senshu K,

Kido T, Sugiyama T, Kijima T, Kim YH, Sakai K, Akutsu T. In

vitro studies of immobilized heparin and sulfonated PU using

epifluorescent video microscopy. ASAIO J 1995;41:389–94.

[49] Ko YG, Kim YH, Park KD, Lee HJ, Lee WK, Park HD, Kim

SH, Lee GS, Ahn DJ. Immobilization of PEG onto polymer

surfaces by ozone oxidation. Biomaterials 2001;22:2115–23.

[50] Lee HJ, Hong J-K, Goo HC, Lee WK, Park KD, Kim SH, Kim

YH. Improved blood compatibility and decreased VSMC

proliferation of surface modified metal grafted with sulfonated

PEG or heparin. J Biomater Sci Polym Ed 2002;13:939–52.


Enhanced blood compatibility of polymers grafted by sulfonated PEO via a negative cilia concept

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