In vitro blood compatibility of functional group-grafted and heparin-immobilized polyurethanes prepared by plasma glow discharge

18 (1997) 1099-1107 0 1997 Elsevier Science Limited Printed in Great Britain. All rights reserved 0142-9612/97/$17.00 Biomaferials

PII

ELSEVIER

SO142-9612

(97)

00035-5

In vitro blood compatibility of functional group-grafted and heparinimmobilized polyurethanes prepared by plasma glow discharge Inn-Kp Kang”, Oh Hyeong Kwon”, Moon Kyu Kim+, Young Moo LeeS and Yong Kiel Sung § ‘Department of Polymer Science and ‘Department of Immunology, Kyungpook National nrversity, Taegu 702-701, Korea: $Department of Industrial Chemistry, Hanyang University, Seoul 133-791, Korea; ‘$’Department of Chemistry, Dongguk University, Seoul 100-715, Korea

Blood compatibilities of functional group-grafted and heparin-immobilized polyurethanes (PUS) were investigated using in vitro thrombus formation, plasma recalcification time (PRT), activated partial thromboplastin time (APTT), platelet adhesion and activation, and peripheral blood mononuclear cell (PBMC) activation. In the experiment with plasma proteins, PRT was shortened on amine groupgrafted PU (PU-NH*) but prolonged on heparin-immobilized polyurethane (PU-Hep) when compared to PU control. APTT was significantly prolonged on PU-Hep, suggesting the binding of immobilized heparin to antithrombin III. The percentage of platelet adhesion was slightly increased by the introduction of functional groups such as carboxylic acid and primary amine on PU surfaces, but significantly decreased by the immobilization of heparin on the same substrate. The percentage of serotonin released from platelets adhered on surface-modified PUS was increased with increase of platelet adhesion. In the PBMC experiment, cells adhered less on heparin-immobilized PUS than on functional group-grafted PUS, and the production levels of tumour necrosis factor mRNAs from the cells stimulated by heparin-immobilized PU (PU-N-Hep) were smaller than those by the other substrates. 0 1997 Elsevier Science Limited. All rights reserved Keywords:

Polyurethanes,

Received 4 September

immobilized

heparin,

in vitro blood compatibility

1996; accepted 8 February 1997

Segmented polyurethane (PU) has been used in a number of blood-contacting devices, such as cathetersl, heart assist pumps’ and
prepolymers with a tertiary amino alcohol. PU containing tertiary amine or ester groups in the sidechain have been also synthesized by Ito et a1.7*8. The second approach is the introduction of a spacer such as diamine and diisocyanate to the urethane linkage (-NHCOO-) of the PU backboneg. An alkyl spacer arm has been attached to medical grade polyetherurethane, which was previously hydrolysed by alkaline aqueous solution, to enhance heparin availability and activitylO. The immobilized heparin retained its ability to bind and inactivate thrombin and Factor Xa. Barbucci et al.*’ have been developing a grafting method of poly(amide-amine) onto the urethane linkage of PU surfaces using hexamethylene diisocyanate as the spacer. The third approach is the graft polymerization of functional monomers such as acrylic acid1’*13, ally1 amine and acryloylbenzotriazole (AB)14 on PU surfaces by plasma glow discharge. The graft polymerization of functional monomers on the surfaces of PUS is a very effective method to increase the immobilizing site. Functional group-grafted PUS have been prepared by the treatment of PU with oxygen plasma glow discharge, followed by graft polymerization of AB and

Correspondence to Dr Inn-Kyu Kang. 1099

Biomaterials

1997. Vol. 18 No. 16

Blood compatibility

1100 a subsequent substitution reaction of AB with sodium hydroxide and ethylenediamine. Heparin-immobilized PU has also been prepared by the coupling reaction of functional group-grafted PUS (PU-NH2, PU-COOH) with heparin14. When a foreign material is exposed to blood, plasma proteins are adsorbed onto the material surface, followed by activation of clotting factors or adhesion and activation of platelets, and finally formation of a fibrin network, i.e. thrombus formation15. It has been pointed out that platelet release reactions involving dense granules and a-granules play an important role in coagulation16. The release reaction triggered by thrombin, collagen and ADP has been investigated17. However, the release reaction can be initiated by the stimulation received from the material surface upon platelet adhesion, and the extent of release reaction may depend on the properties of the material surface and on the nature of the interaction between platelets and material surfaces18,1g. The purpose of this study is to evaluate in vitro blood compatibility of functional group-grafted and heparin-immobilized PUS synthesized previously’4. The blood compatibility of the material was investigated using thrombus formation, plasma time (PRT), activated partial recalcification thromboplastin time (APTT), platelet adhesion and activation, and peripheral blood mononuclear cell (PBMC) activation.

of polyurethane:

1-acryloyl henzotriazole (AB) *

N, N-dimethylaniline

i 4 -_ ; D ft

I.-K. Kang et al.

O--tCHq+ F

=o

PU-AB Figure 1 Schematic diagram showing the formation of acryloylbenzotriazole-grafted polyurethane surfaces (PUAB) by oxygen plasma treatment followed by graft polymerization.

PU+B

MATERIALS AND METHODS Preparation of functional group-grafted and heparin-immobilized PUS The methods for the preparation of functional groupgrafted and heparin-immobilized PUS have been described previously in detail14. Briefly, PU was synthesized from 4,4’-diphenylmethanediisocyanate (MDI), poly(tetramethylene glycol) and ethylenediamine as a chain extender in a mole ratio of 1.3:1:1. The film was prepared by the solvent cast method using lOwt% dimethylformamide solution of PU. Functional group-grafted PUS were prepared by oxygen plasma discharge treatment, followed by graft polymerization of AB and a subsequent substitution reaction of AB with sodium hydroxide and ethylenediamine. The primary amine or carboxylic acid groups grafted onto the surfaces were coupled with heparin (187.5 IUmg-‘, Sigma) using l-ethyl-3-dimethylamidopropyl carbodiimide. The schematic diagrams showing the preparation of AB-grafted and heparin-immobilized PUS are shown in Figures 1 and 2, respectively.

Thrombus formation Human whole blood (30ml) from a healthy volunteer was collected and mixed with an aqueous solution containing anhydrous D-glucose (O.l36M), sodium citrate citric acid dihydrate (0.075 M) and monohydrate (0.0004M) (ACD, 3 ml). The surfacemodified PU film (3 x 3 cm’) was attached to a watch glass (diameter 7.5 cm) and subjected to the thrombus formation test. The test was carried out according to the method reported previously”. Biomaterials 1997, Vol.

18 No. 16

I

PU-COOH

wwz PU-NH2 &*

I

I

I

,

PU-C-Hep PU-N-Hep

II /

I

Figure 2 Schematic diagram showing the formation of heparin-immobilized polyurethane urea surfaces by hydrolysis and transamidation followed by condensation reaction.

Briefly, the ACD-blood (200~1) was placed on the sample and incubated at 37°C in a constant temperature bath. The clotting reaction was started by adding 0.1 M aqueous CaCls solution (20 ~1) to the blood. The sample was shaken gently by hand to mix the chemicals and the blood homogeneously. Distilled water (5 ml) was then added to stop the reaction after an appropriate incubation time (IO40min). The thrombus formed was taken with a spatula and transferred into distilled water (5 ml) in a watch glass, left for 5 min at room temperature, and then placed in 37% aqueous formaldehyde solution (5 ml) to fix the thrombus. The thrombus

Blood compatibility

of polyurethane:

I.-K. Kang et al.

1101

was dried at redu.ted pressure until the sample weight was unchanged. The weight of the thrombus formed relative to that formed on glass was calculated13Z20.

Plasma recalcification

time (PRT)

The human whole blood containing 10% ACD was centrifuged at 30001; for 10min to separate the blood corpuscles, and the platelet-poor plasma (PPP) obtained was used for the PRT experiment?. PPP (300~1) was placed on the sample film (3 x 3 cm”) attached to a watch glass (diameter 4 cm), and incubated statically at 37°C; 0.025 M CaClz aqueous solution (300 ~1) was then added to the PPP and the plasma solution was monitored for clotting by manually dipping a stainlesssteel wire hook coated with silicone into the solution to detect fibrin threads. Clotting times were recorded at the first signs of fibrin formation on the hook. The experiment was carried out in triplicate and a mean value taken.

Activated partial tlhromboplastin time (AP’IT) The sample film (3 x 3 cm’) was attached to a glass (diameter 4cm, height 1.5 cm), and preswelled with distilled water (2 ml). Plasma solution (100~1) and actin activated cephaloplastin reagent (100~1, Baxter Diagnostics Inc., Deerfield, IL, USA) were added to the sample film, followed by addition of 0.025M CaClz solution (100~1) after 5 min incubation. The clotting time of the plasma solution was observed as described in the PRT experiment.

Bioactivity of immobilized heparin The bioactivity of immobilized heparin was measured the reported Chromogenic method as bY previously22~23. Brie-fly, PPP (38~1) was diluted with Tris buffer solution (162~1, pH 8.3) in a vial and heparin-immobilized PU (1 x 1 cm’) was subsequently dipped in the solution. The vial was incubated at 37°C for 3min and O.lml. of thrombin solution (8IUml-I, Sigma 7009, St. Louis, MO, USA) was added and incubated for an additional 90s. Then 0.1 ml of 1.13 x lop3 M Chrom ozym TH solution (Tos-Gly-ProBoehringer Mannheim Biochemica, Arg-pNA, Mannheim, Germany) was added and incubated at 37°C for 310s. The rleaction was terminated by adding 0.3 ml of 50% acetic acid. The samples were monitored spectrophotometrically at 405nm using a Shimadzu UV-2100 spectrophotometer. Heparin standards, between 0.2 and l.OIUml-l (0.1 ml), were dipped in the solution containing PPP (38~1) and Tris buffer (162 ~1). This solution was incubated at 37°C for 3min and thrombin (0.1 ml) was added and incubated for an additional 90s. Chromozym TH was then added and incubated at 37°C for 310s. The samples were subjected to the measurement of UV absorbance at 405nm and the values were used for the construction of a heparin The relative bioactivity was standard curve. calculated by comparing the thrombin neutralization of immobilized heparin with that of free heparin. The schematic diagr.am showing the measurement of the bioactivity of immobilized heparin is shown in Figure 3.

3 min

PU-N

l

37OC

in plasma

P”-N@B

+

D

90 set

370c

h

excess

PU-N@&jB

+

P”-N@D

t D

PU-N@D

Stage

I

+

Hz0

t

t

Tos-Gly-Pro-Arg-pNA (Chromozym

310 set 37OC

.

Tos-Gly-Pro-Arg-OH

Stage

D

TH)

t

1

II

405 nm

Figure 3 Schematic illustration showing the chromogenic method to measure bioactivity of immobilized heparin.

Adhesion and activation of platelets The ACD-blood was centrifuged at 180 g for 10 min to obtain a platelet-rich plasma (PRP). PRP (100~1) was placed on the surface-modified PUS (3 x 3 cm2) at 37°C for 30 and 60 min, respectively. Phosphate-buffered saline (PBS, 6ml) was then added to the PRP to stop further platelet adhesion, and left for 1 min. The number of platelets adhered to the samples was determined by measuring the lactate dehydrogenase (LDH) activity of cells lysed with Triton X-100. A linear relationship was obtained between the LDH activity of aliquots of the cell suspension thus obtained and the number of platelets, which were counted with a haemocytometer. The LDH activity was determined by measuring the initial rate of nicotinamide adenine dinucleotide hydride (NADH) oxidation in the presence of pyruvate24. The assay was carried out in a solution containing sodium phosphate (50 mM), NADH (0.06mM) and pyruvate (0.2m~) at pH 7.5 in the presence of Triton X-100 (O.OZwt%). The NADH oxidation was followed on a Shimadzu UV-2100 spectra hotometer by the decrease in absorbance at 340nm % . Serotonin released from adhering platelets was measured as follows. The surface-modified PU (diameter 1.5 cm) was attached to a 24-well plate and kept at 37°C. PRP (500~1) was then placed on the sample. After 30 or 60min incubation, 0.02 M ethylenediamine tetra-acetic acid (EDTA, 500 ~1) was added to stop further release reaction of platelets. The platelet and plasma layers were transferred to different vessels after centrifugation at 12 000 rpm. 200% Trichloroacetic acid (TCA, ZOO$) was then added to both the platelet and plasma layers to aggregate proteins. The proteins aggregated by TCA Biomaterials

1997,

Vol. 18

No. 16

1102

Blood compatibility

were removed by centrifugation, and the protein-free samples obtained were transferred to a glass tube containing 2 ml of 0-phthalaldehyde solution (10 ml of 0.5% o-phthalaldehyde ethanol + 100ml of 8N hydrochloric acid) and subsequently incubated at 100°C for 10min. The excess TCA was extracted with chloroform (2ml) and the fluorescence intensity of the aqueous layer phase at 475 nm was measured using a Shimadzu RF-5000 spectrofluorophotometer. The amount of serotonin remaining in both the platelets and plasma layers was calculated from a standard calibration curve, which was constructed by the relationship between fluorescence intensity and known concentration of serotonin creatinine sulphate.

Adhesion and activation of peripheral blood mononuclear cells (PBMCs) PBMCs from a healthy human volunteer were prepared from ACD-blood centrifugation (4OOg, 30 min) on Ficoll-Hypaque (Sigma). The cells were washed three times and then resuspended at a concentration of 2 x lo6 cellsml-l with RPMI-1640 medium (Gibco, NY, USA) supplemented with 1% bovine serum albumin. The cells were placed on the surface-modified PUS which were prewetted with RPMI-1640 in 5% CO2 at 37”C, and then the number of cells adhered on each PU was examined by the LDH methodz4 after 2 and 5 h incubation. Interleukin 1-p (IL-l/I) and tumour necrosis factor (TNF) mRNA levels were measured by reverse transcription and polymerase chain reaction (RT-PCR)25V26quantitatively. Total RNA was isolated from cultured PBMCs using RNAzol B (Cinna/Biotecx Laboratories International Inc., Friendswood, USA). Reverse transcription reaction was carried out by using a StrataScript RT-PCR kit with 8 pg of each total RNA (Stratagene, La Jolla, CA, USA). PCR reactions were performed in a total volume of 50~1 containing 0.2 pM of each primer, 2Om~ of dNTPs, 10 n&i Tris-HCI (pH 8.3), 50 mM KCI, 2 mu MgCI,, 1.25 U Taq polymerase (Perkin-Elmer Cetus), and 2 ~1 of cDNA sample. Amplification was done with 30 cycles of 94°C for 1 min, 60°C for 2min and 72°C for 1.5min, and an additional extension cycle at 72°C for 5min. The PCR products were electrophoresed in 1.5% agarose gel. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were assayed on all samples as a control to verify intactness of RNA and efficient cDNA synthesis.

0

10

of polyurethane:

20

I.-K. Kang et al.

30

40

50

Incubation time (min) Figure 4 Thrombus formation on surface-modified polyurethanes as a function of incubation time. Data are presented as mean f standard deviation of three different experiments: PU, 0; PU-AB, a; PU-COOH, A; PU-NH2, 0; PU-CHep, W; PU-N-Hep, 0.

Thrombus formed on the surfaces was gradually increased with increasing incubation time. The amount of thrombus on PU-AB was relatively larger than that on the other substrates. This result suggests that AB grafted on PU surfaces is not promising for blood compatibility. However, thrombus formation was significantly suppressed by the immobilization of heparin on the PU surfaces, as shown in Figure 4 (PUC-Hep and PU-N-Hep). The plasma recalcification time (PRT) of surfacemodified PUS is shown in Figure 5. PRT of PUNI& was shorter than that of the other substrates, as presented in Figure 5. On the other hand, heparinimmobilized PU, PU-N-Hep, showed longer PRTs compared to functional group-grafted PUS (PU-AB, PUNHs, PU-COOH). Plasma clotting factors are generally accelerated by the addition of activated partial thromboplastinz7. The activated partial thromboplastin time (APTT) and bioactivity of immobilized heparins are presented in Table 1. The APTT of PU was not prolonged by the introduction of a functional group (PU-COOH, PU-NHs) but elongated by the immobilization of heparin (PU-C-Hep, PU-N-Hep). This result suggests that antithrombin III (AT III) was bound to immobilized heparin, thus leading to a suppression of

Scanning electron microscopy (SEM) analysis Blood cells adhered on surface-modified PUS were dipped into 0.1 M aqueous sodium cacodylate solution (pH 7.2) at 37°C for 30min. The films were then dehydrated in a graded series of ethanol, and dried in a Hitachi model HCP-2 critical point drier using liquid CO2 as a transition liquid, and sputter-coated with platinum. These samples were observed with a Hitachi S-510 scanning electron microscope.

RESULTS AND DISCUSSION PU-AI

Blood coagulation Figure 4 shows the amount of thrombus formed on the surface-modified PUS as a function of incubation time. Biomaterials

1997,

Vol.

18 No. 16

P".NA* PU-COOA PU-C-By,

PU-N-Aep

Figure 5 Plasma recalcification time of PU and surfacemodified PU films. Data are presented as mean f standard deviation of three different experiments.

Blood

compatibility

Table 1 Substrates

PU PU-AB PU-COOH PU-NH2 PU-C-Hep PU-N-Hep

of polyurethane:

In vitrobioactivity

I.-K.

Kang

of surface-modified

APTT’

(s)

31 fl 2752 29fl 27 f 1 37 f 1 38 ZIZ1

et a/.

1103

PUS Amount heparin

of immobilized (pg cm-*)

1.4 f 0.08 2.0 f 0.13

Bioactivityb

(IUcm-*)

Thrombin

neutralizationC

W)

0.059 f 0.003 0.095 f 0.007

23f1.5 25% 1.9

*Activated partial thromboplastin time. bMeasured by chromogenic assay. ‘Bioactivity ratio of immobili;!ed heparin to free heparin.

thrombin activity’“. The amount of heparin immobilized on PU-N-Hep (2.Opgcm-‘) is larger than that on PU-C-Hep (1.4pg cm-‘), as reported previously14. bioactivity of PU-N-Hep The (0.095 f 0.13 IUcm-*, 25 f 1.9%) was slightly higher than that of PU-C-Hep (0.059 f 0.003, 23 f 1.5%) as determined by the chromogenic method (Table 1). Han et ~1.‘~ have investigated the modification of commercial PU by poly(ethylene oxide) (PEO) grafting and heparin immobilization. They reported that thrombin neutralization of PU-PEO-Hep was around lo-27% irrespective of the chain length of PEO as well as the concentration of immobilized heparin. Park et ~1." also reported the effect of PEO spacers on immobilized heparin bioactivity. They used FXa assay for the determination of heparin bioactivity and the results showed that the bioactivity of immobilized heparin was in the range of 0.0081-0.01061Ucm-2 (5.32-19.09%).

Activation of platelets In a previous study, platelet activation on polypeptide derivatives with different water contact investigated in the presence of angles was inhibit re-uptake of released imipramine to serotonin3’. The results showed that the amount of by the adhered platelets was almost unaffected addition of imipramine and the effect of imipramine on the amount of released serotonin was not observed on all surfaces except very hydrophobic ones. In this study, platelet experiments were carried out in the a,bsence of imipramine. Figure 6 shows the amount of platelets adhered on various modified PUS for 30 and 60min incubation. The amount of platelets adhered on functional groupgrafted PUS such as PU-AB, PU-COOH and PU-NH2 was larger than that on PU control. However, the amount of adhered platelets was significantly decreased on heparin-immobilized PUS (PU-C-Hep, PU-N-Hep). As shown in Table 2, the relative percentage of released serotonin on the surfaceincreased with increasing modified PUS was incubation time of platelets. The percentage of serotonin released from the platelets adhered on functional group-grafted PUS (PU-AB, PU-COOH and PU-NHJ after 60miri incubation was in the range of 44-49%. However, the serotonin release was significantly suppressed on heparin-immobilized PU (20-24%) compared to the other substrates. As shown from the results of Figure 6 and Table 2, serotonin release was suppressed on heparinimmobilized PUS tc which platelets adhered less.

PU

?U-AB PU-COOA

PU-NH, PU-C.Hep PU-N-Acp

Figure 6 Adhesion of platelets on surface-modified ethanes after 30 min (0) and 60min (a) incubation. presented as mean f standard deviation of three experiments.

Table 2 Release of surface-modified PUS Substrate

Glass PU PU-A6 PU-COOH PU-NH2 PU-C-Hep PU-N-Hep ‘Measured

serotonin

Serotonin

from

platelets

released’

(%)

30min

60 min

49i3 34 * 43 f 40 f 43 f 15f4 13f2

62 f 2 41 f2 49 f 3 44f2 46 f 2 24 f 2 20 Z.t2

2 4 3 2

by the o-phthalaldehyde

polyurData are different

adhered

on

method after 30 and 60 min incubation.

Cholakis and Sefton31 have studied the in vitro interactions with a heparin-poly(viny1 platelet alcohol) (PVA) hydrogel. In their results, no difference in platelet reactivity was found between an immobilized heparin containing hydrogel (heparin-PVA) and the hydrogel without heparin (PVA). We have previously reported using polypeptide derivatives with different water contact angles that serotonin release is increased with increasing platelet adhesion3’. On the other hand, Salzman et d3’ have reported that serotonin release is accelerated by a material to which platelets adhered less. Figure 7 shows SEM photographs of platelets adhered to surface-modified PUS after 1 h incubation. The morphological change of platelets on functional group-grafted PUS was slightly larger than PU and Biomaterials

1997, Vol. 18 No. 16

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Blood compatibility

Flgure 7 Scanning electron micrographs of platelets adhered PU-AB; c, PU-COOH; d, PU-NHP; e, PU-C-Hep; f, PU-N-Hep.

on PU and surface-modified

of polyurethane:

I.-K. Kang et al.

PUS after 1 h incubation:

a, PU; b,

heparin-immobilized PUS. In vitro, heparin has been reported to reduce, enhance or have no effect on platelet aggregation, depending upon the system employed33*34.

Activation of peripheral blood mononuclear (PBMCs)

”

PU

PU-AB

PU-COOE

PU-NH,

PU-C-Hep

PU-N-Hep

Figure 8 Adhesion of peripheral blood mononuclear cells on surface-modified PUS after 2 h (D) and 5 h (I) incubation. Data are presented as mean f standard deviation of three different experiments. Biomaterials

1997, Vol. 18 NO. 16

cells

Figure 8 indicates the amount of PBMCs adhered on surface-modified PUS after 2 and 5 h incubation. The amount of PBMCs adhered on functional groupgrafted PUS (PU-AB, PU-COOH, PU-NH2) was larger than that on the PU control, while PBMC adhesion was significantly suppressed on heparin-immobilized PUS (PU-C-Hep, PU-N-Hep). These adhesion patterns of PBMCs on surface-modified PUS are similar to those obtained from the platelet adhesion experiment (Figure 6). SEM photographs of adhering PBMCs on surface-modified PUS are shown in Figure 9. PBMCs

Blood compatibility

of polyurethane:

I.-K. Kang et al.

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Figure 9 Scanning electron micrographs of peripheral blood mononuclear a, PU; b, PU-AB; c, PU-COOH; d, PU-NH*; e, PU-C-Hep; f, PU-N-Hep.

were largely aggregatled on PU and functional groupPU-NH2) upon grafted PUS (PU-AB, PU-COOH, adhesion, while aggregation of the cells was suppressed on heparin-immobilized PUS (PU-C-Hep, PU-N-Hep). These results suggest that the activation of monocytes might lbe generated from their contact with foreign materials and changed depending on the physicochemical properties of the material surfaces. It is considered that the amount of cytokine can be used as a measure for the evaluation of activation of PBMCs when contacting with foreign materials. In this study, the RT-PCR technique35’36 was used to measure the cytokine mRNA level from PBMCs stimulated by surface-modified PUS. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an external control. Figure 10 illustrates the results of RT-PC;R analysis of cytokine mRNAs in PBMCs after 5 h incubation on surface-modified PUS. As shown in Figure 20, all substrates elicited the production of IL-l/?and the level of IL-l/I

cells on surface-modified

PUS after 5 h incubation:

mRNA. TNF mRNA was produced on all samples except PU-N-Hep. However, the difference of the TNF level between PU-C-Hep and PU-N-Hep is uncertain. In conclusion, the activation of plasma proteins and platelets was accelerated on functional group-grafted PUS (PU-AB, PU-COOH, PU-NHJ, but significantly reduced on heparin-immobilized PUS (PU-C-Hep, PUN-Hep), thus leading to a lower thrombus formation. In addition, PBMCs adhered less on heparin-immobilized PUS, and the production level of TNF mRNA from the cells stimulated by PU-N-Hep was smaller than that by the other substrates.

ACKNOWLEDGEMENTS This work was supported by the Korean Ministry of Education Research Fund for Advanced Materials in 1995. Biomaterials

1997, Vol. 18 No. 16

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Blood compatibility a

b

cd

ef

12.

GAPDH 13.

14.

TNF

15.

16. chain transcription-polymerase Figure 10 Reverse reaction analysis of cytokine mRNAs in peripheral blood mononuclear cells stimulated with surface-modified PUS for 5h: a, glass; b, PU; c, PU-COOH; d, PU-NH2; e, PU-C-Hep; f, PU-N-Hep.

17.

18.

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