Ex vivo and in vitro platelet adhesion on RFGD deposited polymers David Kiaei and Allan S. Hoffman* Center for Bioengineering, FL-20, University of Washington, Seattle, Washington 98195 Stephen R. Hansont Scripps Clinic and Research Foundation, La Jolla, California 92037 Clinical applications of small-diameter synthetic vascular grafts are hindered by their highly thrombogenic surfaces. To develop vascular grafts that resist thrombotic occlusion, a radio frequency glow discharge (RFGD) process was employed to modify the surface of existing graft materials. Ultrathin coatings of RFGD polymers of ethylene (E), tetrafluoroethylene (TFE), and hexamethyldisiloxa n e (HMDS) w e r e d e p o s i t e d o n t h e lumen of Dacron grafts. Surfaces were characterized by electron spectroscopy for chemical analysis (ESCA). The effect of glow discharge treatments on plateletgraft interactions was evaluated in an ex vivo baboon s h u n t model. Following placement of a n untreated or RFGDtreated graft in the shunt, deposition of l"Indium-labeled platelets was monitored for 60 min by y camera imaging. Untreated Dacron rapidly accumulated large numbers of platelets, reaching a plateau i n 60 m i n . HMDS- a n d T F E - t r e a t e d Dacron had significantly lower levels of platelet deposition compared to the un-

treated control. In contrast, the ethylene treatment of Dacron augmented platelet deposition, making it the most plateletadherent surface studied. In vitro studies were also performed using untreated and RFGD-t reated poly (ethylene terepht halate) (PET) coverslips. ESCA verified that the surface composition of the untreated and RFGD-treated coverslips were virtually identical t o their untreated and t r e a t e d Dacron g r a f t c o u n t e r p a r t s . Samples w e r e i n c u b a t e d i n w a s h e d baboon platelet suspensions for 2 h at 37°C. Platelet adhesion on the untreated PET was relatively high, and many of the platelets had a completely spread morphology. The HMDS and TFE treatment of PET reduced the number of adherent platelets and prevented platelet spreading on t h e surface. Platelet adhesion and spreading on the ethylene-treated surface was the highest among the four studied. There is a remarkable linear correlation of the ex vivo and i n vitro platelet adhesion data.

INTRODUCTION

Thrombotic occlusion is one of the main causes of early failure of small diameter synthetic vascular grafts.' Platelet interaction with the surface of a synthetic graft is perceived as the primary factor in surface-induced thrombosi~.'-~ Furthermore, there is abundant evidence in the literature that the "To whom correspondence should be addressed. 'Present address: Department of Medicine, Emory University, Atlanta, GA. Journal of Biomedical Materials Research, Vol. 26, 357-372 (1992) C C C 0021-9304/92/0303 57-16$4.00 0 1992 John Wiley & Sons, Inc.

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surface composition of a material in contact with blood greatly influences the extent of platelet adhesion and activation."-"' Therefore, we have directed our efforts toward modifying the surface composition of synthetic vascular grafts in order to prevent platelet-mediated thrombotic occlusion. Among the various surface modification techniques available, radio frequency glow discharge (RFGD) polymerization is the ideal technique for our purpose since it is versatile, clean, simple, and does not alter the bulk properties of the synthetic vascular We have previously shown that a tetraf luoroethylene (TFE) RFGD treatment of Dacron vascular grafts improves their resistance to thrombotic occlusion in an ex vivo baboon shunt m ~ d e l . ' ~ - ' ~ Additionally, a significant reduction in microemboli production in an in vitro recirculation system was noted after the TFE glow discharge treatment of Dacron grafts." The goal of this study is to evaluate platelet interactions with various surfaces prepared by the RFGD technique. Specifically, we have used RFGD to deposit ultrathin coatings of polymers of ethylene (E), 'TFE and hexamethyldisiloxane (HMDS) on the lumen of Dacron grafts. We have chosen these three gases because of their similarities to the repeating units of polyethylene, poly (tetraf luoroethylene), and poly (dimethyl siloxane) which are considered by most to be well-tolerated by blood in arterial flow conditions. Our primary hypothesis is that RFGD treatments of Dacron grafts with these gases would reduce surface-induced platelet adhesion and activation. The effects of the different RFGD treatments on platelet-surface interactions have been evaluated both in an ex vivo baboon shunt model and in an in vitvo platelet adhesion test. The choice of the baboon for this study is based on the similarity of the baboon hemostatic mechanism to humans and the wellcharacterized, chronically patent arteriovenous shunt model which we have The in vitro platelet adhesion was selected because it is widely used by other researchers in this field and allows investigation of platelet interactions with surfaces in the absence of exogenous plasma proteins. Platelet adhesion and morphological studies have been carried out on untreated and RFGD-treated poly (ethylene terephthalate) (PET) coverslips since PET is chemically identical to Dacron vascular grafts. We also chose an in vitro plus an ex vivo test to see if there would be any correlation between the two models. MATERIALS

Microvel double velour Dacron vascular grafts of 4-mm i.d. were supplied by Meadox Medicals, Inc. (Oakland, NJ). PET Thermanox coverslips were purchased from Nunc Inc. (Naperville, IL). The vascular grafts were used as received but the coverslips were cleaned using 15-min ultrasonic treatments in methylene chloride, acetone, and distilled water. Argon (Ar) was obtained from Air Products (Allentown, PA). Ethylene was purchased from Byrne Specialty Gases (Seattle, WA). Tetrafluoroethylene was received from PCR Inc. (Gainesville, FL). Hexamethyldisiloxane was produced by Alfa Products (Danvers, MA).

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METHODS

Glow discharge polymerization The glow discharge reactor system which we used is shown schematically in Figure 1. Dacron vascular grafts were treated in a 135-cm-long, 5-mm i.d., Pyrex reactor. PET coverslips, 11 X 16 mm, were placed horizontally in a 16-mm i.d. reactor to allow uniform treatment of both sides of the substrates. An EN1 Power Systems generator (Model HF-300, Rochester, NY) supplied the radio frequency power at 13.56 MHz. The power was capacitively coupled to the glow discharge reactor via two external copper electrodes. The electrodes traversed the length of the reactor at a predetermined constant speed. The flow rate of gases was monitored by an ULTRAFLO mass flow sensor (Vacuum General Inc., San Diego, CA). The pressure was measured by a Hastings thermocouple vacuum gauge (Model VT-6) connected downstream from the reactor. A Stokes vane pump (Model 009-2, Pennwalt Corp., Philadelphia, PA) evacuated the system to a base pressure of 0.01 mm Hg. The substrates were initiaIly treated with an argon glow discharge (2.6 W, 3 cm3/min, 0.1 mm Hg, 3.3 mm/s). Next, the desired monomer was introduced and the glow discharge was initiated under similar conditions to the argon treatment. HMDS was degassed by freeze-thawing once under vacuum, prior to the initiation of glow discharge, and was introduced in the reactor via a micrometering valve. Therefore, the flow rate of this monomer was not measured. Surface characterization The ESCA spectra of untreated and RFGD-treated Dacron grafts and PET coverslips were obtained on a Surface Science Laboratories SSX-100 ESCA spectrometer at the National ESCA and Surface Analysis Center for Biomedical Problems (NESAC/BIO) at the University of Washington. Survey scans of RF

Matctung Network

Generator

Pressure Gauge

-

Movement of Plarma

Ar

TFE

vacuum Pump

Trap

Figure 1. Schematic diagram of the radio frequency glow discharge apparatus.

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0 to 1000 eV binding energy were run to determine the elemental composition of the surface. High-resolution scans of the carbon Is region (20 eV window) were also recorded and the CH, peak was assigned to 285.0 el? For binding energy referencing of TFE-treated surfaces, the CF2peak was set at 292.0 eV. Water contact angles of surfaces were obtained using distilled water and a Ram6-Hart goniometer (Ram&-HartInc., Mountain Lakes, NJ). Each contact angle value represents the mean 2 standard deviation of at least 10 separate drops placed on three replicates.

Ex vivo platelet deposition

These studies employed normal male baboons (Pupio anubis, 9-11 kg) which were quarantined and observed to be disease-free for 6 weeks prior to use. All procedures were approved by the Institutional Animal Care and Use Committee in accordance with federal guidelines (Guide for the Care and Use of Laboratory Animals, 1986). All animals had a chronic silicone rubber arteriovenous (A-V) shunt placed between the femoral artery and vein, as described e l s e ~ h e r e . Untreated ~ , ~ ~ , ~ ~and RFGD-treated Dacron vascular grafts (10 cm x 4.0 mm i.d.) were incorporated as extension segments of the A-V shunt. The grafts were connected at their proximal and distal ends to 4.0mm i.d. silicone rubber tubing to avoid hemodynamic perturbations at the graft-shunt junctions.22,21 Blood flow rates through the shunt were measured noninvasively using a Doppler ultrasonic flowmeter (L and M Electronics Model 1012, Daly City, CA), and were controlled at 100 mL/min by a clamp placed distal to the graft segment. For imaging studies of platelet thrombus formation, autologous platelets were labeled with 1 mCi of '"Indium-oxine (Amersham Corp., Arlington Heights, IL) as described previously." The labeled cells were injected into recipient animals at least 1 h prior to graft placement in the A-V shunt. Serial 5-min images of platelet accumulation onto the graft segment, beginning at the moment of blood contact, were acquired with a Picker Dyna scintillation camera coupled to a Medical Data Systems A3 image processing system. A high-sensitivity collimator was used to acquire the low-energy peak of "'Indium (172 keV) with a 10% energy window. The radioactivity in regions of interest (5 cm x 15 cm) was measured for both the graft segment and for silicone rubber tubing comprising the proximal portion of the A-V shunt (blood standard). Because there was no measurable deposition of platelets onto the smooth-walled silicone rubber, the blood standard radioactivity was subtracted from that measured in the grafted segment to yield graftdeposited (noncirculating) radioactivity Results are expressed as total deposited platelets (labeled plus unlabeled cells) by dividing the graftdeposited radioactivity (cpm) by the circulating whole blood platelet radioactivity (cpm/mL), and multiplying by the circulating platelet count (platelets/ mL).22Four replicates of the TFE- and HMDS-treated Dacron vascular grafts were studied. In the case of the untreated and E-treated Dacron, the number

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of replicates were increased to 5 and 9, respectively. The data are presented as the mean f standard error of the mean.

In Vitm Platelet adhesion 120 mL of blood from a healthy baboon were withdrawn directly into two disposable plastic syringes containing acid-citrate-dextrose anticoagulant (10% ACD, NIH formula A). Platelet-rich plasma (PRP) was obtained by centrifuging blood at 2009 for 20 min at room temperature. The PRP was separated and centrifuged for 15 min at 1300g to obtain a platelet pellet. The platelet-poor plasma supernatant was removed and the platelet pellet was rinsed with 5 mL of Ringer's-citrated-dextrose (RCD, 102 mM NaC1, 2.8 mM KCl, 1.54 mM CaClz.2Hz0,21.2 mM Na2C6H5O7.2Hz0, 0.5% w/v dextrose, pH = 6.5) solution containing 0.03 mg/mL apyrase (Sigma Chemical Co., St. Louis, MO). The platelet pellet was resuspended in 9.8 mL of RCD plus 0.03 mg/mL apyrase by gently aspirating the suspension using a disposable plastic pipette. Platelets were radiolabeled with "'Indium-tropolone by a modification of the technique developed by Dewanjee et al?4One hundred microliters of tropolone (Sigma) at a concentration of 1 mg/mL were mixed with 100 pCi of "'Indium monochloride solution (New England Nuclear, Boston, MA). The mixture was shaken for 2 min and RCD was added to bring the final volume to 200 pL. The l"Indium-tropolone solution was added to the platelet suspension and incubated for 30 min at room temperature. In order to remove any unincorporated '"Indium, the platelet suspension was centrifuged at 15008 for 15 min forming a pellet. The pellet was rinsed and resuspended in platelet suspending buffer (145 mM NaC1, 2.7 mM KC1,4 mM NaH2P04.H20,1 mM MgC12.6Hz0,2 mM CaClZ.2H20,5 mM HEPES, 5.5 mM dextrose, 0.003 mg/ mL apyrase, pH 7.4). The platelet concentration was determined using a Coulter counter (Coulter Electronics, Hialeah, FL) and adjusted with platelet suspending buffer to 3.75 x 10' platelets/mL. The radioactivity of a 50 p L sample of this suspension was measured with a y counter (Tracor Analytic, Elk Grove, IL) and the specific activity of the solution was calculated. Untreated and RFGD-treated PET coverslips were placed in 4-mL polystyrene cups and incubated in 2 mL of platelet suspending buffer at 37°C. One milliliter of labeled platelet suspension was added to each sample bringing the final platelet concentration to 1.25 x 10' platelets/mL. After 2 h of incubation at 37"C, the samples were removed, dip-rinsed in platelet suspending buffer, and placed in polystyrene tubes containing glutaraldehyde solution (75 mM sodium cacodylate, 3% glutaraldehyde, pH = 7.35 filtered through 0.22 pm filter). The amount of radioactivity associated with each sample was measured with a y counter. Following correction for the background radioactivity, the number of adherent platelets was calculated based on the sample radioactivity, specific activity of the platelet suspension, and the planar surface area of each sample. The data are presented as the mean k standard error of the mean of three replicates for each type of surface.

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The next day, samples were dehydrated using ethanol-water mixtures of increasing ethanol concentration (25, 50, 75, and 100% ethanol). The samples were dried using a liquid CO, critical point drying apparatus (Tousimis Research Corp., Rockville, MD) and sputter-coated with Au-Pd (Desk-1 sputter coater, Denton Vacuum Inc., Cherry Hill, NJ). Samples were examined by scanning electron microscopy (JEOL L td., Tokyo, Japan) and photographs of representative areas were taken. RESULTS A N D DISCUSSION

ESCA surface analysis

Figures 2 through 5 are the high-resolution ESCA carbon 1s spectra of the untreated and RFGD-treated Dacron grafts. The spectra of the RFGD-treated grafts are drastically different from the untreated Dacron. ‘The carbon Is spectrum of the untreated Dacron is composed of three peaks centered at 285, 286.5 and 289 eV (Fig. 2). Consistent with the chemical composition of

299.3

Binding Energy (eV)

279.3

Figure 2. High-resolution ESCA carbon Is spectrum of Dacron 1-

C

E

Q

46

52

2

L 298.6

Binding Energy (eV)

218.6

Figure 3. High-resolution ESCA carbon 1s spectrum of TFE-treated Dacron.

EX VIVO AND IN VlTRO PLATELET ADHESION

298.8

Binding Energy (eV)

363

278.8

Figure 4. High-resolution ESCA carbon Is spectrum of ethylene-treated Dacron.

Atomic Percent

C 57

299.2

Q

s 16

! 27

Binding Energy (eV)

219.2

Figure 5. High-resolution ESCA carbon 1s spectrum of HMDS-treated Dacron.

Dacron, these three peaks have been assigned to the C-C, C - 0 and 0- C= 0 functional groups, respectively. The TFE-treated Dacron (TFE/ Dacron) has a very complex spectrum, indicating considerable molecular fragmentation and rearrangement of the TFE monomer during glow discharge polymerization (Fig. 3). The carbon Is spectrum of TFE/Dacron can be resolved into five peaks centered at 287.5,289,290.5,292, and 294 eV corresponding to C -CF,, CF -C, CF -CF,, CFz, and CF, groups, re~pectively.~~ Similar polymers have been obtained by other researchers following RFGD polymerization of TFE.26The absence of a peak at 285 eV in the carbon Is spectrum of TFE/Dacron attests to the complete coverage of the substrate by the RFGD-deposited polymer. The carbon Is spectrum of the ethylenetreated grafts (E/Dacron) shows a single peak at 285 eV assigned to the C-C and C-H groups (Fig. 4). The complete coverage of the Dacron substrate by the ethylene glow discharge polymerization is indicated by the absence of C -0 and 0 -C =0 peaks of Dacron in the carbon 1s spectrum of E/Dacron. As Figure 5 shows, the carbon Is spectrum of the HMDS-treated grafts (HMDS/Dacron) is composed exclusively of a single peak at 285 eV

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attributed to C-Si as well as C-C and C-H groups. The absence of C - 0 and 0-C=O peaks is indicative of the uniform coverage of the substrate as well as lack of formation of these groups during the RFGD process. This result is in agreement with the work of Coopes and Grie~ser.’~ It is clear from the carbon Is spectra and the surface elemental composition of the treated grafts that the underlying Dacron substrate is completely covered by the RFGD-deposited polymers. Even though the surface composition of Dacron grafts has been modified, examination of the untreated and RFGD-treated grafts by scanning electron microscopy at the magnification of ~10,000 indicates that RFGD-deposited polymers are conformal and the topography of Dacron grafts has not been altered by the RFGD process (pictures not shown). Ex vivo platelet deposition on vascular grafts The results of the ex vivo platelet deposition study are shown in Figure 6. Platelet deposition curves for the untreated and ethylene-treated Dacron are sigmoidal and reach a plateau by 60 min. The number of adherent platelets on the ethylene-treated graft significantly exceeds the number of platelets on the untreated graft almost throughout the blood exposure period. The grafts treated with HMDS and TFE both show markedly reduced platelet deposition over the time course of the study. Although HMDS- and TFE-treated Dacron appear to continue to accumulate platelets after 1 h, the decrease in the slope of the curves suggests platelet deposition is approaching a plateau. In fact, after 90 min of blood exposure, HMDS/Dacron accumulated only 4.75 2 1.2 x 10’ platelets/cm. This amount is 22% higher than the number of platelets deposited after 60 min of blood exposure. In contrast, a 254% increase in platelet deposition is noted for the period between 30-min and 60-min data points. Because platelet depositions on Dacron and E/Dacron

-2

EiDacron

m

w 3

Dacron

HMDSDacron TFEiDacron

0

10

20

30

40

50

60

Time (min)

Figure 6. Ex vivo platelet deposition on RFGD-treated Dacron.

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have both reached a plateau by 60 min and there appeared to be only a slight increase in platelet deposition on TFE- and HMDS-treated Dacron beyond 60 min, data collection was not routinely continued beyond this time point. Didisheim et al. have also observed a reduction in platelet accumulation on TFE-treated Dacron grafts compared to the untreated Dacron grafts.” The authors have attributed this decrease in platelet adhesion (in part) to the reduced wettability and surface area exposed to blood following the TFE treatment. They state that the TFE treatment had transformed the porous, wettable Dacron graft into a non-wettable, functionally nonporous graft.28 We examined the possibility that our observed reduction in platelet accumulation is also due to or affected by the reduced wettability of grafts. To determine wettability, water contact angles of untreated and RFGD-treated PET coverslips were measured. Table I shows that all three RFGD-treated polymers are less wettable than the untreated PET. Therefore, if the reduced wettability of the treated grafts were responsible for the reduced platelet deposition, all three treated grafts should have reduced platelet deposition. However, platelet deposition on E/Dacron is markedly higher than the untreated Dacron (Fig. 6). Furthermore, the void volume of the untreated graft wall interstices is less than 1%of the volume of the graft lumen. If the void volume of the graft wall were filled with blood, then the detected radioactivity for the pores would be less than 1%of the radioactivity of the volume of whole blood within the lumen of the untreated Dacron graft. This estimated amount of radioactivity in the graft wall pores is insignificant compared to the amount of radioactivity detected for the untreated Dacron, which was on the average 47-fold higher (at 1h) than the radioactivity of the volume of blood contained within the graft’s lumen. Clearly, changes in wettability of the fabric leading to changes in blood absorption into the pores cannot account for this great increase, which must reflect the selective and marked accumulation of platelets on the graft surface after it is wetted. Further evidence is provided by another study, where a low porosity woven Dacron was used for the RFGD treatments instead of the highporosity Microvel graft. (The water permeability of the low porosity Dacron is 30 cm3/min cm2compared to 1900 cm3/min cm2for the Microvel Dacron.) The same ranking of the RFGD treatments for the low-porosity weave as for the Microvel was obtained. That is, low platelet accumulation on the HMDS- and TFE-treated grafts was obtained in contrast to the higher levels for the untreated and E-treated Dacron (data not shown). Thus, it is apparent that the reduced wettability of the treated grafts does not strongly influence our reTABLE I Water Contact Angles of RFGD-Treated PET Coverslips Surface PET E/PET HMDS/PET TFEfPET

Water Contact Angle 77 r 90 t 99 105 t

*

2 2 1 5

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sults while the specific surface chemistry of the graft appears to play the critical role in graf t-platelet interactions. It is possible that the differences between this study and the work of Didisheim et al. are due to differences in the experimental techniques. Didisheim and co-workers carried out their experiments in vitro using a parallel plate flow chamber and heparinized canine blood.28In contrast, we have evaluated the materials in an ex vivo baboon shunt model in the absence of anticoagulants. In vitro platelet adhesion on PET coverslips ESCA spectra and surface elemental composition of the untreated and RFGD-treated PET coverslips are virtually identical to the untreated and RFGD-treated Dacron grafts and have been presented in a previous publication.29Examination of the RFGD-treated PET coverslips with a scanning electron microscope revealed that all surfaces are smooth and defect-free, similar to the untreated coverslips. The in vitro platelet adhesion study was carried out for 2 h in contrast to 1 h for the ex vivo platelet deposition study. This decision was based on the fact that the concentration and the diffusion rate of platelets under the conditions of the in vitro study are markedly lower than the ex vivo study. Therefore, in order to allow more time for platelets to diffuse and interact with the test surfaces, the in vitro study was extended to 2 h. The results of the in vitro platelet adhesion study are shown in Figure 7. The highest number of adherent platelets is on the ethylene-treated PET (E/PET). The untreated PET also accumulates a large number of platelets, but less than that of the E/PET. Platelet adhesion on the TFE- and HMDS-treated surfaces are comparable and the lowest among the surfaces studied. The morphology of the platelets on the HMDS- and TFE-treated surfaces is also radically different from the untreated or E/PET. On both TFE- and HMDStreated PET, platelets appear to be mostly round, with a few extended pseudopods [Figs. 8(c,d)]. Spread platelets on these surfaces are rare or completely absent. However, the platelet population on the untreated and E/PET varies

-

12 1

HMDSIPET TFEIPET

PET

EIPET

Figure 7. In vitro platelet adhesion on RFGD-treated PET.

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from round to completely spread. A large number of fully spread platelets are visible on these surfaces [Figs. 8(a, b)]. These in vitro findings indicate that PET and E/PET are highly platelet-reactive surfaces, causing significant platelet adhesion and spreading. In contrast, the TFE- and HMDS-treated surfaces both reduce platelet adhesion and inhibit platelet spreading during the 2 h study.

Ex vivo and in vifro correlation The ex vivo acute platelet deposition levels on these four surfaces agree very closely with the in u i h platelet adhesion results. This finding is rather surprising considering the numerous differences between the two models. In

Figure 8. Scanning electron micrographs of platelets adherent on (a) untreated PET, (b) ethylene-treated PET, (c) TFE-treated PET, and (d) HMDStreated PET. Scale bar = 10 pm.

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(4 Figure 8. (continued)

the ex vivo study, whole blood flows through the porous double velour vascular grafts at arterial wall shear rate. The in uitro study is performed using a washed platelet suspension nearly free of other blood components (e.g., erythrocytes, leukocytes and proteins) on smooth, nonporous coverslips in the absence of flow. Despite these differences, the cross-plot of the ex v i m and in vifro data indicates that there is a remarkably good linear correlation (correlation coefficient = 0.933) between the ranking of the surfaces by the two models (Fig. 9). In this study, we have shown that untreated Dacron accumulates large numbers of platelets. This finding supports previously published results on platelet adhesion and activation by Dacron vascular grafts, in which Dacron was shown to adhere and activate platelets, as measured by platelet factor 4 release in an in vitro recirculation In vivo studies have also shown that Dacron grafts fail due to thrombotic occlu~ion.~ The reduction in both ex uivo and in vitvo platelet adhesion on Dacron grafts and PET coverslips as a result of the TFE glow discharge treatment is

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E-treated

1

0

5

10

15

20

Ex Viva (Platelets/cm x lOE8)

Figure 9. Cross-plot of the ex vivo and in vifro platelet adhesion data.

the latest observation in a series of beneficial effects of this treatment reported by our group. Previous work has shown that the TFE glow discharge treatment reduces acute fibrinogen adsorption from plasma, suppresses emboli production,” and improves the patency rate of Dacron grafts in an ex vivo baboon shunt m ~ d e l . ’ ~Yeh - ’ ~ et al. have also reported favorable blood interactions with materials which have been RFGD-treated with TFE or with a hexafluoroethane-Hzm i ~ t u r e .Utilizing ’~ the same ex vivo baboon shunt model reported here, they have also shown that the TFE glow dischargedeposited polymer is not a platelet consumptive surface. In addition, they have observed a significant reduction in platelet adhesion on hexaf luoroethane-H2 glow discharge-treated Gore-tex grafts in comparison to the untreated control. The significant reduction in platelet adhesion and inhibition of platelet spreading following the HMDS treatment of surfaces reported here, is in agreement with other published work in this area. Coating of hemoperfusiongrade charcoal by RFGD polymerization of HMDS was effective in reducing in vitro platelet retention in a packed column.31Glow discharge polymerization of hexamethylcyclotrisiloxane was beneficial in suppressing ex vivo platelet and leukocyte adhesion on Celgard and Silastic membrane^.^^-^^ Glass surfaces have been RFGD-treated with hexamethylcyclotrisiloxane and several other organosiloxane monomers. In vitro platelet adhesion on the RFGDtreated surfaces was 10-30% less than the untreated glass.3s The enhanced thrombus formation on the E-treated Dacron in comparison to the untreated control is surprising in light of the improvements observed for the other RFGD treatments. The surface of the ethylene-treated Dacron is primarily hydrocarbon. The only element detected by ESCA, other than carbon, is oxygen in the amount of 4%. The source of this oxygen may be postpolymerization reaction of the surface free radicals (generated by the RFGD process) with atmospheric oxygen. Yeh et al. utilized the same ex vivo baboon shunt model reported in this work, and showed that an RFGD-depogited polymer of methane resists thrombus f~rmation.‘~ The elemental composition and the ESCA carbon Is

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spectrum of our ethylene glow discharge-deposited polymer closely resemble those of the RFGD-deposited polymer of methane reported by Yeh et al.23 The reason for the different behavior of these two polymers in contact with blood, despite their similar compositions, is not understood. There are, however, some obvious differences between the two studies that complicate this comparison. Our RFGD treatments were carried out on porous Dacron grafts, whereas the methane RFGD-polymer of Yeh et al. was deposited on smooth-walled silicone rubber. In addition, traces of silicone were detected by ESCA on the surface of the methane RFGD-deposited polymer. The silicone signal may have been due to the underlying substrate if the thickness of the methane RFGD-deposited polymer were less than the sampling depth of ESCA. Alternative explanations include migration of low-molecular-weight silicones through the deposited film to the interface following the RFGDpolymerization of methane on silicone rubber and/or silicone contamination of the RFGD-treated surface by other external sources. No silicone was detected on our ethylene RFGD-treated Dacron. The presence of silicone noted by Yeh et al. on the RFGD-deposited polymer of methane may have contributed to the reduced platelet deposition on that surface, thereby explaining the different behavior of the methane (Yeh et al.) and ethylene glow discharge-treated surfaces (this study). These results clearly indicate that although several RFGD polymers have been shown to resist platelet deposition, not all RFGD-deposited polymers can be regarded as thromboresistant surfaces. Further studies are needed to determine the specific chemical groups on the ethylene glow dischargedeposited polymer responsible for triggering thrombosis. SUMMARY

The effect of surface modification of Dacron vascular grafts on their interactions with platelets has been studied in an ex oivo baboon shunt model. Untreated Dacron grafts were found to be highly platelet adherent. Ethylene glow discharge treatment of grafts caused increased platelet accumulation, whereas TFE and HMDS treatments diminished platelet deposition. Similar effects of these treatments were observed for platelet adhesion in vitvo on PET coverslips. A remarkable linear correlation was obtained between the ex oivo and in vitra platelet adhesion data. These findings provide further evidence that surface composition of a material plays a vital role in its interactions with blood. Furthermore, the resistance to platelet adhesion by the TFE and HMDS glow discharge-deposited polymers may be exploited in developing novel materials and devices for clinical applications. Financial support for this work was provided by NIH Grants HL 33229-03, 04, and HL 31469. We also would like to thank NESAC/BIO (NlH Grant RR 01296) staff for their assistance in obtaining ESCA spectra and the Regional Primate Research Center at the University of Washington (NIH Grant RR 00166) for baboon blood draws.

References 1. J. M. Craver, L. W. Ottinger, R.C. Darling, W. G. Austen, ,and R. R. Linton, "Hemorrhage and thrombosis as early complications of femoropop-

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Received December 4, 1990 Accepted September 23, 1991

Ex vivo and in vitro platelet adhesion on RFGD deposited polymers.

Clinical applications of small-diameter synthetic vascular grafts are hindered by their highly thrombogenic surfaces. To develop vascular grafts that ...
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