Thrombotic Events on Grafted PolyacrylamideSilastic Surfaces as

6 Thrombotic Events on Grafted Polyacrylamide—Silastic Surfaces as Studied in a Baboon

Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch006

ALLAN S. HOFFMAN, THOMAS A. HORBETT, and BUDDY D. RATNER— University of Washington, Departments of Chemical Engineering and Bioengineering, Seattle, WA 98195 STEPHEN R. HANSON and LAURENCE A. HARKER— University of Washington, Department of Medicine, Seattle, WA 98195 LARRY O. REYNOLDS—University of Washington, Department of Nuclear Engineering, Seattle, WA 98195

The interaction of well-characterized grafted polyacrylamideSilastic and Silastic surfaces with blood was studied using an ex vivo, arteriovenous baboon shunt model. The consumption of autologous Cr-labeled blood platelets was measured in the animals with test shunts. A new laser light scattering technique was developed to detect flowing platelet microemboli in real time. This technique was applied to the measurement of the size and number of microemboli generated by biomaterial cannulae in the ex vivo baboon shunt. In vitro protein adsorption studies also were carried out using a new, highly sensitive silver staining technique to visualize proteins separated by sodium dodecyl sulfate gel electrophoresis. The results suggest that the polyacrylamide-Silastic hydrogel surface is more platelet-consumptive than the Silastic surface due to a greater rate of platelet aggregation on and subsequent embolization from the hydrogel surface. 51

A

h y d r o g e l can b e d e f i n e d as a p o l y m e r i c material that has the ability to s w e l l i n water a n d retain a significant fraction (e.g., 10-20%) of water

w i t h i n its structure, b u t that w i l l not dissolve i n water. H y d r o g e l materials resemble, i n t h e i r p h y s i c a l p r o p e r t i e s , l i v i n g tissue m o r e than any other class of synthetic b i o m a t e r i a l . I n particular, t h e i r relatively h i g h water contents and t h e i r soft, r u b b e r y consistency give t h e m a resemblance to l i v i n g soft tissue. O n the basis o f these p r o p e r t i e s , hydrogels have b e e n investigated extensively i n a variety o f b i o m a t e r i a l applications (1-3). 0065-2393/82/0199-0059$06.00/0 ® 1982 American Chemical Society Cooper et al.; Biomaterials: Interfacial Phenomena and Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

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BIOMATERIALS: INTERFACIAL P H E N O M E N A A N D APPLICATIONS

P o l y a c r y l a m i d e h y d r o g e l s are a m o n g the most h i g h l y h y d r a t e d gels, a n d often e x h i b i t water contents r a n g i n g f r o m 60 to 95%. T h e y have b e e n tested for b l o o d c o m p a t i b i l i t y b y a n u m b e r of researchers, i n c l u d i n g o u r o w n g r o u p , using a w i d e variety of i n v i t r o a n d i n v i v o test techniques a n d a n i m a l models (4-11). D e s p i t e these n u m e r o u s studies, no general consensus c o n c e r n i n g the b l o o d c o m p a t i b i l i t y o f p o l y a c r y l a m i d e surfaces exists. T h i s chapter brings together o u r p r e v i o u s a n d most recent studies o f grafted p o l y a c r y l a m i d e surfaces i n contact w i t h b l o o d a n d its c o m p o n e n t s , a n d attempts to reach a general c o n c l u s i o n c o n c e r n i n g t h e b l o o d c o m p a t i b i l i t y o f this i n t e r e s t i n g biomaterial. T h e studies d e s c r i b e d i n this chapter w e r e c o n d u c t e d as four separate Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch006

projects to d e s c r i b e the sequence a n d m e c h a n i s t i c aspects of the short- a n d l o n g - t e r m t h r o m b o t i c events that o c c u r w h e n synthetic materials contact b l o o d . T h e first project was r e s p o n s i b l e for the d e v e l o p m e n t , synthesis, and surface characterization o f a l l materials u s e d for subsequent studies. T h i s assured that a l l investigators w e r e e x a m i n i n g i d e n t i c a l materials a n d that a careful r e c o r d was kept o f the surface structure o f each s p e c i m e n tested. S h o r t - t e r m interactive events after exposure to b l o o d w e r e c o n s i d e r e d i n the second project. T h e s e events i n c l u d e d p r o t e i n d e p o s i t i o n , i n i t i a l c e l l adhesion, a n d t h r o m b u s b u i l d u p . P u r i f i e d baboon proteins (in vitro) a n d baboon b l o o d (both i n v i t r o a n d i n vivo) w e r e u s e d for all e x p e r i m e n t s . T h e baboon was chosen as t h e sole a n i m a l m o d e l for this project because o f its h e m a tological s i m i l a r i t y to h u m a n s (12). T h e t h i r d project e x a m i n e d c h r o n i c events o c c u r r i n g w h e n materials i n t u b u l a r f o r m w e r e c o n n e c t e d to an arteriovenous (AV) shunt i n the b a b o o n . I n particular, platelet c o n s u m p t i o n by the i m p l a n t e d materials r e c e i v e d the most attention. T h e f o u r t h project e x p l o r e d the m e c h a n i s m b y w h i c h the platelets w e r e c o n s u m e d b y materials. A laser light scattering system was d e v e l o p e d a n d u s e d to measure t h e n u m b e r , d e n s i t y , a n d size d i s t r i b u t i o n s of e m b o l i generated b y the A V shunt d u r i n g b o t h the acute a n d c h r o n i c phases of e m b o l i z a t i o n . U s i n g the same materials a n d a relevant, r e p r o d u c i b l e a n i m a l m o d e l a l l o w e d a systematic p i c t u r e of the c o m p l e t e t h r o m b o t i c process to b e assembled.

Experimental Preparation of Polymers and Materials Characterization. Techniques have been developed previously for the grafting of acrylamide to polymer substrates (13). These techniques were adapted so that the luminal surfaces of long tubes could be grafted. Silastic tubing (medical-grade Silastic, Dow Corning Inc., 0.125 in. i.d.) was cleaned by sonicating with Ivory soap solution (0.1%) flowing at 100 mL/min through the tube lumen. Three water rinses with flow and sonication followed. A portion of this tubing was radiation grafted to produce a polyacrylamide hydrogel luminal surface using a flow grafting apparatus described previously (14). (Electrophoresis-grade acrylamide monomer was obtained from Eastman Kodak.) A cupric nitrate solvent solution was used to inhibit homopolymerization during the grafting (15). A radiation dose of 0.25 Mrad was used, and the grafting temperature averaged 48°C over the course of the reaction.

Cooper et al.; Biomaterials: Interfacial Phenomena and Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1982.


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Grafted Polyacrylamide-Silastic Surfaces

61

Graft levels and graft water controls were measured using methods described previously (16). Scanning electron micrographs (SEMs) were taken using a J E O L JSM25 instrument with gold-palladium control specimens. Electron spectroscopy for chemical analysis (ESCA) spectra were taken on a Hewlett-Packard Model 5950B ESCA system. An 0.8-kW monochromatized x-ray beam from an aluminum anode was used for all spectra. An emission from an electron flood gun was used to neutralize static charge buildup on nonconducting polymeric surfaces. All aliphatic carbon Is peaks were assigned a binding energy of 285.0 eV to correct for the energy shift resulting from the electron flood gun. Other element peak positions were shifted an amount corresponding to 285.0 eV (observed carbon Is peak position). Protein Adsorption Studies. Grafted or ungrafted Silastic materials in tubular form, each approximately 100 cm long X 0.3 cm i.d. were used. Baboon plasma was prepared from fresh baboon blood (anticoagulated by drawing it into 0.1 volume of acid citrate dextrose or ACD) by centrifugation, and was stored at — 70°C. The buffer used for prefilling and rinsing was 0.01M citrate, 0.01M phosphate, 0.12M sodium chloride, and 0.02% sodium azide (CPBSz). Adsorbed proteins were eluted with 1% sodium dodecyl sulfate, 0.01M trisphosphate, pH 7 (SDS sample buffer). Immersible molecular sieves (Millipore, Immersible CX) were used to concentrate the eluates. SDS-polyacrylamide slab gel electrophoresis was done with the same buffers and chemicals described previously (17, 18), and with an apparatus obtained from Biorad (Model 220). The gels were 1.5 mm thick and contained 7.5% acrylamide. Staining the proteins with silver was based on a published procedure (19). Adsorption of plasma proteins was studied by pumping 37°C plasma through the tubes (prefilled with degassed buffer at 37°C) for 2 h at 100 mL/min. Rinsing was done by pumping 37°C buffer through the tubes for 1 min. The tube was drained of the buffer, filled with SDS sample buffer, immersed in an ultrasonic bath kept at 50°C, and sonicated for 1 h. The eluate from each tube was then concentrated approximately tenfold and frozen at — 70°C until electrophoresis was done. Measurements of Platelet Consumption. A baboon AV shunt model was used to assess quantitatively the effects of physicochemical properties associated with polyacrylamide grafted substrates. In this model, measurements of platelet consumption using Cr-labeled platelets were steady state, reproducible between test animals, and unaffected by the surgical procedure (8, 10). The methodology involved in the use of AV baboon shunts has been described previously (8,10). We concluded that this primate model simulates arterial thrombotic processes in humans and is suitable for the in vivo evaluation of biomaterial thrombogenesis (10). Laser Light Scattering Detection of Microemboli. To elucidate the mechanism of platelet destruction by grafted polyacrylamide-Silastic surfaces, an optical scattering technique was developed (20) to observe and quantitate the sizes and size distributions of microaggregates in blood produced within the AV baboon shunt. Presumably, any such flowing microaggregates represent platelet microemboli. The optical scattering cuvette with which these measurements were made was placed at the distal tip of the biomaterial test shunt (Figure 1). The cuvette was constructed of a 1.0-mm diameter medical-grade Silastic tube, 5 mm long (Figure 2). Two 3-mm diameter Teflon tubes were used to connect the cuvette to the baboon shunt. The cuvette was encased in an aluminum housing that held three optical fibers in contact with and orthogonal to the Silastic flow tube. Light from a helium-neon laser was focused on one of the optical fibers, and the scattered intensity detected by the orthogonal arrangement of the other fibers was analyzed to determine the size and number density of thromboemboli produced in the shunt. The formation of microaggregates by any particular shunt was determined by a series of optical sizing measurements consisting of 50 data acquisitions (25 60-s measurements were taken during the first hour, beginning immediately after the 51


62


BIOMATERIAL TEST SECTION


FEMORAL ARTERY

VEIN

Figure 1. Schematic of optical scattering cuvette at distal end of shunt.

Figure 2. Detail of optical scattering cuvette.


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initial blood-biomaterial interaction, and 25 data acquisitions, each 120 s long, were taken in the following 2 h). The size distribution of microaggregates from 20-800 μπι in diameter and the number of emboli were measured during each datum acquisition. The system was calibrated using polystyrene microspheres before each experiment. (For details on this technique, see Ref. 20. ) From the size distribution measurements, an equivalent rate of platelet thromboembolization (platelets/day) was calculated assuming that the thromboemboli were spheres composed of close-packed spherical platelets.

Results Material Preparation and Characterization.

T h e effect o f c u p r i c i o n


on a c r y l a m i d e graft l e v e l , as d e t e r m i n e d i n an e x p e r i m e n t separate f r o m t h e t u b i n g p r e p a r a t i o n , is s h o w n i n F i g u r e 3 . B a s e d o n this result, a c u p r i c nitrate c o n c e n t r a t i o n o f 0 . 0 1 M i n t h e solvent was chosen. Graft l e v e l (measured gravimetrically) as a f u n c t i o n of distance f r o m the p u m p is s h o w n i n F i g u r e 4. T h e first 200 c m o f t u b i n g was discarded, r e s u l t i n g i n a l e n g t h o f t u b i n g w i t h a graft l e v e l o f 2.90 ± 0.34 mg/cm . 2

S E M s w e r e u s e d to c o m p a r e , i n a qualitative sense, the surface texture of the a c r y l a m i d e - g r a f t e d Silastic a n d t h e ungrafted Silastic. S i m i l a r surface textures w e r e n o t e d i n b o t h cases ( F i g u r e 5). Because t h e acrylamide graft was d e h y d r a t e d for S E M analysis, t h e graft also was observed i n a h y d r a t e d

Figure 3. Effect of Cu concentration on acrxAamide grafting to 10-mil Silastic (0.375 Mrad). 2


64


3.5 3 Graft Level 2.5 mg/sq cm 2


1.5

0.5 h 0 0

100

200

300

400

500

600

700

800

Distance (cm) from Pump Figure 4. Graft level as a function of length along the shunt for polyacrylamide-Silastic shunts prepared using theflow-graftingapparatus described in Refl 14. state w i t h a light stereomicroscope. T a k i n g into account the resolution l i m i t s of such an i n s t r u m e n t , t h e general surface texture o f b o t h surfaces still appeared to b e s i m i l a r . Graft a n d substrate p o l y m e r surface chemistries w e r e e x a m i n e d b y the E S C A t e c h n i q u e . E S C A spectra are shown i n F i g u r e s 6 and 7. Surface e l e m e n t a l ratios d e t e r m i n e d b y E S C A are c o m p a r e d w i t h those expected, based o n t h e s t o i c h i o m e t r y i n Table I. C l o s e a g r e e m e n t exists b e t w e e n e x p e r i m e n t a l l y d e t e r m i n e d values a n d theory for untreated Silastic. H o w ever, t h e a c r y l a m i d e graft surface is a m i x t u r e o f p o l y a c r y l a m i d e a n d p o l y d i m e t h y l s i l o x a n e . E a r l i e r experiments u s i n g f r o z e n , h y d r a t e d grafts have shown that, i n t h e h y d r a t e d state, a m u c h larger fraction o f the graft surface w i l l b e attributable to p o l y a c r y l a m i d e than i n t h e d e h y d r a t e d state (21 ) (Table I, F i g u r e 8). H o w e v e r , silicon can b e detected even i n t h e h y drated state. F u r t h e r i n f o r m a t i o n o n the surface c h e m i s t r y of these materials can b e obtained by e x a m i n i n g the carbon Is spectra i n F i g u r e s 6 a n d 7. F o r Silastic, only one s y m m e t r i c a l peak w i t h a full w i d t h at half m a x i m u m (fwhm) o f 1.2 e V is expected i n t h e carbon Is s p e c t r u m ; this peak is o b s e r v e d . F o r p o l y a c r y l a m i d e , the c o m p a r i s o n b e t w e e n a c u r v e - r e s o l v e d carbon Is s p e c t r u m o f a p u r e p o l y m e r sample ( F i g u r e 9a) and t h e d e h y d r a t e d graft u s e d i n this study ( F i g u r e 7a) is i n s t r u c t i v e . T h e h i g h e r b i n d i n g energy peak d u e to t h e carbon atom ττ-bonded to oxygen i n t h e a m i d e functionality is almost u n -




HOFFMAN ET AL.

Figure 5. SEMs of (a) Silastic tubing, luminal surface, 100 Χ , and (b) polyacrylamide-Silastic, luminal surface, 100 x (white bar - 100 ^m).




LJ

289

287

285

283

281

- « — BINDING ENERGY (eV) Figure 6a. ESCA C Is spectrum of the luminal surface of Silastic tubing.

I

ι 106 ^

ι ι ι L _ 104 102 100 98 BINDING ENERGY (eV)

Figure 6b. ESCA Si 2 ρ spectrum of the luminal surface of Silastic tubing.


H O F F M A N ET AL.


ο


ο

536

534 532 530 528 BINDING ENERGY (eV)

Figure 6c. ESCA Ο Is spectrum of the luminal surface of Silastic tubing.

&\

UJ

289

287 285 283 281 —BINDING ENERGY (eV)

Figure 7a. ESCA C Is spectrum of the luminal surface of polyacrylamide-Silastic tubing in the dehydrated state.




—

I

1

I

I

I

I

106

104

102

100

98

BINDING ENERGY (eV) Figure 7b. ESCA Si 2 ρ spectrum of the luminal surface of polyacrylamide-Silastic tubing in the dehydrated state.

—ι 405

1 ι ι 1 403 401 399 397 BINDING ENERGY (eV)

Figure 7c. ESCA Ν Is spectrum of the luminal surface of polyacrylamide-Silastic tubing in the dehydrated state.


HOFFMAN ET A L .

6.

69


Table I. E S C A Elemental Ratios for Silastic and Acrylamide-Grafted Silastic Acrylamide-Grafted Acrylamide-Grafted Silastic Silastic Silastic (Dry) (Hydrated, 160K) Theory Experimental C/O C/Si C/N

2.0 2.0 —

Theory

d

2.1 1.7 —

3.0 oc 3.0

Experimental '

Theory

— 2.3 54.9

— °° 3.0

1

a

Experimental* 0.4Γ* 2.5 3.41

Complete coverage of the Silastic by a polyacrylamide film is assumed. ''Data from Figure 7. Data from Ref. 21, see Figure 8. ''The high levels of oxygen in this film indicate that the polymer is hydrated. a


c

detectable at the graft surface. A g a i n , i n the h y d r a t e d state, an increased ratio of this peak to that at 285.0 e V o c c u r r e d ( F i g u r e 8). A comparison of the nitrogen Is signal for the d e h y d r a t e d graft on Silastic ( F i g u r e 7c) and the p u r e p o l y m e r ( F i g u r e 9b) is also i n t e r e s t i n g i n this regard. (This h y d r a t i o n d e h y d r a t i o n effect was discussed i n the previous paragraph.) Protein Adsorption.

Since p r o t e i n adsorption is the earliest event i n

the t h r o m b o t i c response of the b l o o d to p o l y m e r s , the types of proteins adsorbed f r o m p l a s m a to p o l y a c r y l a m i d e - S i l a s t i c and Silastic tubes w e r e e x a m i n e d . A s d e s c r i b e d i n the E x p e r i m e n t a l section, plasma was recir culated t h r o u g h the tubes for 2 h at 37°C and r i n s e d away w i t h buffer. T h e proteins a d s o r b e d to the p o l y m e r s w e r e e l u t e d w i t h S D S , separated b y S D S - p o l y a c r y l a m i d e slab gel electrophoresis, and silver-stained u s i n g a n e w e x t r e m e l y sensitive m e t h o d (19). F i g u r e 10 shows the results of these assays. A m i x t u r e of p r o t e i n s of k n o w n m o l e c u l a r weights was separated on the p o l y a c r y l a m i d e g e l (#3 i n F i g u r e 10). T h e calibration plot obtained w i t h this m i x t u r e was u s e d to assign m o l e c u l a r weights to the u n k n o w n proteins i n the eluates f r o m the test surfaces. A p p r o x i m a t e l y 0.08 μg of each calibration p r o t e i n w e r e n e e d e d to give a v e r y clearly stained p r o t e i n b a n d , w h i c h is o n l y 1 % of the a m o u n t n e e d e d w i t h other stains, such as Coomassie b l u e . I n a d d i t i o n , i n other e x p e r i m e n t s , f i b r i n o g e n , i m m u n o g l o b u l i n G (IgG), a l b u m i n , a n d h e m o g l o b i n m i x t u r e s w e r e separated o n s i m i l a r gels (not shown). F i b r i n o g e n r e m a i n e d at the top of the gel, I g G o c c u r r e d as a b a n d b e t w e e n 102,000 a n d 132,000 D a l t o n s , a l b u m i n o c c u r r e d at 58,000 D a l t o n s , and h e m o g l o b i n d i s p l a y e d a diffuse b a n d c e n t e r e d at 15,000 D a l t o n s . T h e s e m o l e c u l a r weights d e v i a t e d f r o m those usually observed for these proteins i n S D S gel electrophoresis because the d i s u l f i d e bonds w e r e not r e d u c e d i n the experiments r e p o r t e d h e r e . T h e eluate f r o m p o l y a c r y l a m i d e - S i l a s t i c h a d six distinct proteins i n it ( G e l 2 i n F i g u r e 10). T h e most p r o m i n e n t bands had m o l e c u l a r weights of



70


—ι— 295

290 BINDING

285

280

ENERGY (ev)

(a.)

20K 15 Κ -

300

295

290

BINDING ENERGY (ev)

285

280

(b)

Figure 8. ESCA C Is spectra for polyacrylamide-Silastic (21). Key: a, 160 Κ (hydrated) ana b, 303 Κ (dehydrated).



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405

403

401

399

71

397

* — BINDING ENERGY (eV) Figure 9. ESCA spectra of pure polyacrylamide film cast on glass. Key: a, C Is spectrum resolved (using a Du Pont 310 analog curve resolver) into its component peaks; and b, Ν Is spectrum.

132,000, 58,000, a n d 21,000. Bands of l o w e r intensity also o c c u r r e d at 93,000, 70,000, a n d 16,000 D a l t o n s . T h e eluate f r o m Silastic ( G e l 1 i n F i g u r e 10) h a d o n l y three v i s i b l e bands, each o f w h i c h was m u c h less intense than the c o r r e s p o n d i n g bands i n the eluate f r o m p o l y a c r y l a m i d e - S i l a s t i c . T w o of the bands f r o m Silastic oc c u r r e d at 131,000 a n d 58,000 D a l t o n s , a n d h a d approximately the same stain intensity. A t h i r d b a n d o f even l o w e r intensity o c c u r r e d at 95,000 D a l t o n s . M e a s u r e m e n t s o f P l a t e l e t C o n s u m p t i o n . W h e n the extent o f grafting of a c r y l a m i d e o n Silastic was v a r i e d , cannular platelet c o n s u m p t i o n increased


72


131

132

95

93

94

58

70 58

68 53 43


29 21 16

17

Figure 10. Analysis of adsorbed proteins with SDS-polyacrylamide gel electrophoresis. Proteins eluted from Silastic (Gel 1 ) or polyacrylamide-Silastic (Gel 2) after exposure to plasma, and a mixture of known proteins (Gel 3), were separated and stained. The approximate positions, stain intensities, and molecular weights ( x 10 ~ ) are indicated in the diagram. 3

sharply as graft l e v e l i n c r e a s e d f r o m 0 to 1 mg/cm , and r e m a i n e d level thereafter ( F i g u r e 11). T h u s , a l l grafted tubes i n this study w e r e grafted to levels w e l l above 1 mg/cm . 2

2

Table II shows t h e effect o n platelet c o n s u m p t i o n of increasing lengths of the p o l y a c r y l a m i d e - S i l a s t i c t u b i n g . T h e a c r y l a m i d e surface is m u c h m o r e destructive to platelets than is t h e Silastic surface. Table II also shows that platelet c o n s u m p t i o n increases l i n e a r l y w i t h shunt area for b o t h the p o l y a c r y l a m i d e - S i l a s t i c shunts. A series o f shunts w i t h radiation a n d eerie i o n - i n i t i a t e d grafts o f p o l y a c r y l a m i d e o n the l u m i n a l Silastic surface, a n d h a v i n g water contents v a r y i n g b e t w e e n 5 1 - 8 3 % w e r e also s t u d i e d . Table I I I shows these data. T h e platelet c o n s u m p t i o n increases l i n e a r l y w i t h t h e g e l water, such that the ratio o f platelet c o n s u m p t i o n to graft water content remains relatively constant. L a s e r L i g h t S c a t t e r i n g D e t e c t i o n o f M i c r o e m b o l i . T h e optical scattering t e c h n i q u e was u s e d to assess i n v i v o the t h r o m b o e m b o l i c p r o p e n s i t y o f p o l y a c r y l a m i d e - S i l a s t i c a n d Silastic shunts o f v a r y i n g lengths. I n four b a boons, c o n t r o l m e a s u r e m e n t s of the n u m b e r and size d i s t r i b u t i o n of t h r o m b o e m b o l i p r o d u c e d b y t h e 3 5 - c m p r o x i m a l section o f the c h r o n i c a l l y


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25η


ί(3)

2

3

4

5

Graft level (mg/cm ) 2

Figure 11. Platelet consumption vs. graft level of polyacrylamide-Silastic

i m p l a n t e d Silastic shunt w e r e made d u r i n g t h e acute phase o f t h r o m b o e m b o l i z a t i o n . T h i s m e a s u r e m e n t also i n c l u d e d endogenous c i r c u l a t i n g platelet aggregates. T h e baseline value o f (1.75 ± 0.77) Χ 1 0 platelets/day, 9

estimated f r o m these m e a s u r e m e n t s ,

subsequently was subtracted w h e n

calculating t h e total rate o f p r o d u c t i o n o f e m b o l u s mass o f a g i v e n test material. G o o d a g r e e m e n t existed b e t w e e n this baseline value a n d the rate of t h r o m b o e m b o l u s mass p r o d u c e d b y a short 35-cm Silastic b i o m a t e r i a l test section p l a c e d p r o x i m a l to the o p t i c a l cuvette ((2.14 ± 0.07) Χ 1 0 platelets/ 9

day). T h e r e f o r e , t h r o m b o e m b o l i are generated o n the basis o f b i o m a t e r i a l surface area a n d are not effected significantly b y the surgical p r o c e d u r e , t h e connectors, o r a n y flow d i s t u r b a n c e caused b y the optical cuvette. T a b l e II. E f f e c t o f C a n n u l a C o m p o s i t i o n a n d A r e a o n P l a t e l e t C o n s u m p t i o n (8) Exposed Area (cm ) 2

Mean Platelet Survival Time (days)

Cannula Platelet Consumption (platelets/day x 10~ ) 10

Cannula Platelet Consumption/ Unit Area (platelets/cm day x 10 ~ ) 2

Polyacrylamide-grafted Silastic 16.6 41.5 83.0 99.7

4.2 ± 0 . 6 2.6 ± 0 . 2 1.5±0.1 1.8 ± 0 . 4

2.7 ± 1.5 7.5 ± 0 . 7 15.5 ± 1.7 18.2 ± 2 . 3

65.8 131.7

4.7±0.1 4.0 ± 0 . 2

0.8 ± 0 . 3 2.5 ± 0 . 4

16.3 ± 8 . 8 18.0 ± 1.6 18.6 ± 2 . 1 18.2 ± 2 . 3

Silastic 1.3 ± 0 . 3 1.9 ± 0 . 4

Note: Values are mean ± standard error.


H

74


T a b l e III. P l a t e l e t C o n s u m p t i o n b y P o l y a c r y l a m i d e - G r a f t e d Substrates (9) Substrate

Silastic/HEMA* Silastic Silastic

Method of Initiation

Water Content (W) (wt %)

Ce Co rad'n. 60| Co rad'n.

50.8 60.8 83.0

4 +

60i }

}

Platelet Consumption* (PC) (platelets/cm -day x 10 ~ ) 2

H

16.4 ± 3 . 4 (3) 19.0 ± 0 . 8 (18) 27.2 ± 2 . 7 (5)

0.32 0.31 0.33

"Mean values are ± 1 . 0 standard error. The number of studies is in parentheses. ''Silastic pregrafted with 1.74 mg/cm poly(2-hydroxyethyl methacrylate). 2


T h e analysis o f the size a n d n u m b e r o f t h r o m b o e m b o l i generated b y grafts o f p o l y a c r y l a m i d e - S i l a s t i c a n d Silastic cannulae shows a consistent peak p e r i o d o f t h r o m b o e m b o l i z a t i o n o c c u r r i n g i n the first h o u r that is at least 3 - 5 times greater than the steady-state value o c c u r r i n g i n the second a n d t h i r d hours. I n a d d i t i o n , as shunt surface area increases, the average rate o f platelet c o n s u m p t i o n d u e to e m b o l i z a t i o n d u r i n g the steady-state p e r i o d increases l i n e a r l y , for b o t h shunt materials s t u d i e d ( F i g u r e 12). F u r t h e r m o r e , the p o l y a c r y l a m i d e - S i l a s t i c shunt produces significantly m o r e t h r o m b o e m b o l u s mass than the Silastic shunt.

Discussion Previous studies o f the response o f b l o o d to p o l y a c r y l a m i d e surfaces have d e m o n s t r a t e d clear differences relative to other materials, especially silicone r u b b e r . E a r l y reports o f " i m p r o v e d b l o o d c o m p a t i b i l i t y " o f p o l y a c r y l a m i d e w e r e based o n i n v i t r o tests, such as L e e - W h i t e clotting times (5) and s p i n n i n g - d i s c platelet a d h e s i o n results (7). T h i s i m p r e s s i o n was r e i n forced w h e n canine v e n a cava rings w i t h grafted p o l y a c r y l a m i d e - S i l a s t i c surfaces r e m a i n e d patent for 2 weeks, w h i l e untreated Silastic rings were o c c l u d e d w i t h i n 2 h (22, 23). H o w e v e r , w h e n s i m i l a r p o l y a c r y l a m i d e - S i l a s t i c rings w e r e i m p l a n t e d j u s t above the canine renal arteries, n u m e r o u s k i d n e y infarcts w e r e n o t e d (23). L u m i n a l l y grafted p o l y a c r y l a m i d e - S i l a s t i c tubes were i m p l a n t e d s u b s e q u e n t l y as ex v i v o A V shunts i n sheep, and these tubes i n d u c e d significantly greater platelet destruction than d i d the ungrafted Silastic (24). Taken together, these e a r l i e r studies suggested that contrary to the " c o n v e n t i o n a l w i s d o m , " p o l y a c r y l a m i d e surfaces are i n fact t h r o m b o genic, b u t are not strongly t h r o m b o a d h e r e n t . S u c h important differences i n b l o o d responses to p o l y a c r y l a m i d e a n d Silastic surfaces have lead us to examine i n greater d e t a i l the differences i n t h e i r surface properties before, d u r i n g , and after exposure to p l a s m a proteins a n d b l o o d , u s i n g a w i d e range of e x p e r i m e n t a l protocols i n c l u d i n g the ex vivo baboon A V shunt m o d e l . T h e r a d i a t i o n grafting t e c h n i q u e u s e d to prepare the p o l y a c r y l a m i d e Silastic grafts is a practical m e t h o d for i m m o b i l i z i n g the mechanically weak


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75


18-J

20

15-1


15 — POLYACRYLAMIDE-SILASTIC 2

12-

9H

10-

6H 5— 3H

As(cm/)

Figure 12. Rates of production of thromboemboli volume (p /s) and equivalent platelet consumption by embolization (platelets/day) vs. surface area for two biomaterials: Silastic (bottom) and polyacrylamide-Silastic (top). The equivalent steady-state platelet consumption is (3.0 ±0.2) x 10 (platelets/day-cm ) for polyacrylamide-Silastic and (0.8 ±0.1) x 10 (platelets/day-cm ) for Silastic. 3

s

2

8

2


76


p o l y a c r y l a m i d e h y d r o g e l on the strong Silastic support. T h e use of c u p r i c i o n to i n h i b i t a c r y l a m i d e h o m o p o l y m e r i z a t i o n is essential to achieve significant grafting levels of p o l y a c r y l a m i d e . E l e m e n t a l analyses of the grafts show that no significant l e v e l of c o p p e r i o n is r e t a i n e d i n these grafts.


T h e surface c h e m i s t r y of this a c r y l a m i d e graft cannot be e x a m i n e d i n the d e h y d r a t e d state b y E S C A i n a b i o l o g i c a l l y m e a n i n g f u l way (Table I, F i g u r e 8) (see Ref. 21). In the h y d r a t e d state, the graft surface is c o m p o s e d of p o l y a c r y l a m i d e i n t e r m i x e d w i t h p o l y d i m e t h y l s i l o x a n e . T h i s c o m p l e x surface raises some questions as to w h e t h e r these graft surfaces are a good m o d e l for p o l y a c r y l a m i d e . T w o observations support o u r contention that these surfaces are, i n d e e d , v a l i d m o d e l s for p o l y a c r y l a m i d e . F i r s t , silicone r u b b e r is among the most passive of materials we have seen w i t h respect to platelet cons u m p t i o n . T h e r e f o r e , silicone r u b b e r chains i n the graft surface p r o b a b l y w o u l d not i n f l u e n c e the platelet c o n s u m p t i o n of a m o r e c o n s u m p t i v e material such as p o l y a c r y l a m i d e . S e c o n d , baboon platelet consumptivities of a w i d e variety of hydrogels (not j u s t polyacrylamide) grafted l u m e n a l l y i n Silastic and p o l y u r e t h a n e shunts increase l i n e a r l y w i t h h y d r o g e l water content (9). If the silicone chains w e r e affecting the platelet c o n s u m p t i o n process, h y drogels grafted onto two different substrates p r o b a b l y w o u l d not have platelet c o n s u m p t i o n s g o v e r n e d o n l y by graft water content. T h e adsorption of p l a s m a proteins to p o l y m e r s precedes the interaction of b l o o d cells w i t h the surfaces, and therefore, is l i k e l y to be an i m p o r t a n t initial event i n the response of b l o o d to p o l y m e r s (25, 29). A t present, however, little is k n o w n about the adsorbed p r o t e i n layer, even t h o u g h it has b e e n s t u d i e d i n some detail i n recent years (30-36). Because p r o t e i n adsorption f r o m b l o o d plasma is a c o m p e t i t i v e process, differences i n the adsorbed layer on different p o l y m e r substrates c o u l d be a p r i m a r y cause of differences i n t h r o m b o g e n i c i t y . Previous studies of the c o m p o s i t i o n of the adsorbed p r o t e i n layer have e m p l o y e d I - l a b e l e d p r o t e i n a d d e d to plasma (37-39), a n t i b o d y b i n d i n g (34) to detect i n d i v i d u a l proteins, or electrop h o r e t i c analysis of detergent-elutable proteins (17, 33, 35). T h e p r o c e d u r e used i n this study does not r e q u i r e the large surface areas used i n previous work (35), nor does it r e l y on i n c o r p o r a t i o n of radiolabels (36) into adsorbed p r o t e i n . Instead, a staining m e t h o d at least 100-fold m o r e sensitive than these other techniques has b e e n used. 12o

T h e eluates f r o m b o t h p o l y a c r y l a m i d e - S i l a s t i c and Silastic contained a p r o t e i n o c c u r r i n g at the same m o l e c u l a r weight as a l b u m i n (58,000). Relatively large amounts of this p r o t e i n i n p l a s m a give this p r o t e i n a considerable competitive advantage over other p l a s m a proteins, m a k i n g its presence on b i o m a t e r i a l surfaces q u i t e p r e d i c t a b l e (37-39). A l b u m i n adsorption is generally thought to make surfaces less adhesive to platelets. T h e m o r e intense staining o b s e r v e d for a l b u m i n i n the eluate f r o m p o l y a c r y l a m i d e - S i l a s t i c c o m p a r e d to Silastic p r e s u m a b l y indicates that the total amount of a l b u m i n is greater on p o l y a c r y l a m i d e - S i l a s t i c than o n Silastic. I n previous studies,


6.

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77


fluorescein-labeled p r o t e i n uptake b y p o l y a c r y l a m i d e - S i l a s t i c films was used to d i s t i n g u i s h a b s o r p t i o n i n t o the p o l y a c r y l a m i d e gel f r o m adsorption onto the gel surface (40). A l b u m i n d i d not e n t e r the g e l , w h i l e c h y m o t r y p i n o g e n ( M W = 26,000) d i d . T h e r e f o r e , the results p r e s e n t e d here for proteins above this m o l e c u l a r w e i g h t most l i k e l y reflect surface adsorption. T h e greater absolute a d s o r p t i o n o f a l b u m i n o n p o l y a c r y l a m i d e - S i l a s t i c may explain w h y these surfaces w e r e not as t h r o m b o a d h e r e n t as Silastic i n the canine vena cava r i n g a n d r e n a l e m b o l u s

experiments.

T h e eluates f r o m b o t h p o l y a c r y l a m i d e - S i l a s t i c and Silastic also contained a major p r o t e i n at m o l e c u l a r w e i g h t 132,000, w i t h i n the range o b served for the I g G g r o u p . Because this group o f glycoproteins is the next Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch006

most c o m m o n p l a s m a after a l b u m i n , t h e presence o f these molecules o n surfaces is also n o t s u r p r i s i n g (34-36, 38, 39). Recent experiments

ina

n u m b e r of laboratories have suggested an i m p o r t a n t role for leukocytes i n the process of t h r o m b o g e n e s i s o n artificial surfaces (41-43), and have indicated a role for I g G i n m e d i a t i n g the w h i t e c e l l response at foreign interfaces (44, 45). Because f i b r i n o g e n remains at the gel top i n the electrophoresis system used here, w e cannot assess its i m p o r t a n c e because staining artifacts freq u e n t l y occur at t h e g e l top. H o w e v e r , adsorption o f

125

I - f i b r i n o g e n to

p o l y a c r y l a m i d e - S i l a s t i c shunt surfaces f r o m baboon b l o o d i n vivo is m u c h greater than adsorption to Silastic shunt surfaces (46). W h e n the greater adsorption of a l b u m i n to p o l y a c r y l a m i d e - S i l a s t i c (as observed b y the electrophoresis technique) is taken together w i t h the h i g h e r f i b r i n o g e n adsorption o n these surfaces (as observed i n the baboon shunt), o f t h e greater t h r o m b o e m b o l i c

potential o f

p o l y a c r y l a m i d e - S i l a s t i c surfaces may b e p r o p o s e d : Platelets

the

f o l l o w i n g explanation

encountering

this surface may b e c o m e activated b y the h i g h local concentration of f i b r i n o gen at its surface, l e a d i n g to aggregation o n o r near t h e sites o f platelet aggregation. H o w e v e r , these platelet aggregates do not adhere v e r y l o n g or very strongly because o f the h i g h l e v e l of adsorbed a l b u m i n i n the v i c i n i t y . Alternatively,

t h e role o f other

proteins

observed

i n t h e eluate f r o m

p o l y a c r y l a m i d e - S i l a s t i c , such as I g G a n d other m i n o r proteins at molecular weights 93,000, 70,000 (prothrombin?), 21,000, a n d 16,000 (hemoglobin?), may be m o r e i m p o r t a n t than p r e v i o u s l y thought. M o r e extensive study of the p r o t e i n adsorption process clearly is n e e d e d . T h e laser scattering t e c h n i q u e is n e w l y d e v e l o p e d , and p r o b a b l y w i l l be extremely valuable i n q u a n t i t a t i n g the differences i n e m b o l i c events d u r i n g b l o o d - m a t e r i a l interactions, a n d thus p o t e n t i a l l y useful i n e l u c i d a t i n g the mechanisms r e s p o n s i b l e for these differences. F o r example,

t h e v o l u m e o f microaggregates

flowing

through the

cuvette at any t i m e can be estimated f r o m the size d i s t r i b u t i o n , assuming that the microaggregates

a r e c o m p o s e d o f close-packed spherical platelets a n d

that the e m b o l i are n e a r l y spherical. A l t h o u g h these assumptions, at present,


78


are somewhat restrictive, the total v o l u m e s o f e m b o l u s mass estimated f r o m these d i s t r i b u t i o n s c o m p a r e w e l l w i t h measurements

of embolus volume

made w i t h the C o u l t e r c o u n t e r and w i t h e m b o l u s mass estimated b y screen filtration pressure (20). T h e c a n n u l a platelet c o n s u m p t i o n values are shown i n F i g u r e 11. I n c o m p a r i n g F i g u r e s 11 a n d 12, the (optically) estimated platelet c o n s u m p t i o n o f the p o l y a c r y l a m i d e - S i l a s t i c cannulae is greater than the Silastic c a n n u l a o n l y b y a factor o f about 3.0-4.0, w h i l e the C r - l a b e l e d 5 1

platelet t e c h n i q u e showed that the surfaces d i f f e r e d i n platelet c o n s u m p t i o n by a factor o f about 10.0. T h e difference b e t w e e n these two m e a s u r e m e n t techniques may b e d u e to v e r y small o r v e r y large platelet aggregates that are not i n c l u d e d i n the optical scattering measurements; to errors i n the calcuDownloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch006

lations d u e to the a s s u m p t i o n o f spherical platelets a n d m i c r o e m b o l i ; to discrepencies b e t w e e n the t i m e at w h i c h the steady-state

measurements

w e r e made i n the two t e c h n i q u e s (3 h i n the optical t e c h n i q u e vs. several days in the C r survival measurements); and/or to a d d i t i o n a l mechanisms of plateo l

let c o n s u m p t i o n such as platelet lysis o r the destruction of single cells. A d d i t i o n a l factors i n c l u d e the p o s s i b i l i t y o f b o t h r e v e r s i b l y and i r r e v e r s i b l y aggregated m i c r o e m b o l i , a n d may represent another source o f discrepancy b e t w e e n the

o l

C r data a n d the optical s i z i n g measurements.

difference exists b e t w e e n

Although a

the platelet c o n s u m p t i o n for p o l y a c r y l a m i d e -

Silastic estimated o p t i c a l l y a n d that calculated f r o m C r - l a b e l e d platelets, 3 l

good a g r e e m e n t exists b e t w e e n the Silastic platelet c o n s u m p t i o n s calculated by both t e c h n i q u e s . B o t h techniques p r e d i c t a linear increase i n platelet c o n s u m p t i o n (embolization) w i t h an increase i n c a n n u l a surface area.

Conclusions P o l y a c r y l a m i d e - S i l a s t i c h y d r o g e l surfaces are m o r e polar, m o r e waterswollen, a n d m o r e porous to s m a l l e r m o l e c u l e s and proteins than the control Silastic surfaces, a n d they are m o r e platelet c o n s u m p t i v e than Silastic surfaces i n the A V b a b o o n shunt. T h i s increased destruction o f platelets is manifested b y greater f o r m a t i o n a n d s h e d d i n g o f m i c r o e m b o l i (platelet aggregates) f r o m the h y d r o g e l surfaces. T h e p o l y a c r y l a m i d e - S i l a s t i c surfaces adsorb m o r e proteins o v e r a l l than Silastic, a n d i n particular, adsorb m o r e o f both a l b u m i n a n d f i b r i n o g e n . T h u s , w h e n c o m p a r e d to Silastic surfaces, the p o l y a c r y l a m i d e - S i l a s t i c h y d r o g e l surface p r o b a b l y has a c o m b i n a t i o n of lower adherence to platelets (higher a l b u m i n ) b u t h i g h e r activity toward platelet aggregation (higher fibrinogen). T h i s situation c o u l d lead to l o w platelet residence times o n the h y d r o g e l surface c o m b i n e d w i t h a h i g h p r o b a b i l i t y of activation a n d aggregation o n o r near the surface. O n the other h a n d , other m i n o r proteins o b s e r v e d o n the p o l y a c r y l a m i d e - S i l a s t i c surface, b u t not o n the Silastic surface, m a y p o s s i b l y play a significant role i n thrombogenesis and e m b o l i z a t i o n o n the p o l y a c r y l a m i d e h y d r o g e l surfaces.


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79

Acknowledgment T h e authors a c k n o w l e d g e t h e support o f the D e v i c e s a n d Technology B r a n c h o f the N a t i o n a l H e a r t , L u n g , a n d B l o o d Institute o f the N a t i o n a l Institutes o f H e a l t h (Contract N O 1 - H B - 2 9 7 0 a n d G r a n t H L - 2 2 1 6 3 ) .


Literature Cited 1. Hoffman, A. S. In "Polymers in Medicine and Surgery"; Kronenthal, R. L.; Oser, Ζ.; Martin, Ε., Eds.; Plenum: New York, 1975; p. 33. 2. Ratner, B. D.; Hoffman, A.S. In "Hydrogels for Medical and Related Appli cations"; Andrade, J. D., Ed.; ACS Symposium Series No. 31, ACS: Washing ton, DC, 1976, p. 1. 3. Ratner, B. D. In "Biocompatibility of Clinical Implant Materials"; Williams, D. F., Ed.; CRC: Boca Raton, FL, to be published. 4. Halpern, B. D.; Cheng, H.; Kuo, S.; Greenberg, H. In "Proc. Artif. Heart. Prog. Conf.," Hegyeli, R. J., Ed.; Washington, DC, 1969; p. 87. 5. Bruck, S. D. Biomater. Med. DevicesArtif.Organs 1973, 1, 79. 6. Kearny, J. J.; Amara, I.; McDevitt, Ν. B. In "Biomedical Applications of Polymers"; Gregor, H. P., Ed.; Plenum: New York, 1975; p. 75. 7. Leonard, E. et al. "Coordinated Studies of Platelet and Protein Reactions in the Artificial Heart," Annual Report, NO1-HB-3-2910; available as PB-241-333/ AS N.T.I.S., Springfield, VA, Nov. 1975. 8. Hanson, S. R.; Harker, L. Α.; Ratner, B. D.; Hoffman, A. S. J. Lab. Clin. Med. 1980, 95, 289. 9. Hanson, S. R.; Harker, L. Α.; Ratner, B. D.; Hoffman, A. S. Ann. Biomed. Eng. 1979, 7, 357. 10. Harker, L. Α.; Hanson, S. R. J. Clin. Invest. 1979, 64, 559. 11. Ihlenfeld, J. V.; Mathis, T. R.; Barker, Τ. Α.; Mosher, D. F.; Riddle, L. M.; Hart, A. P.; Updike, S. J.; Cooper, S. L. Trans. Am. Soc. Artif. Intern. Organs 1978, 24, 727. 12. Todd, M. E.; McDevitt, E.; Goldsmith, E. J. J. Med. Primatol. 1972, 1, 132. 13. Hoffman, A. S.; Ratner, B. D. J. Rad. Phys. Chem. 1980, 14, 831-840. 14. Ratner, B. D.; Balisky, T.; Hoffman, A. S. J. Bioengineering 1977, 1, 115. 15. Ratner, B. D.; Hoffman, A. S. J. Appl. Polym. Sci. 1974, 18, 3183-3204. 16. Sasaki, T.; Ratner, B. D.; Hoffman, A. S. In "Hydrogels for Medical and Related Applications"; Andrade, J. D., Ed.; ACS Symposium Series No. 31, ACS: Washington, DC, 1976, p. 283. 17. Weathersby, P. K.; Horbett, Τ. Α.; Hoffman, A. S. Trans. Am. Soc. Artif. Intern. Organs 1976, 22, 242. 18. Weber, K.; Osborn, M. J. Biol. Chem. 1969, 244, 4406. 19. Switzer, R. C.; Merrill, C. R.; Shifrin, S. Anal. Biochem. 1979, 98, 231-237. 20. Reynolds, L. O.; Simon, T. L. Transfusion 1980, 20(6), 669-678. 21. Ratner, B. D.; Weathersby, P. K.; Hoffman, A. S.; Kelly, Μ. Α.; Scharpen, L. H. J. Appl. Polym. Sci. 1978, 122, 643-664. 22. Ratner, B. D.; Hoffman, A. S.; Whiffen, J. D. J. Bioeng. 1978, 2, 313-323. 23. Ratner, B. D.; Hoffman, A. S.; Hanson, S. R.; Harker, L. Α.; Whiffen, J. D. J. Polym. Sci., Polymer Symp. 1979, 66, 363. 24. Hoffman, A. S. "Interaction of Natural and Chemically Modified Aortic Tissue Proteins with Blood," NHLBI Annual Report Contract NO1-HV-42970, July, 1975-June, 1976. 25. Salzman, E. W. Bull. N.Y. Acad. Med. 1972, 48, 225. 26. Bruck, S. D. Biomed. Mater. Symp. 1977, 8, 1. 27. Baier, R. E. Ann. N.Y. Acad. Sci. 1977, 283, 17. 28. Baier, R. E.; Dutton, R. C.; J. Biomed. Mater. Res. 1969, 3, 191.



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