14 Thrombogenesis: An Ionic Steric Phenomenon Biomaterials: Interfacial Phenomena and Applications Downloaded from pubs.acs.org by UNIV OF TEXAS AT EL PASO on 10/19/18. For personal use only.
1
S. A. BARENBERG
University of Michigan, Department of Chemical Engineering, Ann Arbor, MI 48109 K. A. MAURITZ 52 Centenial Way, Geneva, OH 44041
A semiempirical/theoretical ionic model was derived to cor relate and interrelate the ultrastructure morphology, surface charge, surface chemistry, and surface molecular motions of a model semicrystalline hydrophobic triblock copolymer to thrombogenesis. This chapter addresses the aspects of ultrastructure order vs. disorder, primary and secondary molecu lar motions, surface and side chain chemistry, thrombo genesis, and the resultant ionic model. This model can be extrapolated to predict the relative thrombogenic responses of various crystalline and semicrystalline hydrophobic polymeric substrates.
I
n d e f i n i n g a n d c o r r e l a t i n g the surface properties of crystalline a n d s e m i crystalline p o l y m e r i c materials to thrombogenesis, the surface free ener
gy, surface charge, ultrastructure m o r p h o l o g y , surface c h e m i s t r y ,
surface
m o l e c u l a r motions, surface topography, c r i t i c a l surface tension, electrical c o n d u c t i v i t y , a n d water content have e v o l v e d as i m p o r t a n t factors (1,2). A p p a r e n t l y , a c o m p l e x i n t e r r e l a t i o n s h i p exists b e t w e e n the surface p r o p e r ties of a material a n d t h r o m b o g e n e s i s . T h i s chapter presents a s e m i e m p i r i c a l / theoretical epitaxial m o d e l that correlates a n d interrelates the ultrastructure m o r p h o l o g y , surface m o l e c u l a r motions, and i o n i c and steric o r d e r of a m o d e l triblock c o p o l y m e r to thrombogenesis. W e w o r k e d w i t h t h e p r e c e p t that m o r p h o l o g i c a l l y o r d e r e d p o l y m e r i c systems, of g i v e n side chain c h e m i s t r y , can sequester ions, w h i c h can sub sequently serve as an i o n i c array/template for o r d e r e d p r o t e i n adsorption, for example, epitaxial crystallization. A d d i t i o n a l l y , w e addressed t h e effect o f side chain m o t i o n of the substrate o n the epitactic process based, i n part, o n 1
Present address: Ε. I. duPont de Nemours and Co., Inc., Experimental Station, Wilming ton, DE 19898 0065-2393/82/0199-0195$06.00/0 ® 1982 American Chemical Society
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our p r e v i o u s w o r k {3,4). A c o m p r e h e n s i v e r e v i e w o f p o l y m e r epitactic p r o cesses is g i v e n b y M a u r i t z (5). T h e p o l y m e r i c system u s e d i n this study was a semicrystalline h y d r o p h o b i c t r i b l o c k c o p o l y m e r s y n t h e s i z e d and characterized i n o u r laboratories w
(6). T h e salient aspects o f this t r i b l o c k c o p o l y m e r are (1) the ultrastructure m o r p h o l o g y o f the c o p o l y m e r (long-range o r d e r vs. no order) can be c o n t r o l l e d t h r o u g h s o l u t i o n casting f r o m selected solvents; (2) the onset o f secondary side c h a i n m o t i o n occurs b e t w e e n 1 2 ° - 2 5 ° C ; (3) the side c h a i n m o t i o n can be r e s t r i c t e d b y casting the c o p o l y m e r s f r o m a n o n p r e f e r e n t i a l solvent; (4) the p e n d a n t side c h a i n groups are p r o j e c t i n g n o r m a l to the h e l i c a l backbone, a n d (5) the c o p o l y m e r can b e e x t r a c o r p o r e a l ^ evaluated w i t h strict control of morphology and molecular motion.
Experimental The polymer used in this study was a triblock copolymer A B A of poly[(-ybenzyl-L-glutamate) (acrylonitrile/butadiene) (7-benzyl-L-glutamate)], polyf(BLG) (ATBN)(BLG)] (6). x
y
x
Solutions (0.1% and 1.0% w/v) of the copolymer were prepared from dioxane and chloroform, which were preferential and nonpreferential solvent systems, respectively. The dynamic mechanical measurements were done using an inverted torsion (braid) pendulum (7). Morphological and electron diffraction studies (not presented) were done on thin films of the copolymer as cast onto carbon-coated glass slides. The thin films were vapor-stained with osmium tetroxide. Thin films of the copolymer were cast from dioxane and chloroform onto carboncoated slides. The films were then placed in contact with isotonic saline for 4 h, and were washed in deionized distilled water. A carbon film was subjected to this same saline treatment to serve as the experimental control. Conventional and scanning transmission electron microscopy (CTEM and STEM) were done on a J E O L 100C electron microscope. The scanning (secondary emission) electron microscopy was done on a J E O L U3. The x-ray dispersion analysis was done on a J E O L 100C (in the STEM mode) using a Princeton Gamma Tech detector. The count times were on the order of 20 min at 30,000, 50,000, and 100,000 magnification. The animals used in the extracorporeal studies (8) were conditioned male dogs. Access to the animals' circulatory system was via chronic shunts surgically implanted into the neck. The shunt was anastomosed to the carotid artery and jugular vein. Blood flow through the shunt was 1 L/min. Twenty-four hours prior to the experiment, the platelets were labeled with C r and human T-labeledfibrinogen,and were injected into the dog. No anticoagulants were used prior to and/or during the experiment. The morphological studies were done on unlabeled dogs. Experiments were carried out for periods of 5, 10, and 60 min at a shear rate of 150 s" . At the completion of the test the exposed shafts (not used in the radiolabeled studies) were placed in buffered glutaraldehyde. The shafts were post-fixed and critical-point dried (9). The critical-point-dried coatings were removed, embedded in 51
12
1
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Spurr (via propylene oxide) and ultramicrotomed. The unstained (osmocated) thin sections were carbon-coated and examined by C T E M and STEM.
Results T h e c o p o l y m e r m o r p h o l o g y ( F i g u r e 1), as cast f r o m dioxane, e x h i b i t e d a l a m e l l a r m o r p h o l o g y , w i t h the acrylonitrile/butadiene ( A T B N ) layers on the o r d e r of m a g n i t u d e of 15 n m thick; the alternating b e n z y l - L - g l u t a m a t e ( B L G ) layers w e r e o n the o r d e r of 50 n m thick. T h e c o p o l y m e r m o r p h o l o g y ( F i g u r e 2), as cast f r o m c h l o r o f o r m , e x h i b i t e d a homogeneous m o r p h o l o g y w i t h the A T B N m i d b l o c k d o m a i n s on the o r d e r of m a g n i t u d e of 15 n m . W h e n the above c o p o l y m e r cast films w e r e exposed to isotonic saline and/or d e i o n i z e d d i s t i l l e d water, the above o b s e r v e d morphologies r e m a i n e d intact ( F i g u r e 3). W h e n the c o p o l y m e r s w e r e cast d i r e c t l y onto isotonic saline and/or d e i o n i z e d d i s t i l l e d water ( F i g u r e 4), the resultant morphologies w e r e m o d i f i e d i n that the dioxane-cast c o p o l y m e r e x h i b i t e d a phase-separated m o r p h o l o g y c o m p l e x e d w i t h the "salt," whereas the chloroform-cast exhibited a homogeneous "salt"-complexed morphology. T h e x-ray d i s p e r s i o n analysis of the c o p o l y m e r s exposed to the saline solutions i n d i c a t e d ( F i g u r e s 5 a n d 6) that the c o p o l y m e r s sequestered the s o d i u m a n d c h l o r i n e ions. H o w e v e r , d u e to the l o w concentrations, given the limits o f i n s t r u m e n t a l r e s o l u t i o n , t h e location o f the ions c o u l d not b e mapped.
Figure 1. TEM of the phase-mixed, (chloroform-cast) osmium vapor-stained triblock copolymer. The stained (dark) regions are the ATBN midblock segments of the block copolymer and the unstained regions are the BLG portions of the block copolymer.
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Figure 2. TEM of the phase-separated, (dioxane-cast) osmium vapor-stained triblock copolymer. The stained (dark) regions are the ATBN midblock segments and the unstained regions are the BLG segments of the block copolymer.
Figure 3. TEM of the phase-mixed (chloroform-cast) copolymer exposed to isotonic saline, washed and vapor-stained with osmium tetroxide.
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Figure 4. TEM of the phase-mixed copolymer cast from chloroform directly onto isotonic saline and vapor-stained with osmium tetroxide.
T h e d i e l e c t r i c (6) a n d d y n a m i c m e c h a n i c a l measurements ( F i g u r e 7) o f the c o p o l y m e r as cast f r o m dioxane a n d c h l o r o f o r m revealed a d i s p e r s i o n m a x i m u m at 298 Κ a n d a split d i s p e r s i o n at 298 a n d 315 K , respectively. T h e 298 Κ d i s p e r s i o n m a x i m u m has b e e n ascribed (10) to the onset of m o t i o n of the B L G side chains; the onset o f m o l e c u l a r m o t i o n o f the B L G backbone occurs at 408 Κ (11). T h e r e f o r e , the o n l y m o l e c u l a r motions o c c u r r i n g i n the c o p o l y m e r s over t h e p h y s i o l o g i c a l t e m p e r a t u r e range affecting h e m o c o m patibility are those that can b e a s c r i b e d to the onset of side chain m o t i o n of B L G . T h e single d i s p e r s i o n m a x i m u m for the dioxane-cast c o p o l y m e r infers unrestricted
side
chain
motion
(dynamic),
whereas
t h e split
maxima
(chloroform-cast) infers r e s t r i c t e d side chain m o t i o n (static). T h i s change, then, infers that at p h y s i o l o g i c a l temperatures, i n conjunction w i t h the m o r phological results, t h e m o t i o n o f the side chains i n the dioxane-cast phaseseparated c o p o l y m e r is u n r e s t r i c t e d a n d occurs i n an o r d e r e d steric array, whereas i n t h e chloroform-cast c o p o l y m e r the side chain m o t i o n is restricted in a d i s o r d e r e d steric array. T h i s t y p e of side chain m o t i o n has b e e n postu lated p r e v i o u s l y b y M e r r i l l (12) a n d d e m o n s t r a t e d b y B a r e n b e r g (3) to i n f l u ence the i n i t i a l s o r p t i o n o f the p l a s m a protein(s) a n d subsequent interaction w i t h b l o o d . T h e s e i n t e r a c t i o n effects i n conjunction w i t h the extracorporeal experiments a n d i o n s e q u e s t e r i n g results are discussed i n terms o f the d e r i v e d ionic m o d e l . W h e n the c o p o l y m e r s w e r e exposed to canine b l o o d (figures are not presented,
see Ref. 4) f o r 5 m i n , t h e d i s o r d e r e d c o p o l y m e r
surface
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BIOMATERIALS: INTERFACIAL P H E N O M E N A A N D APPLICATIONS
Figure 5. X-ray dispersion spectra of the phase-mixed (chloroform-cast) copolymer (Figure 3) exposed to isotonic saline and washed. The peaks correspond to sodium, osmium, and chlorine.
(chloroform-cast) consisted of platelets with no other type of observable deposition occurring. However, when the phase-separated copolymer (dioxane-cast) was exposed under the same conditions, a proteinaceous deposition was observed in addition to aggregates of adhering platelets. After 10 min exposure to canine blood, the platelets appeared to spread and flatten on the disordered copolymer surface forming a confluent type of sublayer. Whether these are in fact platelets has yet to be shown. In contrast, the phase-separated copolymer surface exhibited a large thrombus primarily composed of platelets, fibrin, and red blood cells. When the copolymers were exposed to canine blood for 60 min, the disordered surface developed a limited pseudoneointima, whereas the phase-separated copolymer surface developed a large thrombus, with no signs of becoming limited. These series of experiments were replicated using different dogs and substrate shafts (9), that is, stainless steel and polypropylene, with consistent reproducibility. The above exposed copolymers were ultramicrotomed in cross section. The disordered surface exhibited a loosely bound 50-nm osmophilic layer,
Figure 6. X-ray dispersion spectra of the phase-mixed copolymer (Figure 4) cast directly onto isotonic saline, washed, and vapor-stained. The peaks correspond to sodium, osmium, ana chlorine.
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Thrombogenesis
A τ
À · >A"
'k •A' Ο · Ο ·
ο · ο · ο.
Β
*···ο·οθ
L
I
ι
ι
ι
1
100
120
140
160
18 0
ι 2 00
ι
ι
2 20
2 40
ι 260
I 2 80
I 30 0
I
>
32 0
TEMPERATURE °Κ
Figure 7. Dynamic mechanical spectra of the copolymer cast from dioxane (o) and chloroform (·) onto glass braids. Arrows A, A', and A" indicate the onset of side chain motion ( 10); B, interfacial relaxation (3); and C, the ,lass transition of the ATBN segment (3). The chloroform-cast copolymer is ispersion-split (A and A") relative to the single maxima for the dioxane-cast system, indicating restricted and unrestricted side chain motion, respectively.
J
whereas t h e phase-separated c o p o l y m e r e x h i b i t e d a tightly b o u n d 10-nm o s m o p h i l i c layer ( F i g u r e s 8 a n d 9).
Discussion T h e s e results indicate that an epitaxial m o d e l can be d e r i v e d to correlate the above data. T h e m o d e l takes into account the differences b e t w e e n t h e two types of secondary m o l e c u l a r motions o b s e r v e d , the ultrastructural dif ferences b e t w e e n t h e o r d e r e d (phase-separated) and d i s o r d e r e d (phasemixed) m o r p h o l o g i e s o f the c o p o l y m e r s , t h e electronegativity o f the side
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BIOMATERIALS: INTERFACIAL P H E N O M E N A A N D APPLICATIONS
Figure 8. TEM of the phase-mixed (chloroform-cast) copolymer, in cross section, exposed to canine blood. Note the loosely bound osmophilic layer on the surface (250,000 x
Figure 9. TEM of the phase-separated (dioxane-cast) copolymer, in cross section, exposed to canine blood. Note the tightly bound osmophilic layer (345,000 χ
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chain c a r b o n y l oxygens, the i o n i c c l u s t e r i n g effects due to the above electro negativity, a n d the surface c h e m i s t r y of the c o p o l y m e r s . T h e m o d e l does not take into account any effects d u e to s t r e a m i n g potential and/or local surface p H differences (13,14), two v e r y i m p o r t a n t areas that n e e d to b e addressed. T h e m o d e l is d i v i d e d into four parts: (1) the d e f i n i t i o n of the surface to be interfaced w i t h b l o o d , (2) the m o d e of the p l a s m a protein(s) and/or electro lyte a d s o r p t i o n , (3) relaxation m o t i o n o f t h e b l o o d interfacing side chain groups, a n d (4) p r o t e i n d e n a t u r a t i o n and/or l i q u i d crystalline order. T h e first aspect o f t h e m o d e l is to define t h e surface that is to b e interfaced w i t h b l o o d , that is, o r d e r e d vs. d i s o r d e r e d . I n the m o d e l system used i n this study, t h e phase-separated (dioxane-cast) c o p o l y m e r w o u l d b e d e f i n e d as a sterically o r d e r e d array, a n d the p h a s e - m i x e d (chloroform-cast) c o p o l y m e r as a sterically d i s o r d e r e d array. T h e second aspect o f the m o d e l incorporates t h e two types o f a d s o r p t i o n o c c u r r i n g c o n c u r r e n t l y at the sur face: (1) that of the p l a s m a protein(s) a n d (2) that o f either ions and/or p o l y electrolytes. T h e s o r p t i o n o f the protein(s) occurs, i n part, b y a two-step process. T h e proteins w o u l d i n i t i a l l y h y d r o p h o b i c a l l y b o n d to the exposed p h e n y l groups i n a metastable state, a n d subsequently surface-diffuse f r o m this metastable state to a l o w e r free-energy state located i n the potential trough b e t w e e n t h e b e n z y l ester side chains (in the case for o u r m o d e l copolymers). T h e s e p r o t e i n s w o u l d t h e n align i n an o r d e r e d fashion, i n the case o f the phase-separated c o p o l y m e r , w i t h t h e h y d r o p h i l i c p o r t i o n o f the sorbed p r o t e i n b e i n g exposed to t h e b l o o d interface. I n the case of a disor d e r e d surface, that i s , t h e chloroform-cast c o p o l y m e r , the sorbed p r o t e i n w o u l d not sorb i n an o r d e r e d fashion n o r f o r m an o r d e r e d type of surface. T h e t h i r d aspect o f the m o d e l takes into account the relaxational m o t i o n o f the side chains a n d the specific side c h a i n c h e m i s t r y . I n our case, the B L G side chains c o n t a i n e d a n electronegative c a r b o n y l oxygen a n d were i n either a d y n a m i c o r static state, d e p e n d i n g o n the solvent system f r o m w h i c h the c o p o l y m e r was cast, dioxane o r c h l o r o f o r m , respectively. T h e side chain m o b i l i t y o f the surface interface may facilitate i o n i c diffusion b e t w e e n the side chains, a n d the e l e c t r o n e g a t i v i t y of the c a r b o n y l oxygens w o u l d , there fore, i m m o b o l i z e these cations. A d d i t i o n a l l y , since the side chains are i n an o r d e r e d p a c k i n g m o d e (15), t h e existence of a relatively o r d e r e d subsurface cationic array m i g h t b e h y p o t h e s i z e d (assuming that the cations are reason ably i m m o b i l i z e d ) . A t this step, t h e n , t h e differences b e t w e e n the effects o f surface c h e m istry a n d secondary m o l e c u l a r motions as dictated b y m o r p h o l o g i c a l o r d e r can b e o b s e r v e d o n t h r o m b o g e n e s i s . I n the case o f the sterically o r d e r e d substrate, i n c o n j u n c t i o n w i t h the subsurface cationic array, the sorbed p r o tein(s) can assume a paracrystalline state, a n d subsequently b e subjected to further pertubations. H o w e v e r , i n t h e case of the d i s o r d e r e d substrate, t h e sorbed proteins c a n n o t assume a paracrystalline state a n d , therefore, w i l l not, p e r se, b e subjected to any f u r t h e r conformational changes.
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In the final state of the m o d e l two events may occur: (1) the adsorbed protein(s) may u n d e r g o denaturation as a result of the subsurface h y d r o p h i l i c cationic array, causing the h y d r o p h i l i c p o r t i o n of the p r o t e i n to partially b u r y itself i n b e t w e e n the side chains, thus exposing the h y d r o p h o b i c p o r t i o n of the p r o t e i n c h a i n to the b l o o d interface, a n d (2) the adsorbed protein(s) may be a l i g n e d on the substrate i n an o r d e r e d paracrystalline and/or l i q u i d crystalline fashion, w h i c h c o u l d serve as an o r d e r e d template to the other plasma proteins. T h i s t h e n w o u l d result i n a d e v e l o p i n g i o n o m e r i c clot. P r e l i m i n a r y e v i d e n c e (16) indicates that the latter does occur. I n the final case of the n o n t h r o m b o g e n i c m o d e l , n e i t h e r a steric nor a subsurface cationic array exists;
therefore,
the
adsorbed
protein(s)
will
remain
in their
native
c o n f o r m a t i o n , p r e s u m a b l y w i t h the h y d r o p h i l i c portions e x t e n d i n g into the biological interface. T h i s m o d e l is d e p i c t e d i n F i g u r e 10 for the m o d e l c o p o l y m e r u s e d i n this study. A d d i t i o n a l e v i d e n c e for this m o d e l r e c e n t l y has b e e n r e p o r t e d b y F i l i s k o (17).
In his studies on the heats of adsorption of proteins, F i l i s k o observed
an e x o t h e r m i c heat, indicative of surface o r d e r i n g , rather than an e n d o t h e r m
Figure 10. (A) Schematic of the BLG portion, in cross section, of the copolymer to be interfaced with blood and (B) initial mode of sorption upon contact with blood. Key: R , cations and/or electrolytes; O, sorbing proteinaceous elements; *, metastable state of the initial sorption; and **, stable state. Continued on next page. +
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Figure 10. (C) Schematic of the hydrophobic bonding of the adsorbed protein(s) and the mode of ionic clustering. This ionic clustering the ionic subsurface template for surface ordering. Key: R, ions and/or electrolytes and O , adsorbed protein(s). (Ό) Denaturation of the adsorbed layer. Continued on next page. in the h y d r a t e d state. A d d i t i o n a l l y , N y i l a s (18) r e p o r t e d s i m i l a r c a l o r i m e t r i c results i n his h y d r a t e d a d s o r p t i o n studies. W h e n F i l i s k o a n d N y i l a s d i d t h e i r studies i n t h e d e h y d r a t e d state, they b o t h observed e n d o t h e r m s . P r e sumably, a p r i o r i , the differenées b e t w e e n the e x o t h e r m i c a n d e n d o t h e r m i c results can be a t t r i b u t e d , i n part, to the presence of the buffer that may have b e e n substrate-sequestered a n d may have served as an i o n i c template. T h e synopsis o f the m o d e l is p r e s e n t e d i n F i g u r e 11. I n essence, the predicative flow chart works b y d e f i n i n g (1) the p o l y m e r backbone and side chain c h e m i s t r y a n d c o n f o r m a t i o n i n the h y d r a t e d and d e h y d r a t e d solid state, (2) the surface c r y s t a l l i n i t y of the p o l y m e r , (3) the p r i m a r y (segmental backbone) a n d secondary (side chain) m o l e c u l a r motions of the p o l y m e r relative to p h y s i o l o g i c a l t e m p e r a t u r e s , (4) the surface ultrastructure m o r phology, a n d (5) the ionic/steric o r d e r and/or d i s o r d e r of the surface and its ability to d e v e l o p an o r d e r e d and/or d i s o r d e r e d array that may serve (or not) as a template for o r d e r e d p r o t e i n a d s o r p t i o n . I f the surface appears as an o r d e r e d i o n i c steric array, the m o d e l p r e d i c t s that a t h r o m b o g e n i c response w i l l result. H o w e v e r , i f the surface appears as a d i s o r d e r e d array, the m o d e l predicts that o n l y a l i m i t e d t h r o m b o g e n i c response w i l l occur, g i v e n l i m i t e d
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Figure 10. (E) Schematic of the immobilized cations forming the subsurface cationic array either ionically bonded directly to the carbonyl oxygens of the BLG side chains or through H-bonding of the bound water. side chain m o b i l i t y . T h e a p p l i c a b i l i t y of this m o d e l to other p o l y m e r i c systems can be o b s e r v e d i n the works of P i c h a (19), H e l m u s (20), a n d L y m a n (21). T h e r e f o r e , i n s u m m a r y , the interaction between the i m m e d i a t e substrate surface a n d h y d r o p h o b i c groupings appears to contribute to the overall adsorption process. H o w e v e r , the d y n a m i c state of the side chains w o u l d
initial sorption mode of bonding « see model
ι =
y
-
LIMITED THROMBUS
Hydrophobic
ι
Hydrophobic
I*
subsurface disorder ( ionic)
subsurface order (ionic)
I*
Static Random Surface
Static Epitaxial Surface
r-—phase\ seporated ordered phasei ιmixed disordered—|
Figure 11. Synopticflowchart of the proposed model indicating the complexity and interactive factors needed to consider in defining the surface to he interfaced with blood, followed by the proposed (Figure 10) mode of protein adsorption, and ultimately, the thrombogenic response. See text for a stepwise description of the model andflowchart.
»
GROSS THROMBUS
I
=
Hydrophobic / Hydrophilic
I
-
ι-
subsurface disorder (ionic)
Dynamic Random Surface
Hydrophobic
subsurface order (ionic)
ι-
Dynamic Epitaxial Surface
1
Ultrastructure Morphology
Ultraslricture Morphology
Γ
« > physiological Λ > physiological
m >physiological ^
