Originally published January, 1996
JAMES M. LAMBERT AND KEVIN G. TINGEY
Polyurethanes of similar bulk composition-- all using the same diisocyanate, chain extender, and soft-segment type--were synthesized in microparticle form. This homologous series of polyurethanes, which varied in morphology and surface structure, was prepared to evaluate the significance of composition-independent parameters on blood/materials interactions. This study of the homologous polyurethane series showed that although bulk composition is maintained, material properties do vary with soft-segment block size.
The polymer microparticles were exposed to plasma and blood to assess their biocompatibility. Platelets and significantly depleted proteins were identified, and correlations to material structure evaluated. Variations in the adsorbed protein profiles substantiated the hypothesis that polyurethanes of similar structure manifest different surfaces. In this report, evidence is presented which demonstrates that surface chemistry and structure are differentiated in segmented polyurethanes (SPUs) of constant bulk composition, establishing the significance of surface features in determining protein and platelet interaction with polyurethane materials.
Intrinsic biological responses in the vascular system occur readily upon exposure to a foreign body. Such responses include protein adsorption, denaturation, and desorption; cellular interaction, release, and spreading; and immunologic probing, interaction, and activation. This complex and multifactor response may result synergistically in thrombosis, cellular encapsulation, and activated immunological attack--reactions dependent on the interaction of cellular elements and proteins with the surface of synthetic materials. Very small chemical and morphological variations in the surface character of a polymer will affect the adsorption profile of interacting plasma proteins.
Materials in contact with blood are exposed to a mixture of protein adsorbates that dominates the available surface area as a dynamically forming surface layer, activated or passivated by the underlying synthetic surface. The concentration of the adsorbate and the conformation of the constituent proteins will determine the adsorption and activation of subsequently interacting proteins and platelets.
The character of the adsorbed protein layer that mediates subsequent events is thought to be dependent on the properties of the substrate surface. In this study, a homologous series of polyurethane microparticles featuring a constant ratio of hard segment to soft segment was produced to evaluate the significance of composition-independent parameters on blood/materials interactions. The polyurethanes varied in soft-segment molecular weight, and thus length, morphology, and surface structure. The microparticles were exposed to plasma and blood to assess their biocompatibility. Plasma composition was evaluated both before and after polymer exposure in order to determine the significant material factors affecting protein adsorption and ascertain the composition profile of the adsorbed protein layer.
MATERIALS AND METHODS
Polyurethane Materials. A series of polyether-urethane block copolymer microbeads, varying in poly(tetramethylene oxide) soft-segment molecular weight from 650 to 2900 amu, were prepared using a method described elsewhere.1 The polyurethanes were of a constant hard-segment concentration of 65 weight percent and were designated as PU-65-650 to PU-65-2900, indicative of hard-segment weight percent and soft-segment block molecular weight. Although bulk concentration was held constant, the surface concentration and phase morphology did vary among the polymers in the series, resulting in a relative maximum in phase separation and soft-segment surface concentration at intermediate soft-segment block molecular weight. Table I summarizes the polymers used in the study and their properties. (Tables not yet available on-line.)
Protein Adsorption. The specific adsorption of proteins was studied using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) according to the procedures of Ho, et al.2 The method entails the analysis of the variation of optical densities of specific silver-stained protein spots on PAGE slab gels. The proteins, which originated from a mixture of plasma pooled from human donors, were loaded onto the gel following exposure to biomaterial particles. Depleted plasma solution concentrations were compared with unexposed plasma controls to obtain adsorbed quantities.
Following the 4-hour exposure time, an aliquot of the supernatant plasma dilution was loaded onto one of 20 polyacrylamide tube gels in the presence of a purified protein standard of constant concentration, so as to normalize gel-to-gel variations in staining. The tube gels were electrophoresed, separating the peptides by their isoelectric points (ranging from 3 to 10). The protein-containing tube gels were then layered onto slab gels to separate the proteins into the second--molecular weight--dimension. Gel staining followed methods described elsewhere.3
The stained gels were then imaged using a charge-coupled device (Photometrics LTD, Tucson, AZ) and the optical densities of individual proteins were determined mathematically using Image 1.26 densitometry software (W. Rasband, NIH). The percent depletion was determined by normalizing the optical density of each individual protein spot to the standard protein optical density from each gel. The normalized optical density of the depleted protein was then compared with the normalized optical density of that same protein from a slab gel used to separate and evaluate the protein from the unexposed plasma control. Proteins evaluated included albumin, IgG, apo A1 and A4 lipoproteins, fibrinogen, antitrypsin, transferrin, a2 and b haptoglobins, complement C3, b glycoproteins, hemopexin, antithrombin, antichymotrypsin, and prealbumin.
Platelet Adhesion. Platelet adhesion was measured using the method of Grevelink, et al.4 Autologous platelets were removed from the bloodstream and labeled with 111indium. The radiolabeled platelets were then returned to the bloodstream after being mixed with a small portion of heparinized blood. Platelet adhesion was measured by exposing polyurethane microparticles to the blood containing excess radiolabeled platelets. After 1 hour of exposure time, the radioactivity of a sample of blood was measured and compared with a sample of similar size drawn from blood unexposed to the polyurethane particles. The percent depletion is indicative of the concentration of adsorbed platelets.
Analysis of the microparticles produced for this experiment indicated that although the polyurethanes have the same hard-to-soft-segment ratio, their bulk and surface properties varied with the soft-segment precursor block molecular weight. Not surprisingly, the domain sizes varied as a function of the block size. Differential scanning calorimetry (DSC) was used to evaluate the bulk hard- and soft-segment-phase purity of the block copolymer (see Table I). PU-65-650 and PU-65-2900 showed little indication of soft-segment thermal transitions, and hard-segment transitions were shifted from 10° to 25° lower. The intermediate block sizes, PU-65-1000 and PU-65-2000, exhibited strong thermal transitions consistent with the constituent homopolymers and indicative of phase purity.
Electron spectroscopy for chemical analysis (ESCA) indicated that the low-energy, highly flexible amorphous soft segment in these polyurethane particles is enhanced at the air or vacuum surface relative to the ordered, inflexible, higher-energy hard segment. Intermediate-block-size polymers demonstrated a significant, though incomplete, enhancement of soft segment at the surface, which was not observed to the same extent with very short or very long soft-segment polyurethanes. The multidomain surface is modeled in Figure 1.
Contact-angle and inverse-chromatography investigations demonstrated that probe molecules--including water, bimolecular gases, and peptides--differentiate the surface of the various polyurethanes in the series. Variation in the elution times indicated stronger polar interactions with the PU-65-650 and PU-65-2900 copolymers, consistent with higher surface hard-segment concentration. The contact-angle hysteresis shows that a heterogeneous surface of hard and soft domains does exist on all copolymers studied in this series.
Based on the polymer analyses conducted, surface and interfacial models of the PU-65-650, PU-65-1000, PU-65-2000, and PU-65-2900 polyurethanes were developed. Models are shown in Figure 1. Intermediate-sized, well-phase-separated domains that are depleted in surface hard segment are observed for the PU-65-1000 and PU-65-2000 polyurethanes.
The characterized polyurethane particles were exposed to bovine and human plasma to evaluate the effect of the subtle surface and bulk polymer differences observed on the individual copolymer protein adsorption profiles. A sampling of the protein adsorption on each copolymer is shown in Table II. The results of the experiment indicate that the proteins do differentiate surfaces and adsorb in unique profiles on each of the similar polyurethane surfaces. Fibrinogen, hemopexin, and lipoprotein were strongly attracted by most surfaces. Albumin, IgG, and antithrombin III (AT III) were not heavily depleted from solution.
It has been broadly accepted that the establishment of a protein overlayer precedes the adhesion of platelets in blood-exposed biomaterials. This protein profile largely determines how platelets adhere to the protein-treated surface. Table III presents the adhesion of platelets to the particles after brief exposure. As observed in Table III, the platelets also differentiate the protein-coated surfaces. Only poor correlations between the adsorption of individual proteins and platelet adhesion were observed. The best correlation appears to be between total protein adsorption and platelet adhesion.
To identify key correlations between material properties and measures of biocompatibility, a tatra plot was constructed after the method of Andrade, et al.5 Because biocompatibility is not a simple, single-factor relationship, the tatra plot was chosen as the most effective means of graphically representing significant biocompatibility parameters. The plot has four two-axis quadrants, which roughly separate the data into biological response, morphology, composition, and surface adsorption. For this study, the following parameters were chosen: hard-segment domain size from stoichiometry and crystal structure; melt temperature from DSC; bulk hard segment from infrared spectroscopy; surface-chemistry ratios from ESCA; cosine * from contact-angle measurements on cast films; peptide retention from inverse liquid chromatography; protein adsorption from electrophoresis; and platelet adhesion using radiolabeled platelets.
Assuming that the model accurately defines the significant parameters, the trace of a polyurethane sample with low protein and platelet interactions--a good biological response--should be close to the origin in the biological-response quadrant (the east and northeast axes). This was observed for PU-65-1000 and PU-65-2000, as shown in Figure 2. By observing the shape of the tracing in the other quadrants, a predictor of biocompatibility can be developed. The current series of experiments indicated that intermediate-sized hard-segment domains with high phase purity are less thrombogenic in blood interaction than are those polyurethanes with larger or smaller hard-segment domains or less phase purity. The surface of polyurethane biomaterials should be well phase-separated and low in hard-segment concentration at the surface, and should exhibit intermediate contact-angle values.
The mechanism for reduced protein adsorption and platelet retention may be explained as a combination of two hypotheses. Proteins are complex multidomain copolymers whose behavior is strongly determined by their conformation. The domains of a single protein are frequently quite different energetically. On a rigid homopolymer, only one type of surface is available for interaction with the divergent protein domains. If the protein environment does not coincide with the energetics of the protein, the conformation of the protein may change to minimize the total energy of the system. Accommodation of a multidomain protein may best be achieved by presenting a multidomain surface, with which the protein may interact without forcing a conformational change.
A second explanation of the diminished biological adsorption on well-phase-separated polyurethane substrates is that the dynamic substrate itself changes surface structure to accommodate its newly acquired protein environment. The substrate may harmlessly adjust to the energy of the adsorbed protein, rather than allowing the protein to be activated through its conformational change as it accommodates the substrate.
Both of these hypotheses suggest that well-phase-separated copolymers of intermediate soft-segment length (PU-65-1000 and PU-65-2000) may be most effective in reducing protein and platelet interaction at the surface.
The characterization of the polyurethane microbead surfaces indicated that although the materials studied are of similar bulk composition, their bulk and surface properties may differ. These subtle variations in surface composition and morphology can be differentiated via protein and platelet adsorption.
Based upon these studies, we have observed that both protein and platelet adsorption decrease with increasing phase purity. This indicates the significance of polymer morphology on the in vitro interaction of polyurethanes with blood.
Small, but significant, amounts of hard-segment phase at the surface act to minimize the concentration of both total adsorbed protein and adhered platelets. The pure-phase homopolymer had higher protein and platelet adsorption than did the higher-phase-purity PU-65-1000 and PU-65-2000 copolymers, indicating that a range of surface hard-segment concentrations may be important to optimize the structure of blood-compatible polyurethane applications.
The correlation between protein and platelet adhesion also indicates the mediating nature of protein layers on platelet adsorption. The microphase-separated structure of polyurethanes is manifest through the protein layer and is important in determining platelet response to a foreign material. Specific protein trends were also observed: high lipoprotein, fibrinogen, and hemopexin concentration correlates with low platelet adhesion. The adsorption of lipoprotein is enhanced on heterogeneous SPU surfaces. Short soft-segment block sizes result in large amounts of adsorbed fibrinogen but low numbers of adhered platelets. Large soft-segment block sizes result in lower amounts of adsorbed fibrinogen and higher amounts of adherent platelets.
This work was funded by a grant from Becton Dickinson Vascular Access. Significant technical input was provided by Joseph D. Andrade, Vladimir Hlady, and Karin Caldwell, all from the Department of Bioengineering at the University of Utah. Chi-Hu Ho assisted in the two-dimensional electrophoresis experiments.
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2. Ho CH, Hlady V, Nyquist G, et al., "Interaction of Plasma Proteins with Heparinized Gel Particles Studied by High-Resolution Two-Dimensional Gel Electrophoresis," J Biomed Mater Res, 25:423443, 1991.
3. Burgess-Cassler A, Johansen JJ, Santek DA, et al., "Computerized Quantitative Analysis of Coomasie-Blue Stained Serum Proteins Separated by Two-Dimensional Electrophoresis," Clin Chem, 35:22972304, 1989.
4. Grevelink JM, Wu F, Kolff WJ, et al., "Study of Platelet Function in a Calf with Artificial Ventricles Attached to an Ex Vivo Shunt," Trans Am Soc Artif Intern Organs, 32:177180, 1986.
5. Andrade JD, Hlady V, and Wei AP, "Adsorption of Complex Proteins at Interfaces," Pure and Appl Chem, 84:17771781, 1992.
James M. (Mick) Lambert, PhD, a staff scientist with the Bard Urological Division of C.R. Bard, Inc. (Covington, GA), has worked in the disposable medical products industry for approximately 10 years. He is listed as inventor or coinventor on seven U.S. patents. Kevin G. Tingey, PhD, is a senior scientist with Becton Dickinson Vascular Access (Sandy, UT), a division of Becton Dickinson and Co.